A method for separating and purifying single-cell blue algae based on enrichment of paramecium and application thereof

Abstract

The application discloses a single-cell blue algae separation and purification method based on rotifer enrichment and application, and the method comprises the following steps: placing a single-cell blue algae solution into a sterile BG11 conical flask; adding a rotifer solution with a known concentration into the sterile BG11 conical flask containing the single-cell blue algae solution to obtain a mixed solution of the rotifer and the single-cell blue algae; placing the sterile BG11 conical flask containing the mixed solution of the rotifer and the single-cell blue algae in a first culture environment for culture to obtain a blue algae and rotifer enrichment solution; transferring the blue algae and rotifer enrichment solution to a sterilized BG11 agar plate for plate coating treatment, and inverting the BG11 agar plate in a second culture environment for culture until a blue algae colony without bacteria is formed; and expanding the blue algae colony without bacteria to obtain single-cell blue algae without bacteria. The application can greatly shorten the time for traditional separation and culture of single-cell blue algae, and can prevent bacterial pollution from an external environment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a single-cell cyanobacteria separation and purification method based on enrichment of paramecium and application thereof. BACKGROUND

[0002] Cyanobacteria are photosynthetic autotrophic prokaryotes, are producers in nature, participate in carbon, nitrogen and phosphorus cycles in nature, and are rich in phycocyanin and can be used as health products, having great research potential and application value. However, it is difficult to purify single-cell cyanobacteria at present, because single-cell cyanobacteria are sensitive to antibiotics, and it is difficult to use antibiotics to kill bacteria to screen cyanobacteria. Moreover, single-cell cyanobacteria have a long growth cycle, and traditional methods such as plate coating or streaking purification of cyanobacteria often need repeated coating or streaking culture, and the time consumed is quite long. The method of using lysozyme to enzymatically digest bacteria to separate cyanobacteria needs to use drug resistance test on bacteria, which is relatively complex, is not suitable for bacteria not sensitive to lysozyme in the environment, and may have the risk of repeated bacterial contamination. Using high-end instruments (flow cytometry sorting, single-cell Raman sorting, etc.) also needs to sort a proper amount of single-cell cyanobacteria to expand culture for 1-2 growth cycles to obtain a certain amount of cyanobacteria free of bacteria, but the instrument is relatively expensive and requires a sterile environment. SUMMARY

[0003] To solve the technical problems in the prior art, the present application provides a single-cell cyanobacteria separation and purification method based on enrichment of paramecium and application thereof, which can accurately solve the above-mentioned technical problems in the prior art.

[0004] Specifically, the present application includes the following contents.

[0005] In a first aspect of the present application, a single-cell cyanobacteria separation and purification method based on enrichment of paramecium is provided, which comprises the following steps:

[0006] Step 1: Provide a solution of single-celled cyanobacteria in the logarithmic growth phase and place the single-celled cyanobacteria solution into a sterile BG11 conical flask; Step 2: Provide a Paramecium solution of known concentration and add the Paramecium solution to the sterile BG11 conical flask containing the single-celled cyanobacteria solution from Step 1, resulting in a Paramecium density of 0.5-2 ce. Step 3: Place the sterile BG11 conical flask containing the Paramecium and single-celled cyanobacteria mixed solution from Step 2 in the first culture environment for cultivation until the number of bacteria is less than half the number of single-celled cyanobacteria. At this point, a cyanobacteria and Paramecium enrichment solution with Paramecium enrichment is obtained, wherein the bacteria are microorganisms other than Paramecium and single-celled cyanobacteria. Step 4: Transfer the cyanobacteria and Paramecium enrichment solution from the sterile BG11 conical flask in Step 3 to a sterile BG11 agar plate for plate coating. Invert the BG11 agar plate after plate coating and cultivate it in the second culture environment until all Paramecium on the BG11 agar plate disappears and a bacterial-free cyanobacteria colony is formed. Step 5: Expand the bacterial-free cyanobacteria colony obtained in Step 4 in an environment free from external contamination to obtain the required number of bacterial-free single-celled cyanobacteria.

[0007] Optionally, the density of the single-celled cyanobacteria solution in the logarithmic growth phase in step one is 1 × 10^ 7 -1×10^ 9 ce ll / mL.

