Single-cell cyanobacteria separation and purification method based on paramecium enrichment and application

Abstract

The invention discloses a paramecium enrichment-based single-cell cyanobacteria separation and purification method and application. The method comprises the following steps: putting a single-cell cyanobacteria solution into a sterile BG11 conical flask; the method comprises the following steps: adding a paramecium solution with known concentration into a sterile BG11 conical flask filled with a single-cell cyanobacteria solution to obtain a mixed solution of paramecium and single-cell cyanobacteria; placing the sterile BG11 conical flask filled with the mixed solution of paramecia and single-cell blue-green algae in a first culture environment for culture to obtain a blue-green algae and paramecia enriched solution; transferring the blue-green algae and paramecium enrichment solution to a sterilized BG11 agar plate for plate coating treatment, and inversely placing the BG11 agar plate in a second culture environment for culture until blue-green algae falling without infectious microbes is formed; and carrying out expanding culture on the bacterial-free cyanobacteria colony to obtain bacterial-free single-cell cyanobacteria. The method can greatly shorten the time of traditional isolated culture of single-cell cyanobacteria, and can prevent bacterial pollution of the external environment.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for separating and purifying single-cell cyanobacteria based on Paramecium enrichment and an application thereof. Background Art

[0002] Cyanobacteria are photosynthetic autotrophic prokaryotic microorganisms, producers in nature, and participate in the carbon, nitrogen and phosphorus cycles in nature. Cyanobacteria are rich in phycocyanin and can be used as health products, with great research potential and application value. However, it is currently difficult to purify single-cell cyanobacteria. Since single-cell cyanobacteria are sensitive to antibiotics, it is difficult to use antibiotics to kill bacteria and screen cyanobacteria. In addition, the growth cycle of single-cell cyanobacteria is long. Traditional methods such as plate coating or streak purification often require repeated coating or streak culture, which takes a long time. The method of using lysozyme to enzymolyze bacteria to separate cyanobacteria requires the use of drug resistance tests on bacteria, which is relatively complicated and is not applicable to bacteria that are insensitive to lysozyme in the environment. At the same time, there may be a risk of repeated bacterial contamination. Using high-precision instruments (flow cytometer sorting, single-cell Raman sorting, etc.), it is also necessary to sort an appropriate amount of single-cell cyanobacteria and expand them for 1-2 growth cycles to obtain a certain amount of bacteria-free cyanobacteria, but the instrument is relatively expensive and the use environment requires sterility. Summary of the invention

[0003] In order to solve the technical problems in the prior art, the present invention provides a method for separating and purifying unicellular cyanobacteria based on Paramecium enrichment and its application, which can accurately solve the above-mentioned technical problems in the prior art.

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

[0005] The first aspect of the present invention provides a method for separating and purifying single-cell cyanobacteria based on Paramecium enrichment, which comprises:

[0006] Step 1: provide a single-cell cyanobacteria solution in a logarithmic growth phase, and place the single-cell cyanobacteria solution in a sterile BG11 conical flask; Step 2: provide a paramecium solution of known concentration, and add the paramecium solution into the sterile BG11 conical flask containing the single-cell cyanobacteria solution in step 1, to obtain a paramecium density of 0.5-2ce ll / mL of a mixed solution of paramecium and unicellular cyanobacteria; step three, placing the sterile BG11 conical flask containing the mixed solution of paramecium and unicellular cyanobacteria in step two in a first culture environment for cultivation until the number of bacteria is less than half of the number of unicellular cyanobacteria, and at this time, a cyanobacteria and paramecium enriched solution with complete paramecium enrichment is obtained, wherein the bacteria are microorganisms other than paramecium and unicellular cyanobacteria; step four, transferring the cyanobacteria and paramecium enriched solution in the sterile BG11 conical flask in step three to a sterile BG11 agar plate for plate coating treatment, and inverting the BG11 agar plate after the plate coating treatment in a second culture environment for cultivation until all the paramecium on the BG11 agar plate disappears and a cyanobacteria colony without foreign bacteria is formed; step five, expanding the cyanobacteria colony without foreign bacteria in step four in an environment to prevent external pollution to obtain a required number of unicellular cyanobacteria without foreign bacteria.

