Yeast and Molasses Examining the Effect of Food Concentration on Fermentation

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1 15 Examining the Effect of Food oncentration on Fermentation All cells need energy, and the most common form of energy used by cells is ATP. The full name given to ATP by chemists is adenosine triphosphate. ATP is a molecule that contains the monosaccharide, ribose which is a pentose sugar. Bonded to ribose is the nitrogenous base, adenine. This is the same nitrogenous base found in DNA and RNA. Also attached to ribose are three phosphate groups. Two of the bonds connecting the phosphates are considered high-energy meaning that the phosphate groups are easily removed. This is due to the negative charges found on the phosphate groups. One or more of these phosphate groups can transfer to another molecule. Remove one phosphate from ATP, and the molecule becomes adenosine diphosphate or ADP. Remove two phosphates from ATP and the molecule becomes adenosine monophosphate or AMP. Adenine N N N N O O O Energy-rich bonds O - O - O - O P O P O P O O O 3 Phosphates O Ribose Many biochemical reactions require the addition of phosphates to one or more reactants in order to proceed. The addition of these phosphates increases the energy content of the molecule. An example of this is glucose-phosphate. Glucose-phosphate has more energy than just plain glucose because of the addition of a phosphate group. ells need a plentiful supply of ATP. Once the ATP is used and converted into ADP, the cells need to regenerate ATP by adding a phosphate to ADP. Since energy was released during the breaking of the phosphate bond, the reverse process of forming a phosphate bond requires an energy input. Laying the Foundation in Biology 457

2 15 You might be wondering where the cell will obtain this energy. This energy comes from the cell breaking down or oxidizing high-energy food molecules like glucose, sucrose, or other such organic compounds. Glucose has considerably more energy than ATP. One molecule of ATP has approximately 16 kcal/mole whereas glucose has approximately 680 kcal/mole. You may ask, why not use glucose instead of ATP as the cell energy currency? The reason that glucose cannot be used directly as an energy source is that glucose has too much energy. Releasing it all at once is like putting a match into a gas can. The cell cannot handle the release of all that energy at once as the increase in temperature would denature the enzymes and kill the cell. So instead, cell respiration releases the energy in small amounts at a time and this involves many steps. Another analogy that illustrates using glucose as a direct energy source in the cell involves using a thousand dollar bill to buying gasoline. You have money but it is unusable money, and the bill has to be taken to the bank and exchanged for useable money such as ten-dollar bills. ATP then represents ten-dollar bills. The cell can use ATP directly, easily. The chemical equation below summarizes the process of cellular respiration. There are three major parts to cell respiration. The three parts to cellular respiration are glycolysis, the Kreb s cycle, and chemiosmosis. Glycolysis occurs in the cytoplasm of the cell. The major events in the process of glycolysis are the following: During the process of glycolysis no oxygen is required. The other 34 molecules of ATP are made in the Krebs cycle and chemiosmosis. These processes occur in the inner compartment of the mitochondria. It is at the very end of these processes that oxygen is needed. Respiration is often referred to as aerobic respiration because oxygen is required. If there is no oxygen present, then the Krebs cycle and chemiosmosis will not occur. If oxygen is not present, then glycolysis can continue to make pyruvic acid and two molecules of ATP. After all, the synthesis of two ATP molecules is better than making none. The limiting factor in this process is having enough NAD to make the NAD. In order to regenerate NAD, several additional steps are needed. These additional steps complete the process known as fermentation. There are several types of fermentation. The most common types of fermentation are lactic acid fermentation and alcohol fermentation. Lactic acid fermentation is performed by muscle cells when muscles are vigorously contracting and are unable to obtain enough oxygen. Yeast cells, plant cells and certain bacteria perform alcoholic fermentation in the absence of oxygen. The alcohol produced is ethanol and during this reaction a molecule of carbon dioxide is released. 458 Laying the Foundation in Biology

3 15 This lab exercise uses yeast cells to investigate the relationship between fermentation and food concentrations. If yeast cells have more food available, will they produce more energy and ultimately more ATP? Yeast cells undergo alcoholic fermentation and as a result produce carbon dioxide as a byproduct. This lab uses the amount of carbon dioxide released as an indicator for the amount of fermentation occurring. Below are the final steps added to glycolysis to complete the process of fermentation. PURPOSE In this activity you will investigate the process of fermentation and its relationship to the availability of food for respiration. Laying the Foundation in Biology 459