[0008] Optionally, the first and second culture environments are characterized by a photosynthetic photon flux density ranging from 36 to 90 μmol / m³. 2 •s, light-dark cycle ratio ranges from 16:8 to 12:12, temperature range is 24-26℃.

[0009] Optionally, providing a Paramecium solution of known concentration includes: first pouring a Paramecium solution of unknown concentration into a centrifuge tube, then adding anhydrous ethanol to the centrifuge tube to kill the Paramecium in the solution; placing the centrifuge tube after killing the Paramecium under a stereomicroscope to count them, and calculating the Paramecium concentration corresponding to the Paramecium solution based on the number of killed Paramecium, thus obtaining a Paramecium solution of known concentration.

[0010] Optionally, the step of transferring the cyanobacterial and paramecium enrichment solution in the sterile BG11 conical flask from step three onto a sterilized BG11 agar plate for plate coating includes: providing a BG11 agar plate containing 1.8% agar; transferring the sterile BG11 conical flask to a sterile laminar flow hood and shaking the cyanobacterial and paramecium enrichment solution in the sterile BG11 conical flask; and extracting the shaken cyanobacterial and paramecium enrichment solution onto the sterilized BG11 agar plate for plate coating.

[0011] Optionally, the step of expanding the culture of the sterile cyanobacteria obtained in step four in an environment free from external contamination to obtain the required quantity of sterile single-celled cyanobacteria includes: providing a stereo microscope, EP centrifuge tubes and tissue culture tubes containing BG11 solution, and sterilized BG11 culture medium; transferring the stereo microscope, the EP centrifuge tubes containing BG11 solution, the tissue culture tubes, and the sterilized BG11 culture medium to a clean bench for ultraviolet sterilization for 25-35 minutes; and transferring the BG11 agar plate from step four to the clean bench. The clean bench was cleaned, and the BG11 agar plate on the clean bench was wiped and sterilized with a 75% alcohol swab. Under a stereomicroscope, the sterile cyanobacterial colony on the BG11 agar plate was picked up with a 1mL sterile needle and transferred to the EP centrifuge tube containing BG11 solution. Under conditions that prevent external contamination, the sterile cyanobacterial colony in the EP centrifuge tube was transferred to a tissue culture tube containing BG11 solution for expansion culture for 15-25 days to obtain the required number of sterile single-celled cyanobacteria.

[0012] Optionally, the step of transferring the sterile cyanobacterial colonies from the EP centrifuge tube to a tissue culture tube containing BG11 solution under conditions preventing external contamination and culturing for 15-25 days to obtain the required number of sterile single-celled cyanobacteria includes: transferring the sterile cyanobacterial colonies from the EP centrifuge tube to the tissue culture tube containing BG11 solution under conditions preventing external contamination; sealing the tissue culture tube containing the sterile cyanobacterial colonies with a sealing film in a sterile environment of a laminar flow hood; and then transferring the sealed tissue culture tube to... The contents are transferred to a sterile beaker; the opening of the beaker is covered with a bacterial breathable membrane, and the beaker with the membrane is placed in a third culture environment for 15-25 days to obtain the required number of bacteria-free single-celled cyanobacteria; when entering the subculture stage, the tissue culture tubes containing the required number of bacteria-free single-celled cyanobacteria are transferred to a sterile laminar flow hood, and the tissue culture tubes are sterilized by wiping them with 75% alcohol swabs. The sterile single-celled cyanobacteria in the tissue culture tubes are then diluted to a preset multiple and transferred to new sterile tissue culture tubes containing BG11 solution for further culture.

[0013] Optionally, the third culture environment is characterized by a photosynthetic photon flux density ranging from 36 to 90 μmol / m²·s, a light-dark cycle ratio ranging from 16:8 to 12:12, and a temperature range of 24 to 26°C.

[0014] Optionally, in step three, the sterile BG11 conical flask containing the mixed solution of Paramecium and single-celled cyanobacteria from step two is placed in the first culture environment for 2-5 days; in step four, the BG11 agar plate after plate coating is inverted in the second culture environment for 10-21 days.

