Immobilization of Yeast cells using Sodium alginate

Aim:

To learn the technique of immobilizing yeast cells in alginate beads

Introduction:

Immobilization is a technique for the combination of a biocatalyst in an insoluble support matrix. The matrix is usually a high molecular weight polymer such as polyacrylamide, starch, cellulose, etc. The advantage of immobilizing enzymes or cells over free cells is to increase their stability and efficiency. The immobilized enzymes or cells can also be recovered at the end of the reaction and can be used repeatedly.

Principle:

In 1916 two scientists named Nelson and Griffin discovered that invertase (an enzyme) shows the same activity when absorbed on a solid surface as when uniformly distributed throughout the solution. This was the first discovery of enzyme immobilization technique. An enzyme is usually immobilized onto an inert, insoluble material e.g. Calcium alginate. This is produced by the reaction of a mixture of Sodium alginate solution with Calcium chloride. These beads provide increased resistance to changes in conditions such as pH or temperature. They also allow enzymes to be held in place throughout a reaction, following which they are easily separated from the products and may be used again - a far more efficient process and so are widely used in industry for enzyme catalyzed reactions. Whole cell immobilization is an alternative to enzyme immobilization. 

Basically, immobilization of live cells is very similar to the enzyme counterpart. In the past, various cells have been immobilized: bacteria, yeasts, fungi, plant tissues, mammalian tissues, and insect tissues. Once the cells are immobilized, the cell viability must be concomitantly sustained over a long period of time. The lower microorganisms (bacteria, yeasts, and fungi) can be easily immobilized with a number of methods: entrapment, ion exchange adsorption, porous ceramics, and even covalent bonding.

 Most of the principles involved in enzyme immobilization are directly applicable to cell immobilization. There are five methods for immobilization of enzymes or cells.

1. Adsorption: It is a method which involves electrostatic interaction such as Van der waals forces, ionic and hydrogen bonding between the enzymes or cells and the support matrix.

2. Covalent Binding: This method involves formation of covalent bonds between the enzymes or cells and the support matrix. The bond is normally formed between the functional groups present on the 4 surface of the support and functional groups belonging to amino acid residues on the surface of the enzyme.

3. Entrapment: In this method the enzyme molecules are mixed with a polyionic polymer material and then crosslinking of the polymer with multivalent cations in an ion exchange reaction to form a lattice structure that traps the enzymes or cells.

4. Encapsulation: This can be achieved by enveloping the enzymes or cells within various forms of semipermeable membranes.

5. Crosslinking: This involves joining of enzymes or cells with each other to form large three dimensional complex structures and can be achieved by physical or chemical methods without any support system.

Requirements:

Measuring cylinder, Conical flask, beaker, Dropper

Yeast Cells in YPD broth/ SD Broth

4 % Sodium Alginate (2 grams sodium alginate suspended  in 50 ml of water 50 ml of distilled water. Heat to dissolve and sterilize by autoclaving)

Calcium chloride (1.5 % solution)

Procedure:

Day 1:

1. Inoculate a single colony of Yeast cell from the Agar (YPD/SDA) plate  in 50 ml of YPD   

   broth.

2. Incubate at 30^oC shaker overnight at 200 rpm.

Day 2:

1. In a 50 ml centrifuge tube take 20 ml of the yeast culture and to it add 20 ml of the 4% Sodium alginate solution. Put the cap tightly.

2. Mix the culture with Sodium alginate solution properly.

3. Take 50 ml of Calcium chloride solution in a conical flask.

4. With a 1 ml pipette, take the alginate and yeast culture mix and add drop wise to the Calcium chloride solution. While adding make sure that the flask is swirled gently.

5. Leave the immobilised yeast cell beads to harden in the Calcium chloride solution for 5–10 minutes. The alginate will be ionically cross-linked by the calcium ions.

6. Isolate the beads after discarding the solution.

Observation and Result:

            Checked for the formation of proper beads. Beads were hard and spherical.

Ref: 

http://himedialabs.com/TD/HTBC001.pdf

https://www.researchgate.net/publication/46256453_Comparison_and_Suitability_of_Gel_Matrix_for_Entrapping_Higher_Content_of_Enzymes_for_Commercial_Applications