Microalgae culture method

Abstract

The invention relates to a microalgae culture method, which comprises the following steps: carrying out first culture on a first microalgae species, and separating the cultured material to obtain a first culture clear liquid; a second microalgae species and the first culture clear liquid are mixed, second culture and third culture are sequentially carried out, and the third culture is carried out in the presence of a grease inducer. According to the method disclosed by the invention, the grease content of a microalgae product can be remarkably improved on the premise of not influencing the microalgae biomass accumulation level, and the utilization value of microalgae biomass is improved.

Description

Technical Field

[0001] The present disclosure relates to the field of microalgae biotechnology, and in particular, to a microalgae cultivation method. Background Art

[0002] Photosynthesis is the process by which organisms use light energy as an energy source to absorb CO2 and convert it into organic matter. It is an efficient means of CO2 fixation that has evolved in nature over billions of years. In the context of a green economy, the recycling of resources has become an important force in supporting the sustainable development of human society. How to make high-value use of the microalgae biomass obtained from fixing CO2 has become an indispensable part of microalgae carbon fixation technology. Fats are mainly used as energy storage substances and components of cell structures in microalgae cells. Among them, triglycerides are the main form of energy storage in cells, while the biomembrane composed of phospholipids and glycolipids is the structural basis for the existence of microalgae cells. The fatty acid chain length in microalgae cells is usually between C 12 ~C 18 The properties of the fatty acid esters obtained are similar to those of diesel, so it is also called microalgae biodiesel. Obtaining fatty acids from the total lipids of microalgae, and then obtaining fatty acid esters through esterification, can be used as fuel. Increasing the total lipid content of microalgae is conducive to improving the economic efficiency of microalgae bioenergy.

[0003] At present, the main methods for increasing the oil content of microalgae are to use high light intensity or nitrogen-deficient culture environments, genetically modify algae species, and add exogenous substances to the microalgae culture system to make the oil content in microalgae cells higher than that in normal culture methods. The above methods, especially the addition of exogenous substances, will affect the growth of microalgae. Therefore, it is still necessary to find a method to increase the oil content of microalgae cells without affecting the accumulation of microalgae biomass. Summary of the invention

[0004] The purpose of the present disclosure is to provide a microalgae cultivation method to significantly increase the total lipid content of microalgae cells without affecting the accumulation of microalgae biomass.

[0005] In order to achieve the above object, the present disclosure provides a microalgae cultivation method, which comprises the following steps:

[0006] Carrying out a first culture of the first microalgae species and separating the cultured material to obtain a first culture supernatant;

[0007] The second microalgae species is mixed with the first culture supernatant and subjected to second culture and third culture in sequence, wherein the third culture is carried out in the presence of a lipid inducer.

[0008] Optionally, the first microalgae species is selected from at least one of Chlorella pyrenoidosa, Chlorella vulgaris and Chlorella ellipsoidea;

[0009] The second microalgae species is selected from at least one of Chlorella vulgaris, Chlorella ellipsoidea, Scenedesmus obliquus and Monopterus punctatus.

[0010] Optionally, the first microalgae species is a Chlorella pyrenoidosa species, and the second microalgae species is a Chlorella vulgaris species.

[0011] Optionally, the first culture conditions include: temperature of 25-32°C, light intensity of 0-15000lx, light cycle of 8-16h, stirring speed of 50-350rpm, algae liquid pH of 7-8, and culture time of 48-96h.

[0012] Optionally, the separation is ultrafiltration separation, and the conditions of the first ultrafiltration separation include: a molecular weight cutoff of 5 to 100 kDa.

[0013] Optionally, the weight ratio of the second microalgae species to the first culture supernatant is 1:(5-100).

[0014] Optionally, the time ratio of the second culture to the third culture is 1:(1-10).

