Science Demonstration

-Fermentation by YeastBy: Christine L. Case, Ed.D., Skyline College

Objective
The purpose of this experiment is to give students the opportunity to learn about
fermentation first hand. Using baker's yeast (Saccharomyces cerevisiae),the students will
address some of the problems faced by Pasteur, Weizmann, and present-day fermentation
specialists. The yeast carry out an alcohol fermentation when grown anaerobically; in the
presence of air, they make carbon dioxide and water by aerobic respiration. Students will
have fun with this experiment as the balloons expand.

Time
First day
Preparation and inoculation of culture media-50 minutes. If culture media are prepared in
advance they can be refrigerated (up to 24 hours) or sterilized for indefinite storage. To
sterilize, prepare materials in a Mason jar and put in a pressure cooker at 121C at 15 psi
for 15 minutes.
Second day 3-7 days later: After growth has occurred-50 minutes.

Materials

Each pair of students should prepare two different bottles of media.

Bottles (any size) half-filled with the following:
o Water
o Sugar-water (1 part table sugar to 4 parts water)
o Starch-water (1:4 mashed potato flakes in water, use the broth)
o Sugar-protein water (1 part commercial beef broth and 1 part sugar-water)
o Protein-water (commercial beef/chicken broth diluted to half strength or
commercial glucose-yeast extract broth)
o Balloons to fit mouths of bottles
o Fruit or leaves- These can be picked from the garden or purchased at the
supermarket.
Knife and cutting board, mortar and pestle, blender or food processor
Baker's yeast
Optional: Hydrometer
Optional: Raisons
Optional: Bacto Sabouraud dextrose broth (Difco) can be substituted for sugarprotein water. It is available from Carolina, Ward's, and Fisher.

Procedure
First day
1. Add a pinch of baker's yeast to each bottle. Cover the mouth of the bottle with a
balloon. Record the appearance of the broth. Optional: Record the specific gravity of the
broth. As alcohol is produced, the specific gravity will decrease. Incubate the bottle at
room temperature until growth has occurred.
2. Cut the fruit or leaves into small pieces to make one-fourth to one-half the volume of
liquid media. You could use mortar and pestle, blender, or food processor for this.
3. Place the plant materials in a bottle of protein-water. Add a pinch of baker's yeast to
each bottle. Cover the mouth of the bottle with a balloon. Record the appearance of the
broth. Record the appearance of the broth. Incubate the bottle at room temperature until
growth has occurred.
4. Optional: Add a raison to each bottle (to increase gas production).
5. Use different fruits or leaves to see if yeast growth is affected.
6. Repeat step 1 without the balloon. The bottle can be covered with aluminum foil or a
cotton stopper to keep out dust. Did the yeast grow? Was alcohol produced?
Second day
1. Record the appearance of the broth after incubation.
2. Was gas produced?
3. Determine the specific gravity of the broth.
4. Make a table of class results. Show the following in the table:

Media
Type of fruit/leaves
Appearance of broth on first day
Specific gravity on first day
Appearance of broth on day ___
Amount of gas produced
Specific gravity on day __

Questions
1. What caused the balloon to inflate?

2. What was the purpose of the balloon?

3. Which substrate resulted in the best yeast growth? The most product?
4. Do yeast require a nitrogen source to grow?
5. How did air affect yeast growth? Product (CO2) production?
6. In 1861, Pasteur wrote that, in the absence of oxygen, yeast were obtaining their
oxygen from the sugar molecules. Is this true?

Activity 2:
Biotechnology Across The Curriculum
The purpose of these activities is to encourage students to integrate their new knowledge
and not keep it compartmentalize it into such things as "this is science," "this is history,"
and "this is math."

Work With A Social Science Teacher

Ask the students to read about and discuss the histories of Palestine and Israel during this
century. Have them list similarities and differences between Israel and the United States.
Encyclopedias are good sources of information on:
Balfour
Israel
Oslo Agreement (Time Magazine Sept. 13, 1993, p. 50)
Palestine
Zionism

Work With An English Teacher

Young people need to know that it is important to get training in something that can
provide a job and a career. They often don't realize that people in the news started out
with career goals that had little to do with their current notoriety. Moreover, young people
see many successful people in sports and music who did not complete their education.
Since most of us don't possess or develop that level of skill, we need to look for role
models who have successful, productive lives as a result of their education. For example,
astronaut Sally Ride earned a Ph.D. in physics and African-American astronaut Mae
Jemison graduated from medical school. My students who are aspiring writers are
surprised to learn that Michael Crichton completed medical school and John Grisholm,
law school. My aspiring politicians are surprised to learn that President Jimmy Carter was

a nuclear engineer; President Woodrow Wilson was a historian and president of Princeton
University; and Supreme Court Justice Oliver Wendell Holmes studied medicine and was
dean of the Harvard Medical School. And, quarterback Steve Young completed law
school. Ask students to look up famous people, current and historical, to see their
education and training.
The biography section of the annual Britannica Book of the Year is a good source of
information.

