2nd Edition Introductory Biology
Student Manual
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Introductory Biology Lab Manual
© 2013 eScience Labs, LLC. All rights reserved. This material may not be reproduced, displayed, modified, or distributed, in whole or in part, without the express prior written permission of eScience Labs. Appropriate citation(s) must accompany all
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3
Lab 9
Cellular Respiration
Cellular Respiration
Learning Objectives
x Compare and contrast aerobic respiration to anaerobic respiration including the role of ADP and
ATP
x Relate glucose to pyruvate during the process of glycolysis
x Explain the role of the citric acid cycle and oxidative phosphorylation in the process of aerobic respiration
x Explain the role of fermentation in the process of anaerobic respiration
Introduction
Cellular respiration refers to the process of converting chemical energy existing within organic molecules,
such as sugars, into a form which can be immediately used by organisms. This process is required for both
aerobic (organisms which have oxygen available) and anaerobic (organisms which do not have oxygen available) organisms, but it is executed differently based on the compounds and structures local to the host. However, both processes share the common goal of harvesting biological energy stored in fuel molecules (such
as glucose) and convert this energy into ATP. The reaction is written as follows:
C6H12O6
(glucose)
+
6 O2
(oxygen)
Æ
6 CO2
+
6 H2O +
(carbon dioxide)
energy
(water)
ADP and ATP
Adenosine triphosphate, or ATP, is often referred to as the
energy currency of the cell. It is used in the majority of cellular
functions including active transport across a membrane, cell
structure maintenance, DNA and RNA synthesis, protein synthesis, etc. However, even though this molecule is extremely important, sometimes more ATP is required than is available.
When ATP levels become too low, a special protein signals the
cell to begin respiration.
Respiration occurs in anaerobic and aerobic organisms, therefore, there is more than one pathway used to produce ATP.
However, a high level analysis can conclude that in both types
of organisms, insufficient concentrations of ATP can trigger a
series of metabolic reactions which ultimately replenish the local
ATP supply. These reactions are primarily catabolic; in other
Figure 1: ATP (above) consists of the nucleotide adenine, a 5-sided sugar (ribose), and
three phosphates. Each is bound to three
oxygen molecules. Cleavage of one phosphate molecule from ATP yields the amount
of energy (calories) found in one peanut.
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Cellular Respiration
words, they break down big molecules into smaller pieces. Free energy is released when the molecules are
broken down, which can be sequentially used in each proceeding step. Through a series of oxidation reactions, the cell converts carbohydrates into carbon dioxide and water. It also transforms adenosine diphosphate (ADP) into ATP by adding a free phosphate molecule to ADP. This set of reactions provides a constant source of energy for the cell, as long as all the critical components for the reactions are available.
Glycolysis
The first stage of respiration is called glycolysis. Glycolysis occurs with or without oxygen and takes place in
the cytoplasm. During glycolysis, glucose is broken down to provide energy for the cell. The glycolytic pathway is highly conserved amongst organisms, and is found in all living organisms.
Glucose contains high energy bonds which release electrons (energy) when broken. Through this process,
the six-carbon glucose molecule is broken down into two, three-carbon molecules called pyruvic acid
(pyruvate). Though the bonds holding pyruvate together contain a great deal of potential energy, this step
yields little energy. Only two ATP molecules and two nictotinamide adenine dinucleotide (NADH) molecules
are produced during glycolysis.
Aerobic Respiration vs. Fermentation
The next stage of respiration depends on if the organism is aerobic or anaerobic; or, in the case of multicellular organisms such as plants, if oxygen is available to the cell. In an aerobic environment, the citric acid cycle
begins. Conversely, if an anaerobic environment exists, fermentation or anaerobic respiration takes place.
Plants primarily engage in aerobic respiration. However, in rare cases such as extremely wet soil which results in waterlogged roots, anaerobic respiration may also occur in plants. The steps involved in aerobic respiration, anaerobic respiration, and fermentation vary, but all involve the manipulation of electrons and serve
to perpetuate the availability of energy for the cell.
