Algae fermentation culture method

Abstract

The invention provides an algae fermentation culture method, which relates to the technical field of bioengineering, and comprises the following steps: selecting microalgae seeds; constructing an index fed-batch model corresponding to the selected microalgae seeds; inoculating the microalgae seeds into a fermentation tank of a Basal culture medium; culturing according to the index fed-batch model under a dark condition; after culturing under a dark condition, adding plant hormones and a nitrogen source to continuously culture to obtain the microalgae. According to the algae fermentation culture method provided by the invention, the growth of microalgae and the accumulation of xanthophyll are facilitated through index fed-batch fermentation, and after the index fed-batch fermentation culture, IAA high-nitrogen combined treatment is matched, so that the xanthophyll yield and yield of the microalgae in a fermentation tank can be effectively improved.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to an algae fermentation and cultivation method. Background Art

[0002] Microalgae are considered to be a very promising source of lutein because, compared with marigold, the only natural source of lutein available, they grow very fast, have a high lutein yield, are highly adaptable to various environments, and are not restricted by seasons. Microalgae can be cultivated in three culture modes: autotrophic, heterotrophic, and polytrophic. Combining the three culture modes, the heterotrophic mode is the most conducive to large-scale cultivation of microalgae for lutein production. In addition, the conditions suitable for microalgae growth and lutein production are often not consistent. For some conditions where the suitable growth and lutein accumulation are quite different, in order to obtain the maximum lutein yield, the two-step method has become the most commonly used method.

[0003] Batch culture is a common culture method in the laboratory. However, batch culture may cause the initial nutrient concentration to be too high and inhibit cell growth or its physiological activities, or cause the nutrient concentration to decrease due to the consumption of nutrients by cells, thereby limiting the further growth of cells and the synthesis of target products. Compared with batch culture, fed-batch (flow-batch) culture can improve the efficiency of cell growth and accumulation of metabolites by intermittently or continuously supplementing the nutrients required by cells. In addition, although flow-batch culture has the advantages of easy control, low cost, high cell density and elimination of substrate inhibition, however, the nutrient composition and proportion of the flow-batch, the volume and time of the feed as important limiting factors affecting the efficiency of the flow-batch culture, are difficult to be accurately determined. Summary of the invention

[0004] In view of the shortcomings of the prior art described above, the object of the present invention is to provide an algae fermentation culture method for solving the problems in the prior art that batch culture may cause the initial nutrient concentration to be too high and inhibit cell growth or its physiological activities, and the nutrient concentration is reduced due to the consumption of nutrients by the cells, thereby limiting the further growth of the cells and the synthesis of the target product, and the nutrient composition and ratio of the feed, the volume and time of the feed are important limiting factors affecting the efficiency of the feed culture, and it is difficult to accurately determine the problem.

[0005] To achieve the above-mentioned purpose and other related purposes, the present invention provides an algae fermentation and cultivation method, comprising the following steps: selecting microalgae seeds; constructing an exponential fed-batch model corresponding to the selected microalgae seeds; inoculating the microalgae seeds into a fermentation tank of Basal culture medium; culturing according to the exponential fed-batch model under dark conditions; after culturing under dark conditions, adding plant hormones and nitrogen sources to continue culturing to obtain microalgae.

[0006] In one embodiment of the present invention, an exponential fed-batch model corresponding to the selected microalgae seeds is constructed, comprising: inoculating the microalgae seeds in a 250 mL conical flask containing 100 mL of fresh modified Basal medium in advance, and culturing in a shaker at 28°C and 180 rpm and in the dark for 48 hours, sampling every 4 hours from the 12th hour, and determining the feeding model corresponding to the exponential fed-batch model.

[0007] In one embodiment of the present invention, the feed model corresponding to the exponential feeding model is Among them, V FS is the flow acceleration rate, Y XS is the ratio of cell biomass to substrate concentration, μ is the specific growth rate of cells, t is the culture time, C FS is the nutrient concentration in the fed-batch medium.

[0008] In one embodiment of the present invention, sampling is performed every 4 hours starting from the 12th hour to determine the feeding model corresponding to the exponential fed-batch model, including: measuring the biomass and the consumption of glucose, urea and phosphate every 4 hours from the 12th hour to the 48th hour of the culture, and calculating the ratio Y of the cell biomass to the upper substrate concentration. XS and the specific growth rate μ of the cells; according to the initial culture solution volume V0 and the initial biomass X0 in the fermenter, the feeding model is determined based on glucose feeding, and the feeding amounts of urea and phosphate are obtained in turn.

