CN111500464A - Method for producing lutein by first mixotrophic-later autotrophic microalgae - Google Patents
Method for producing lutein by first mixotrophic-later autotrophic microalgae Download PDFInfo
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- KBPHJBAIARWVSC-RGZFRNHPSA-N lutein Chemical compound C([C@H](O)CC=1C)C(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\[C@H]1C(C)=C[C@H](O)CC1(C)C KBPHJBAIARWVSC-RGZFRNHPSA-N 0.000 title claims abstract description 57
- 229960005375 lutein Drugs 0.000 title claims abstract description 57
- KBPHJBAIARWVSC-XQIHNALSSA-N trans-lutein Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2C(=CC(O)CC2(C)C)C KBPHJBAIARWVSC-XQIHNALSSA-N 0.000 title claims abstract description 57
- FJHBOVDFOQMZRV-XQIHNALSSA-N xanthophyll Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2C=C(C)C(O)CC2(C)C FJHBOVDFOQMZRV-XQIHNALSSA-N 0.000 title claims abstract description 57
- 239000001656 lutein Substances 0.000 title claims abstract description 54
- 235000012680 lutein Nutrition 0.000 title claims abstract description 53
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- 238000004519 manufacturing process Methods 0.000 title claims abstract description 22
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- 229910052799 carbon Inorganic materials 0.000 claims abstract description 49
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- 230000008569 process Effects 0.000 claims description 11
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- 238000012258 culturing Methods 0.000 claims description 9
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical group [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 8
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- 239000001632 sodium acetate Substances 0.000 claims description 8
- 235000017281 sodium acetate Nutrition 0.000 claims description 8
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- 235000010344 sodium nitrate Nutrition 0.000 claims description 8
- 230000006698 induction Effects 0.000 claims description 6
- 241000195649 Chlorella <Chlorellales> Species 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 5
- 235000015097 nutrients Nutrition 0.000 claims description 5
- 235000008210 xanthophylls Nutrition 0.000 claims description 4
- 230000008376 long-term health Effects 0.000 claims description 2
- 230000001737 promoting effect Effects 0.000 claims 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 16
- 230000001276 controlling effect Effects 0.000 description 11
- 230000007774 longterm Effects 0.000 description 5
- 229910002651 NO3 Inorganic materials 0.000 description 4
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 235000005881 Calendula officinalis Nutrition 0.000 description 3
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- C12N1/12—Unicellular algae; Culture media therefor
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Abstract
The invention relates to a method for producing lutein by using first mixotrophic-second autotrophic microalgae, which comprises the following steps of 1) a mixotrophic growth stage, wherein microalgae seed liquid is connected into a photobioreactor containing a fermentation culture medium, when the dissolved oxygen value in a system is increased to 3-7 mg/L, organic carbon source solution is fed, the concentration of the organic carbon source is enabled to reach 1-3 g/L, the rapid growth of algae cells is promoted in a mixotrophic mode, 2) when the biomass of the algae cells reaches 3.0-7.0 g/L, the feeding of the organic carbon source is stopped, the algae cells enter an autotrophic stage, and the algal cell lutein is induced to accumulate in an autotrophic mode.
Description
Technical Field
The invention belongs to the field of microbial fermentation processes, and particularly relates to a method for producing lutein by using mixotrophic-autotrophic microalgae.
Background
Lutein is an oxygen-containing carotenoid and is widely applied to industries such as food, feed additives, health care products, medicines and the like due to the characteristics of strong oxidation resistance, strong inflammation resistance, strong coloring and the like, the commercially available lutein is mainly derived from Marigold, but the problems of slow growth speed, seasonal harvesting, high labor cost, large occupied arable land resources and the like exist when the Marigold is used for producing the lutein, and the like, in recent years, the microalgae for producing the lutein is more and more concerned (L in, J.H., lTtT transfer = L &l/T &gTtTtTtTtree, D.J., Chang, J.S.,2015. L utein production of lutein: Marigold colors, Bioresource, Technology, 184, 421 o, etc.).
