Microalgae culture medium and microalgae culture method

Abstract

The invention provides a microalgae culture medium and a microalgae culture method, and belongs to the technical field of algae culture, the microalgae culture medium provided by the invention comprises a nitrogen source and a salt agent, the mass ratio of the nitrogen source to the salt agent is 1: (0.7-1.3), and the nitrogen source comprises a phytic acid compound, amino acid and putrescine. When the microalgae culture medium provided by the invention is used for culturing microalgae, the growth rate, the total lipid accumulation rate and the unsaturated fatty acid content of the microalgae can be effectively improved, and the microalgae culture medium provided by the invention is not easy to cause eutrophication of a water body and is suitable for large-scale culture of the microalgae.

Description

Technical Field

[0001] The present invention relates to the technical field of algae cultivation, and in particular to a microalgae culture medium and a microalgae cultivation method. Background Art

[0002] In the field of large-scale cultivation of microalgae, the supply mode and proportion control of key nutrients such as nitrogen, phosphorus, and iron are the core factors that determine biomass accumulation and target product synthesis. Traditional microalgae culture media generally use inorganic nitrogen sources (such as sodium nitrate, urea) and phosphates as the main nutrient sources. Although these ingredients can be quickly absorbed by microalgae, they have two major systemic defects: 1. Uncontrollable nutrient release: Inorganic nitrogen and phosphorus are very easy to dissolve in water, resulting in a sudden increase in concentration in a short period of time. When the absorption rate of microalgae is lower than the nutrient supply rate, excess inorganic nitrogen (NH4⁺, NO3⁻) and phosphorus (PO4³⁻) will be discharged into natural water bodies with the culture solution, causing algal blooms to consume dissolved oxygen and block the air. Sunlight inhibits the growth of submerged plants, ultimately leading to the collapse of aquatic ecosystems. 2. Element synergy is unbalanced. Existing culture medium formulas are mostly based on empirical ratios and lack the ability to match the dynamic metabolic needs of elements such as nitrogen, phosphorus, and iron. For example, although high concentrations of nitrate can promote the initial growth of microalgae, they will inhibit the activity of tryptophan synthase, resulting in insufficient accumulation of intracellular antioxidants (such as carotenoids); and improper selection of iron forms (such as Fe³⁺, which is easily hydrolyzed and precipitated) will limit chlorophyll synthesis, further reducing the efficiency of light energy utilization.

[0003] While recent studies have attempted to improve nutrient utilization efficiency by optimizing nitrogen-to-phosphorus ratios or adding slow-release fertilizers, the following bottlenecks remain: Poor adaptability of organic nitrogen sources: Some approaches use composite organic nitrogen sources such as peptone and yeast extract, but these sources are complex and have unstable degradation rates, which can easily lead to ammonia nitrogen accumulation in late cultivation. Therefore, developing a microalgae culture medium formulation that combines precise nutrient release, synergistic element enhancement, and environmental friendliness has become a key technical challenge in addressing the risk of eutrophication and achieving efficient and sustainable microalgae cultivation. Summary of the Invention

[0004] The problem to be solved by the present invention is how to overcome the problem that the existing microalgae culture medium uses too much inorganic nitrogen and phosphorus, which easily causes eutrophication of water bodies and restricts the growth of microalgae.

[0005] To solve the above technical problems, the first aspect of the present invention provides a microalgae culture medium, which includes a nitrogen source and a salt agent, wherein the mass ratio of the nitrogen source to the salt agent is 1:(0.7~1.3), and the nitrogen source includes phytic acid compounds, amino acids and putrescine.

[0006] The microalgae culture medium provided by the present invention can provide stable nitrogen release for microalgae through quality control of nitrogen source and salt agent, combined with phytic acid compounds as a slow-release nitrogen source, reduce nitrogen element fluctuations in the microalgae culture system in the short term, and avoid the eutrophication risk caused by excessive nitrogen and phosphorus in traditional culture media. The role of putrescine is to improve the stress resistance of microalgae.

[0007] Preferably, the phytic acid compound is sodium phytate, and the amino acids include lysine, glycine, and tryptophan. The microalgae culture medium includes, by mass, 10-20 parts of sodium phytate, 1-4 parts of lysine, 2-5 parts of glycine, 3-8 parts of tryptophan, and 0.01-0.03 parts of putrescine.

