Chlorella cultivation method

Abstract

The present disclosure relates to a method for culturing Chlorella, comprising the following steps: culturing a first Chlorella strain to obtain a first culture solution; culturing a second Chlorella strain in the presence of the first culture solution to obtain a second culture solution; mixing flue gas absorption liquid with the second culture solution to obtain a third culture solution; wherein the flue gas absorption liquid contains hydrogen peroxide and nitric acid. The present disclosure can fix inorganic nitrogen in the flue gas absorption liquid into Chlorella biomass, significantly increase the chlorophyll content of Chlorella, and improve the utilization value of Chlorella.

Description

Technical Field

[0001] This disclosure relates to the field of microalgae biotechnology, and more specifically, to a method for culturing Chlorella. Background Technology

[0002] Chlorophyll is the main pigment in plants that fix CO2 during photosynthesis. The chlorophyll molecule contains porphyrin, whose structure is very similar to that of porphyrin in human heme. Chlorophyll helps increase hemoglobin levels in the human body, improves oxygen supply, and has anti-fatigue effects. Modern medicine has found that chlorophyll can promote granulation tissue formation in ulcers and accelerate wound healing. In addition, chlorophyll also has antioxidant, blood pressure-lowering, blood lipid-lowering, antibacterial, and cardiovascular-improving effects. Currently, there are many health supplements on the market with chlorophyll as the main functional ingredient, making it an important category in the health supplement market.

[0003] Chlorella is a eukaryotic organism belonging to the Chlorophyta phylum. It boasts advantages such as rapid growth and high chlorophyll content, making it an important raw material for chlorophyll production. Chlorophyll can be divided into several subtypes based on subtle differences in its specific molecular structure. The chlorophyll in Chlorella cells is mainly chlorophyll a and chlorophyll b. The chlorophyll content (combined chlorophyll a and chlorophyll b) in Chlorella cells is typically around 0.5% to 2%. Increasing the chlorophyll content of Chlorella is beneficial for increasing the yield of chlorophyll extracts, reducing production costs, and improving the economic benefits of algal-derived chlorophyll health products.

[0004] Currently, the main method to increase the chlorophyll content of Chlorella is to add exogenous substances to the Chlorella culture system, resulting in a higher chlorophyll content in the Chlorella cells compared to normal culture methods. For example, CN201310004747.X discloses adding ether sugars from biomass pyrolysis or detoxified cellulose pyrolysis solution to the Chlorella culture medium. CN201810200594.9 discloses adding soybean sprout extract to the Chlorella culture medium. CN201811416377.X discloses adding a regulatory substance composed of sodium selenite, indomethacin, inositol, or FeCl3 to the Chlorella culture medium. The substances used in these methods to increase the chlorophyll content of Chlorella are sometimes derived from biomass extracts, and their composition fluctuates depending on the source of the raw materials and the production batch, making it difficult to consistently increase chlorophyll content. Others have complex compositions and use ingredients not permitted for use in food, increasing production costs and posing potential safety risks. Summary of the Invention

[0005] The purpose of this disclosure is to provide a method for culturing Chlorella to significantly increase the chlorophyll content of Chlorella.

[0006] To achieve the above objectives, this disclosure provides a method for culturing Chlorella, which includes the following steps:

[0007] The first Chlorella species was cultured for the first time to obtain the first culture medium;

[0008] The second Chlorella species was cultured in the presence of the first culture medium to obtain the second culture medium.

[0009] The flue gas absorbent is mixed with the second culture medium for a third culture to obtain a third culture medium; wherein the flue gas absorbent contains hydrogen peroxide and nitric acid.

[0010] Optionally, the first Chlorella species and the second Chlorella species are each independently selected from at least one of Chlorella pyrenoidosa, Chlorella vulgaris, Chlorella ellipsoidosa, Chlorella ehrlich, Chlorella kJ, and Chlorella thermophila.

