Solid culture medium for microalgae separation culture and microalgae separation method

Abstract

The invention provides a solid culture medium for microalgae separation culture and a microalgae separation method, and belongs to the technical field of algae separation, the solid culture medium provided by the invention comprises the following components: 30-40 g / L of agar, 0.2 g / L of KNO3, 0.02 g / L of KH2PO4, 0.005 g / L of FeSO4. 7H2O, 0.02 g / L of EDTANa2, 0.0005 g / L of MnSO4, 0.04 g / L of Na2SiO3, 6 [mu] g / L of vitamin B1, and 50 ng / L of vitamin B12. When the solid culture medium and the separation method provided by the invention are used for separating the microalgae, the separation time of the microalgae can be greatly shortened, the purity of the microalgae is improved, high-quality microalgae resources are provided for subsequent research, large-scale culture and application of the microalgae, and the solid culture medium and the separation method have wide application prospects and important practical values in the related fields of the microalgae.

Description

Technical Field

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

[0002] Microalgae are of great value in the fields of bioenergy, food, medicine, etc. However, there are many problems with their separation technology: traditional microalgae separation methods are often inefficient. When separating microalgae from complex water samples, it is difficult to accurately obtain a single and high-purity microalgae strain. For example, plate streaking culture is prone to dense and mixed colony growth, making it difficult to distinguish the target microalgae from other impurity microorganisms; the water droplet separation method requires each water sample to be drawn, and a single drop of liquid with less algae is taken and placed in the prepared seawater for cultivation. This process requires multiple operations, which is time-consuming and difficult to ensure that there are no impurities mixed in the microalgae droplets selected each time. It is greatly affected by the proficiency and randomness of the operation, and it is also difficult to obtain high-purity microalgae. In addition, there is a lack of standardized procedures for the picking of microalgae, which results in the low purity of the separated microalgae, which cannot meet the needs of scientific research and production for high-quality microalgae. Therefore, there is an urgent need for an efficient, accurate and pure separation method for microalgae. Summary of the invention

[0003] The technical problem to be solved by the present invention is: how to improve the purity of microalgae obtained by separation of microalgae.

[0004] In order to solve the above technical problems, the first aspect of the present invention provides a solid culture medium for the isolation and cultivation of microalgae, wherein the 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, 0.04 g / L Na2SiO3, 6 μg / L vitamin B1, 50 ng / L vitamin B 12 .

[0005] The above solid culture medium has similar components to the basic culture medium for microalgae culture and has a high concentration adaptability, which can ensure that the microalgae have a good growth state and a high survival rate during the isolation and subsequent early culture stages, and provide a stable and suitable nutritional support system for the transition of microalgae from a complex water environment to a single pure strain culture.

[0006] A second aspect of the present invention provides a method for separating microalgae, comprising the following steps:

[0007] S1: diluting the microalgae culture solution with the microalgae separation dilution solution to obtain a microalgae dilution solution;

[0008] S2: taking the microalgae dilution obtained in step S1, and coating it on a plate having the solid culture medium for microalgae isolation and culture as described in the first aspect;

[0009] S3: placing the coated plate in an incubator for separation and culture;

[0010] S4: After the culture is completed, a single colony is picked up and transferred to a plate having the solid culture medium for microalgae isolation and culture as described in the first aspect for purification culture. The microalgae isolation is completed.

[0011] In the current microalgae separation technology, the direct coating culture method is prone to dense and mixed colony growth, making it difficult to accurately distinguish the target microalgae from impurity microorganisms, resulting in low separation purity, while the liquid separation method is cumbersome, time-consuming and greatly affected by human factors, making it difficult to stably obtain high-purity microalgae. The microalgae separation method provided by the present invention combines the advantages of the direct coating method and the liquid separation method, and can quickly and accurately separate single microalgae colonies.