In aerobic respiration, pyruvate molecules are shuttled from the cytoplasm into the mitochondria to prepare
for the citric acid cycle. An enzyme complex called pyruvate dehydrogenase complex produces acetylcoenzyme A (acetyl-CoA) by initially breaking down pyruvate to a two-carbon acetyl group and carbon dioxide. The acetyl group is then transferred to coenzyme A. Coenzyme A then acts as a carrier for the twocarbon acetyl group and transfers the acetyl group to the citric acid cycle.
The Citric Acid Cycle
The citric acid cycle, also known as the Krebs cycle or tricarboxylic acid cycle (TCA cycle), is an eight step
process in which the acetyl group is oxidized to carbon dioxide. The citric acid cycle engages both anabolic
and catabolic reactions to construct and break down large molecules. During this process, two important
electron shuttle molecules [flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD+)]
are reduced forming FADH2 and NADH. These molecules shuttle electrons to the next step of aerobic respiration, the electron transport chain.
128
Cellular Respiration
The electron transport chain, ETC, performs the final series of biochemical reactions. Ultimately, the
ETC functions to regenerate oxidized molecules (coenzymes) from their reduced state so that other glucose molecules can be converted to energy through future rounds of respiration. To accomplish this objective, electrons (and hydrogen ions) from FADH2 and NADH are transferred to enzyme complexes embedded in the mitochondria’s inner membrane. These complexes act as electron acceptors, passing the electrons from one complex to the next. Since oxygen has a very high affinity for electrons, aerobic respiration
is the most efficient means of producing ATP (up to 34 moleculesare generated per round).
Figure 2: The electron transport chain.
Fermentation
Eukaryotic and prokaryotic organisms use fermentation
in anaerobic conditions. In these cases, the citric acid
cycle and the ETC are not used. Instead, pyruvate is the
final substrate. These molecules are metabolized in the
cytoplasm to produce alcohol or lactic acid, depending
on the organism. The main function of fermentation is the
reduction of NADH to NAD+. This contributes to the propagation of the glycolytic pathway, which allows ATP production to continue. Recall that glycolysis nets two ATP
molecules per glucose molecule. Fermentation allows for
the generation of much less ATP per glucose molecule
than aerobic respiration because the citric acid cycle and
the ETC are not engaged.
?
Did You Know...
Yeast has been used to make leavened bread for
centuries. When yeast undergoes fermentation,
carbon dioxide is trapped between gluten and
causes the bread to
rise. Ethanol, another byproduct of yeast
fermentation, generates the alcohol content in beer, and the
carbon dioxide provides effervescence.
129
Cellular Respiration
Figure 3: Anaerobic vs. aerobic respiration charts. The green area represents the cytoplasm of the cell where glycolysis and anaerobic respiration occur. The blue area represents the inside of the mitochondria where aerobic respiration occurs.
Industrial Applications
Fermentation has many industrial applications, many of which are evident within the food industry. For example, the sour or tart taste of yogurt and sauerkraut is due to bacteria in the Lactobacillus genus. Lactobacillus perform lactate fermentation in which pyruvic acid (from glycolysis) is reduced directly to the final end
product, lactic acid. Another example of industrial fermentation focuses on yeast. Yeast, a unicellular eukaryote, has been used to make leavened (rising) bread for centuries. When yeast undergoes fermentation,
carbon dioxide is trapped between gluten and causes the bread to rise. Ethanol is another byproduct of fermentation and is responsible for the alcohol content in beer. Carbon dioxide produced through fermentation
is responsible for the effervescence in beer.
Respirometers
Scientists may use respirometers to measure the rate of respiration. This device capitalizes on the ideal gas
law for its function. The ideal gas law applies to those gases in which all collisions between atoms or molecules are perfectly elastic and no intermolecular forces exist. The internal energy is completely kinetic, and
any change in energy will be accompanied by a change in temperature, volume, and pressure. The relationship is defined by the ideal gas law equation:
PV = nRT
130
Cellular Respiration
In this equation, P is the absolute pressure, V is the volume, R is the universal gas constant (8.3145 J/mol °
K), and T is the absolute temperature (Kelvin). Respirometers typically consist of a device to absorb carbon
dioxide produced through respiration and measure oxygen uptake by displacement of fluid in a tube connected to the sealed chamber. Since the carbon dioxide is absorbed, air will be taken in through the tube
(displacing the fluid) to maintain equal pressure within the chamber.
