High-density culture method of chrysophyceae and application of chrysophyceae in production of chrysophyceae laminarin

Abstract

The invention discloses a method for producing chrysophyceae laminarin through high-density culture of chrysophyceae and an application of the chrysophyceae laminarin. The culture method comprises the following steps: (1) culturing a chrysophyceae species under low-temperature and low-light conditions, and then transferring the chrysophyceae species into a seed culture medium for culturing to obtain activated chrysophyceae species; (2) placing the activated algae species obtained in the step (1) under a dark condition for shake culture to obtain dark adaptation algae species; and (3) inoculating the dark adaptation algae seeds into a fermentation tank, and adding a basic culture medium and a fed-batch culture medium for fermentation culture. By selecting the nitrogen source and accurately controlling the ammonium concentration, the double improvement of the biomass and the yield of the chrysophyceae laminarin is realized, the adverse effect of the high-concentration nitrogen source on growth is avoided, and the culture efficiency and the product economy are improved.

Description

Technical Field

[0001] This invention relates to the field of industrial biotechnology, and in particular to a method for high-density cultivation of golden algae and its application in the production of golden algae kelp polysaccharides. Background Technology

[0002] Golden algae ( Poterioochromonas malhamensis Belonging to the phylum Chrysophyta and the genus *Cyclocarya*, *Chrysophyta* is a cell wall-less, mixed-nutrient, single-celled flagellated algae with high application value in aquaculture, environmental remediation, and synthetic biomanufacturing. In aquaculture, *Chrysophyta*, as a nutrient-rich microalga, can be used as initial feed or feed additives for fish, shrimp, and shellfish, improving the survival rate and disease resistance of aquatic animal larvae. In environmental remediation, *Chrysophyta* can engulf bacteria and harmful microorganisms such as *Microcystis aeruginosa*, offering potential advantages in water purification and algal bloom control. In synthetic biomanufacturing, *Chrysophyta* can accumulate large amounts of *Chrysophyta laminarin* polysaccharides in its vacuoles, which possess multiple functions including antibacterial, anti-inflammatory, antiviral, antitumor, immune-enhancing, blood sugar-regulating, and cholesterol-lowering effects, and are widely used in functional foods and pharmaceuticals. The realization of these applications all relies on a low-cost, high-concentration, and stable supply of *Chrysophyta laminarin* biomass; while the production of functional foods and pharmaceuticals requires high-purity, high-yield *Chrysophyta laminarin* polysaccharides. Therefore, developing a low-cost, high-density, and stable method for cultivating golden algae is key to promoting the industrial application of golden algae.

[0003] Existing golden algae cultivation technologies mainly include two categories: photosynthetic autotrophic culture and heterotrophic culture. Photosynthetic autotrophic culture relies on light, has a long cultivation cycle, and typically results in low biomass concentrations, making it difficult to meet the industrial demand for high-density biomass. While heterotrophic culture can achieve higher biomass in a shorter time, it often relies on expensive organic nitrogen sources, such as yeast extract and liver extract powder, significantly increasing production costs. Furthermore, existing fed-batch heterotrophic culture methods largely borrow from the fermentation processes of other microalgae or microorganisms, failing to fully consider the metabolic characteristics of golden algae, leading to poor batch-to-batch stability and large fluctuations in product content and yield. In some cultivation processes, unreasonable nitrogen source supply strategies may result in either incorrect nitrogen source selection, excessive nitrogen inhibiting product synthesis, or insufficient nitrogen limiting cell growth, making it difficult to simultaneously achieve high biomass and high product content.

[0004] In summary, existing technologies generally suffer from problems such as high production costs, insufficient biomass concentration, low product content and yield, and poor batch stability. There is an urgent need for a high-density cultivation method that optimizes the nitrogen supply mode for the metabolic characteristics of golden algae, so as to reduce production costs, improve the biomass of golden algae and the production efficiency of golden algae kelp polysaccharides, and achieve stable industrial production. Summary of the Invention

[0005] In view of the defects in the prior art, the present application provides a high-density culture method of golden algae and application thereof in production of laminarin from golden algae.