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

[0008] Optionally, the first culture environment and the second culture environment are: the photosynthetic photon flux density range is 36-90umol / m 2 ·s, the light-dark cycle ratio ranged from 16:8 to 12:12, and the temperature ranged from 24 to 26°C.

[0009] Optionally, providing a Paramecium solution of known concentration includes: first pouring a Paramecium solution of unknown concentration to be tested into a centrifuge tube, and then adding anhydrous ethanol into the centrifuge tube to kill the Paramecium in the Paramecium solution to be tested; placing the centrifuge tube after killing the Paramecium under a stereo microscope for counting, and calculating the Paramecium concentration corresponding to the Paramecium solution according to the number of killed Paramecium to obtain a Paramecium solution of known concentration.

[0010] Optionally, the step of transferring the cyanobacteria and paramecium enriched solution in the sterile BG11 conical flask in step three to a sterilized BG11 agar plate for plate spreading treatment comprises: providing a BG11 agar plate containing 1.8% agar; transferring the sterile BG11 conical flask to a sterile clean bench, and shaking the cyanobacteria and paramecium enriched solution in the sterile BG11 conical flask; and extracting the shaken cyanobacteria and paramecium enriched solution onto the sterilized BG11 agar plate for plate spreading treatment.

[0011] Optionally, the step of dropping the sterile cyanobacteria in step 4 in an environment to prevent external contamination for expansion culture to obtain a desired number of sterile unicellular cyanobacteria comprises: providing a stereo microscope, an EP centrifuge tube and a tissue culture tube filled with a BG11 solution, and a sterilized BG11 culture solution; transferring the stereo microscope, the EP centrifuge tube and the tissue culture tube filled with the BG11 solution, and the sterilized BG11 culture solution to an ultra-clean bench for ultraviolet sterilization for 25-35 minutes; transferring the BG11 agar plate in step 4 to the ultra-clean bench; The clean bench is cleaned and the BG11 agar plate on the clean bench is wiped and sterilized with a 75% alcohol cotton ball; under a stereo microscope, a 1 mL sterile needle is used to pick the bacteria-free cyanobacteria colony on the BG11 agar plate and transfer it to the EP centrifuge tube filled with BG11 solution; under the condition of preventing external contamination, the bacteria-free cyanobacteria colony in the EP centrifuge tube is transferred to a tissue culture tube filled with BG11 solution for expansion culture for 15-25 days to obtain the required number of bacteria-free unicellular cyanobacteria.

[0012] Optionally, under the condition of preventing external contamination, transferring the bacteria-free cyanobacteria colony in the EP centrifuge tube to a tissue culture tube filled with BG11 solution for culturing for 15-25 days to obtain a required number of bacteria-free unicellular cyanobacteria, comprising: under the condition of preventing external contamination, transferring the bacteria-free cyanobacteria colony in the EP centrifuge tube to the tissue culture tube filled with BG11 solution; in the sterile environment of a clean bench, sealing the tissue culture tube containing the bacteria-free cyanobacteria colony with a sealing film, and then transferring the sealed tissue culture tube to the sterile environment of a clean bench; into a sterile beaker; using a bacterial breathable membrane to cover the opening of the beaker, and placing the membrane-covered beaker in a third culture environment for culture for 15-25 days to obtain the required number of bacteria-free unicellular cyanobacteria; when entering the subculture stage, the tissue culture tube with the required number of bacteria-free unicellular cyanobacteria is transferred to a sterile clean bench, and the tissue culture tube is wiped with a 75% alcohol cotton ball for sterilization, and the bacteria-free unicellular cyanobacteria in the sterilized tissue culture tube is diluted to a preset multiple, and then transferred to another new sterile tissue culture tube filled with BG11 solution for culture.

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

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

[0015] The second aspect of the present invention provides an application of a single-cell cyanobacteria separation and purification method based on Paramecium enrichment according to any of the above items in the fields of single-cell resistance gene detection, cyanobacteria carbon, nitrogen and phosphorus cycle research and cyanobacteria resource utilization.

