Fermentation with Yeast Background

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The University of Oklahoma *

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1124

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Biology

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Feb 20, 2024

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Biology 1124 2024 edition Background Bakers yeast ( Saccharomyces cerevisiae ) is a eukaryotic single celled fungus that undergoes cellular respiration to produce ATP. Sugars are the primary food source of yeast cells and yeast are able to metabolize some sugars, but not others. In order for cells to make use of a food source, the cell must be able to transport the food across its membrane and produce enzymes capable of breaking the food’s bonds in a useful way. For millennia humans have used yeast to metabolize sugars in grain to make bread, and in fruit juice to make wine. In addition to these roles, yeast have recently taken on an increased importance in the production of ethanol based bio- fuels. Yeast digest the sugars in corn starch to produce ethanol that is blended with gasoline for use in automobiles. Ongoing research in the areas of bio-fuel and beverage production are hoping to determine if other sugars can be efficiently fermented by yeast. Yeast can metabolize sugar in two ways, aerobically, with the aid of oxygen, or anaerobically, without oxygen. In both cases, carbon dioxide, CO 2 , is produced. Measuring the rate at which this gas is produced will allow you to determine the metabolic rate of yeast. During this investigation you will design an experiment to address the question “why do yeast cells metabolize some sugars faster than others?” Aerobic and anaerobic respiration All organisms require energy to function. This energy is derived from reactions that break the bonds in carbohydrates to release ATP. When oxygen is available, organisms use the process of aerobic cellular respiration to break down glucose to carbon dioxide and water in a long series of steps, producing about 36 ATP molecules in the process. When oxygen is not present it is still possible to extract some Fermentation and Metabolism 1 By the end of this lab you should be able to: 1. Compare and contrast two types of respiration. 2. Explain how CO2 production can be used to measure yeast metabolism. 3. Describe the difference between monomers and polymers. 4. Explain why we are using 0.3M solutions of monosaccharides, and 0.15M solutions of disaccharides. 5. Perform an experiment to determine which sugar source is metabolized best. 6. Communicate your findings with a Lab Summary. CELLULAR RESPIRATION AND METABOLISM Why do yeast cells metabolize some sugars faster than others?

Biology 1124 2024 edition energy from the breakdown of glucose, but the process is much less efficient, producing only 2 ATP molecules that can be used to perform work in cells. In this case, only the fi r s t s t a g e o f c e l l u l a r respiration, called glycolysis, is able to function. In glycolysis, each molecule of glucose, a 6-carbon sugar, produces two molecules of pyruvate, a 3-carbon sugar, along with a net gain of 2 ATP molecules. Fermentation occurs when cells metabolize sugars in the absence of oxygen. This anaerobic respiration allows yeast to perform a modified version of glycolysis, producing NADH and ATP, along with the waste products of carbon dioxide and the alcohol ethanol. Yeast have mitochondria capable of producing ATP by aerobic respiration, but frequently perform alcoholic fermentation even when oxygen is plentiful! The sugar food source used by cells may constitute a single carbohydrate molecule (monosaccharides), or can be made from two or more molecules (disaccharides and polysaccharides). However not all sugar sources are used equally as well, which leads to the question under investigation today: Why do yeast cells metabolize some sugars at a different rate than others? Some polysaccharides can be rather large. Do yeast have the ability to transport these large molecules into their cells? Some carbohydrates are made from monomers other than glucose. Are yeast able to produce the multiple enzymes required to digest a variety of carbohydrate molecules? Your hypothesis should address one of these two components of carbohydrate digestion. What materials are available in lab? It has been established that yeast can metabolize glucose (a monosaccharide) and sucrose (a disaccharide made from glucose and fructose), but scientists are wondering if other types of sugars can be metabolized equally as well. In this investigation you will feed yeast with stock solutions water, 0.3M glucose , 0.15M refined sucrose (table sugar), and 0.3M lactose (see figure on next page). Lactose is found in dairy products and is a disaccharide like sucrose, but is made from one glucose and one galactose molecule. Will yeast be able to metabolize this disaccharide just like any other, providing an additional carbohydrate Fermentation and Metabolism 2