[0015] In a second aspect, the present invention provides an application of the method for isolating and purifying single-celled cyanobacteria based on Paramecium enrichment as described in any of the preceding claims in the fields of single-cell resistance gene detection, cyanobacterial carbon, nitrogen and phosphorus cycle research, and cyanobacterial resource utilization.

[0016] The technical effects of this invention include, but are not limited to:

[0017] In a first aspect, a known concentration of Paramecium solution is placed in a logarithmic growth phase unicellular cyanobacteria solution environment. By utilizing the Paramecium's habit of preying on bacteria, the ratio of unicellular cyanobacteria to fine algae is increased, thereby enriching unicellular cyanobacteria.

[0018] In the second aspect of this invention, single-celled cyanobacteria are cultured on BG11 agar plates. During the culture process, bacteria in the treated single-celled cyanobacteria solution on the BG11 agar plates form single colonies. Then, the single-celled cyanobacteria grow on the bacterial colonies, compete with the bacteria and undergo community succession. Some single-celled cyanobacteria will occupy the bacterial colonies, forming single-celled cyanobacteria colonies free from surrounding bacteria. At this time, the purified single-celled cyanobacteria colonies can be picked and expanded under the assistance of a stereomicroscope to obtain bacteria-free single-celled cyanobacteria.

[0019] In a third aspect of the present invention, in the sterile environment of a laminar flow hood, a sealing film is used to seal tissue culture tubes containing bacterial-free cyanobacterial colonies, and then the sealed tissue culture tubes are transferred to a sterile beaker. This method of sealing tissue culture tubes in a beaker can prevent contamination by bacteria from the external environment.

[0020] This invention has a good purification effect on single-celled cyanobacteria, low cost, and quick results. It can separate single-celled cyanobacteria and bacteria with only one plate coating, which can greatly shorten the time of traditional separation, purification and culture of single-celled cyanobacteria. It can also stably subculture and prevent contamination by bacteria in the external environment. Attached Figure Description

[0021] Figure 1 The flowchart illustrates the implementation of the method for isolating and purifying single-celled cyanobacteria based on Paramecium enrichment provided in this embodiment of the invention.

[0022] Figure 2 These are photomicrographs (4×10) of the growth of single-celled cyanobacteria after coating on BG11 agar plates in this embodiment of the invention. Figures a and b show the growth of Synechocystis plating before and after treatment with Paramecium solution, respectively. Synechocystis without Paramecium treatment grows densely on BG11 agar plates, and it is difficult to find single algal colonies. The algal colonies are rough around the edges. However, Synechocystis enriched with Paramecium can be found with smooth single algal colonies. The distance between the algal colonies and bacterial colonies is relatively far, which can achieve the selection of sterile single algal colonies for expansion.

[0023] Figure 3 This is a diagram of the living environment for culturing single-celled cyanobacteria in an embodiment of the present invention. The 500mL beaker is covered with a bacterial breathable membrane as the first line of defense against external bacteria. A sealing film is used to tightly seal the connection between the cap of the tissue culture tube and the glass (tissue culture tube body) to prevent bacterial contamination of its opening.

[0024] Figure 4 The figures show the bacterial growth of single-celled cyanobacteria (FACHB-905, FACHB-898, Synechocystis M1, Synechocystis M2, and Synechocystis Z2) before and after purification in this embodiment of the invention, uniformly spread on LB solid medium and cultured at 37°C for 7 days. The CK group (control group) showed bacterial growth in the single-celled cyanobacteria before purification, while the purified single-celled cyanobacteria showed no bacterial growth on the plates, indicating that the purified single-celled cyanobacteria were free of contaminating bacteria, thus proving the reliability of the purification method of this invention.

[0025] Figure 5 This image shows the sequencing results of purified single-celled cyanobacteria (FACHB-905, FACHB-898, Synechocystis M1, Synechocystis M2, and Synechocystis Z2) according to an embodiment of the present invention. The abundance of all single-celled cyanobacteria samples is above 99.6%, indicating that the purified single-celled cyanobacteria are sterile, proving the reliability of the Paramecium enrichment method for plate purification of single-celled cyanobacteria. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is described. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail. Unless otherwise stated, “%” means percentage based on weight.

[0029] Those skilled in the art will understand that other steps or operations may be included before, after, or between any steps in this embodiment, for example, to further optimize and / or improve the method described in this invention.