[0006] The present application provides a high-density culture method of golden algae, comprising the following steps: (1) after the golden algae spores are cultured under the conditions of a temperature of 15-35℃ and a light intensity of 10-300 μmol·m -2 ·s -1 , the spores are transferred to a seed culture medium for culture to obtain activated spores; (2) the activated spores obtained in step (1) are cultured under light-proof conditions for 3-6 days to obtain dark-adapted spores; (3) the dark-adapted spores obtained in step (2) are inoculated into a fermenter, a basic culture medium is added for culture, and then a feeding culture medium is added for fed-batch fermentation culture.

[0007] In some embodiments, the golden algae spores are Poterioochromonas malhamensis .

[0008] In some embodiments, the feeding culture medium uses ammonium acetate as the only nitrogen source, and the absolute concentration of ammonium ions in the fermentation culture is 5-500 mg / L.

[0009] Preferably, the absolute concentration of ammonium ions in the fermentation culture is 150-300 mg / L; during the fermentation process, the concentration of ammonium ions is controlled at 150-300 mg / L, which can not only ensure the nitrogen required for the synthesis of amino acids and nucleotides by cells, but also avoid problems such as acidification and increase of osmotic pressure caused by excessive ammonium ions, so as to realize rapid accumulation of the biomass of golden algae and laminarin from golden algae.

[0010] In some embodiments, the seed culture medium in step (1) comprises the following components: glucose 5-25 g / L, yeast extract 1-5 g / L, beef liver infusion powder 1-5 g / L, potassium dihydrogen phosphate 0.1-2.0 g / L, and magnesium sulfate heptahydrate 0.1-2.0 g / L.

[0011] Preferably, the seed culture medium in step (1) comprises the following components: glucose 10 g / L, yeast extract 3 g / L, beef liver infusion powder 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate heptahydrate 0.5 g / L.

[0012] In the present application, by optimizing the seed culture medium formula, it is ensured that the cells can quickly adapt during the static culture and the shaking culture process, and the lag phase of inoculation is reduced.

[0013] In some embodiments, in step (3), the pH of the fermentation culture is 3.0-9.0; preferably 5.8-6.2.

[0014] In some embodiments, in step (3), the temperature of the fermentation culture is 10-35°C; preferably 25-30°C.

[0015] In some embodiments, in step (3), the fermentation culture time is 3-8 days; preferably 4-7 days.

[0016] In some embodiments, in step (3), the rotation speed of the fermentation culture is 100-400 rpm, and the relative dissolved oxygen content during the fermentation culture is maintained at 5-30%.

[0017] In some embodiments, in step (3), the inoculation density of the dark-adapted algae is 5-30% of the total volume of the fermentation system.

[0018] In some embodiments, in step (3), the basic culture medium comprises the following components: glucose 5-20 g / L, ammonium chloride 1-4 g / L, potassium dihydrogen phosphate 0.3-1.5 g / L, magnesium sulfate heptahydrate 0.5-2.0 g / L, CaCl2·2H2O mother liquor 0.5-2 mL / L, FeCl3·6H2O mother liquor 0.5-2 mL / L, A5 mother liquor 0.5-2 mL / L, and vitamin Mix mother liquor 0.5-2 mL / L; Preferably, the basic culture medium comprises the following components: glucose 20 g / L, ammonium chloride 1 g / L, potassium dihydrogen phosphate 0.3 g / L, magnesium sulfate heptahydrate 0.5 g / L, CaCl2·2H2O mother liquor 0.5 mL / L, FeCl3·6H2O mother liquor 0.5 mL / L, A5 mother liquor 0.5 mL / L, and vitamin Mix mother liquor 0.5 mL / L; The feeding medium comprises the following components: glucose 100-400 g / L, ammonium acetate 10-80 g / L, potassium dihydrogen phosphate 2-10 g / L, magnesium sulfate heptahydrate 2-10 g / L, CaCl2·2H2O mother liquor 5-20 mL / L, FeCl3·6H2O mother liquor 5-20 mL / L, and A5 mother liquor 5-20 mL / L; Preferably, the feeding medium comprises the following components: Glucose 400 g / L, ammonium acetate 30 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate heptahydrate 10 g / L, CaCl2·2H2O mother liquor 20 mL / L, FeCl3·6H2O mother liquor 20 mL / L and A5 mother liquor 20 mL / L; The CaCl2·2H2O mother liquor comprises CaCl2·2H2O 147 g / L; The FeCl3·6H2O mother liquor comprises FeCl3·6H2O 6.3 g / L, EDTA-2Na 8.8 g / L; The A5 mother liquor comprises H3BO3 1.94 g / L, ZnSO4·7H2O 0.04 g / L, MnCl2·4H2O g / L, Na2MoO4·2H2O 0.012 g / L, Co(NO3)2·6H2O 0.0244 g / L; The vitamin Mix mother liquor comprises vitamin B1 0.75 g / L and vitamin B12 0.25 g / L.

