Yeast Fermentation Experiment

The purpose of this lab was to observe fermentation in yeast with different carbohydrates at different temperatures and cellular respiration of lima beans with different quantities of the necessary reactants. It was observed that fermentation reacted at a slower rate than cellular respiration. Also it was observed that fermentation was most efficient with glucose at a temperature of 37°C. It was determined that respiration was most efficient 150µL of DPIP, 150µL mitochondrial suspension and 200µL of succinate. The slower rate of fermentation is caused by it lacking the electron transport chain that is present in cellular respiration.

Introduction

Cellular respiration is the process cells use to convert and breakdown organic substances, such as glucose, to release energy that the cell can use to function. Glucose is an important part of this reaction because it is what all of the products are derived from, without it there would be no respiration; it also the most efficient carbohydrate to use. Cellular respiration has multiple parts in which redox reactions are utilized to create ATP. Respiration occurs in different parts of the cell at certain parts in the process. The reaction begins in the cytoplasm but will eventually move to the mitochondria. These pathways allow the ATP to be produced in the cell where it is needed and therefore will not need to be transported elsewhere in the cell. Oxygen is necessary in cellular respiration making this an aerobic reaction. Oxygen is necessary to “pull” electrons down the electron transport chain at the end of respiration. This results in H2O molecules being formed as well as ATP. The following equation describes the full reaction of cellular respiration C6H12O6 + 6O2 è 6CO2 + 6H2O + energy (approx. 36 ATP). It is clear to see that oxygen is an important part of this process, this I why most organisms need a steady supply of oxygen (Upadhyaya).

When oxygen is not available respiration is still necessary so that an organism has energy to function. This type of respiration is called fermentation. Organisms that do not have access to oxygen must utilize fermentation. Fermentation begins as one part of cellular respiration called glycolysis. This part of cellular respiration does not use oxygen but uses redox reactions to create small amount of ATP. Yeast is an example of an organism that can switch between respiration and fermentation. The fermentation of yeast is observed in this lab. The type of fermentation utilized by yeast is called alcohol fermentation because one of the products of this process is alcohol. This process is described by the following equation. C6H12O6 è 2C2H5OH + 2CO2 + energy (2 ATP) (Upadhyaya). Another type of fermentation is lactic acid fermentation where the by-product is lactic acid. This reaction often happens in muscle cells that need to generate energy much faster, like during exercise. Animals generally use cellular respiration but will use fermentation if they need to, while only organisms without access to oxygen will use fermentation all of the time. This lab was conducted in two separate parts, the first observing fermentation in east and the second observing cellular respiration in lima bean mitochondria (Upadhyaya). It is important to understand these two processes so that we know how organisms acquire and use the energy necessary for them to live. The two parts of this lab this lab observed these two types of metabolic reactions under different conditions with different quantities of reactants. The different quantities of reactants were expected to change the reaction rates in some way. With the fermentation reactions the lower temperature was expected to slow or hinder the reaction while the higher temperature was expected to speed up the reaction. Also the fermentation reaction was observed with three different carbohydrates in an attempt to determine which is the best for fermentation of yeast. The respiration portion of this lab observed the rates of respiration for different quantities of the reactants. The rate of reaction was determined by using a spectrophotometer. The reactant DPIP is blue before respiration takes place. However, during the reaction DPIP acts as an oxidizing agent and accepts an electron from succinate during a redox reaction. A redox reaction is when one molecule gives up and electron to another molecule thus reducing it and oxidizing itself. As this reduction of DPIP occurs the chemical changes from to colorless. Using the spectrophotometer the absorbance of DPIP can be measured thus determining how much of the reaction has taken place.

It can be hypothesized that for part 1 the glucose at 37 degrees will be the most efficient because glucose is the best carbohydrate for respiration and 37 degrees is very close to optimum temperature. For part 2 it is hypothesized that the sample with the higher amount of succinate will be the most efficient because it will have the highest number of electrons to give to DPIP.

Materials and Methods

Three different solutions and a control (water) were used for this part of the lab, all of the solutions were each kept at three different temperatures, 4°C, ~25°C (room temperature) and 37°C. The researchers used a pipette to place 15mL of each solution into 50mL beakers. The researchers then measured out .5 grams of yeast and mixed with each solution. After the yeast was thoroughly mixed in the solutions the mixture was quickly transferred into the fermentation tube. The tube was then inverted to be sure all of the air had escaped. The 4°C tubes were placed in the refrigerator while the 37°C tubes were placed in an incubator. Every five minutes for forty minutes the amount of CO2 formed in the tube was measured.

