Chlorella culture method

Abstract

The invention relates to a chlorella culture method which comprises the following steps: carrying out first culture on a first chlorella species to obtain a first culture solution; performing second culture on a second chlorella strain in the presence of the first culture solution to obtain a second culture solution; mixing a flue gas absorption solution with the second culture solution, and carrying out third culture to obtain a third culture solution; wherein the flue gas absorption liquid contains hydrogen peroxide and nitric acid. According to the invention, inorganic nitrogen in the flue gas absorption liquid can be fixed into the chlorella biomass, the chlorophyll content of chlorella is obviously improved, and the utilization value of chlorella is improved.

Description

Technical Field

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

[0002] Chlorophyll is the main pigment for plants to fix CO2 through photosynthesis. Chlorophyll molecules contain porphyrin, whose structure is very similar to the porphyrin in human hemoglobin. Chlorophyll helps the human body increase its hemoglobin content, improves the body's oxygen supply, and has an anti-fatigue effect. Modern medicine has found that chlorophyll can promote the regeneration of granulation tissue in ulcer wounds and accelerate wound healing. In addition, chlorophyll also has antioxidant, blood pressure lowering, blood lipid lowering, antibacterial, and cardiovascular function improvement effects. At present, there are many health products on the market with chlorophyll as the main functional ingredient, which is an important category in the health product market.

[0003] Chlorella is a eukaryotic organism belonging to the Chlorophyta. It has the advantages of fast growth and high chlorophyll content, and is an important raw material for the production of chlorophyll. Chlorophyll can be divided into multiple subtypes according to the subtle differences in its specific molecular structure. The chlorophyll in Chlorella cells is mainly chlorophyll a and chlorophyll b. The chlorophyll content in Chlorella cells (chlorophyll a and chlorophyll b combined) is usually around 0.5% to 2%. Increasing the chlorophyll content of Chlorella is conducive to increasing the yield of chlorophyll extracts, reducing production costs, and improving the economic benefits of algae-derived chlorophyll health products.

[0004] At present, the main method for increasing the chlorophyll content of Chlorella is to add exogenous substances to the Chlorella culture system so that the chlorophyll content in Chlorella cells is higher than that in the normal culture method. For example, CN201310004747.X discloses adding ether sugars from biomass pyrolysis or detoxified cellulose pyrolysis liquid to the Chlorella culture solution. CN201810200594.9 discloses adding soybean sprout extract to the Chlorella culture solution. CN201811416377.X discloses adding a regulating substance composed of sodium selenite, indomethacin, inositol or FeCl3 to the Chlorella culture solution. Some of the substances used in these methods to increase the chlorophyll content of Chlorella are derived from biomass extracts, and their components fluctuate with the source of raw materials and production batches, making it difficult to stably achieve the effect of increasing the chlorophyll content; some have complex compositions and use ingredients that are not allowed to be used in food, which increases production costs and also poses potential safety risks. Summary of the invention

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

[0006] In order to achieve the above object, the present disclosure provides a method for culturing Chlorella, which comprises the following steps:

[0007] Carrying out a first culture of the first Chlorella species to obtain a first culture solution;

[0008] Carrying out a second culture of the second Chlorella species in the presence of the first culture solution to obtain a second culture solution;

[0009] The flue gas absorption liquid is mixed with the second culture liquid for a third culture to obtain a third culture liquid; wherein the flue gas absorption liquid 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 ellipsoidea, Chlorella elsdenii, Chlorella kasseri and Chlorella thermophila.

[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, culture time of 48-96h, and algae liquid pH of 6.0-8.5.

[0012] Optionally, the method further comprises: subjecting the first culture solution to a first ultrafiltration separation, and subjecting the obtained filtrate to rotary evaporation to obtain a first culture concentrate, and using the first culture concentrate for the second culture;

[0013] The conditions of the first ultrafiltration separation include: a molecular weight cut-off of 5 to 100 kDa;

[0014] The rotary evaporation conditions include: temperature of 20 to 70° C., pressure of -0.05 MPa to -0.1 MPa, and time of 0.1 to 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 to 32° C., a light intensity of 0 to 15,000 lx, a light cycle of 8 to 16 h, a stirring speed of 50 to 350 rpm, and a culture time of 0.5 to 24 h.

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

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

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

[0020] Optionally, the method further comprises: adding the flue gas absorption liquid into the second culture liquid in a feeding manner; the flow acceleration of the flue gas absorption liquid is 1 to 10 mL / min based on each liter of the second culture liquid.

