A culture medium for linear circular algae and a cultivation method thereof

Abstract

This invention discloses a linear circular sieve algae culture medium, comprising: NH4HCO3, K2HPO4, Na2SiO3·9H2O, Na2EDTA, FeCl3, MgSO4·7H2O, CaCl2·2H2O, Tween-80, CuSO4·5H2O, ZnSO4·7H2O, CoCl2·6H2O, MnCl2·4H2O, H3BO3, vitamin B1, and vitamin B2. 12 Biotin and SeO2 are the preferred components and proportions of the culture medium in this scheme. After testing, the culture medium can significantly improve the growth rate of Cyclosporium rotundum and reduce the empty cell shell rate.

Description

Technical Field

[0001] This invention relates to the field of bioculture technology, specifically to a linear spherical algae culture medium and a method for culturing spherical algae using the medium. Background Technology

[0002] *Coscinodiscus lineatus*, belonging to the phylum Coscinodia, class Centromerdia, order Coscinodiscales, and genus *Coscinodiscus*, is a marine diatom. Its biological characteristics include disc-shaped cells with large pores on the shell surface, arranged in a hexagonal, radial pattern. It is one of the most common planktonic diatoms and a primary food source for marine larvae of fish, shrimp, and shellfish. Studies have found that this algae is high in protein and rich in unsaturated fatty acids, making it an excellent initial food source for blood clam larvae, significantly improving their survival rate.

[0003] Currently, the culture media used in the large-scale cultivation of *Cyclocarya linearis* are mainly f / 2 medium with added silicate and seawater MAV mother liquor with added silicate. These media have problems such as slow growth rate, low algal cell density, and easy empty cell shells, which seriously restrict the large-scale cultivation of *Cyclocarya linearis* and need to be improved. Summary of the Invention

[0004] To address at least one of the aforementioned technical deficiencies, the present invention provides the following technical solution:

[0005] This application discloses a culture medium for linear circular sieve algae, comprising: NH4HCO3 100-150 mg / L, K2HPO4 20-30 mg / L, Na2SiO3·9H2O 20-50 mg / L, Na2EDTA 40-50 mg / L, FeCl3 5-10 mg / L, MgSO4·7H2O 100-200 mg / L, CaCl2·2H2O 100-200 mg / L, Tween-80 0.01-0.05 v / v%, CuSO4·5H2O 0.03-0.05 mg / L, ZnSO4·7H2O 0.03-0.05 mg / L, CoCl2·6H2O 0.01-0.03 mg / L, MnCl2·4H2O 0.03-0.07 mg / L, and H3BO3. 0.02-0.05 mg / L, Vitamin B1 0.1-0.3 mg / L, Vitamin B 12 0.3-0.5 mg / L, Biotin 0.3-0.5 mg / L, SeO2 0.05-0.2 mg / L.

[0006] The optimal composition and ratio of the culture medium in this scheme have been tested and found to significantly improve the growth rate of *Cyclocarya paliurus*, increase algal cell density, and reduce the empty cell rate.

[0007] Further, the following concentrations are listed: NH4HCO3 120-140 mg / L, K2HPO4 25-30 mg / L, Na2SiO3·9H2O 40-50 mg / L, Na2EDTA 40-50 mg / L, FeCl3 7-10 mg / L, MgSO4·7H2O 150-180 mg / L, CaCl2·2H2O 150-180 mg / L, Tween-80 0.03-0.05 v / v%, CuSO4·5H2O 0.05 mg / L, ZnSO4·7H2O 0.04 mg / L, CoCl2·6H2O 0.03 mg / L, MnCl2·4H2O 0.05 mg / L, H3BO3 0.05 mg / L, Vitamin B1 0.2 mg / L, Vitamin B2... 12 0.4 mg / L, Biotin 0.4 mg / L, SeO2 0.1 mg / L.

[0008] The second aspect of this application discloses a method for culturing *Cyclocarya linearis*, comprising the above-mentioned culture medium, adding the culture medium to sterilized seawater in a certain proportion, and then inoculating *Cyclocarya linearis* in the logarithmic growth phase for culturing.

[0009] For the above-mentioned culture medium, tests have confirmed that the appropriate inoculation time of *Cyclocarya paliurus* helps to promote the cultivation of *Cyclocarya paliurus*.

[0010] For different culture environments, there are various sterilization methods, such as autoclaving and disinfectant sterilization. As for the method of adding culture medium, it can be added in the form of stock solution or mother solution.

[0011] Furthermore, the culture is carried out in a natural environment. The filtered seawater is disinfected with chlorine dioxide. After 24 hours, a neutralizing agent is added to neutralize the disinfected seawater, and then the culture medium is added.

