Microalgae culture medium and microalgae preservation method

Abstract

The invention provides a microalgae culture medium and a microalgae preservation method, relates to the technical field of algae culture, and particularly provides a brand-new microalgae preservation liquid culture medium and a microalgae preservation solid culture medium, and develops a brand-new microalgae biphasic preservation method by using the microalgae preservation liquid culture medium and the microalgae preservation solid culture medium. The preservation method comprises the following steps: inoculating microalgae species in an exponential growth phase into a microalgae preservation solid culture medium, culturing for 5-10 days, and adding a microalgae preservation liquid culture medium into the microalgae preservation solid culture medium inoculated with the microalgae species to form a semi-solid culture medium, the semi-solid culture medium is subjected to biphasic preservation under natural light at 2-10 DEG C. The preservation method can effectively prolong the preservation time of microalgae species, ensures the preservation effect, and has wide application prospects.

Description

Technical Field

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

[0002] Microalgae has the advantages of small planting coverage area, simple equipment requirements, fast growth rate, low consumption, and high nutritional value. The preservation of unicellular algae species has always been an important factor restricting the normal supply and long-term preservation of unicellular algae in factory-based seedling production. At present, the methods for preserving algae species include: liquid preservation method and solid preservation method. In actual production, the liquid preservation method is usually used. It is simple to operate, but the cycle is short, it is easy to be contaminated, and the preservation conditions are difficult to control, which can easily cause the preservation of algae species to fail. Although the solid preservation method is not easy to be contaminated, the culture cycle is relatively long, and the solid culture medium is prone to cracking due to easy evaporation of water and depletion of nutrients. Therefore, in the current microalgae cultivation, there is an urgent need for a microalgae preservation method that has a long preservation time, can avoid contamination by miscellaneous bacteria, and is easy to operate. Summary of the invention

[0003] Based on the technical difficulties in the current cultivation and preservation of microalgae, the purpose of the present invention is to provide a microalgae preservation method with a long preservation time, the ability to avoid contamination by miscellaneous bacteria, and easy operation.

[0004] To achieve the above objectives, the first aspect of the present invention provides a microalgae preservation liquid culture medium, which comprises the following components: 0.2 g / L KNO3, 0.02 g / L KH2PO4, 0.005 g / L FeSO4·7H2O, 0.02 g / L EDTANa2, 0.0005 g / L MnSO4, and 0.04 g / L Na2SiO3.

[0005] A second aspect of the present invention provides a microalgae preservation solid culture medium, which comprises the following components: 30-40 g / L agar, 0.2 g / L KNO3, 0.02 g / L KH2PO4, 0.005 g / L FeSO4·7H2O, 0.02 g / L EDTANa2, 0.0005 g / L MnSO4, and 0.04 g / L Na2SiO3.

[0006] The third aspect of the present invention provides a method for preserving microalgae, which specifically comprises the following steps: inoculating a microalgae species in an exponential growth phase into the microalgae preservation solid culture medium described in the second aspect, adding the microalgae preservation liquid culture medium described in the first aspect to the microalgae preservation solid culture medium inoculated with the microalgae species after culturing for 5 to 10 days to form a semi-solid culture medium, and placing the semi-solid culture medium in a two-phase preservation at 2 to 10° C. and natural light.

[0007] In the current microalgae preservation technology, liquid preservation has a short preservation period and is easily contaminated, and the agar culture medium in the solid preservation method is easy to dry and crack. The present invention provides a new two-phase preservation method based on the liquid preservation method and the solid preservation method. The preservation method combines the advantages of the solid preservation method that is not easily contaminated and the liquid preservation method that has a long preservation time. It is a new preservation method with low cost, simple operation and long preservation time.

[0008] Preferably, the culture conditions of the microalgae species in the microalgae preservation solid culture medium are: culture temperature of 20-25° C., and light intensity of 3000-5000 Lx.

[0009] Preferably, the light-dark cycle during the two-phase storage is 12h:12h.

[0010] A fourth aspect of the present invention provides a microalgae growth liquid culture medium, which comprises the following components: 0.1 g / L KNO3, 0.01 g / L KH2PO4, 0.0025 g / L FeSO4·7H2O, 0.01 g / L EDTANa2, 0.00025 g / L MnSO4, and 0.02 g / L Na2SiO3.

[0011] A fifth aspect of the present invention provides a solid culture medium for the growth of microalgae, comprising the following components: 30-40 g / L agar, 0.1 g / L KNO3, 0.01 g / L KH2PO4, 0.0025 g / L FeSO4·7H2O, 0.01 g / L EDTANa2, 0.00025 g / L MnSO4, and 0.02 g / L Na2SiO3.

[0012] The microalgae growth culture medium provided by the present invention also includes a liquid culture medium and a solid culture medium, and its core components are the same as those of the microalgae preservation culture medium, with a small concentration difference, thereby ensuring the survival rate of microalgae preservation or recovery.