At present, the culture mode for producing lutein by utilizing microalgae mainly comprises autotrophy, heterotrophy and mixotrophy, the autotrophy culture of microalgae is a common mode for producing lutein, and the important roles of lutein in absorbing and transmitting light energy, quenching triplet chlorophyll, avoiding photooxidation damage of photosynthetic mechanism and the like (Jahns, P., holzwitter, A.R., 2012, the role of the xanthophyll cycle and of luminescence in photoprocessing II. BBA-Bioenergetics 1817(1), 182 and 193) are mainly played, so that algae cells can accumulate higher lutein content under autotrophy condition (Chen, J.H., Chen, C.Y., Chang, J.S., 2017a, L tissue production with long-term and tissue strain of luteinChlorella sorokinianaMB-1 undersexotropic growth. Journal of the Taiwan Institute of Chemical Engineers,79: 66-73.) but under autotrophic conditions, the growth rate of algal cells is relatively slow, limiting their commercial use to some extentng, C.H., Ng, I.S., Lee, D.J.,Chang, J.S., 2018. A highly efficient two-stage cultivation strategy forlutein production using heterotrophic culture ofChlorella sorokinianaMB-1-M12 Bioresource Technology, 253: 141-147) and under mixotrophic conditions, microalgae growth has the metabolic advantages of autotrophy and heterotrophy, and aerobic respiration and photosynthesis can be carried out by using both organic carbon sources and inorganic carbon sources under illumination conditionsChlorella sorokinianaMB-1 undermixotrophic growth. Journal of the Taiwan Institute of Chemical Engineers,79: 66-73; Chen, C.Y., Ho, S.H., Liu, C.C., Chang, J.S., 2017b. Enhancinglutein production withChlorella sorokinianaMb-1 by optimizing acetate and nitrate concentrations of acetic acid and microorganisms, 79: 88-96, acetate, glucose and glycerol are the most common organic carbon sources in microalgae growth, wherein the problem of contamination of mixotrophic processes is alleviated because acetate is relatively inexpensive and rarely used by bacteria, and is suitable for outdoor large-scale cultivation (Chen, C.Y., Ho, S.H., &lTtTrandtransfer = L "&gTtL &lTt/T &gTtI, C.C., Chang, J.S., 2017b. Enhancing protein production with its ownChlorella sorokinianaMb-1 bytopting acetate and nitrate concentrations of under mixotpic growth. journal of the Taiwan Institute of Chemical Engineers,79: 88-96.) addition of acetate in appropriate amounts promotes algal cell growth, but at too high a concentration can inhibit algal cell growth (Chen, C.Y., Ho, S.H., &. lTtT transfer = L "&. gTtL &. lTtT/T gTtI, C.C., Chang, J.S., 2017b. Enhancing viral reduction withChlorella sorokinianaMb-1 by optimizing acetate and nitrate control scheme under the need of mixotrophic growth. Journal of the Taiwan Institute of chemical Engineers,79: 88-96). In addition, acetate also inhibits the synthesis of proteins required for photosynthesis to some extent, thereby reducing the rate of photosynthesis, which in turn affects algal cell lutein accumulation (Chen, c.y., Ho, S).H., Liu,C.C., Chang, J.S., 2017b. Enhancing lutein production withChlorella sorokinianaMb-1 by optimizing acetate and nitrate controls undersexxographic growth. Journal of the Taiwan Institute of Chemical Engineers,79: 88-96). Therefore, when the microalgae uses acetate as an organic carbon source to produce lutein, a fed-batch culture strategy is considered to be adopted to control the concentration of the acetate in a culture solution, so that the adverse effects of high-concentration acetate on the growth of algae cells and the accumulation of lutein are avoided. However, at present, the acetate in the culture solution needs to be measured by devices such as liquid chromatography and ion chromatography, so that a certain hysteresis exists in the detection result, the consumption condition of the acetate in the culture solution cannot be known in real time, the fed-batch process of the acetate cannot be well regulated, and the influence on the culture process is generated. Until now, no mature culture method has been available to rapidly monitor the concentration of organic carbon source in the process of microalgae mixotrophy and to solve the technical problems of microalgae lutein accumulation and cell growth at the same time.