[0016] The technical effects of the present invention include but are not limited to:

[0017] In a first aspect of the present invention, a Paramecium solution of known concentration is placed in a unicellular cyanobacteria solution environment in a logarithmic growth phase, and the habit of Paramecium to prey on bacteria is utilized to increase the number ratio of unicellular cyanobacteria and microalgae, thereby enriching unicellular cyanobacteria.

[0018] In a second aspect of the present invention, a BG11 agar plate is used to coat and culture unicellular cyanobacteria. During the culture process, the bacteria in the treated unicellular cyanobacteria solution on the BG11 agar plate form a single colony, and then the unicellular cyanobacteria grow on the bacterial colony, forming a competitive relationship with the bacteria and undergoing community succession. Some unicellular cyanobacteria will occupy the bacterial colony to form a unicellular cyanobacteria colony with no foreign bacteria around. At this time, the purified unicellular cyanobacteria colony can be picked up with the assistance of a stereo microscope for expansion culture to obtain unicellular cyanobacteria without foreign bacteria.

[0019] According to a third aspect of the present invention, in a sterile environment of a clean bench, a sealing film is used to seal a tissue culture tube containing a blue algae colony without foreign bacteria, and the sealed tissue culture tube is then transferred to a sterile beaker. The culture method of sealing the tissue culture tube in a beaker can prevent contamination by bacteria from the external environment.

[0020] The present invention has good purification effect on unicellular cyanobacteria, low cost and quick effect. It only needs one plate coating to separate unicellular cyanobacteria and bacteria, which can greatly shorten the time of traditional separation, purification and cultivation of unicellular cyanobacteria, and can stably subculture and prevent contamination of bacteria in the external environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A flowchart of the implementation of the method for separating and purifying single-cell cyanobacteria based on Paramecium enrichment provided in an embodiment of the present invention;

[0022] Figure 2 Figure 1 is a microscopic photograph (4×10) of the growth of single-cell cyanobacteria after coating on a BG11 agar plate in an embodiment of the present invention. Figure a and Figure b are the growth conditions of Synechocystis before and after treatment with a Paramecium solution, respectively. The Synechocystis that was not treated with Paramecium grew densely on the BG11 agar plate, and it was difficult to see single algae colonies. The surroundings of the algae colonies were rough. However, after enrichment with Paramecium, single algae colonies with smooth surroundings could be found for the Synechocystis. The distance between the algae colonies and the bacterial colonies was relatively far, so it was possible to select sterile single algae colonies for expansion.

[0023] Figure 3 This is a diagram of the living environment for culturing unicellular cyanobacteria in an embodiment of the present invention, wherein the 500 mL small beaker is covered with a bacterial breathable membrane as the first line of defense to isolate the bacteria outside, and a sealing film is used to tightly cover the connection between the lid of the tissue culture tube and the glass (tissue culture tube body) to prevent bacteria from contaminating the seam;

[0024] Figure 4 The single-cell cyanobacteria (FACHB-905, FACHB-898, Synechocystis M1, Synechocystis M2 and Synechocystis Z2) before and after purification in the embodiment of the present invention are evenly coated on LB solid culture medium and cultured in an environment at a temperature of 37° C. for 7 days. Among them, the single-cell cyanobacteria in the CK group (control group) before purification have bacterial growth, and no bacteria grow on the plate of the single-cell cyanobacteria after purification, indicating that the purified single-cell cyanobacteria have no foreign bacteria, proving that the purification method of the present invention is reliable;

[0025] Figure 5 The results of sequencing the purified unicellular cyanobacteria (FACHB-905, FACHB-898, Synechocystis M1, Synechocystis M2 and Synechocystis Z2) in the present invention are shown in Figure 1. The abundance of all unicellular cyanobacteria samples is above 99.6%, indicating that the purified unicellular cyanobacteria are sterile, proving that the method of enriching unicellular cyanobacteria with Paramecium and purifying them on a plate is reliable. DETAILED DESCRIPTION

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

[0027] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail. Unless otherwise specified, "%" is a percentage based on weight.