4 15 MATERIALS Part I 100 ml graduated cylinder 8 ea graduated centrifuge tubes 17 x 120 mm (0.1 ml graduations) stock yeast solution 5 % molasses solution 20 % molasses solution 60 % molasses solution 100 % molasses solution Part II scissors 8 ea test tubes 25 x 200 mm (70 ml) test tube rack 4 ea # 4 test tube stoppers 10 % molasses solution 40 % molasses solution 80 % molasses solution distilled water glue stick PROEDURE PART I 1. Formulate a hypothesis that explains the relationship between the amount of energy produced through fermentation and increasing amount of food available. State your hypothesis on your student answer page. 2. Obtain the above materials and label the large test tubes Add 40 ml of the indicated molasses solution to each of the following test tubes: # 1 0 % molasses/ only distilled water # 2 5 % molasses # 3 10 % molasses # 4 20 % molasses # 5 40 % molasses # 6 60 % molasses # 7 80 % molasses # % molasses 4. Add 10 ml of stock yeast solution to each test tube. 5. Stopper each tube and shake thoroughly to ensure a uniform mixture. 6. Remove the stopper and rinse the stopper with water. 460 Laying the Foundation in Biology

5 15 7. Obtain a small centrifuge tube and notice that it has markings on the sides of the tube in ml. The small tube needs to be filled with the yeast-molasses solution. Do this by inverting the small centrifuge tube and sliding it into the large test tube. Then, put the large stopper into the large tube and hold it on its side. When the small tube is completely filled with the suspension, slowly move the large tube back to its upright position. If there is any air bubble in the small tube, repeat the procedure until no air is present. 8. Allow the tubes to sit for 24 hours. After 24 hours, measure the gas in the centrifuge tube for each tube by observing the amount of gas in the tube using the gradations on the side of the centrifuge tube. Record this in the data table. ollect data for each group in the classroom and average the data. 9. Graph your data and the class-averaged data. Optional These calculations can be done on a TI-83 calculator by doing the following: 1. To make a data table press, the select EDIT (Figure 1) and press. Notice that there are columns or lists to record data. Laying the Foundation in Biology 461

6 15 Figure 1 Figure 2 2. To clear a list that might have data in it, put the cursor at the very top of the list so that the name of the column is highlighted. Press followed by the. 3. Now enter the percent of molasses in L1 starting at 0 for the first entry and ending at 100 percent for the last entry. In L2, record the amount of carbon dioxide collected. In L3, record the class average for the amount of carbon dioxide collected. (Figure 2). To view this graphically, press,,. At this time make sure to put your cursor on PLOT 1 and press. Then move the cursor down to ON and press. Both PLOT 1 and ON should be highlighted. All other plots should be inactivated. ighlight the line graph (Figure 3) and press. 4. ighlight the Xlist and press, [L1]. 5. ighlight the Ylist and press, [L2]. 6. ighlight the box symbol for the Mark and press (Figure 3). Figure 3 Figure Laying the Foundation in Biology

7 15 7. To put a second line on the graph, position the cursor on Plot 2 and press. Move the cursor down to ON and press. Both PLOT 2 and ON should be highlighted. ighlight the line graphs (Figure 4) and press. 8. ighlight the Xlist and press, [L1]. 9. ighlight the Ylist and press, [L3]. 10. ighlight the cross hairs symbol for the Mark and press (Figure 4). 11. Press, then press. A graph should appear that will have automatically adjusted the axes so that they fit the window. (Figures 5 and 6). Figure 5 Figure 6 It may appear that there is only one line on the graph if your data is close to the class average. To demonstrate that both lines are present press and then use the and to differentiate between the points. Look at the figures above. In figure 5, the data point, Y=14.9, is class averaged data and in figure 6, the data point Y=15, is the student s data. PART II Below are the steps of glycolysis and fermentation. As you read about the steps of fermentation, look at the accompanying diagram on the student answer sheet. You will notice that there are names of products and descriptions of the reaction missing. The empty boxes indicate missing items. At the end of the diagram is a list of missing parts. Using scissors and a glue stick, fill in the missing items to make the biochemical pathway complete. Be sure to place either the missing step or molecule in the correct sequence. 1. A phosphate is added to glucose. It comes from ATP. This phosphate increases the amount of energy of glucose. Laying the Foundation in Biology 463