Disclosure of Invention
In order to make up the defects of the prior art, the invention provides a fermentation process for producing lutein by mixotrophic-autotrophic microalgae, which can realize timely feeding control of an organic carbon source in the process of mixotrophic microalgae so as to promote the rapid growth of algae cells, and further can ensure that the algae cells can rapidly accumulate lutein under the autotrophic induction condition by switching the culture mode. By adopting the fermentation process, the rapid growth of the algae cells can be realized in a short culture period, and the algae cells can also accumulate high-content lutein, so that the technical problem that the accumulation of the lutein in the microalgae is contradictory to the growth of the cells is solved, and a novel method can be provided for the production of the lutein in the microalgae.
The technical scheme of the invention comprises the following steps:
1) and a long-term health preserving stage: inoculating the microalgae seed liquid into a photobioreactor filled with a fermentation medium, and controlling certain culture conditions for fermentation culture; when the dissolved oxygen value in the culture system rises to a certain concentration, starting to add an organic carbon source solution in a pulse flow manner; meanwhile, when the concentration of the nitrogen source in the culture solution is reduced to a certain concentration, the pulse flow is started to add the fermentation culture medium concentrated solution which does not contain the organic carbon source, and the rapid growth of the algae cells is promoted in a mixotrophic mode.
2) Autotrophic induction stage: stopping feeding the organic carbon source solution when the biomass of the algae cells reaches a certain concentration, and enabling the algae cells to enter an autotrophic stage from a mixotrophic stage; controlling a certain culture condition to carry out autotrophic culture, and starting pulse flow adding of a fermentation culture medium concentrated solution without containing an organic carbon source when the concentration of a nitrogen source in a culture solution is reduced to a certain concentration, so as to induce the accumulation of the xanthophyll in the algae cells in an autotrophic mode.
In the step 1), the fermentation medium is BG11 medium, wherein the concentration of sodium nitrate is adjusted to be 0.5-1.0 g/L, the concentration of an additionally added organic carbon source is 1-3 g/L, and the organic carbon source is sodium acetate.
In the step 1), the culture conditions are that the initial concentration of algae cells in the reactor is controlled to be 75-200 mg/L after inoculation, the temperature is controlled to be 25-35 ℃ in the culture process, and the illumination intensity is controlled to be 100-750 mu mol/m2And/s, keeping the concentration of carbon dioxide in the introduced gas at 1-4%, controlling the aeration amount at 0.02-0.2 VVM, and culturing for 3-5 days.
In step 1), whenever the dissolved oxygen value in the culture system rises to 3-7 mg/L, the organic carbon source solution of 200-400 g/L is fed in to make the concentration of the organic carbon source in the culture solution reach 1-3 g/L.
In the step 1) and the step 2), when the concentration of the nitrogen source in the culture solution is reduced to 50-100 mg/L, feeding a fermentation culture medium concentrated solution which does not contain an organic carbon source and is concentrated by 50-100 times, so that the concentration of the nutrient source reaches the initial concentration.
In the step 2), when the biomass of the algae cells reaches 3.0-7.0 g/L, stopping feeding the organic carbon source solution.
In the step 2), the culture conditions are as follows: the temperature is controlled to be 25-35 ℃, and the illumination intensity is controlled to be 100-750 mu mol/m2And/s, keeping the concentration of carbon dioxide in the introduced gas at 1-4%, controlling the aeration amount at 0.05-0.5 VVM, and culturing for 4-7 days.
In the step 2), the biomass concentration can reach 3.5-10.0 g/L, the lutein yield is 30-120 mg/L, and the lutein yield is 4.5-10.0 mg/L/d.
The photobioreactor is a closed flat plate photobioreactor, a column type photobioreactor or a pipeline type photobioreactor.
The algae species are microalgae of Chlorella.
