High-density culture medium and application thereof

Abstract

The invention provides a high-density culture medium and application thereof, and belongs to the field of algae culture, the high-density culture medium provided by the invention comprises a salt agent, and the salt agent comprises the following components: 212-24 mg / L of CO (NH2); the concentration of KH2PO4 is 0.75 to 1.25 mg / L; 0.50 mg / L to 1.00 mg / L of FeC6H5O7; 10 mg / L of Na2EDTA; the concentration of MnSO4 is 0.25 mg / L; the concentration of Na2SiO3 is 20mg / L; and 6-7 [mu] g / L of vitamin. When the culture medium and the culture method are used for culturing the scenedesmus diformosus, the advantages of high growth speed, high yield, high total lipid accumulation speed and increased unsaturated fatty acid content are achieved, the culture density, the total lipid accumulation speed and the unsaturated fatty acid content of the scenedesmus diformosus can be effectively improved, and the culture medium and the culture method are suitable for large-scale culture.

Description

Technical Field

[0001] The invention relates to the field of algae cultivation, and in particular to a high-density culture medium and application thereof. Background Art

[0002] Scenedesmus obliquus belongs to the genus Scenedesmus, class Centricae, order Chlorococcales, family Scenedesmaceae, and phylum Chlorophyta. It is widely distributed at home and abroad, and is usually composed of 4 or 8 cells, with a cell length of 16 to 23 μm and a width of 3 to 5 μm. At present, the cultivation of Scenedesmus obliquus has problems such as slow growth of algal cells, long doubling time, slow total lipid accumulation, and low content of unsaturated fatty acids.

[0003] In the cultivation of Scenedesmus dimorphus, the supply of nitrogen, phosphorus and iron directly affects the growth and metabolism of algae. The right amount of nitrogen can promote protein and nucleic acid synthesis, increase growth rate and total lipid content. Phosphorus is a component of cellular nucleic acids and energy molecules, and its appropriate concentration helps to increase total lipid accumulation. Iron ensures the stability of its cell structure, the conduct of photosynthesis, and the proper absorption of silicon. By carefully adjusting the ratio and concentration of nitrogen, phosphorus and iron, the cultivation conditions of Scenedesmus dimorphus can be optimized, and problems such as slow growth rate, long doubling time, and insufficient lipid accumulation can be solved to improve the cultivation effect. This requires careful experimental design and monitoring to ensure that the algae obtain appropriate nutritional support at different growth stages. Summary of the invention

[0004] The purpose of the present invention is to provide a culture medium with reasonable proportions and suitable for high-density culture of Scenedesmus dimorphus.

[0005] To achieve the above object, the present invention provides a high-density culture medium in a first aspect. The high-density culture medium includes a salt agent, and the salt agent includes the following components:

[0006]

[0007]

[0008] Preferably, the vitamins include vitamin B1 and vitamin B12 in a mass ratio of (100-150):1.

[0009] Preferably, the high-density culture medium also includes water, and the mass ratio of water to salt is (800-1000):1.

[0010] Furthermore, the second aspect of the present invention provides an application of the high-density culture medium of the first aspect, wherein the application comprises using the high-density culture medium for culturing Scenedesmus dimorphus.

[0011] Preferably, when the high-density culture medium is used for culturing Scenedesmus dimorphus, the culture light intensity is 30 to 50 μmol / (m 2 ·s), the culture temperature was 24-26℃, and the light-dark cycle was 12h:12h.

[0012] Compared with the prior art, the present invention has the following beneficial effects: the existing culture medium for dimorphic Scenedesmus does not determine the use of nitrogen salts, phosphorus salts and iron salts, and does not provide suitable concentrations for use. The present invention uses suitable nitrogen, phosphorus and iron salts and corresponding concentration agents to increase the concentration of dimorphic Scenedesmus cells, accelerate total lipid accumulation and increase the content of unsaturated fatty acids, so as to obtain a higher culture density, a higher total lipid accumulation rate and a higher unsaturated fatty acid content. Cultivating dimorphic Scenedesmus using the culture medium and culture method of the present invention has the advantages of fast growth rate, high yield, fast total lipid accumulation and increased unsaturated fatty acid content, can effectively improve the culture density, total lipid accumulation rate and unsaturated fatty acid content of dimorphic Scenedesmus, and is suitable for large-scale cultivation. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The experimental results of the effects of three culture media on algae cell concentration in Example 4, Comparative Example 1 and Comparative Example 2 in the specific implementation of the present invention;

[0014] Figure 2 This is a comparison result diagram of the K values ​​of three culture media in Example 4, Comparative Example 1, and Comparative Example 2 in a specific implementation manner of the present invention. DETAILED DESCRIPTION

[0015] In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation method and typical parameters of the present invention, and are not used to limit the parameter range described in the present invention. Reasonable changes derived therefrom are still within the scope of protection of the claims of the present invention.

