Lab Exercises/Demonstrations ........
A Simple Laboratory Exercise for Ethanol
Production by Immobilized Bakery Yeasts
(Saccharomyces cerevisiae)
Diana L. Vullo and Mónica B. Wachsman
ABSTRACT: This laboratory experiment was
designed for Chemistry, Food Technology,
Biology, and Chemical Engineering under-
graduate students. This laboratory experi-
ence shows the advantages of immobilized
bakery yeasts in ethanol production by al-
coholic fermentation. The students were
able to compare the ethanol production
yields by free or calcium alginate entrapped
cells and appreciated the easy separation
of the final product and the biocatalyst re-
utilization.
© 2005 Institute of Food Technologists
Introduction
The immobilized enzyme is defined as “the enzyme physically confined or local-
ized in a certain defined region of space with retention of its catalytic activity, which
can be used repeatedly and continuously.” In some cases, it is not necessary to use
purified enzymes. Immobilization of cells or organelles serves to incorporate multi-
enzyme systems. Furthermore, enzyme-rich cells can themselves be immobilized
and the industrial process operated continuously. Applications of immobilized bio-
catalysts in bioprocessing include (1) the production of useful compounds by ste-
reospecific reactions, (2) the production of energy by biological reactions, (3) the
selective treatments of pollutants to solve environmental problems, (4) the analysis
of various compounds with high sensitivity and specificity, and (5) the manufacture
of new drugs, artificial organs, and so forth (Tanaka and Kawamoto 1999).
The immobilization methods can be classified into 4 categories: carrier-binding,
cross-linking, entrapping, and a combination of these 3 methods. An ideal carrier
should have adequate functional groups for immobilization of the biocatalyst. Lack
of toxicity and economic feasibility should be considered in food technologies.
Several natural polysaccharides, such as alginates, ␬-caraggenan, agar, and agar-
ose, are excellent gel materials and are widely used for entrapment of biocatalysts
(Kierstan and Bucke 1977; Vullo 2003).
Alginic acid is a natural polymer found in marine algae. The chemical structure is
shown in Figure 1 (Davis and others 2003). The fact that free carboxylic groups are
repeated in the macromolecule makes them accessible to divalent cations such as
Ca2+, and the formation of coordination complexes occurs in the gelation process.
Monovalent cation salts are soluble in water and commonly used to increase vis-
cosity in the food industry; therefore, students must pay close attention to sodium
alginate solubilization and the viscosity of the solution.
Yeasts are very important and the most extensively used microorganisms in in-
dustry are shown in Table 1. They are cultured for the cells themselves, for cell
components, and for the end products that they produce during alcoholic fermen-
tation. Large-scale fermentation by yeasts is responsible for the production of etha-
nol for industrial purposes, but yeast is better known for its role in the manufacture
of alcoholic beverages: beer, wine, and liquors. The alcoholic fermentation is an
anaerobic process that takes place in presence of excess sugar (Madigan and oth-
ers 2002).
This laboratory exercise was developed for Chemistry, Food Technology, Biology,
and Chemical Engineering undergraduate students in Industrial Microbiology
courses.
The objectives of this laboratory exercise are as follows:
1. To produce spheres of immobilized bakery yeasts in a calcium alginate gel.
2. To explain the mechanism of Saccharomyces cerevisiae entrapment in calcium
alginate.
3. To compare the alcoholic production kinetics of free and immobilized bakery
yeasts.
MS 20050075 Submitted 2/2/05, Revised 3/9/05, Accepted 3/17/05. The authors are with Area
Microbiología, Dept. de Química Biológica, Facultad de Ciencias Exactas y Naturales, Univ. de
Buenos Aires, Piso 4, Pabellón II, Ciudad Univ., (1428) Buenos Aires, Argentina. Direct inquiries
to author Vullo (E-mail: dvullo@qb.fcen.uba.ar).
Vol. 4, 2005—JOURNAL OF FOOD SCIENCE EDUCATION 53
JFSE: Journal of Food Science Education
4. To determine the ethanol production yield in immobilized and Table 1—Industrial uses of yeasts and yeast products
free yeast fermentation systems. Industrial useProduct a
Production of yeast cells Baker’s yeast for bread making
Dried food yeast for food
Experimental Procedure supplements
Dried feed yeast for animal
Immobilization of bakery yeast by feeds
entrapment in calcium alginate gel Yeast products Yeast extract for culture media
A polymeric matrix was prepared using sodium alginate. For B Vitamins, vitamin D
this purpose, 300 mL of distilled water was prewarmed at 60 °C, Enzymes for food industry,
and 4.5 g of sodium alginate (Sigma Chemical Co., St. Louis, Mo.) invertase, galactosidase
was added with continuous stirring until every clot had been dis- Biochemicals for research,
ATP, NAD+, RNA
solved. Commercial pressed bakery yeast (CALSA, Compañia Ar-
gentina de Levaduras S.A.I.C., Buenos Aires, Argentina, approxi- Fermentation products from yeast Ethanol for industrial alcohol
Glycerol
mately 8 × 109 colony-forming units in Sabouraud agar plates/g), Beverage alcohol Beer
11.25 g, was mechanically suspended in 300 mL of the previous- Wine
ly prepared 1.5% sodium alginate solution. Figure 2 shows the Distilled beverages Whiskey
dropping system used for yeast immobilization. An Erlenmeyer Brandy
flask was filled with the yeast suspension and then emptied by Vodka
gravity, drop by drop, into a 0.1 M CaCl2 solution. The drop vol- Rum
ume was calibrated to be 0.04 mL, using an appropriate diameter a ATP = Adenosine triphosphate; NAD+ = Nicotinamide adenine dinucleotide; RNA

tube. The calibration was performed by passing distilled water = Ribonucleic acid.

through the tube and weighing a fixed number of water droplets.