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

[0030] Example 1

[0031] See also Figure 1 , the following is based on Figure 1 Provide explanation.

[0032] 1. Paramecium enriches unicellular cyanobacteria

[0033] (1) First, place the purchased Paramecium solution in a dark room for 4 days, then take 250uL of the shaken Paramecium solution and place it in a 1.5mL centrifuge tube. Kill the Paramecium in the Paramecium solution with 750uL of anhydrous ethanol, and count it under a stereo microscope to calculate the Paramecium concentration.

[0034] (2) Take 100 mL of five single-cell cyanobacteria solutions (FACHB-905, FACHB-898, Synechocystis M1 solution, Synechocystis M2 solution, and Synechocystis Z2 solution) cultured in BG11 liquid medium and in the logarithmic growth phase and place them in five 150 mL sterile BG11 conical flasks, where the density of each single-cell cyanobacteria solution is 1×10^ 7 -1×10^ 9 ce ll / mL, then add the paramecium solution into each 150mL sterile BG11 conical flask to make the density of paramecium in each single-cell cyanobacteria solution be 0.5-2ce ll / mL;

[0035] In this step, five 150 mL sterile BG11 conical flasks containing a mixed solution of Paramecium and unicellular cyanobacteria were placed in a photosynthetic photon flux density range of 36-90 μmol / m 2 ·s, cultured in a culture environment with a light-dark cycle ratio range of 16:8-12:12 and a temperature range of 24-26℃ for 2-5 days, and the number of bacteria in 5 single-cell cyanobacteria solution environments was examined under a microscope every day (bacteria are microorganisms other than Paramecium and single-cell cyanobacteria). When the number of bacteria examined under a microscope is approximately less than half of the number of cyanobacteria (at this time, the growth rate of bacteria is no longer faster than that of single-cell cyanobacteria), the enrichment of Paramecium is completed.

[0036] 2. Plate separation and purification

[0037] (1) Transfer five 150 mL sterile BG11 conical flasks to a sterile clean bench, gently shake the single-cell cyanobacteria solution in the five 150 mL sterile BG11 conical flasks, use a 1.8% BG11 agar plate, extract 100 uL of the treated single-cell cyanobacteria solution in each of the five 150 mL sterile BG11 conical flasks for plate coating, and invert the five 150 mL sterile BG11 conical flasks at a photosynthetic photon flux density range of 36-90 umol / m 2 ·s, light-dark cycle ratio range of 16:8-12:12, temperature range of 24-26℃, culture for 10-21 days, single algae colony appears, such as Figure 2 shown.

[0038] 3. Transfer and culture of unicellular cyanobacteria

[0039] (1) Move the stereo microscope to a clean bench, prepare sterilized BG11 culture medium, 1.5 mL EP centrifuge tubes, and transfer 40 mL tissue culture tubes to the clean bench for UV sterilization for 30 min.

[0040] (2) The BG11 agar plate with unicellular cyanobacteria was transferred to a clean bench and sterilized by wiping with 75% alcohol cotton balls. Under a stereo microscope (4×10), a 1 mL sterile needle was used to carefully select a single colony of unicellular cyanobacteria and transfer it to a sterile EP tube containing 100 uL BG11, and then transferred to a tissue culture tube containing 10 mL sterile BG11. The photosynthetic photon flux density ranged from 36 to 90 μmol / m 2 ·s, a light-dark cycle ratio range of 16:8-12:12, and a temperature range of 24-26°C for 15-25 days to obtain the required number of bacteria-free unicellular cyanobacteria.

[0041] 4. Measures to prevent external pollution

[0042] (1) Before culturing the unicellular cyanobacteria in the tissue culture tube, seal the joint between the lid and the glass of the tissue culture tube containing the purified unicellular cyanobacteria with a sealing film in the sterile environment of the clean bench, transfer it to a sterile beaker, and cover the beaker mouth with a bacterial breathable membrane. 2 ·s, light-dark cycle ratio range of 16:8-12:12, temperature range of 24-26℃, such as Figure 3 shown.