The invention has the following remarkable advantages: the method fully utilizes the characteristic of aerobic respiration in the process of mixotrophic microalgae cultivation, namely, the dissolved oxygen value shows a rapid rising trend when the concentration of acetate in the culture solution is exhausted, and the dissolved oxygen value shows a gradual falling trend after the organic carbon source is supplemented, so that the timely feeding control of the organic carbon source can be realized by monitoring the change of the dissolved oxygen value in real time. The invention also makes full use of the characteristics of the microalgae that can realize the mixotrophic rapid growth and the autotrophic inducement of the lutein accumulation, adopts the first mixotrophic-second autotrophic mode to realize the rapid growth of the algae cells and ensure that the algae cells accumulate high-content lutein, and has short fermentation period and simple production process. Meanwhile, the method is also suitable for improving the production method of related products of various chlorella and can obviously improve the industrialization prospect of producing microalgae-based products by utilizing the chlorella.
Drawings
FIG. 1 is a graph showing the variation of biomass concentration, sodium nitrate concentration and lutein yield of Chlorella vulgaris in example 1.
FIG. 2 is a graph showing the change in the dissolved oxygen concentration and the sodium acetate concentration in the culture solution in example 1.
FIG. 3 is a HP L C profile of the xanthophyll assay of example 1.
Detailed Description
The embodiments of the present invention will be described in detail below with reference to the accompanying drawings: in the present example, experiments were performed on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of the present invention is not limited to the following examples.
The microalgae adopted in the specific embodiment of the invention are all chlorella.
Example 1
1) And a long-term maintenance stage, namely inoculating microalgae seed liquid into a 50L column type photobioreactor filled with a fermentation culture medium, whereinThe concentration of sodium nitrate in the fermentation medium is 0.75 g/L, the concentration of sodium acetate is 1 g/L, the initial concentration of algae cells in the reactor is controlled to be 100 mg/L after inoculation, the temperature is controlled to be 33 ℃ during the culture process, and the illumination intensity is controlled to be 350 mu mol/m2The concentration of carbon dioxide in the gas is kept at 2.5%, the aeration rate is controlled at 0.02 VVM, every time the dissolved oxygen value in the culture system is increased to 6 mg/L, 400 g/L g of organic carbon source solution is fed in, so that the concentration of the organic carbon source in the culture solution is 1 g/L, and every time the concentration of the nitrogen source in the culture solution is reduced to 75 mg/L, 100 times of concentrated fermentation medium concentrated solution which does not contain the organic carbon source is fed in, so that the concentration of the nutrient source reaches the initial concentration, the algae cells are promoted to grow rapidly by a mixotrophic mode after 3 days of culture, wherein the fermentation medium is BG11 medium.
2) An autotrophic induction stage, stopping feeding organic carbon source solution when the biomass of algae cells reaches 3 g/L, allowing the algae cells to enter autotrophic stage from mixotrophic stage, wherein the temperature is controlled at 33 deg.C and the illumination intensity is controlled at 350 μmol/m2And/s, keeping the concentration of carbon dioxide in the introduced gas at 2.5%, controlling the aeration rate at 0.05 VVM, starting to feed a concentrated fermentation medium solution not containing an organic carbon source every time the concentration of the nitrogen source in the culture solution is reduced to 75 mg/L, culturing for 4 days, and inducing the accumulation of lutein in the algal cells by an autotrophic mode.
In the whole fermentation culture process, sampling is carried out at regular intervals to determine biomass concentration, sodium nitrate concentration, sodium acetate concentration and lutein content, wherein the biomass concentration is determined by a cell dry weight method, the sodium nitrate concentration is determined by an ultraviolet spectrophotometry method, the sodium acetate concentration is determined by a total organic carbon analyzer, and the lutein content is determined by an HP L C method, according to a fermentation process curve shown in a figure 1, the biomass concentration is 3.5 g/L, the lutein yield is 33.6 mg/L, and the lutein yield is 4.7 mg/L/d after fermentation culture for 7 days, according to a fermentation process curve shown in a figure 2, in a long-term simultaneous culture period, when the acetate concentration in a culture solution is exhausted, the dissolved oxygen value is in a rapid rising trend, and after an organic carbon source is supplemented, the dissolved oxygen value is in a gradual falling trend, according to an HP L C spectrum shown in a figure 3, and through comparison with the retention time of HP L C of a corresponding standard product, the lutein peak emergence time is about 14.7 min.