[0009] In one embodiment of the present invention, the feed amounts of glucose, nitrate and phosphate in the fed-batch culture medium are rounded to a molar ratio of 37:7:2.

[0010] In one embodiment of the present invention, the selected microalgae seeds are C. protothecoides CS-41.

[0011] In one embodiment of the present invention, the microalgae seeds are inoculated into a fermenter containing Basal medium, comprising: inoculating the microalgae seeds at 10% (v / v) into a 2L fermenter containing 1000mL of fresh modified Basal medium, and culturing at 28°C, 300-700rpm shaking on a shaker and in dark conditions, wherein the pH of the modified Basal medium is 6.1.

[0012] In one embodiment of the present invention, the culture is carried out under dark conditions according to the exponential feeding model, including: under dark conditions, glucose, nitrate and phosphate are supplemented into the fermentation tank according to the feeding model corresponding to the exponential feeding model for culture.

[0013] In one embodiment of the present invention, after culturing under dark conditions, plant hormones and nitrogen sources are added to continue culturing to obtain microalgae, including:

[0014] After culturing under dark conditions for the sixth day, IAA and urea were added, and fermentation was stopped on the eighth day to obtain the microalgae.

[0015] In one embodiment of the present invention, the IAA added is 200 mM and the urea added is 3.6 g / L.

[0016] As described above, an algae fermentation and cultivation method of the present invention has the following beneficial effects: exponential feeding fermentation is beneficial to the growth of microalgae such as C. protothecoides CS-41, and at the same time, due to the continuous depletion and replenishment of glucose in this mode, the lutein content can be stimulated to increase slowly, which is also beneficial to the accumulation of lutein, and is a more favorable fermentation mode for producing lutein. At the same time, after the exponential feeding fermentation, for example, when the culture reaches the plateau phase, the lutein yield can be greatly improved by using IAA high nitrogen combined treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Shown is a flow chart of the algae fermentation culture method of the present invention.

[0018] Figure 2 It is a schematic diagram showing the growth curve of C. protothecoides CS-41 in a preferred embodiment of the present invention.

[0019] Figure 3 Shown is a schematic diagram of the relationship between glucose, urea and phosphate and biomass consumption in a preferred embodiment of the present invention.

[0020] Figure 4 It is a schematic diagram of the growth curve of C. protothecoides CS-41 under different fermentation modes in a preferred embodiment of the present invention.

[0021] Figure 5 It is a schematic diagram showing the growth and glucose consumption of C. protothecoides CS-41 during batch fermentation in a preferred embodiment of the present invention.

[0022] Figure 6 It is a schematic diagram showing the growth and glucose consumption of C. protothecoides CS-41 during the exponential feeding fermentation process in a preferred embodiment of the present invention.

[0023] Figure 7 It is a schematic diagram showing the growth and glucose consumption of C. protothecoides CS-41 during high-concentration substrate fermentation in a preferred embodiment of the present invention.

[0024] Figure 8 It is a schematic diagram showing the lutein content of C. protothecoides CS-41 under different fermentation modes in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0025] The following is an explanation of the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the features in the following embodiments and the embodiments can be combined with each other without conflict. It should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention. The test methods for which specific conditions are not indicated in the following examples are usually carried out under conventional conditions or according to the conditions recommended by the manufacturers.

[0026] See also Figures 1 to 8 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention, so they have no substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the effects and purposes that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0027] See also Figure 1 The present invention provides an algae fermentation culture method, comprising the following steps:

[0028] Step S10: selecting microalgae seeds;

[0029] Step S20: constructing an exponential fed-batch model corresponding to the selected microalgae seeds;

[0030] Step S30: inoculating the microalgae seeds into a fermentation tank containing Basal culture medium;

[0031] Step S40: culturing in the dark according to the exponential fed-batch model;

[0032] Step S50: After culturing under dark conditions, plant hormones and nitrogen sources are added to continue culturing to obtain microalgae.