[0011] Optionally, the conditions for the first culture include: a temperature of 25–32°C, a light intensity of 0–15000 lx, a light cycle of 8–16 h, a stirring speed of 50–350 rpm, a culture time of 48–96 h, and a pH value of 6.0–8.5 for the algal solution.

[0012] Optionally, the method further includes: performing a first ultrafiltration separation on the first culture medium, and then rotary evaporating the obtained filtrate to obtain a first culture concentrate, and using the first culture concentrate for the second culture;

[0013] The conditions for the first ultrafiltration separation include: a molecular weight cutoff of 5–100 kDa;

[0014] The conditions for rotary evaporation include: a temperature of 20–70°C, a pressure of -0.05 MPa to -0.1 MPa, and a time of 0.1–5 h.

[0015] Optionally, the weight ratio of the second Chlorella species to the first culture concentrate is 1:(0.5-10).

[0016] Optionally, the conditions for the second culture include: a temperature of 25–32°C, a light intensity of 0–15000 lx, a light cycle of 8–16 h, a stirring speed of 50–350 rpm, and a culture time of 0.5–24 h.

[0017] Optionally, the flue gas absorbent is obtained by contacting and absorbing flue gas with the absorbent; the NO content in the flue gas is less than 50% by volume.

[0018] Based on the total weight of the flue gas absorbent liquid, the content of hydrogen peroxide is 0.2-5% by weight, and the content of nitric acid is 5-20% by weight;

[0019] The weight ratio of the flue gas absorption liquid to the second culture medium is 1:(10-1000).

[0020] Optionally, the method further includes: adding the flue gas absorbent liquid to the second culture medium in a fed-batch manner; the fed-batch rate of the flue gas absorbent liquid is 1 to 10 mL / min based on the second culture medium per liter.

[0021] Optionally, the conditions for the third culture include: H2O2 concentration of 1–50 mg / L, NO3- concentration of 1–50 mg / L. - The concentration should not exceed 5 g / L, the pH value should be 6.0 to 8.5, the temperature should be 25 to 32℃, the light intensity should be 0 to 15000 lx, the photoperiod should be 8 to 16 h, the stirring speed should be 50 to 350 rpm, and the incubation time should be 48 to 96 h.

[0022] Optionally, the method further includes: performing a second ultrafiltration separation on at least a portion of the third culture medium, and using the resulting filtrate as the first culture medium for the second culture;

[0023] The conditions for the second ultrafiltration separation include a molecular weight cutoff of 5–100 kDa.

[0024] Through the above technical solution, this disclosure can fix inorganic nitrogen in flue gas absorbent into Chlorella biomass, significantly increasing the chlorophyll content of Chlorella and enhancing its utilization value. This method has the advantages of simple operation, low cost, and strong practicality.

[0025] Other features and advantages of this disclosure will be described in detail in the following detailed description section. Detailed Implementation

[0026] The following provides a detailed description of specific embodiments of this disclosure. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of this disclosure.

[0027] This disclosure provides a method for culturing Chlorella, the method comprising the following steps:

[0028] The first Chlorella species was cultured for the first time to obtain the first culture medium;

[0029] The second Chlorella species was cultured in the presence of the first culture medium to obtain the second culture medium.

[0030] The flue gas absorbent is mixed with the second culture medium for a third culture to obtain a third culture medium; wherein the flue gas absorbent contains hydrogen peroxide and nitric acid.

[0031] This disclosure uses Chlorella culture medium (i.e., the first culture medium) to cultivate Chlorella species, which can increase the catalase content in Chlorella cells. Then, it is further cultured with flue gas absorbent containing hydrogen peroxide and nitric acid. This not only removes inorganic nitrogen from the flue gas absorbent but also rapidly decomposes hydrogen peroxide, thereby significantly increasing the chlorophyll content of the produced Chlorella and improving the utilization value of Chlorella.