[0016] It should be noted that the endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0017] Unless otherwise defined, all terms, symbols and other scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with conventionally understood meanings are defined herein for the purpose of clarification or ease of reference, and such definitions herein should not be construed as indicating significant differences from conventional understandings in the art. The technical methods described or cited herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents and instruments is carried out in accordance with the protocols and parameters given by the manufacturer.

[0018] Example 1

[0019] The high-density culture medium of the dimorphic scenedesmus is prepared in proportion; the culture medium includes the following components: CO(NH2)2 24 mg / L; KH2PO4 1.25 mg / L; FeC6H5O7 0.75 mg / L; Na2EDTA 10 mg / L; MnSO4 0.25 mg / L; Na2SiO3 20 mg / L; vitamin 6.05 μg / L. The vitamins include vitamin B1 and vitamin B12; the added amount of vitamin B1 is 6 μg / L; the added amount of vitamin B12 is 50 ng / L.

[0020] The prepared high-density culture medium is added dropwise into the disinfected fresh water to prepare a Scenedesmus dimorphus culture medium for use; the mass ratio of the high-density culture medium to the fresh water is 1:1000.

[0021] The dimorphic Scenedesmus in the growth period was inoculated into the dimorphic Scenedesmus culture medium of step S2 for cultivation, and then inoculated into the GXZ intelligent light incubator for cultivation. The cultivation conditions were: the light intensity was 40 μmol / (m 2 ·s), the culture temperature was 25°C, and the light-dark cycle was 12h:12h.

[0022] Example 2

[0023] The high-density culture medium of the dimorphic scenedesmus is configured in proportion; the culture medium includes the following components: CO(NH2)2 12 mg / L; KH2PO4 0.75 mg / L; FeC6H5O7 0.50 mg / L; Na2EDTA 10 mg / L; MnSO4 0.25 mg / L; Na2SiO3 20 mg / L; the vitamin addition amount is 6 μg / L; wherein the vitamin B12 addition amount is 100 ng / L.

[0024] The high-density culture medium is added dropwise into the disinfected fresh water to prepare a Scenedesmus dimorphus culture medium for use; the mass ratio of the high-density culture medium to the fresh water is 1:1000.

[0025] The dimorphic Scenedesmus in the growth period was inoculated into the dimorphic Scenedesmus culture medium of step S2 for cultivation, and then inoculated into the GXZ intelligent light incubator for cultivation. The cultivation conditions were: the light intensity was 30 μmol / (m 2 ·s), the culture temperature was 24°C, and the light-dark cycle was 12h:12h.

[0026] Example 3

[0027] The high-density culture medium of the dimorphic Scenedesmus is prepared in proportion; the culture medium comprises the following components: CO(NH2)2 24 mg / L; KH2PO4 1.25 mg / L; FeC6H5O7 1.00 mg / L; Na2EDTA 10 mg / L;

[0028] MnSO4 0.25mg / L; Na2SiO3 20mg / L; vitamins 7μg / L; the vitamins include vitamin B1 and vitamin B12, wherein the added amount of vitamin B12 is 120ng / L.

[0029] The high-density culture medium is added dropwise into the sterilized fresh water to prepare a Scenedesmus dimorphus culture medium for later use;

[0030] The mass ratio of the high-density culture medium to the fresh water is 1:1000.

[0031] The dimorphic Scenedesmus in the growth period was inoculated into the dimorphic Scenedesmus culture medium of step S2 for cultivation, and then inoculated into the GXZ intelligent light incubator for cultivation. The cultivation conditions were: the light intensity was 50 μmol / (m 2 ·s), the culture temperature was 26°C, and the light-dark cycle was 12h:12h.

[0032] The following provides embodiments and comparative examples combined with actual data to describe the above technical solutions of the present invention:

[0033] In the following embodiments, including 3 # The composition and proportion of the culture medium and f / 2 culture medium are as follows:

[0034] 3 # Culture medium:

[0035] NaNO3 100mg / L, KH2PO4 10mg / L, FeSO4 2.5mg / L, MnSO4 0.25mg / L, Na2SiO3 20mg / L, Na2EDTA10mg / L, VB1 6μg / L, VB 12 50ng / L.