[0033] It is not difficult to find through the above steps that through step S10: selecting microalgae seeds, it can be seen that in order to increase the lutein production of microalgae, in the process of seed selection, algae species with higher lutein content can be selected as an indicator for algae species selection. Through step S20: constructing an exponential flow addition model corresponding to the selected microalgae seeds, it can be seen that before fermenting and culturing the microalgae seeds, an exponential flow addition model belonging to the microalgae seeds is constructed according to the selected microalgae seeds. The constructed exponential flow addition model can further determine the nutrient supplementation during the microalgae seed cultivation process. Through step S30: inoculating the microalgae seeds in a fermentation tank of Basal culture medium, it can be seen that after selecting the microalgae seeds, the microalgae seeds are further inoculated in a fermentation tank of Basal culture medium for cultivation. Through step S40: culturing in accordance with the exponential feed model under dark conditions, it can be seen that in the culturing process, by supplementing the nutrient in accordance with the exponential feed model, the culturing is carried out by the exponential feed fermentation method, so that the nutrients such as glucose in the fermentation process are replenished after being exhausted, thereby stimulating the slow increase of the content of the lutein, which is the most favorable fermentation mode for producing lutein. Through step S50: after culturing in dark conditions, adding plant hormones and nitrogen sources to continue culturing, and culturing to obtain microalgae, it can be seen that after culturing using the exponential feed fermentation method, when reaching the plateau period, the total carotenoid content and lutein content can be effectively increased by further adding plant hormones and nitrogen sources to continue culturing.

[0034] In step S10, the selected microalgae seeds are C.protothecoides CS-41. In this embodiment, in the process of seed selection, in order to increase the lutein production, the microalgae seeds can select C.protothecoides CS-41 which is more suitable for producing lutein. Moreover, the growth of C.protothecoides CS-41 is not affected by the nitrogen source concentration, but the lutein content increases with the increase of the nitrogen source concentration. Of course, in order to achieve the purpose of additionally supplementing the nitrogen source to increase the lutein production, the microalgae seeds can also select other types of algae such as P.kessleri HH2.

[0035] In step S20, an exponential fed-batch model corresponding to the selected microalgae seeds is constructed, including:

[0036] Step S201: Inoculate the microalgae seeds in advance into a 250 mL conical flask containing 100 mL of fresh modified Basal medium, and culture in a shaker at 28°C and 180 rpm in the dark for 48 hours. Sampling is performed every 4 hours from the 12th hour to determine the feeding model corresponding to the exponential fed-batch model.

[0037] In one embodiment of the present invention, in order to accurately measure the nutrient consumption in the culture medium, a feeding model corresponding to the exponential fed-batch model is established. C. protothecoides CS-41 can be cultured in a shake flask, and the consumption of biomass and nutrients such as glucose, urea and phosphate are measured every 4 hours during the culture period of 12 to 48 hours. Thus, based on the consumption of nutrients, the feeding model corresponding to the exponential fed-batch model is further determined.

[0038] Preferably, the feed model corresponding to the exponential feed model is Among them, V FS is the flow acceleration rate, Y XS is the ratio of cell biomass to substrate concentration, μ is the specific growth rate of cells, t is the culture time, C FS is the nutrient concentration in the fed-batch medium.

[0039] In a preferred embodiment of the present invention, during the construction of the exponential feed model, the exponential feed model is obtained based on the substrate consumption model, aiming to balance the concentration of nutrients. This equation can be expressed as

[0040] Where S, X and P are the substrate concentration, cell biomass and extracellular product concentration (g / L), the feed concentration of Fs flow addition (g / mL / h), and Y XS and Y PS are the cell biomass and extracellular product concentrations divided by the substrate concentration (g / g), and t is the culture time (h). When the substrate concentration in the culture medium remains unchanged, dS / dt = 0, that is, At the same time, during the exponential growth period of prokaryotic Chlorella, the concentration of extracellular products is very low, i.e. So the formula can be simplified to Where, dX / dt = μ × X0 × e μt It represents the amount of algae added per unit mass per hour. Substitute it into the formula get For the convenience of feeding operation, the volume is converted into a measurement index of feeding, and the formula is can be further converted into Among them, V FS is the flow rate (mL / h), V0 is the initial culture volume (mL), C FSis the nutrient concentration in the fed-batch medium (g / L).

