Microalgae culture method

Abstract

The invention relates to the technical field of microalgae culture, and discloses a microalgae culture method. The invention relates to a microalgae culture method, which comprises the following steps: (1) in the presence of a fat degradation inhibitor, culturing microalgae species to obtain a microalgae solution; (2) separating, adsorbing and degerming the algae solution to obtain a culture solution; and (3) returning the culture solution to the step (1) for reuse. The method can effectively control the generation of organic matters with a growth inhibition effect in the microalgae culture process, improve the production rate and reduce the culture water consumption.

Description

Technical Field

[0001] This invention relates to the technical field of microalgae cultivation, and more specifically to a method for microalgae cultivation. Background Technology

[0002] Large-scale microalgae cultivation requires substantial water resources. Reusing the culture medium can reduce the demand for freshwater and nutrients. Therefore, recycling the culture medium after algal cell harvesting is crucial for the economic feasibility and environmental sustainability of the microalgae industry. However, after a period of cultivation, the culture medium deteriorates significantly. Compared to fresh culture medium, recycling inhibits algal growth, reduces biomass yield, and may even halt further cultivation. The main reason for culture medium deterioration is likely that microalgae produce and secrete organic matter into the culture environment during their own metabolism, inhibiting algal growth. These metabolic products are called extracellular organic matter. This extracellular organic matter mainly includes: ① organic matter secreted into the culture medium from within or on the surface of microalgae cells; ② protocellular organic matter that enters the culture medium after microalgae die and lyse. The harmful effect of extracellular organic matter is primarily its inhibitory effect on microalgal growth. Generally, extracellular organic matter of microalgae is mainly composed of fatty acids, polysaccharides, and proteins, primarily originating from intracellular and cell membrane components of microalgae.

[0003] Different types of microalgal growth inhibitors exhibit different growth-inhibiting mechanisms. For example, fatty acids can damage plasma membranes, including mitochondrial and chloroplast membranes, leading to impaired cellular energy supply and acquisition, reduced respiratory and photosynthetic activity, and even cell lysis due to membrane damage. Polysaccharides can cause cell aggregation, affecting nutrient uptake from the environment and slowing cell growth. Humic acid may impair intracellular catalase, leading to hydrogen peroxide accumulation and oxidative damage. Furthermore, some inhibitors also participate in intracellular signal transduction processes, inducing programmed cell death.

[0004] The presence of growth inhibitors in microalgae circulating culture media significantly limits its practical reuse. This is particularly true for heterotrophic and multitrophic microalgae cultivation. With the addition of organic matter, microalgae cells grow rapidly, reaching very high cell densities in a short time. Consequently, extracellular organic matter in the culture medium accumulates to high levels, exerting a far more pronounced inhibitory effect on microalgae growth than in autotrophic cultivation. Therefore, to achieve culture medium recycling and reduce water consumption in multitrophic microalgae cultivation, the key lies in reducing the secretion of growth-inhibiting organic matter and removing growth inhibitors from the culture medium. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of inhibiting microalgae growth and increasing water consumption after recycling microalgae culture medium in the existing technology, and to provide a microalgae cultivation method that can effectively control the generation of organic matter with growth-inhibiting effect during microalgae cultivation and reduce cultivation water consumption.

[0006] To achieve the above objectives, the present invention provides a method for cultivating microalgae, wherein the method includes the following steps:

[0007] (1) Microalgae were cultured in the presence of lipid degradation inhibitors to obtain algal solution;

[0008] (2) The algal solution is separated, adsorbed and sterilized to obtain a culture medium;

[0009] (3) Return the culture medium to step (1) for reuse.

[0010] The method provided by this invention involves adding a lipid degradation inhibitor during microalgae cultivation to promote lipid synthesis, inhibit the production of free fatty acids and fatty acid degradation products, and prevent the accumulation of substances with microalgae growth-inhibiting effects in the culture medium. In addition, the method further removes the substances with microalgae growth-inhibiting effects through adsorption, thereby more effectively relieving the inhibitory effect on microalgae growth after recycling the microalgae culture medium, increasing the number of culture medium recycling cycles, thereby reducing the water consumption for preparing the culture medium and lowering the water consumption for microalgae cultivation. Detailed Implementation

[0011] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0012] This invention provides a method for cultivating microalgae, wherein the method includes the following steps:

[0013] (1) Microalgae were cultured in the presence of lipid degradation inhibitors to obtain algal solution;

[0014] (2) The algal solution is separated, adsorbed and sterilized to obtain a culture medium;

[0015] (3) Return the culture medium to step (1) for reuse.

