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The synthesis of ATP is a very complex process but is absolutely necessary for living organisms. This is due to ATP’s ability to drive cellular work. Looking at each of the four major steps to cellular respiration in depth gives a further understanding of how ATP is acquired by the cells. The first major step to cellular respiration is Glycolysis. In this step the six-carbon sugar, glucose, undergoes a series of chemical transformations. In the end, it is converted into two molecules of pyruvate, a three-carbon organic molecule.
In these reactions, ATP is made, and NAD+ is converted into NADH. At the end of glycolysis 2 ATP are produced per molecule of glucose. In the second step, pyruvate oxidation, each of the pyruvates from glycolysis goes into the mitochondrial matrix-the innermost compartment of the mitochondria. In the mitochondria, it is converted into a two-carbon molecule bound to Coenzyme A, known as acetyl CoA. The Krebs Cycle is then run twice producing 6 NADH, 2 FADH2, and 4 CO2.
The electron carriers, NADH and FADH2, then move to the mitochondrial cristae.
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Here, they are oxidized by highly electronegative oxygen causing them to release the electrons that they were holding. The electrons that were released are then used to power the proton pumps which pump H+ into the intermembrane and create an electrochemical gradient. This gradient allows them to reenter the matrix using ATP synthase. ATP synthase uses this process to power a rotor and phosphorylates ADP. This process is known as the electron transport chain.
This has a much greater ability to produce ATP in comparison to fermentation. The ETC produces a maximum of 38 ATP, usually producing 34-36, while fermentation produces a mere 2 ATP (Freeman et al.).
The three experiments are focused on the pathways organisms take to produce ATP, specifically cellular respiration and fermentation. The first experiment is taking a closer look into the Krebs Cycle, “By using a suspension of mitochondria (prepared from lima beans) we will monitor the activity of one enzymatic reaction in the citric acid cycle, the conversion from succinate to fumarate. This reaction reduces FAD to FADH2, which produces electrons” (Malinoski 67). By providing an alternate electron acceptor, a dye called DCPIP (dichlorophenol-indophenol), we can monitor the reduction of the dye. This is due to the fact that the dye will snatch the generated electrons. Since DCPIP is blue in its oxidized state and colorless in its reduced state, we can watch this reaction by measuring the reduction of DCPIP using a spectrophotometer. We will be able to see the rate of cellular respiration by measuring the percent transmittance, as DCPIP is further reduced it becomes more clear. Given this information, it is appropriate to hypothesize that if there is more succinate with the mitochondrial solution, then there will be more reduction leading to a more transparent solution. In the second experiment, we will change the temperatures from 0°C, to 22.8°C, to 45°C, to 60°C, and 100°C of yeast solution containing glucose.
In doing so, it will be possible to see yeast’s ability to generate ATP and have CO2 as a byproduct by measuring its volume. Therefore, it is appropriate to hypothesize that if we changed the temperature to 45°C then more CO2 will be produced thus making fermentation more efficient. This is because this temperature is not too hot and will not denature the yeast and is not too cold for the yeast to react effectively. The third experiment is focused on different sources of carbohydrates and their effect on fermentation. The experiment uses Galactose, Sucrose, Fructose, Maltose, Lactose, and Lactose+Lactaid solutions to measure the CO2 differences between them. The volume of CO2 produced will ultimately tell us the efficiency of fermentation of yeast with different sugar sources. Therefore, it is possible to hypothesize that if disaccharides like Sucrose and Lactose+Lactaid are added to the solution as a sugar source then more CO2 will be produced. The yeast will be able to break up the disaccharides and use glucose. Glucose will increase the rate of fermentation as it is the main energy source in almost all living things.