[0041] In step S201, sampling is performed every 4 hours starting from the 12th hour to determine the feeding model corresponding to the exponential feeding model, including:

[0042] From 12 hours to 48 hours of cultivation, the biomass and the consumption of glucose, urea and phosphate were measured every 4 hours, and the ratio of cell biomass to substrate concentration Y was calculated. XS and the specific growth rate μ of the cell;

[0043] According to the initial culture liquid volume V0 and the initial biomass X0 in the fermenter, the feeding model is determined based on glucose feeding, and the feeding amounts of urea and phosphate are obtained in turn.

[0044] In one embodiment of the present invention, in order to accurately measure the formula The parameters in the experiment were obtained by culturing C.protothecoides CS-41 microalgae seeds in a shake flask. The biomass and the consumption of nutrients such as glucose, urea and phosphate were measured every 4 hours from 12 to 48 hours. The ratio of cell biomass to substrate concentration Y was calculated. XS and the specific growth rate μ of the cells. In addition, according to the initial culture volume and initial inoculation biomass in the fermenter. Based on glucose feeding, that is, C FS , determine the feeding equation. Finally, the feeding model V is obtained FS .

[0045] In a preferred embodiment of the present invention, Y is calculated by culturing C. protothecoides CS-41 in a shake flask and measuring the biomass and the consumption of glucose, urea and phosphate every 4 hours during the period from 12 hours to 48 hours. XS =0.5257g / g, μ = 0.049h -1 In addition, the initial culture volume V0 in the fermenter is 1200 mL, and the initial inoculated biomass X0 is 0.4 g / L. Based on glucose feeding, that is, C FS =500g / L, determine the feeding equation. Finally, the feeding model V FS =0.09023×exp(0.05t).

[0046] Preferably, the feed amounts of glucose, nitrate and phosphate in the fed culture medium are rounded to a molar ratio of 37:7:2.

[0047] See also Figure 2 and Figure 3 , Figure 2Figure 2 shows the growth curve of biomass over time when C. protothecoides CS-41 was cultured in shake flasks. Figure 3 The relationship between glucose, urea and phosphate and biomass consumption is determined by determining the linear relationship between biomass growth and glucose, urea and phosphate consumption when the urea and phosphate feed amounts are determined. The linear relationship is obtained through a quasi-linear equation. The relationship between glucose and biomass is y = -10.462x + 67.12 (R2 = 0.99), that is, for every increase of 1g of biomass, 10.462mmol of glucose is consumed. Similarly, the relationship between urea and phosphate and biomass is y = -1.9638x + 19.946 (R 2 =0.98) and y = -0.5636x + 13.539 (R 2 =0.93). The molar ratio of glucose, nitrate and phosphate in the feed medium was rounded to 37:7:2, and after conversion to mass, the feed medium composition included: glucose 500 g / L, potassium nitrate 94.6 g / L, potassium dihydrogen phosphate 27 g / L.

[0048] In step S30, the microalgae seeds are inoculated into a fermentation tank containing a Basal medium, comprising:

[0049] The microalgae seeds were inoculated into a 2L fermentor containing 1000mL of fresh modified Basal medium at 10% (v / v), and placed in a shaking table at 28°C, 300-700rpm and cultured in the dark, with the pH of the modified Basal medium being 6.1. In the present embodiment, C.protothecoides CS-41 can be fermented at high density in a fermentor. The shaking table can be a constant temperature shaking table of DHZ-DA, the oscillator can be a vortex oscillator of 88880018, and the fermentor can be a BioFlo 115.

[0050] Furthermore, the improved Basal medium includes KH2PO4, MgSO4·7H2O, EDTA·Na2, H3BO3, mgCaCl2·2H2O, FeSO4·7H2O, ZnSO4·7H2O, MnCl2·4H2O, CuSO4·5H2O, Co(NO3)2·6H2O, and MoO3.

[0051] Preferably, the mass concentration ratio of KH2PO4, MgSO4·7H2O, EDTA·Na2, H3BO3, mgCaCl2·2H2O, FeSO4·7H2O, ZnSO4·7H2O, MnCl2·4H2O, CuSO4·5H2O, Co(NO3)2·6H2O, and MoO3 is 1.25:1.0:0.5:0.1142:0.111:0.0498:0.0882:0.0142:0.0157:0.0049:0.0071.