[0036] f / 2 medium:

[0037] NaNO3 74.8mg / L, NaH2PO4 4.4mg / L, Na2SiO3·9H2O 20mg / L, “f / 2” trace element solution 1mL / L, “f / 2” vitamin solution 1mL / L.

[0038] Among them, "f / 2" trace element solution: ZnSO4·4H2O 23mg / L, MnCl2·4H2O 178mg / L, CuSO4·5H2O 10mg / L, FeC6H5O7·5H2O 3.9mg / L, Na2MoO4·2H2O 7.3mg / L, CoCl2·6H2O12mg / L, Na2EDTA4.35mg / L.

[0039] "f / 2" Vitamin Solution: V B1 100mg / L,V B12 0.5mg / L, biotin 0.5mg / L.

[0040] Example 4

[0041] S1: Prepare 1L of the following mother solution:

[0042] CO(NH2)2 24g / L, KH2PO4 1.25g / L, FeC6H5O7 0.75g / L, Na2EDTA10g / L, MnSO4 0.25g / L, Na2SiO3 20g / L, V B1 6mg / L,V B12 50μg / L.

[0043] S2: Add the mother solution to the disinfected fresh water at a ratio of 1:1000 to prepare the following fresh water culture medium:

[0044] CO(NH2)2 24mg / L, KH2PO4 1.25mg / L, FeC6H5O7 0.75mg / L, Na2EDTA10mg / L, MnSO40.25mg / L, Na2SiO3 20mg / L, V B1 6μg / L, V B12 50ng / L.

[0045] S3: Take the dimorphic Scenedesmus in the logarithmic growth phase and inoculate it into the culture medium for cultivation. Place it in the GXZ intelligent light incubator for cultivation. The light intensity is 40 μmol / (m 2 ·s), 25℃, light-dark cycle 12h:12h, no aeration, test period 7d. At the beginning and end of the experiment, algae cells were counted using a hemocytometer.

[0046] Comparative Example 1:

[0047] Use 3 # The culture medium was used as a control culture medium and compared with the Scenedesmus dimorphus culture medium of Example 4. The experimental conditions were: cultured in a GXZ intelligent light incubator with a light intensity of 40 μmol / (m 2 ·s), 25℃, light-dark cycle 12h:12h, no inflation, test period 7d.

[0048] Comparative Example 2:

[0049] The f / 2 culture medium was used as a control culture medium for comparison with the Scenedesmus dimorphus culture medium of Example 4. The experimental conditions were: cultured in a GXZ intelligent light incubator with a light intensity of 40 μmol / (m 2 ·s), 25℃, light-dark cycle 12h:12h, no inflation, test period 7d.

[0050] Statistical results, comparing the effects of three culture media on algal cell concentration (see Figure 1 ), and compare the K values ​​of the three culture media (see Figure 2 ), and compared the color of the algal suspension on the fifth day among the three culture media.

[0051] like Figure 1 As shown in Figure 2, with the increase of culture time, the increase rate of algal cell concentration in the culture medium of Example 4 is much higher than that of Example 3. # The algae cell concentration in the medium and f / 2 medium, and the highest algae cell concentration in the medium of Example 4 is much higher than that of 3 # The maximum concentration of algal cells in the culture medium and f / 2 culture medium. Figure 2 As shown, by comparing the growth rates, it can be found that the K value of the culture medium in Example 4 is also much higher than that of 3. # Therefore, it is proved that the culture medium of the present invention has the advantages of rapid algae proliferation, early entry into the logarithmic growth phase, shortened culture time, long stable period and good stability.

[0052] Although the disclosure is disclosed as above, the protection scope of the disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A high-density culture medium, characterized in that The high density culture medium includes a salt solution comprising the following components:

2. The high-density culture medium according to claim 1, characterized in that The vitamins include vitamin B1 and vitamin B12 in a mass ratio of (100-150):

1.

3. The high-density culture medium according to claim 1, characterized in that The high-density culture medium also includes water, and the mass ratio of water to salt agent is (800-1000):

1.

4. A use of the high-density culture medium according to any one of claims 1 to 3, characterized in that: The high-density culture medium is used for culturing Scenedesmus dimorphus.

5. The use according to claim 4, characterized in that When the high-density culture medium is used for culturing Scenedesmus dimorphus, the culture light intensity is 30 to 50 μmol / (m 2 ·s), the culture temperature was 24-26℃, and the light-dark cycle was 12h:12h.

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