[0030] Example 1

[0031] Please see Figure 1 The following is based on Figure 1 Please provide an explanation.

[0032] 1. Paramecium enriches single-celled cyanobacteria

[0033] (1) First, place the purchased paramecium solution in a dark room for 4 days. Then, take 250uL of the well-shaken paramecium solution and place it in a 1.5mL centrifuge tube. Use 750uL of anhydrous ethanol to kill the paramecium in the solution. Count the paramecium under a stereomicroscope and calculate the paramecium concentration accordingly.

[0034] (2) Take 100 mL of each of the following solutions (FACHB-905, FACHB-898, Synechocystis M1 solution, Synechocystis M2 solution, and Synechocystis Z2 solution) containing bacteria and cultured on BG11 liquid medium in the logarithmic growth phase, and place them in five 150 mL sterile BG11 Erlenmeyer flasks. The density of each single-celled cyanobacterial solution is 1 × 10^ 7 -1×10^ 9 Then, add the Paramecium solution to each 150 mL sterile BG11 conical flask to make the density of Paramecium in each single-celled cyanobacterial solution 0.5-2 ce ll / mL;

[0035] In this step, five 150mL sterile BG11 conical flasks containing a mixed solution of Paramecium and single-celled cyanobacteria are placed in a photosynthetic photon flux density range of 36-90 μmol / m 2 The bacteria were cultured for 2-5 days in a culture environment with a light-dark cycle ratio of 16:8-12:12 and a temperature range of 24-26℃. The bacterial count in the solution of 5 single-celled cyanobacteria was examined daily under a microscope (the bacteria were microorganisms other than Paramecium and single-celled cyanobacteria). When the number of bacteria under a microscope was less than half that of the number of cyanobacteria (at which point the growth rate of bacteria could no longer keep up with the growth rate of single-celled cyanobacteria), the enrichment of Paramecium was completed.

[0036] 2. Plate separation and purification

[0037] (1) Transfer the five 150mL sterile BG11 conical flasks to a sterile laminar flow hood. Gently shake the single-celled cyanobacteria solution in each flask. Using a 1.8% BG11 agar plate, extract 100µL of the treated single-celled cyanobacteria solution from each of the five 150mL sterile BG11 conical flasks for plate coating. Invert the five 150mL sterile BG11 conical flasks on a surface with a photosynthetic photon flux density ranging from 36 to 90µmol / m². 2 ·s, with a light-dark cycle ratio ranging from 16:8 to 12:12, and a temperature range of 24-26℃, after culturing for 10-21 days, single algal colonies appeared, such as Figure 2 As shown.

[0038] 3. Transfer culture of single-celled cyanobacteria

[0039] (1) Transfer the stereomicroscope to the laminar flow hood, prepare sterilized BG11 culture medium, 1.5mL EP centrifuge tubes, and 40mL tissue culture tubes to the laminar flow hood for UV sterilization for 30min.

[0040] (2) Transfer the BG11 agar plates containing single-celled cyanobacteria to a clean bench and sterilize by wiping with 75% alcohol swabs. Under a stereomicroscope (4×10), carefully select single-celled cyanobacteria colonies using a 1mL sterile needle and transfer them to a sterile EP tube containing 100µL of BG11, then to a tissue culture tube containing 10mL of sterile BG11. The photosynthetic photon flux density range is 36-90µmol / m 2 Cultivate for 15-25 days in a culture environment with a light-dark cycle ratio of 16:8-12:12 and a temperature range of 24-26℃ to obtain the required number of bacteria-free single-celled cyanobacteria.

[0041] 4. Measures to prevent external pollution

[0042] (1) Before culturing the single-celled cyanobacteria in the tissue culture tube, seal the connection between the cap and the glass of the tissue culture tube containing the purified single-celled cyanobacteria in a sterile environment of a laminar flow hood with sealing film, transfer it to a sterile beaker, and cover the mouth of the beaker with a bacterial breathable membrane. The photosynthetic photon flux density range is 36-90 μmol / m 2 Cultured in an environment with a light-dark cycle ratio ranging from 16:8 to 12:12 and a temperature range of 24-26℃, such as... Figure 3 As shown.