[0012] Different from the existing "direct coating method", the separation method provided by the present invention first dilutes the microalgae culture solution to obtain a microalgae dilution solution, and then takes the dilution solution and operates on a solid culture medium plate used for microalgae separation and culture. This method is not simply to spread the algae sample directly, but to pre-place the dilution process, which can effectively regulate the concentration of algae cells and avoid excessive density of subsequent colonies. In addition, there are subsequent steps such as transferring the cultured single colony to the same solid culture medium plate for purification and culture, which cooperates with the purpose of accurately separating single microalgae colonies, making the entire separation process more scientific and efficient.

[0013] The separation method provided by the present invention has better separation effect than the existing direct coating method, and there are three main reasons: first, there is a pre-dilution step. The present invention uses a microalgae separation diluent to dilute the microalgae culture solution, reduce the concentration of microalgae cells, and make the microalgae cells on the plate after coating sparsely distributed, avoiding dense and mixed colonies, which is conducive to the accurate separation of single colonies. The existing coating often lacks this key link, and most of the undiluted samples are directly coated, making it difficult to distinguish the colonies; second, an optimized solid culture medium specifically used for microalgae separation and culture is used, and its components and ratios are finely adjusted, which can not only create a suitable growth environment for microalgae and promote the reproduction of target microalgae, but also inhibit the growth of impurity microorganisms and improve the separation effect; third, combined with the purification and culture step, after coating, a single colony is picked and transferred to the same solid culture medium plate for purification, which deeply removes impurity microorganisms, ensures the purity of microalgae, and achieves efficient separation.

[0014] Preferably, in step S1, the microalgae separation dilution solution is an aqueous solution containing 0.85% NaCl and 0.01 g / L sodium citrate.

[0015] The microalgae separation diluent provided by the present invention can effectively disperse the microalgae in the water sample, prevent the microalgae from agglomerating, and reduce the interference of impurities on the separation of the microalgae.

[0016] Preferably, in step S3, the environmental parameters for separation and culture are: culture temperature of 20°C, culture light intensity of 3000Lx, and culture time of 3 to 5 days.

[0017] Preferably, in step S4, the environmental parameters of the purification culture are: culture temperature 20°C, light intensity 3000Lx, and light-dark cycle 12h:12h.

[0018] The present invention has the beneficial effects as follows: the present invention optimizes the composition of the microalgae solid culture medium and the process of microalgae separation. The solid culture medium for microalgae separation and culture provided by the present invention in combination with the microalgae separation method provided by the present invention can form clear and easily distinguishable single colonies of microalgae in the coating culture stage. The microalgae separation method provided by the present invention can more accurately separate target microalgae from complex water samples, reduce the contamination of miscellaneous algae and other microorganisms, provide high-quality microalgae resources for subsequent research, large-scale culture and application of microalgae, and has broad application prospects and important practical value in microalgae-related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A comparison chart of the time required for separating Cyclotella by the separation method provided in Example 1, the separation method provided in Comparative Example 1, and the separation method provided in Comparative Example 2;

[0020] Figure 2 A comparison chart of the time required for separating Crown of Spiny Algae by the separation method provided in Example 2, the separation method provided in Comparative Example 1, and the separation method provided in Comparative Example 2;

[0021] Figure 3 This is a comparison chart of the time required to separate Nitzschia closterium using the separation method provided in Example 3, the separation method provided in Comparative Example 1, and the separation method provided in Comparative Example 2. DETAILED DESCRIPTION

[0022] 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.

[0023] 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.

[0024] A specific embodiment of the present invention provides a solid culture medium for isolation and cultivation of microalgae, wherein the 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, 0.04 g / L Na2SiO3, 6 μg / L vitamin B1, 50 ng / L vitamin B 12 .

[0025] A specific embodiment of the present invention also provides a method for separating microalgae, comprising the following steps:

[0026] S1: diluting the microalgae culture solution with the microalgae separation dilution solution to obtain a microalgae dilution solution;

[0027] S2: taking the microalgae dilution obtained in step S1 and applying it on a plate having the aforementioned solid culture medium for microalgae isolation and culture;

[0028] S3: placing the coated plate in an incubator for separation and culture;

[0029] S4: After the culture is completed, a single colony is picked and transferred to a plate having the aforementioned solid culture medium for microalgae isolation and culture for purification culture. The microalgae isolation is completed.