[0015] Optionally, the second culture is aeration stirring culture, and the conditions of the second culture include: based on each liter of algae liquid, the CO2 ventilation volume is 0.1-3L / h, the temperature is 25-32°C, the light intensity is 5000-15000lx, the light cycle is 12-16h, the stirring speed is 50-350rpm, the pH value of the algae liquid is 7-8, and the culture time is 24-48h;

[0016] The third culture is aeration and stirring culture, and the conditions of the third culture include: based on each liter of algae liquid, the CO2 ventilation volume is 0.1-3L / h, the temperature is 25-32°C, the light intensity is 5000-15000lx, the light cycle is 12-16h, the stirring speed is 50-350rpm, the algae liquid pH is 7-8, and the culture time is 48-120h.

[0017] Optionally, the oil inducer is one or more selected from carbonyl cyanide-3-chlorophenylhydrazone, peracetic acid, 2-mercapto-1-methylimidazole, diphenyl iodonium chloride and phenylalanine-arginine-β-naphthylamide.

[0018] Optionally, the oil inducer is carbonyl cyanide-3-chlorophenylhydrazone and / or peracetic acid.

[0019] Optionally, the final concentration of the oil inducer is 0.1-5.0 mmol / L.

[0020] Through the above technical scheme, the present disclosure first cultures the first microalgae species, and then cultures the second microalgae species with the first culture supernatant, which can induce the antioxidant system in the cells of the second microalgae species, so that when the oil inducer is subsequently added, it can not only avoid oxidative damage that affects biomass accumulation, but also fully accept the effect of the oil inducer to improve the oil synthesis ability, thereby significantly improving the oil content of the microalgae product without affecting the microalgae biomass accumulation level, and improving the utilization value of the microalgae biomass. The method disclosed in the present disclosure can simultaneously obtain the first microalgae biomass with a high protein content and the second microalgae biomass with a high oil content, and has the advantages of simple operation, strong practicality, and a wide range of applications.

[0021] Other features and advantages of the present disclosure will be described in detail in the following detailed description. DETAILED DESCRIPTION

[0022] The specific embodiments of the present disclosure are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure, and are not used to limit the present disclosure.

[0023] The present invention provides a method for cultivating microalgae, which comprises the following steps:

[0024] Carrying out a first culture of the first microalgae species and separating the cultured material to obtain a first culture supernatant;

[0025] The second microalgae species is mixed with the first culture supernatant and subjected to second culture and third culture in sequence, wherein the third culture is carried out in the presence of a lipid inducer.

[0026] The present invention first cultivates the first microalgae species, and then uses the first culture supernatant to cultivate the second microalgae species, which can induce the antioxidant system in the cells of the second microalgae species, so that when the oil inducer is added later, it can not only avoid oxidative damage that affects biomass accumulation, but also fully accept the effect of the oil inducer to improve the oil synthesis ability, thereby significantly improving the oil content of the microalgae product without affecting the microalgae biomass accumulation level, and improving the utilization value of the microalgae biomass. Using the method of the present invention, the first microalgae biomass with a high protein content and the second microalgae biomass with a high oil content can be obtained at the same time, which improves the scope of application of the present invention. When the first microalgae species is subjected to the first cultivation under high nitrogen conditions, it is also conducive to further obtaining microalgae biomass with a high protein content, which effectively solves the defect that high protein and high fat biomass cannot be obtained at the same time in the traditional microalgae cultivation method. The method has the advantages of simple operation, strong practicality, and a wide range of applications.

[0027] According to the present disclosure, the first microalgae species can be selected from at least one of Chlorella pyrenoidosa, Chlorella vulgaris and Chlorella ellipsoidea; the second microalgae species can be selected from at least one of Chlorella vulgaris, Chlorella ellipsoidea, Tetradesmus obliquus and Monoraphidium sp. In a preferred embodiment, the first microalgae species is Chlorella pyrenoidosa, which has a high protein content; the second microalgae species is Chlorella vulgaris, which has a high fat content; this is conducive to further improving the protein and total fat content of the microalgae product.