[0019] The present application maintains the moderate growth of the algal species in the initial stage of fermentation by using a low concentration of nitrogen source and an appropriate amount of mineral salts, and avoids excessive metabolic burden. The high concentration of glucose and ammonium acetate in the feeding medium provides continuous carbon and nitrogen supply, meeting the requirements of exponential cell expansion under high-density conditions. The trace element mother liquor and vitamin Mix maintain the photosynthetic pigment synthesis, metal enzyme activity and coenzyme balance of the cells under high-density conditions.

[0020] The present application introduces differential medium formulations at different culture stages, precisely regulates the carbon-nitrogen ratio and trace element supply, and enables the chrysophyte to maintain high activity and yield under high-density conditions.

[0021] The present application also provides the use of the high-density culture method in the preparation of chrysophyte biomass or chrysophyte polysaccharide. The chrysophyte biomass refers to the total dry weight of the harvested chrysophyte, which contains various cell components, including polysaccharides, lipids, proteins, pigments, minerals and trace elements, etc. The chrysophyte polysaccharide includes laminarin.

[0022] Compared with the prior art, the present application achieves the following technical effects: 1. The present application realizes the rapid accumulation of chrysophyte biomass and laminarin by optimizing the medium components at different culture stages and reasonably controlling the ammonium concentration during fermentation. The chrysophyte biomass reaches 64 g / L at the 6th day of culture, with an average production rate of 10.67 g / L / d; the laminarin yield reaches 42.73 g / L, with an average yield of 7.12 g / L / d, which is about 1.8 times higher than the maximum yield of the prior art, showing a significant high-yield property.

[0023] 2、The feed medium used in the present application does not involve the addition of expensive organic nitrogen sources, does not rely on multiple vitamins, and does not require the addition of pure oxygen during operation, significantly simplifying the culture process, reducing equipment and operation complexity, and reducing production costs by about 85% compared to existing methods. Overall, the present application provides a simple, economical and efficient gold algae high-yield culture method suitable for industrial amplification, which can significantly improve the production efficiency and economic benefits of gold algae biomass and laminarin. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be considered as limiting the scope. For those skilled in the art, other related drawings can also be obtained without creative labor.

[0025] Figure 1 The gold algae culture scheme flowchart of the present application.

[0026] Figure 2 The gold algae batch culture results of the nitrogen source replacement process of Example 2 of the present application.

[0027] Figure 3 The gold algae fed-batch fermentation culture results of the nitrogen source optimization process of Example 3 of the present application.

[0028] Figure 4 The ammonium ion concentration curves of each group in the gold algae culture process under the control of the ammonium ion concentration of Example 4 of the present application.

[0029] Figure 5 The biomass concentration changes of the gold algae fed-batch culture process under the control of the ammonium ion concentration of Example 4 of the present application.