[0043] (2) When the unicellular cyanobacteria in the tissue culture tube enters the subculture stage, the tissue culture tube with the required number of bacteria-free unicellular cyanobacteria is transferred to a sterile clean bench, the tissue culture tube is wiped with a 75% alcohol cotton ball for sterilization, and the sterilized bacteria-free unicellular cyanobacteria in the tissue culture tube is diluted to a preset multiple (preferably 1 / 10), and then transferred to another new sterile tissue culture tube filled with BG11 solution for culture.

[0044] 5. Purification verification

[0045] (1) In a sterile clean bench, 100uL of the original solution of five types of single-cell cyanobacteria (FACHB-905, FACHB-898, Synechocystis M1, Synechocystis M2 and Synechocystis Z2) without purification was evenly spread on five LB solid culture media, and then inverted in an incubator and cultured at 37°C for 7 days to observe the culture effect of the single-cell cyanobacteria before purification.

[0046] In a sterile clean bench, 100uL of 5 purified unicellular cyanobacteria stock solutions (FACHB-905, FACHB-898, Synechocystis M1, Synechocystis M2 and Synechocystis Z2) were evenly spread on 5 LB solid culture media, then inverted in an incubator and cultured at 37°C for 7 days to observe the culture effect of the purified unicellular cyanobacteria.

[0047] (2) 2 mL of stock solution of five unicellular cyanobacteria (FACHB-905, FACHB-898, Synechocystis M1, Synechocystis M2, and Synechocystis Z2) that had been passaged three times and were in the logarithmic growth phase were extracted and analyzed according to EZNA According to the requirements of the Bacterial DNA Kit, the blue algae DNA was extracted and purified, and the sample was sent for sequencing to detect 16S (V3-V4 region, 338F_806R). The test results are as follows Figure 4 shown.

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

Claims

1. A method for separating and purifying single-cell cyanobacteria based on Paramecium enrichment, characterized in that: include: Step 1, providing a single-cell cyanobacteria solution in a logarithmic growth phase, and placing the single-cell cyanobacteria solution into a sterile BG11 conical flask; Step 2, providing a paramecium solution of known concentration, and adding the paramecium solution into the sterile BG11 conical flask containing the single-cell cyanobacteria solution in step 1, to obtain a mixed solution of paramecium and single-cell cyanobacteria with a paramecium density of 0.5-2 cel l / mL; Step 3, placing the sterile BG11 conical flask containing the mixed solution of paramecium and unicellular cyanobacteria in step 2 in a first culture environment for culture until the number of bacteria is less than half of the number of unicellular cyanobacteria, thereby obtaining a cyanobacteria and paramecium enriched solution with complete paramecium enrichment, wherein the bacteria are microorganisms other than paramecium and unicellular cyanobacteria; Step 4, transferring the cyanobacteria and paramecium enrichment solution in the sterile BG11 conical flask in step 3 to a sterile BG11 agar plate for plate coating, and inverting the BG11 agar plate after the plate coating process in a second culture environment for culture until all the paramecium on the BG11 agar plate disappears and a cyanobacteria colony free of foreign bacteria is formed; Step 5: The bacteria-free cyanobacteria obtained in step 4 are placed in an environment to prevent external contamination and cultured to obtain the required number of bacteria-free unicellular cyanobacteria.

2. The method for separating and purifying single-cell cyanobacteria based on Paramecium enrichment according to claim 1, characterized in that: The density of the single-cell cyanobacteria solution in the logarithmic growth phase in step 1 is 1×10^ 7 -1×10^ 9 cel l / mL.

3. The method for separating and purifying single-cell cyanobacteria based on Paramecium enrichment according to claim 1, characterized in that: The first culture environment and the second culture environment are: the photosynthetic photon flux density range is 36-90umol / m 2 ·s, the light-dark cycle ratio ranged from 16:8 to 12:12, and the temperature ranged from 24 to 26°C.

4. The method for separating and purifying single-cell cyanobacteria based on Paramecium enrichment according to claim 1, characterized in that: The method of providing a Paramecium solution with a known concentration comprises: 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; The centrifuge tube after killing the Paramecium is placed under a stereo microscope for counting, and the Paramecium concentration corresponding to the Paramecium solution is calculated according to the number of killed Paramecium to obtain a Paramecium solution of known concentration.

