Cellular Respiration

Introduction

The reactions within cells which result in the synthesis of ATP using energy stored in glucose are referred to as cellular respiration. Aerobic respiration requires oxygen as the final electron acceptor. Fermentation does not require oxygen.

The equation for aerobic respiration is below.

$\mathrm{C_6H_{12}O_6 + 6O_2 → 6CO_2 + 6 H_2O + 36\: or\: 38\: ATP}$

In aerobic respiration (equation above) glucose is completely broken down to CO2 + H2O but during fermentation, it is only partially broken down. Much of the energy originally available in glucose remains in the products produced. Plant and fungal cells produce alcohol as a result of fermentation and animal cells produce lactic acid. The equation for alcohol fermentation is below.

$\mathrm{C_6H_{12}O_6 → 2CO_2 + 2C_2H_5OH + 2\: ATP}$

Notice from the above equations that aerobic respiration produces much more ATP per glucose molecule than fermentation.

Fermentation

We will investigate fermentation by measuring the amount of carbon dioxide produced by yeast. The rate of cellular respiration is proportional to the amount of CO2 produced (see the equation for fermentation above).

In this experiment, we will measure the rate of cellular respiration using either distilled water or one of four different food sources.

First, create a hypothesis regarding the rate of cellular respiration for each of the different food sources listed in the step below. The words "gas bubble" or something similar should be used in your hypothesis.

Fill each of five small test tubes one-half full with the solutions listed below. Each tube should be filled to exactly the same level.

• Tube 1 - glucose (a monosaccharide)
• Tube 2 - fructose (a monosaccharide)
• Tube 3 - sucrose (a disaccharide)
• Tube 4 - starch
• Tube 5 - distilled water

Use a dropper to finish filling each tube with a thoroughly-mixed yeast suspension. Be sure to mix the yeast suspension immediately before adding it to the tubes. The tube should be filled as full as possible while holding it over a sink. Carefully invert a larger tube and place it over each of the smaller tubes containing the yeast suspension. Push the smaller tube all the way into the larger tube using your finger or a pencil and then invert both tubes so that the opening of the larger tube is up. Be sure that the small tube is pushed to the top of the larger tube before inverting it.

Below: Tubes containing yeast and a sugar solution are inverted so that CO2 produced by the yeast can collect.

 Click on the image to view an enlargement. Press "back" to return here.

Place the five test tubes in a 37 degree incubator and record the time.

The tubes should be checked approximately every 5 minutes to observed the size of the gas bubble that accumulates in the small tube.

The experiment should be stopped when the gas bubble in any of the tubes is approximately one half the length of the tube. Record the time when the experiment is terminated.

Record the time when the tubes were removed from the incubator.

 The level of the liquid can be seen through the sides of the test tubes. Click on the image to view an enlargement. Press "back" to return here.

After the tubes are removed from the incubator, hold each tube over a sink and quickly invert them as shown below. Use your finger or a pencil to keep the small tube in place while inverting so that the liquid inside the small tube remains in the small tube. Lift the larger tube off of the smaller tube and set the smaller tube in a test tube rack. Repeat this procedure with the other tubes.

The size of the gas bubble produced by the yeast can be measuring the amount of liquid remaining in the tube and subtracting it from the total volume of the tube. Measure the amount of liquid in each of the tubes with a 10 ml graduated cylinder and record that value in your notebook.

Obtain an empty, small tube and measure its volume using a 10 ml graduated cylinder. With this number you can calculate the volume of gas produced in each tube by subtraction as described above. Perform this calculation for each tube and enter the values in your notebook.

5) Which food source produced in the highest rate of cellular respiration? Which food source produced the slowest rate? Explain your results for tube 5 (distilled water).

Below: The level of liquid in each of the tubes below is indicated with a blue line. Notice that each of the sugars (glucose, fructose, and sucrose) produced approximately the same amount of CO2. Sucrose is expected to produce CO2 at a slower rate because it is a disaccharide and must first be converted to glucose by the cell. Fructose is easily converted to glucose by yeast cells. Yeast cells in water produced little CO2 because they do not have a source of sugar.

Aerobic Respiration

We will measure the rate of aerobic cellular respiration in beans by measuring the volume of O2 consumed using the apparatus shown below. The apparatus consists of two test tubes with stoppers and a graduated pipette inserted into each stopper. A colored liquid is placed in each of the pipettes. When the volume of gas in the test tube changes, the liquid in the pipette will move.