[0012] Furthermore, the cells were cultured indoors with a light-to-dark ratio of 10-14 h and a light intensity of 35-42 μmol / (m²). 2 •s), the culture temperature is 20-26℃.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] 1. The preferred culture medium formula of this invention can promote the rapid growth of round sieve algae cells, with low cell empty shell rate, and significant effect, making it suitable for large-scale culture applications. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a schematic diagram showing the curve changes of *Circular sieve algae* cell density over time in Examples 1, 2, and the comparative example.

[0017] Figure 2 These are graphs showing the empty shell rate of *Circular sieve algae* cells in Examples 1, 2, and the comparative example.

[0018] Figure 3 These are schematic diagrams showing the changes in cell density of *Circular sieve algae* over time in Examples 3, 4, and the comparative examples.

[0019] Figure 4 These are data graphs showing the empty shell rate of *Cyclocarya paliurus* cells in Examples 3, 4, and the comparative example; Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0021] The implementation plan is divided into two parts: the first part is indoor culture; the second part is outdoor culture.

[0022] I. Indoor Culture

[0023] Example 1

[0024] 1. Prepare a linear sieve algae culture medium with the following formula using fresh water: NH4HCO3 120mg / L, K2HPO4 25mg / L, Na2SiO3·9H2O 40mg / L, Na2EDTA 40mg / L, FeCl3 7mg / L, MgSO4·7H2O 150mg / L, CaCl2·2H2O 150mg / L, Tween-80 0.03v / v%, CuSO4·5H2O 0.05mg / L, ZnSO4·7H2O 0.04mg / L, CoCl2·6H2O 0.03mg / L, MnCl2·4H2O 0.05mg / L, H3BO3 0.05mg / L, Vitamin B1 0.2mg / L, Vitamin B2 12 Prepare a stock solution by adding 0.4 mg / L of biotin, 0.4 mg / L of seO2, and 0.05 mg / L of seO2 (at a ratio of 1:1000).

[0025] 2. Culture in a 500ml Erlenmeyer flask, add 200ml of filtered seawater and sterilize in a 121℃ autoclave. Then add the above-mentioned culture medium in proportion. During the experiment, logarithmic growth phase *Cyclocarya linearis* was inoculated into the culture medium at the standard inoculation rate. The light-to-time ratio was 12:12, and the light intensity was 40 μmol / (m²). 2 •s), the incubator temperature was controlled at 24℃, and the cell density of *Lycopodium lineare* was recorded every morning at 9:00 AM. See details. Figure 1 .

[0026] Example 2

[0027] 1. Prepare the following culture medium for *Alternaria lineari* using fresh water: NH₄HCO₃ 140 mg / L, K₂HPO₄ 30 mg / L, Na₂SiO₃·9H₂O 50 mg / L, Na₂EDTA 50 mg / L, FeCl₃ 10 mg / L, MgSO₄·7H₂O 180 mg / L, CaCl₂·2H₂O 180 mg / L, Tween-80 0.05 v / v%, CuSO₄·5H₂O 0.05 mg / L, ZnSO₄·7H₂O 0.04 mg / L, CoCl₂·6H₂O 0.03 mg / L, MnCl₂·4H₂O 0.05 mg / L, H₃BO₃ 0.05 mg / L, Vitamin B1 0.2 mg / L, Vitamin B₂ 12 Prepare a stock solution by adding 0.4 mg / L of biotin, 0.4 mg / L of seO2, and 0.1 mg / L of seO2 (at a ratio of 1:1000).

[0028] 2. The cultivation method is the same as in Example 1.

[0029] Comparative Example

[0030] Two culture media, f / 2 with sodium silicate and MAV with sodium silicate, were used as control media. Except for the different media components, the results were the same as in Example 1. For details comparing the results with the example, please refer to [link to example]. Figure 1 .

[0031] II. Outdoor cultivation

[0032] Example 3

[0033] 1. Prepare the following culture medium for *Lycopodium clavatum* using fresh water: NH₄HCO₃ 120 mg / L, K₂HPO₄ 25 mg / L, Na₂SiO₃·9H₂O 40 mg / L, Na₂EDTA 40 mg / L, FeCl₃ 7 mg / L, MgSO₄·7H₂O 150 mg / L, CaCl₂·2H₂O 150 mg / L, Tween-80 0.03 v / v%, CuSO₄·5H₂O 0.05 mg / L, ZnSO₄·7H₂O 0.04 mg / L, CoCl₂·6H₂O 0.03 mg / L, MnCl₂·4H₂O 0.05 mg / L, H₃BO₃ 0.05 mg / L, Vitamin B1 0.2 mg / L, Vitamin B₂ 12 Prepare a stock solution by adding 0.4 mg / L of biotin, 0.4 mg / L of seO2, and 0.05 mg / L of seO2 (at a ratio of 1:1000).

