Instructor OVERVIEW Sugar Metabolism with Yeast (Method 2 Ethanol Sensor) Yeast can metabolize sugars in two ways, aerobically, with the aid of oxygen, or anaerobically, without oxygen. In both cases, carbon dioxide, CO 2, is produced. The rate that CO 2 is produced is referred to as the rate of respiration. If sugars are readily available, baker s yeast (Saccharomyces cerevisiae) prefers to metabolize glucose and other sugars anaerobically through fermentation, producing ethanol in addition to CO 2. This is commonly referred to as the Crabtree effect. 1 Figure 1 In the Preliminary Activity, your students will use an Ethanol Sensor to determine the rate of fermentation of glucose by yeast. A student handout for the Open Inquiry version of the Preliminary Activity can be found at the end of this experiment During the subsequent Inquiry Process, your students will first find out more about sugars, fermentation, aerobic and anaerobic respiration, and yeast using the course textbook, other available books, and the Internet. They will then generate and investigate researchable questions dealing with the fermentation of sugars by yeast. 1 Fraenkel, D.G. Yeast Intermediate Metabolism, 1 st ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2011. Vernier Software & Technology 1
LEARNING OUTCOMES In this inquiry experiment, students will Identify variables, design and perform the experiment, collect data, analyze data, draw a conclusion, and formulate a knowledge claim based on evidence from the experiment. Obtain graphic representations of fermentation rate. Determine fermentation rate by yeast while using different sugars. Determine which sugars can be used as a food source by yeast. THE INQUIRY PROCESS Suggested Time to Complete the Investigation Inquiry Phase Open Inquiry Guided Inquiry I Preliminary Activity 35 minutes 35 minutes II Generating Researchable Questions (Omitted in Guided Inquiry Approach) 15 minutes 0 minutes III Planning 10 minutes 10 minutes IV Carrying Out the Plan 50 minutes 50 minutes V Organizing the Data 10 minutes 10 minutes VI Communicating the Results 10 minutes 10 minutes VII Conclusion 5 minutes 5 minutes MATERIALS Make the following materials available for student use Vernier data-collection interface yeast suspension Vernier data-collection program magnetic stir bar Vernier Ethanol Sensor magnetic stir plate plumber s tape Beral pipettes #6 split stopper four 15 ml conical tubes 250 ml fermentation chamber 0.3 M lactase enzyme solution 0.3 M glucose, fructose, and galactose sugar solutions 0.15 M glucose, sucrose, maltose, and lactose sugar solutions 2
I. Preliminary Activity This inquiry begins with an activity to reinforce prior knowledge of the use of Vernier data-collection technology and to introduce a method for collecting fermentation rate data. Sample Results Answers to the Questions Figure 2 Fermentation of glucose by yeast 1. Perform a linear fit on the graph. Record the slope of the line, m, as the fermentation rate (in ppm/min). Answers will vary. The fermentation rate determined in the Sample Results above is 135.7 ppm/min. 2. List three common sugars, other than glucose. Answers will vary. Fructose (fruit sugar or levulose) and galactose are two common monosaccharides. Sucrose (table sugar, contains a glucose and a fructose unit), maltose (malt sugar, contains two glucose units), and lactose (milk sugar, contains a glucose and a galactose unit) are three common disaccharides. 3. List three factors that could possibly affect fermentation rates of sugars by yeast. Answers will vary. Some factors are ph, yeast strain, yeast concentration, sugar used, sugar concentration, and number of sugar monomer units per molecule. 4. List at least one researchable question concerning the fermentation of sugars by yeast. Answers will vary. See the Researchable Questions list below for some possible answers. II Generating Researchable Questions Note: Researchable questions are assigned by the instructor in the Guided Inquiry approach. See page xiii in the Doing Inquiry Experiments section of Investigating Biology Through Inquiry for a list of suggestions for generating researchable questions. Some possible researchable questions for this experiment are listed below: Recommended for Open Inquiry or Guided Inquiry (sample results provided) How do the fermentation rates of various monosaccharides compare? How do the fermentation rates of various disaccharides compare? 3