[0016] The method provided by this invention involves adding a lipid degradation inhibitor during microalgae cultivation to promote lipid synthesis, inhibit the production of free fatty acids and fatty acid degradation products, and prevent the accumulation of substances with microalgae growth-inhibiting effects in the culture medium. In addition, the method further removes the substances with microalgae growth-inhibiting effects through adsorption, thereby more effectively relieving the inhibitory effect on microalgae growth after recycling the microalgae culture medium, increasing the number of culture medium recycling cycles, thereby reducing the water consumption for preparing the culture medium and lowering the water consumption for microalgae cultivation.

[0017] In this invention, there is no particular limitation on the specific species of microalgae. The method provided by this invention is particularly suitable for the cultivation of high-protein microalgae. Preferably, in step (1), the microalgae species is an oil-based microalgae species, further preferably selected from at least one of Chlorella pyrenoidosa, Chlorella vulgaris, Chlorella ellipsoidea, and Scenedesmus spicata, and even more preferably selected from Chlorella pyrenoidosa. Using Chlorella pyrenoidosa species within the above-preferred range, due to its high protein content, is beneficial for further improving the utilization value of microalgal biomass.

[0018] In this invention, a fat degradation inhibitor is introduced to promote the synthesis of lipids and inhibit the production of free fatty acids and fatty acid degradation products, thus preventing the substances with microalgae growth-inhibiting effects from remaining in the culture medium. Preferably, in step (1), the fat degradation inhibitor is selected from at least one of orlistat, 1-(2,3,4-trimethoxybenzyl)piperazine, amiodarone, aloxacillin, and lansoprazole, more preferably from at least one of orlistat, 1-(2,3,4-trimethoxybenzyl)piperazine, and amiodarone, and even more preferably orlistat. Using orlistat as a fat degradation inhibitor is beneficial for inhibiting the production of free fatty acids and fatty acid degradation products, preventing the accumulation of substances with microalgae growth-inhibiting effects in the algal culture medium, and improving the value of microalgae cultivation.

[0019] In this invention, preferably, the final concentration of the lipid degradation inhibitor in the algal solution is 5-100 nmol / L, more preferably 20-60 nmol / L. Controlling the final concentration of the lipid degradation inhibitor helps to inhibit the production of free fatty acids and fatty acid degradation products, avoids the accumulation of substances that inhibit microalgae growth in the algal solution, and improves the value of microalgae cultivation.

[0020] In this invention, by first culturing the microalgae strain and then introducing a lipid degradation inhibitor for a second culture, lipid synthesis can be promoted and the production of free fatty acids and fatty acid degradation products can be inhibited. Preferably, in step (1), the culture includes: first culturing the microalgae strain, and then adding a lipid degradation inhibitor for a second culture. The advantage of this preferred embodiment is that it facilitates the rapid entry of microalgae into the logarithmic growth phase, increases the rate of lipid accumulation, and avoids the adverse inhibitory effect of the lipid degradation inhibitor on the initial growth of microalgae after inoculation.

[0021] In this invention, there is no particular limitation on the cultivation method described in step (1). The cultivation methods conventionally defined in the art can be applied to this invention, such as open cultivation or closed cultivation, preferably closed cultivation.

[0022] In this invention, preferably, the cultivation is carried out in a closed bioreactor.

[0023] In this invention, the first and second cultures can be carried out under microalgae culture conditions conventionally defined in the art, and this invention does not impose any particular limitation on them. Preferably, in step (1), the conditions for the first and second cultures each independently include: a temperature of 25-32℃, a light intensity of 0-15000 lx, a light cycle of 8-16 h, a stirring speed of 50-350 rpm, and a pH value of 7-8 for the algal solution.