[0052] In this embodiment, the modified Basal medium ( / L): 1.25g KH2PO4, 1.0g MgSO4·7H2O, 0.5g EDTA·Na2, 114.2mg H3BO3, 111mg CaCl2·2H2O, 49.8mg FeSO4·7H2O, 88.2mg ZnSO4·7H2O, 14.2mg MnCl2·4H2O, 15.7mg CuSO4·5H2O, 4.9mg Co(NO3)2·6H2O, 7.1mg MoO3. Compared with Kuhl medium and BG11 medium, the modified Basal medium is more conducive to the growth of C.protothecoides CS-41 algae species. The modified Basal medium in this embodiment is three times concentrated than the basic Basal medium (boric acid is the initial concentration), the initial glucose concentration is 50g / L, and the initial urea concentration is 4.5g / L.

[0053] In step S40, culturing is performed under dark conditions according to the exponential fed-batch model, including:

[0054] Under dark conditions, glucose, nitrate and phosphate are added to the fermenter according to the feeding model corresponding to the exponential feeding model for cultivation.

[0055] Comparative Example 1

[0056] Batch culture (fermenter): C. protothecoides CS-41 microalgae seeds were inoculated at 10% (v / v) in a 2L fermenter filled with 1000mL fresh modified Basal medium, pH 6.1, 28°C, 300-700rpm in the dark. 200mM IAA and 3.6g / L urea were added on the 3rd day. Fermentation was stopped on the 5th day.

[0057] Comparative Example 2

[0058] High-concentration substrate culture (fermenter): C. protothecoides CS-41 microalgae seeds were inoculated at 10% (v / v) in a 2L fermenter filled with 1000 mL of fresh concentrated modified Basal medium, pH 6.1, 28°C, 300-700 rpm in the dark. Glucose and urea were supplemented when sugars were exhausted.

[0059] See also Figure 4 , showing the growth of C.protothecoides CS-41 in three different fermentation modes: batch fermentation, exponential feeding fermentation, and high-concentration substrate feeding fermentation. Due to the limitation of substrate, the maximum biomass of batch fermentation can only reach 6.3g / L, while exponential feeding fermentation and high-concentration substrate feeding fermentation can accumulate 52.7g / L and 52.4g / L of biomass, respectively. Although there is no difference in the maximum biomass, the growth rate of C.protothecoides CS-41 in the exponential feeding fermentation process is faster, the specific growth rate is greater, and it reaches the plateau phase 36h faster than the high-concentration substrate feeding fermentation mode. This proves the superiority of the exponential feeding fermentation model, which can ensure that the cells continue to grow at the maximum specific growth rate as much as possible.

[0060] See also Figures 5 to 7 , showing the growth curves and glucose consumption / supplement curves under three fermentation modes. Figure 6 It can be seen that during the exponential feeding fermentation, the supplementary nutrients increased exponentially with the exponential growth of C. protothecoides CS-41, and the supplementary nutrients could be fully consumed until 108h. Therefore, the growth curve was smoother. However, the growth curve of the high-concentration substrate feeding fermentation produced a larger slope only when the substrate was supplemented twice.

[0061] See also Figure 8 , showing the changes in lutein content of C.protothecoides CS-41 under three fermentation modes. In batch fermentation, glucose was exhausted on the second day, so the lutein content increased significantly on the third day. Similarly, in the high-concentration substrate fermentation process, sugar was exhausted on the fifth and sixth days, and the lutein content also increased on the sixth, seventh and eighth days. Exponential fed-batch fermentation is a more favorable fermentation mode for producing lutein because its glucose is constantly exhausted and replenished, which can stimulate a slow increase in lutein content.

[0062] In step S50, after culturing under dark conditions, plant hormones and nitrogen sources are added to continue culturing to obtain microalgae, including:

[0063] After culturing under dark conditions for the sixth day, IAA and urea were added, and fermentation was stopped on the eighth day to obtain the microalgae.

[0064] Preferably, the IAA added is 200 mM and the urea added is 3.6 g / L.

