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Lab 8: Cellular Respiration
OBJECTIVES:
 Understand the major events of glucose catabolism (cellular respiration): glycolysis, the citric
acid cycle and oxidative phosphorylation.
 Compare and contrast aerobic and anaerobic respiration.
 Measure the relative production of carbon dioxide by plants and animals.
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INTRODUCTION:
All living organisms have evolved mechanisms to obtain energy needed to fuel biological
functions, including growth, metabolism and maintenance. These mechanisms include a series of
biochemical reactions, collectively referred to as cellular respiration. During this process,
organic molecules (e.g. glucose) are enzymatically broken down, releasing energy stored in the
bonds of adenosine triphosphate (ATP), which cells use to perform metabolic functions.
Energy flow in biological systems occurs through oxidation-reduction or redox reactions
where electrons are transferred from one molecule to another. Recall from Lab 4 that reduction
is the acquisition of electrons or hydrogen atoms while oxidation is the loss of electrons or
hydrogen atoms. During cellular respiration, electrons are removed from glucose (i.e. oxidized)
and some of the released energy is stored as ATP. The dozens of redox reactions that take place
during respiration use electron acceptors for energy transfer. Two of the most important electron
acceptors are nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide
(FAD+), derived from niacin (Vitamin B3) and riboflavin (Vitamin B2), respectively. These
molecules are reduced to NADH and FADH2 when they acquire electrons, which they transfer to
other molecules to generate ATP. Depending on which molecule serves as the final electron
acceptor, the process is aerobic or anaerobic. In aerobic respiration, the final electron acceptor
is oxygen while in anaerobic respiration the final electron acceptor can be inorganic
compounds (other than oxygen) such as nitrates, sulfates, or ethanol (Fig. 1).
Figure 1. Comparison of redox reactions in aerobic and anaerobic respiration
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Cellular respiration (Fig. 2) can be divided into four stages:
(1) Glycolysis
(2) Pyruvate oxidation
(3) Kreb’s cycle
(4) Electron Transport Chain and Chemiosmosis
Figure 2. An Overview of Aerobic Respiration
Glucose catabolism begins with glycolysis in the cytoplasm (Fig. 3). Glycolysis includes
a series of reactions where each entering glucose molecule (6 carbons) is split into 2 molecules
of pyruvate (3 carbons). In total, glycolysis yields 4 ATP molecules, however, 2 ATPs are used
for the priming reactions that initiate glycolysis. Thus, a net of 2 ATPs are generated for the
entire process. In addition, 2 NADH molecules are reduced from NAD+ during this stage.
If oxygen is present, the processes will begin with pyruvate oxidation. During this
phase, each pyruvate molecule generated from gylcolysis enters the mitochondria and is
converted into carbon dioxide (CO2), which is released as a side-product, and forms acetyl, a 2carbon sugar that joins with coenzyme A to form acetyl-CoA. More importantly, this process
also reduces NAD+ to NADH, which can be used to generate ATP. During aerobic respiration,
acetyl-CoA enters the Krebs cycle (also known as the citric acid cycle). For every turn of the
Krebs cycle, one ATP molecule is produced and multiple NAD+ and FAD+ molecules are
reduced to NADH and FADH2, respectively. The final products of the Krebs cycle per glucose
molecule include: 2 ATP, 2 FADH2, 6 NADH, and 4 CO2.
In the final stage of cellular respiration (Fig. 3), the electrons carried by FADH2 and
NADH are transferred through a series of transmembrane proteins known as the electron
transport chain (ETC), creating a proton gradient that is used to drive ATP synthesis. Each
molecule of NADH yields 3 ATPs while each FADH2 generates 2 ATPs, resulting in an overall
production of 32 ATPs in this stage.
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Intermembrane space
Pyruvate from
cytoplasm
H+
H+
e–
NADH
H+
1. Electrons are harvested
and carried to the transport
system.
Acetyl-CoA
e–
NADH
Krebs
cycle
2. Electrons provide
energy to pump
protons across the
membrane.
H2O
FADH2
3. Oxygen joins with
protons to form water.
1
2
O2
O2
+ 2H+
CO2
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2
ATP
H+
ATP
Mitochondrial matrix
4. Protons diffuse back
in, driving the synthesis
of ATP.
ATP
synthase
Figure 3. Overview of ETC and Chemiosmosis
Conversely, in the absence of oxygen (anaerobic respiration), the pyruvate molecules
produced during glycolysis do not enter the Kreb’s cycle but undergo fermentation instead.
