Hillsborough Community College - Ybor City Campus 1025C Laboratory Exercise 6: The Process of Fermentation Introduction

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1 Hillsborough Community College - Ybor City Campus 1025C Laboratory Exercise 6: The Process of Fermentation Introduction Cellular Respiration Cellular respiration is the release of energy from carbohydrates and other molecules. Energy from these reactions is used to synthesize ATP molecules which are used to energize a large variety of cellular reactions. Aerobic cellular respiration requires oxygen (O2) and involves the complete breakdown of glucose to produce carbon dioxide (CO2) and water (H2O). With the exception of the process of glycolysis, aerobic cellular respiration occurs within cell organelles called mitochondria. Figure 1. Aerobic Cellular Respiration Glycolysis Glycolysis is the starting point of cellular respiration and occurs in the cytosol of the cell. It is the breakdown of glucose to two pyruvate (pyruvic acid) molecules with the net production of two ATP molecules. Glycolysis is universally found in all organisms and likely evolved before the mitochondrial citric acid cycle and the oxygen-requiring electron transport system used by cells in aerobic cellular respiration. Glycolysis is anaerobic; it does not require oxygen. Glucose 2 ADP Fructose diphosphate NAD + PGAL PGAL NAD + NADH NADH 2 ADP + 2 P 2 ADP + 2 P Pyruvic Acid Pyruvic Acid Figure 2. Glycolysis 48

2 The Process of Fermentation Fermentation is the anaerobic release of food energy. It occurs when there is no or insufficient oxygen available to support aerobic cellular respiration. Human muscle cells can make ATP with and without oxygen. They have enough ATP to support intense activities, such as quick sprinting, for about 3 seconds. A secondary supply of energy (creatine phosphate) can keep muscle cells energized for about another 10 seconds. To keep functioning beyond that, muscles must then generate ATP by the anaerobic process of fermentation. Glycolysis is the metabolic pathway that provides ATP during fermentation. Pyruvic acid is reduced by NADH, producing NAD +, which keeps glycolysis going. In human muscle cells, lactic acid (C3H6O3) is a principal product of fermentation. 2 NAD + Glucose 2 ADP + 2 P 2 NADH Pyruvic Acid 2 NADH 2 NAD + 2 Lactic Acid Figure 3. Lactic Acid Fermentation Fermentation in Microorganisms Various types of microorganisms perform fermentation. Many bacteria carry out lactic acid fermentation. Yeast cells carry out a slightly different type of fermentation pathway. This pathway produces carbon dioxide and ethyl alcohol ( CO2 and C2H5OH). 2 NAD + Glucose 2 ADP + 2 P 2 NADH Pyruvic Acid 2 NADH 2 NAD + 2 CO2 released 2 Ethyl Alcohol Figure 4. Alcoholic Fermentation The food industry uses microbial fermentation, both lactic acid and alcoholic, to produce a large variety of food products. The list includes such foods as bread, beer and wine, yogurt, some cheeses, pepperoni and salami, soy sauce, sauerkraut and many, many others. The science of fermentation is known as zymology. 49