[0032] According to this disclosure, the first and second Chlorella species can be species of the genus Chlorella. Specifically, the first and second Chlorella species are each independently selected from at least one of the following: Chlorella pyrenoidosa, Chlorella vulgaris, Chlorella ellipsoidea, Chlorella emersonii, Chlorella kesslerii, and Chlorella thermophila. In a preferred embodiment, the first and second Chlorella species are each independently Chlorella vulgaris and / or Chlorella pyrenoidosa, capable of converting inorganic nitrogen in the flue gas absorbent into organic nitrogen (mainly protein) with extremely high efficiency, and have very high application value. Preferably, the first and second Chlorella species are the same Chlorella species.

[0033] According to this disclosure, the first culture can be carried out under conventional Chlorella culture conditions and using a culture medium well known in the art. Specifically, the conditions for the first culture may include: a temperature of 25–32°C, a light intensity of 0–15000 lx, a photoperiod of 8–16 h, a stirring speed of 50–350 rpm, a culture time of 48 h–96 h, and the use of a pH adjuster (such as 5% nitric acid) to control the pH of the algal solution to 6.0–8.5. In one 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·H2O 14.2 mg / L, Co(NO3)2·6H2O 15.7 mg / L, ZnSO4·7H2O 88.2 mg / L, Na2MoO4·2H2O 11.92 mg / L, and H3BO3 114.2 mg / L. The inoculation amount of the first Chlorella species (i.e., the weight ratio of the first Chlorella species to the culture medium used in the first culture) can be 1:(5-100). During the first culture, individual culture medium components can be added as needed, as long as each component is within the above-mentioned content range. The above-mentioned culture medium can provide sufficient nutrition for the growth of Chlorella, and the resulting first culture medium is more conducive to increasing the catalase content in Chlorella cells when used for the second culture.

[0034] The first culture medium can act as a catalase inducer, promoting an increase in the catalase content and activity in Chlorella cells, thereby enhancing the decomposition of hydrogen peroxide entering the cells during subsequent culture and improving tolerance to flue gas absorbent containing hydrogen peroxide. Using the first culture medium for the second culture of Chlorella eliminates the need for additional inducers, improving the growth of Chlorella under flue gas absorbent conditions without significantly adversely affecting its growth and reducing costs.

[0035] In one specific embodiment, the method further includes: when the optical density of the Chlorella cells in the first culture medium is OD 680When the temperature exceeds 80°C, the first culture medium is subjected to a first ultrafiltration separation, and the resulting filtrate is rotary evaporated to obtain a first culture concentrate, which is then used for the second culture. The conditions for the first ultrafiltration separation may include: using an ultrafiltration module with a molecular weight cutoff of 5–100 kDa; the conditions for the rotary evaporation may include: a temperature of 20–70°C, a pressure of -0.05 MPa to -0.1 MPa, and a time of 0.1–5 h. This embodiment is beneficial for further enhancing the hydrogen peroxide decomposition capacity of Chlorella cells.

[0036] According to this disclosure, the weight ratio of the second Chlorella species to the first culture medium can be 1:(5-100), preferably 1:(10-50), and more preferably 1:(10-20). When using the first culture concentrate, the weight ratio of the second Chlorella species to the first culture concentrate is 1:(0.5-10), preferably 1:(1-5), to further promote the increase of catalase content in Chlorella cells.

[0037] The second culture can be carried out under conventional Chlorella culture conditions and using a culture medium well-known in the art. Specifically, the conditions for the second culture may include: a temperature of 25–32°C, a light intensity of 0–15000 lx, a photoperiod of 8–16 h, a stirring speed of 50–350 rpm, and a culture time of 0.5–24 h. The culture medium used in the second 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·H2O 14.2 mg / L, Co(NO3)2·6H2O 15.7 mg / L, ZnSO4·7H2O 88.2 mg / L, Na2MoO4·2H2O 11.92 mg / L, and H3BO3 114.2 mg / L. The weight ratio of the second Chlorella species to the culture medium used in the second culture can be 1:(5-100). During the second culture process, individual culture medium components can be added as needed, as long as the content of each component is within the above-mentioned range. The culture medium used in the second culture can be the same as or different from the culture medium used in the first culture, but it is preferred to be the same culture medium.