[0024] In this invention, preferably, the pH value of the algal solution is controlled by a pH adjuster. This invention does not have any particular limitation on the type and amount of pH adjuster, as long as the pH value of the algal solution is 7-8. For example, the pH adjuster can be nitric acid with a mass fraction of 5%.

[0025] In this invention, preferably, in step (1), the first culture time is 0-36h, and more preferably 12-36h.

[0026] In this invention, preferably, in step (1), the second culture time is 12-120h, and more preferably 40-120h.

[0027] In this invention, in step (1), the first culture and the second culture are each carried out independently in the culture medium.

[0028] In this invention, there are no particular limitations on the composition of the culture medium. Any culture medium conventionally defined in the art can be applied to this invention. For example, carbon sources and nitrogen sources can be added to water as the culture medium, glucose can be used as the carbon source, and nitric acid can be used as a nitrogen source supplement and pH adjuster to ensure that the culture system is rich in nitrogen source, thereby enabling the microalgae species to grow rapidly in large quantities and maintain a high protein content. Preferably, the culture medium contains the following components: carbon source 1-30 g / L, nitrogen source 200-10000 mg / L, phosphorus source 200-2000 mg / L, magnesium source 200-2000 mg / L, calcium source 50-500 mg / L, iron source 20-200 mg / L, trace elements 30-1200 mg / L, chelating agent 100-2000 mg / L, such as glucose 1-30 g / L, NaNO3 200-10000 mg / L, K2HPO4 200-2000 mg / L, Na2EDTA (chelating agent) 100-1000 mg / L, MgSO4·7H2O 200-2000 mg / L, CaCl2·2H2O 50-500 mg / L, CuSO4·5H2O 5-200 mg / L, FeCl3·7H2O 20-200 mg / L, MnCl2·H2O 5-200 mg / L, Co(NO3)2·6H2O 5-200 mg / L, ZnSO4·7H2O 5-200 mg / L, Na2MoO4·2H2O 5-200 mg / L, H3BO3 5-200 mg / L, of which CuSO4·5H2O, MnCl2·H2O, Co(NO3)2·6H2O, ZnSO4·7H2O, Na2MoO4·2H2O, and H3BO3 are trace elements.

[0029] In this invention, preferably, in step (1), the volume ratio of the microalgae species to the culture medium is 1:5-100, and the volume of the culture medium refers to the volume of water in the culture medium.

[0030] In this invention, in a preferred embodiment, the method further includes: in step (1), the components lost during the cultivation of microalgae can be replenished to meet the requirements of the content of each component in the original culture medium (the original culture medium can be understood as the fresh culture medium before cultivation).

[0031] In this invention, there is no particular limitation on the separation method in step (2). Preferably, in step (2), the separation is ultrafiltration separation.

[0032] In this invention, there are no particular limitations on the conditions for ultrafiltration separation, as long as the microalgal cells and macromolecular organic matter in the algal solution can be retained, and the ultrafiltrate is free of microalgal cells and macromolecular organic matter. Preferably, the conditions for ultrafiltration separation include a molecular weight cutoff of 5-100 kDa.

[0033] In this invention, preferably, the ultrafiltration separation is performed within an ultrafiltration module. This invention does not specifically limit the type of ultrafiltration module, as long as the molecular weight cutoff of the ultrafiltration module meets the above-mentioned requirements. Those skilled in the art can select the appropriate module based on actual needs. In this invention, the degree of ultrafiltration separation is not specifically limited. Preferably, ultrafiltration separation can be stopped when the filtrate volume reaches 50-95% of the algal solution volume, yielding a superfine ultrafiltrate.

[0034] In this invention, preferably, in step (2), the adsorption is carried out in the presence of an adsorbent. The adsorbent further removes substances that inhibit microalgae growth, thereby more effectively relieving the inhibitory effect on microalgae growth after recycling the microalgae culture medium, increasing the number of times the culture medium is recycled, thus reducing the water consumption for preparing the culture medium and lowering the water consumption for microalgae cultivation.

