An Observation of Aerobic Cellular Respiration Under Varying Conditions

Objective: The purpose of this lab was to observe and analyse the process of Aerobic Cellular Respiration, and the effects that an increase in temperature has on the rate of consumption of O2. The experiment was done same way twice with the exception of a change in temperature. When the temperature of the water was raised the rate of O2 consumption increased. The higher the temperature, the faster O2 is consumed.

Introduction: the Process and Mechanism of Aerobic Cellular Respiration.

ATP (Adenosine Triphosphate), is the main source of energy for living organisms. Energy is stored in the structure of ATP’s three phosphate tails. The energy released from the removal of a Phosphate from ATP powers the majority of metabolic processes in any organism. When a phosphate group is removed, ATP is converted to ADP (Adenosine DiPhosphate); Therefore, there is always a constant cycling between ATP and ADP in all metabolic processes.

In all energy producing Metabolic Processes, one step remains constant; Glycolysis, the breakdown of the sugar molecule for conversion to energy, as summarized by the following equation:

C6H12O6 + 6 O2--> 6 CO2+ 6 H2O + 36 ATP

Aerobic Respiration will be the focus of this investigation. Aerobic respiration is comprised of several reactions in four steps; Glycolysis, Synthesis of Acetyl CoA, the Krebs/Citric Acid Cycle, and the electron transport chain.

Cellular Respiration; An overview.

A=Glycolysis, B=Acetyl CoA synthesis/Krebs cycle, and C=Electron Transport Chain.

Step One; Glycolysis -

In Glycolysis, Glucose is broken down into two pyruvates in the cytoplasm of the cell, each containing 3 Carbons. Two molecules of ATP are required for a cell to start Glycolysis, and four ATP are yielded; therefore, there is a net gain of two ATP and two NADH.

In the presence of oxygen, Aerobic respiration follows Glycolysis, because this process produces the most energy in the form of ATP. The derivatives of Glucose are then broken down in a series of another four steps.

Steps Two and three; Acetyl CoA Synthesis and the Krebs/Citric Acid Cycle-

The Pyruvate produced during Glycolysis proceeds to the mitochondria of the cell. During this Process, the Pyruvate is converted to Acetyl CoA, a Coenzyme used primarily in respiration to convey carbon atoms for oxidation, and in the synthesis and oxidation of fatty acids. When Acetyl CoA is synthesized, a molecule of Carbon Dioxide (CO2) and one NADH molecule are produced. The Acetyl CoA is further broken down in the Krebs Cycle, where for each Acetyl CoA broken down, two CO2 are produced; the energy from that reaction is then stored in ATP. Some of the energy is passed on in the form of electrons via NAD+ and FAD, which are subsequently reduced to NADH and FADH2. Overall there is a net gain of 8 NADH, 2 FADH2, and 2 ATP (keep in mind that the Krebs cycle is completed two times- one for each Pyruvate).

Left; The Synthesis of Acetyl CoA. Right; The Krebs Cycle.

Step Four; The Electron Transport Chain-

As shown in the first figure, The FADH2 and NADH produced in the previous three steps are utilized in the electron transport chain to synthesize more ATP. The NADH and FADH2 transport electrons and hydrogen atoms from to the electron transport chain, a series of proteins in the Mitochondrial Membrane. Once the electron carriers are stripped of their hydrogens and electrons, they return to other sites of the cell as NAD+ and FADH to repeat the process. The electrons from the carriers is passed along the proteins of the Electron transport chain, the energy released shuttles the hydrogen ions into the intermembrane space of the mitochondria, as shown below. The ions create a gradient as they amass, and the resulting pressure creates a flow of ions through the channel protein ATP Synthase. The rush of ions through the Protein powers it and drives the production of ATP; ATP Synthase adds another phosphate group to ADP, thus synthesizing ATP. The electron transport chain produces 16 ATP for each Pyruvate and its resulting electron carriers; since their are two pyruvates to glucose, there is a net gain of 32 ATP.