[0038] According to this disclosure, the flue gas absorbent liquid is obtained by contacting and absorbing flue gas with the absorbent liquid. The flue gas refers to the gas produced during the combustion of fossil fuels such as coal, and its main component is nitrogen oxides (NOx). x Flue gas typically requires denitrification treatment to meet standards before being discharged. The absorbent liquid is used to remove NO from the flue gas. x The oxidized form is HNO3, fixed in the liquid phase, and can be an aqueous solution containing nitric acid or other oxidants (such as H2O2, O3, etc.). This disclosure does not impose special limitations on the specific operating methods and conditions for the absorption of flue gas and absorbent liquid, as long as the flue gas absorbent liquid contains hydrogen peroxide and nitric acid. For example, the conditions for contact absorption may include: the absorbent liquid is sprayed from top to bottom at a rate of 20–200 L / h, the flue gas is introduced from bottom to top at a flow rate of 100–2000 L / h, and the absorption temperature is 10–40°C. Continuing the third culture using the flue gas absorbent liquid not only removes inorganic nitrogen from the absorbent liquid but also utilizes the acidity of nitric acid to adjust the pH of the culture system, ensuring that the pH of the algal solution is within a suitable growth range. Furthermore, using a nitric acid-containing flue gas absorbent liquid instead of sodium nitrate in the commonly used culture medium as the nitrogen source can avoid the adverse effects of sodium ion accumulation in the algal solution on the growth of Chlorella.

[0039] In a preferred embodiment, the NO content in the flue gas is less than 50% by volume to further improve the denitrification effect. The content of hydrogen peroxide and nitric acid in the flue gas absorbent can be adjusted within a wide range. Specifically, based on the total weight of the flue gas absorbent, the hydrogen peroxide content can be 0.2-5% by weight, the nitric acid content can be 5-20% by weight, and the remainder is water; preferably, based on the total weight of the flue gas absorbent, the hydrogen peroxide content is 0.2-1.0% by weight, the nitric acid content is 7-10% by weight, and the remainder is water.

[0040] The weight ratio of the flue gas absorbent to the second culture medium can be adjusted within a wide range, for example, it can be 1:(10-1000). To further optimize the denitrification effect and increase the chlorophyll content of Chlorella, the preferred weight ratio of the flue gas absorbent to the second culture medium is 1:(90-500). The flue gas absorbent and the second culture medium can be mixed by various conventional methods. In one embodiment, the method further includes: adding the flue gas absorbent to the second culture medium by a feed-feed method. Based on the second culture medium per liter, the feed-feed rate of the flue gas absorbent can be 1-10 mL / min, and the timing of starting and stopping the feed-feed is determined according to the pH value.

[0041] In a preferred embodiment, the conditions for the third culture may include: the H2O2 concentration in the mixture of the flue gas absorbent and the second culture medium is 1-50 mg / L, and the NO3 concentration is... - The concentration should not exceed 5 g / L, the pH value should be 6.0 to 8.5, the temperature should be 25 to 32℃, the light intensity should be 0 to 15000 lx, the photoperiod should be 8 to 16 h, the stirring speed should be 50 to 350 rpm, and the incubation time should be 48 to 96 h.

[0042] The third culture medium can be separated and dried using conventional methods in the art, such as centrifugation, to obtain the Chlorella product. The obtained Chlorella product has a high chlorophyll content; specifically, the chlorophyll a content can be 20–30 mg / g, the chlorophyll b content can be 10–15 mg / g, and the total chlorophyll content can be 30–45 mg / g.

[0043] Furthermore, the method may further include: when the optical density of the Chlorella cells in the third culture medium is OD 680 When the temperature reaches >60°C, at least a portion of the third culture medium is subjected to a second ultrafiltration separation, and the resulting filtrate is used as the first culture medium for the second culture. In this way, returning a portion of the third culture medium to the preceding step enables the cyclic culture of *Chlorella vulgaris*. The conditions for the second ultrafiltration separation may include using an ultrafiltration module with a molecular weight cutoff of 5–100 kDa.

