Determination of the Rates and Optimal Temperature for Aerobic Respiration and Photosynthesis. Lab report

(See Figure 1 ). Figure 1 : Eukaryotic glucose metabolism. (Iwasa, J and Marshall, W (2016) Karp’s Cell and Molecular Biology, 8 th edition. Wiley. P. 173) We will be examining the rate of aerobic respiration in two organisms: yeast ( Sacchromyces cerevisiae ) and Euglena . The terminal electron acceptor of the electron transport chain of aerobic respiration is oxygen (see Figure 2 ). Therefore, by determining the rate at which oxygen is utilized by the cell under different conditions, we can monitor the effects of these variables on the overall rate of respiration. Figure 2: Mitochondrial components of the electron transport chain for oxidative phosphorylation and ATP production. (Urry, et al. (2019) Campbell: Biology in Focus, 3 rd edition, page 156) In the broadest sense, photosynthesis is the process whereby cells absorb the energy of visible light, and use this energy to synthesize organic compounds. The most important light absorbing pigment in this process is chlorophyll . In eukaryotes, chlorophyll is found embedded in the thylakoid membranes of chloroplasts . The overall process is shown in the familiar summary equation for photosynthesis:

6CO 2 + 6H 2 O – light → C 6 H 12 O 6 + 6O 2 Of course, this equation oversimplifies the actual chemistry of the process. The Hill reactions (often referred to as the “light reactions” ) provide the primary step in photosynthesis. It photolyzes water, producing oxygen, and traps energy in the form of ATP and reduced electron carriers (NADPH). This reaction occurs in the thylakoid membranes of chloroplasts. (See Figure 3 on the following page ).

I n the second phase of photosynthesis, which occurs in the stroma of the chloroplast, the energy-rich products of the Hill reactions (NADPH and ATP) are used for the synthesis of organic molecules in the Calvin cycle . These reactions do not have a direct dependence on light, and are often referred to as the “ dark reactions ”. Do not confuse this as being equivalent to “dark conditions”! In these reactions, carbon dioxide is reduced (or "fixed") to produce carbohydrates Because the products of the Hill reactions (ATP and NADPH) are the reactants in the Calvin cycle, and the products of the Calvin cycle (ADP and NADP + ) are reactants in the Hill reactions, these two processes are interdependent . This means that neither set of reactions can occur in the absence of the other for long. Therefore, despite being often referred to as the “dark reactions”, the Calvin cycle will cease to function in the absence of light.

2 Figure 3. An overview of the light-dependent (Hill) reactions. (By Somepics - Own work, CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=38088695) In this lab, we will be focusing on how alterations in temperature influence the rate of aerobic respiration in both yeast and Euglena . As aerobic respiration is largely dependent on enzymatic reactions, we would expect that the rate of oxygen use will be altered in a manner similar to that seen for most enzymatic activity. That is, as temperature increases to the optimal temperature for enzymatic function, the rate will increase, as more reduced electron carriers will be produced. However, once temperatures increase to those which result in the denaturation of the relevant enzymes, and hence a loss of function, the rate of oxygen usage should decrease as reduced electron carriers are no longer produced, and thus the ETC is no longer active. In this laboratory exercise, we will measure the rate of aerobic respiration as a function of temperature using an oxygen electrode . We will also measure the rate of photosynthesis in Euglena cells as a function of temperature using the oxygen electrode. You will measure the rate of oxygen evolution in “ light conditions ” to determine the observed rate of photosynthesis . However, as Euglena also contain mitochondria, they will utilize aerobic respiration for the production of ATP, regardless of whether light is present or not. Therefore, we will also measure the rate of oxygen consumption in “ dark conditions ” to determine the rate of aerobic respiration. By adding the absolute rates of oxygen consumption in the dark and oxygen evolution in the light we can determine the actual rate of photosynthesis . We will also add a proton ionophore, CCCP, to our assays in order to examine it’s effect on the rates of aerobic respiration and photosynthesis. CCCP functions to dissipate the proton gradient established by the electron transport chains of both processes, but it 3 has a differential effect in our cellular systems. These observations will be provided with you along with the data for these experiments, and an explanation of why this occurs will need to be included in your final lab report.