 The assembled respirometer apparatus. Click on the photograph to view an enlargement.

Oxygen consumption cannot be measured simply by putting beans in the test tubes because beans are also producing CO2. Any change in gas volume will be due to both O2 consumption and CO2 production. In order to minimize the confounding effect of CO2, KOH will be added to the tubes. It reacts with CO2 to form solid potassium carbonate. The solid will not have a measurable increase in the volume inside the tubes.

$\mathrm{CO_2 + 2KOH → K_2CO_3 + H_2O}$

Temperature will also affect the measurement of gas consumption because gasses expand when they warm and contract when they cool. This effect will be minimized by keeping the respirometer tubes immersed in water at room temperature. Water temperature changes slowly, so the water will minimize temperature fluctuations inside the tubes.

Create a hypothesis regarding the movement of dye in the tube containing germinated bean seeds. Record this hypothesis in your notebook.

Obtain the materials for assembling the respirometer apparatus. A tank containing water at room temperature will be needed to hold the three respirometer tubes. Two large test tubes with stoppers and graduated pipettes will also be needed.

Push a small wad of cotton to the bottom of each of two respirometer tubes. The cotton should occupy approximately 2 cm of space on the bottom of the tube. Use a dropper to add 15% KOH solution to the cotton in each tube. Use enough KOH to saturate the cotton but not enough to pour out of the test tube. Use the same amount of KOH in each tube. Be careful not to let KOH come in contact with the sides of the test tube because it will kill the bean seeds.

Push a small wad of dry cotton on top of the KOH-saturated cotton in each tube. This will prevent KOH from coming in contact with the bean seeds and killing them.

Fill one of the tubes half full with germinated bean seeds and record the number of bean seeds used.

The second tube will remain empty to measure the effect of temperature changes.

Assemble each apparatus and place the tubes in the water tank. Be sure that the rubber stopper is inserted tightly into the test tube. The valve in the stopper of each tube should be kept open so that air can move through the stopper and into or out of the tube.

 The valve shown at the right should be open while you add the marker dye.

Before adding the marker dye, double-check to see that the valve on top of the stopper is open and that air can pass through. Sometimes the rubber tube that passes through the valve remains squeezed shut even though the valve is open. Be sure that you can see an opening through the tube.

After the respirometer tube is inserted into the water tank, use 200 ul micropipette to push a small amount of colored liquid into the tip of each of the graduated pipettes as shown below. The glass pipette is marked with milliliter marks but the narrow tip does not have any marks. The dye should be pushed far enough into the pipette that it is in the area where there are milliliter marks. This will enable you to read how far the dye moves from the beginning of the experiment to the end. The dye in the control tube (the tube without beans) should be inserted far enough so that outward movement can be measured if the temperature changes.

8) Record the time that the respirometer is set up and the dye is in place. You will be ready to begin the experiment after the tubes have been in the water for 10 minutes.

Before beginning the experiment, be sure that the dye in the pipette is in a region of the pipette that has calibration marks. This is important because you will measure the amount of movement of the dye. Also, check the rubber stoppers to be sure that they are firmly inserted into the test tubes.

After the respirometer has been idle for 5 minutes, close the valve in the top of each of the tubes. After the valves are closed, any air movement into the tubes will cause the dye in the pipette to move inward. After the valves are closed, record the position of the dye and continue to record its position every 10 minutes for a total of thirty minutes.

When you are finished, do not disassemble the apparatus. It will be needed in the temperature experiment below.

Effect of Temperature

In the experiment below, you will use the respirometer that does not contain bean seeds from the apparatus above and expose it to hot and cold temperatures. Create hypotheses regarding the movement of the liquid in the respirometer tube when it is placed in cold water and when it is warmed. Record these hypotheses in your notebook.

Place the respirometer in a beaker of ice water to observe movement of the fluid in the pipette. Remove the tube from the icewater before the marker dye moves all the way into the tube. Next, warm the tube by wrapping your hand around it. What happened in each case? Record your observations in your notebook.

Questions

10) What is the function of using a respirometer without any bean seeds in this experiment?

11) What two gasses are involved in aerobic respiration? How should they have affected the dye in the tube that contained germinated bean seeds? To answer this question, review the equation for cellular respiration and review the discussion of KOH above.

12) Explain how temperature fluctuations affect the apparatus and how these were corrected.Your answer to this question should mention the use of subtraction to correct for temperature change.