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LAB
3: RESPIRATION
QUESTIONS/OBJECTIVES OF LABORATORY:
·
What is
fermentation? How (and why) do cells use fermentation?
·
How do
plants/animals use aerobic respiration? How do they balance it with
photosynthesis?
·
Where do the
processes of respiration (aerobic and anaerobic) occur?
RELEVANT READING :Chapter
7 (pp. 125-144) in text.
INTRODUCTION:
·
What is respiration?
As you already know,
respiration is, in a sense, the “flip side” of the photosynthetic reactions
that we studied in the last laboratory session. Just as photosynthesis
involves a series of anabolic (biosynthetic) reactions that
build organic molecules, namely glucose, respiration is a set of
catabolic (biodegradative) reactions that break down glucose or
other macromolecules to produce energy (in the form of ATP) and molecules
that may serve as intermediates in other anabolic/catabolic reactions.
ATP (adenosine triphosphate) is the standard “currency” or energy
source that drives most metabolic reactions. Respiration also produces
carbon dioxide and water, though these are wasteful byproducts rather than
desirable end products.
A simplified overall
formula for the chemical reactions of respiration would be:
C6H12O6
+ 6 H2O + 6 O2
à
6 CO2 + 12 H2O + energy
Remember that we will use
the term “respiration” here to refer to the enzymatic, metabolic processes
of cell respiration. However, the large-scale gas exchange of
pulmonary respiration (i.e., inhalation and exhalation of lungs) is of
course a necessary consequence of cellular respiration. We need to take in O2
because our cells need O2 (as a final electron acceptor
when oxidizing—essentially combusting or “burning”—fuels such as glucose).
At the same time our lungs need to rid our body of CO2 and H2O,
the waste products of cellular respiration.
Cellular respiration has
two stages. The first is glycolysis. This is an anaerobic
process (i.e., it does not require O2), occurring in the
cytoplasm of both aerobic and anaerobic cells. In other words, glycolysis
can proceed with or without oxygen present. Glycolysis literally refers to
the “glucose splitting” in which a six-carbon glucose molecule is split into
a pair of three-carbon pyruvate molecules, yielding 2 ATP (net) and 2
reduced NADH nucleotide coenzymes.
When oxygen is
unavailable, this conversion of glucose to pyruvate is the sole source of
cellular energy. The pyruvate is, however, further metabolized through a
second series of reactions in an anaerobic process called fermentation
(which merely perpetuates glycolysis).
There are two types of
fermentation: one in which pyruvate is converted to ethyl alcohol (a.k.a.
ethanol) with the release of CO2, and one in which pyruvate
is converted to lactate. Neither fermentation generates additional
ATP, so that anaerobic respiration always yields only the 2 ATP from
glycolysis, but both types of fermentation oxidize NADH to produce
NAD+, allowing glycolysis to continue. A major difference
between the two types of fermentation is that lactate fermentation is
reversible. Lactate is generally soon converted to lactic acid, but both
are three-carbon compounds that can readily (for a cost) be made back into
pyruvate. Alcohol fermentation is a non-reversible process: it involves
release of CO2 and production of the two-carbon molecule ethanol.
The reason our bodies undergo lactate fermentation is that it is reversible,
although it would not be very good to produce alcohol in our exercising
tissues either!
Still, anaerobic
respiration is a rare occurrence for large, complex organisms like
vertebrate animals because it yields little energy from the metabolite. Thus
we are obligate aerobes—we need O2—since O2 is
needed to further metabolize the pyruvate produced by glycolysis to yield
significant sources of energy. This second stage of respiration involves the
Krebs cycle (a.k.a. citric acid or tricarboxylic acid cycle) and the
electron transport system (ETS). Both of these components of
aerobic respiration occur in mitochondria. Together glycolysis and
the Krebs cycle break down pyruvate to release all the carbon atoms (as CO2),
and in so doing they load up electrons on nucleotide coenzymes to make the
reduced NADH and FADH2.