Biology 1124 2024 edition source for the bio-fuel and brewing industries? To determine the rate of yeast cellular respiration, you will be provided a carbon dioxide probe and a 250 mL bottle in which the carbohydrate source and a yeast solution can be mixed. You can use Logger Pro software to keep track of the amount of CO 2 produced during each trial. What unit does the “M” represent? The sugar solutions you will have to work with in lab today are: 0.3 M glucose (monosaccharide) solution, 0.15 M refined sucrose, raw sucrose, and lactose (disaccharides) solutions. You may be wondering why the concentrations of these sugar solutions are different? Isn’t it important to keep sugar concentrations the same in an experiment like this? Yes! The unit “M” represents molarity which is a measure of concentration based on the number of solute molecules in that solution. Remember that a disaccharide is made of two monosaccharide molecules. Using half the number of disaccharide molecules in solution actually makes the total number of monosaccharide molecules equal between the 0.15 M disaccharide and 0.3 M monosaccharide solutions. Fermentation and Metabolism 3 Lactose

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Biology 1124 2024 edition In Lab: Data Collection and Sampling Protocols 1. Connect your Carbon Dioxide (CO 2 ) sensor to the LabQuest box connected to your lab computer. Make sure the switch on the probe is set to the Low setting and that units are displayed as parts per million (PPM) in the Logger Pro software. Let the probe warm up for 2 minutes at which point your probe should read between 300-800 PPM . If you have a CO 2 reading that is a significantly lower or higher PPM before you begin your trial, please alert your TA. Calibration of the CO 2 probe may be needed. 2. Set up your data sampling regime in Logger Pro by clicking on the “clock” icon. a. Collect CO 2 data for 15 minutes with 10 samples per minute. 3. Pipet 5.0 mL of yeast suspension into a 10 mL graduated cylinder, then pour into a 250 mL respiration chamber. Important : The yeast suspension must be removed from the middle of a yeast source that is being stirred by a magnetic stirrer at a constant stirring speed. 4. Pipet 5.0 mL of test solution (water, glucose, sucrose, etc.) into a 10 mL graduated cylinder, then pour the liquid into a 250 mL respiration chamber. 5. Swirl the test solution-yeast mixture for five seconds to ensure thorough mixing. 6. Using a gentle twisting motion, quickly place the shaft of the CO 2 Gas Sensor into the opening of the respiration chamber. 7. Start data collection. ( PRO TIP: Logger Pro will continue collecting data even if you switch applications to Word allowing you to write your Lab Summary while data is being collected!) a. Data collection will stop automatically after 15 minutes. b. Choose “Store Latest Run” from the Experiment menu to collect more data using the same sampling regime. c. Rename each trial by double clicking on title of the data collection table in the Logger Pro window. You can give each trial a unique name (i.e. “Glucose trial 1”) so that data is easier to keep organized for analysis. 8. When data collection has finished, remove the CO 2 Gas Sensor from the respiration chamber. Rinse the respiration chamber with water and then empty it. Make sure that all traces yeast have been washed out. 9. During the initial minutes of the data-collection period, the sugar was transported across the membrane of the yeast cells where enzymes began catalyzing the metabolism of glucose, which produces CO 2 gas. This process takes time to happen. Fermentation and Metabolism 4

Biology 1124 2024 edition Accordingly, the first five minutes of the data-collection period will not be used to determine the respiration rate. 10.Click and drag over the 5-15 minute portion of your graph so that the first 5 minutes of the trials are excluded. Use the linear fit button in the Logger Pro toolbar to perform a linear fit on the 5–15 minute portion of the graph. Record the slope of the line, m , as the respiration rate (in ppm/min) in the table below. 11. Repeat steps 3-10 for each additional test solution trial. You will complete 4 trials total. When you are completely finished with all of your data collection, wash the chamber with soap and water, do a final rinse with DI water, and place the chamber on a drying rack. Data Collection Create a table similar to this one to keep track of your team data. Independent variable Condition type Rates of CO2 production Fermentation and Metabolism 5

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