1
NCBE, University of Reading
www.eurovolvox.org
Aim
To provide an introduction to the techniques of cell
immobilisation and the quantitative study of fermentation.
Introduction
Entrapment within calcium alginate is the most widely-used technique
for immobilising cells. It is especially suited to living cells as it tends
not to damage them. Applications of this versatile method include
immobilisation of cells in bioreactors, entrapment of plant protoplasts
and plant embryos (‘artificial seeds’) for micropropagation,
immobilisation of hybridomas for the production of monoclonal
antibodies, and the entrapment of enzymes and drugs (see table).
The cells or enzymes to be entrapped are first mixed with
a solution of sodium alginate. This is then dripped into a
solution containing multivalent cations (usually Ca
+
). The
droplets form spheres as they fall, entrapping the cells in a
three-dimensional lattice of ionically cross-linked alginate.
Copyright © Dean Madden,
Dean Madden
National Centre for Biotechnology Education, University of Reading
Science and Technology Centre, Reading RG BZ UK | E: D.R.Madden@reading.ac.uk
Immobilised yeast
Immobilisation of yeast in calcium alginate beads
134567
Electron micrograph of yeast cells
immobilised in calcium alginate.
Bud scars are visible on some of
the cells.
PHOTOGRAPH COURTESY DUNCAN CASSON
2
NCBE, University of Reading
www.eurovolvox.org
Immobilised yeast
Equipment and materials
Needed by each person or group
Equipment
mL plastic syringe (without a needle)
Small beakers or disposable plastic cups,
mL conical flask
Bung to fit flask, bored to take a fermentation lock
Fermentation lock
Tea strainer
Glass stirring rod
Materials
% sodium alginate solution, mL
. % calcium chloride solution, mL
Bakers’ yeast, dried, . g
% sucrose solution, mL
Universal indicator solution, mL, diluted with
mL of distilled or deionised water
For measuring carbon dioxide evolved (optional)
Equipment
mL conical flask
Bung, to fit conical flask, bored and fitted with a delivery tube
mL measuring cylinder
mL beaker
Materials
% sodium chloride solution, about litre
(ordinary table salt is adequate)
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Copyright © Dean Madden,
Note
All solutions must be made up
using distilled or deionised water.
Sodium alginate is not readily
soluble, and requires both warm
water and stirring to dissolve it.
Cells Product or purpose Cells Product or purpose
Bacteria Algae
Erwinia rhapontici Isomaltulose Botryococcus braunii Hydrocarbons
Pseudomonas denitrificans Cleaning of drinking water Plant cells
Zymomonas mobilis Ethanol Chatharanthus roseus Alkaloids for cancer therapy
Cyanobacteria Various species Artificial seeds
Anabena sp. Ammonia Plant protoplasts Cell handling, microscopy
Fungi Mammalian cells
Kluyveromyces bulgaricus Hydrolysis of lactose Hybridomas Monoclonal antibodies
Saccharomyces cerevisiae Ethanol Islets of Langerhans Insulin/implantation
Saccharomyces bajanus Champagne production Fibroblasts or lymphomas Interferons (
a
or
b
)
Examples of uses of alginate-immobilised cells. After Smidsrød and Skjåk-Bræk (1990).
3
NCBE, University of Reading
www.eurovolvox.org
Immobilised yeast
Fig. Fig. Fig.
Fig. Fig. Fig.
Procedure
Mix the dried yeast with mL of distilled water in a small beaker.
Cover and leave to rehydrate for minutes at room temperature.
Add ml of sodium alginate solution to
the yeast suspension. Stir well.
Draw up some of the yeast/alginate mixture into a syringe.
Add it, a drop at a time, to the calcium chloride solution.
1
2
3
Copyright © Dean Madden,
Leave the immobilised yeast cell beads to harden in the
calcium chloride solution for – minutes. The alginate
will be ionically cross-linked by the calcium ions.
Separate the beads from the solution using the tea strainer.
