By: Christine L. Case, Ed.D., Skyline College
-Fermentation by Yeast-
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.
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 121°C at 15 psi for 15 minutes.
3-7 days later: After growth has occurred-50 minutes.
- Each pair of students should prepare two different bottles of media.
- Bottles (any size) half-filled with the following:
- Sugar-water (1 part table sugar to 4 parts water)
- Starch-water (1:4 mashed potato flakes in water, use the broth)
- Sugar-protein water (1 part commercial beef broth and 1 part sugar-water)
- Protein-water (commercial beef/chicken broth diluted to half strength or commercial glucose-yeast extract broth)
- Balloons to fit mouths of bottles
- 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 sugar-protein water. It is available from Carolina, Ward's, and Fisher.
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?
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:
- 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 __
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?
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:
- Oslo Agreement (Time Magazine Sept. 13, 1993, p. 50)
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.
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 121°C 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
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
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 35°C, 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).
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).
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