Rachel Putri Utomo Block F, Honors Biology Mr.

Kevin Quick 19th February 2014 Effects of Sucrose Concentration On Cell Respiration In Yeast Abstract This lab investigates the effects of Sucrose concentration on cell respiration in yeast. Yeast produces ethyl alcohol and CO2 as a byproduct of anaerobic cellular respiration, so we measured the rate of cellular respiration by the amount of CO2 produced per minute. The results show a trend wherein increased concentrations of sucrose increase the rate of cellular respiration. Introduction All living cells require energy in order to proceed with cellular processes such as active transportation, and the synthesis of molecules. ATP (Adenine Tri-Phosphate) is a molecule, which provides energy in a form that cells can use for such cellular processes. Cellular respiration is the process in which cells produce this energy to survive. It occurs in the mitochondria of the cell and is is vital for the survival of most organisms because cells cannot use the energy in glucose until it is stored in ATP. In the presence of oxygen, organisms can respire aerobically. The balanced chemical equation for aerobic respiration is: C6H12O6 + 6O2 ! 6 CO2 + 6 H2O + ~ 36-38 ATP In the process of aerobic respiration, C6H12O6 is first broken down into 2 3-Carbon molecules called pyruvate or pyruvic acid through the process of Glycolysis, which literally means, Sugar decomposition. A net of 2 ATP is produced during Glycolysis. When oxygen is available, these 2 pyruvates move on to the Krebs cycle and electron transport chain to produce the remaining 34-36 ATP.

Fig 1.1: An overview of Aerobic Cell Respiration (http://www.phschool.com/science/biology_place/biocoach/images/cellresp/glucover.gif)

In the process of anaerobic respiration, C6H12O6 is also broken down into 2 pyruvates through the process of Glycolysis. However because oxygen is unavailable, instead of the Krebs cycle and the electron transport system occurring, fermentation occurs Lacate fermentation in animals, or Alcoholic Fermentation in yeast. Like in aerobic respiration, the co-enzyme NAD+ will need to keep re-generating to continue making ATP in anaerobic respiration. Unlike aerobic respiration however, NAD+ is regenerated by alcoholic fermentation in anaerobic respiration in yeast, producing ethyl alcohol which is also known as ethanol or C2O5OH, the same form of alcohol used in alcoholic beverages such as beerand CO2. The formula for anaerobic respiration in yeast is: C6H12O6 ! 2 CH3CH2OH + 2 CO2 + 2 ATP Yeast is a facultative anaerobe, meaning that its cells are able to make ATP through aerobic and anaerobic respiration (Shimomura-Shimizu, 2009). This experiment explores the effect of varying sucrose concentrations on the rate of anaerobic cell respiration in yeast. Hypothesis Yeast is a facultative anaerobe meaning that it is capable of making ATP by aerobic respiration when Oxygen is available, but can also switch to aerobic respiration when it is not so it will first respire aerobically until the test-tube is sealed with the rubber stopper. The rubber stopper with the measuring mechanism (an airline tube with one end attached to the hole of the rubber stopper and the other end joined to a syringe, or simply a gas syringe) will stop the inflow of air, forcing the yeast to respire anaerobically after all the oxygen in the airtight test-tube has been used up. The amount of carbon dioxide gas produced reflects the rate of cell respiration because CO2 is a byproduct of anaerobic cell respiration in yeast (as well as ethyl alcohol). So an increase in carbon dioxide production means an increase in the rate of cellular respiration. If the amount of substrate, sucrose, is increased then the rate of cellular respiration and carbon dioxide production will also increase. This is because an increase in the availability of the substrate sucrose will allow more yeast cells to use the substrate for cellular respiration in the mitochondriaand the more yeast cells working on cellular respiration at a given time, the more ATP and CO2 it produces at a given time. In other words, higher sucrose concentrations should promote an increase in cellular respiration rates. Materials 4 Airline Tubes 4 Syringes (No Needle) 4 One-Holed Rubber Stopper 4 Test Tubes 4g Yeast (1g in each of the 4 test-tubes) 3g Sucrose (varying amounts in each of the 4 test-tubes) 140mL Warm Water (35mL in each of the 4 test-tubes) 0.4g Salt (0.1g for each of the 4 test-tubes) Styrofoam test-tube holder Procedure I. Preparing The Gas Measuring Mechanism/ Make-shift Gas Syringe

1. Cut an airline tube about the length of your index finger. 2. Secure one end of the tube onto the tip of a needless syringe 3. Secure the other end of the tube into the hole located on the rubber stopper II. Cell Respiration In Yeast 1. Measure and add 1g of yeast into 4 of the test tubes. 2. Measure and add 0.1g of salt into 4 of the test tubes. 3. Measure and add 0.5g, 1.0g, and 1.5g of sucrose into 3 of the test tubes. Do not add sucrose into the 4th test tube because this will be the control. Lightly shake the test tube to mix the contents together. 4. Measure 35mL of warm water and add them into each of the 4 test tubes at about roughly the same time. It is essential that the water is warm. Do not seal the test tube. 5. Wait 5 minutes. During these 5 minutes, set all 4 of the syringe plungers on the gas measuring mechanism at 2mL. 6. After 5 minutes have passed, quickly seal all the test tubes with the rubber stopper on the gas measuring mechanism. Make sure it is airtight and secured properly by lightly pushing down the syringe plunger. If the plunger rises after being pushed, it is secured properly. 7. Wait 1 minute. During the minute, shake each of the test tubes so that the contents mix and dissolve well. 8. In 1-minute increments, note down the position of the plunger for each of the 4 test tubes (i.e.: Plunger is at 3.4mL mark on the scale for 0.5g of sucrose at 7 minutes). After taking note, lightly push the plunger down again so that the probability of the plunger getting stuck in the following minutes decreases. 9. Do this for 10 minutes. Results -./)#$%.0%12+%34.5)6$5%78%9$2+*%&#:,% ;>?<%=)64.+$% @>;<%=)64.+$% @>?<%=)64.+$% A% A% A% A% A% A>@% A>A% A>B% A% A>A% A>C% D% A% A>C% A>E% D>@% A% A>E% A>F% D>C% A% A>B% D% D>G% A% D% D>E% C>E% A% D>?% C% ?>?% A% D>B% C>E% E>G% A% C>D% ?>A% B>A% A% ?% E>A% F>C%