[0021] Optionally, the third culture conditions include: H2O2 concentration of 1-50 mg / L, NO3 - The concentration does not exceed 5g / L, the pH value is 6.0-8.5, the temperature is 25-32°C, the light intensity is 0-15000lx, the light cycle is 8-16h, the stirring speed is 50-350rpm, and the culture time is 48-96h.

[0022] Optionally, the method further comprises: subjecting at least a portion of the third culture fluid to a second ultrafiltration separation, and using the obtained filtrate as the first culture fluid for the second culture;

[0023] The conditions for the second ultrafiltration separation include: a molecular weight cut-off of 5 to 100 kDa.

[0024] Through the above technical solution, the present invention can fix inorganic nitrogen in the flue gas absorption liquid into the Chlorella biomass, and significantly increase the chlorophyll content of Chlorella, thereby improving the utilization value of Chlorella. The method has the advantages of simple operation, low cost, and strong practicality.

[0025] Other features and advantages of the present disclosure will be described in detail in the following detailed description. DETAILED DESCRIPTION

[0026] 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.

[0027] The present invention provides a method for culturing Chlorella, which comprises the following steps:

[0028] Carrying out a first culture of the first Chlorella species to obtain a first culture solution;

[0029] Carrying out a second culture of the second Chlorella species in the presence of the first culture solution to obtain a second culture solution;

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

[0031] The present invention adopts a Chlorella culture solution (i.e., a first culture solution) to culture Chlorella species, which can increase the catalase content in Chlorella cells, and then continues to culture with a flue gas absorption solution containing hydrogen peroxide and nitric acid, which can not only remove inorganic nitrogen in the flue gas absorption solution, but also quickly decompose hydrogen peroxide, thereby significantly increasing the chlorophyll content of the produced Chlorella and improving the utilization value of Chlorella.

[0032] According to the present disclosure, the first chlorella algae species and the second chlorella algae species can be algae species of the genus Chlorella. Specifically, the first chlorella algae species and the second chlorella algae species are each independently selected from at least one of Chlorella pyrenoidosa, Chlorella vulgaris, Chlorella ellipsoidea, Chlorella emersonii, Chlorella kesslerii, and Chlorella thermophila. In a preferred embodiment, the first chlorella algae species and the second chlorella algae species are each independently Chlorella pyrenoidosa and / or Chlorella vulgaris, and can convert the inorganic nitrogen in the flue gas absorption liquid into organic nitrogen (mainly protein) with extremely high efficiency, and have very high application value. The first chlorella algae species and the second chlorella algae species are preferably the same chlorella algae species.

[0033] According to the present disclosure, the first culture can be carried out under conventional Chlorella 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 6.0 to 8.5. 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·H2O 14.2 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 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), 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 of Chlorella, and the obtained first culture solution is more conducive to increasing the catalase content in Chlorella cells when used in the second culture.

[0034] The first culture solution can be used as a catalase inducer to increase the catalase content and activity in the Chlorella cells, thereby enhancing the decomposition of hydrogen peroxide entering the cells during subsequent culture and improving tolerance to the flue gas absorption liquid containing hydrogen peroxide. The first culture solution is used to carry out the second culture of the Chlorella species without adding additional inducers, which can improve the growth of Chlorella under the condition of using the flue gas absorption liquid, and at the same time will not have a significant adverse effect on the growth of Chlorella, and reduces costs.

[0035] In a specific embodiment, the method further comprises: when the optical density of the Chlorella cells in the first culture solution is OD 680>80, subjecting the first culture solution to a first ultrafiltration separation, and subjecting the obtained filtrate to rotary evaporation to obtain a first culture concentrate, and using the first culture concentrate for the second culture. Wherein, the conditions for the first ultrafiltration separation may include: using an ultrafiltration component with a molecular weight cutoff of 5 to 100 kDa for the first ultrafiltration separation; the conditions for the rotary evaporation may include: a temperature of 20 to 70°C, a pressure of -0.05 MPa to -0.1 MPa, and a time of 0.1 to 5 h. This embodiment is conducive to further improving the hydrogen peroxide decomposition ability of Chlorella cells.

[0036] According to the present disclosure, the weight ratio of the second Chlorella species to the first culture solution can be 1:(5-100), preferably 1:(10-50), and more preferably 1:(10-20). When the first culture concentrate is used, 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 the catalase content in Chlorella cells.