[0030] Figure 6 The laminarin yield changes of the gold algae fed-batch culture process under the control of the ammonium ion concentration of Example 4 of the present application. DETAILED DESCRIPTION

[0031] In order to better understand the present application by those skilled in the art, the technical solutions in the embodiments of the present application are described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should be within the scope of the present application.

[0032] The present application relates to a kind of high-density culture of chrysophyte method of producing laminarin, the culture method is fed-batch fermentation culture, and the characteristics of its culture mode are as follows: ① dark adaptation is carried out to the chrysophyte of light standing culture, and fermentation algal seed preparation is carried out in dark shaking incubator;② screening is carried out in the nitrogen source of fed-batch fermentation culture, and ammonium acetate is selected as the only nitrogen source in the feed medium;③ the addition concentration of ammonium acetate in the feed medium is regulated during the process of feeding culture;④ according to the change of ammonium concentration in the fermentation culture system, the feeding is adjusted, and the absolute concentration of ammonium is maintained at different levels.According to the method of the present application, the biomass concentration of chrysophyte can reach 64-65g / L at the 6th-7th day of culture, the content of laminarin is up to 65.99% of the dry weight of cells, and the yield reaches 42.73g / L.Compared with the control group, the selection of ammonium acetate and the control of ammonium concentration make the growth rate of chrysophyte significantly faster.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, etc. used can be obtained from commercial channels.

[0034] The technical solution involved in the present application is shown in Figure 1, which is specifically divided into five steps.

[0035] The three kinds of culture media involved in the present application are seed culture medium, basic culture medium and feed medium, and the components are shown in Tables 1-4. The culture media are sterilized by high-pressure steam before use, and the sterilization conditions are 121℃ for 20 minutes.

[0036] Table 1 Seed culture medium

[0037] Table 2 Basic culture medium

[0038] Table 3 Mother liquor formula

[0039] Table 4 Feed medium (wherein the mother liquor formula is the same as Table 3)

[0040] Example 1 Activation and preparation of chrysophyte algal seed 1. Activation of algal seed: 30 mL of chrysophyte liquid algal seed which was low-temperature and low-light (18℃, 50 μmol m -2 s -1 ) standing culture for 15 days was inoculated into a 1L triangular flask containing 400 mL of seed culture medium, and placed in a low-light shaking incubator for shaking culture. The culture conditions were temperature 26℃, light intensity 25 μmol m -2 s-1 The shaking speed was 160 rpm, and the incubation time was 5 days.

[0041] 2. Algal strain preparation: Take 30 mL of the activated golden algae seed and inoculate it into a 1 L Erlenmeyer flask containing 400 mL of seed culture medium. Place the flask in a dark shaker for incubation at 26℃ and 160 rpm for 3 days. Dark adaptation of the golden algae is achieved through incubation in the dark.

[0042] Example 2 Batch culture of golden algae under different nitrogen sources Dark-acclimatized golden algae were inoculated into 1L shake flasks containing 400mL seed culture medium and cultured in a dark shaker at 26℃ and 160rpm for 4 days. The control group used the same seed culture medium formulation, with yeast extract and bovine liver extract as nitrogen sources. In the experimental groups, ammonium chloride, urea, ammonium acetate, and potassium nitrate were used as the sole nitrogen sources, replacing yeast extract and bovine liver extract, respectively, and each of the four stock solutions was added at 0.5mL / L according to the stock solution formulation in Table 4. Except for the control group, which used 3g / L yeast extract and 1g / L bovine liver extract as nitrogen sources, the nitrogen molar concentrations in the other experimental groups were kept consistent: ammonium chloride 1g / L, urea 0.56g / L, ammonium acetate 1.44g / L, and potassium nitrate 1.89g / L.

