TITLE: Cyanobacteria Encapsulation in Sepiolite-Alginate Beads
AUTHORS: Jorge   Fernández Méndez, celiamm  

[DESCRIPTION]
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. 

[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. 

[STEPS]
SECTION: Solutions preparation
1. 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. 

1.1. 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
    
1.2. 2:5 alginate:sepiolite
To produce 10 mL of mix, follow same steps as 4:1.
        -2.5 mL alginate 8 % (v/v)
        -5 mLsepiolite 10 % (v/v) 
        -2.5 mL of distilled water

1.3. 1:5 alginate:sepiolite 
To produce 10 mLof mix, follow same steps as 4:1.
         -1.25 mL alginate 8 % (v/v)
        -5 mL sepiolite 10 % (v/v)
        -3.75 mLdestilled water

SECTION: Cyanobacteria
2. 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). 
SECTION: Cyanobacteria
3. Centrifuge the cyanobacteria medium in the Falcon. At 8000 rpm, 10 min 

SECTION: Cyanobacteria
4. 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. 
SECTION: Beads
5. 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. 
SECTION: Beads
6. 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. 
SECTION: Beads
7. Let the beads into the CaCl2 10 minThen, 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.