Fermentation by Yeast

Purpose

To observe the effect on fermentation of various factors such as temperature, pH, and concentration of the reactant sugar.

Safety

1. Wear protective goggles throughout the laboratory.

Procedure 1 (Effect of Different Concentrations of Sugar on Fermentation Rate)

1. Obtain three 125-ml Erlenmeyer flasks and label them 1,2, and 3.

2. Add to each of the three flasks 2.5 g of yeast, accurately weighed.

3. Weigh accurately 1.0 g of sugar for flask #2 and 2.5 g of sugar for flask #3.

4. Prepare 350 ml of distilled water, heated to 37°C. Add 100 ml of the water to each of the three flasks. Swirl the contents gently to mix.

5. Attach a deflated spherical balloon over the mouth of each flask. Secure the balloon with both a rubber band and masking tape that seals the balloon's opening to the neck of the flask.

6. Wrap the flasks with insulating material provided by your teacher (alternative is to place in a Styrofoam container or incubator set to 37°C).

7. After 30 minutes, measure the circumference of each balloon, using a piece of string, a narrow ribbon, or a paper strip that can be marked.

8. Repeat step # 7 after 60 and 90 minutes from the start of the experiment.

9. Record the data in a properly labeled data table.

Procedure 2 (Effect of Temperature on Fermentation Rate)

1. Set up three new flasks labeled 4, 5, and 6.

2. To each flask, add 2.5 g of sugar and 2.5 g of yeast.

3. Prepare water for three different temperatures: 5°C, room temperature, and 37°C. Add 100 ml of cold water (5°C) to flask #4, 100 ml of room temperature water to flask #5, and 100 ml of 37°C water to flask #6. Gently swirl the contents of the flask to mix.

4. Attach a deflated balloon to the mouth of each flask and secure with a rubber band and masking tape.

5. To maintain the various temperatures of the individual flasks, place each in a water bath of the proper temperature using ice or water of the desired temperature.

6. After 30 minutes, measure the circumference of each balloon. Repeat the measurements at 60 and 90 minutes.

Procedure 3 (Effect of pH on Fermentation Rate)

1. Set up three flasks labeled 7, 8, or 9.

2. To each flask, add 2.5 g of sugar.

3. Prepare 350 ml of water at a temperature of 37°C. Divide the water into three equal volumes and place in separate 150-ml beakers labeled either 7, 8, or 9.

4. To establish a pH of 2 in beaker #7, add 3 drops of Universal indicator (or use pH paper to test), then add 0.1 M acetic acid solution, drop by drop from a Beral pipet while stirring until you reach a pH of 2 as indicated by the color of the indicator (or pH paper).

5. To establish a pH of 6 in beaker #8, add 3 drops of universal indicator (or use pH paper). Then add 0.1M acetic acid solution, drop by drop while stirring, until you reach a pH of 6 as indicated by the color of the solution (or the pH paper).

6. To establish a pH of 11 in the water of beaker #9, add 3 drops of Universal indicator (or use pH paper), then add solid sodium carbonate, Na₂CO₃, in small amounts (size of a match head) using a spatula. Stir to dissolve, noting color of solution. Continue to add the sodium carbonate until a pH of 11 is reached.

7. Add 2.5 g of yeast to each flask.

8. Check the temperatures of the three different solutions and reheat if necessary before measuring out 100 ml of each solution and adding to the individual flasks.

9. Swirl the contents of the flasks to mix.

10. Attach a deflated balloon to each flask and seal with a rubber band and masking tape.

11. Place the flasks in a water bath or incubator maintained at 37°C.

12. After 30 minutes, measure the circumference of each balloon. Repeat the measurements at 60 and 90 minutes.

13. At the end of the experiment, measure the pH again with pH paper or note the color of the universal indicator in the solutions.

Data Analysis

1. For each variable used, determine from your circumference measurements which variable had the greatest affect on the activity of the yeast. (What gas is responsible for inflating the balloons?)

2. How would you test for the effect of combining all the variables in one flask? What would be the setup for your control? Would there be more than one control?

3. What do you predict would happen if the temperature in the flask is increased beyond 37°C? What is special about the temperature of 37°C? Why not 30°C or 40°C?

4. Was there any change in pH of the solutions as measured with pH paper or the change in the color of the universal indicator. If so, how would you explain such a change?

5. How could you verify your explanation in #4 through experimentation? What chemical changes are taking place in the fermentation? What new substances are produced? How can you chemically verify their presence? How could you chemically determine if they have an effect on the pH of a solution?

6. Ethyl alcohol is a product of fermentation. Why do you think the fermentation process cannot produce a concentration of alcohol beyond 12%?