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Date Created: 10/10/16
The Presence of Succinate and its Effect On the Rate of Cellular Respiration & The Effect of Alternate Sugar Sources and Incubation Temperatures on Carbon Dioxide Generation Michael McPadden Biology 110727 TA: Lauren Harrison TA: Deaneira Lakheram September 27, 2016 Hypotheses Aerobic Respiration If more succinate is present in the solution, then it follows that the rate of cellular respiration will be higher. Anaerobic Respiration If the incubation tubes are left at 60 degrees Celsius, then they will have more CO2 production than any other temperature. Fermentation If fructose is used as the carbohydrate, then this will have the greatest effect on an increase of CO2 production during fermentation. Introduction Cells are dependent on oxygen and aerobic respiration to produce the energy the cell needs to survive. Cellular respiration is the way in which cells produce a maximum of 38 ATP molecules used for energy. The first step in cellular respiration, glycolysis, occurs in the cytoplasm, while the citric acid cycle and electron transport chain occur in the mitochondria. In the first part of the experiment the conversion of succinate to fumarate is monitored as one of the enzymatic reactions in the citric acid cycle. During cellular respiration succinate is oxidized to create fumarate. One of the ways we measure this reaction is by providing an alternate electron acceptor, which is a dye called DCPIP. During the oxidation process DCPIP is reduced when it “snatches” the generated electrons (Lombard). When this happens the dye changes from blue in its oxidized state, to colorless in its reduced state. Using the spectrophotometer we can try to accurately account for this reaction. When cells don’t have the oxygen required for cellular respiration, fermentation takes place instead, generating 2 ATP. After glycolysis, if oxygen is not present, then fermentation reactions will proceed to produce this ATP. The goal of this experiment is to test and monitor the generation of carbon dioxide by yeast cells when they are placed with different sugars and in pre determined incubation temperatures. After creating a series of nested tubes we can determine how both incubation temperature and alternative sugar sources affect the reaction. Results Shown in “Figure 1” is the line graph version of the “Table 132” results of the aerobic respiration. The raw data from this part of the experiment is used to show the how succinate forms in to fumarate using a mitochondrial fraction made from lima beans. Tube 1 was our “blank” tube, used to set the standard for transmittance and read “100%” on the spectrophotometer. Tubes 2 through 5 all contained 1 mM of DCIP, while tube 4 contains additional succinate to add to the reaction. Tube 2 started at 22.1% and finished the 30 minutes with 48.9%. Tube 3 also started at 22.1% but increased more than tube 2, finishing the 30 minutes at 67.9%. Tube 4 first read 24% transmittance and finished the 30 minutes at 75.4% Lastly tube 5 started with a higher transmittance of 33.8% and finished with 46.5%. Tube 4 had the greatest increase in transmittance. Tube 4 was also the tube that had 1 mL more succinate than the others and helps prove the hypothesis, “If more succinate is present in the solution, then it follows that the rate of cellular respiration will be higher.” “Figure 2” represents the initial gas volume, final gas volume, and the total volume of CO2 produced. At 0 degrees Celsius the gas volume increased from 1 mL to 1.5 mL for an total of .5 mL. At room temperature the initial gas volume started at 1.1 mL and increased to 2.2 mL for a total of 1.1 mL. At 45 degrees Celsius the volume of CO2 increased from 1.6 mL to 5.8 mL for a total production of 4.2 mL. At 60 degrees Celsius the initial gas volume increased from 1.3 mL to 5 mL for a total increase of 3.7 mL. Lastly the boiled yeast increased from 2 mL to 2.7 mL for a total increase of .7 mL. The table disproves my hypothesis “If the incubation tubes are left at 60 degrees Celsius, then they will have more CO2 production than any other temperature.” This was disproven because the increase in CO2 production was greatest at 45 degrees Celsius instead of 60 degrees. “Figure 3” shows the increase of CO2 volume based on the type of carbohydrate used. Galactose was the first carbohydrate tested and increased from 1.5mL to 1.8 mL for a total of .3 mL. sucrose increased from 1.3 mL to 4.4 mL for a total of 3.1 mL. Fructose