Without oxygen, pyruvate cannot enter the remaining steps of aerobic respiration and must be
used differently - either ethanol or lactic acid is produced. Along the way, NADH that was
created in glucose catabolism is oxidized back to NAD+, pyruvate is reduced and broken down,
and a small quantity of ATP is produced. There are two main types of fermentation reactions: (1)
ethanol fermentation and (2) lactic acid fermentation. Ethanol fermentation occurs in
organisms such as yeast (Fig. 4a) which have been used for food and alcoholic beverage
production. Lactic acid fermentation occurs in animal cells. For example, when oxygen is not
readily available to muscle tissue, the muscle cells use lactic acid fermentation to produce ATP
(Fig. 4b). Build up of lactic acid is the primary cause of muscle fatigue, often experienced during
strenuous exercise. Overall, the anaerobic process yields a net of 2ATP, an 18-fold decrease in
ATP production (per glucose molecule) compared to aerobic respiration.
a)
b)
Figure 4. Anaerobic respiration: a) alcohol fermentation and b) lactic acid fermentation
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Figure 5 summarizes the basic differences between the products of aerobic and anaerobic
respiration. While all three types of cellular respiration produce carbon dioxide (CO2), water
(H2O), energy (ATP) and heat, they do so at different efficiencies. Both types of anaerobic
fermentation produce a net total of 2 ATP since they only undergo glycolysis. In contrast,
during aerobic respiration, up to 38 ATP molecules are produced through the continuous redox
reactions of glycolysis, pyruvate oxidation, Krebs cycle, and ETC stages of glucose metabolism.
Thus, aerobic respiration, when compared to anaerobic respiration, is a much more efficient
process for ATP production.
Aerobic respiration:
C6H12O6 + 6O2
6CO2 + 6H2O +ATP +Heat
Anaerobic fermentation (plants and some microbes):
C6H12O6
(Glucose)
2C2H5OH + 2CO2 +ATP + Heat
(Ethanol)
Anaerobic fermentation (animals and some microbes):
C6H12O6
2CH3CHOHCOOH +ATP + Heat
(Lactic Acid)
Figure 5. Equations for different forms of cellular respiration
Question:
List the advantages and disadvantages of respiring anaerobically.
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TASK 1 - Demonstration of Anaerobic Respiration
Anaerobic organisms, including bacteria and yeast, produce energy in the absence of oxygen via
anaerobic respiration. In this pathway, glucose is catabolized to 2 pyruvate molecules during
glycolysis, which is reduced to either lactic acid or ethanol and CO2 during fermentation. In this
exercise you will demonstrate CO2 production during anaerobic fermentation by yeast. You will
examine the effects of sugar type on the fermentation process.
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Sugars are simple carbohydrates. Also called saccharides, they come in two forms:
monosaccharides and disaccharides. Monosaccharides have the chemical formula
C6H12O6
and Disaccharides have the chemical formula
C12H22O11. However, many different
configurations exist for each of the two kinds. These different configurations of atoms are
called isomers. Isomers of sugars are important to life because organisms have evolved various
enzymes to access the energy in each form. Some organisms are therefore better at getting at
some forms of sugar than other forms because of the enzymes that they can use. Today you
will test the effects of 5 different sugars on yeast fermentation: glucose, sucrose, fructose,
maltose and xylose. Glucose, fructose, and xylose are all monosaccharides, while maltose and
sucrose are both disaccharides. Glucose is a simple sugar found in plants and is one of the
main products of photosynthesis. Fructose is also a plant-based simple sugar. Sucrose is
consists of glucose and fructose bonded together to form a disaccharide. Xylose is a sugar first
isolated from wood and is derived from hemicellulose, a component plant cell walls and fibers.
Maltose is a disaccharide formed from two glucose molecules. It is the sugar produced when
amylase breaks down starch.
glucose
xylose
fructose
sucrose
maltose
Figure 6. Chemical structure of five sugars
Develop a Hypothesis:
Given what you already know about anaerobic respiration, state what you expect to happen over
time in the experiment (Hint: consider the products of anaerobic respiration).
Do you expect to observe a difference between the sugars? Propose an explanation. Write your
hypotheses (Ho and Ha) in the space provided below.