[0028] According to the present disclosure, the first culture can be carried out under conventional microalgae culture conditions in the art and using a culture medium well known in the art. Specifically, the first culture conditions may include: a temperature of 25 to 32°C, a light intensity of 0 to 15000 lx, a light cycle of 8 to 16 hours, a stirring speed of 50 to 350 rpm, a culture time of 48 hours to 96 hours, and a pH regulator (such as 5% nitric acid by mass) is used to control the pH value of the algae solution to 7 to 8.

[0029] The first culture can use glucose as a carbon source and nitric acid as a nitrogen source supplement and pH regulator to ensure that the nitrogen source in the culture system is abundant, so that the first microalgae species grows rapidly in large quantities and maintains a high protein content. In a specific embodiment, the culture medium used for the first culture may include the following components: glucose 0-30 g / L, NaNO3 200-10000 mg / L, K2HPO4 200-2000 mg / L, Na2EDTA 500 mg / L, MgSO4·7H2O 1000 mg / L, CaCl2·2H2O 111 mg / L, CuSO4·5H2O 15.7 mg / L, FeCl3·7H2O 49.8 mg / L, MnCl2·H2O14.2 mg / L, Co(NO3)2·6H2O 15.7 mg / L, ZnSO4·7H2O 88.2 mg / L, Na2MoO4·2H2O11.92 mg / L, and H3BO3 114.2 mg / L. The inoculation amount of the first microalgae species (i.e., the weight ratio of the first microalgae species to the culture medium used in the first culture) can be 1:(5-100), and individual culture medium components can be supplemented as needed during the first culture process, as long as the content of each component is within the above range. The above culture medium can provide sufficient nutrition for the growth and protein accumulation of the first microalgae species.

[0030] In one embodiment, when the optical density of the microalgae cells of the material after the first culture is OD 680 >80, the cultured material is separated to obtain a first culture supernatant and a first microalgae product, and the first culture supernatant is used for subsequent culture. The separation may be ultrafiltration separation, and the conditions for the ultrafiltration separation may include: using an ultrafiltration component with a molecular weight cutoff of 5 to 100 kDa for the ultrafiltration separation. The obtained first microalgae product has a high protein content, specifically, the protein content of the first microalgae product may be 30 to 50% by weight.

[0031] According to the present disclosure, the weight ratio of the second microalgae species to the first culture supernatant may be 1:(5-100), preferably 1:(5-20).

[0032] The second culture and the third culture can be carried out under conventional microalgae culture conditions in the art. In one embodiment, the time ratio of the second culture to the third culture can be 1:(1-10) in hours to further increase the oil content of the microalgae product. The second culture and the third culture can be CO2 ventilation and stirring culture. Specifically, the conditions of the second culture may include: based on each liter of algae liquid, the CO2 ventilation volume is 0.1~3L / h, the temperature is 25~32℃, the light intensity is 5000~15000lx, the light cycle is 12~16h, the stirring speed is 50~350rpm, the pH value of the algae liquid is 7~8, and the culture time is 24~48h; the conditions of the third culture may include: based on each liter of algae liquid, the CO2 ventilation volume is 0.1~3L / h, the temperature is 25~32℃, the light intensity is 5000~15000lx, the light cycle is 12~16h, the stirring speed is 50~350rpm, the pH value of the algae liquid is 7~8, and the culture time is 48~120h.

[0033] The oil inducer is used to promote the production of oil in microalgae cells. In one embodiment, the oil inducer is one or more selected from carbonyl cyanide-3-chlorophenylhydrazone, peracetic acid, 2-mercapto-1-methylimidazole, diphenyl iodonium chloride and phenylalanine-arginine-β-naphthylamide. Preferably, the oil inducer is carbonyl cyanide-3-chlorophenylhydrazone and / or peracetic acid. When the oil inducer is carbonyl cyanide-3-chlorophenylhydrazone and peracetic acid, the ratio of the molar concentration of the carbonyl cyanide-3-chlorophenylhydrazone and peracetic acid can be 1: (0.1~10). The above-mentioned types of oil inducers are beneficial for significantly increasing the total lipid content of microalgae cells without affecting the accumulation of microalgae biomass. The oil inducer can achieve the oil induction effect when used in a small amount. Specifically, the final concentration of the oil inducer can be 0.1~5.0mmol / L, preferably 0.5~2.0mmol / L.