[0008] Preferably, the salt agent includes ferric citrate, sodium metasilicate and manganese sulfate.

[0009] The function of ferric citrate is to enable microalgae to absorb iron more efficiently, improve the bioavailability of iron, and promote the synthesis and photosynthesis efficiency of chlorophyll; sodium metasilicate can effectively enhance the mechanical strength of microalgae cells, and manganese sulfate is used as a cofactor to activate the superoxide dismutase of microalgae to reduce the oxidative stress of microalgae.

[0010] Preferably, the microalgae culture medium comprises, by mass, 0.5-1 parts of ferric citrate, 22-30 parts of sodium metasilicate, and 0.1-0.5 parts of manganese sulfate.

[0011] Preferably, the microalgae culture medium also includes vitamin B 12 .

[0012] Vitamin B 12 As a methyl transfer coenzyme, it promotes carbon skeleton synthesis and improves the accumulation efficiency of lipids and carbohydrates.

[0013] Preferably, the microalgae culture medium further comprises water, and the mass ratio of the water to the salt agent is (30,000-50,000):1.

[0014] The microalgae culture medium provided by the present invention ensures that the ion concentration of the culture medium is appropriate, and can effectively promote the balanced absorption of medicinal substances by microalgae cells.

[0015] A second aspect of the present invention provides a microalgae cultivation method, wherein the microalgae cultivation method uses the microalgae culture medium described in the first aspect to cultivate microalgae.

[0016] As a preferred culture condition, the light intensity is 30-50 μmol / (m 2 ·s), the culture temperature was 24-26°C, and the light-dark cycle was 12h:12h.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The microalgae culture medium provided by the present invention uses suitable nitrogen sources, phosphorus sources, iron sources, and corresponding preparations to increase the microalgae cell concentration, accelerate total lipid accumulation, and increase the unsaturated fatty acid content, thereby achieving a high culture density, total lipid accumulation rate, and unsaturated fatty acid content. The present invention uses organic nitrogen and phosphorus fertilizers to effectively control the total amount of nitrogen and phosphorus in the water body, avoiding eutrophication of the water body. Using the culture medium and culture method provided by the present invention to cultivate microalgae, the microalgae has the advantages of high growth rate, large output, rapid total lipid accumulation, and increased unsaturated fatty acid content. It can also effectively control the total amount of nitrogen and phosphorus in the water body, reducing the risk of eutrophication of the water body, and is suitable for large-scale cultivation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of measuring the algal cell concentration in the culture medium after 7 days of culture in the three culture media in Example 4 of the present invention are as follows; Figure 2 These are the measurement results of the K value of algae growth rate after culturing algae in the three culture media in Example 4 of the present invention. DETAILED DESCRIPTION

[0019] To make the above-mentioned objects, features, and advantages of the present invention more clearly understood, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only intended to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter ranges described in the present invention. Reasonable variations derived therefrom are still within the scope of protection of the claims of the present invention.

[0020] It should be noted that the endpoints of the ranges and any values ​​disclosed herein 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 endpoints of each range, the endpoints of each range and 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 considered to be specifically disclosed herein.

[0021] As described in the background art, current microalgae culture media have the disadvantages of an unreasonable nitrogen-to-phosphorus ratio and easily lead to eutrophication of water bodies. To overcome the above-mentioned disadvantages, a specific embodiment of the present invention provides a microalgae culture media, which includes a nitrogen source and a salt agent, and the mass ratio of the nitrogen source to the salt agent is 1: (0.7~1.3). The nitrogen source includes phytic acid compounds, amino acids and putrescine.

[0022] In the above embodiment, the phytic acid compound is sodium phytate, and the amino acids include lysine, glycine, and tryptophan. The microalgae culture medium includes, by mass, 10 to 20 parts of sodium phytate, 1 to 4 parts of lysine, 2 to 5 parts of glycine, 3 to 8 parts of tryptophan, and 0.01 to 0.03 parts of putrescine.

[0023] In the above embodiment, the salt agent includes ferric citrate, sodium metasilicate and manganese sulfate.