[0035] In this invention, there is no particular limitation on the type of adsorbent; any adsorbent conventionally defined in the art is applicable. Preferably, the adsorbent is selected from at least one of activated carbon, diatomaceous earth, XAD-2 resin, XAD-4 resin, XAD-7HP resin, XAD-8 resin, and D001 resin, more preferably activated carbon, and even more preferably granular activated carbon. The above-mentioned adsorbent can adsorb organic matter with microalgae growth inhibitory effects in the ultrafiltration solution onto the surface of the adsorbent, thereby significantly reducing the content of such organic matter in the treated solution and further relieving the inhibitory effect of repeated reuse of the culture medium on microalgae growth.

[0036] In this invention, preferably, the adsorption is carried out in an adsorbent-filled adsorbent. This invention does not particularly limit the specific type of adsorbent, but preferably it can be a chromatography column.

[0037] In this invention, there is no particular limitation on the amount of adsorbent used. Preferably, the amount of adsorbent accounts for 30-90% of the volume of the adsorber.

[0038] In this invention, the purified liquid after adsorption treatment is subjected to microfiltration sterilization to remove bacteria from the purified liquid. The apparatus used for sterilization in this invention is not particularly limited. Preferably, in step (2), the sterilization is performed in a microfiltration assembly.

[0039] In this invention, preferably, the average pore size of the microfiltration component is 0.01-0.2 μm.

[0040] In this invention, there is no particular limitation on the specific type of microfiltration component. Any filter conventionally defined in the art that can achieve filtration and sterilization and meets the above-mentioned average pore size range can be applied to this invention, such as a sterile filter.

[0041] In this invention, the culture medium obtained after sterilization is returned to step (1) to inoculate microalgae species to start the next round of microalgae culture.

[0042] In this invention, there is no particular limitation on the method of introducing microalgae species. For example, it can be method one: take out all the culture medium, sterilize the reactor and then introduce microalgae species. At this time, the microalgae species refers to fresh microalgae species of the same type as the microalgae species in step (1). Alternatively, it can be method two: take out part of the culture medium and do not sterilize the reactor. At this time, the microalgae species is the remaining microalgae species after taking out part of the algal liquid in step (1). In the preferred case, method one is selected.

[0043] In this invention, preferably, the culture medium is reused 1-5 times in step (3).

[0044] In this invention, preferably, the volume ratio of the reused culture medium to the microalgae seed in step (3) is 50-2:1. It should be noted that when the microalgae seed is introduced by method one, the volume of the microalgae seed here refers to the volume of the fresh microalgae seed; when the microalgae seed is introduced by method two, the volume of the microalgae seed here refers to the volume of the remaining microalgae seed in step (1).

[0045] The present invention will be described in detail below through embodiments.

[0046] The microalgae used in the examples are: Chlorella proteoglycans (FACHB-1516), Chlorella vulgaris (FACHB-2338), Sphaerophyta monophylla (FACHB-1975), and Scenedesmus spicata (FACHB-1221), all of which are preserved in the Freshwater Algae Culture Collection of the Chinese Academy of Sciences.

[0047] Optical density (OD) 680 The testing method is as follows: Use a 752 UV-Vis spectrophotometer with a detection wavelength of 680 nm and select absorbance (A) mode. Add deionized water to a 1 cm glass cuvette, place it in the spectrophotometer's detection chamber, close the detection chamber lid, and press the 100%T (0%A) button. When the absorbance reading reaches 0.000, remove the cuvette, discard the deionized water, add algal solution, place it in the detection chamber, close the detection chamber lid, and read the absorbance value A. If the A value is not within the range of 0.2-0.9, dilute the algal solution by a dilution factor of N until the A value falls within the range of 0.2-0.9, and use this as the detection result. Calculate the optical density (OD) of the algal solution according to the following formula. 680 ) and relative growth rate.

[0048] Optical density (OD) 680 )=A×N

[0049] Specific growth rate μ=(lnOD) 680 t2 -lnOD 680 t1 ) / (t2-t1)

[0050] Among them, OD 680 t1 The OD of the algal solution at time t1 680 OD 680 t2 The OD of the algal solution at time t2 680 .

[0051] The water consumption per unit of biomass is obtained by dividing the amount of water used to prepare the culture medium by the amount of biomass obtained from the culture. The volume of water used to prepare the culture medium is the volume of the culture medium.