[0034] The material obtained after the third culture is separated to obtain a second microalgae product with a higher total lipid content. Specifically, the total lipid content of the second microalgae product can be 30-40% by weight.

[0035] In the present disclosure, the first culture, the second culture, and the third culture can be carried out in a bioreactor. The bioreactor can be an open reactor or a closed reactor with an artificial light source.

[0036] The present disclosure is further described in detail below in conjunction with embodiments, but the present disclosure is not limited thereto.

[0037] The microalgae species used in the embodiments are the protein nucleus Chlorella species deposited in the Freshwater Algae Seed Bank of the Chinese Academy of Sciences and numbered FACHB-1516; the protein nucleus Chlorella species deposited in the Freshwater Algae Seed Bank of the Chinese Academy of Sciences and numbered FACHB-1560; the common Chlorella species deposited in the Freshwater Algae Seed Bank of the Chinese Academy of Sciences and numbered FACHB-2338; the common Chlorella species deposited in the Freshwater Algae Seed Bank of the Chinese Academy of Sciences and numbered FACHB-2723; the ellipsoidal Chlorella species deposited in the Freshwater Algae Seed Bank of the Chinese Academy of Sciences and numbered FACHB-962, and the monospinous algae species deposited in the Freshwater Algae Seed Bank of the Chinese Academy of Sciences and numbered FACHB-1975.

[0038] The test method for optical density (OD680) is as follows: Use a 752 UV-Vis spectrophotometer, detect at a wavelength of 680nm, and select the absorbance (A) mode. Add deionized water to a 1cm glass cuvette, place it in the spectrophotometer detection tank, close the detection chamber cover, and press the 100% T (0% A) button. When the absorbance reading is 0.000, take out the cuvette, discard the deionized water in it, add algae solution, place it in the detection tank, close the detection chamber cover, and read the absorbance value A. If the A value is not within the range of 0.2 to 0.9, dilute the algae solution with a dilution factor of N until the A value is within the range of 0.2 to 0.9 as the test result. Calculate the optical density (OD680) of the algae solution according to the following formula.

[0039] Optical density (OD680) = A × N

[0040] Example 1

[0041] (1) First training

[0042] In a closed bioreactor, 500 mL of Chlorella vulgaris FACHB-1516 algae was added to 5 L of the first culture medium, the pH of the algae solution was controlled at 7.5±0.5, and the first culture was carried out for 72 h at 28°C, light intensity of 6000 lx, light cycle of 12 h, and stirring at 200 rpm. The culture medium composition is glucose 30g / L, NaNO3 5000mg / L, K2HPO4 1500mg / L, Na2EDTA500mg / L, MgSO4·7H2O 1000mg / L, CaCl2·2H2O 111mg / L, CuSO4·5H2O 15.7mg / L, FeCl3·7H2O 49.8mg / L, MnCl2·H2O 14.2mg / L, Co(NO3)2·6H2O 15.7mg / L, ZnSO4·7H2O 88.2mg / L, Na2MoO4·2H2O 11.92mg / L, H3BO3 114.2mg / L. Glucose and other nutrients are supplemented as needed during the culture process. When the algae solution OD 680>80, the culture was stopped, and the material after the first culture was ultrafiltered and separated using an ultrafiltration component with a molecular weight cutoff of 30 kDa. The ultrafiltration was stopped when the volume of the filtrate reached 80% of the volume of the original algae liquid. The ultrafiltered concentrated algae liquid was centrifuged at 10,000 rpm, and the centrifuged supernatant was combined with the ultrafiltration supernatant to obtain the first culture supernatant.