[0030] The function of step S4 in the above embodiment is that after the previous microalgae separation and culture step, the obtained colony still has a low purity. At this time, a single colony is picked and transferred to a solid culture medium plate for microalgae separation and culture for purification culture, which can allow a single microalgae cell to grow to form a single pure colony, and obtain a pure culture with a single genetic background and consistent physiological characteristics. This is convenient for subsequent accurate research on the biological characteristics, physiological metabolism, genetic characteristics, etc. of microalgae, and can ensure that various experiments such as growth curve determination, oil content analysis, photosynthetic efficiency research, etc. are not interfered by miscellaneous algae or microorganisms, ensuring that the experimental results are accurate and reliable. In addition, the purified microalgae can also be stored for a long time on a solid culture medium plate, which is convenient to use, and can maintain its genetic stability and physiological characteristics, reduce the risk of strain degeneration or mutation, and the separation of microalgae is completed.

[0031] In some specific embodiments, the microalgae separation diluent used in step S1 is an aqueous solution containing 0.85% NaCl and 0.01 g / L sodium citrate, which serves to disperse the microalgae in the water sample, prevent them from agglomerating, and reduce the interference of impurities on the separation of microalgae.

[0032] In step S1 and step S2 of some specific embodiments, the dilution multiple of the microalgae culture solution and the amount of liquid used for coating are flexibly adjusted according to actual conditions.

[0033] In step S3 of some specific embodiments, the environmental parameters of the separation culture are: culture temperature of 20° C., culture light intensity of 3000 Lx, and culture time of 3 to 5 days.

[0034] In step S4 of some specific embodiments, the environmental parameters of the purification culture are: culture temperature 20° C., light intensity 3000 Lx, and light-dark cycle 12h:12h.

[0035] The technical scheme of the present invention is further illustrated by specific examples below. 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, the terms with conventionally understood meanings are defined herein for the purpose of clarification or convenience of reference, and such definitions herein should not be understood to represent 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 schemes and parameters given by the manufacturer.

[0036] Example 1

[0037] Cyclotella isolation

[0038] The microalgae separation diluent used in the present invention is an aqueous solution containing 0.85% NaCl and 0.01g / L sodium citrate; the solid culture medium used for microalgae separation and culture comprises the following components: 30-40g / L agar, 0.2g / L KNO3, 0.02g / L KH2PO4, 0.005g / L FeSO4·7H2O, 0.02g / L EDTANa2, 0.0005g / L MnSO4, 0.04g / L Na2SiO3, 6μg / L vitamin B1, 50ng / L vitamin B 12 .

[0039] S1: using the microalgae separation dilution solution to gradedly dilute the Cyclotella culture solution at a volume ratio of 1: (8-12) to obtain a Cyclotella dilution solution;

[0040] S2: taking 100 μL of the Cyclotella dilution obtained in step S1 and spreading it on a plate having the aforementioned solid culture medium for isolation and culture of microalgae;

[0041] S3: Place the coated plate in an incubator for isolation and culture. The temperature for isolation and culture is 20°C and the light intensity is 3000Lx. After 3 days of culture, complete and obvious colonies grow on the culture medium.

[0042] S4: After the cultivation is completed, a single Cyclotella colony is picked up and transferred to a plate having the aforementioned solid culture medium for microalgae isolation and cultivation for purification and cultivation. The Cyclotella isolation is completed. The temperature for purification and cultivation is 20°C, the light intensity is 3000Lx, and the light-dark cycle is 12h:12h.