[0052] Example 1

[0053] (1) Microalgae culture

[0054] In a closed bioreactor, 500 mL (OD) of Chlorella pyrenoidosa FACHB-1516 algal seed was taken. 680 =32.5) was added to 5L of culture medium, and the pH of the algal solution was controlled at 7.5±0.5. The algae were cultured at 28℃, light intensity of 6000lx, light cycle of 12h, and stirring at 200rpm. The culture medium consisted of 30 g / L glucose, 5000 mg / L NaNO3, 1500 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. After 24 hours of the first culture, filtered and sterilized orlistat solution was added, bringing the final concentration of orlistat in the algal solution to 40 nmol / L, for the second culture. During the cultivation process, glucose and other nutrients are supplemented as needed. Algal culture samples are taken at the beginning of the first cultivation (h0) and at the end of the second cultivation (h120) to measure OD. 680 Value, calculate the specific growth rate.

[0055] (2) Culture medium treatment

[0056] After a total culture time of 120 hours, cultivation was stopped, and the algal solution in the reactor was removed. Ultrafiltration was performed using an ultrafiltration unit with a molecular weight cutoff of 30 kDa. Ultrafiltration was stopped when the volume of the filtrate reached 85% of the original algal solution volume. The ultrafiltrate was then passed through a filter filled with granular activated carbon (Beijing Bailingwei Technology, particle size 8-20 mesh, specific surface area 1200 m²). 2 A chromatography column with a flow rate of / g) was used, and the ultrafiltrate flow rate was 200 mL·min. -1 The chromatography column had an inner diameter of 2 cm, a length of 70 cm, and an activated carbon packing height of 50 cm. The ultrafiltrate was circulated for adsorption for a total treatment time of 2 hours. The treated supernatant was then sterilized by passing it through a sterile filter with a pore size of 0.1 μm at a flow rate of 200 mL / min. -1 .

[0057] (3) Culture medium reuse

[0058] The empty closed bioreactor was sterilized, and the sterilized culture medium was introduced into the reactor, followed by inoculation with Chlorella proteoglycans FACHB-1516. The volume ratio of the algae to the reused culture medium was 1:10. After supplementing with glucose, NaNO3, K2HPO4, and other trace element nutrients, the culture was continued according to the method in (1). The culture medium was recycled twice.

[0059] Example 2

[0060] Microalgae were cultured according to the method of Example 1, except that in step (1), the final concentration of orlistat in the algal solution was 5 nmol / L, and the other conditions were the same as in Example 1.

[0061] Example 3

[0062] Microalgae were cultured according to the method of Example 1, except that in step (1), the final concentration of orlistat in the algal solution was 100 nmol / L, and the other conditions were the same as in Example 1.

[0063] Example 4

[0064] Microalgae were cultured according to the method of Example 1, except that in step (1), the lipid degradation inhibitor was amiodarone, the final concentration of amiodarone in the algal solution was 60 nmol / L, and other conditions were the same as in Example 1.

[0065] Example 5

[0066] Microalgae were cultured according to the method in Example 1, except that in step (2), the adsorbent was XAD-7HP resin (Amberlite XAD-7HP macroporous adsorption resin with a characteristic pore size of 45 nm and a specific surface area of ​​500 m²). 2 / g), other conditions are the same as in Example 1.

[0067] Example 6

[0068] The microalgae were cultured according to the method of Example 1, except that in step (1), the algae used was Scenedesmus fusiforme FACHB-1221 (OD). 680 =28.7), and other conditions are the same as in Example 1.

[0069] Example 7

[0070] The microalgae were cultured according to the method of Example 1, except that in step (1), the volume of the algal seed was 100 mL, glucose was 10 g / L, NaNO3 was 2000 mg / L, K2HPO4 was 500 mg / L, the final concentration of orlistat in the algal solution was 20 nmol / L, the first culture time was 12 h, the second culture time was 96 h, and in step (3), the volume ratio of the algal seed to the reused culture medium was 1:5.

[0071] Comparative Example 1

[0072] Microalgae were cultured according to the method of Example 1, except that orlistat solution was not added in step (1).

[0073] Comparative Example 2

[0074] Microalgae were cultured according to the method of Example 2, except that in step (1), orlistat solution was not added and in step (2), adsorption treatment was not performed.

