Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Use a heavy-duty pin or needle (such as a dissection pin) to make four holes in a diamond pattern around the indentation in the middle of each fermentation tube cap. Use a piece of paper towel or rubber gloves to provide a more secure grip while inserting and removing the pin or needle.
2. Prepare a 0.030 M solution of glucose by adding 5.95 g of glucose to 1000 mL of water. Heat this solution to 35–40 °C and maintain this temperature throughout the lab period.
3. To prepare a 0.030 M fructose solution, dissolve 5.40 g of fructose in 1000 mL of distilled water.
4. To prepare a 0.015 M maltose solution, dissolve 5.40 g of maltose in 1000 mL of distilled water.
5. To prepare a 0.015 M fructose solution, dissolve 5.13 g of fructose in 1000 mL of distilled water.
6. Make two copies of the fermentation tube reader card template and cut around each tube. For long-term use, laminate the tube reader cards.

Safety Precautions

Use caution when handling hot glassware. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. All student-produced procedures must be reviewed by an instructor. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. All leftover solutions from the introductory activity may be rinsed down the drain with excess water according to Flinn Suggested Disposal Method #26b. Extra sugar solutions may be stored in a lab refrigerator for short periods of time. Sugar water is growth media for microbes, so keep refrigerated and bring to room temperature before resuming the lab. For any chemical not specifically listed in the materials, but used in the inquiry portion of the lab, please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply before students begin experimentation.

Lab Hints

• Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. The introductory activity and data compilation can reasonably be completed in one 50-minute class period. The prelaboratory assignment may be completed before coming to lab. The inquiry laboratory activity, including time for designing and performing the experiment, can reasonably be completed in one to two class periods.
• Four types of sugars are included for students to explore sugar type and respiration rate. Salt concentration and pH are other popular variables for inquiry.
• If students want to test the effect of ethanol concentration on respiration rate, non-denatured ethanol must be used. Denatured ethanol, even at low concentrations, will kill the yeast. Consult with risk management and your administration before using non-denatured (drinking) ethanol in your classroom.
• The first ten minutes after combining the glucose suspension and the yeast will usually not show the same respiration rate as the last twenty minutes and this “activation” time may vary between types of yeast.
• The glucose reader cards may be shared between two groups.
• The fermentation tubes and caps may be cleaned and reused. Use alcohol to remove the permanent marker.

Teacher Tips

• This lab integrates two important concepts—artificial selection and respiration. It may be helpful to review these concepts before completing this lab.
• Students can graph their results and determine the line of best fit and slope of that line in order to report a respiration rate in volume of carbon dioxide produced per minute.
• Disaccharides and monosaccharides are processed differently by different yeasts. Enzymes must cleave the disaccharides and convert them to glucose before glycolysis can begin. Students could investigate the differences in chemical structure of different sugars as an extension to integrate structure and function.
• Students could visit a bakery or invite a baker to speak about the characteristics of yeast and how yeast is controlled in the baking process.
• Students could research the history of fermentation as a way to preserve food prior to refrigeration and modern agriculture.
• Evolution of Yeast Using a CO₂ Gas Sensor—Guided-Inquiry Laboratory Kit (Catalog No. FB2128) collects the same type of data using a Vernier CO₂ Gas Sensor.
• Aerobic Respiration and Fermentation Made Easy (Catalog No. FB1750) explores yeast’s ability to carry out aerobic as well as anaerobic respiration and is a good companion experiment.
• Yeast on the Job—Student Laboratory Kit (Catalog No. FB1436) is a good follow-up lab that explores four major life processes— maintaining homeostasis, respiration, enzyme-controlled reactions and reproduction.

Answers to Prelab Questions

1. What are the reactants and products of fermentation?

   Glucose (C₆H₁₂O₆) is the reactant. Carbon dioxide (CO₂) and ethanol (C₂H₅OH) are the products.

2. Define rate of respiration. How will this be determined in the experiment?

   The rate of respiration is the amount of glucose that is converted to carbon dioxide and ethanol in a given amount of time. This experiment measures the amount of carbon dioxide in a closed container and the amount of glucose remaining in the suspension. Carbon dioxide is one of the two products of anaerobic respiration and the amount of carbon dioxide gas is directly correlated to the rate of respiration. The decrease in glucose concentration indicates that glucose is being consumed during the experiment, showing that respiration is occurring.

3. The three strains of yeast used in this experiment have been bred for different purposes. Describe one adaptation that might be found in each of the different strains of yeast.

   Student answers will vary. Make sure they have one reasonable answer for each strain. Baker’s yeast must withstand high temperatures, tolerate doughy substrates, metabolize sugars quickly, and not produce by-products that negatively affect the taste of the product. Brewer’s yeast must metabolize starch that has been steeped from barley malt, tolerate moderate levels of ethanol and substrates with a variety of chemical profiles, and have a moderate metabolic rate. Wine yeasts must be able to metabolize high levels of sugar including fructose from grapes and tolerate high levels of ethanol. They generally are active under cool temperatures over a long period of time.