[0043] Example 2

[0044] Isolation of Crown of Thorns

[0045] The microalgae separation diluent used in the present invention is an aqueous solution containing 0.85% NaCl and 0.01g / L sodium citrate; the solid culture medium used for microalgae separation and culture comprises the following components: 30-40g / L agar, 0.2g / L KNO3, 0.02g / L KH2PO4, 0.005g / L FeSO4·7H2O, 0.02g / L EDTANa2, 0.0005g / LMnSO4, 0.04g / LNa2SiO3, 6μg / L vitamin B1, 50ng / L vitamin B 12 .

[0046] S1: using the microalgae separation dilution solution to gradually dilute the crown of acanthus culture solution according to a volume ratio of 1: (8-12) to obtain a crown of acanthus dilution solution;

[0047] S2: taking 100 μL of the Crown of spinulosa dilution obtained in step S1, and spreading it on a plate having the aforementioned solid culture medium for isolation and culture of microalgae;

[0048] S3: Place the coated plate in an incubator for isolation and culture. The temperature for isolation and culture is 20°C and the light intensity is 3000Lx. After 3 days of culture, complete and obvious colonies grow on the culture medium.

[0049] S4: After the cultivation is completed, a single colony of Crown of Spinosa is picked up and the colony is transferred to a plate having the aforementioned solid culture medium for microalgae isolation and cultivation for purification and cultivation. The Crown of Spinosa is isolated and the purification and cultivation temperature is 20°C, the light intensity is 3000Lx, and the light-dark cycle is 12h:12h.

[0050] Example 3

[0051] Nitzschia closterium isolated

[0052] The microalgae separation diluent used in the present invention is an aqueous solution containing 0.85% NaCl and 0.01g / L sodium citrate; the solid culture medium used for microalgae separation and culture comprises the following components: 30-40g / L agar, 0.2g / L KNO3, 0.02g / L KH2PO4, 0.005g / L FeSO4·7H2O, 0.02g / L EDTANa2, 0.0005g / LMnSO4, 0.04g / LNa2SiO3, 6μg / L vitamin B1, 50ng / L vitamin B 12 .

[0053] S1: using the microalgae separation dilution liquid to gradually dilute the culture liquid of Nitzschia closterium at a volume ratio of 1: (8-12) to obtain a dilution liquid of Nitzschia closterium;

[0054] S2: taking 100 μL of the Nitzschia closterium dilution obtained in step S1, and spreading it on a plate having the aforementioned solid culture medium for isolation and culture of microalgae;

[0055] S3: Place the coated plate in an incubator for isolation and culture. The temperature for isolation and culture is 20°C and the light intensity is 3000Lx. After 3 days of culture, complete and obvious colonies grow on the culture medium.

[0056] S4: After the cultivation is completed, a single colony of Nitzschia closterium is picked up and transferred to a plate having the aforementioned solid culture medium for microalgae isolation and cultivation for purification culture. The isolation of Crown of Spiny Algae is completed. The temperature of the purification culture is 20°C, the light intensity is 3000Lx, and the light-dark cycle is 12h:12h.

[0057] Comparative Example 1

[0058] Separation of Cyclotella, Crown of Thorns, and Nitzschia Closterium by Water Droplet Separation

[0059] Specific steps: Use a small beaker to hold the diluted algae sample, insert the micropipette into the algae sample, extract the micropipette and let the excess algae drip out, then contact the tube mouth with the disinfected slide to leave small water droplets on the slide, drop 3 to 4 drops on a slide and keep a certain distance apart, and then observe under a microscope. During the observation process, for Cyclotella, look for Cyclotella cells that are disc-shaped, have an annular cell wall structure, and are not mixed with other organisms in the water droplets; for Crown of Thorns, it is necessary to identify its unique morphological algae cells; for Crescent Nitzschia, it is necessary to find crescent-shaped algae cells. If it is found that there is only one type of algae cell that needs to be separated in the water droplet, use a pipette to absorb the culture solution and flush the water droplet into the test tube, plug the mouth of the test tube with a cotton plug, and culture it under appropriate conditions. The key to this separation method is that the algae sample should be appropriately diluted, diluted to about 1-2 algae cells in each water droplet, and the water droplet size should be appropriate, so that all or most of the water droplets can be seen under a low-power microscope, and the observation should be accurate and rapid.