[0044] In this disclosure, the first, second, and third cultures can be carried out in a bioreactor. The bioreactor can be an open reactor or a closed reactor with an artificial light source.

[0045] The disclosed technical solution is simple and easy to implement, can significantly increase the chlorophyll content in Chlorella cells, and is compatible with other cultivation condition optimizations. It can be easily integrated into existing Chlorella production technologies and has good prospects for promotion and application.

[0046] The present disclosure will be further described in detail below with reference to the embodiments, but this does not constitute a limitation on the present disclosure.

[0047] In this embodiment, the Chlorella species are Chlorella proteoglycans species FACHB-1516 and Chlorella ellipsoides species FACHB-962, both of which are preserved in the Freshwater Algae Culture Collection of the Chinese Academy of Sciences.

[0048] The method for testing optical density 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 to 0.9, dilute the algal solution by a dilution factor of N until the A value falls within the range of 0.2 to 0.9, and use this as the detection result. Calculate the optical density (OD680) of the algal solution according to the following formula.

[0049] Optical density (OD680) = A × N

[0050] Example 1

[0051] (1) Preparation of catalase inducer

[0052] In an open bioreactor, the seed culture of Chlorella proteoglycans was added to 3L of culture medium and cultured for 72 hours at 28℃, light intensity of 6000lx, light cycle of 12h, and stirring at 200rpm. The pH of the algal solution was 7.0. 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. The weight ratio of the first Chlorella species to the culture medium was 1:10. Glucose and other nutrients were supplemented as needed during the cultivation process. When the OD of the algal solution... 680 After 80 minutes, the algal solution was removed and separated using an ultrafiltration unit with a molecular weight cutoff of 30 kDa. Ultrafiltration was stopped when the volume of the filtrate reached 80% of the original algal solution volume. The filtrate was then added to a rotary evaporator and subjected to reduced pressure rotary evaporation for 1.5 hours in a 40°C water bath at -0.095 MPa. Rotary evaporation was stopped when the volume of the concentrate decreased to 0.1 times the original volume of the filtrate, yielding the first culture concentrate.

[0053] (2) Preparation of flue gas absorbent

[0054] Flue gas from the hydrogenation catalyst roasting process is fed into the oxidation absorption tower through the bottom inlet, where it comes into countercurrent contact with the absorbent liquid fed in from the top for oxidation absorption. The absorption tower is filled with stainless steel θ-ring packing, with a packing layer height of 30 cm. The flue gas NO content is 810 ppm (0.081 vol%), and the NO2 content is 679 ppm (0.0679 vol%). The absorbent liquid spray rate is 60 L / h, the flue gas flow rate is 778 L / h, and the temperature is 27℃. The flue gas absorbent obtained at the bottom of the tower is an aqueous solution containing nitric acid and hydrogen peroxide, with the nitric acid content being 8 wt% and the hydrogen peroxide content being 0.4 wt%.

[0055] (3) Chlorella culture

[0056] Prepare a culture medium with the same composition as in step (1). Add the Chlorella proteoglycans inoculum to 3L of culture medium, and add 0.1 times the volume of the first culture concentrate. The weight ratio of Chlorella proteoglycans inoculum to the first culture concentrate is 1:1. Perform a second culture for 12 hours at 28℃, light intensity of 6000 lx, photoperiod of 12h, and shaking at 200 rpm to obtain the second culture medium. Then, add flue gas absorbent solution to the second culture medium by feeding. Based on the second culture medium, the feeding rate of the flue gas absorbent solution is 5 mL / min, and the weight ratio of the added flue gas absorbent solution to the second culture medium is 1:400, so that the H2O2 concentration in the algal solution is 10 mg / L and NO3- is 10 mg / L. - The concentration was 3.72 g / L, the pH was 6.75, and the third culture was carried out for 48 h under the same temperature, light intensity and cycle and stirring conditions to obtain the third culture medium.