[0043] like Figure 2 The results showed that when using organic nitrogen sources such as yeast extract and bovine liver extract powder, the maximum biomass concentration of *Chlorella vulgaris* during batch culture was 3.68 g / L; when using ammonium acetate as the sole nitrogen source, the maximum biomass concentration was 4.5 g / L; and when using ammonium chloride, urea, and potassium nitrate as the sole nitrogen sources, the maximum biomass concentrations were 1.82 g / L, 1.28 g / L, and 1.07 g / L, respectively. These results confirm that relatively inexpensive nitrogen sources such as ammonium acetate and ammonium chloride can be used to replace expensive organic nitrogen sources for the cultivation of *Chlorella vulgaris*.

[0044] Example 3: Fermentation culture of golden algae 400 mL of dark-acclimatized golden algae were inoculated into a 7L fermenter containing 2.5L of basal culture medium for fed-batch culture. The culture temperature was 26℃, the aeration rate was 3 L / min, the initial aeration speed was 100 rpm, and the aeration speed range was 100~350 rpm. During the culture, the relative dissolved oxygen was maintained at 20% by increasing the aeration speed, and the pH was adjusted by sodium hydroxide and phosphoric acid to maintain a stable pH of 6.0±0.2. During the fed-batch culture, the culture medium was added to the fermenter at a certain flow rate using a peristaltic pump, and the culture time was 7 days.

[0045] Example 4 Fermentation culture of Chlorella under different nitrogen source conditions Using the fed-batch fermentation conditions of Example 3, urea, ammonium chloride, and ammonium acetate were respectively used as the only nitrogen source to prepare the feed medium. During the preparation process, the molar concentration of nitrogen elements was ensured to be consistent under different forms of nitrogen source, i.e., the urea addition concentration was 12 g / L, the ammonium chloride addition concentration was 21 g / L, and the ammonium acetate addition concentration was 30 g / L. During the fed-batch fermentation culture process, the above-mentioned three kinds of feed media were respectively added to culture Chlorella, and the culture time was 7 days.

[0046] The results are shown in Figure 3 After 7 days of culture, the dry weight of Chlorella under different culture conditions was determined by the constant weight method: 3000 g of a certain volume of algae liquid was centrifuged for 5 min, the supernatant was discarded, the soluble salt in the precipitate was removed by washing, 200 μL of pure water was used to resuspend the precipitate, and the whole was transferred to a pre-weighed GF / C filter membrane, which was then dried to constant weight, and the total weight was accurately weighed. The difference between the two masses was the dry weight of the cells in the volume of algae liquid.

[0047] The results show that when ammonium acetate is used as the only nitrogen source to prepare the feed medium, the maximum biomass concentration of Chlorella is 46.3 g / L; when ammonium chloride is used, it is 27.7 g / L; and when urea is used, it is 17.5 g / L; indicating that ammonium acetate is the optimal nitrogen source for the fermentation culture of Chlorella of the present application.

[0048] Example 5 Effect of different ammonium ion concentrations on the growth of Chlorella Using the optimal nitrogen source ammonium acetate screened in Example 4 as the only nitrogen source, ammonium acetate concentrations of 40 g / L, 30 g / L, and 20 g / L were respectively prepared to prepare the feed medium, which was sterilized at 121°C for 20 min and cooled to room temperature for standby under the culture conditions of Example 2. The initial addition of the base medium in the fermenter was 2.5 L, and the dark-adapted algae seed was 400 mL. The fermentation conditions were temperature 26°C, aeration rate 3 L / min, initial stirring speed 100 rpm, and the stirring speed was gradually increased to not more than 350 rpm according to the dissolved oxygen level to maintain the dissolved oxygen content at 20%; the pH value was automatically adjusted using sodium hydroxide and phosphoric acid, and maintained at 6.0±0.2.

[0049] During the fermentation culture process, the ammonia nitrogen concentration in the fermentation broth was monitored by ultraviolet spectrophotometry. The addition rate of the feed medium was controlled so that the ammonium ion concentration in the fermentation system was in the high concentration (>350 mg / L, control group 1), medium concentration (150~350 mg / L, experimental group), and low concentration (<150 mg / L, control group 2) range.