Example 2
1) And a long-term stage of health preservation, namely inoculating the microalgae seed liquid into a 50L flat-plate photobioreactor filled with a fermentation culture medium, wherein the concentration of sodium nitrate in the fermentation culture medium is 1 g/L, the concentration of sodium acetate in the fermentation culture medium is 1 g/L, controlling the initial concentration of algae cells in the reactor to be 200 mg/L after inoculation, controlling the temperature to be 35 ℃ and the illumination intensity to be 750 mu mol/m in the culture process2The concentration of carbon dioxide in the gas was maintained at 4.0% and the aeration rate was controlled at 0.2 VVM, and every time the dissolved oxygen value in the culture system was increased to 7 mg/L, 400 g/L g of the organic carbon source solution was fed so that the concentration of the organic carbon source in the culture solution became 2 g/L, and every time the concentration of the nitrogen source in the culture solution was decreased to 100 mg/L, 100-fold concentration of the fermentation medium concentrate containing no organic carbon source was fed so that the concentration of the nutrient source became the initial concentration.
2) An autotrophic induction stage, stopping feeding organic carbon source when the biomass of algae cell reaches 7 g/L, allowing the algae cell to enter autotrophic stage from mixotrophic stage, and culturing at 35 deg.C and 750 μmol/m2And/s, keeping the concentration of carbon dioxide in the introduced gas at 4.0%, controlling the ventilation amount at 0.5 VVM, starting to feed a fermentation medium concentrated solution which does not contain an organic carbon source every time the concentration of a nitrogen source in the culture solution is reduced to 100 mg/L, culturing for 7 days, and inducing the accumulation of lutein in the algae cells in an autotrophic mode, wherein the fermentation medium is BG11 medium.
The detection method is the same as that in example 1, the fermentation culture is carried out for 12 days, the biomass concentration is 10 g/L, the lutein yield is 120 mg/L, and the lutein yield is 10.0 mg/L/d.
Example 3
1) And a long-term stage of cultivation, namely inoculating the microalgae seed liquid into a 300L pipeline type photobioreactor filled with a fermentation culture medium, wherein the concentration of sodium nitrate in the fermentation culture medium is 0.5 g/L, the concentration of sodium acetate is 3 g/L, controlling the initial concentration of algae cells in the reactor to be 75 mg/L after inoculation, controlling the temperature to be 25 ℃ and the illumination intensity to be 100 mu mol/m in the cultivation process2The concentration of carbon dioxide in the gas is kept at 1.0%, the aeration rate is controlled at 0.1 VVM, every time the dissolved oxygen value in the culture system is increased to 3 mg/L, 200 g/L g of organic carbon source solution is fed in, so that the concentration of the organic carbon source in the culture solution is 3 g/L, and every time the concentration of the nitrogen source in the culture solution is reduced to 50 mg/L, the fermentation medium concentrated solution which does not contain the organic carbon source and is concentrated by 50 times is fed in, so that the concentration of the nutrient source reaches the initial concentration, and the algae cells are cultured for 5 days to promote the rapid growth of the algae cells in a mixotrophic mode.
2) An autotrophic induction stage, stopping feeding organic carbon source when the biomass of algae cell reaches 3 g/L, allowing the algae cell to enter autotrophic stage from mixotrophic stage, and culturing at 25 deg.C and 100 μmol/m of illumination intensity2And/s, keeping the concentration of carbon dioxide in the introduced gas at 1.0%, controlling the ventilation amount at 0.5 VVM, starting to feed a fermentation medium concentrated solution which does not contain an organic carbon source every time the concentration of a nitrogen source in the culture solution is reduced to 50 mg/L, culturing for 5 days, and inducing the accumulation of lutein in the algae cells in an autotrophic mode, wherein the fermentation medium is BG11 medium.
