Shipment of Cyanobacteria by Agarose Gel Embedding (SCAGE)—A Novel Method for Simple and Robust Delivery of Cyanobacteria

Materials and reagents

Biological materials

1. Synechocystis sp. PCC6803, glucose-tolerant (GT) [9]

2. Anabaena (Nostoc) sp. PCC7120 [10]

Reagents

1. NaNO₃ (Sigma-Aldrich, CAS: 7631-99-4)

2. K₂HPO₄ (water-free) (Sigma-Aldrich, CAS: 7758-11-4)

3. MgSO₄·7H₂O (AppliChem, CAS: 10034-99-8)

4. CaCl₂·2H₂O (Merck, CAS: 10035-04-8)

5. Na₂-EDTA·2H₂O (Merck, CAS: 6381-92-6)

6. Na₂CO₃ (Merck, CAS: 497-19-8)

7. Fe(III)-citrate (Sigma-Aldrich, CAS: 3522-50-7)

8. Citric acid (Roth, CAS: 77-92-9)

9. H₃BO₃ (Merck, CAS: 10043-35-3)

10. MnCl₂·4H₂O (Roth, CAS: 13446-34-9)

11. ZnSO₄·7H₂O (Fluka, CAS: 7758-99-8)

12. Na₂MoO₄·2H₂O (Sigma-Aldrich, CAS: 10102-40-6)

13. CuSO₄·5H₂O (Fluka, CAS: 7758-99-8)

14. Co(NO₃)₂·6H₂O (Sigma-Aldrich, CAS: 10026-22-9)

15. NaHCO₃ (Fisher Chemica, CAS: 144-55-8)

16. Agarose low melt (Roth, CAS: 39346-81-1)

Solutions

1. BG11 medium (see Recipes)

2. 5% (w/v) agarose low melting (ALM) solution (see Recipes)

Recipes

1. BG11 medium (modified after Rippka et al. [11]):

   1. Prepare 200 mL of each stock solution (1–7) from Table 1, 100 mL of trace element solution (Table 2), and 50 mL of 1 M NaHCO3 solution (Table 3). Use Milli-Q water for each solution.

   2. Stock solutions 1–7 and the trace element solution are sterilized by autoclaving (121 °C, 15 psi, 20 min), while the 1 M NaHCO₃ solution is sterilized by filter sterilization (0.22 μm).

      Note: Store solutions 1–6 at room temperature and solution 7 [Fe(III)-citrate & citric acid] protected from light at room temperature. Store trace solution at 4 °C and 1 M NaHCO₃ at -20 °C.

   3. To prepare 1 L of BG11 (1×), add 5 mL of each stock solution 1–7 (200×), 1 mL of the trace element solution (1,000×), and up to 995 mL of Milli-Q water. Sterilize by autoclaving (121 °C, 15 psi, 20 min). Add 5 mL of 1 M NaHCO₃ solution before use.

      Note: For the modified BG11 medium, conduct the following adjustments:

      1. Na₂CO₃: 0.04 g/L (originally 0.02 g/L).

      2. Ferric ammonium citrate was replaced by Fe(III)-citrate.

   
   

   Table 1. Stock solutions (200×) for BG11 medium

   Solution	Chemical	Molar mass M (g/mol)	Molarity c (mM) in 200× stock solution	Mass concentration β (g/L) in 200× stock solution	Molarity c (mM) 1× medium	Mass concentration β (g/L) in 1× medium	Mass m (g) in 200 mL stock solution
   1	NaNO3	84.99	35230	300	17.65	1.5	60
   2	K2HPO4 (water-free)	174.18	35.9	6.25	0.18	0.03125	1.25
   3	MgSO4·7H2O	246.48	60.9	14.8	0.3	0.075	3
   4	CaCl2 ·2H2O	147.02	49.0	7.2	0.24	0.036	1.44
   5	Na2-EDTA·2H2O	372.24	0.5	0.2	0.003	0.001	0.04
   6	Na2CO3	105.99	75.5	8	0.38	0.04	1.6
   7	Fe(III)-citrate	244.94	4.9	1.2	0.024	0.006	0.24
   8	Citric acid	192.13	6.2	1.2	0.031	0.006	0.24

   
   