These molecules in turn
donate their electrons to a chain of electron carriers in the complexly
folded cristae (inner mitochondrial membrane). As the electrons
bounce along this chain they drop to lower energy levels, and this released
energy is used to pump protons (H+ ions) from the
matrix (inner mitochondrial compartment) to the outer compartment
(intermembrane space). The accumulated protons then flow passively, along
their concentration (chemical) and charge (electrical) gradient, through an
ATP synthase (a.k.a. synthetase) channel protein (in the cristae) to
phosphorylate ADP and generate ATP. This proton flow (linked to electron
transport) is called chemiosmosis. This particular type of
chemiosmotic phosphorylation is called oxidative phosphorylation. And
what of the electrons stripped from glucose, which are carried by the
coenzymes and then “bounced” along the ETS? These ultimately end up on
oxygen. Oxygen, the final electron acceptor, combines with protons as well
to create metabolic water.
Water is considered a
“waste” byproduct of aerobic respiration, and we release much of it via
exhalation of air from the lungs (the body also rids excess water from the
skin and kidneys in sweat and urine). However, water is of course a very
useful molecule for nearly all biological reactions—it is absolutely
essential for life—and in many environments water is in very short supply.
Hence this metabolic water (produced in mitochondria) may be a very
significant source of water, especially in desert animals. Some flour
beetles rely so heavily on metabolic water production that they do not
normally drink water. They make all the water they need themselves.
Unlike anaerobic
respiration, aerobic respiration yields many ATP: 2 from the Krebs cycle and
an additional 32 (or in rare cases 34) from the ETS. Much of the original
energy in glucose (686 kcal/mol) is of course lost as heat. After all,
that’s what the second law of thermodynamics is all about. Only 36 ATP are
normally generated during glycolyis + aerobic respiration, which translates
to roughly 252 kcal. This means that only 37% of the potential chemical
energy in glucose is captured, and the remaining 63% is lost as heat.
However, 37% efficiency is actually quite good when compared with the
combustion that occurs in gasoline engines and other mechanical devices,
which typically extract only 20-30% of the available energy of a fuel.
Remember too that glucose
is not the only fuel that can be metabolized. Your body stores glycogen
(animal starch), a long chain polymer of glucose, in your muscles and liver,
and this is easily broken into glucose, but your diet also includes lipids,
proteins, and other molecules. These can be “plugged into” anaerobic and
aerobic respiration at various places, and they can be converted to glucose
or other metabolic intermediates. Not only do you degrade such molecules,
but your body also builds other macromolecules via several anabolic
(synthetic) pathways.
EXERCISES: A. How do yeast cells
ferment substrates?
Fermentation is an
anaerobic process that converts pyruvate or pyruvic acid into other organic
compounds while regenerating NAD+ needed for glycolysis to
continue. Fermentation does not in itself create ATP. Your cells
undergo lactate fermentation to perpetuate glycolysis during brief bursts of
strenuous activity (e.g., sprinting) when all available O2 is
quickly used up in your cells.
In yeast cells, however,
fermentation yields ethyl alcohol and CO2. The released CO2.
helps baking bread rise and provides carbonation for alcoholic beverages
such as beer and champagne. The CO2 can also be observed—and
measured—as evidence of the fermenting process.
Yeasts are simple
unicellular organisms related to mushrooms, molds, and mildew. They are
heterotrophic and obtain their nutrition from such sources as grapes or
grain (like barley). Yeasts are facultative anaerobes—that is, they can live
in aerobic or anaerobic environments, though they prefer aerobic conditions.
Under anaerobic conditions, they ferment their “food” source.
NOTE:
There are several different types of fermentation that can be used,
including single glass tubes with upright arms or arrangements with a
pair of glass tubes nested together or connected by flexible tubing. The
instructions below refer to the first kind, but listen carefully and pay
attention in the lab to see if another kind will be used and if so, how
they are to be set up.
1.
Obtain five glass
fermentation tubes. Label them 1-5 or put them (in order) in a rack.
2.
Add 20 ml of one of
the following solutions: Tube 1—distilled water; 2—10% glucose; 3—10%
galactose; 4—10% sucrose; 5—10% starch. These four solutions (and the
distilled water) should be warm. [The volumes may be changed—listen
carefully for instructions!]
3.
Put 1 ml of yeast
solution (a stock solution of actively growing yeast culture) into each of
the five tubes and mix thoroughly.
4.
Rotate the tube to
fill the closed “upright arm” with fluid. Your instructor can demonstrate.
5.