Activity 3: Industrial Fermentation Objective

This experiment is a more technical approach to fermentation. It deals with some of the
questions that must be answered before a product can be produced for sale. Large
volumes of cultures are needed for industrial fermentations but you can't go from a petri
plate directly to a bioreactor. The inoculum for the bioreactor must be built up to the
correct number of cells and correct stage of growth of the cells. In industry, considerable
time and money is spent on this stage of preparing the cells for production.

Time
Bacto Dextrose Broth (Difco) can be obtained from Carolina, Ward's and Fisher.
Dextrose broth contains 1.3% protein and 0.5% glucose. Culture media must be sterilized
in a pressure cooker or autoclave at 121C at 15 psi for 15 minutes. Cotton or foam plugs
should be used to stopper the tubes and flasks.
First day: 30 minutes
Daily readings: 15 minutes

Materials
Students can work in groups of 2-4 students; each group should have:
one tube or flask of culture media.
A 24-hour broth culture of bacteria; Escherichia coli or Bacillus sp.
Sterile test tube containing 5 ml nutrient broth
Sterile 125-ml flask containing 25 ml broth
Sterile 125-ml flask containing 50 ml broth
Sterile 250-ml flask containing 100 ml broth
Sterile 250-ml flask containing 100 ml broth with a magnetic stir bar
pH paper or pH meter
Sterile, one-piece plastic droppers

Procedure

1. Inoculate the nutrient broth tube with approximately 0.5 ml of bacterial culture.
Inoculate the each of the nutrient broth flasks with 5 ml of bacterial culture.
2. Record the starting pH and (optional) turbidity of the culture vessel.
a. If you use pH paper: aseptically remove a few drops of the culture medium and place
on the pH paper. If you use a pH meter: place 1 ml of the culture medium in a small
beaker and add enough 5 - 10 ml distilled water.
b. Turbidity Scale
You can measure the amount of bacterial growth using the turbidity scale. Hold each
culture container against the scale and record the lowest value you can see clearly
through the broth.
10

20

30

40

50

60

70

80

90

100

Safety: Discard the used culture and glassware in disinfectant. Wash the pH probe with
alcohol and distilled water.
3. Incubate each nutrient broth. Growth will be faster at 35C, however room temperature
can be used. Incubate the flask with the stir bar on a magnetic stirrer. This flask will be
aerated.
4. Record the pH and turbidity of each broth every day for a week.
5. Make a graph showing the relation between time (on the x-axis) and pH and turbidity
(on the y-axis).

Questions
1. Which container produced the best growth?
2. Why did the pH of the culture medium change?
3. What factors affected the growth?
4. If your bacterium makes the desired product at pH 7.0, what would you do to maintain
this pH?
5. If you need to grow 10,000 liters of bacteria to get enough product to sell, can you
inoculate a 10,000-bioreactor from a test tube of bacteria? Briefly explain your answer.
6. Some of the bacterial products listed in Tables 1 and 2 are injected into people to treat
diseases. Why doesn't the injection cause an infection?

Answers to questions for Activity 1

1. Carbon dioxide produced by the yeast

2. The balloon trapped the CO2, as a way to measure product produced, and kept air (O2)
out of the bottle.
3. Yeast growth can be determined by looking at the cloudiness of the broth and the
amount of sediment (yeast) that collects in the bottom of the bottle. The amount of
product can be determined by looking at the size of the balloon and/or by determining the
specific gravity.
4. The yeast do require nitrogen and should grow better with some protein in the broth.
5. Yeast grow better in the presence of oxygen because they can completely oxidize the
sugar. Complete oxidation provides more energy from the sugar molecules. The yeast
should not ferment the sugar in the presence of oxygen.
6. This is not true. The yeast were using an organic molecule (acetaldehyde) as the final
electron acceptor instead of molecular oxygen.

Answers to questions for Activity 3

1. The aerated flask should grow best.
2. As the bacteria grew they started using the sugar for fermentation creating acids. Later,
they used the proteins which resulted in alkaline products (e.g., ammonia).
3. Growth is affected by many factors including nutrients, temperature, oxygen, pH, and
growth itself. In this experiment the bacteria changed the pH of their environment. 4. The
pH needs to be adjusted by addition of acids or bases
. 5.A small inoculum in a large volume of medium will take too long to grow therefore
the cell numbers need to be increased step-wise. Cells are first grown in a test tube, then
the 5-ml from the tube will be used to inoculate a 100-ml broth. This is continued until a
large enough inoculum is grown.
6. The products must be removed from the cells and culture media and chemically
purified. One common method of purification is chromatography. (See the
Chromatography Science Seminar).