[0065] In one embodiment of the present invention, after C.protothecoides CS-41 is cultured by exponential feeding fermentation, IAA and urea may be added when C.protothecoides CS-41 reaches a plateau phase, etc., to further stimulate lutein accumulation and increase the total carotenoid content. 200mM IAA and 3.6g / L urea are the most favorable conditions for C.protothecoides CS-41. After exponential feeding fermentation, IAA high nitrogen combined treatment can increase the lutein content from 2.07mg / g to 3.27mg / g, an increase of 58%, and the lutein yield reaches 163mg / L, an increase of 56.7% compared with the untreated 104mg / L; the final lutein yield is 20.38mg / L / d, which greatly improves the lutein yield of this algae species.

[0066] In summary, the present invention is beneficial to the growth of microalgae such as C.protothecoides CS-41 through exponential feeding fermentation. At the same time, due to the continuous depletion and replenishment of glucose in this mode, the lutein content can be stimulated to increase slowly, which is also beneficial to the accumulation of lutein, and is a more favorable fermentation mode for producing lutein. At the same time, after the exponential feeding fermentation, for example, when the culture reaches the plateau phase, the lutein yield can be greatly improved by using IAA high nitrogen combined treatment. Therefore, the present invention effectively overcomes the various shortcomings of the prior art and has a high industrial utilization value.

[0067] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for fermenting algae, characterized in that: The steps include: Selecting microalgae seeds; constructing an exponential fed-batch model corresponding to the selected microalgae seeds; Inoculating the microalgae seeds into a fermentation tank containing Basal medium; Cultivating in the dark according to the exponential fed-batch model; After culturing under dark conditions, plant hormones and nitrogen sources are added to continue culturing to obtain microalgae.

2. The algae fermentation and cultivation method according to claim 1, characterized in that: Construct an exponential fed-batch model corresponding to the selected microalgae seeds, including: The microalgae seeds were inoculated in advance in a 250 mL conical flask containing 100 mL of fresh modified Basal medium, and cultured in a shaker at 28°C and 180 rpm in the dark for 48 hours. Samples were taken every 4 hours from the 12th hour to determine the feeding model corresponding to the exponential feeding model.

3. The algae fermentation and cultivation method according to claim 2, characterized in that: The feeding model corresponding to the exponential feeding model is: Among them, V FS is the flow acceleration rate, Y XS is the ratio of cell biomass to substrate concentration, μ is the specific growth rate of cells, t is the culture time, C FS is the nutrient concentration in the fed-batch medium.

4. The algae fermentation and cultivation method according to claim 2, characterized in that: Sampling was performed every 4 hours starting from the 12th hour to determine the feeding model corresponding to the exponential feeding model, including: From 12 hours to 48 hours of cultivation, the biomass and the consumption of glucose, urea and phosphate were measured every 4 hours, and the ratio of cell biomass to substrate concentration Y was calculated. XS and the specific growth rate μ of the cell; According to the initial culture liquid volume V0 and the initial biomass X0 in the fermenter, the feeding model is determined based on glucose feeding, and the feeding amounts of urea and phosphate are obtained in turn.

5. The algae fermentation and cultivation method according to claim 4, characterized in that: The feed amounts of glucose, nitrate and phosphate in the fed-batch culture medium were rounded to a molar ratio of 37:7:

2.

6. The algae fermentation and cultivation method according to claim 1, characterized in that: The selected microalgae seeds were C. protothecoides CS-41.

7. The algae fermentation and cultivation method according to claim 1, characterized in that: The microalgae seeds are inoculated into a fermentation tank containing a Basal medium, comprising: The microalgae seeds were inoculated at 10% (v / v) into a 2 L fermenter filled with 1000 mL of fresh modified Basal medium, and cultured at 28° C., 300-700 rpm shaking in a shaker in the dark, with the pH of the modified Basal medium being 6.

1.

8. The algae fermentation and cultivation method according to claim 7, characterized in that: Cultivation is performed under dark conditions according to the exponential fed-batch model, including: Under dark conditions, glucose, nitrate and phosphate are added to the fermenter according to the feeding model corresponding to the exponential feeding model for cultivation.

9. The algae fermentation and cultivation method according to claim 1, characterized in that: After culturing under dark conditions, plant hormones and nitrogen sources are added to continue culturing to obtain microalgae, including: After culturing under dark conditions for the sixth day, IAA and urea were added, and fermentation was stopped on the eighth day to obtain the microalgae.

10. The algae fermentation and cultivation method according to claim 9, characterized in that: The IAA added was 200 mM and the urea added was 3.6 g / L.

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