[0013] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a liquid culture medium and a solid culture medium for preserving microalgae, and on this basis, develops a new two-phase preservation method for preservation. The microalgae preservation method provided by the present invention adds a liquid culture medium in an appropriate proportion on the basis of the traditional solid preservation method. The liquid culture medium not only ensures the humidity of the solid culture medium, but also has a sustained release effect of nutrients, which can effectively prevent the culture medium from drying out due to water evaporation or nutrient depletion of the solid culture medium. Furthermore, the preservation time of the microalgae species can be extended to ensure the preservation effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1This is a comparison chart of the survival status of Crown of Thorns algae after being preserved for 24 months using the dual-phase preservation method provided by the present invention and the liquid preservation method;

[0015] Figure 2 This is a comparison chart of the survival of Nitzschia closterium after being preserved for 24 months using the dual-phase preservation method provided by the present invention and the liquid preservation method;

[0016] Figure 3 The following is a comparison chart of the survival status of Thalassiosira species after being preserved for 24 months using the preservation method provided by the present invention and the liquid preservation method. DETAILED DESCRIPTION

[0017] 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.

[0018] 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.

[0019] 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.

[0020] Example 1

[0021] Crown of spinulosa algae collection

[0022] Prepare liquid culture medium for growth according to the following substance concentrations: 0.1 g / L KNO3, 0.01 g / L KH2PO4, 0.0025 g / L FeSO4·7H2O, 0.01 g / L EDTANa2, 0.00025 g / L MnSO4, and 0.02 g / L Na2SiO3.

[0023] Solid culture medium for preservation was prepared according to the following substance concentrations: 30-40 g / L agar, 0.2 g / L KNO3, 0.02 g / L KH2PO4, 0.005 g / L FeSO4·7H2O, 0.02 g / L EDTANa2, 0.0005 g / L MnSO4, and 0.04 g / L Na2SiO3.

[0024] Solid culture medium for preservation was prepared according to the following substance concentrations: 0.2 g / L KNO3, 0.02 g / L KH2PO4, 0.005 g / L FeSO4·7H2O, 0.02 g / L EDTANa2, 0.0005 g / L MnSO4, and 0.04 g / L Na2SiO3.

[0025] The above culture medium was sterilized at a pressure of 6.8 kg, a temperature of 120° C., and a time of 30 min, and then the solid culture medium to be preserved was cooled and solidified.

[0026] The algae species of Crown of Spinosa were inoculated into a solid culture medium for preservation, and cultured in an incubator at 20°C and 3000Lx light intensity for 10 days. Several algae colonies were randomly selected and examined under a microscope to confirm that there was no contamination.

[0027] Add 3 times the volume of liquid culture medium for preservation to the above-mentioned solid culture medium for preservation inoculated with Crown of Spinosa species to form a semi-solid culture medium, and carry out two-phase preservation at 10°C, natural light, and a light-dark ratio of 12h:12h. Take samples regularly to observe the health and contamination of the algae.

[0028] Example 2

[0029] Collection of Nitzschia closterium

[0030] Prepare liquid culture medium for growth according to the following substance concentrations: 0.1 g / L KNO3, 0.01 g / L KH2PO4, 0.0025 g / L FeSO4·7H2O, 0.01 g / L EDTANa2, 0.00025 g / L MnSO4, and 0.02 g / L Na2SiO3.

[0031] Solid culture medium for preservation was prepared according to the following substance concentrations: 30-40 g / L agar, 0.2 g / L KNO3, 0.02 g / L KH2PO4, 0.005 g / L FeSO4·7H2O, 0.02 g / L EDTANa2, 0.0005 g / L MnSO4, and 0.04 g / L Na2SiO3.

[0032] Solid culture medium for preservation was prepared according to the following substance concentrations: 0.2 g / L KNO3, 0.02 g / L KH2PO4, 0.005 g / L FeSO4·7H2O, 0.02 g / L EDTANa2, 0.0005 g / L MnSO4, and 0.04 g / L Na2SiO3.

[0033] The above culture medium was sterilized at a pressure of 6.8 kg, a temperature of 120° C., and a time of 30 min, and then the solid culture medium to be preserved was cooled and solidified.

[0034] The Nitzschia crescentica algae species were inoculated into a solid culture medium for preservation and cultured in an incubator at 20°C and 3000Lx light intensity for 10 days. Several algae colonies were randomly selected and examined under a microscope to confirm that there was no contamination.

[0035] Add 3 times the volume of liquid culture medium for preservation to the above-mentioned solid culture medium for preservation inoculated with the crescent-shaped algae species to form a semi-solid culture medium, and carry out two-phase preservation at 10°C, natural light, and a light-dark ratio of 12h:12h. Take samples regularly to observe the health and contamination of the algae.

[0036] Example 3

[0037] Thalassiosira species collection

[0038] Prepare liquid culture medium for growth according to the following substance concentrations: 0.1 g / L KNO3, 0.01 g / L KH2PO4, 0.0025 g / L FeSO4·7H2O, 0.01 g / L EDTANa2, 0.00025 g / L MnSO4, and 0.02 g / L Na2SiO3.