The detection method is the same as that in example 1, the fermentation culture lasts for 10 days, the biomass concentration is 4.5 g/L, the lutein yield is 45 mg/L, and the lutein yield is 4.5 mg/L/d.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (10)
1. A method for producing lutein by mixotrophic-autotrophic microalgae is characterized by comprising the following steps:
1) and a long-term health preserving stage: inoculating the microalgae seed liquid into a photobioreactor filled with a fermentation medium, and controlling certain culture conditions for fermentation culture; when the dissolved oxygen value in the culture system rises to a certain concentration, feeding an organic carbon source solution to ensure that the organic carbon source in the culture solution reaches a certain concentration; meanwhile, when the concentration of the nitrogen source in the culture solution is reduced to a certain concentration, feeding a fermentation culture medium concentrated solution which does not contain an organic carbon source, and promoting the rapid growth of algae cells in a mixotrophic mode;
2) autotrophic induction stage: stopping feeding the organic carbon source solution when the biomass of the algae cells reaches a certain concentration, and enabling the algae cells to enter an autotrophic stage from a mixotrophic stage; controlling a certain culture condition to carry out autotrophic culture, and starting to feed a fermentation culture medium concentrated solution without containing an organic carbon source when the concentration of a nitrogen source in a culture solution is reduced to a certain concentration, so as to induce the accumulation of the xanthophyll in the algae cells in an autotrophic mode.
2. The method for producing lutein by using mixotrophic-autotrophic microalgae according to claim 1, wherein the fermentation medium in step 1) is BG11 medium, wherein the concentration of sodium nitrate is adjusted to 0.5-1.0 g/L, the concentration of the additional organic carbon source is 1-3 g/L, and the organic carbon source is sodium acetate.
3. The method for producing lutein by using mixotrophic-autotrophic microalgae according to claim 1, wherein the culture conditions in step 1) are that the initial concentration of algal cells in the reactor is controlled to be 75-200 mg/L after inoculation, the temperature is controlled to be 25-35 ℃ and the illumination intensity is controlled to be 100-750 μmol/m during the culture process2And/s, keeping the concentration of carbon dioxide in the introduced gas at 1-4%, controlling the aeration amount at 0.02-0.2 VVM, and culturing for 3-5 days.
4. The method for producing lutein by using autotrophic microalgae according to claim 1, wherein in step 1), every time the dissolved oxygen value in the culture system increases to 3-7 mg/L, the organic carbon source solution of 200-400 g/L is fed so that the concentration of the organic carbon source in the culture solution is 1-3 g/L.
5. The method for producing lutein by using autotrophic microalgae according to claim 1, wherein in step 1) and step 2), whenever the concentration of nitrogen source in the culture solution is reduced to 50-100 mg/L, the fermentation medium concentrated solution which does not contain organic carbon source and is concentrated by 50-100 times is fed to the culture solution to make the concentration of nutrient source reach the initial concentration.
6. The method for producing lutein by using mixotrophic-autotrophic microalgae according to claim 1, wherein in the step 2), the organic carbon source solution is stopped from being fed when the biomass concentration of the algal cells reaches 3.0-7.0 g/L.
7. The method for producing lutein by using mixotrophic-postautotrophic microalgae according to claim 1, wherein in step 2), the culture conditions are as follows: the temperature is controlled to be 25-35 ℃, and the illumination intensity is controlled to be 100-750 mu mol/m2And/s, keeping the concentration of carbon dioxide in the introduced gas at 1-4%, controlling the aeration amount at 0.05-0.5 VVM, and culturing for 4-7 days.
8. The method for producing lutein by using mixotrophic-autotrophic microalgae according to claim 1, wherein in the step 2), the biomass concentration is 3.5-10.0 g/L, the lutein yield is 30-120 mg/L, and the lutein yield is 4.5-10.0 mg/L/d.
9. The method for producing lutein by mixotrophic-autotrophic microalgae according to claim 1, wherein the photobioreactor is a closed flat plate photobioreactor, a column photobioreactor or a tube photobioreactor.
10. The method for producing lutein by mixotrophic-postautotrophic microalgae according to claim 1, wherein said microalgae is of the genus Chlorella.
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