   Table 2. Trace element solution (1,000×) for BG11 medium

   Chemical	Molar mass M (g/mol)	Molarity c (mM) in 1,000× stock solution	Mass concentration β (g/L) in 1,000× stock solution	Molarity c (μM) 1× medium	Mass m (mg) in 100 mL stock solution
   H3BO3	61.83	46.26	2.86	46.26	286
   MnCl2·4H2O	197.91	9.15	1.81	9.15	181
   ZnSO4·7H2O	287.54	0.77	0.222	0.77	22.2
   Na2MoO4·2H2O	241.95	1.61	0.39	1.61	39
   CuSO4·5H2O	249.68	0.32	0.079	0.32	7.9
   Co(NO3)2·6H2O	291.03	0.17	0.0494	0.17	4.9

   
   

   Table 3. 1 M NaHCO₃ stock solution for BG11 medium

   Chemical	Molar mass M (g/mol)	Molarity c (mM)	Mass concentration β (g/L) in 1 M stock solution	Molarity c (mM) 1× medium	Mass concentration β (g/L) in 1× medium	Mass m (g) in 100 mL stock solution
   NaHCO3	84.01	1,000	84	5	0.42	8.4

   
   

2. 5% (w/v) agarose low melting (ALM) solution

   To prepare 100 mL of 5% (w/v) ALM solution, add 5 g of ALM in 100 mL of BG11 medium. Sterilize by autoclaving (121 °C, 15 psi, 20 min). Store at room temperature.

Laboratory supplies

1. Pipette tips with filter (Mettler Toledo, type RAININ)

2. Serological pipette, 5 mL (Sarstedt, catalog number: 86.1253.001)

3. Conical centrifuge tubes, 15 mL (Sarstedt, catalog number: 62.554.502)

4. Disposal bags (Roth, catalog number: E706.1)

5. Grease-proof paper Profissimo (DM-Drogerie Markt, catalog number: 470799)

6. Rubber band “Alco 758” 80 × 4 mm (Lyreco, catalog number: 5.112.077)

7. Steristopper (VWR, catalog number: HERE1013700)

8. Plastic cuvettes (Sarstedt, catalog number: 67.742)

9. Erlenmeyer wide-neck flask DIN ISO 24450, 100 mL (VWR, catalog number: 214-1131)

10. Laboratory bottle, 1 L (VWR, catalog number: 215-1517P)

11. Beaker, 800 mL (Roth, catalog number: X694.1)

12. Aluminum foil (Roth, catalog number: 1399.1)

13. Sterile indicator strip (Roth, catalog number: XC20.1)

14. Multimark overhead marker permanent (Faber-Castell, catalog number: 151304)

Equipment

1. Pipettes (Mettler Toledo, model: Pipet-Lite XLS series)

2. Pipetboy (VWR, catalog number: 613-4438)

3. Rotary shaker (GFL, catalog number: GFL-3017)

4. OSRAM LUMILUM DE LUX Daylight lamp (OSRAM, catalog number: L58W/954)

5. Biosafety cabinet (Bioquell, model: ABS1500CLS2-MK2)

6. Microwave (OK, model: OMW 330 D-M)

7. Centrifuge 5804 R (Eppendorf, catalog number: 5804 R)

8. Centrifuge rotor S-4-72 (Eppendorf, catalog number: S-4-72)

9. Photometer (Thermo Scientific, model: Spectronic Helios model δ)

10. Foil welding apparatus (BOSCH, catalog number: FG16-0717901002)

Procedure

Note: All steps should be carried out under sterile conditions.

1. Cultivation of cyanobacteria

   1. Put the Steristopper (paper plug) on top of clean Erlenmeyer flasks and cover the Erlenmeyer flasks with grease-proof paper. Secure the grease-proof paper with a rubber band.

   2. Sterilize the prepared Erlenmeyer flask by autoclaving (121 °C, 15 psi, 20 min) and let it cool to room temperature.

   3. Open the Erlenmeyer flask under sterile conditions and inoculate Synechocystis sp. PCC6803 GT or Anabaena (Nostoc) sp. PCC7120 in 50 mL of BG11 medium. Close the Erlenmeyer flask after inoculation.

   4. Grow Synechocystis sp. PCC6803 GT or Anabaena (Nostoc) sp. PCC7120 under continuous light illumination (~ 40 μmol m^-2·s^-1) at 120 rpm and 28 °C until the stationary phase (OD₇₅₀ > 4) is reached.




2. Preparation for gel embedding of cyanobacteria

   1. Sterilize disposable bags in a beaker covered with aluminum foil by autoclaving (121 °C, 15 psi, 20 min) and let them cool down to room temperature.

