Patrick McCrystal Cellular Respiration: From O2 to CO2 Purpose: This lab provided insight to the process of cellular respiration

and how it is affected by temperature in both germinating and dormant pea seeds. Cellular respiration is an ATP-producing catabolic process in which the electron receiver is an inorganic molecule. It is the release of energy from organic compounds by chemical oxidation in the mitochondria within each cell. Carbohydrates, proteins, and fats can all be metabolized, but cellular respiration usually involves glucose: C6H12O6 + 6O2 → 6CO2 + 6H2O + 686 Kcal of energy/mole of glucose oxidized. Cellular respiration involves glycolysis, the Krebs cycle, and the electron transport chain. Glycolysis is a catabolic pathway that occurs in the cytosol and partially oxidizes glucose into two pyruvate (3-C). The Krebs cycle occurs in the mitochondria and breaks down a pyruvate (Acetyl-CoA) into carbon dioxide. These two cycles both produce a small amount of ATP by substrate-level phosphorylation and NADH by transferring electrons from substrate to NAD+. The Krebs cycle also produces FADH2 by transferring electrons to FAD. The electron transport chain is located at the inner membrane of the mitochondria and accepts energized electrons from enzymes that are collected during glycolysis and the Krebs cycle, and couples this exergonic slide of electrons to ATP synthesis or oxidative phosphorylation. This process produces most of the ATP. Cellular respiration can be measured in two ways: the consumption of O2 (how many moles of O2 are consumed in cellular respiration) and production of CO2 (how many moles of CO2 are produced in cellular respiration). PV = nRT is the formula for the inert gas law, where P is the pressure of the gas, V is the volume of the gas, n is the number of molecules of gas, R is the gas constant, and T is the temperature of the gas in degrees K. This law shows several important things about gases. If temperature and pressure are kept constant then the volume of the gas is directly proportional to the number of molecules of the gas. If the temperature and volume remain constant, then the pressure of the gas changes in direct proportion to the number of molecules of gas. If the number of gas molecules and the temperature remain constant, then the pressure is inversely proportional to the volume. If the temperature changes and the number of gas molecules is kept constant, then either pressure or volume or both will change in direct proportion to the temperature. Methods: During the lab, we prepared both a room temperature and a 10oC water bath. Then, we filled a 50 mL graduated cylinder halfway with water. We added 25 germinating peas and determined the amount of water that was displaced. Then we removed the peas, placed them on a paper towel, refilled the graduated cylinder, and added glass beads to the graduated cylinder until the volume was equivalent to that of the expanded germinating peas. We removed the beads, refilled the graduated cylinder, added 25 non-germinating peas, and then added more glass beads until the volume was once again equal to the germinating peas’ volume. After all that was done, we prepared another set of peas and beads for the last 3 respirometers. Assembly of the respirometers was the next step. We obtained 6 vials, stoppers, and graduated pipettes. Then we placed a wad of absorbent cotton in the bottom of each vial and, using a pipette, saturated the cotton with about 2-3 mL of 15 % KOH. We then placed a layer of non-absorbent cotton on top of the KOH-soaked cotton in order to protect the peas from the KOH. We placed the first set of germinating peas, dry peas and beads, and beads alone in vials 1, 2, and 3, and the second set in vials 4, 5, and 6, then placed the stoppers in each vial. We made slings out of masking tape in

order to hold the pipettes out of the water for the 10 minute equilibration period and placed the vials on them (1, 2, and 3 in the room temperature bath, 4, 5, and 6 in the 10oC bath). The 10 minute period was necessary to ensure that a difference in temperature between the air in the vial and the water would not skew our results. Once the vials were properly adjusted, we lowered them into the water. Thankfully, the water did not rush into the respirometer, which would have indicated a leak. We then recorded the reading on the pipette at set time periods. Results: Measurement of O2 Consumption by Soaked and Dry Pea Seeds at Room Temperature and 10˚C Actual Temp (oC) 21 20 20 20 20 9 Beads Alone Reading at time X .87 .9 .9 .87 .86 .86 -.03 -.03 0 .01 Diff. Germinating Peas Reading Diff. at time X .9 .79 .7 .62 .5 .85 .11 .2 .28 .4 .14 .23 .28 .49 Corrected Diff.∆ Dry Peas and Beads Reading Diff. at time X .89 .9 .89 .89 .89 .89 -.01 0 0 0 .02 .03 0 -.01 Corrected Diff.∆