[0039] Solid culture medium for preservation was prepared according to the following substance concentrations: 30-40 g / L agar, 0.2 g / L KNO3, 0.02 g / L KH2PO4, 0.005 g / L FeSO4·7H2O, 0.02 g / L EDTANa2, 0.0005 g / L MnSO4, and 0.04 g / L Na2SiO3.

[0040] Solid culture medium for preservation was prepared according to the following substance concentrations: 0.2 g / L KNO3, 0.02 g / L KH2PO4, 0.005 g / L FeSO4·7H2O, 0.02 g / L EDTANa2, 0.0005 g / L MnSO4, and 0.04 g / L Na2SiO3.

[0041] The above culture medium was sterilized at a pressure of 6.8 kg, a temperature of 120° C., and a time of 30 min, and then the solid culture medium to be preserved was cooled and solidified.

[0042] The Thalassiosira species were inoculated into a solid culture medium for preservation and cultured in an incubator at 20°C and 3000Lx light intensity for 10 days. Several algal colonies were randomly selected and examined under a microscope to confirm that there was no contamination.

[0043] Add 3 times the volume of liquid culture medium for preservation to the aforementioned solid culture medium for preservation inoculated with Thalassiosira species to form a semi-solid culture medium, and carry out two-phase preservation at 10°C, natural light, and a light-dark ratio of 12h:12h. Take samples regularly to observe the health and contamination of the algae.

[0044] Comparative Example 1

[0045] Use the existing liquid preservation medium to directly preserve Crown of Thorns, Nitzschia Closterium, and Thalassiosira, and take samples regularly to observe the health and contamination of the algae. And compare with the corresponding algae data in Examples 1 to 3. Figures 1 to 3 shown.

[0046] like Figure 1 As shown, with the increase of storage time, the amount of Crown of Acanthium in the storage medium of Example 1 was much higher than that in the ordinary liquid culture medium after eight months.

[0047] like Figure 2 As shown, with the increase of storage time, the number of Nitzschia closterium in the storage medium of Example 2 was much higher than that in the ordinary liquid culture medium after eight months.

[0048] like Figure 3 As shown, with the increase of storage time, the amount of Thalassiosira in the storage medium of Example 3 was much higher than that in the ordinary liquid culture medium after eight months.

[0049] In addition, the algae body conditions and the growth of miscellaneous bacteria observed in the example group were better, which proved that the preservation method of the present invention has the advantages of long preservation time and low growth of miscellaneous bacteria for microalgae, and can effectively prolong the preservation time of microalgae species and improve the preservation effect. The base in this method has a sustained release effect of nutrients and can prevent the evaporation and cracking of water in the culture medium, which is suitable for the long-term preservation of microalgae species.

[0050] 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 liquid culture medium for preserving microalgae, characterized in that: The microalgae preservation liquid culture medium comprises the following components: 0.2 g / L KNO3, 0.02 g / L KH2PO4, 0.005 g / L FeSO4·7H2O, 0.02 g / L EDTANa2, 0.0005 g / L MnSO4, and 0.04 g / L Na2SiO3.

2. A solid culture medium for preserving microalgae, characterized in that: The microalgae preservation solid culture medium comprises the following components: 30-40 g / L agar, 0.2 g / L KNO3, 0.02 g / L KH2PO4, 0.005 g / L FeSO4·7H2O, 0.02 g / L EDTANa2, 0.0005 g / L MnSO4, and 0.04 g / L Na2SiO3.

3. A method for preserving microalgae, characterized in that: The following steps are involved: The microalgae preservation solid culture medium according to claim 2 is inoculated with microalgae species in the exponential growth phase. After culturing for 5 to 10 days, the microalgae preservation liquid culture medium according to claim 1 is added to the microalgae preservation solid culture medium inoculated with the microalgae species to form a semi-solid culture medium. The semi-solid culture medium is placed at 2 to 10° C. and stored in two phases under natural light.

4. The preservation method according to claim 3, characterized in that: The culture conditions of the microalgae species in the microalgae preservation solid culture medium are: the culture temperature is 20-25° C. and the light intensity is 3000-5000 Lx.

5. The preservation method according to claim 3, characterized in that: The light-dark cycle during the two-phase storage was 12h:12h.

6. The preservation method according to claim 3, characterized in that: The volume of the microalgae preservation liquid culture medium added is 2 to 3 times that of the microalgae preservation solid culture medium.

7. A liquid culture medium for growing microalgae, characterized in that: The microalgae growth liquid culture medium comprises the following components: 0.1 g / L KNO3, 0.01 g / L KH2PO4, 0.0025 g / L FeSO4·7H2O, 0.01 g / L EDTANa2, 0.00025 g / L MnSO4, and 0.02 g / L Na2SiO3.

8. A solid culture medium for growing microalgae, characterized in that: The microalgae growth solid culture medium comprises the following components: 30-40 g / L agar, 0.1 g / L KNO3, 0.01 g / L KH2PO4, 0.0025 g / L FeSO4·7H2O, 0.01 g / L EDTANa2, 0.00025 g / L MnSO4, and 0.02 g / L Na2SiO3.

Images