How do the fermentation rates of monosaccharides and disaccharides compare? Which sugars can be fermented by yeast? How does the fermentation rate of lactose solution containing lactase compare to the fermentation rate of a lactose solution containing no lactase? Recommended for Open Inquiry or Guided Inquiry (no sample results provided) How does yeast concentration affect fermentation rate? How does boiling the yeast affect fermentation rate? How does salinity affect the fermentation rate of glucose by yeast? How does the fermentation rate of 0.15 M sucrose (containing a glucose and a fructose unit) compare to that of a solution 0.15 M in glucose and fructose? How does the fermentation rate of 0.15 M lactose (containing a glucose and a galactose unit) compare to that of a solution 0.15 M in glucose and galactose? How does the fermentation rate of 0.15 M maltose (containing two glucose units per molecule) compare to that of 0.30 M glucose? Recommended for Advanced Students (no sample results provided) How does ph affect the rate of fermentation of glucose by yeast? Can yeast metabolize artificial sweeteners? How does sugar concentration affect fermentation rate? How does the previous exposure of yeast to galactose affect galactose fermentation rate? How does the previous exposure of yeast to sucrose affect sucrose fermentation rate? Students should choose a researchable question that addresses the learning outcomes of your specific standards. Be sure to emphasize experimental control and variables. (Instructors using the Guided Inquiry approach select the researchable questions to be investigated by their students. We encourage you to assign multiple researchable questions because this strategy enhances student interaction and learning during phases IV VII.) III Planning During this phase students should formulate a hypothesis, determine the experimental design and setup, and write a method they will use to collect data. The plan should list laboratory safety concerns and specify how they will be addressed during the investigation. Circulate among the student groups asking questions and making helpful suggestions. IV Carrying Out the Plan During this phase, students use their plan to carry out the experiment and collect data. Circulate among the student groups asking questions and making helpful suggestions. V Organizing the Data Student groups or individuals should organize their data using graphs, tables, and/or charts that appropriately communicate the outcome of their experiment. They can prepare these using Logger Pro, LabQuest, Graphical Analysis TM for ipad, PowerPoint, or other method. 4
VI Communicating the Results During this important and exciting phase of the inquiry process, student groups present their research results using oral presentations, poster presentations, or journal club formats. Interaction among groups researching different questions leads to a better understanding of the process of fermentation. VII Conclusion Using your notes recorded during the Communicating the Results phase, summarize the group results for the experiment and tell how they will fit into the upcoming instruction. VIII Assessment Scientific inquiry assessment may take on many forms. SAMPLE RESULTS Student results will vary depending on experimental design. Comparing Sugar Fermentation Rates Table 1: Monosaccharide Fermentation Rates Sugar Molecular formula Respiration rate (ppm/min) fructose C 6 H 12 O 6 126.4 glucose C 6 H 12 O 6 137.1 galactose C 6 H 12 O 6 11.91 water (control) H 2 O 13.54 Figure 3 Fermentation rates of monosaccharides This investigation addresses the question, How do the fermentation rates of various monosaccharides compare? In this investigation, the fermentation rates of 0.30 M solutions of 5
the monosaccharides listed were determined using the Preliminary Activity procedure. The yeast suspension, made using Fleischmann s Active Dry Yeast, was maintained at room temperature after activation and prior to use. Yeast cannot utilize all monosaccharides equally well. Fructose and glucose gave the largest fermentation rates at 126.4 and137.1 ppm/min, respectively. Galactose, in contrast, is apparently used to a minimal extent by yeast under the conditions of this investigation. Sugar Table 2: Disaccharide Fermentation Rates Molecular formula Fermentation rate (ppm/min) sucrose C 12 H 22 O 11 55.98 maltose C 12 H 22 O 11 43.00 lactose C 12 H 22 O 11 14.00 water (control) H 2 O 7.76 Figure 4 Fermentation rates of disaccharides This investigation addresses the question, How do the fermentation rates of various disaccharides compare? In this investigation, the fermentation rates of 0.15 M solutions of the disaccharides listed were determined using the Preliminary Activity procedure. The yeast suspension, made using Fleischmann s Active Dry Yeast, was maintained at room temperature after activation and prior to use. Yeast apparently cannot utilize all disaccharides equally well. Sucrose and maltose gave the largest fermentation rates at 55.98 and 43.00 ppm/min, respectively. Lactose, in contrast, is apparently used to a negligible extent by yeast under the conditions of this investigation. 6