The Electron Transport Chain.

In the last step of the electron transport chain, oxygen comes in; once the electrons reach the end of the pathway, they are passed to O2, resulting in H2O. Oxygen enables the electron transport chain to function; without it there to pick up electrons, no more electrons would be able to pass through the chain from NADH and FADH2, crippling the entire system.

Hypothesis: If the temperature is raised, the rate of consumption of O2 will increase.

• Independent Variables; Time and Temperature.
• Dependent Variable; Volume
• Controlled Variable; Beads.

Two separate procedures were created for two models. Both models were completely identical, with the exception of raised temperature in model 2.

Model 1 serves as the control group for Model 2, the baseline on whose data Model 2 could be conducted and analyzed on. All data was collected into the following table, one for each model;

Respirometer 1: Germinating Peas. Respirometer 2: Beads

Degrees C. Time (Min) V of Pipet ΔV Corrected ΔV V of Pipet ΔV

24 0 - - -

24 5

24 10

24 15

24 20

ΔV= Volume at time zero - Volume at time of Current reading.

Corrected ΔV= ΔV for respirometer 1 - ΔV of respirometer 2.

Respirometers were used to model Cellular Respiration, using Germinating peas and KOH Solution. During the experiment, the respirometer was completely submerged in a water bath at room temperature; when the peas respired, oxygen was consumed and Carbon

Dioxide released. the KOH solution altered the Equilibrium produced by the respiration between the Oxygen and CO2, based on the following reaction;

CO2 + 2KOH = K2CO3 + H2O

Because CO2 is produced from the reaction with KOH as oxygen is used in Respiration, the volume of gas will decrease. Due to the nature of the reaction, these chemicals and respirometers were used to model Cellular Respiration.

The Experiment

Materials

Tray of room temperature water

25 Germinating Peas

Plastic Beads

2 Respirometers

50 mL Graduated Cylinder

3 Absorbent cotton balls

Nonabsorbent cotton balls 2 Rubber Stoppers

15% KOH solution

dropping Pipet

forceps

Paper

Stopwatch/timer

Thermometer

Procedure

1. Set up the water baths. The water bath will buffer the respirometers against temperature changes during the experiment.
2. Place a sheet of paper in the bottom of the water bath. This makes the Graduated pipet easier to read.
3. Place a thermometer in each tray. Observe the thermometer to make sure the temperature of the water is stable.
4. Prepare the peas and beads.
5. for respirometer one, put 25 mL of water in your 50 mL graduated tube. Drop in the 25 Germinating peas. Determine the volume of water that has been displaced, which is equivalent to the volume of peas. Record the volume of the peas, and place them on a paper towel.
6. For Respirometer two, refill the graduated tube with 25 mL of water. Add enough beads to equal the volume of the germinating peas. Remove these beads and place them on a paper towel.
7. Prepare samples.
8. place an absorbent cotton ball in the bottom of each respirometer vial.
9. Use a dropping pipet to saturate the cotton with 2 mL of 15% KOH solution. Do not get
10. Place a small wad of dry, nonabsorbent cotton on top of the KOH soaked cotton ball to prevent the KOH solution from contacting the peas, The amount of cotton and KOH must be the same for both respirometers.
11. Place 25 germinating peas in the vial of respirometer 1.
12. Place the equivalent volume of beads in the vial of respirometer 2.
13. Insert a stopper filled with a calibrated pipet into each respirometer vial. The stopper must fit tightly. If the respirometers leak, you must start over.
14. Place the set of respirometers in the water bath with the pipet tips resting on the lip of the tray. Wait 5 minutes before proceeding, to allow time for the respirometers to reach thermal equilibrium with the water. If they begin to leak, you must start over.
15. After the equilibration period, immerse all the respirometers completely in the water bath. Position them so that you can read the scale on each of the pipets.
16. Allow the respirometers to equilibrate for another 5 minutes.
17. Observe the initial volume reading on the scale to the nearest .01 mL. Record the data in table 1 for time zero. Also, observe and record the temperature of the water. Repeat your observations for both the two samples. Every 5 minutes for 20 minutes, record the temperature and take readings of the volume of the air in each of the two pipets. Record the data in table 1 and complete the calculations necessary to complete the table.