[0043] (2) When the single-celled cyanobacteria in the tissue culture tube enter the passage stage, the tissue culture tubes containing the required number of bacteria-free single-celled cyanobacteria are transferred to a sterile laminar flow hood. The tissue culture tubes are then sterilized by wiping them with 75% alcohol swabs. After the sterilized tissue culture tubes are diluted to a preset multiple (preferably 1 / 10), they are transferred to new sterile tissue culture tubes containing BG11 solution for further cultivation.

[0044] 5. Purification Validation

[0045] (1) In a sterile laminar flow hood, 100 μL of unpurified stock solutions of five single-celled cyanobacteria (FACHB-905, FACHB-898, Synechocystis M1, Synechocystis M2 and Synechocystis Z2) were evenly spread on five LB solid culture media. The media were then inverted in an incubator and cultured at 37°C for 7 days to observe the culture effect of single-celled cyanobacteria before purification.

[0046] In a sterile laminar flow hood, 100 μL of each of the five purified single-celled cyanobacterial stock solutions (FACHB-905, FACHB-898, Synechocystis M1, Synechocystis M2, and Synechocystis Z2) was evenly spread onto five LB solid culture media. The media were then inverted in an incubator and cultured at 37°C for 7 days to observe the culture effect of the purified single-celled cyanobacteria.

[0047] (2) Take 2 mL of stock solution of five single-celled cyanobacteria (FACHB-905, FACHB-898, Synechocystis M1, Synechocystis M2 and Synechocystis Z2) after three subcultures and in the logarithmic growth phase, and process them according to EZNA. According to the requirements of the Bacterial DNA Kit, after extracting and purifying cyanobacterial DNA, the sample is sent for sequencing to detect 16S (V3-V4 region, 338F_806R). The test results are as follows. Figure 4 As shown.

[0048] Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.

Claims

1. A method for isolating and purifying single-celled cyanobacteria based on Paramecium enrichment, characterized in that, include: Step 1: Provide a solution of single-celled cyanobacteria in the logarithmic growth phase and place the single-celled cyanobacteria solution into a sterile BG11 conical flask; Step 2: Provide a Paramecium solution of known concentration and add the Paramecium solution to the sterile BG11 conical flask containing the single-celled cyanobacteria solution from Step 1 to obtain a mixed solution of Paramecium and single-celled cyanobacteria with a Paramecium density of 0.5-2 cells / mL. Step 3: Place the sterile BG11 conical flask containing the mixed solution of Paramecium and single-celled cyanobacteria from Step 2 into the first culture environment for culture until the number of bacteria is less than half of the number of single-celled cyanobacteria. At this point, a solution of cyanobacteria and Paramecium enrichment is obtained, wherein the bacteria are microorganisms other than Paramecium and single-celled cyanobacteria. Step four: Transfer the cyanobacteria and paramecium enrichment solution from the sterile BG11 conical flask in step three to a sterile BG11 agar plate for plate coating. After the plate coating is completed, invert the BG11 agar plate in the second culture environment for incubation until all paramecia on the BG11 agar plate disappear and a bacterial-free cyanobacteria colony is formed. Step 5: Place the bacteria-free cyanobacteria from Step 4 into an environment that prevents external pollution for propagation to obtain the required number of bacteria-free single-celled cyanobacteria. In step one, the density of the single-celled cyanobacteria solution in the logarithmic growth phase is 1 × 10^ 7 -1´10^ 9 cell / mL; The first and second culture environments are characterized by a photosynthetic photon flux density ranging from 36 to 90 μmol / m³. 2 •s, light-dark cycle ratio ranges from 16:8 to 12:12, temperature range is 24-26℃.

2. The method for isolating and purifying single-celled cyanobacteria based on Paramecium enrichment according to claim 1, characterized in that, The provision of a paramecium solution of known concentration includes: First, pour the Paramecium solution of unknown concentration into a centrifuge tube, and then add anhydrous ethanol into the centrifuge tube to kill the Paramecium in the Paramecium solution. Centrifuge tubes after killing paramecia were placed under a stereomicroscope for counting, and the paramecium concentration corresponding to the paramecium solution was calculated based on the number of killed paramecia, thus obtaining a paramecium solution of known concentration.