[0024] More specifically, the microalgae culture medium includes, by mass, 0.5-1 parts of ferric citrate, 22-30 parts of sodium metasilicate, and 0.1-0.5 parts of manganese sulfate.

[0025] In the above embodiment, the microalgae culture medium also includes vitamin B 12 .

[0026] In the above embodiment, the microalgae culture medium further includes water, and the mass ratio of the water to the salt agent is (30,000-50,000):1.

[0027] More specifically, vitamin B 12 The content is 0.001~0.003mg / mL.

[0028] Another embodiment of the present invention further provides a microalgae cultivation method, wherein the microalgae cultivation method uses the aforementioned microalgae culture medium to cultivate microalgae under the following culture conditions: a light intensity of 30 to 50 μmol / (m 2 ·s), the culture temperature was 24-26°C, and the light-dark cycle was 12h:12h.

[0029] The technical solutions of the present invention are further described below in conjunction with specific embodiments. 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 conventional understandings are defined herein for the purpose of illustrating or facilitating reference. 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 provided by the manufacturers.

[0030] Example 1 Prepare the microalgae culture medium in the following proportions: sodium phytate (C6H60 24 P6Na 12 ) 15 mg / L; Lysine (Lys) 2.5 mg / L; Glycine (Gly) 3.5 mg / L; Tryptophan (Try) 5 mg / L; Putrescine (Put) 0.02 mg / L; FeC6H5O7 0.75 mg / L; Vitamin B 12 0.001mg / L; Na2SiO3 25 mg / L; MnSO40.25mg / L.

[0031] The above culture medium is labeled as M1, and the above culture medium can be used as a culture medium for Thalassiosira wiseri.

[0032] Thalassiosira wiseri in the growth period was inoculated into the medium marked as M1 for cultivation, and then cultured in a GXZ intelligent light incubator. The culture conditions were: light intensity of 30 μmol / (m 2 ·s), the culture temperature was 24°C, and the light-dark cycle was 12h:12h.

[0033] Example 2 Prepare the microalgae culture medium in the following proportions: sodium phytate (C6H60 24 P6Na 12 ) 10 mg / L; Lysine (Lys) 1 mg / L; Glycine (Gly) 2 mg / L; Tryptophan (Try) 3 mg / L; Putrescine (Put) 0.01 mg / L; FeC6H5O7 0.5 mg / L; Vitamin B 12 0.002mg / L; Na2SiO3 22mg / L; MnSO40.1mg / L.

[0034] The above culture medium is labeled as M2, and the above culture medium can be used as a culture medium for Thalassiosira wiseri.

[0035] Thalassiosira wiseri in the growth phase was inoculated into the medium labeled M2 for cultivation, and then cultured in a GXZ intelligent light incubator. The culture conditions were: light intensity of 40 μmol / (m 2 ·s), the culture temperature was 25°C, and the light-dark cycle was 12h:12h.

[0036] Example 3 Prepare the microalgae culture medium in the following proportions: sodium phytate (C6H60 24 P6Na 12 ) 20mg / L; Lysine (Lys) 4mg / L; Glycine (Gly) 5mg / L; Tryptophan (Try) 8mg / L; Putrescine (Put) 0.03mg / L; FeC6H5O 71mg / L; Vitamin B 12 0.003mg / L; Na2SiO3 30mg / L; MnSO40.5mg / L.

[0037] The above culture medium is labeled as M3, and the above culture medium can be used as a culture medium for Thalassiosira wiseri.

[0038] Thalassiosira wiseri in the growth phase was inoculated into the medium labeled M3 for cultivation, and then cultured in a GXZ intelligent light incubator. The culture conditions were: light intensity of 50 μmol / (m2 ·s), the culture temperature was 26°C, and the light-dark cycle was 12h:12h.

[0039] Comparative Example 1 Self-developed culture medium 3 # Thalassiosira wissensis was cultured in the following culture medium: the light intensity was 40 μmol / (m 2 ·s), the culture temperature was 25℃, the light-dark cycle was 12h:12h, no aeration, and the experimental period was 7d.