The spectrophotometer was turned on and set to read % transmittance at 600nm. 6 test tubes were labeled B1, B2, 1, 2, 3 and 4. The researchers prepared the two blanks, B1 and B2, according to the table below. After the blanks were prepared the spectrophotometer was blanked with B1 and the other four test tubes were prepared according the table, adding the succinate last. After the succinate was added parafilm was placed over the four test tubes and they were shaken more two seconds. Test tubes 1, 2 and 3 were placed into the spectrophotometer after it was blanked with B1. The spectrophotometer was then blanked with B2 and test tube four placed in the spectrophotometer. This process of testing the test tubes was repeated every five minutes for thirty minutes.

Figure 1- This figure shows how each of the 6 test tubes was prepared for the cellular respiration reactions.

Results

Results of the fermentation reactions:

Figure 2 (Effect of Food Source and Temperature on CO2 Production)- This table shows the results of the fermentation reactions.

Figure 3- This graph shows the results of the reaction for each food course at 37 degrees Celsius.

Figure 4- This graph shows the results of the reaction for each food course at 25 degrees Celsius.

Figure 5- This graph shows the results of the reaction for each food course at 4 degrees Celsius.

These four figures represent the same information only in different forms. Figure 2 shows that by far the most efficient conditions for the yeast fermentation was with glucose at 37 degrees. The figure also shows that the control samples with only water did not have any kind of reaction. Also it is clear that the colder samples did not ferment very well either.

Results of the cellular respiration reactions.

Figure 6 (Results of Cellular Respiration Reactions)- This table shows each sample’s % transmittance at wavelength 600nm for forty minutes.

Figure 7- This graph shows % transmittance for each sample (1, 2, 3 and 4) over the forty minutes that the reactions took place.

Figure 8 (Initial Reaction Rates of Cellular Respiration)- This figure shows the initial rates of reaction by finding the slope of the lines on the graph.

The first two figures show that the contents of sample 3 produced the most efficient reaction given it had the highest % transmittance. Also it is clear that sample 4 had almost no continuing reaction.

Discussion

Based on the results of part 1 of this lab it easy to see that the most efficient condition for fermentation of yeast is glucose at 37 degrees. This result happened because glucose is the main fuel source for respiration. Glycolysis, which is utilized by fermentation, works best and most efficiently with the sugar glucose. Starch and sucrose, although they do work for fermentation, do not ferment as well resulting in far less ATP or CO2 being produced. The temperature the reaction takes place at also plays a major role in the amount of reaction. Fermentation often occurs inside in the body of an organism, which is generally a warmer environment; therefore the optimum conditions for any kind of respiration would be in warmer environments. The reaction simply did not take place at the near freezing level because chemical reactions generally need heat energy to take place efficiently (Clark).

In the cellular respiration part of this lab samples 1, 2 and 3 all contained equal parts mitochondrial suspension and DPIP. However, samples 3 and 2 were much more successful in their reactions with 3 being the most successful. The difference between these three samples was their level of succinate, with 3 having the highest, 2 having the second highest and 1 having the least. This proves that the amount of succinate is very important in the reaction of cellular respiration. This supports the hypothesis because it was hypothesized that the sample with the highest succinate would react the most because it would have the most ability to be oxidized and give up electrons to DPIP. The fourth sample was by far the least reactive. This is due to the fact that sample 4 contained no mitochondrial suspension meaning there was no strong environment for the reaction to take place. Overall it can be concluded that for yeast fermentation warmer temperatures with glucose are the most efficient and larger amount of succinate are useful for cellular respiration.

Question From Lab Manual

The addition of succinate was crucial in the reduction of DPIP. Without it, as in sample 1, there was very little reaction compared to samples 2 and 3, which contained large amounts of succinate.

It is evident that the mitochondria were respiring. If the mitochondria were not respiring then the DPIP would not have undergone reduction. DPIP only changes in color when it undergoes reduction. Therefore, if DPIP were not being reduced the % transmittances readings would not have changed. So, the fact that the transmittance readings continually changed means that the DPIP must have been reduced meaning a reaction was taking place.

Succinate was being oxidized because it was giving up and electron to DPIP. Redox reactions occur when one particle gives up an electron to another particle. The particle that gives up the electron is oxidized and is called the reducing agent while the particle that accepts the electron is reduced and is called the oxidizing agent (Tro).

Had rat mitochondria from rat muscle been used instead on lima beans the results for the reaction would be much higher. This is because muscle cells are required to produce a lot more energy than lima beans.

Errors/Future Experiments

Possible errors could have occurred in the form of human error during measuring or transferring chemicals from on place to another. Also contamination of the carbohydrate samples, or any other chemicals, could have occurred by improper pipette usage. Error may have occurred when inverting the fermentation tube and not allowing all of the air to escape. For future experiments it may be useful to include higher temperatures for the fermentation part in order to find the optimum temperature for fermentation. Also higher levels of succinate could be used to create the absolute most efficient cellular respiration.