Observe the
fermentation tubes every ten minutes for 1½ hours or more. In the table (in
your worksheet) record the amount of gas produced. The gas-collecting part
of our tubes lack graded volumetric or linear markings, but you can measure
(in mm) with a ruler the part of each tube that fills with gas. [If you were
instructed to add blue dye to each tube, also record the color intensity of
the yeast suspension. It should turn yellow as CO2 is produced.]
6.
Answer the
questions on your worksheet. Why do some tubes (and not others) produce gas?
Can you explain the differences in the amount of CO2 gas
production? What do you conclude about the importance of the type of sugar
used as a food source for fermenting yeasts?
B. How does aerobic
respiration occur in plants?
People often forget that plants, like animals, carry out aerobic
respiration. As you recall the overall chemical formula for respiration, you
will see that the amount of CO2 made is directly proportional to
the amount of O2 consumed. By chemically removing the CO2
gas produced during respiration one can indirectly measure the amount of
oxygen used in cellular respiration. In
this exercise you will compare the relative amounts of O2 used by
germinating and non-germinating pea seeds at various temperatures. The CO2
produced will be removed using KOH (potassium hydroxide) according to the
following formula:
2KOH + CO2
à
K2CO3 + H2O You
can perform this exercise at various temperatures to demonstrate the effect
of temperature on respiration. You will be assigned to small groups to carry
out the experiment at different temperatures. All groups will share their
data at the end of the activity.
1.
Place 50 ml of
water in a 100 ml graduated cylinder. Place 10 germinating pea seeds in the
cylinder and measure the volume in ml (x ml), then figure the total volume
of the pea seeds (x –50 ml). Remove the seeds and dry them on a paper towel.
2.
Place another 50 ml
of water in the cylinder and add 25 nongerminating pea seeds. [Note: normal
seeds can be placed in bleach, etc., to “kill” them so they will not
respire.] Add glass beads to the cylinder to reach volume x ml (the same
volume as in step 1). [The volume of these seeds and beads is thus equal to
the volume of the germinating seeds.] Remove the seeds and beads and dry
them on a paper towel.
3.
Once again, place
50 ml of water in the cylinder, and add glass beads until you reach volume x
ml. These glass beads will be used as a control. Remove and dry them on a
paper towel.
4.
Place a ball of
absorbent cotton in the bottom of each of the three vials of the respiration
apparatus. Saturate the cotton with KOH, but be careful not to drip any of
this chemical on the sides of the vials. Next place a ball of nonabsorbent
cotton on top of the KOH. Label the vials 1-3 or keep them organized so that
you remember which is which.
5.
Place the
germinating seeds in vial #1, the nongerminating seeds and beads in vial #2,
and the beads alone (the control) in vial #3.
6.
Insert a rubber
stopper/pipette apparatus into each vial and make sure you have a good seal.
Place a heavy nut or washer (as weight) over the end of each pipette and lay
the three vials on their sides in the water bath at your assigned temp. The
weight will keep them submerged. Note:
Prop the ends of the pipettes over the edge of the bath and allow the setup
to equilibrate for five minutes. When you are ready to begin the test
immerse the rest of the pipette in water. Water
will enter the pipette and stop. As the seeds use the O2 in the
vial, CO2 will precipitate (be soaked up by the KOH) and the
level of water will sink in the pipette.
7.
Let the setup
equilibrate for another 2-3 minutes, then record the water level every 5
minutes for 20 minutes Try to maintain your waterbath at the assigned
temperature, because changes in temperature will affect the volume of gases
in the vials. Use your control to correct for any temperature or pressure
changes. When finished, post your data on the chalkboard.
Record the class data and graph your results in the worksheet.
C. How is respiration
balanced with photosynthesis?
It’s easy to see, when
looking at the overall balanced equations of photosynthesis and respiration,
that the end products of one series of reactions are simply the reactants of
the other.
The P/R ratio
compares the rate of Photosynthesis to the rate of Respiration
(not Products and Reactants!). Knowing this ratio can
help to explain what happens in a plant at different times in its life. For
instance, the P/R ratio is different for a corn plant during spring, summer,
and fall. It is also different for day and night. Animals, of course, can
only engage in respiration (though their respiratory rates certainly vary
for different times of the day or year).