3. The method for isolating and purifying single-celled cyanobacteria based on Paramecium enrichment according to claim 1, characterized in that, The step of transferring the cyanobacteria and paramecium enrichment solution from the sterile BG11 conical flask in step three onto a sterilized BG11 agar plate for plate coating includes: BG11 agar plates containing 1.8% agar are provided; Transfer the sterile BG11 conical flask to a sterile laminar flow hood and shake the cyanobacteria and paramecium enrichment solution in the sterile BG11 conical flask. The cyanobacteria and paramecium enrichment solution, after being shaken and mixed, was transferred onto a sterilized BG11 agar plate for plate coating.

4. The method for isolating and purifying single-celled cyanobacteria based on Paramecium enrichment according to claim 1, characterized in that, The step of cultivating the bacteria-free cyanobacteria obtained in step five in an environment free from external contamination to obtain the required quantity of bacteria-free single-celled cyanobacteria includes: Provides a stereo microscope, tissue culture tubes, sterilized BG11 culture medium, and EP centrifuge tubes containing sterilized BG11 culture medium; The stereomicroscope, the tissue culture tube, the sterilized BG11 culture medium, and the EP centrifuge tube containing the sterilized BG11 culture medium were transferred to a clean bench for ultraviolet sterilization for 25-35 minutes. Transfer the BG11 agar plate from step four to the laminar flow hood, and wipe the BG11 agar plate on the laminar flow hood with a 75% alcohol swab to sterilize it. Under a stereomicroscope, a 1 mL sterile needle was used to pick up the bacterial-free cyanobacterial colony on the BG11 agar plate and transfer it to the EP centrifuge tube containing sterilized BG11 culture medium. Under conditions that prevent external contamination, the bacteria-free cyanobacterial colonies in the EP centrifuge tubes are transferred to tissue culture tubes containing sterilized BG11 culture medium and cultured for 15-25 days to obtain the required number of bacteria-free single-celled cyanobacteria.

5. The method for isolating and purifying single-celled cyanobacteria based on Paramecium enrichment according to claim 4, characterized in that, Under conditions preventing external contamination, the bacteria-free cyanobacterial colonies in the EP centrifuge tubes are transferred to tissue culture tubes containing sterilized BG11 culture medium and cultured for 15-25 days to obtain the required quantity of bacteria-free single-celled cyanobacteria, including: Under conditions that prevent external contamination, the bacteria-free cyanobacterial colony in the EP centrifuge tube is transferred to the tissue culture tube containing sterilized BG11 culture medium. In the sterile environment of the laminar flow hood, the tissue culture tube containing the bacterial-free cyanobacterial algae colony is sealed with a sealing film, and then the sealed tissue culture tube is transferred to a sterile beaker. The opening of the beaker is covered with a bacterial breathable membrane, and the beaker with the membrane is placed in a third culture environment for 15-25 days to obtain the required number of bacteria-free single-celled cyanobacteria. When entering the passage stage, the tissue culture tubes containing the required number of sterile single-celled cyanobacteria are transferred to a sterile laminar flow hood. The tissue culture tubes are then sterilized by wiping them with 75% alcohol swabs. After the sterile single-celled cyanobacteria in the tissue culture tubes are diluted to a preset multiple, they are transferred to other new sterile tissue culture tubes containing sterilized BG11 culture medium for further cultivation. The third culture environment is characterized by a photosynthetic photon flux density ranging from 36 to 90 μmol / m³. 2 •s, light-dark cycle ratio ranges from 16:8 to 12:12, temperature range is 24-26℃.

6. The method for isolating and purifying single-celled cyanobacteria based on Paramecium enrichment according to claim 1, characterized in that, In step three, the sterile BG11 conical flask containing the mixed solution of Paramecium and single-celled cyanobacteria from step two is placed in the first culture environment for 2-5 days; in step four, the BG11 agar plate after plate coating is inverted in the second culture environment for 10-21 days.

7. The application of the single-celled cyanobacteria isolation and purification method based on Paramecium enrichment according to any one of claims 1-6 in the fields of cyanobacterial cell resistance gene detection, cyanobacterial carbon, nitrogen and phosphorus cycle research and cyanobacterial resource utilization.

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