[0050] The ammonium concentration curve is shown in Figure 4As shown, the initial ammonium concentration was 250-260 mg / L, and the ammonium concentration decreased initially and then increased after the first day of culture. The ammonium concentration of the control group 1 was greater than 300 mg / L on the 3rd to 4th day after feeding, reached a maximum of 812.5 mg / L on the 6th day, and decreased to 692.5 mg / L on the 7th day. The ammonium concentration of the control group 2 slowly accumulated on the 3rd to 4th day after feeding, and reached 144 mg / L on the 7th day. The ammonium concentration of the experimental group accumulated on the 3rd to 4th day after feeding, reached a maximum of 353 mg / L on the 6th day, and decreased to 207 mg / L on the 7th day.

[0051] The biomass concentration change is shown in FIG. 3. Figure 5 As shown, the control group 1 reached a maximum biomass of 55.75 g / L on the 5th day, growth stagnated on the 6th day, and decreased to 49.5 g / L on the 7th day; the biomass of the control group 2 continued to increase, and reached 64 g / L on the 7th day; the experimental group reached a maximum of 64.75 g / L on the 6th day, which was higher than that of the control group 1 and the control group 2. High ammonium supply led to growth inhibition of the golden algae in the later stage and a decrease in biomass; low ammonium supply could maintain continuous growth, but the overall biomass was slightly lower than that of the medium ammonium group; medium ammonium supply not only reached the highest biomass of 64.75 g / L on the 6th day, but also maintained a high final biomass, indicating that moderate ammonium levels are conducive to prolonging the high-efficiency growth period and obtaining higher final biomass.

[0052] Golden algae cells at different culture stages were collected synchronously in fed-batch fermentation culture, freeze-dried, and then the laminarin content in the golden algae cells was determined by enzymatic hydrolysis.

[0053] The steps for determining the laminarin content in the golden algae cells by enzymatic hydrolysis are as follows: 1. Take 10 mg of freeze-dried golden algae powder, add 1 mL of dd-H2O, and warm in a water bath at 50°C for 30 min.

[0054] 2. Centrifuge at 4000 g for 15 min at 18°C to collect the supernatant.

[0055] 3. Take 100 μL of the supernatant, add 900 μL of 100 mM sodium acetate buffer (pH 4.0 containing 1 mg / mL BSA), 1 μL of β-1,3-D-glucosidase and exo-β-1,3-glucanase mixed solution, and 1 μL of glucosidase, and hydrolyze at 40°C for 4 h at 60 rpm / min. The hydrolyzed solution is filtered with a 3 kDa ultrafiltration membrane, diluted, and the glucose concentration in the enzymatic hydrolysis product is determined by HPLC to calculate the glucan content.

[0056] The results are shown in FIG. 4. Figure 6As shown, the control group 1 is high concentration ammonium control (> 350 mg / L), after 5 days of culture, the laminarin production reaches the maximum of 34.58 g / L, slightly decreases on the 6th day, and decreases to 23.53 g / L on the 7th day. The control group 2 is low concentration ammonium control (< 100 mg / L), the laminarin accumulates during the culture process, and reaches the maximum concentration of 28.56 g / L on the 7th day. The experimental group is middle concentration ammonium control (150-350 mg / L), the laminarin obtains the maximum yield of 42.73 g / L on the 6th day of culture, and slightly decreases to 34.24 g / L on the 7th day. It is shown that too high ammonium level will inhibit the late accumulation of laminarin and lead to yield decrease; low concentration supply can sustain the accumulation of laminarin, but the peak value is low; middle concentration ammonium supply not only makes the peak value (42.73 g / L) of laminarin higher than that of other groups, but also can maintain a high level at the end.

[0057] The fermentation performance comparison under different ammonium control strategies in the embodiment is shown in Table 5.