During this exercise you
will allow organisms to engage in their normal biochemical processes of
photosynthesis and respiration. Sampling O2 and CO2
content of the environment of these organism(s) will indicate whether (and
in what proportion) these reactions have occurred. Please note that these
reactions will yield only tiny amounts of end products. The more careful you
are in following test procedures (for dissolved O2 and CO2),
the better your results will be!
NOTE:
The exercise is experimental and “open-ended.” It will provide
experience in data-gathering and show that experimental work sometimes
yields results that are difficult to interpret.
In this exercise, working
in groups, you will:
A.
Determine dissolved
oxygen and carbon dioxide in “aged” water. You will do this both to get
baseline data as well as to get practice with these complex tests.
B.
Set up control
containers of aged water and three experimental containers: plants in light,
plants in the dark, and fish.
C.
Determine dissolved
oxygen and dissolved carbon dioxide of the control and the three
experimental containers at the end of an hour.
D.
Use collected data
to answer questions.
NOTE:
Your instructor may substitute other tests to determine dissolved O2
and CO2 content.
D. Where does
respiration occur?
Your
instructor may have an additional exercise in which you will observe and
study mitochondria in cells of a celery stalk, in which case an additional
set of instructions (and questions) will be provided. Also, look carefully
at the electron micrographs of mitochondria.
CONCLUSIONS:
Materials for Cellular Respiration
Lab
A:
Fermentation tubes and racks 4
drops methylene blue 500
ml each of: All on slide warmer at 37 degrees Celsius.
distilled water
10% glucose,
10% galactose
10% sucrose
10% starch 1 ml
Yeast soln
Rulers 20 ml
Graduated cylinders 10ml
pipets and pumps Lab
B: 20 ml
Graduated Cylinders 100
ml Graduated Cylinders peas,
½ killed in bleach, others in water approximately 2 pounds
absorbent cotton balls
non-absorbent cotton
15%
KOH glass
beads
washers
Parafilm
Scissors
Respirometers Water
Bath at 37 degrees Celsius
Thermometer
Petroleum Jelly (may
need more respirometers set up if class size is large) 1mL
pipets (glass
rubber stoppers with hole to fit pipets large
test tubes Lab
C: Fish
Elodea Large
beakers Lamps Denim
Micro covers DO
Kits CO2
Kits Large
Test Tubes and Racks
Parafilm and Scissors #4
rubber stoppers to fit test tubes
REFERENCES/SOURCES:
Enger, E.D., A.H. Gibson,
J.R. Kormelink, F.C. Ross, R.J. Smith, C.H. Borgman, and R.H. Northrup.
1988. Concepts in Biology Laboratory Manual, 5e. Wm. C. Brown.
Helms, D.R., C.W. Helms,
R.J. Kosinski, and J.R. Cummings. 1998. Biology in the Laboratory,
3e. New York : W.H. Freeman.
Kull, R.C., Jr. 1992.
Revised Laboratory Manual to Accompany The Nature of Life (Postlethwait
& Hopson), 2e. McGraw-Hill.
Wilke, A.O. 1996.
Exploring Biology Today. Mosby-Year Book, Inc.
Name:
A. How do yeast cells
ferment substrates?
·
Record your data
for the yeast fermentation experiment in the table below. Enter the amount,
if any, of gas produced, best expressed as a measurement of the distance
from the surface of the fluid to the tip of the tube, and the color change
(if any).
·
Why do some tubes
(and not others) produce gas? Can you explain the differences in the amount
of CO2 gas production?
·
What do you
conclude about the importance of the type of sugar used as a food source for
fermenting yeasts?
B. How does aerobic
respiration occur in plants?
·
Remember to use
your control to correct for any temperature or pressure changes. When
finished, post your group data on the chalkboard. Record class data in the
following table:
·
Graph the O2
consumption of germinating and nongerminating pea seeds versus time for each
temperature tested (put temperature on the X axis and O2
consumption on the Y axis).
·
What effect did
temperature have on seed respiration? Explain…
·
Compare respiratory
rates of germinating/nongerminating seeds. Was there a difference? Why.
C. How is respiration
balanced with photosynthesis?
·
Enter your data in
the following table:
D. Where does
respiration occur? [Optional exercise]
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Yeast
use both aerobic and anaerobic respiration. Why do they prefer aerobic
respiration?