[0037] The second culture can be carried out under conventional Chlorella culture conditions in the art and using a culture medium well known in the art. Specifically, the second culture conditions may include: the second culture conditions 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, and a culture time of 0.5 to 24 hours. The culture medium used for 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, Na2MoO4·2H2O11.92 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), and individual culture medium components can be supplemented as needed during the second culture process, as long as the content of each component is within the above 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, and is preferably the same culture medium.

[0038] According to the present disclosure, the flue gas absorption liquid is obtained by contacting and absorbing the flue gas. The flue gas refers to the gas generated by the combustion of fossil fuels such as coal, and its main component is nitrogen oxides (NO x ), usually need to be denitrated to meet the standards before discharge. The absorption liquid is used to remove NO in the flue gas. x Oxidized to HNO3 and fixed in the liquid phase, it can be an aqueous solution containing nitric acid or other oxidants (such as H2O2, O3, etc.). The present disclosure has no special restrictions on the specific operation methods and conditions for absorbing flue gas and absorption liquid, as long as the flue gas absorption liquid contains hydrogen peroxide and nitric acid. For example, the conditions for contact absorption may include: the absorption liquid is sprayed from top to bottom, the spraying volume is 20 to 200L / h, the flue gas is introduced from bottom to top, the flow rate is 100 to 2000L / h, and the absorption temperature is 10 to 40°C. The use of the flue gas absorption liquid to continue the third culture can not only achieve the removal of inorganic nitrogen in the flue gas absorption liquid, but also use the acidity of nitric acid to adjust the pH of the culture system to ensure that the pH value of the algae liquid is within a suitable growth range. In addition, the use of a flue gas absorption liquid containing nitric acid instead of sodium nitrate in a common culture medium as a nitrogen source can also avoid the adverse effects of the accumulation of sodium ions in the algae liquid 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 denitration effect. The content of hydrogen peroxide and nitric acid in the flue gas absorption liquid can be adjusted in a wide range. Specifically, based on the total weight of the flue gas absorption liquid, the content of hydrogen peroxide can be 0.2-5% by weight, the content of nitric acid can be 5-20% by weight, and the balance is water; preferably, based on the total weight of the flue gas absorption liquid, the content of hydrogen peroxide is 0.2-1.0% by weight, the content of nitric acid is 7-10% by weight, and the balance is water.

[0040] The weight ratio of the flue gas absorption liquid to the second culture liquid can be adjusted in a wide range, for example, it can be 1:(10~1000). In order to further optimize the denitrification effect and increase the chlorophyll content of Chlorella, the weight ratio of the flue gas absorption liquid to the second culture liquid is preferably 1:(90~500). The flue gas absorption liquid and the second culture liquid can be mixed in various conventional ways. In one embodiment, the method further includes: adding the flue gas absorption liquid to the second culture liquid in a flow addition manner, and the flow acceleration of the flue gas absorption liquid can be 1~10mL / min per liter of the second culture liquid, and the timing of starting and stopping the flow addition is determined according to the pH value.

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

[0042] The third culture solution can be subjected to conventional methods in the art, such as centrifugal separation and drying, to obtain a 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 solution is OD 680 >60, subjecting at least a portion of the third culture fluid to a second ultrafiltration separation, and using the resulting filtrate as the first culture fluid for the second culture. In this way, returning a portion of the third culture fluid to the previous step can achieve a cyclic culture of Chlorella. The conditions for the second ultrafiltration separation may include: using an ultrafiltration component with a molecular weight cutoff of 5 to 100 kDa for the second ultrafiltration separation.

[0044] 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.

[0045] The technical solution disclosed in the present invention is simple and easy to implement, can significantly increase the chlorophyll content in Chlorella cells, is compatible with the optimization of other culture conditions, can be easily embedded in the existing Chlorella production technology, and has a good prospect for promotion and application.

[0046] The present disclosure is further described in detail below in conjunction with embodiments, but the present disclosure is not limited thereto.

[0047] In the embodiment, the Chlorella species is the Chlorella pyrenoidosa species deposited in the Freshwater Algae Seed Bank of the Chinese Academy of Sciences and numbered FACHB-1516, and the Chlorella ellipsoidea species deposited in the Freshwater Algae Seed Bank of the Chinese Academy of Sciences and numbered FACHB-962.

[0048] The test method for optical density is: 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.