Time (Min) Initial0 0-5 0-10 0-15 -20 Initial0 0-5 0-10 0-15 0-20

10 .84 .02 .79 .04 .02 Oxygen Comsumption of.06 Germinating.87 Peas, Non -0 10 .85 .01 .1 .9 -.01 Germinating Peas,.74 Glass Beads at 20˚C and -.02 and .11 10 .87 .01 .69 10˚C .16 .17 .92 -.03 -.02 10 .89 -.03 .65 .2 .23 .95 -.06 -.03

Beads Alone 20˚ Germinating Peas 20˚ Dry Peas and Beads 20˚ Beads Alone 10˚ Germinating Peas 10˚ Dry Peas and Beads 10˚

1 Oxygen Consumption 0.8 0.6 0.4 0.2 0 0 0-5 0-10 Time 0-15 -20

It was necessary to compare the reading from the peas with the reading from the beads because the beads served as a control variable, therefore, the beads experienced no change in gas volume. Germinating seeds have a higher metabolic rate and need more oxygen for growth and survival. Non-germinating peas, though alive, need to consume far less oxygen in order to survive. The KOH absorbed the carbon dioxide and caused it to form a precipitate at the bottom of the vial, preventing it from changing the pressure in the vial. When the peas underwent cellular respiration, they consumed oxygen and released carbon dioxide, which reacted with the KOH in the vial, resulting in a decrease of gas in the pipette. The water moved into the pipette because the pressure in the pipette lessened. Calculations: Condition Germinating Peas/ 10 oC Germinating Peas/ 20 oC Dry Peas/ 10 oC Dry Peas/ 20 oC Calculations (0.85-0.65) 20 min. (0.9-0.5) 20 min. (0.89-0.95) 20 min. (0.89-0.89) 20 min. Rate in mL O2/ minute .01 .02 -.003 0

Conclusion: The lab demonstrated many important things relating to cellular respiration. It showed that the rates of cellular respiration are greater in germinating peas than in non-germinating peas.

It also showed that temperature and respiration rates are directly proportional; as temperature increases, respiration rates increase as well. Because of this fact, the respirometers placed in the water at 10 oC displayed a lower rate of cellular respiration than the respirometers placed in the room temperature water. The non-germinating peas consumed far less oxygen than the germinating peas. This is because, though germinating and non-germinating peas are both alive, germinating peas require a larger amount of oxygen to be consumed so that the seed will continue to grow and survive. In the lab, CO2 made during cellular respiration was removed by the potassium hydroxide (KOH) and created potassium carbonate (K2CO3). It was necessary that the carbon dioxide be removed so that the change in the volume of gas in the respirometer was directly proportional to the amount of oxygen that was consumed. The result was a decrease in gas volume within the tube, and a related decrease in pressure in the tube. The respirometer with just the glass beads served as a control group that did not undergo cellular respiration. Numerous errors could have occurred throughout the lab. The temperature of the baths may have been allowed to fluctuate, which would change the temperature in the vials. The amounts of peas, beads, KOH, and cotton may have varied from vial to vial. Air may have been allowed to creep into the vial via a leaky stopper or poorly sealed pipette. The vials may have not properly equilibrated, and students could have read the pipettes either too soon or too late. Students may have misread pipettes. KOH could have come into contact with the sides of the vials when it was being dropped onto the cotton. Mathematical inaccuracies may have occurred when completing the table.

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