PROCEDURE Basic procedure for calibrating and using the oxygen probe Your lab instructor will demonstrate the proper procedure for calibrating and using the oxygen electrode. The temperatures and times may differ somewhat depending on which organism you are using, but the basic procedure of calibrating the machines at the different temperatures is the same. See Appendix E: Use of the Oxygen Electrode and Data Analysis for a full explanation of this process.

1. First calibrate the oxygen machine at your starting temperature using the solution found in the tube within the water bath. Be certain that the system has reached the appropriate temperature by checking the thermometer in the water bath. When working with the electrode, it is essential that you avoid piercing the membrane at the bottom of the chamber. Always turn the stir bar off prior to emptying the chamber. Always place the tip of the plastic Pasteur pipette against the side of the chamber .

2. Once you have completed your calibration , d rain the fluid from the reaction chamber with the plastic pipette. Mix the cell culture . Add 2.0 ml to the reaction chamber with a pipette. Leave the top off the chamber at this point. Start the stirrer without adjusting the speed. Adjust the placement of the pen on the chart recorder to allow for accurate measurement (see specific procedure). Then, place the chamber stopper into the reaction cell by slowly screwing it downwards, until the fluid of the culture can be seen just moving up the capillary tube inside the stopper. Stretch a small piece of Parafilm over the top of the stopper, to prevent any casual leakage in the system out of the capillary tube. Check for the presence of an air bubble in the chamber and readjust the chamber stopper until you are confident there isn’t one.

3. Set the chart to record the change in oxygen fo r the specified time .

4. At the conclusion of the run, stop the chart from recording, remove the chamber stopper, stop the stir bar, remove the cells, and wash the chamber three times with distilled water.

4 i. Measuring the rate of aerobic respiration in yeast.

1. Prepare a beaker containing 500 mL of tap water at approximately 40 o C . Add the contents of the provided vial, which contains 1 g of sucrose and 0.5 g of yeast . Stir well to dissolve and allow to sit at room temperature for 10 minutes .

2. Check to make sure that the spectrophotometer is set to 600 nm . Use tap water to BLANK the spectrophotometer and then, after mixing your yeast culture well, determine the absorbance. You should obtain an OD of approximately 0.80 to 1.00 .

3. As we will be comparing data obtained by different groups at different times, it will be necessary to ensure that we have standardized our experiment across days. In order to achieve this, we will need to determine the cell number in the cultures used that day. At 600 nm, an OD of 1.00 is equal to approximately 1 x 10 7 cells/mL . This information is then used to prepare a 20 mL culture of 0.75 x 10 7 cells/mL prior to running the experiment, which ensures that the number of cells utilized by all groups are approximately equal. U se the C 1 V 1 = C 2 V 2 equation (See Lab 2 if you need a refresher on this!) 4. Follow the basic procedure provided on page 35. Calibrate the oxygen machine at 20 o C . After adding your 2.0 mL of yeast culture, reposition the pen on the chart recorder to approximately 70 units by gently bubbling air into the culture. Once the chamber has been properly sealed, record the rate of oxygen consumption for 4 minutes .

5. At the conclusion of the run, stop the chart from recording, remove the chamber stopper, suck out the cells, and wash the chamber three times with distilled water and r epeat the entire measurement procedure at the same temperature with a fresh 2 ml sample of cells from the stock culture . Average your results and record them for that temperature.

6. Adjust the water bath to 3 0 o C . Monitor with the thermometer until this temperature is reached. Re-calibrate the oxygen electrode and chart at this new temperature , using the same procedure you followed before. Do replicate determinations of oxygen utilization at this temperature, using fresh cells for each run ( swirl the flask!! ), as described previously.

7. Inform your lab instructor when you are at the end of your second trial at 3 0 o C. They will add the carbonyl cyanide m-chlorophenyl hydrazone (CCCP) into the reaction chamber for you with an injection syringe. Monitor the effects of CCCP for another 4 min . It is essential that at the end of this run you are extremely thorough when rinsing out the chamber (wash out 6 times and be sure to clean the stopper as well!), as any remaining drug may contaminate the readings obtained in your remaining trials.

