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Cells need energy to do work to carry out metabolic processes that keep them alive and functioning. This energy is stored in the form of ATP, which stands for adenosine triphosphate. All plant and animal cells use a process known as cellular respiration to make ATP from carbohydrates. Plant cells also have a process known as photosynthesis that they use to fixate carbon into carbohydrates using energy from the sun. However, even in plant cells, the most important energy contribution is made by cellular respiration, since ATP formed during photosynthesis is used primarily to fixate the carbon atoms for later use in glycolysis (a step of cellular respiration). Cellular respiration can either be carried out aerobically (in the presence of oxygen) or anaerobically (in the absence of oxygen) (Miyazaki, 85). This lab is concerned with testing the anaerobic pathway, namely alcohol fermentation.
Alcohol fermentation occurs after glycolysis, instead of the Krebs cycle. The pyruvate molecules made out of the break down of glucose in glycolysis are used to create carbon dioxide and ethanol, meanwhile restoring NAD+ molecules for reuse in glycolysis. Since glycolysis makes 2 ATP molecules per glucose molecule, the process of fermentation allows the continuous glycolysis of glucose molecules to form two molecules of ATP. In this lab, we use yeast as the cellular systems through which we will monitor fermentation. (Miyazaki, 88) Since CO2 is produced during fermentation, we measure the volume change caused by this CO2 to measure this process. Hypothetically, the gas produced by the yeast in the glucose/yeast mixture should displace the water in a test tube by a certain amount as fermentation proceeds. This displacement will be measured in millimeters. We also predict that varying pH, temperature, yeast concentration, and type of sugar will affect how quickly and successfully the fermentation process will be carried out. The more extreme the change in pH or temperature is, the slower fermentation will proceed, and the larger the concentration, the quicker it will proceed. In this case, the variables are pH, temperature, yeast concentration, and type of sugar.
The first experiment involved setting up a standard protocol, so that we could become familiar with the apparatus that measures the water displacement by CO2 and simultaneously see the affect of yeast concentration on fermentation rate. The apparatus consisted of three test tubes, which would have the reaction mixtures, connected via a tube and rubber stopper to another test tube which was partially filled with water, inverted and submerged into water inside a beaker. The end of the tube inside the inverted test tube lets out CO2 that was generated in the test tube with the yeast/glucose mixture. This set up prevents air from getting into the tube that the CO2 is going into since the tube is submerged in water.
There were a total of 3 test tubes with reaction mixtures, each with a different concentration of yeast. The first test tube we prepared contained 0 mL of yeast, 3 mL of 40% glucose solution, and 4 mL of water. The second contained 1 mL of yeast, 3 mL of 40% glucose solution, and 3 mL of water. The third contained 3 mL of yeast, 3 mL of 40% glucose, and 1 mL of water. As you can see, all total volumes were equal at 7mL per test tube. The actual method did not involve us mixing all ingredients into the test tubes at once. First, each test tube was given the glucose and water. Only after the entire apparatus was set up did we add yeast to each tube and immediately plugged them up with the rubber stopper so as not to lose any CO2 that was produced. Every 5 minutes we took measurements of how much the water has been displaced in millimeters in the CO2 collection test tube.
Following the conclusion of the standard protocol, we set up another experiment using the same set up. This time, however, instead of varying the yeast concentration, we performed the experiment in different pH levels. The first test tube mixture contained 1mL of yeast, 3mL of 40% glucose solution, and 3mL of a pH 10 buffer. The second test tube contained the same amounts of yeast and glucose, but had 3mL of pH 8 buffer instead of pH 10. The yeast was added at the last second, test tubes were plugged up, and measurements were taken every 5 minutes.
We had set up the standard protocol expecting to see if yeast concentrations would affect the fermentation rate. Based on the graphs and data tables, it seems that it did indeed. Obviously, the tube with no yeast at all produced no CO2. However, the tube with 1mL of yeast had a lower rate of fermentation than the tube with 3mL of yeast. The 1 mL of yeast tube had a slope of .72 while the 3mL yeast tube had a slope of 4.84. The slope of test tube 1 which contained 0 mL of yeast is 0, as there was no fermentation occurring at all.
The experiment we designed was set up to show the effect of pH on fermentation rate. We predicted that a more extreme pH would cause the fermentation rate to decrease. Our prediction was confirmed by the data. Test tube 1 had a pH 10 buffer, and its slope as seen in the graph below is 1.0743. Test tube 2 with the pH 8, however, shows a higher slope of 1.8114.
With respect to the standard protocol experiment, there is one main reason why the data turned out as it did. The yeast in the test tubes was what caused the fermentation to occur. The more yeast, the more fermentation. Consequently ââ‚¬" the more fermentation, the more CO2 produced, and the more water displacement. The tube with the most yeast volume (tube 3 with 3mL of yeast) showed the greatest rate of fermentation. Clearly, this is because there is more yeast to ferment the glucose.
As for the experiment we designed, we predicted that a more extreme pH would cause a slower rate of fermentation. This was indeed the case as shown by the graph for our designed experiment. I speculate that the reason for the slow rate of fermentation in a highly basic pH is that an excess of OH- ions causes a disruption in some of the reactions involving the NADH molecules. Fermentation is supposed to use up NADH molecules and return them back as NAD+ for use in the glycolysis stage. (Miyazaki, 87). I think this cycle may be disrupted when high concentrations of OH- ions are present.
In conclusion, according to our experiment, fermentation is greatly affected by yeast concentration and pH levels. Whereas raising concentration of yeast increases rate of fermentation, increasing pH far past 7 decreases rate of fermentation. The yeast concentration variation seemed to have a greater effect, but this is probably because we increased concentration by 3 times, whereas the pH was only changed from 8 to 10.