Nov 30, 2021
Cyanobacteria Encapsulation in Sepiolite-Alginate Beads
- 1Universidad Complutense de Madrid;
- 2Universidad Autónoma de Madrid
Protocol Citation: Jorge Fernández Méndez, celiamm 2021. Cyanobacteria Encapsulation in Sepiolite-Alginate Beads. protocols.io https://dx.doi.org/10.17504/protocols.io.bwqgpdtw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: July 19, 2021
Last Modified: November 30, 2021
Protocol Integer ID: 51688
Abstract
Cyanobacteria can be encapsulated in biohybrid materials, with components such as biopolymers and clays, as in this case. With alginate (biopolymer) and sepiolite (clay that provides better biocompatibility), spheres of about 0.2 mm in diameter are created by using calcium chloride to form a structure in which the cells can be embedded.
This material, despite being transparent and resistant, encounters problems in guaranteeing the correct diffusion of the materials and sufficient photosynthetic activity.
The beads are easily synthesised with mixtures of alginate and sepiolite, and by forming the spheres with CaCl2, following a two steps syntesis.
Materials
- Alginic acid salt (high molecular weight)
- Sepiolite
- Destilled water
- Powdered CaCl2
- Cyanobacteria in BG-11
- BG-11
- EtOH to wash in sterility
- Glasses (it could be crystal glasses or plastic glasses, it is not relevant). It is convenient to use little ones
- Balance
- Flow hood or similar to ensure sterility
- Centrifuge
- Micropipette
- Syringe with medium needle
- Volumetric flask
- Falcon
Safety warnings
Working in sterility with cyanos, and take care at the lab as normally it is done; no extra caution are needed.
Before start
It is recommendable to use cyanobacteria at OD 0,6 or similar. With less OD, it is more difficult to have encapsulated cyano in beads.
Solutions preparation
Solutions preparation
IMPORTANT: the amounts used are the experimental ones did it experimentally, it could change.
Having different options to do the alginate: sepiolite solutions, best of all are explained.
First, produce a 8 % (v/v) suspension of alginate. It is IMPORTANT to mention that it is difficult to disolve it. To do it:
-Take a glass (recomendable to use a plastic glass), with a magnetic stirrer, put it on a heating plate and add 50 mL of water.
-Heat the water at aprox. 65 °C .
-Weight 2 grams of alginc acid salt
-Add the alginic acid salt with stirring and heat. It is Important to add it slowly. Otherwise, it formed 'clots'.
-when all the alginic acid has been added, put off the heat and let it stirring overnight.
Then, to do the 10 % (v/v) sepiolite solution:
-Weight 5 grams of sepiolite (NOTE: take care of sepiolite, it is like dust) and put it on a 50 mL volumetric flask
-Add water up to the mark in the flask. Move slowly and it is ready to use
It is also necessary to have a 10 % (v/v) CaCl2 solution.
4:1 alginate: sepiolite
To produce 10 mL of mix:
-Take5 mL of alginate 8 % (v/v) in a plastic bottle with stirring
-Add 1 mL of 10 % (v/v) sepiolite and 4 mL of water
-Stirring until it is completely uniform
NOTE: it is recommendable to take out the magnetic stirrer
2:5 alginate:sepiolite
To produce 10 mL of mix, follow same steps as 4:1.
-2.5 mL alginate 8 % (v/v)
-5 mL sepiolite 10 % (v/v)
-2.5 mL of distilled water
1:5 alginate:sepiolite
To produce 10 mL of mix, follow same steps as 4:1.
-1.25 mL alginate 8 % (v/v)
-5 mL sepiolite 10 % (v/v)
-3.75 mL destilled water
Cyanobacteria
Cyanobacteria
Having the cyanobacteria culture, work in a flow hood or other methods to guarantee sterility.
Take Falcon and add 2/3 of cyano in BG-11. With a pellet of only one Falcon, it is possible to produce a little bottle of beads (maybe 20/30 beads).
Centrifuge the cyanobacteria medium in the Falcon. At 8000 rpm, 00:10:00
10m
Add 3.75 mL of BG-11 into the Falcon to resuspend the cyano pellet.
NOTE: to make 2:5 beads, it is better to add a little bit more of BG-11, to make it easier to disolve with the alginate: sepiolite solution.
Beads
Beads
10m
10m
Add 2/3 of cyano-BG-11 with 1/3 of mix alginate: sepiolite. Note: in the case of 2:5, it could be better to add more of cyano--BG-11. Mix it in the Falcon, or maybe in a bottle.
When it is mixed, take it with a sirynge and a needle. Having the CaCl2 solution in a bottle, add the syringe content. NOTE: add it at medium speed. If it is added to slowly, it will form deformed beads; and add it to quickly is not recommended.
Let the beads into the CaCl2 00:10:00 Then, take out the beads and put it on BG-11. To take out, it is possible to use a strainer, or do it with a spoon.
10m