8. Repeat the procedure from steps 4 & 5 at 40 o C, 50 o C, and 60 o C, performing replicate trials at each temperature. You do not need to have CCCP added at these temperatures. Remember to recalibrate the machine at each temperature.

5 ii. Measuring the rate of aerobic respiration in Euglena .

1. You need to determine the concentration of the liquid culture of Euglena sp . using the haemocytometer. To do so, place a coverslip on the haemocytometer and add a drop of Euglena culture and a drop of iodine to immobilize/fix the cells. Focus on the haemocytometer using a bright field microscope at 40x magnification. Count the 4 corners and the middle square as shown below. If cells are found on the borders, be consistent and count only those that are on the same two of the four lines. Add the 5 areas and mulitply by 50,000 to obtain the number of cells/mL. An image of what you will see under the microscope is shown below.

2. Use this information and the C 1 V 1 = C 2 V 2 equation (See Lab 2 if you need a refresher on this!) to prepare a 20 mL culture at a concentration of 1.5 x 10 7 cells/mL.

3 . Follow the same procedure as in Part i above (Steps 4-8) using your Euglena culture. The only alteration is that you must remember to leave your culture in the dark, and that you must wrap the reaction chamber with the provided black cloth. This ensures that the cells will only be utilizing aerobic respiration, rather than photosynthesis.

iii. Measuring the rate of photosynthesis in Euglena 1. Use the same culture as provided for Part ii steps 1 & 2 to prepare a 20 mL culture at a concentration of 1.5 x 10 7 cells/mL.

2 . Follow the basic procedure provided on page 35. Calibrate the oxygen machine at 20 o C . After adding your 2.0 mL of Euglena culture, reposition the pen on the chart recorder to approximately 30 units by gently bubbling nitrogen into the culture. Once the chamber has been properly sealed, immediately turn on the desk lamp and place it as close to the reaction chamber as possible. Record the rate of oxygen production for 3 minutes in the light .

3. After 3 minutes, immediately turn off the lamp, swing it out of the way, and cover the reaction chamber securely with the provided black cloth. Record the rate of oxygen utilization for 2 minutes in the dark.

4. At the conclusion of the run, stop the chart from recording, remove the chamber stopper, stop the stir bar, remove the cells, and wash the chamber three times with distilled water and r epeat the entire measurement procedure at the same temperature with a fresh 2 ml 6 sample of cells from the stock culture . Record the average of your trials (average light and average dark) for later analysis.

5. Adjust the water bath to 3 0 o C . Monitor with the thermometer until this temperature is reached. Re-calibrate the oxygen electrode and chart at this new temperature , using the same procedure you followed before. Do replicate determinations of oxygen production and utilization at this temperature using fresh cells for each run ( swirl the flask!! ), as described previously.

6. Once you have completed your second trial at 3 0 o C, set up a third trial, and i nform your lab instructor . They will add the CCCP into the reaction chamber for you with an injection syringe. Monitor the effects of CCCP for another 3 min in the light. It is essential that at the end of this run you are extremely thorough when rinsing out the chamber (wash out 6 times and be sure to clean the stopper as well!), as any remaining drug may contaminate the readings obtained in your remaining trials.

7. Repeat the procedure at 40 o C, and 50 o C, performing replicate trials at each temperature. You do not need to have CCCP added at these temperatures. Remember to recalibrate the machine at each temperature.

iv. Microscopy of Euglena and yeast cells It is essential in any study which utilizes live cells for them to be accessed to ensure that they appear healthy. A wet mount of each of the cultures should be prepared and accessed by phase contrast microscopy at 400x magnification. Do the cells for each culture demonstrate the expected shape? Are the Euglena generally active and demonstrating motility? Can you find any actively dividing yeast in the culture?

Please view the Oxygen Electrode video prior to the Lab 4A Tutorial . A more in depth description of the use of the oxygen electrode, as well as the procedure for data analysis is found in Appendix E: Use of the Oxygen Electrode and Data Analysis . Further explanation on interpretation will be provided in the Lab 4A Tutorial.

Lab 4 Report Lab 4A will be combined with 4B into a formal lab report worth 10% of your final grade and due by 11:59 PM on November 12 th . You should review the General Lab Report Guidelines, as well as the Lab 4 Report Requirements available in the Lab 4 folder on Nexus.

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