[0040] 3 # The culture medium includes the following components: NaNO3 100 mg / L; KH2PO4 10 mg / L; FeSO4 2.5 mg / L; MnSO4 0.25 mg / L; Na2SiO3 20 mg / L; Na2EDTA 10 mg / L; VB1 6 μg / L; VB 12 50 ng / L.

[0041] Comparative Example 2 Thalassiosira wissensis was cultured in f / 2 medium under the following conditions: light intensity of 40 μmol / (m 2 ·s), the culture temperature was 25℃, the light-dark cycle was 12h:12h, no aeration, and the experimental period was 7d.

[0042] f / 2 culture medium includes: NaNO3 74.8 mg / L; NaH2PO4 4.4 mg / L; Na2SiO3·9H2O 20 mg / L; "f / 2" trace element solution 1 mL / L; "f / 2" vitamin solution 1 mL / L. The "f / 2" trace element solution contains: ZnSO4·4H2O 23 mg / L; MnCl2·4H2O 178 mg / L; CuSO4·5H2O 10 mg / L; FeC6H5O7·5H2O 3.9 mg / L; Na2MoO4·2H2O 7.3 mg / L; CoCl2·6H2O 12 mg / L; and Na2EDTA 4.35 mg / L. The "f / 2" vitamin solution contains: V B1 100 mg / L; V B12 0.5 mg / L; biotin 0.5 mg / L.

[0043] Example 4 Comparative Example 2, Comparative Example 1 and Comparative Example 2 were cultured for 7 days and the algae cell concentration in the culture medium was measured. The experimental results are as follows: Figure 1 As shown. Figure 1 It can be seen that with the increase of culture time, the increase rate of algal cell concentration in the culture medium of Example 2 is much higher than that of Example 3.# The concentration of algal cells in the culture medium and f / 2 culture medium was much higher than that in the culture medium of Example 2. # The highest algal cell concentration values ​​in the culture medium and f / 2 culture medium are shown, which proves that the performance of the culture medium in Example 2 is far superior to that of the existing culture medium.

[0044] The algae growth rate K values ​​of Example 2, Comparative Example 1 and Comparative Example 2 were measured respectively. The results are as follows: Figure 2 As shown. Figure 2 It can be seen that the K value of the algae cultured in the culture medium in Example 2 is also much higher than 3 # The culture medium and f / 2 culture medium are shown to have the advantages of rapid algae proliferation, early entry into the logarithmic growth phase, shortened culture time, long stable period and good stability.

[0045] Although the present disclosure is disclosed as above, the protection scope of the present 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 present disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A microalgae culture medium, characterized in that: The microalgae culture medium comprises a nitrogen source and a salt agent, wherein the mass ratio of the nitrogen source to the salt agent is 1:(0.7-1.3), and the nitrogen source comprises phytic acid compounds, amino acids and putrescine.

2. The microalgae culture medium according to claim 1, wherein The phytic acid compound is sodium phytate, and the amino acids include lysine, glycine, and tryptophan. Calculated by mass, the microalgae culture medium includes 10 to 20 parts of sodium phytate, 1 to 4 parts of lysine, 2 to 5 parts of glycine, 3 to 8 parts of tryptophan, and 0.01 to 0.03 parts of putrescine.

3. The microalgae culture medium according to claim 1, wherein , the salt agent includes ferric citrate, sodium metasilicate and manganese sulfate.

4. The microalgae culture medium according to claim 3, wherein The microalgae culture medium comprises, by mass, 0.5 to 1 parts of ferric citrate, 22 to 30 parts of sodium metasilicate, and 0.1 to 0.5 parts of manganese sulfate.

5. The microalgae culture medium according to claim 1, wherein The microalgae culture medium also includes vitamin B 12 .

6. The microalgae culture medium according to claim 1, wherein The microalgae culture medium also includes water, and the mass ratio of the water to the salt agent is (30,000-50,000):

1.

7. A microalgae cultivation method, characterized in that: Microalgae are cultured using the microalgae culture medium according to any one of claims 1 to 6.

8. The microalgae cultivation method according to claim 7, wherein: The culture conditions were: light intensity of 30~50μmol / (m 2 ·s), the culture temperature was 24-26°C, and the light-dark cycle was 12h:12h.

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