In this way, all the suspension turned into immobilized yeast
spheres with 107 cells per bead. After 15 min, the beads were
washed 4 times with distilled water to eliminate Ca2+ excess.
Alcoholic fermentation of glucose
For fermentation kinetics evaluation, a special device was used
(Figure 3). It was completely constructed in borosilicate glass. This
fermentor was designed to allow only the CO2 release. The H2SO4
trap retained only some ethanol molecules that could escape to the
gas phase from the fermentation mixture (Vullo and others 2000).
The fermentation process was developed as follows: One batch
fermentor was filled with 4000 beads. The other was loaded with
6 g of bakery yeast, which represents the amount yeast entrapped
in the beads. Then 200 mL of the fermentation medium was add-
ed to each fermentor. After connecting the Erlenmeyer flask to the
trap filled with the H2SO4 solution, the fermentors were immedi-
ately weighed and the initial weight was registered. Subsequently,
at 1-h intervals at 35 °C incubation without shaking, the weight
loss, assumed as CO2 mass, was determined. The ⌬mass (the fer-
mentor mass at time n minus the fermentor mass at time 0) was
plotted against incubation time.
Summary of reagents and equipment
for each team of students
4.5 g of sodium alginate (Sigma) in 300 mL of distilled water
600 mL of 0.1 M CaCl2 in distilled water
11.25 g of commercial bakery yeast (8 × 109 colony-forming units
in Sabouraud agar plates/g)
2 fermentors
100 mL H2SO4 0.1 M

Figure 2—Immobilization of Saccharomyces cerevisiae by entrapment

in calcium alginate gels.

Figure 1—Chemical structure of the alginate matrix: (a) alginate mono-

mers; (b) the alginate polymer chain sequences of the alginate poly-
mers (Davis and others 2003). Figure 3—Fermentation device used for ethanol production.

54 JOURNAL OF FOOD SCIENCE EDUCATION—Vol. 4, 2005 Available on-line at: www.ift.org

Ethanol production by immobilized yeasts . . .
500 mL fermentation medium: glucose 80 g/L, (NH4)2SO4 4 g/L,
MgSO4.7H2O 1 g/L, KH2PO4 2g; pH = 4.5
Thermostatic incubator
Magnetic stirrer
Plastic tubes
Balance
General glass laboratory materials
Results and Conclusions
The results presented subsequently are the experimental data
obtained by 2 groups of students belonging to the year 2004
course in General and Industrial Microbiology. This subject is
compulsory for a chemistry career in the Buenos Aires Univ. The
students worked in teams of 4, guided by the authors. Each team
worked independently in the whole laboratory experience.
Figure 4 shows the glucose fermentation kinetics, measuring
the CO2 mass loss as a function of incubation time. The ethanol
production rate is equal to the CO2 delivering rate, according to
the stoichiometry of the involved reaction:
Figure 4—Ethanol production kinetics: experimental data obtained by
students. Teams A and B, course 2004. 䊏 = by immobilized yeasts; 䉱 =
by free yeasts.
Available on-line at: www.ift.org
The students could compare both kinetics and determine that,
using free or immobilized yeasts, the rates were of the same order.
Team A obtained an ethanol stoichiometric yield of 78.8% for free
cells and 83.3% for immobilized cells, and Team B obtained
55.1% and 76.9%, respectively. The stoichiometric yield was cal-
culated as follows:
Initial glucose mass: 16 g (200 mL of 80 g/L fermentation medium)
Ethanol mass obtained by stoichiometry (ms): 8.2 g
Ethanol mass experimentally obtained (me):
(CO2 mass/CO2 molecular weight) × Ethanol molecular weight
Yield = (me/ms) × 100
These results indicate that the immobilization technique in the
alginate matrix is a good system to develop industrial fermenta-
tions. The students appreciated the most important advantages of
this process, which are the easy separation of the final product
and the biocatalyst reutilization.
Evaluating Questions
1. What happened to the alginate and Ca2+? Explain the fact that
the spheres are easily disrupted when just formed, while after 15
min in CaCl2 solution they are not.
2. How would you calibrate the drop volume of the sodium algi-
nate-yeast suspension?
3. How will you estimate the number of 4000 beads?
4. How do you calculate the free yeast mass you must weigh for
the non-immobilized fermentation system equivalent to the 4000
beads used in the immobilized system? Explain and support your
answer.
5. Explain how the immobilized yeast situated in the middle of an
alginate bead can metabolize glucose.
6. How do you calculate the total yield of ethanol produced after
6 hours? What food analysis technique might you use to confirm
whether your calculations are accurate? Explain and support your
answer.
7. Explain why there is a difference in the rate of alcohol pro-
duced by free and immobilized yeasts. In your answer, take into
account the fact that free yeasts appear to have a greater surface
area exposed to the fermentation mixture than the immobilized
yeasts but there is less alcohol produced.
8. Describe other immobilization methods that could be used in
the food industry.
References
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Kierstan M, Bucke C. 1977. The immobilization of microbial cells, subcel-
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Madigan MT, Martinko JM, Parker J. 2004. Brock. Biology of microorgan-
isms. 10th ed in Spanish. Madrid, Spain: Pearson Edu: Prentice Hall Intl.
p 957-85.
Tanaka A, Kawamoto T. 1999. Cell and enzyme immobilization. In: De-
main AL, Davies J, editors. Manual of industrial microbiology and bio-
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al Atlante S.R.L., Buenos Aires, Argentina. p 221-5.
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