Sample Data

Answers to Questions

1. Hold a “reader card” next to your fermentation tubes and line up the markings. Compare the marks on the tubes to the “reader card” to determine the volume of CO₂ produced at each time interval and record in the data table above.

   See the sample data table.

2. Create a graph showing the relationship between time and CO₂ gas volume for each yeast type.

   See the sample graph.

3. Determine the average change in CO₂ per minute using the data collected from 10 minutes to 30 minutes for each yeast type. Record under Notes and Observations in the data table.

   See the sample data table.

4. Obtain data from two groups that investigated the type of yeast you did not and data from one group that investigated the same type of yease you did. Record the change in glucose and CO₂ of their second trials below.

   Student answers wil vary.

5. Describe any differences in the respiration rates (volume of carbon dioxide produced per minute) of the different types of yeast.

   Baker's yeast has the highest respiration rate, followed by Brewer's yeast and wine yeast. The glucose level decreased most quickly with the Baker’s and then the Brewer’s yeasts and more slowly with the wine yeast.

6. In the Notes and Observations section of the data table, record any interesting observations as well as any actions that were done that were not in the written procedure.

   See the sample data table.

7. Explain why there may be differences between your data and another group’s data for the same yeast type using these observations.

   Accept all reasonable answers. Individual organisms may vary in their metabolism, just as different humans have different metabolic rates. The method of data collection leads to small losses of solution during the experiment, which may vary slightly. The amount of carbon dioxide that stays in solution may vary as well. The reading of the tube is somewhat imprecise because of the formation of bubbles. Therefore, variation of some degree is normal. Yeast is sensitive to temperature changes, so a small temperature change may impact the results.

8. List factors that could possibly affect the evolution of different strains of yeast.

   Accept all reasonable answers. Environmental (substrate) conditions including temperature, pH, presence of ethanol and presence of salt. Ability to metabolize various types of carbohydrates. This is usually done by enzymes, so the evolution of enzymes with the ability to break down varies types of carbohydrates is very crucial to yeast function.

Teacher Handouts

11317_Teacher1.pdf

References

Black, S., Moore, R., Haugen, H., ed. Biology Labs That Work: The Best of How-To-Do-Its, National Association of Biology Teachers: Reston, VA. 2000, Vol. II. pp. 47–50.

Melville, J.M., Collins, M., and D. Volz. Investigation 22, Evolution of Yeast in Investigating Biology through Inquiry. Vernier Software & Technology, 2012.

Introduction

Just as farmers select the most fit plants and animals to breed, bakers, brewers and vintners select specific strains of the yeast Saccharomyces spp. to produce baked goods and fermented beverages. The specific strains of yeast are all good fermenters, meaning they metabolize glucose into carbon dioxide and ethanol, but each strain has specific characteristics that make it desirable for a particular product such as bread, beer or wine. When the baker, brewer or vintner select a specific strain of yeast, they are participating in artificial selecton, which is defined as the process of selecting individuals with particular characteristics for breeding.

Concepts

• Artificial selection
• Anaerobic respiration
• Fermentation
• Evolution
• Respiration rate

Background

Through both natural and artificial selection, hundreds of varieties of yeast have evolved to have adaptations that help them survive in many different environments. These yeasts vary in inherited traits that allow them to break down specific types of sugars to produce ethanol and carbon dioxide as a result of fermentation. Depending on the other ingredients and conditions for fermentation, yeast varieties may have other beneficial traits, such as a high tolerance for heat, acid, alcohol or salt. For example, the yeast that is used in baking has a high tolerance for heat and salt while the yeast strains used to make beer and wine tolerate acidic environments and high ethanol concentrations.

Alcoholic fermentation is a form of anaerobic respiration, which is yeast’s preferred method of metabolizing glucose. In alcoholic fermentation, the yeast obtain energy for metabolic processes when glucose (C₆H₁₂O₆) is broken down into carbon dioxide (CO₂) and ethanol (C₂H₅OH). See Equation 1.

Baker’s yeast produces carbon dioxide gas, which makes dough rise while the ethanol evaporates during cooking. In beer and wine making, the ethanol stays in solution, while most of the carbon dioxide gas comes out of solution and into the air.

Carbohydrates—including sugars—must be converted to glucose before fermentation can occur. Glucose is the reactant in fermentation (Equation 1). Enzymes facilitate this conversion by reducing the amount of energy required to start the reaction. For example, sucrose is a disaccharide that reacts with water to form glucose and fructose. See Equation 2. An organism must have a set of enzymes to convert different types of carbohydrates into glucose. Each type of enzyme breaks down one type of carbohydrate, as in the example of invertase breaking down sucrose.