Table 3: Fermentation Rates of Lactose With and Without Lactase Sugar Fermentation rate (ppm/min) lactose 14.00 lactose with lactase enzyme 57.99 water (control) 7.76 Figure 5 Fermentation rates of lactose with and without lactase This investigation addresses the question, How does the fermentation rate of lactose solution containing lactase compare to the fermentation rate of a lactose solution containing no lactase? In this investigation, the fermentation rates of 0.15 M solutions of lactose and lactose in the presence of lactase enzymes were determined using the Preliminary Activity procedure. The yeast suspension, made using Fleischmann s Active Dry Yeast, was maintained at room temperature after activation and prior to use. Yeast apparently cannot utilize lactose without first breaking it down with the lactase enzyme. Lactose on its own had a fermentation rate of 14.00 ppm/min, while lactose in the presence of lactase had a fermentation rate of 57.99 ppm/min. TIPS 1. To prepare the yeast suspension, dissolve 7 g (1 package) of dried yeast for every 120 ml of distilled water. Incubate the suspension in 37 40 C water for at least 10 minutes. After activation, the yeast suspension can be maintained at room temperature. For best results, prepare the yeast suspension at least one hour before use. The yeast suspension can also be prepared 24 hrs before use. Prepare the suspension as directed above and then add 5 g of glucose to the solution. Place the yeast suspension in a beaker and place on a magnetic stirrer. Maintain the suspension at a constant stirring speed overnight. 7
2. To ensure consistent yeast suspension concentration, make the yeast suspension available in a beaker situated on a magnetic stirrer. Maintain a constant stirring speed as students remove portions. Important: The yeast portions must be removed from the middle of the stirred yeast source. 3. The normal operating temperature range of the Ethanol Sensor is 20 30 C. All of the Sample Results were determined at room temperature. The Ethanol Sensor does not have temperature compensation built in so all experiments should be done at the same temperature. 4. If suitable pipets and pipet bulbs are not available, graduated Beral pipets can be substituted. 5. The stopper included with the Ethanol Sensor is slit to allow easy application and removal from the probe. Remove the stopper from the sensor sideways by pulling it off through the slit, not by sliding the stopper off the bottom. Doing the latter will result in the tip coming off and potentially being lost. When students are placing the probe in the fermentation chamber, they should gently twist the stopper into the chamber opening. 6. The Ethanol Sensor relies on the diffusion of gases into the probe shaft. Students should allow a couple of minutes between trials so that gases from the previous trial will have exited the probe shaft. 7. Water can be removed from and added to a water bath with a 20 ml syringe or a small dipper. 8. Preparation of solutions (prepare all solutions in distilled water): 0.30 M glucose requires 5.40 g of anhydrous glucose, C 6 H 12 O 6, per 100 ml of solution. Alternatively, 5.95 g of glucose monohydrate, C 6 H 12 O 6 H 2 O, per 100 ml of solution. 0.30 M fructose requires 5.40 g of fructose, C 6 H 12 O 6, per 100 ml of solution. 0.30 M galactose requirees 5.40 g of galactose, C 6 H 12 O 6, per 100 ml of solution. 0.15 M glucose requires 2.70 g of anhydrous glucose, C 6 H 12 O 6, per 100 ml of solution. Alternatively, 2.97 g of glucose monohydrate, C 6 H 12 O 6 H 2 O, per 100 ml of solution. 0.15 M lactose requires 5.40 g of lactose monohydrate, C 12 H 22 O 11 H 2 O, per 100 ml of solution. 0.15 M maltose requires 5.40 g of maltose monohydrate, C 12 H 22 O 11 H 2 O, per 100 ml of solution. 0.15 M sucrose requires 5.13 g of sucrose, C 12 H 22 O 11, per 100 ml of solution. 9. More information about the sensor used in this Investigation, as well as tips for optimal performance, can be found in the sensor s user manuals available for download from the Vernier web site, www.vernier.com/sensors. 10. The plans that your students submit for approval should list laboratory safety concerns, including chemical safety concerns, and specify how they will address these safety concerns during their investigations. 8