Place the beads in a sugar solution in a conical flask. Stopper
a flask with a bung that has been fitted with a fermentation
lock. If universal indicator is added to the fermentation lock, the
indicator will change colour as carbon dioxide is produced.
4
5
6
Ca
+
—a
4
NCBE, University of Reading
www.eurovolvox.org
Immobilised yeast
OPTIONAL Measuring carbon dioxide production
Stopper the flask with a bung to which a
delivery tube has been fitted.
Maintain the flask at – °C. Collect the gas that
is produced over a % solution of sodium chloride.
Carbon dioxide will not dissolve in this solution.
Record the volume of gas that has been collected
at convenient intervals. Plot the results on a graph,
showing the volume of gas evolved against time.
7
8
9
Copyright © Dean Madden,
Fig. Fig. Fig.
Safety guidelines
The build-up of gas within glass vessels could be dangerous.
Ensure that the flasks are adequately vented.
Preparation and timing
Immobilised yeast cells can be prepared in – minutes. The
sodium alginate takes some time to dissolve, so the solution is best
prepared before the lesson. If you wish to store sodium alginate
solution for more than a few days, it is advisable to autoclave
it. To prevent excessive depolymerisation of the alginate chains,
however, it is advisable to raise the pH to – before autoclaving.
Troubleshooting
Because sodium alginate is difficult to dissolve, it may help to leave
the alginate to dissolve overnight. If you wish to try additional
activities using buffer solutions, please note that any containing
phosphate, citrate or EDTA should be avoided, as these will cause
the alginate matrix to dissolve ( mM sodium citrate or phosphate
buffer at pH can be used to recover cells from the beads).
5
NCBE, University of Reading
www.eurovolvox.org
Immobilised yeast
Additional investigations
Ordinary bakers’ yeast, Saccharomyces cerevisiae, is unable
to ferment the sugar lactose. The enzyme
b
-galactosidase
breaks down lactose to glucose and galactose. Yeast that is
co-immobilised with this enzyme is able to grow in a medium
that contains lactose. Of the two sugars formed by enzyme
action, glucose is used preferentially. Once supplies of this sugar
have been exhausted, the yeast adjusts its metabolism and the
other breakdown product of lactose, galactose, is utilised. The
activity of the yeast is readily-monitored simply by measuring the
volume of gas (carbon dioxide) evolved during the fermentation.
Different sugar solutions, incubation temperatures or types of
yeast e.g., wine-makers’ or bakers’ yeast, may be compared.
Other sources of information
Publications
Smidsrød, O. and Skjåk-Bræk, G. () Alginate as an immobilization
matrix for cells. Trends in Biotechnology () –.
Practical fermentation: A guide for schools and colleges by John Schollar
and Bene Watmore () Society for General Microbiology,
Reading. ISBN: x.
Two 20-page booklets describing 14 practical investigations
of fermentation. The Teacher’s guide includes ideas for
extension activities and specimen results. The booklets
may be downloaded from: http://www.ncbe.reading.
ac.uk/NCBE/PROTOCOLS/fermentation.html
Immobilized biocatalysts: An introduction by Winnifred Hartmayer
() Springer-Verlag, Heidelberg. ISBN: .
A useful handbook with numerous practical protocols.
Immobilised enzymes and cells: a practical approach by
Jonathan Woodward [Ed] () Oxford University
Press, Oxford. ISBN: .
An academic laboratory manual describing methods
of immobilising enzymes and cells.
Web site
EIBE Unit : Microbes and molecules
http://www.eibe.info
Suppliers
Sodium alginate may be purchased from school chemical
suppliers. It is also used in food production, so may
be available from food industry suppliers.
Acknowledgement
This practical protocol was adapted from a practical protocol by
Dean Madden, that was first published in EIBE Unit 1: Microbes
and molecules (see link above). The Volvox project is funded under
the Sixth Framework Programme of the European Commission.
1
2
Copyright © Dean Madden,