[0034] 2. Culture was carried out in a 20-ton tank. Filtered seawater was added and disinfected with chlorine dioxide. After two days, the remaining chlorine was neutralized with sodium thiosulfate. Then, the above-mentioned culture medium was added to it in the same proportion as in Example 1. During the experiment, *Cyclocarya linearis* in its logarithmic growth phase (in the same proportion as in Example 1) was inoculated into the culture medium and cultured under natural light. The cell density of *Cyclocarya linearis* was recorded every morning at 9:00 AM. See details... Figure 2 .

[0035] Example 4

[0036] 1. Prepare the following culture medium for *Alternaria lineari* using fresh water: NH₄HCO₃ 140 mg / L, K₂HPO₄ 30 mg / L, Na₂SiO₃·9H₂O 50 mg / L, Na₂EDTA 50 mg / L, FeCl₃ 10 mg / L, MgSO₄·7H₂O 180 mg / L, CaCl₂·2H₂O 180 mg / L, Tween-80 0.05 v / v%, CuSO₄·5H₂O 0.05 mg / L, ZnSO₄·7H₂O 0.04 mg / L, CoCl₂·6H₂O 0.03 mg / L, MnCl₂·4H₂O 0.05 mg / L, H₃BO₃ 0.05 mg / L, Vitamin B1 0.2 mg / L, B 12 Prepare a stock solution by adding 0.4 mg / L of biotin and 0.1 mg / L of SeO2 (1:1000 ratio).

[0037] 2. The cultivation method is the same as in Example 3.

[0038] Comparative Example

[0039] Two culture media, f / 2 with sodium silicate and MAV with sodium silicate, were used as control media. Except for the different media components, the results were the same as in Example 3. For details comparing the results with the examples, please refer to [link to example]. Figure 2 .

[0040] refer to Figure 1 , Figure 3 It can be seen that, compared with f / 2 medium and MAV medium, the medium of the present invention can more rapidly increase the cell proliferation of *Cyclocarya linearis*, and the highest concentration of algal cells reached is also significantly higher than that of the control group f / 2 medium and MAV medium by 30-50%.

[0041] from Figure 2 , Figure 4 It can be seen that, compared with f / 2 medium and MAV medium, the cell empty shell rate of *Lycopodium lineare* was significantly reduced when using the medium of the present invention.

[0042] It can be considered that the culture effect obtained by the culture medium of the present invention after fine-tuning of the components is significantly better than expected, which is more conducive to the rapid proliferation of algal cells and the accumulation of biomass, and is suitable for large-scale culture applications.

[0043] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principle of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A linear circular sieve algae ( Coscinodiscus lineatus A method for cultivating ( ) including a culture medium, characterized in that: The culture medium was added to sterilized seawater in a specific ratio, followed by inoculation with logarithmic growth phase *Cyclocarya filamentosa*. The culture was then carried out indoors under a light-drying ratio of 10-14 h and a light intensity of 35-42 μmol / (m²). 2 •s), the culture temperature is 20-26℃; The culture medium includes: NH4HCO3 100-150 mg / L, K2HPO4 20-30 mg / L, Na2SiO3·9H2O 20-50 mg / L, Na2EDTA 40-50 mg / L, FeCl3 5-10 mg / L, MgSO4·7H2O 100-200 mg / L, CaCl2·2H2O 100-200 mg / L, Tween-80 0.01-0.05 v / v%, CuSO4·5H2O 0.03-0.05 mg / L, ZnSO4·7H2O 0.03-0.05 mg / L, CoCl2·6H2O 0.01-0.03 mg / L, MnCl2·4H2O 0.03-0.07 mg / L, H3BO3 0.02-0.05 mg / L, Vitamin B1 0.1-0.3 mg / L, Vitamin B 12 0.3-0.5 mg / L, Biotin 0.3-0.5 mg / L, SeO2 0.05-0.2 mg / L.

2. The method for culturing linear circular sieve algae as described in claim 1, characterized in that: NH4HCO3 120-140 mg / L, K2HPO4 25-30 mg / L, Na2SiO3·9H2O 40-50 mg / L, Na2EDTA 40-50 mg / L, FeCl3 7-10 mg / L, MgSO4·7H2O 150-180 mg / L, CaCl2·2H2O 150-180 mg / L, Tween-80 0.03-0.05 v / v%, CuSO4·5H2O 0.05 mg / L, ZnSO4·7H2O 0.04 mg / L, CoCl2·6H2O 0.03 mg / L, MnCl2·4H2O 0.05 mg / L, H3BO3 0.05 mg / L, Vitamin B1 0.2 mg / L, Vitamin B 12 0.4 mg / L, Biotin 0.4 mg / L, SeO2 0.1 mg / L.

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