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Conclusion The results of the experiment reflect the hypothesis, which predicted that an increase in the concentration of the substrate (Sucrose) increases the rate anaerobic of cell respiration in yeast cells. This is because an increase in substrate availability allows more cells to use up the substrate for respiration, thereby increasing the amount of its byproduct CO2. In this experiment, the increase in sucrose availability allows more yeast cells to use up the sucrose in order to proceed with cell respiration, which ultimately produces more of the byproduct CO2. It is notable to mention that the formula for anaerobic respiration calls for a monosaccharide (C6H12O6), but the disaccharide sucrose (C12H22O11) is instead used in place of the monosaccharide. The

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disaccharide sucrose is essentially made of the two monosaccharaides Glucose (C6H12O6) and Fructose (C6H12O6), and can therefore be broken down into these two monosaccharaides through the process of hydrolysis. In addition to hydrolysis, yeast also produces its own digestive enzymes, which assist in the breakdown of sucrose. It is also important to mention that disaccharides increase the reaction time of cellular respiration because it needs to be broken down before it can be used, as opposed to monosaccharaides, which can readily be used by cells to respire. This fact, however, does not affect the results because all the yeast was submerged in sucrose concentrations, which means that there was no variety in the type of substrate and therefore was a fair experiment. The yeast submerged in 1.5g of sucrose, the solution with the highest sucrose concentration in this experiment, produced the highest volume of gas in a time period of 10 minutes. The yeast submerged in the solution with the second highest sucrose concentration (1.0g of Sucrose) produced the second highest volume of CO2, and the yeast submerged in the solution with the third highest sucrose concentration produced the third highest volume of CO2. The yeast submerged in 0.5g of sucrose, the fourth highest sucrose concentration produced the least amount of CO2 instead of the second least because the control, the yeast submerged in 0g of Sucrose solution, did not produce any CO2. The control did not produce any CO2 because no substrate was available for cell respiration to occur. Since C6H12O6 (or in this experiment C12H22O11) was unavailable, the yeast cells were unable to proceed with the first step of anaerobic cell respirationglycolysiswhere C6H12O6 is required to make 2 pyruvate molecules. The entire cell respiration process is strictly conditional, so if C6H12O6 is unavailable, then the entire cell respiration process will not occur. Concentrations above 0% and below 4.3% (1.5g of Sucrose in 35 mL of water) show a positive increase in the rate of cellular activity. Hence, in conclusion, an increase in sucrose concentration (or simply substrate concentration) increases the rate of cell respiration because more cells are able to use that substrate to respire. Evaluation A miniscule amount of salt was added to the solution in order to retard the rate of cellular respiration. Anaerobic cellular respiration occurs very quickly so we would be unable to record the amount of CO2 produced in longer periods of time (like 10 minutes in this experiment) due to limits in the measuring apparatus, which can only record up to 10mL of gas. This factor does not change the overall results of the experiment because each test tube was fed equal amounts of salt, making it a fair experiment. The procedure called for warm water because it would contribute to molecular energy for the yeast cells. If hot water were used, denaturing would occur in the yeast cells, killing the yeast (because it will not be able to proceed with cellular respiration). Using cold water would also kill inactivated yeast, or simply significantly retard the rate of cellular respiration. Other than CO2, yeast also produces ethyl alcohol as byproduct, which happens to be toxic to yeast. High concentrations of ethyl alcohol can retard the rate of cell respiration in yeast, or even lead to cell death (which is what happens when beer is made). The concentration of ethyl alcohol, however, does not significantly affect this experiment because you would need 14% of ethyl alcohol concentration to kill the yeast cells (Ackalnd, 2012) This 14% of ethyl alcohol concentration cannot be achieved in a 10 minute time period with the given sucrose concentrations, so this does not affect the results.

This experiment only tested concentrations above 0% and below 4.3%, which show an increase in cell respiration with an increase in sucrose concentration. Other concentrations above 4.3% have not been tested, so whether the same trend applies to these concentration is something unclear as of now.

References Ackland, T. (2012, March 5). Fermentation. Home Distillation of Alcohol. Retrieved February 18, 2014, from http://homedistiller.org/wash/ferment Shimomura-Shimizu, M. (2009, October 18). Yeast Based Sensors. National Center for Biotechnology Information. Retrieved February 20, 2014, from http://www.ncbi.nlm.nih.gov/pubmed/20087724