3 Name: Hillsborough Community College - Ybor Campus 1025C Laboratory Exercise 6: The Process of Fermentation Aerobic cellular respiration for the production of ATP occurs in most cells, and requires oxygen for effective completion. The process is therefore said to be aerobic (overall), even though its first stage (glycolysis) is anaerobic (occurs without oxygen). When oxygen is insufficient or unavailable, organisms that are able to must shunt to an anaerobic pathway and perform an alternative process of ATP production. One such anaerobic alternative is known as fermentation. Yeasts are tiny, unicellular fungi that live wherever sugars are plentiful. Yeasts process sugar via aerobic cell respiration, which requires oxygen. If their environment becomes anaerobic, they have an alternative system for sugar processing that does not require oxygen alcoholic fermentation. Alcoholic fermentation by yeasts is the basis for the production of some foods, such as beer and leavened bread. In an anaerobic environment, yeasts will produce carbon dioxide and ethyl alcohol by alcoholic fermentation. Using this laboratory procedure we will determine the relative rates of metabolism by yeasts that have been placed in a variety of substrates and in aerobic and anaerobic environments by measuring the production of carbon dioxide. PROCEDURE 1. Work in teams of four persons to perform this laboratory exercise. Each team member must select at least one of five possible test solutions to set up: Solution 1 Solution 2 Solution 3 Solution 4 Solution 5 20 ml water plus.05 g yeast 20 ml 10% glucose or sucrose solution plus 0.5 g yeast 20 ml 10% glucose or sucrose solution plus 0.5 g yeast, shaken vigorously for 1 minute 20 ml 1% starch solution plus 0.5 g yeast 20 ml 1% starch solution, plus 5 ml of 0.5% amylase plus 0.5 g yeast 2. For each solution, add a 20 ml sample of one of the three (we use 10% glucose twice and 1% starch twice) test solutions to a small beaker (#5 will have a total of 25 ml) according to the table above. 3. Transfer solution 3 to a large test tube, stopper the tube and shake it vigorously for one minute to dissolve air into the solution; this increases its oxygen content and provides a more aerobic environment for the yeasts. Transfer the shaken solution into a beaker. 4. Measure and add.5 g of yeast to each of your beakers. 5. Stir each solution gently for several minutes to allow the yeast to dissolve and pour each solution into one fermentation tube. 6. Carefully tip the fermentation tube so the tail of the tube completely fills with the solution. 7. Mark the fermentation tube s base with a wax pencil for identification purposes (there will be many tubes in the incubator). 8. Place the tube in the incubator for 15 minutes per the instructor s direction. 9. After 15 minutes remove the tube from the incubator and use the scale on the tail of the tube to measure the gas volume of above the level of the solution. This represents the volume of CO2 gas produced by the yeast. 10. Place the fermentation tube back into the incubator for an additional 15 minutes. 11. Repeat the measuring process for the volume of gas produced. 12. Place the fermentation tube back into the incubator for the final 15 minutes. 13. Repeat the measuring process for the volume of gas produced. 50

4 14. Record your team s observations in Table 1. Solution Table 1: Volume of CO2 Generated vs. Time Contents of the Volume of CO2 (ml) Fermentation Tube 0 MIN 15 MIN 30 MIN 45 MIN 1 20 ml water 2 20 ml 10% sugar 3 20 ml 10% sugar, shaken 4 20mL 1% starch 5 20 ml 1% starch + 5 ml 0.5% amylase 15. Plot the data from Table 1 to and observe the lines the plots produce to visually compare the rates of CO2 production by yeasts in the several solutions. Be sure to include a key to the graph Graph 1 Volume of CO2 vs. Time Volume of CO2 (ml) Time (Minutes) 51

5 16. In Table 2 below, provide a plausible explanation, based on the tube contents and treatment, of the results you observed. Table 2: Explanation of Observations Solution 1 (water) Solution 2 (sugar) Solution 3 (sugar, shaken) Solution 4 (starch) Solution 5 (starch + amylase) 52

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7 Hillsborough Community College - Ybor Campus 1025C Laboratory Exercise 6: The Process of Fermentation Review Questions Yeasts have the capability to degrade glucose both aerobically (in the presence of O2) and anaerobically (in the absence of O2). In each case, one of the products of respiration is CO2 (aerobic respiration produces H2O + CO2 and anaerobic respiration (fermentation) by yeasts produces ethyl alcohol, C2H5OH + CO2). The solutions we are using in this exercise actually contain a small amount of dissolved O2 from their contact with the atmosphere. As the yeasts use this oxygen to degrade glucose, their watery environment becomes progressively more anaerobic. One of the key issues here is that aerobic respiration produces much more energy per glucose molecule (~3) than does anaerobic respiration (). Another way to look at this is yeasts will use glucose much more slowly to satisfy their energy needs in the presence of O2 because they can extract a lot of energy from each glucose molecule or, conversely, yeasts will use glucose much more quickly in the absence of O2 because they can extract only a small amount of energy from each glucose molecule. Use this fact and your findings in this laboratory exercise to explain the following observation, made by the famous French microbiologist Louis Pasteur, on the making of wine: When yeast cells are added to a sealed vat of grape juice, the alcohol content of the juice rises very slowly - until the dissolved oxygen in the juice is gone. When the last trace of oxygen disappears, however, the alcohol content of the juice increases quickly and dramatically. Explain this phenomenon below, which is known as the Pasteur effect. 54

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