   2. Prepare 5% (w/v) ALM solution (see Recipes).




3. Gel embedding

   1. Collect 15 mL of liquid bacterial culture in a conical reaction tube and harvest cells by centrifugation at 4000× g for 10 min at room temperature. Discard the supernatant and resuspend cells in 2 mL of BG11 medium.

   2. Simultaneously, heat the 5% (w/v) ALM suspension until a clear, homogenous solution is obtained.

      Caution: The flask containing the ALM solution will be hot and even a boiling delay could be observed. Open the cap of the bottle slightly. Use appropriate safety measures to prevent burning yourself.

   3. Let the solution cool down to approximately 30 °C under the sterile hood.

   4. Once the appropriate temperature is reached, add 3 mL of the 5% (w/v) ALM solution to the resuspended cyanobacterial strain (obtained in step C1) and mix through careful pipetting, resulting in the cyanogel.

      Note: Re-use the serological pipette to mix the components to save time before the cyanogel is solidified. The concentration of ALM in the resulting cyanogel will be 3% (w/v).

   5. Open the disposable bag under sterile conditions and transfer the cyanogel into the bag by slow and careful pipetting.

   6. Situate the disposable bag containing the gel on a flat surface and apply a slight amount of pressure with your hands on the upper side of the bag to evenly distribute the cyanogel within the bag. A smooth plane should be obtained (8 cm × 20 cm × ≤1 mm), which can then be sealed with the foil welding machine (section D).




4. Sealing the cyanogel

   1. Place the bag between the contacts of a foil welding machine and close the machine for 3 s.

      Caution: Be cautious when pressing the machine down since the contacts will get hot. Take measures to prevent burning yourself.

   2. Open the apparatus again and wait for 5 s.

      Note: Removing the cyanogel before letting it cool may result in deformation or breakage of the bag. Usually, 5 s is enough to let the sealed bag cool down.

   3. Label the bag appropriately, e.g., with a permanent marker, for later identification of the cyanobacterial strain contained within.




5. Packaging of embedded cyanobacteria

   1. Package the embedded cyanobacteria in an appropriate container (e.g., an envelope) and send it to the location of your choice.

      Note: Depending on the destination of your cyanobacterial samples, different regulations regarding labeling may apply.




6. Recovering of cyanobacteria out of the cyanogel

   1. Prepare 50 mL of BG11 medium in an Erlenmeyer flask under sterile conditions.

   2. Open the received cyanogel and squeeze the gel out of the disposal bag into the BG11 medium.

      Note: Use a sterile pair of scissors for cutting an edge of the cyanogel for easier and more precise gel squeezing.

   3. Let the cyanobacteria grow under dimmed light conditions (flask wrapped with a paper towel) of ~20 µmol m^-2·s^-1 at 120 rpm for 24 h at 28 °C.

   4. After 24 h of incubation, remove the paper towel and cultivate the cyanobacteria under standard cultivation conditions.

Validation of protocol

For validation of the protocol presented here, the procedure was performed with two different cyanobacterial strains [Synechocystis sp. PCC6803 and Anabaena (Nostoc) sp. PCC7120]. Synechocystis and Anabaena (Nostoc) were cultivated under standard conditions until an OD₇₅₀ of 9.3 and 5.2 was reached, respectively. 25 mL of culture was centrifuged and resuspended in 8 mL of BG11 medium; 2 mL of resuspended cyanobacteria were mixed with 3 mL of BG11 medium and used as a control group. Triplicates of 2 mL culture were mixed with 3 mL of 5% (w/v) ALM and cast into disposable bags. Bags were sealed with a foil welding machine and were stored for one week in a non-transparent, padded envelope at room temperature in the dark to simulate packaging shipment (Figure 1).

Figure 1. Disposal bags filled with cyanobacteria. Left: Synechocystis sp. PCC6803 GT; right: Anabaena (Nostoc) sp. PCC7120 embedded in 5% (w/v) ALM [cyanogel, end concentration 3% (w/v) ALM] before and after shipment simulation.

After one week of shipment simulation, regeneration of cyanobacterial strains was performed according to the described method. Full recovery of both strains was reached after 14 days of cultivation (Figure 2).

Figure 2. Recovery of cyanobacteria over 14 days. Left: Synechocystis sp. PCC6803 GT; right: Anabaena (Nostoc) sp. PCC7120.

Acknowledgments

This project was conducted with the support of the Korea Evaluation Institute of Industrial Technology (KEIT) grant funded by the Korean government (MOTIE) (No. RS-2022-00155902) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2021R1I1A3055799).

Competing interests

The authors have no conflicts of interest to declare.

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