?
Did You Know...
Cells require more energy during physical activity than when the
body is at rest. Humans rely on aerobic respiration to provide this
energy. However, some physical activities (such as a strenuous
workout) may create an anaerobic environment if the oxygen supply
depletes before the demand is removed. When this occurs, lactate
fermentation will begin to provide additional energy without the use
of oxygen. Lactic acid is a 3-carbon molecule. Therefore, most of
the energy remains and the only energy produced is the two ATP
molecules generated from glycolysis. The buildup of lactic acid can
cause muscles to feel sore and stiff. This feeling persists until the
lactic acid is removed from the muscles by diffusion into the blood.
Definitions
x Aerobic Respiration: Oxidation of molecules to produce energy in the presence of oxygen.
x Anaerobic Respiration: Oxidation of molecules to produce energy in the absence of oxygen.
x ATP: Adenosine triphosphate; the main energy source of the cell.
x Citric Acid Cycle (The Krebs Cycle): The step following glycolysis in which acetyl groups produced using pyruvate molecules are oxidized yielding CO2, ATP, H2O and reduced electron shuttle molecules.
x Fermentation: A metabolic pathway used in anaerobic respiration which reduces NADH to NAD+
x Glycolysis: The initial process of energy metabolism for anaerobic and aerobic organisms which
produces two ATP, two NADH, and two pyruvate molecules.
x Oxidation: The loss of electrons.
x Oxidation State: The degree of oxidation of an atom.
x Pyruvate (Pyruvic Acid): A three-carbon molecule generated through glycolysis.
x Reduction: Gain of electrons.
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Cellular Respiration
Pre-Lab Questions
1. Why is cellular respiration necessary for living organisms?
2. Why is fermentation less effective than respiration?
3. What is the purpose of glycolysis?
4. How many ATP molecules are produced in aerobic respiration, fermentation, and glycolysis?
Experiment 1: Fermentation by Yeast
Yeast cells produce ethanol, C2H6O, and carbon dioxide, CO2, during alcoholic fermentation. In this experiment, you will measure the production of CO2 to determine the rate of anaerobic respiration in the presence
of different carbohydrates with a simplified respirometer.
Materials
(4) 250 mL Beakers
Ruler
15 mL 1% Glucose Solution
15 mL 1% Sucrose (Sugar) Solution, C12H22O11
(1) 100 mL Graduated Cylinder
Test Tube Rack
Measuring Spoon
1 Yeast Packet
1 g. Packets of Equal®, Splenda®, and Sugar
*Stopwatch
Permanent Marker
*Warm Water
3 Pipettes
5 Respirometers (two test tubes that fit into each
other – 5 plastic and 5 glass; see Figure 4).
132
* You Must Provide
Cellular Respiration
Note: Sucrose (a disaccharide) is made up of glucose and fructose. Glucose is a monosaccharide.
Figure 4: Four respirometers. Note how the smaller, plastic test tube is inverted
into the larger test tube. You will create five respirometers in this experiment.
Procedure
1. In this experiment, you will mix yeast with sugar, Equal, and Splenda. Before you begin, develop a hypothesis predicting what will happen when the sugar/sweeteners are mixed with yeast. Example, will fermentation occur? Why or why not? Record your hypothesis in the Post-Lab Questions section.
2. Use the permanent marker to label three 250 mL beakers as Equal®, Splenda®, and Sugar.
3. Empty the Equal®, Splenda®, and Sugar packets into the corresponding beakers.
4. Fill the Equal® and Splenda® beakers to the 100 mL mark with tap water.
5. Fill the Sugar beaker to the 200 mL mark.
6. Mix each beaker thoroughly by pipetting the solution up and down. Each beaker now contains a 1% solution. Set these aside for later use.