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Procedure:
a. Your TA will prepare one class control j-tube using only yeast and water, following
the instructions below.
b. Check with your TA to determine which three sugars you will prepare. Obtain 3 Jtubes, 3 large beakers, and 3 small flasks. Label each beaker and flask with the sugar
types you have been assigned. Fill all large beakers with water from the 38˚C water
bath.
c. Weigh out 1g of each sugar and three 1g portions of yeast. Add 35ml of warm tap
water to each flask. Add the sugars to the appropriately labeled flask and mix to
dissolve.
d. Add 1g of yeast to each flask, mix well (no lumps!) and pour the contents into a Jtube and place into the appropriately labeled beaker. Make sure the closed end of the
J-tube is completely filled with liquid! Note the time or start a timer. Try to set up all
the j-tubes at approximately the same time, utilizing three group members.
e. Replace the warm water in both beakers every 15 minutes to maintain temperature.
f. Quantify the amount of CO2 produced by measuring the height of the gas bubble in
the J-tube from the top of the closed end to the liquid-gas interface. Record this data
every 5 minutes in Table 1.
g. Allow the fermentation process to proceed for 30 minutes. Once the experiment is
complete, copy your team’s data onto the board for the rest of the class.
h. Fill out the remainder of Table 1 using the class data. Calculate the average bubble
height for each time point.
Table 1:
Height of gas bubble (mm)
Sugar
Replicate
0 min
5 min
10 min
1
2
Glucose
3
Average
1
2
Xylose
3
Average
6
15 min
20 min
25 min
30 min
1
2
Fructose
3
Average
1
2
Maltose
3
Average
1
2
Sucrose
3
Average
Control
Questions:
1. Was there a noticeable increase in the amount of CO2 produced for all treatments over
time?
2. How does gas bubble height relate to the amount of carbon dioxide produced during
fermentation?
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3. Using the graph paper provided, plot the change in CO2 over time for each treatment
using the averaged values. Make sure you label your axes and the line representing
each treatment.
4. What happens to CO2 levels with different carbohydrates? Explain the observed
differences (or lack there of) based on your knowledge of the provided sugars.
5. Based on your results, can you explain which ingredients are essential for
fermentation to occur and why?
6.
Overall, what can you conclude about your hypotheses? Explain.
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TASK 2 - Demonstration of Aerobic Respiration
When eukaryotic organisms respire they release CO2, which can combine with H20 to form
carbonic acid (H2CO3). In general, acidic solutions such as H2CO3 (pH less than 7) have a larger
concentration of H+ ions while basic ones (pH greater than 7) contain more OH- ions (Fig. 6). An
indicator, such as bromothylmol blue (BTB) can be used to detect changes in pH resulting from
CO2 production during cellular respiration. In a basic solution, BTB is blue, in neutral solutions
it turns green, and in acidic solutions it is yellow. During this task you will examine aerobic
respiration by comparing the pH of water containing actively respiring animal (snails) and plant
(Elodea) cells.
Figure 6. pH Scale
Questions:
a. What major energy producing process is characteristic of plant cells but not animal cells?
b. What gas is consumed in this process?
Develop a Hypothesis:
Considering the set-up for this procedure (Table 2), state what you expect to occur when
comparing respiratory rates in animals vs. plants in the light and dark. Write your hypotheses (Ho
and Ha) in the space provided below.
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Procedure:
(Note: Since this task requires a 24-hour incubation, your TA will prepare these materials ahead
of time.)
1. Fill 8 test tubes with tap water (see Table 2).
2. Obtain the organisms listed in Table 2 and add each organism to the corresponding tube.
3. Add 10 drops of BTB and stopper to prevent gas exchange with the atmosphere.
4. Place tubes 1-4 under a grow light and tubes 5-8 in the dark. Leave undisturbed for 24
hours.
5. Record your results in Table 2.
Table 2:
Tube #
1
2
3
4
5
6
7
8
Independent
Beginning
Color
variables
Color
Prediction
0 snails, 0 Elodea, light
blue
2 snails, light
blue
2 Elodea, light
blue
2 snails, 2 Elodea, light
blue
0 snails, 0 Elodea, dark
blue
2 snails, dark
blue
2 Elodea, dark
blue
2 snails, 2 Elodea, dark
blue
Color
Result
Questions:
1. Based on your results, what can you conclude about your hypotheses? Explain.
2. Use your knowledge of the behavior of CO2 in water to explain what happened to the pH
in each tube.
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2. How do snails affect the level of CO2 in the water? Does light or dark have an effect?
3. How does Elodea affect the level of CO2 in the water? Does light or dark have an effect?
4. What tube best represents a balanced system? Explain.
5. What would you expect to happen to the pH in a tube if the snail died shortly after being
placed inside? What about the Elodea?
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