Note; The procedure for Model 2 is identical with the

exception of heating the water bath before hand.

Results

Procedure 1

Respirometer 1: Germinating Peas. Respirometer 2: Beads

Degrees C. Time (Min) V of Pipet ΔV Corrected ΔV V of Pipet ΔV

24 0 .9 - - .9 -

24 5 .8 .1 .7 .8 .1

24 10 .7 .2 .61 .81 .09

24 15 .71 .19 .62 .81 .09

23 20 .57 .33 .51 .84 .06

ΔV= Volume at time zero - Volume at time of Current reading.

Corrected ΔV= ΔV for respirometer 1 - ΔV of respirometer 2.

Note; All volume is expressed in Milliliters.

Model 1 showed a rather linear rate for both Respirometers; until the 10 minute mark, the O2 was consumed at more or less the same rate. After that point, O2 consumption increased dramatically for respirometer 1 in comparison to Respirometer 2.

As shown in the graph above, close to the middle of the 20 minute test period, the volume of oxygen in respirometer 1 rose ever so slightly from .7 to .71 mm, then continued to decrease. Similarly, in Respirometer 2, the volume showed an increase from .8 to .81 mm. These increases occurred most probably because as the oxygen consumption rate rose, the levels grew sufficient enough to meet the cell's needs; Thus, the oxygen levels rose due a temporary lapse in respiration, and decreased again as the cell’s supply needed replenishment.

Procedure 2

Respirometer 1: Germinating Peas. Respirometer 2: Beads

Degrees C. Time (Min) V of Pipet ΔV Corrected ΔV V of Pipet ΔV

35 0 1 - - 1 -

35 5 .5 .5 .35 .85 .15

35 10 .35 .65 .4 .75 .25

35 15 0 1 .5 .5 .5

34 20 0 1 .4 .4 .6

ΔV= Volume at time zero - Volume at time of Current reading.

Corrected ΔV= ΔV for respirometer 1 - ΔV of respirometer 2.

Note; All volume is expressed in Milliliters.

As predicted, the rate of O2 consumption practically skyrocketed compared to Model 1, proving our hypothesis and shedding light on the results of Model 1; due to the increased temperature, and subsequently the faster rate of production, Oxygen was consumed and used fast enough to prevent a slight rise in O2 levels and keep the consumption rate steadily downward, until all O2 was consumed at a very early stage (see graph below).

Respirometer 2 showed a considerably lower rate of respiration in both models 1 and 2 compared with Respirometer 1; as a tube filled with inorganic plastic beads, not very much O2 was consumed by the respirometer.

Conclusion

As shown by the data exhibited by the investigation, temperature regulates and increases the rate of Aerobic Cellular Respiration. Over the course of two 20 minute time intervals, both models showed that with the increase of time and temperature, the rate of O2 consumption increased; as time went on and the process gained momentum, the rate of respiration increased and thus volume decreased, and as temperature increased, volume decreased even more dramatically. As shown by the upward shift in volume in Model 1 (see graph), higher temperature also moderates the process of Respiration; the temperature keeps the process running at a constant, quicker rate- thus, the process is more intensive and draining, keeping volume at a consistent decrease.

While conclusive and accurate overall, the results obtained are somewhat irregular; due to some faulty laboratory conditions, the results could not be 100% accurate. However, with more precise scientific equipment in a more controlled and sterile laboratory setting, any inaccuracies could be avoided. Additionally, human error is to be blamed; in the middle of testing Model 2, the group was forced to evacuate due to a fire drill, putting our timing off by approximately two minutes.

All results indeed confirmed and abided by the principles of Cellular Respiration, and support all current theories.