7. Completely fill the smallest tube with tap water and invert the larger tube over it. Push the small tube up
(into the larger tube) until the top connects with the bottom of the inverted tube. Invert the respirometer
so that the larger tube is upright (there should be a small bubble at the top of the internal tube).
Note: Repeat Step 6 several times as practice. Strive for the smallest bubble possible. When you feel
comfortable with this technique, empty the test tube(s) and proceed to Step 7.
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Cellular Respiration
8. Use the permanent marker to label the fourth 250 mL beaker as Yeast.
9. Fill this beaker with 175 mL of warm tap water. The exact temperature does not matter, but the water
should be warm to the touch.
10. Open the yeast package, and use the measuring spoon to measure and pour 1 tsp. yeast into the beaker. Pipette the solution up and down until all of the yeast has dissolved into solution.
Note: Make sure the yeast solution remains homogenous before each test tube is filled in the proceeding steps. The yeast concentration is fairly saturated, and yeast may precipitate out of solution
if the beaker rests for an extended period of time.
11. Use the permanent marker to label the big and small test tubes as 1, 2, 3, 4, and 5.
12. Use the 100 mL graduated cylinder to measure and pour 15 mL of the following solutions into the corresponding small test tubes:
Tube 1: 1% Glucose Solution
Tube 2: 1% Sucrose Solution
Tube 3: 1% Equal® Solution
Tube 4: 1% Splenda® Solution
Tube 5: 1% Sugar Solution
Note: Rinse the graduated cylinder between each measurement.
13. Fill the remaining volume in each small tube to the top with the yeast solution.
14. Slide the corresponding larger tube over the small tube and invert it as practiced in Step 6. This will mix
the yeast and sugar/sweetener solutions.
15. Place the respirometers in the test tube rack, and use a ruler measure the initial air space in the rounded bottom of the internal tube. Record these values in the Table 1.
16. Allow the test tubes to sit in a warm place (approximately 30 °C) for two hours. Placement suggestions
include: a sunny windowsill, atop (not in!) a warm oven heated to approximately 85 °C (185 °F on an
oven setting), or under a very bright (warm) light.
17. At the end of the respiration period, use your ruler to measure the final gas height (total air space) in the
tube. Record this data in Table 1.
Table 1: Yeast Fermentation Data
Tube
134
Initial Gas Height (mm)
Final Gas Height (mm)
Net Change
Cellular Respiration
Post-Lab Questions
1. Include your hypothesis from Step 1 here. Be sure to include at least one piece of scientific reasoning in
your hypothesis to support your predictions.
2. Did you notice a difference in the rate of respiration between the various sugars? Did the artificial sugar
provide a good starting material for fermentation?
3. Was anaerobic fermentation occurring? How do you know (use scientific reasoning)?
4. If you observed respiration, identify the gas that was produced. Suggest two methods you could use for
positively identifying this gas.
5. Hypothesize why some of the sugar or sweetener solutions were not metabolized, while others were. Research the chemical formula of Equal® and Splenda® and explain how it would affect yeast respiration.
6. How do the results of this experiment relate to the role yeast plays in baking?
7. What would you expect to see if the yeast cell metabolism slowed down? How could this be done?
8. Indicate sources of error and suggest improvement (for example, what types of controls could be added?).
135
Cellular Respiration
Experiment 2: Aerobic Respiration in Beans
We will evaluate respiration in beans by comparing carbon dioxide production between germinated and nongerminated beans. As shown in the balanced equation for cellular respiration, one of the byproducts is CO2
(carbon dioxide):
C6H12O6 + 6 H2O + 6 O2 Æenergy + 6 CO2 +12 H2O
We will use a carbon dioxide indicator ( bromothymol blue) to show oxygen is being consumed and carbon
dioxide is being released by the beans. Bromothymol blue is an indicator that turns yellow in acidic conditions, green in neutral conditions, and blue in basic conditions. When carbon dioxide dissolves in water,
carbonic acid is formed by the reaction:
H2O + CO2 Æ H2CO3
resulting in the formation of this weak acid. If an indicator such as bromothymol blue is present, what do you
think would happen? (Hint - what color would the indicator change to?)