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

[0050] Example 1

[0051] (1) Preparation of catalase inducer

[0052] In an open bioreactor, the protein nucleus Chlorella algae seed solution was added to 3L culture medium, and the first culture was carried out for 72h at 28°C, light intensity of 6000lx, light cycle of 12h, and stirring at 200rpm. The pH value of the algae solution was 7.0. The culture medium composition is glucose 30g / L, NaNO3 5000mg / L, K2HPO4 1500mg / L, Na2EDTA 500mg / L, MgSO4·7H2O1000mg / 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. The weight ratio of the first Chlorella algae to the culture medium is 1:10. Glucose and other nutrients are supplemented as needed during the culture process. When the algae solution OD 680 >80, take out the algae liquid, use the ultrafiltration component with a molecular weight cutoff of 30kDa to separate the algae liquid, and stop ultrafiltration when the volume of the filtrate reaches 80% of the original algae liquid volume. Add the filtrate to the rotary evaporator, and perform vacuum rotary evaporation for 1.5 hours in a 40℃ water bath and -0.095MPa pressure. Stop rotary evaporation when the volume of the concentrate is reduced to 0.1 times the original volume of the filtrate to obtain the first culture concentrate.

[0053] (2) Preparation of flue gas absorption liquid

[0054] The flue gas from the hydrogenation catalyst roasting process is sent into the oxidation absorption tower from the air inlet at the bottom of the tower, so that it is countercurrently contacted with the absorption liquid sent from the top of the tower for oxidation absorption. The absorption tower is equipped with stainless steel θ ring packing, and the packing layer height is 30cm. The flue gas NO content is 810ppm (0.081 volume%), and the NO2 content is 679ppm (0.0679 volume%). The absorption liquid spray volume is 60L / h, the flue gas flow rate is 778L / h, and the temperature is 27°C. The flue gas absorption liquid obtained at the bottom of the tower is an aqueous solution containing nitric acid and hydrogen peroxide, wherein the nitric acid content is 8% by weight and the hydrogen peroxide content is 0.4% by weight.

[0055] (3) Cultivation of Chlorella

[0056] Prepare a culture medium with the same ingredients as step (1), take the protein-nuclear Chlorella algae liquid and add it to 3L of culture medium, and add the first culture concentrate with a volume of 0.1 times the volume of the culture medium, the weight ratio of Chlorella algae to the first culture concentrate is 1:1. Carry out a second culture for 12 hours at 28°C, a light intensity of 6000lx, a light cycle of 12h, and an oscillation of 200rpm to obtain a second culture solution. Then, add the flue gas absorption liquid to the second culture solution in a feeding manner, with the flow acceleration of the flue gas absorption liquid being 5mL / min per liter of the second culture solution as the basis, and the weight ratio of the added flue gas absorption liquid to the second culture solution being 1:400, so that the H2O2 concentration in the algae solution is 10mg / L, and NO3 - The concentration is 3.72 g / L, the pH value is 6.75, and the third culture is carried out for 48 hours under the same temperature, light intensity and cycle and stirring conditions to obtain a third culture solution.

[0057] When the optical density of the Chlorella cells in the third culture solution is OD 680 >60, 80 wt% of the algae liquid was led out of the reactor, and the algae liquid was separated using an ultrafiltration component with a molecular weight cutoff of 30 kDa, and the filtrate was returned to the reactor for the second culture.

[0058] Example 2

[0059] The method of Example 1 is used to culture Chlorella, except that in step (3), the weight ratio of Chlorella species to the first culture concentrate is 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 the Chlorella species to the first culture concentrate was 1:10.

[0062] Example 4

[0063] The method of Example 1 was used to culture Chlorella vulgaris, except that in step (3), the weight ratio of the flue gas absorption liquid to the second culture liquid was 1:600, the concentration of H2O2 in the algae liquid was 20 mg / L, and the concentration of NO3 - The concentration is 3.92g / L and the pH value is 6.33.

[0064] Example 5

[0065] The method of Example 1 was used to culture Chlorella vulgaris, except that in step (3), the weight ratio of the flue gas absorption liquid to the second culture liquid was 1:80, the concentration of H2O2 in the algae liquid was 50 mg / L, and the concentration of NO3 - The concentration is 4.52g / L and the pH value is 6.07.

[0066] Example 6

[0067] The chlorella was cultured according to the method of Example 1, except that in step (2), the NO content of the flue gas was 1220 ppm (0.122 volume %), and the NO2 content was 1456 ppm (0.1456 volume %). The absorption liquid spraying 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% by weight, and the hydrogen peroxide content was 5.7% by weight.