When an enzyme called sucrase binds to sucrose, the sucrose molecule changes shape and destabilizes. Once destabilized, the activation energy that is required to start the reaction is reduced. (The adaptation of yeast strains to have different sets of enzymes allows them to use different food sources.) For example, grapes contain three sugars that wine yeast can usually ferment—glucose, fructose and sucrose. Enzymes for converting sucrose and fructose are present in the wine yeast. If this type of yeast was used to brew beer, the results may not be as favorable because one of the prime sugars in beer making is maltose. The adaptation of yeast strains occur through natural selection and artificial selection. Wild yeast strains continue to adapt to environmental conditions while domestic yeast strains adapt to the specific conditions they are exposed to during controlled fermentation.

Experiment Overview

In Part A, a fermentation chamber is assembled and used to determine the rate of respiration for baker’s yeast and for brewer’s or wine yeast. In addition, glucose concentration will be measured at the beginning and end of the experiment to determine the concentration of glucose in the original solution. The yeast varieties will be characterized by their ability to convert glucose into carbon dioxide and ethanol. The production of carbon dioxide will be measured over time, giving the rate of respiration.

In Part B, research, design and perform an experiment to determine how various factors affect respiration rate.

Materials

Prelab Questions

1. What are the reactants and products of fermentation?
2. Define rate of respiration. How will this be determined in the experiment?
3. The three strains of yeast used in this experiment have been bred for different purposes. Describe one adaptation that might be found in each of the different strains of yeast.

Safety Precautions

Use caution when handling hot glassware. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. All student-produced procedures must be reviewed by an instructor. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Part A. Introductory Activity

1. Prepare a water bath by adding 150 mL of water to a 250-mL beaker. Place a thermometer in the beaker and heat the beaker on a hot plate until the water is 40 °C. Use heat-resistant gloves or tongs to remove the beaker and place it on a ceramic fiber square. Monitor the temperature throughout the experiment. If the temperature drops to 30 °C, place the beaker back on the hot plate until it reaches 40 °C again and then remove it. You may need to do this a few times.
2. Add about 10 mL of glucose solution to each of the two fermentation tubes.
3. Dip a separate glucose test strip into each solution and then remove the strips. Wait 30 seconds, then compare the color of the test strips with the color chart provided. Based on the color, record the glucose concentrations in the data table on the Evolution of Yeast Worksheet. These values are the initial glucose concentrations. At this time, also record the temperature of the water bath.
4. Add 0.4 g of baker’s yeast to the first fermentation tube and 0.4 g of either brewer’s or wine yeast to the second fermentation tube. Cap both tubes. Cover the holes in both lids and gently shake to thoroughly mix the yeast.
5. Remove the caps and add glucose solution until there is a liquid bubble above the lip of each tube. Doing so eliminates air bubbles from the tubes.
6. Cap the tubes. It is typical for a few drops of the mixture to come out through the holes.
7. Turn the tubes upside down and place them in the water bath (see Figure 1). Begin timing.
   {11317_Procedure_Figure_1}
8. After 5 minutes, carefully lift each tube and use a dark permanent marker to mark the top of the liquid in both fermentation tubes. Make the marks where the liquid ends and the foam begins. Record the temperature in the data table. Note: Keep the tubes upside down while marking.
9. Continue to mark the tubes and record the temperature every five minutes for 30 minutes for both tubes. Remember to keep the temperature of the water bath between 30–40 °C.
10. After 30 minutes, remove the tubes from the beaker, uncap and dip a glucose test strip into each of the suspensions. Wait 30 seconds, then compare the color of the test strips with the color chart provided. Based on the color, record the glucose concentrations in the data table as the final glucose concentrations. Calculate the change in glucose concentrations and record these values under Notes and Observations in the data table.
11. Complete the questions on the Evolution of Yeast Worksheet before continuing to Part B.

Part B. Guided-Inquiry Design

1. Consider the following questions while reflecting upon your knowledge of respiration and yeast.
   1. How might changing a variable in this experiment, such as the type of sugar, temperature or pH, affect the rate of respiration?
   2. What are the environmental conditions the yeast encounter when used to make bread? How are these conditions different from those encountered when making food or beverages?
   3. What are the differences among the types of sugar?
   4. What is the optimal temperature for yeast?
2. Choose a variable to explore and design an experiment to measure the affects this variable has on the respiration rate of yeast. Follow the steps below to create your experiment.
   1. Develop a testable hypothesis.
   2. Discuss and design a controlled experiment to test the hypothesis. Include a detailed procedure based on the introductory activity that collects data to test your hypothesis.
   3. List any safety concerns or precautions that will be taken to protect yourself, your classmates and your instructor during the experiment. Make changes to increase protection and decrease risk.
   4. Determine how you will collect and record raw data.
   5. Determine how you will analyze the data to test your hypothesis.
   6. Review your hypothesis, safety precautions, procedure, data tables and proposed analysis with your instructor prior to beginning your experiment.
   7. Run your experiment. Once the experiment and analysis are complete, evaluate whether the experimental evidence supports, refutes or provides no information concerning the hypothesis.
   8. Make suggestions for revisions to the experiment or hypothesis.
3. Consult your instructor for proper disposal of materials used in both the introductory and inquiry portions of the lab.

Student Worksheet PDF

11317_Student1.pdf