Materials
(6) 250 mL Beakers
1 Pipette
24 mL Bromothymol Blue Solution, C27H28Br2O5S
6 Rubber Bands (Large. Contains latex; please
handle with gloves if you have a latex allergy.)
100 Kidney Beans
6 Medicine Cups (small, clear, plastic cups)
15 cm Parafilm®
*Water
*Paper Towels
Permanent Marker
100 Pinto Beans
*You Must Provide
Procedure
1. Label two of the 250 mL beakers as Soaked: Pinto and Soaked: Kidney.
2. Fill each beaker with 200 mL water.
3. Count and transfer 50 pinto beans into the Soaked: Pinto beaker. Then, count and transfer 50 kidney
beans into the Soaked: Kidney beaker. Allow the beakers to rest for 24 hours.
4. After 24 hours have passed, carefully strain the water from each beaker.
5. Place two paper towels on a flat work surface. Use the permanent marker to label one paper towel as
Soaked: Pinto and the second as Soaked: Kidney.
6. Pour the soaked beans onto paper towels, keeping them sorted by bean type.
7. Label the remaining beakers as Dry: Pinto, No Pinto, Dry: Kidney, and No Kidney.
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Cellular Respiration
8. Place several layers of moist paper towels at the bottom of all six, 250 mL beakers.
9. Place 50 pre-soaked pinto beans into the Soaked: Pinto beaker, 50 unsoaked pinto beans into the Dry:
Pinto beaker, and no beans into the No Pinto beaker.
10. Place 50 pre-soaked kidney beans into the Soaked: Kidney, 50 unsoaked kidney beans into the Dry: Kidney beaker, and no beans into the No Kidney beaker.
11. Dispense four mL of bromothymol blue solution into each of the six measuring cups. Then, place one
cup inside each beaker (Figure 5).
12. Stretch Parafilm® across the top of each beaker. Secure with a rubber band to create an air-tight seal.
Note: If your Parafilm® seal breaks, plastic wrap
(such as Saran® wrap) can be used as a replacement.
13. Place the beakers on a shelf or table, and let sit undisturbed at room temperature.
14. Record the initial color of the bromothymol solutions
and observe the jars at 30 minute intervals for three
hours. Record the color of the bromothymol blue in
Tables 2 and 3.
Figure 5: The image above shows what the beans,
15. Let the beans and the jar sit overnight. Record your beaker, and measuring cup set-up should look like.
final observations in Tables 2 and 3.
137
Cellular Respiration
Table 2: Bromothymol Blue Color Change Over Time for Pinto Bean Trial
Time
Pre-Soaked Pinto Beans
Dry Pinto Beans
No Pinto Beans
0 min
30 min
60 min
90 min
120 min
150 min
180 min
24 hours
Table 3: Bromothymol Blue Color Change Over Time for Kidney Bean Trial
Time
0 min
30 min
60 min
90 min
120 min
150 min
180 min
24 hours
138
Pre-Soaked Kidney Beans
Dry Kidney Beans
No Kidney Beans
Cellular Respiration
Post-Lab Questions:
1. How did the color of the bromothymol blue solution in each beaker change over time in each condition?
2. What is the mechanism driving the bromothymol blue solution color change?
3. What can be inferred from the color change of the bromothymol blue solution?
4. What evidence do you have to prove that cellular respiration occurred in the beans? Explain your answer.
5. What are the controls in this experiment, and what variables do they eliminate? Why is it important to
have a control for this experiment?
6. If this experiment were conducted at 0 °C, what difference would you see in the rate of respiration? Why?
7. Would you expect to find carbon dioxide in your breath? Why?
8. What else could you incorporate into this experiment to verify that the gas is responsible for the color
change? Design an experiment that shows the steps required.
139