8 15 2. Atoms are rearranged and glucose 6-phosphate is turned into fructose 6-phosphate. 3. Another phosphate is added to fructose 6-phosphate. It comes from ATP. This phosphate increases the amount of energy of fructose. 4. Fructose 1-6 bisphosphate has so much energy and is so unstable that this hexose will be cleaved into two trioses, phosphoglyceraldehyde or PGAL and dihydroxacteone phosphate. 5. Atoms are rearranged and dihydroxyacetone is turned into a second phosphoglyceraldehyde PGAL molecule. From this point in time, everything is multiplied by a factor of two because there are two trioses. 6. ydrogens are stripped from phosphoglyceraldedhyde and transferred to NAD +. In addition, an inorganic phosphate group is added to the molecule. This phosphate group comes from the cytoplasm. This produces 1, 3-biphosphoglyceric acid Biphosphoglyceric acid transfers one of its phosphates to ADP to make ATP. This produces 3- phoshoglyceric acid. 8. The phosphate group found on carbon number three is transferred to carbon number two. This produces 2-phosphoglyceric acid. 9. A dehydration reaction occurs as a water molecule is removed to form phosphoenolpyruvic acid or PEP. 10. Phosphoenolpyruvic acid transfers it phosphate to ADP to form ATP. This produces pyruvic acid. If oxygen is present then the pyruvic acid is used in the Kreb s cycle. If NO oxygen is present, then fermentation occurs. 11. Lactic acid fermentation- ydrogens are transferred to pyruvic acid from NAD to form lactic acid and NAD +. Alcoholic fermentation- ydrogens are transferred to pyruvic acid from NAD to form lactic acid and NAD + and a molecule of carbon dioxide is removed. 464 Laying the Foundation in Biology

9 15 Name Period Examining the Effect of Food oncentration on Fermentation YPOTESIS DATA AND OBSERVATIONS Amount of O 2 ollected (ml) Test Tube # Percent of Molasses 0 % 5 % 10 % 20 % 40 % 60 % 80 % 100 % Individual Team Data Team # 1 Team # 2 Team # 3 Team # 4 Team # 5 Team # 6 Team # 7 lass Average Laying the Foundation in Biology 465

10 15 DIAGRAM OF FERMENTATION O 2 O O O O O 2 O P O O O O O P 2 Glucose O O Glucose 6-phosphate O 2 1. A phosphate is added to glucose. It comes from ATP. This phosphate increases the amount of energy of glucose. 2. Atoms are rearranged and glucose 6-phosphate is turned into fructose 6-phosphate. O O O 466 Laying the Foundation in Biology

11 15 O P 2 O O 2 O O O Fructose 6-phosphate 3. Another phosphate is added to fructose 6-phosphate. It comes from ATP. This phosphate increases the amount of energy of fructose. O P 2 O O P 2 O O O Fructose 1-6 bisphosphate O 2 =O O P 2 Dihydroxyacetone phosphate =O O 2 O P Phosphoglyceraldehyde 5. Atoms are rearranged and dihydroxyacetone phosphate is turned into phosphoglyceraldehyde, PGAL. From This point in time, everything is multiplied by a factor of two because there are two trioses. Laying the Foundation in Biology 467

12 Laying the Foundation in Biology

13 15 Laying the Foundation in Biology 469

14 Laying the Foundation in Biology

15 15 ere are the missing items to make glycolysis and fermentation complete: Laying the Foundation in Biology 471

16 15 ONLUSION QUESTIONS 1. What happened to the amount of gas produced as the amount of molasses increased? 2. What percentage of molasses produced the greatest amount of energy? Does this support your hypothesis? 3. Design an experiment based on this protocol that would investigate the effect of temperature on fermentation. 472 Laying the Foundation in Biology

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