Measuring Respiration of Germinating and Non-germinating Peas

Measuring Respiration of Germinating and Non-germinating Peas
By: Krunal Patel

Introduction

          Living cells require transfusions of energy from outside sources to perform their many tasks – for example, assembling polymers, pumping substances across membranes, moving, and reproducing (Campbell, and Reece 162). Heterotrophs obtains its energy for its cells by eating plants that makes it own food (Autotrophs); some animals feed on other organisms that eat plants. The most beneficial catabolic pathway in an organism is cellular respiration, in which oxygen and glucose are consumed and where carbon and water become the waste products. The purpose of cellular respiration is to convert glucose into ATP(energy) for the organism. Respiration consists of glycolysis, the Krebs Cycle, and the oxidative  phosphorylation. Glycolysis, which occurs in the cytosol, breaks the six carbon glucose molecule into two pyruvates. During this stage two ATP and two NADH molecules are made. The next step in respiration is the Krebs cycle. The Krebs cycle uses the two pyruvates made during glycolysis and converts them to Acetyl-CoA and carbon dioxide to make three NADH, one FADH₂,  and two CO₂ through redox reactions, and goes to the Electron Transport Chain. ATP is also formed during the Krebs cycle (Campbell, and Reece 166). Since two pyruvates are made during glycolysis, the Krebs cycle repeats two times to produce four CO₂, six NADH, two FADH₂, and two ATP (Campbell, and Reese 166). The last stage in cellular respiration is the Oxidative phosphorylation Electron Transport. The Oxidative phosphorylation occurs in the inner membrane of the mitochondria. The electron transport chain is powered by electrons from electron carrier molecules NADH and FADH₂ (Campbell, and Reese 166). As the electrons flow through the electron chain, the loss of energy by the electrons is used to power the pumping of electrons across the inner membrane. At the end of the electron transport chain, the electrons from the inner membrane bind to two flowing hydrogen ions to form water molecules. The protons, outside the inner membrane, flow down the ATP gradient and make a total of thirty two ATP (Campbell, and Reese 166).

In this experiment, an apparatus called a respirometer is used. A respirometer is a tool used to observe exactly how much oxygen was consumed by the peas and the glass beads. Since the carbon dioxide produced is removed by reaction with potassium hydroxide (Forming K₂CO₃ + H₂O as shown below), as oxygen is used by cellular respiration the volume of gas in the respirometer will decrease. As the volume of gas decreases, water will move into the pipet. This decrease of volume, as read from the scale printed on the pipet, will be measured as the rate of cellular respiration (Cell Respiration).

CO₂ + 2KOH —> K₂CO₃ +H₂O

The purpose of this lab was to measure the rate of cellular respiration. There are three ways to measure the rate of cellular respiration. These three ways are by measuring the consumption of oxygen gas, by measuring the production of carbon dioxide, or by measuring the release of energy during cellular respiration (Respiration). In order to measure the gases, the general gas law must be understood. The general gas law state: PV=nRT 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 (Respiration). The rate of respiration of germinating and non-germinating peas in this experiment was determined by the consumption of oxygen. Potassium Hydroxide (KOH) was used to alter the equilibrium. KOH removed the carbon dioxide and oxygen was used by cellular respiration thus decreasing the gas in the respirometer. The rate of respiration in germinating peas was compared to the rate of the non-geminating peas. These peas were placed in two different temperatures: 10ºC and 23ºC.

The hypothesis of this lab states that if the peas are germinated then the rate of cellular respiration will be higher in both room temperature and cold temperature. If the temperature of water is cooler than room temperature, then the process of cellular respiration of the peas will decline.

Materials

v  Room-Temperature Water Bath                      Nonabsorbent Cotton

v  Cold Water Bath                                             15% Potassium Hydroxide (KOH) Solution

v  Container of Ice                                              Dropping Pipets

v  Paper (White or Lined)                                   Forceps

v  Water                                                              Thermometers

v  Germinating Peas                                            Stopwatch (Timer or Clock)

v  Nongerminating Peas                                      Calculators (Optional)

v  Glass Beads                                                    Absorbent Cotton Balls

v  Respirometers                                                 Graduated Tube

Procedure

Setup of Respirometers and Water Baths

There are two water baths (trays of water) to buffer the respirometers against temperature change and to provide two temperatures for testing: room temperature and a colder temperature (Approx. 10°C). Place of sheet of paper in the bottom of each water bath. This will make the graduated pipet easier to read. Next, place a thermometer in each tray. If necessary, add ice to the cold-temperature tray to further cool the water to get it as close to 10°C as possible. While waiting for the cold- water temperature to stabilize at 10°C, prepare the three respirometers to test at room temperature, and prepare an identical set of three respirometers to test at the colder temperature.

Prepare Peas and Glass Beads

Respirometer 1: Put 25 mL of H₂O in your 50-mL graduated plastic tube. Drop in 25 germinating peas. Determine the volume of water that is displaced (equivalent to the volume of peas). Record the volume of the 25 germinating peas. Remove these peas and place them on a paper towel.