[0058] Table 5. Fermentation performance comparison under different ammonium control strategies

[0059] When the ammonium concentration is at a high level, the chlorella cell is large, and the laminarin content is high, but the cell is under high stress, the cell number decreases in the late culture period, the cell ruptures, and the biomass is low; when the ammonium is at a low level, the laminarin content is low, the chlorella cell is small, but the cell number is more, and the high biomass concentration can be obtained. Only when the ammonium concentration of the fermentation culture system is in a moderate range, the chlorella can reach a balance in cell size, cell number and laminarin content, and the maximum chlorella biomass concentration and laminarin yield can be obtained.

[0060] The above only describes the preferred embodiments of the present application, and is not used to limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for high-density cultivation of chrysophyte, characterized by, Comprising the following steps: (1) the chrysophyte algae are cultured at a temperature of 15-35℃ and a light intensity of 50-200 μmol·m -2 -1 After 10-20 days, the chrysophyte algae are transferred to a seed culture medium to obtain activated algae, and the chrysophyte algae account for 5-20 % v / v of the seed culture medium.​ (2) The activated algae obtained in step (1) is cultured under lightless condition for 3-6 days to obtain dark-adapted algae; (3) The dark-adapted algae obtained in step (2) is inoculated into a fermenter, and a basic medium is added for culture, and then a feeding medium is added for fermentation culture; The feeding medium takes ammonium acetate as the only nitrogen source, and the absolute concentration of ammonium ion in the fermentation culture is 5-500 mg / L.

2. The high-density culture method according to claim 1, characterized by, The seed medium in step (1) comprises the following components: Glucose 5-25 g / L, yeast extract 1-5 g / L, beef liver infusion powder 1-5 g / L, potassium dihydrogen phosphate 0.1-2.0 g / L, magnesium sulfate heptahydrate 0.1-2.0 g / L.

3. The high density culture method according to claim 1, characterized by, In step (3), the pH of the fermentation culture is 3.0-9.

0.

4. The high density culture method according to claim 1, characterized by, In step (3), the temperature of the fermentation culture is 10-35℃.

5. The high density culture method according to claim 1, characterized by, In step (3), the fermentation culture time is 3-8 days.

6. The high density culture method according to claim 1, characterized by, In step (3), the rotation speed of the fermentation culture is 100-400 rpm, and the relative dissolved oxygen content is maintained at 5-30%.

7. The high density culture method according to claim 1, characterized by, In step (3), the inoculation density of the dark-adapted algae is 5-30% of the total volume of the fermentation system.

8. The high density culture method according to claim 1, characterized by, In step (3), the basic medium comprises the following components: Glucose 5-20 g / L, ammonium chloride 1-4 g / L, potassium dihydrogen phosphate 0.3-1.5 g / L, magnesium sulfate heptahydrate 0.5-2.0 g / L, CaCl2·2H2O mother liquor 0.5-2 mL / L, FeCl3·6H2O mother liquor 0.5-2 mL / L, A5 mother liquor 0.5-2 mL / L, and vitamin Mix mother liquor 0.5-2 mL / L; The feeding medium comprises the following components: Glucose 100-400 g / L, ammonium acetate 10-80 g / L, potassium dihydrogen phosphate 2-10 g / L, magnesium sulfate heptahydrate 2-10 g / L, CaCl2·2H2O mother liquor 5-20 mL / L, FeCl3·6H2O mother liquor 5-20 mL / L, and A5 mother liquor 5-20 mL / L; The CaCl2·2H2O mother liquor comprises CaCl2·2H2O 147 g / L; The FeCl3·6H2O mother liquor comprises FeCl3·6H2O 6.3 g / L, EDTA-2Na 8.8 g / L; The A5 mother liquor comprises H3BO3 1.94 g / L, ZnSO4·7H2O 0.04 g / L, MnCl2·4H2O g / L, Na2MoO4·2H2O 0.012 g / L, Co(NO3)2·6H2O 0.0244 g / L; The vitamin Mix mother liquor comprises vitamin B1 0.75 g / L and vitamin B12 0.25 g / L.

9. The use of the high-density culture method according to any one of claims 1-8 in the preparation of Chrysophyte biomass, Chrysophyte polysaccharide, or Chrysophyte oil.

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