Fermentation Lab
Lab Topic 6
Alcoholic Fermentation
Written by Nickie Cauthen, Clayton State University Morrow, GA
Objectives
1. Define the terms in bold type.
2. Understand the process of alcoholic fermentation.
3. Determine the independent and dependent variables in the experiment.
4. Understand why CO2 is measured in alcoholic fermentation
5. Understand why and how the rate of alcoholic fermentation can be altered.
6. Interpret the data generated in the experiment.
Introduction
Organisms are open systems allowing them to take in components from the environment and convert them to a form of energy that can be used by the cell.
Many organisms undergo a process called cellular respiration to produce energy in the form of adenosine triphosphate, or ATP. ATP is then used to power most cellular activities. The following general formula describes this process:
C6H12O6 + 6O2 6CO2 + 6H2O + ATP
Glucose is broken down in a series of chemical reactions that release small, manageable amounts of energy. This allows the cells to capture the energy more efficiently than if the glucose were broken down in one single reaction. Glucose is broken down in a series of chemical reactions that begin in the cytoplasm of the cell.
The reactants are then transferred to the mitochondria where the process is continued. The process that occurs in the cytoplasm of the cell is called glycolysis and does not require oxygen, meaning it is an anaerobic process. This portion of the process makes 2 net ATP molecules per molecule of glucose. In the mitochondria of the eukaryotic cell, oxygen is employed to more efficiently harness the energy stored in the glucose molecules. The use of oxygen, an aerobic process, allows the cell to make a total of 34-38 ATP molecules per molecule of glucose.
When oxygen is available, cells tend to produce ATP in the mitochondria since this process produces more ATP than glycolysis alone. Some organisms, like yeast and the muscle cells of animals, have the ability to continue to produce ATP when oxygen is no longer available by a process called fermentation. During this process in yeast, the glucose is broken down by glycolysis in the cytoplasm and 2 net ATP, 2 NADH, and 2 molecules of pyruvate are produced. Since oxygen is not present, pyruvate, the product that results at the end of glycolysis, is converted to ethanol and CO2. The NADH is converted to NAD+ and H+ and the ATP is used for cellular work. The general formula below shows the conversion of glucose to 2 molecules of pyruvate, 2 molecules of NADH, and 2 net ATP in glycolysis. In yeast cells when oxygen is absent, fermentation occurs to convert the 2 molecules of pyruvate (produced in glycolysis) to 2 molecules of ethanol and 2 molecules of CO2 and to convert the 2 molecules NADH (also produced in glycolysis) to 2 molecules of NAD+ and 2H+.
C6H12O6 2 C3H4O3 + 2 net ATP + 2 NADH 2 C3H5OH + 2CO2 + 2 NAD+ + 2 H+
(glucose) (pyruvate) (ethanol)
Glycolysis Fermentation
Fermentation allows NADH that is produced during glycolysis to be recycled back to NAD+, providing NAD+ for the additional rounds of glycolysis. This process, called alcoholic fermentation, allows a cell to continue to produce ATP and meet its energy needs when oxygen is no longer available.
In today’s lab we will investigate alcoholic fermentation in yeast. Certain types of yeast are economically important and pay roles in the food and beverage industries. For example, yeast uses alcoholic fermentation to convert sugars found in barley to ethanol to make beer. The CO2 that is produced when the yeast converts glucose to ATP makes yeast breads rise. You will be studying variables that affect the rate of alcoholic fermentation in yeast.
Exercise 6.1
Alcoholic Fermentation
In this laboratory exercise you will set up conditions that are favorable for alcoholic fermentation in yeast and test the effect of the concentration of yeast on the rate of alcoholic fermentation. You will measure the rate of alcoholic fermentation by collecting the CO2 that is produced by the yeast as a byproduct of alcoholic fermentation. The more CO2 that is produced the faster the rate of alcoholic fermentation.
Hypothesis: Write a hypothesis that describes the effect of using different sugar sources on alcoholic fermentation. Write this hypothesis on your report sheet (number 1).
Procedure:
1. After running water from a faucet for 1-2 minutes, fill the pot or baking dish with 2 inches of hot tap water. The water should be approximately 50°C. Use a thermometer to monitor the temperature throughout the experiment. Change the water out every half hour to keep it warm.
2. Yeast solution – Add 1 packet of baker’s yeast to 1 cup of warm water. Stir for at least one minute. Place in the warm water bath.
3. In a separate cup, add 1/3 cup of light corn syrup and 2/3 cup of warm water. Stir for at least one minute and place in warm water bath.
4. In another cup, add 3 teaspoons of table sugar to 1 cup of warm water. Stir for at least one minute and place in warm water bath.
5. In another cup, add 3 teaspoons of artificial sweetener to 1 cup of warm water. Stir for at least one minute and place in warm water bath.
6. Label each empty plastic water bottle with a different sugar source.
7. Add 1/3 cup of the sugar source to each respective bottle.
8. Add1/3 cup of yeast solution to each bottle. Cap and invert several times to mix.
9. Identify negative control – Fill a fourth water bottle with the appropriate substances to make a negative control experiment.
10. Uncap all the water bottles and stretch a balloon over the top. Make sure the balloon is mostly deflated.
11. Place all water bottles in the warm water bath. This is time zero (0 minutes) for all samples; record 0 cm for Bottles 1, 2, 3 in the 0 minutes column in Table 6.3 on the report sheet.
12. If fermentation occurs in the water bottle, CO2 will be released, and it will inflate the balloon. To ensure accurate results, confirm that the balloon fits tightly and monitor the temperature of the water bath. Temperatures between 35 and 45°C are sufficient. If the temperature dips, gradually replace water in the bath with hot water from the tap.
13. At 10-minute intervals measure the inflation of each balloon by wrapping a piece of string around it, making a mark where the string overlaps with the beginning of the string and the holding the string next to a ruler. Record the measurement in cm in Table 6.3 on your report sheet. Do this for each of your samples for 60 minutes. Measure from the line that you mark each time a measurement is taken.
Lab 6-1: Background Information and Protocol
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