Temperature, Carbs, Succinate, and Cellular Respiration

This report discusses how different factors impact the fermentation and aerobic respiration reactions that occur in everyday cellular respiration. Redox reactions are essential in aerobic respiration, especially in succinate to fumarate, which is measured in absorbance level of colorless, reduced DCPIP solutions as they act as the electron acceptors in this experiment. Using a spectrophotometer, an absorbance level is obtained to represent the amount of DCPIP that has been reduced from previous redox reactions. Enzyme properties are also important in the fermentation, if there is a lack of oxygen in aerobic respiration, as specific factors of enzymes allow them to operate at an optimal rate.

At optimal speed, more CO2 will be produced at a specific temperature or carbohydrate, both factors are tested in this experiment.

The experiments tested is vital to understand the fundamentals of cellular respiration and how it functions to help perform everyday activities. If the amount of succinate is increased in a mitochondrial suspension solution, then the absorbance level of the solution will increase because the redox reactions occuring in the model Kreb Cycle allows DCPIP to be clear in the reduced state.

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If the temperature of yeast solution is increased, then the volume of CO2 produced will also increase until a certain temperature then decrease because enzymes in yeast denature at higher temperatures. If different carbohydrates are placed in yeast solutions, then the volume of CO2 produced will vary between molecules because each carbohydrate has unique chemical structures that react with yeast.

Materials & Methods The experiments performed followed the procedures given in the BIO 1107 Lab Manual (Lombard 69-70) (Lombard 75-76) Results After experimentation, results of the continuous assay of the reduced DCPIP solutions show Tube 4 had the highest increase in absorbance level followed by Tube 3 then Tube 2, after the 30 minutes elapsed.

The positive control in Tube 1 and negative control in Tube 5 had a relatively horizontal trend. In the experimentation of yeast solution in incubation temperature, results show the solution in 0oC produced 0mL of CO2 while room temperature, 22.6oC produced 1.3mL, 45oC produced 4.8mL, 60oC produced 2.9mL, and 100oC produced 0mL. The trend in results indicate there was an increase in CO2 produced until 45oC and then decreases back to 0mL. In the experimentation of yeast solution with alternate carbohydrate sources, results show galactose producing 0mL of CO2, sucrose producing 4.1mL, fructose producing 4.3,mL maltose producing 1.1mL, lactose producing 1.3mL, and lactose + lactaid producing 4.9mL.

The trend in results indicates a higher volume of CO2 produced with the monosaccharides than the disaccharide carbohydrates. Discussion After inspection of the trends in the results of each experiment, the data collected can be used to support all three hypotheses discussed in the introduction. In the continuous assay of DCPIP solutions, it is confirmed by the trend in Tube 4 compared to the other tubes that as succinate levels increase in a solution of mitochondrial suspension, DCPIP will be more reduced, turning colorless therefore having a higher absorbance level. In regular, aerobic cellular respiration, the process to create the electron carrier FADH2 resides in the Citric Acid Cycle. This reaction uses the redox reactions of succinate to fumarate as discussed in the introduction. Succinate, in the presence of a mitochondrial suspension will be oxidized to fumarate while molecule FAD will accept the electron and become reduced to FADH2.

At this point, FADH2 would be transferred to the Electron Transport Chain to be oxidized back into FAD, but in this experiment, a DCPIP dye in introduced as an alternate electron acceptor. This is how the amount of FADH2 is created because as FADH2 is being reduced, DCPIP intercepts FADH2 and oxidizes it back to FAD. The DCPIP dye is then reduced; reduced DCPIP dye is colorless, so the more DCPIP that gets reduced, the more transparent the solution will appear. The transparency can then be measured using a spectrophotometer, the more transparent a solution appears, the higher the absorbance level will be recorded. In the experiment conducted, Figure 1 shows a steeper slope for Tube 4 than any other. This is due to the solution in Tube 4 having a higher volume of succinate, 0.2mL compared to Tube 3 which only has 0.1mL. Having more succinate in the tube, more reactions occur to produce FADH2 which in turn can reduce more DCPIP.

The final absorbance after 30 minutes are 58.1 for Tube 4 and 49.8 for Tube 3. This shows a significant difference of 8.3, showing how much DCPIP got reduced in each solution. This can conclude that as the volume of succinate increases, the volume of reduced DCPIP will also increase. In the case there is a lack of oxygen for aerobic respiration, the process of fermentation will take place but for this experiment, in the form of yeast. Two experiments were performed in order to test the rate of fermentation, one with temperature as the independent variable and the other with carbohydrate sources. In the first experiment with temperature, the hypotheses can be supported in the results, 45oC was the temperature where CO2 was produced, therefore is the optimal point for a reaction for yeast. The trend in the data was explained as an increase from 0mL of CO2 in 0oC to a maximum of 4.8mL in 45oC and then dropping back down to 0mL in 100oC. This is due to the fact that yeast fermentation is a substrate-enzyme reaction. The environment of enzymes has to be very specific in order to operate at an optimal speed, this includes the temperature. As temperature increases, molecules will move faster, allowing more interactions between substrate and enzyme to occur, creating more CO2 as one of the products as seen in Figure 2 with the increase from 0mL to 1.3 mL.

The reaction will eventually reach a maximum rate at which temperature is ideal for the most interactions to occur seen at 45oC, but as the temperature keeps increasing, enzymes will denature because of their specific protein structure (Freeman 183). This will essentially kill the yeast, halting all reactions seen at 100oC when there was no CO2 produced at all. This can conclude that as temperature increases, the trend of CO2 produced will increase until a certain point, then decrease. Another predicted factor that affects yeast fermentation is the alternate source of carbohydrates. As predicted in the hypothesis, different types of carbohydrates yielded different volumes of CO2. From the results of the experiment, the trend is that monosaccharides produce more CO2 than disaccharides. Again, this is due to the fact that enzymes are very specific, even in the way they receive the substrate, if it does not fit in the active site of the enzyme, the reaction will not occur. Yeast fermentation usually uses glucose as the carbohydrate for the reaction, a monosaccharide.

If a disaccharide is introduced to the enzyme, because it is too big of a molecule, it will require extra energy and time to break the disaccharide back into monomers to use for fermentation. From the results of the alternate carbohydrate source experiment, maltose and lactose only produced 1.1 and 1.3mL of CO2 respectively, not very high compared to a monosaccharide like fructose which produced 4.3mL. The irregularities within the experiment are the results of sucrose and lactose + Lactaid which produced similar if not higher volumes of CO2 with 4.1mL for sucrose and 4.9mL for lactose + Lactaid. The reasoning for this is yeast naturally has an invertase content (White 1), which is basically sucrase that can break sucrose into its monosaccharide components, glucose, and fructose. Since yeast already has an enzyme to break down the disaccharide, and glucose is one of its components, it can quickly be used in fermentation. The process is similar for lactose + Lactaid. Lactaid is an enzyme that breaks down lactose into its monosaccharides, glucose and galactose like sucrose and sucrose. When compared to just lactose, it is a huge difference with 1.3mL being produced in lactose, but when lactaid is also present, the volume of CO2 increases to 4.9mL.

The data indicate that all types of carbohydrates can be used in yeast fermentation, but the major difference is how much time it takes each carbohydrate to go through the process due to enzyme specificity to glucose. A potential drawback in the experiment is the result of galactose. In the experiment, the yeast solution with galactose produced no CO2 at all which is not accurate since galactose is a monosaccharide and is the most similar to glucose being structural isomers. There must have been an error in the mixing of the galactose yeast solution, or the enzymes may have denatured due to misplacement in an extreme temperature environment.