5. The method for separating and purifying single-cell cyanobacteria based on Paramecium enrichment according to claim 1, characterized in that: The step of transferring the cyanobacteria and paramecium enrichment solution in the sterile BG11 conical flask in step 3 to a sterilized BG11 agar plate for plate coating includes: Provide BG11 agar plates containing 1.8% agar; Transfer the sterile BG11 conical flask to a sterile clean bench, and shake the cyanobacteria and paramecium enrichment solution in the sterile BG11 conical flask; The shaken cyanobacteria and paramecium enrichment solution was extracted and placed on the sterilized BG11 agar plate for plate spreading.

6. The method for separating and purifying single-cell cyanobacteria based on Paramecium enrichment according to claim 1, characterized in that: The step of placing the sterile cyanobacteria in step 4 in an environment to prevent external contamination for expansion treatment to obtain the required number of sterile unicellular cyanobacteria comprises: Provide stereo microscope, EP centrifuge tubes and tissue culture tubes filled with BG11 solution, and sterilized BG11 culture medium; Transfer the stereo microscope, the EP centrifuge tube containing the BG11 solution, the tissue culture tube and the sterilized BG11 culture solution to a clean bench for ultraviolet sterilization for 25-35 minutes; Transfer the BG11 agar plate in step 4 to the clean bench, and wipe and sterilize the BG11 agar plate on the clean bench with a 75% alcohol cotton ball; Under a stereo microscope, use a 1 mL sterile needle to pick the bacteria-free cyanobacteria colony on the BG11 agar plate and transfer it to the EP centrifuge tube containing the BG11 solution; Under the condition of preventing external contamination, the bacteria-free cyanobacteria colony in the EP centrifuge tube is transferred to a tissue culture tube filled with BG11 solution for culturing for 15-25 days to obtain the required number of bacteria-free unicellular cyanobacteria.

7. The method for separating and purifying single-cell cyanobacteria based on Paramecium enrichment according to claim 6, characterized in that: Under the condition of preventing external contamination, the bacteria-free cyanobacteria colony in the EP centrifuge tube is transferred to a tissue culture tube filled with BG11 solution for culturing for 15-25 days to obtain the required number of bacteria-free unicellular cyanobacteria, including: Under the condition of preventing external contamination, the bacteria-free cyanobacteria colony in the EP centrifuge tube is transferred to the tissue culture tube containing the BG11 solution; In the sterile environment of the clean bench, the tissue culture tube containing the bacteria-free cyanobacteria colony is sealed with a sealing film, and then the sealed tissue culture tube is transferred to a sterile beaker; Covering the opening of the beaker with a bacterial breathable membrane, and placing the beaker covered with the membrane in a third culture environment for culturing for 15-25 days to obtain a desired amount of bacteria-free unicellular cyanobacteria; When entering the subculture stage, the tissue culture tube with the required number of bacteria-free unicellular cyanobacteria is transferred to a sterile clean bench, the tissue culture tube is wiped with a 75% alcohol cotton ball for sterilization, and the sterilized bacteria-free unicellular cyanobacteria in the tissue culture tube is diluted to a preset multiple and then transferred to another new sterile tissue culture tube filled with BG11 solution for culture.

8. The method for separating and purifying single-cell cyanobacteria based on Paramecium enrichment according to claim 7, characterized in that: The third culture environment is: photosynthetic photon flux density range is 36-90umol / m 2 ·s, the light-dark cycle ratio ranged from 16:8 to 12:12, and the temperature ranged from 24 to 26°C.

9. The method for separating and purifying single-cell 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 unicellular cyanobacteria in step two is placed in the first culture environment for culture for 2-5 days; in step four, the BG11 agar plate after the plate coating treatment is inverted in the second culture environment for culture for 10-21 days.

10. An application of the single-cell cyanobacteria separation and purification method based on Paramecium enrichment according to any one of claims 1 to 9 in the fields of single-cell resistance gene detection, cyanobacteria carbon, nitrogen and phosphorus cycle research and cyanobacteria resource utilization.

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