Respirometer 2: Refill the graduated tube to 25 mL with H₂O. Drop 25 dry, nongerminating peas into the graduated cylinder. Next, add enough glass beads to equal the volume of the germinating peas. Remove the nongerminating peas and beads and place them on a paper towel.

Respirometer 3: Refill the graduated tube to 25 mL with of H₂O. Add enough glass beads to equal the volume of the germinating peas. Remove these beads and place them on a paper towel.

The independent variable is the type of peas (Germinated or Nongerminated) and the temperature (Room or Cold Temperature). The dependent variable is the consumption of oxygen from all 6 respirometers. The control group is respirometer three from both temperatures that consists of only glass beads.

Respirometer Assembly

This requires three respirometers for room-temperature testing and three respirometers for cold-temperature testing.

To assemble a respirometer, place an absorbent cotton ball in the bottom of each respirometer vial. Use a dropping pipet to saturate the cotton with 2 mL of 15% KOH. (Caution: Avoid skin contact with KOH. Be certain that the respirometer vials are dry on the inside. Do not get KOH on the sides of the respirometer.) Place a small wad of dry, nonabsorbent cotton on top of the KOH- soaked absorbent cotton. The nonabsorbent cotton will prevent the KOH solution from contacting the peas. It is important that the amount of cotton and KOH solution be the same for each respirometer.

• Place 25 germinating peas in the respirometer vial(s) 1.
• Place 25 dry peas and beads in your respirometer vial(s) 2.
• Place beads only in your respirometer vial(s) 3.

Insert stopper fitted with a calibrated pipet into each respirometer vial. The stopper must fit tightly. If the respirometers leak during the experiment, you will have to start over.

Placement of Respirometers in Water Baths

Place a set of respirometers (1, 2, and 3) in each water bath with their pipet tips resting on lip of the tray. Wait five minutes before proceeding. This is to allow time for the respirometers to reach thermal equilibrium with the water. If any of the respirometers begins to fill with water, the experiment will have to restarted.

After the equilibrium period, immerse all respirometers (including pipet tips) in the water bath. Position the respirometers so that it’s easy to read the scales on the pipets. The paper should be under the pipets to make reading them easier. Do not put anything else into the water bath or take anything out until all readings have been completed.

Take Readings

Allow the respirometers to equilibrate for another five minutes. Then, observe the initial volume reading on the scale to the nearest 0.01 mL. Record the data in Table 1 for Time 0. Also, observe and record the temperature. Repeat your observations and record them every five minutes for 20 minutes.

(Cell Respiration)

Results/Data Collection

Table 1: Respiration of Peas at Room Temperature

*All values are in mL except °C and Time

Table 2: Respiration of Peas at 10° C

*All values are in mL °C and Time

Discussion/Conclusion

The results of this lab show that the germinating peas had consumed more oxygen at a faster rate than the non-germinating peas and the beads had. The non-germinating peas and the beads showed to consume barely any oxygen at all. In this lab, the germinating peas respiration rate proved to be faster than the respiration rate of non-germinating peas. Finally, this experiment showed that respiration rates increase as the temperature increases. Concluding that temperature and respiration rates are directly proportional and have a direct relationship to each other. From this experiment, it can also be concluded that the germinating peas that were undergoing the process of cellular respiration had a much higher oxygen consumption rate than the consumption rate in non-germinated peas and the glass beads. The non-germinating peas shows hardly any consumption of oxygen. Since the germinating peas are germinating or sprouting, they require a more extensive amount of energy or ATP. This allows them to have high oxygen consumption rates or respiration rates in this experiment. In addition to the germinating peas, the non-germinating peas, are not germinating so because of this they do not need significant amount of ATP production. Therefore, the non-germinating peas have a significantly low rate of respiration in comparison with the germinating peas. The rate at which they respire was most prevalent in the first respirometer since they were all germinating peas. The data table and graph accurately depict this idea or trend with the germinating and non-germinating peas.

Numerous errors could have occurred during the lab. Miscalculation of the glass beads which went into the respirometer, and hence would ruin the controlled results. The seals on the respirators may not have been completely air-tight which may have caused a leak and therefore oxygen would have been lost altering the data.  The temperature may have been slightly off in the water baths. There was also the problem of reading the scales on the pipets which could have lead to improper measurements of the water position.

To improve this experiment, more accurate and precise instruments can be used such as a advanced pipet which has scales that are easy to read. Also a completely air tight respirometer instead of using petroleum jelly which water can still leak in.

Literature Cited

Campbell, Neil A., and Jane B. Reece. Biology. Eighth Ed. San Francisco: Pearson Benjamin Cummings, 2008. Print.

Cell Respiration. AP Biology Laboratory 5: Carolina Biological Supply Co., 2005. Print.

“Respiration.” StudyMode.com. StudyMode.com, 06 2011. Web. 06 2011. http://www.studymode.com/essays/Respiration-713319.html