increased from 1.8 mL to 4.2 mL for a total of 2.4 mL. Maltose increased from 2.0 mL to 3.0 mL. Lactose didn’t increase at all and instead stayed at the gas volume of 1.1 mL. Lastly, Lactose and Lactaid had the most CO2 produced with a total of 3.4 mL produced, increasing from 1.1 mL to 4.5 mL. Percent Transmittance at 600 nm 120 100 80 1 2 60 Transmittance (% T) 3 4 40 5 20 0 1.0 5.0 10.0 15.0 20.0 25.0 30.0 Time (Minutes) Figure 1 Effect of Incubation on Fermentation 7 5.8 6 5 5 4.2 3.7 4 initial gas Volume of Carbon Dioxide (mL) 3 2.7 final gas 2.2 2 volume produced 2 1.5 1.6 1.3 1 1.1.1 1 0.5 0.7 0 Temperature (Celsius) Figure 2 Effect of Carbohydrate on Fermentation 5 4.4 4.5 4.5 4.2 4 3.5 3.4 3.1 3 3 2.4 2.5 2 Initial Gas 2 1.8 1.8 Volume of Carbon Dioxide (mL) 1.1.3 Final Gas 1.5 11.1.1 Volume Produced 1 0.5 0.3 0 0 Carbohydrate Figure 3 Conclusion In our experiment we used succinate because it converts to fumarate. This reaction produces free electrons that normally enter the electron transport chain. However we used DCPIP as an alternate electron acceptor (Lombard). As a result we can monitor the reduction of the DCPIP to see the reactions taking place. We use a spectrophotometer to quantify these results through a measurement of the light that is transmitted. We measure transmittance because it informs us on how much of the wavelength was able to pass through the solution. My hypothesis was, “If more succinate is present in the solution, then it follows that the rate of cellular respiration will be higher.” On figure 1 it is clearly shown that tube 4 increased to have the greatest transmittance percentage. Tube 4 was also the tube with additional succinate. As we can see the additional succinate helped cellular respiration as it had the greatest conversion from the blue oxidized DCPIP to the colorless DCPIP. We know this because of the spectrophotometer reading of 75.4%, which was higher than all the other tubes, showing that it has the most DCPIP conversion occurring. The temperature affects CO2 production in yeast in a way that simulates a bell curve. When looking at figure 2 you can see how the CO2 production increases to the final product point of 5.8 mL before decreasing back down as the temperature continues to increase. 45 degrees Celsius was the median temperature we used in the experiment. This temperature proved to be the most optimal and had the greatest volume of CO2 produced at 4.2 mL. At 60 degrees Celsius the CO2 production wasn’t too far behind at 3.7 mL. My hypothesis stated, “If the incubation tubes are left at 60 degrees Celsius, then they will have more CO2 production than any other temperature.” This proved to be wrong and 45 degrees Celsius was the better choice. After completing the experiment it seems safe to say that at 0 degrees Celsius, room temperature, and 100 degrees Celsius, not much CO2 is produced in comparison to both 45 and 60 degrees Celsius. Different carbohydrates were used to test the effect on fermentation. The only sugar that did not ferment at all was lactose. Galactose did not ferment well but we did record an increase of .3 mL of CO2 for this sugar. My hypothesis was “If fructose is used as the carbohydrate, then this will have the greatest effect on an increase of CO2 production during fermentation.” My hypothesis proved to be false because both Lactose and Lactaid, and Sucrose had a higher CO2 volume produced. However, Fructose was a close third place behind these two sugars. Sucrose and Fructose both worked and produced 3.1 mL and 2.4 mL. Maltose and Lactose both didn’t produce much CO2 but when Lactose and Lactaid were combined it produced the greatest amount of CO2 out of any of the sugars. Lactaid was used as an enzyme and helped convert lactose (Types of Sugars). One of the sources of error we experienced was not blanking the spectrophotometer initially. As a result, this negatively influenced our data for the first minute of aerobic respiration experiment. We were able to work through this by comparing data with another group and figuring out about what percentage the tubes should have been. Works Cited A. E. (2006). Introduction. Retrieved September 27, 2016, from http://www.salisbury.edu/wac/excellence/ellison/ellison.htm Lombard, K., Terry, T., & Milinoski, C. (n.d.). Principles Of Biology I (LaboratoryManual). Macmillan Learning. Types of Sugars. (n.d.). Retrieved September 27, 2016, from http://www.ivyroses.com/HumanBiology/Nutrition/TypesofSugar.php
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