[0043] (2) Second and third training

[0044] Take 4L of the first culture supernatant, add 400mL of common Chlorella FACHB-2723 algae species (the weight ratio of the second microalgae species to the first culture supernatant is 1:10), and culture under the conditions of temperature 28°C, light intensity 10000lx, light cycle 12h, stirring speed 150rpm, and ventilation stirring gas volume of 1L / h per liter of algae liquid. CO2 is introduced into the algae liquid to control the pH of the algae liquid at 7.5±0.5. After the second culture for 24 hours, the lipid inducer carbonyl cyanide-3-chlorophenylhydrazone is added to the final concentration of 0.1mmol / L, and the third culture is continued under the same conditions for 96h.

[0045] Example 2

[0046] The microalgae were cultured according to the method of Example 1, except that in step (2), the final concentration of carbonyl cyanide-3-chlorophenylhydrazone was 2.0 mmol / L.

[0047] Example 3

[0048] The microalgae were cultured according to the method of Example 1, except that in step (2), the final concentration of carbonyl cyanide-3-chlorophenylhydrazone was 5.0 mmol / L.

[0049] Example 4

[0050] The microalgae were cultured according to the method of Example 2, except that in step (2), the lipid inducer was peracetic acid.

[0051] Example 5

[0052] The microalgae were cultured according to the method of Example 2, except that in step (2), the lipid inducer was carbonyl cyanide-3-chlorophenylhydrazone and peracetic acid, wherein the molar concentration ratio of carbonyl cyanide-3-chlorophenylhydrazone to peracetic acid was 1:1.

[0053] Example 6

[0054] The microalgae were cultured according to the method of Example 2, except that in step (2), the lipid inducer was phenylalanine-arginine-β-naphthamide.

[0055] Example 7

[0056] Microalgae are cultured according to the method of Example 2, except that the first microalgae species used in step (1) is Chlorella vulgaris FACHB-2338; and the second microalgae species used in step (2) is Chlorella pyrenoidosa FACHB-1560.

[0057] Example 8

[0058] Microalgae were cultured according to the method of Example 2, except that the first microalgae species used in step (1) was Chlorella vulgaris FACHB-962, and the second microalgae species used in step (2) was Monoacanthus fascicularis FACHB-1975.

[0059] Example 9

[0060] The microalgae were cultured according to the method of Example 2, except that in step (2), the second culture time was 16 h, and the third culture time was 12 h.

[0061] Example 10

[0062] The microalgae were cultured according to the method of Example 2, except that in step (2), 4 L of the first culture supernatant was taken and 40 mL of Chlorella vulgaris FACHB-2723 algae species was added (the weight ratio of the second microalgae species to the first culture supernatant was 1:100).

[0063] Comparative Example 1

[0064] The microalgae were cultured according to the method of Example 2, except that in step (2), no lipid inducer was added.

[0065] Comparative Example 2

[0066] The microalgae were cultured according to the method of Example 2, except that step (1) was not performed, and in step (2), the first culture supernatant was not used, but the second microalgae species were cultured using a culture medium containing the following components: 5.6 g / L glucose, 2630 mg / L NaNO3, 760 mg / L K2HPO4, 500 mg / L Na2EDTA, 1000 mg / L MgSO4·7H2O, 111 mg / L CaCl2·2H2O, 15.7 mg / L CuSO4·5H2O, 49.8 mg / L FeCl3·7H2O, 14.2 mg / L MnCl2·H2O, 15.7 mg / L Co(NO3)2·6H2O, 88.2 mg / L ZnSO4·7H2O, 11.92 mg / L Na2MoO4·2H2O, and 114.2 mg / L H3BO3.

[0067] Comparative Example 3

[0068] The microalgae were cultured according to the method of Example 2, except that in step (2), the second culture was not performed, but the third culture was performed directly, that is, 4 L of the first culture supernatant was taken, 400 mL of Chlorella vulgaris FACHB-2723 algae species were added (the weight ratio of the second microalgae species to the first culture supernatant was 1:10), and the lipid inducer carbonyl cyanide-3-chlorophenylhydrazone was added to a final concentration of 0.1 mmol / L, and the culture was carried out under the conditions of temperature 28°C, light intensity 10000 lx, light cycle 12 h, stirring speed 150 rpm, ventilation stirring gas volume of 1 L / h per liter of algae liquid, CO2 was introduced into the algae liquid, the pH of the algae liquid was controlled at 7.5±0.5, and the third culture was performed for 96 h.