In the first experiment, the results conveyed that the presence of the mitochondrial suspension made from Lima Beans 0.5 mL, 0.2 mL succinate, 4.0 mL phosphate buffer, and 0.3 mL 1mM DCPIP (Tube 4) resulted in the greatest transmittance. This means that it had the greatest reduction over time, specifically shown as it went from 13.4% initial to 84.7% final transmittance. The other test tubes were 1 (0.0 mL DCPIP, 0.5 mL Mitochondrial suspension, 0.0 mL Succinate), 2 (0.3 mL DCPIP, 0.5 mL Mitochondrial suspension, 0.0 mL Succinate), 3 (0.3 mL DCPIP, 0.5 mL Mitochondrial Suspension, 0.1 mL Succinate), and 5 (0.3 mL DCPIP, 0.0 mL Mitochondrial Suspension, 0.1 mL Succinate). Test tube 1 was kept as the control and measured 100% throughout the entirety of the experiment. It is also important to note that test tube 5 had the lowest transmittance by the end of the experiment, making it the darkest tube.
In the second experiment, the yeast was able to produce the most CO2 when it was stored in temperatures of 45 degrees Celsius and 60 degrees Celsius. The volumes of CO2 produced by each of the tubes were .5 mL of 1(0 °C), 1.0 mL of 2 (22.8 °C), 4.5 mL of 3 (45 °C), 3.5 mL of 4 (60 °C), and 1.0 mL of 5 (100 °C).
The third experiment generated results in which the fermentation of sugars by yeast varied depending on the ability to break down the sugar. Lactose and Galactose produced very little CO2 as they both yielded 1.0 mL and 0.2 mL respectively. Also Maltose produced 2.6 mL, Fructose produced 3.5 mL, Sucrose produced 4.0 mL and the combination of Lactose + Lactaid Produced 5.0 mL of CO2.
In the first experiment focusing highly on the Krebs Cycle, it can be seen that throughout all of the test tubes the transmittance increased as time increased. This is mainly due to the reaction between the mitochondrial suspension and succinate. The more succinate that was in the tube, the higher the transmittance. This is shown in tube 4 as it had the highest percent increase among the tubes, from 13.4% to 84.7% at 30 minutes. Also, while tube 2 did not have the highest percent increase, its transmittance still increased from 11% to 25.8% at 30 minutes. This increase of transmittance is due to succinate being available to be oxidized and serve as the reducing agent for FAD to create FADH2. DCPIP then functions as the oxidative phosphorylation chain and becomes increasingly more clear. This data and reasoning supports the stated hypothesis that the more succinate there is in the tube with the mitochondrial suspension, the more reduction and transmittance.
In the second experiment regarding temperature change and yeast fermentation efficiency, it was evident that the 45°C tube had the highest CO2 production with a volume of 4.5 mL. This shows that the most optimal temperature for yeast fermentation is somewhere between 45 and 60 degrees as 60 degrees was the next most effective temperature with 3.5 mL of CO2 produced. This supported the stated hypothesis that 45 degrees would be the most effective for yeast fermentation as it is not too warm for the protein to become denatured and it is not too cold for the yeast to react slowly.
The third experiment involving different carbohydrate solutions resulted in Lactose+Lactaid and Sucrose producing the highest volume of CO2 with values of 5.0 mL and 4.0 mL. These findings support my hypothesis that if disaccharides like Sucrose Lactose+Lactaid are added to the solution as a sugar source then more CO2 will be produced. In a study done by Morrison Rogosa, he found similar results to the experiment we performed when he states, “The lactose-adapted strain, as was to be expected, fermented lactose at a faster rate than either of the other two adapted strains. The fermentation of lactose was complete within 55 hours. At this time 0.25 per cent of sugar remained unfermented in the glucose-galactose mixture” (Rogosa 421).
Sources of error can be present in these studies as both my lab partner and I may have misread readings. Also, the amount of time each test tube was in each temperature may have differed from tube to tube. In order to improve these studies, more trials could be performed to determine if the data is truly accurate.
Analysis of Cellular Respiration Using Mitochondrial Suspension and Fermentation. (2021, Dec 20). Retrieved from https://studymoose.com/analysis-of-cellular-respiration-using-mitochondrial-suspension-and-fermentation-essay
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