[0057] When the optical density of Chlorella cells in the third culture medium is OD 680 When the temperature reaches >60°C, 80% by weight of the algal solution is discharged from the reactor, and the algal solution is separated using an ultrafiltration module with a molecular weight cutoff of 30 kDa. The resulting filtrate is then returned to the reactor for a second culture.

[0058] Example 2

[0059] Chlorella was cultured according to the method of Example 1, except that in step (3), the weight ratio of Chlorella species to the first culture concentrate was 1:0.5.

[0060] Example 3

[0061] Chlorella was cultured according to the method of Example 1, except that in step (3), the weight ratio of Chlorella species to the first culture concentrate was 1:10.

[0062] Example 4

[0063] Chlorella was cultured according to the method of Example 1, except that in step (3), the weight ratio of flue gas absorbent to the second culture medium was 1:600, the H2O2 concentration in the algal solution was 20 mg / L, and the NO3 concentration was... - The concentration was 3.92 g / L, and the pH value was 6.33.

[0064] Example 5

[0065] Chlorella was cultured according to the method of Example 1, except that in step (3), the weight ratio of flue gas absorbent to the second culture medium was 1:80, the H2O2 concentration in the algal solution was 50 mg / L, and the NO3 concentration was... - The concentration was 4.52 g / L, and the pH value was 6.07.

[0066] Example 6

[0067] Chlorella was cultured according to the method of Example 1, except that in step (2), the NO content in the flue gas was 1220 ppm (0.122 vol%) and the NO2 content was 1456 ppm (0.1456 vol%). The absorption liquid spray rate was 240 L / h and the flue gas flow rate was 80 L / h. The flue gas absorption liquid obtained at the bottom of the tower was an aqueous solution containing nitric acid and hydrogen peroxide, wherein the nitric acid content was 6.4 wt% and the hydrogen peroxide content was 5.7 wt%.

[0068] Example 7

[0069] Chlorella was cultured according to the method of Example 1, except that Chlorella ellipsoides was used instead of Chlorella proteoglycans in Example 1.

[0070] Comparative Example 1

[0071] Chlorella was cultured according to the method of Example 1, except that in step (3), the first culture concentrate was not added during the second culture.

[0072] Comparative Example 2

[0073] Chlorella was cultured according to the method of Example 1, except that in step (3), the first culture concentrate was not added during the second culture, but γ-aminobutyric acid with a final concentration of 500 mg / L was added as a catalase inducer.

[0074] Comparative Example 3

[0075] Chlorella was cultured according to the method of Example 1, except that in step (3), flue gas absorbent was not added during the third culture, but a nitric acid solution with a concentration of 8% by weight (i.e., without hydrogen peroxide) was added.

[0076] Test Implementation Examples

[0077] The catalase activity of the third culture medium obtained from the examples and comparative examples, as well as the chlorophyll content of the Chlorella products, were detected, and the results are listed in Table 1.

[0078] Catalase activity assay: 40 mL of algal culture was placed in a centrifuge tube and centrifuged at 5000 rpm for 10 min. The supernatant was discarded, and the cells were resuspended in distilled water and centrifuged at 5000 rpm for 10 min. The supernatant was discarded, and the fresh weight of the obtained Chlorella product was weighed. The catalase activity (fresh weight) of the Chlorella product was determined using a catalase assay kit (purchased from Solarbio, trade number BC0205).

[0079] Chlorophyll extraction and detection method: Place 40 mL of algal solution in a centrifuge tube and centrifuge at 5000 rpm for 10 min. Discard the supernatant, resuspend the cells in distilled water, and centrifuge at 5000 rpm for 10 min. Discard the supernatant, dry in a 105℃ oven to constant weight, and weigh the dry weight m (g) of the microalgae sample. Place 40 mL of algal solution in a centrifuge tube and centrifuge at 5000 rpm for 10 min. Discard the supernatant, resuspend the cells in distilled water, and centrifuge at 5000 rpm for 10 min. Discard the supernatant, add 20 mL of methanol, vortex to mix, and allow to stand for 10 min for extraction. Centrifuge at 5000 rpm for 10 min, collect the supernatant, and use a 752 UV-Vis spectrophotometer to detect the absorbance A at 666 nm and 645 nm. 666 and A 645 Calculate the chlorophyll a, chlorophyll b, and total chlorophyll content (dry weight) using the following formula:

[0080] Chlorophyll a content = (12.7 × A) 666 -2.69×A 645 )×0.02 / m

[0081] Chlorophyll b content = (22.9 × A) 645 -4.68×A 666 )×0.02 / m

[0082] Total chlorophyll content = (8.02 × A) 666 +20.21×A 645 )×0.02 / m

[0083] Table 1

[0084]

[0085] As shown in Table 1, the method disclosed in this paper can significantly increase the chlorophyll content of Chlorella.

[0086] The preferred embodiments of this disclosure have been described in detail above. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0087] 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, this disclosure will not describe the various possible combinations separately.

[0088] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. A method for culturing Chlorella, characterized in that, The method includes the following steps: The first Chlorella species was cultured for the first time to obtain the first culture medium; When the optical density of Chlorella cells in the first culture medium is OD680>80, the first culture medium is subjected to first ultrafiltration separation, and the resulting filtrate is rotary evaporated to obtain the first culture concentrate; The second Chlorella species is cultured in the presence of the first culture concentrate to obtain a second culture solution; the weight ratio of the second Chlorella species to the first culture concentrate is 1:(0.5~10). The flue gas absorbent is mixed with the second culture medium for a third culture to obtain a third culture medium; wherein the flue gas absorbent contains hydrogen peroxide and nitric acid.

2. The method according to claim 1, wherein, The first Chlorella species and the second Chlorella species are each independently selected from at least one of Chlorella pyrenoidosa, Chlorella vulgaris, Chlorella ellipsoidosa, Chlorella ehrlich, Chlorella kJ, and Chlorella thermophila.

3. The method according to claim 1, wherein, The conditions for the first culture include: temperature of 25~32℃, light intensity of 0~15000 lx, light cycle of 8~16 h, stirring speed of 50~350 rpm, culture time of 48~96 h, and pH of algal solution of 6.0~8.

5.

4. The method according to claim 1, wherein, The conditions for the first ultrafiltration separation include: a molecular weight cutoff of 5~100 kDa; The conditions for rotary evaporation include: a temperature of 20~70℃, a pressure of -0.05 MPa to -0.1 MPa, and a time of 0.1~5 h.

5. The method according to claim 1, wherein, The conditions for the second culture include: temperature of 25~32℃, light intensity of 0~15000 lx, light cycle of 8~16 h, stirring speed of 50~350 rpm, and culture time of 0.5~24 h.

6. The method according to claim 1, wherein, The flue gas absorbent is obtained by contacting and absorbing flue gas with the absorbent; the NO content in the flue gas is less than 50% by volume. Based on the total weight of the flue gas absorbent liquid, the hydrogen peroxide content is 0.2-5% by weight, and the nitric acid content is 5-20% by weight. The weight ratio of the flue gas absorption liquid to the second culture medium is 1:(10~1000).

7. The method according to claim 1, wherein, The method further includes: adding the flue gas absorbent liquid to the second culture medium by a feed rate; the feed rate of the flue gas absorbent liquid is 1~10 mL / min based on the second culture medium per liter.

8. The method according to claim 1 or 7, wherein, The conditions for the third culture include: H2O2 concentration of 1~50 mg / L, NO3... - The concentration should not exceed 5 g / L, the pH value should be 6.0~8.5, the temperature should be 25~32℃, the light intensity should be 0~15000 lx, the photoperiod should be 8~16 h, the stirring speed should be 50~350 rpm, and the incubation time should be 48~96 h.

9. The method according to claim 1, wherein, The method further includes: performing a second ultrafiltration separation on at least a portion of the third culture medium, and using the resulting filtrate as the first culture medium for the second culture; The conditions for the second ultrafiltration separation include a molecular weight cutoff of 5-100 kDa.

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