[0069] Comparative Example 4

[0070] (1) Autotrophic cultivation of Chlorella

[0071] Sterile BG11 liquid culture medium was added to a 2L sterile conical flask, and the added volume was 1L to prepare BG11 liquid culture medium. Then, the purified Chlorella vulgaris preserved in the laboratory and liquid activated was inoculated into the BG11 liquid culture medium, and the inoculation density was OD 700 =0.05(about 1.1×10 6 cells / mL), and finally at a temperature of 25°C and a light intensity of 70 μmol / m - 2 s -1 Cultivated under continuous aeration conditions.

[0072] (2) Oxidant-induced treatment

[0073] With 700nm as the scanning wavelength, a standard curve between OD value and biomass was established. By measuring the OD value of the liquid culture medium, the concentration of biomass in the liquid culture medium was calculated. In the actual culture process, the OD value of the liquid culture medium can be measured in real time using a spectrophotometer to determine the growth period of the microalgae. 700 Reach 1.5 (about 1.5×10 7 cells / mL), a solution containing methyl viologen, an oxidant, was added dropwise to the BG11 liquid culture medium at a rate of 1.5 mL / min, and the BG11 liquid culture medium containing the oxidant was stirred uniformly at a speed of 70 rpm using a magnetic stirrer with a sterile rotor. When the final concentration of methyl viologen was 0.15 mM, the addition of the solution containing the oxidant was stopped, and the autotrophic culture of Chlorella was continued for 24 hours.

[0074] Test Example

[0075] The total lipid content and total lipid yield of the second microalgae product obtained in the above examples and comparative examples, as well as the protein content and protein yield of the first microalgae product (a microalgae product obtained by comparative examples 2 and 4) were detected. The results are listed in Table 1.

[0076] Total lipid content detection method: Take 1L of the material after the third culture, centrifuge at 10000rpm, take out the algae mud obtained by centrifugation, dry it in an oven at 105℃ to constant weight, and obtain the second microalgae product, weighed as W0. Take about 2.0g of the dried biomass of the second microalgae product, weigh it as W1, put it into a filter paper bag, and weigh it as W2. Put the filter paper bag containing biomass into a Soxhlet extractor, add methanol: chloroform with a volume ratio of 2:1, and extract at 90℃ for 6h. After the extraction, take out the filter paper bag, evaporate the solvent to constant weight in a fume hood, and weigh it as W3. Calculate the total lipid content and total lipid yield of microalgae biomass according to the following formula.

[0077] Total fat content (%) = [(W3-W2) / W1] × 100%

[0078] Total lipid yield (g / L) = [(W3-W2) / W1] × W0

[0079] Protein content detection method: Take 1L of the material after the first culture, centrifuge at 10000rpm, take out the algae mud obtained by centrifugation, dry it in an oven at 105℃ to constant weight, and obtain the first microalgae product, weighed as W0'. Take a small amount of dry biomass of the first microalgae product, place it in a mortar, add liquid nitrogen and grind it into fine powder. Weigh 0.2g of powder, add 10mL of water, shake and mix evenly, and freeze-thaw twice with liquid nitrogen. Add 80μL of 50% NaOH solution (mass ratio of algae powder to NaOH 5:1), mix evenly and boil for 30min, invert and mix once every 5min. After cooling to room temperature, ultrasonically treat it in an ultrasonic cleaner for 10min. Centrifuge at 5000rpm for 5min, dilute the supernatant appropriately (so that the protein mass concentration is about 10-120mg / L), the dilution factor is F, and the protein concentration is measured by Bradford method.