[0068] Example 7

[0069] The Chlorella was cultured according to the method of Example 1, except that the Chlorella ellipsoidea species was used to replace the Chlorella pyrenoidea species 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 was added at a final concentration of 500 mg / L as a catalase inducer.

[0074] Comparative Example 3

[0075] The Chlorella vulgaris was cultured according to the method of Example 1, except that in step (3), no flue gas absorbing liquid was added during the third culture, but a nitric acid solution with a concentration of 8 wt % (i.e., without hydrogen peroxide) was added.

[0076] Test Example

[0077] The catalase activity of the third culture solution obtained in the example and the comparative example and the chlorophyll content of the Chlorella product were detected, and the results are listed in Table 1.

[0078] Catalase activity detection method: 40 mL of algae solution was placed in a centrifuge tube and centrifuged at 5000 rpm for 10 min. The supernatant was discarded, 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 detected using a catalase detection kit (purchased from Solebao, product number BC0205).

[0079] Chlorophyll extraction and detection method: Take 40mL of algae solution and place it in a centrifuge tube, and centrifuge it at 5000rpm for 10min. Discard the supernatant, resuspend the cells with distilled water, and centrifuge it at 5000rpm for 10min. Discard the supernatant, dry it in an oven at 105℃ to constant weight, and weigh the dry weight of the microalgae sample m(g). Take 40mL of algae solution and place it in a centrifuge tube, and centrifuge it at 5000rpm for 10min. Discard the supernatant, resuspend the cells with distilled water, and centrifuge it at 5000rpm for 10min. Discard the supernatant, add 20mL of methanol, vortex mix, and let it stand for 10min. Centrifuge at 5000rpm for 10min, aspirate the supernatant, and use a 752 UV-visible spectrophotometer to detect the absorbance A at 666nm and 645nm 666 and A 645 , Calculate chlorophyll a, chlorophyll b and total chlorophyll content (dry weight) according to 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 can be seen from Table 1, the chlorophyll content of Chlorella can be significantly increased by adopting the method disclosed in the present invention.

[0086] 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.

[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, the present disclosure will not further describe various possible combinations.

[0088] 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 Chlorella, characterized in that: The method comprises the following steps: Carrying out a first culture of the first Chlorella species to obtain a first culture solution; Carrying out a second culture of the second Chlorella species in the presence of the first culture solution to obtain a second culture solution; The flue gas absorption liquid is mixed with the second culture liquid for a third culture to obtain a third culture liquid; wherein the flue gas absorption liquid 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 ellipsoidea, Chlorella elsdenii, Chlorella kasseri and Chlorella thermophila.

3. 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, culture time of 48-96h, and algae liquid pH of 6.0-8.

5.

4. The method according to claim 1, wherein: The method further comprises: subjecting the first culture solution to a first ultrafiltration separation, and subjecting the obtained filtrate to rotary evaporation to obtain a first culture concentrate, and using the first culture concentrate for the second culture; The conditions of the first ultrafiltration separation include: a molecular weight cut-off of 5 to 100 kDa; The rotary evaporation conditions include: temperature of 20 to 70° C., pressure of -0.05 MPa to -0.1 MPa, and time of 0.1 to 5 h.

5. The method according to claim 4, wherein: The weight ratio of the second Chlorella species to the first culture concentrate is 1:(0.5-10).

6. The method according to claim 1, wherein: The second culture conditions include: temperature of 25-32° C., 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.

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

8. The method according to claim 1, wherein: The method further includes: adding the smoke absorption liquid into the second culture liquid in a flow addition manner; based on each liter of the second culture liquid, the flow acceleration of the smoke absorption liquid is 1 to 10 mL / min.

9. The method according to claim 1 or 8, wherein: The third culture conditions include: H2O2 concentration of 1-50 mg / L, NO3 - The concentration does not exceed 5g / L, the pH value is 6.0-8.5, the temperature is 25-32°C, the light intensity is 0-15000lx, the light cycle is 8-16h, the stirring speed is 50-350rpm, and the culture time is 48-96h.

10. The method according to claim 1, wherein: The method further comprises: subjecting at least a portion of the third culture fluid to a second ultrafiltration separation, and using the obtained filtrate as the first culture fluid to perform the second culture; The conditions for the second ultrafiltration separation include: a molecular weight cut-off of 5 to 100 kDa.

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