[0075] The specific growth rate and water consumption per unit biomass for fresh culture medium and recycled culture medium are shown in Table 1.

[0076] Table 1

[0077]

[0078] Note: The specific growth rate of fresh culture medium refers to the specific growth rate of the culture medium measured at the end of the second culture without the culture medium being reused, and it serves as a control sample.

[0079] As shown in Table 1, the cultivation method of the present invention can significantly reduce the inhibitory effect of the culture medium on microalgae growth after two reuses and reduce the overall water consumption for microalgae production. As can be seen from Example 1 and Comparative Example 1, when no lipid degradation inhibitor is added, the specific growth rate of the fresh culture medium in Example 1 is lower than that in Comparative Example 1. However, the specific growth rate of the culture medium after two reuses is much higher than that in Comparative Example 1. This indicates that the cultivation method of the present invention can significantly reduce the generation of free fatty acids and other lipid degradation products during microalgae cultivation, avoid the accumulation of substances with microalgae growth inhibitory effects in the culture medium, improve the production rate of microalgae cultivation, and reduce water consumption.

[0080] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for cultivating microalgae, characterized in that, The method includes the following steps: (1) Microalgae were cultured in the presence of lipid degradation inhibitors to obtain algal solution; (2) The algal solution is separated, adsorbed and sterilized to obtain a culture medium; (3) Return the culture medium to step (1) for reuse.

2. The method according to claim 1, wherein, In step (1), the microalgae species is an oily microalgae species, preferably selected from at least one of Chlorella pyrenoidosa, Chlorella vulgaris, Chlorella ellipsoidosa, and Scenedesmus cuspidatum.

3. The method according to claim 1 or 2, wherein, In step (1), the lipid degradation inhibitor is selected from at least one of orlistat, 1-(2,3,4-trimethoxybenzyl)piperazine, amiodarone, aloxacillin and lansoprazole, preferably selected from at least one of orlistat, 1-(2,3,4-trimethoxybenzyl)piperazine and amiodarone.

4. The method according to claim 3, wherein, In step (1), the fat degradation inhibitor is provided by a solution containing the fat degradation inhibitor; Preferably, in step (1), the final concentration of the lipid degradation inhibitor in the algal solution is 5-100 nmol / L, and more preferably 20-60 nmol / L.

5. The method according to any one of claims 1-4, wherein, In step (1), the cultivation includes: first culturing the microalgae strain, and then adding a lipid degradation inhibitor for a second culture; Preferably, in step (1), the conditions for the first culture and the second culture each independently include: a temperature of 25-32℃, a light intensity of 0-15000lx, a light cycle of 8-16h, a stirring speed of 50-350rpm, and a pH value of 7-8 for the algal solution. Preferably, in step (1), the first culture time is 0-36h, more preferably 12-36h; Preferably, in step (1), the second culture time is 12-120h, and more preferably 40-120h.

6. The method according to claim 5, wherein, In step (1), the first culture and the second culture are each carried out independently in the culture medium; Preferably, the culture medium contains the following components: carbon source 1-30 g / L, nitrogen source 200-10000 mg / L, phosphorus source 200-2000 mg / L, magnesium source 200-2000 mg / L, calcium source 50-500 mg / L, iron source 20-200 mg / L, trace elements 30-1200 mg / L, and chelating agent 100-2000 mg / L; Preferably, in step (1), the volume ratio of the microalgae species to the culture medium is 1:5-100.

7. The method according to any one of claims 1-6, wherein, In step (2), the separation is ultrafiltration separation, and the preferred conditions for ultrafiltration separation include: a molecular weight cutoff of 5-100 kDa.

8. The method according to any one of claims 1-7, wherein, In step (2), the adsorption is carried out in the presence of an adsorbent, preferably selected from at least one of activated carbon, diatomaceous earth, XAD-2 resin, XAD-4 resin, XAD-7HP resin, XAD-8 resin and D001 resin.

9. The method according to any one of claims 1-8, wherein, In step (2), the sterilization is carried out in a microfiltration module, preferably with an average pore size of 0.01-0.2 μm.

10. The method according to any one of claims 1-9, wherein, In step (3), the volume ratio of the reused culture medium to the microalgae species is 50-2:1.

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