[0080] Staining solution: 100 mg Coomassie Brilliant Blue G250, add 50 mL ethanol, 100 mL 85% phosphoric acid and 600 mL water, filter with filter paper after fully dissolving, and add water to the filtrate to make up to 1 L.

[0081] Take 1 mL of the diluted sample, add 5 mL of staining solution, mix well, and measure the absorbance at 595 nm using a spectrophotometer. Use bovine serum albumin (BSA) to prepare a solution with a known protein concentration and make a standard curve. Calculate the protein content and yield of the dried microalgae sample according to the following formula:

[0082] Protein content (%) = (153.54 × A 595 ﹣8.0862)×F×0.01×100% / 200

[0083] Protein yield (g / L) = protein content (%) × W0'

[0084] Where: A 595 is the absorbance of the test sample at a wavelength of 595nm; F is the dilution factor.

[0085] Table 1

[0086]

[0087] As can be seen from Table 1, the method disclosed herein can significantly increase the total lipid content and total lipid yield of the second microalgae product, while being conducive to obtaining a first microalgae product with a higher protein content.

[0088] The preferred embodiments of the present disclosure are described in detail above; however, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0089] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0090] In addition, various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. A method for cultivating microalgae, characterized in that: The method comprises the following steps: Carrying out a first culture of the first microalgae species and separating the cultured material to obtain a first culture supernatant; The second microalgae species is mixed with the first culture supernatant and subjected to second culture and third culture in sequence, wherein the third culture is carried out in the presence of a lipid inducer.

2. The method according to claim 1, wherein: The first microalgae species is selected from at least one of Chlorella pyrenoidosa, Chlorella vulgaris and Chlorella ellipsoidea; The second microalgae species is selected from at least one of Chlorella vulgaris, Chlorella ellipsoidea, Scenedesmus obliquus and Monopterus.

3. The method according to claim 1 or 2, wherein: The first microalgae species is Chlorella pyrenoidosa species, and the second microalgae species is Chlorella vulgaris species.

4. The method according to claim 1, wherein: The first culture conditions include: temperature of 25-32°C, light intensity of 0-15000lx, light cycle of 8-16h, stirring speed of 50-350rpm, algae liquid pH of 7-8, and culture time of 48-96h.

5. The method according to claim 1, wherein: The separation is ultrafiltration separation, and the conditions of the first ultrafiltration separation include: a molecular weight cut-off of 5 to 100 kDa.

6. The method according to claim 1, wherein: The weight ratio of the second microalgae species to the first culture supernatant is 1:(5-100).

7. The method according to claim 1, wherein: The time ratio of the second culture to the third culture is 1:(1-10).

8. The method according to claim 1 or 7, wherein: The second culture is aeration and stirring culture, and the conditions of the second culture include: based on each liter of algae liquid, the CO2 ventilation rate is 0.1-3L / h, the temperature is 25-32°C, the light intensity is 5000-15000lx, the light cycle is 12-16h, the stirring speed is 50-350rpm, the pH value of the algae liquid is 7-8, and the culture time is 24-48h; The third culture is aeration and stirring culture, and the conditions of the third culture include: based on each liter of algae liquid, the CO2 ventilation volume is 0.1-3L / h, the temperature is 25-32°C, the light intensity is 5000-15000lx, the light cycle is 12-16h, the stirring speed is 50-350rpm, the algae liquid pH is 7-8, and the culture time is 48-120h.

9. The method according to claim 1, wherein: The oil and fat inducer is one or more selected from carbonyl cyanide-3-chlorophenylhydrazone, peracetic acid, 2-mercapto-1-methylimidazole, diphenyl iodonium chloride and phenylalanine-arginine-β-naphthylamide.

10. The method according to claim 1 or 9, wherein: The oil and fat inducer is carbonyl cyanide-3-chlorophenylhydrazone and / or peracetic acid.

11. The method according to claim 1, wherein: The final concentration of the oil inducer is 0.1-5.0 mmol / L.

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