[0060] Comparative Example 2

[0061] Separation of Cyclotella, Crown of Thorns, and Nitzschia Closterium by streaking

[0062] Specific steps: When using the streak method to separate Cyclotella, Crown Algae and Nitzschia crescentica, first sterilize the inoculation loop thoroughly on an alcohol lamp. After the inoculation loop cools down, dip it in the algae sample and streak it flat on the surface of the culture medium.

[0063] For Cyclotella, because its cells are disc-shaped and have a ring-shaped cell wall structure, when streaking for the first time, the algal cells are densely distributed in the streaking area, making it difficult to separate individual cells. However, after several subsequent streakings, as the number of algal cells on the inoculation loop gradually decreases, there is a chance to separate single or small aggregated Cyclotella communities; Crown of Thorns has its own unique morphology. During the initial streaking, a large number of Crown of Thorns cells accumulate in one place. Subsequently, multiple streakings are used to allow the algal cells carried by the inoculation loop to decrease gradually, and then dispersed Crown of Thorns communities are obtained in the subsequent streaking areas; The cells of Crescent Nitzschia are crescent-shaped. At the beginning of streaking, the algal cells aggregate. After repeated operations, the Crescent Nitzschia cells are separated from each other with the help of the subsequent streaking area, and finally an independent Crescent Nitzschia community is separated for subsequent cultivation and research.

[0064] Comparing the separation and purification time required for separating the corresponding microalgae in Examples 1 to 3 and Comparative Examples 1 to 2, the relevant results are as follows Figures 1 to 3 As shown, Figure 1 This is a comparison chart of the time required for separating Cyclotella by the separation method provided in Example 1, the separation method provided in Comparative Example 1, and the separation method provided in Comparative Example 2. It can be seen that the separation method provided by the present invention can greatly shorten the separation time of Cyclotella; Figure 2This is a comparison chart of the time required for separating Crown of Spinosa by the separation method provided in Example 2, the separation method provided in Comparative Example 1, and the separation method provided in Comparative Example 2. It can be seen that the separation method provided by the present invention can significantly shorten the separation time of Crown of Spinosa; Figure 3 This is a comparison chart of the time required for separating Nitzschia closterium by the separation method provided in Example 1, the separation method provided in Comparative Example 1, and the separation method provided in Comparative Example 2. It can be seen that the separation method provided by the present invention can significantly shorten the separation time of Nitzschia closterium.

[0065] 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 solid culture medium for isolation and cultivation of microalgae, characterized in that: The 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, 0.04 g / L Na2SiO3, 6 μg / L vitamin B1, 50 ng / L vitamin B 12 .

2. A method for separating microalgae, characterized in that: The following steps are involved: S1: diluting the microalgae culture solution with the microalgae separation dilution solution to obtain a microalgae dilution solution; S2: taking the microalgae dilution obtained in step S1 and coating it on a plate having the solid culture medium for microalgae isolation and culture according to claim 1; S3: placing the coated plate in an incubator for separation and culture; S4: After the culture is completed, a single colony is picked and transferred to a plate having the solid culture medium for microalgae isolation and culture according to claim 1 for purification culture, and the microalgae isolation is completed.

3. The microalgae separation method according to claim 2, characterized in that: In the step S1, the microalgae separation dilution solution is an aqueous solution containing 0.85% NaCl and 0.01 g / L sodium citrate.

4. The microalgae separation method according to claim 2, characterized in that: In step S3, the environmental parameters for separation and culture are: culture temperature of 20° C., culture light intensity of 3000 Lx, and culture time of 3 to 5 days.

5. The microalgae separation method according to claim 2, characterized in that: In step S4, the environmental parameters of the purification culture are: culture temperature 20°C, light intensity 3000Lx, and light-dark cycle 12h:12h.

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