Download -1 Respiration and Fermentation Respiration is the process of

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Respiration and Fermentation
Respiration is the process of breaking down complex molecules to simpler
molecules and converting the chemical energy released in the process to another form of
chemical energy - ATP. ATP can be utilized in anabolic reactions to manufacture more
complex molecules from less complex molecules. For example, using energy from ATP,
amino acids can be joined together to make proteins.
Respiration can be divided into 3 steps; 1) glycolysis, 2) the Krebs cycle, and 3)
electron transport. Each step along the way involves both oxidation and reduction
reactions as electrons are removed from one molecule as it is oxidized and transferred to
electron acceptors that become reduced. In aerobic respiration, oxygen is the ultimate
electron acceptor. These reactions can be summarized in the following equation:
C 6 H 12 O6 + 6 O 2  6 CO 2 + 6 H 2 O
In glycolysis, NAD+ is reduced to produce NADH as Glyceraldehyde-3-phosphate
is oxidized. The net products of glycolysis are 2 ATPs, 2NADHs, and 2 Pyruvates. If
Oxygen or some other electron acceptor is not available, the next two steps in respiration
cannot occur. Under these conditions, some organisms can regenerate oxidized NAD+ in
fermentation reactions. In fermentation, the ultimate electron acceptor (oxidizer) is not
oxygen, but part of the original molecule. Two types of fermentation are common,
alcohol and lactic acid fermentation. In alcohol fermentation, pyruvate is broken down to
carbon dioxide (CO2) and 2 acetaldehyde. The 2 acetaldehyde is then reduced by
accepting electrons from NADH thus regenerating the cells supply of NAD+. This can be
summarized in the following two equations:
H 3 C - C - COOH
H 3C - C - H

O
H 3C - C - H
O
+CO2
O
H 3C - C - H 2
+NADH + H + 
|
+ NAD+
OH
Alcohol fermentation is used in the production of alcoholic beverages, production of
ethanol as a fuel, and in the making of bread. During bread making, the CO2 produced is
trapped in bubbles making the holes and spaces in bread. A major component of the
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delightful aroma of baking bread is actually ethanol that is evaporating from the bread as
it bakes.
Lactic acid fermentation is used to produce buttermilk and yogurt and is an
integral part of the pickling process. In this type of fermentation, pyruvate is the final
electron acceptor.
In this laboratory, we will observe CO2 production during alcohol fermentation
and aerobic respiration as well as O2 consumption during respiration. In addition, heat
produced as a byproduct of these reactions will be measured.
Exercise 1: Fermentation
1.
Obtain 6 10 ml test tubes, number them 1 - 6 and load 1 ml of the following into
each: Tube 1, 5 % Glucose; Tube 2, 5 % fructose, Tube 3, 5 % Sucrose; Tube 4, 5
% Galactose; Tube 5, distilled water; and Tube 6, 5 % Glucose.
2.
Add 1 ml of 0.1 g/ml yeast suspension to every tube except Tube 6. To Tube 6
add 1 ml of heat killed 0.1 g/ml yeast suspension. Make sure that the contents of
each tube are mixed well. You may want to note that tubes 5 and 6 are controls.
3.
Obtain 6 1 ml pipettes. Fill one pipette to the very top with the contents of Tube 1
then seal the pointed end of the pipette with parafilm.
4.
Invert the pipet into Tube 1 so that the blunt end is in what remains of the mixture
you made and the pointed end
is sticking out of the top of the
test tube.
5.
Repeat what you did with
Tube 1 to the rest of the tubes
and then place them in the
incubator. Make sure that they
are clearly marked so that you
can retrieve them later.
6.
Check the volume of CO2 that
collects at the top of each
pipette every 30 min. for the
rest of the laboratory. You will want to note which sugars worked best for the
production of CO2 by yeast.
Exercise 2: CO2 produced by aerobic respiration
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1.
Obtain 5 150 ml beakers and number them 1 - 5.
2.
Get a spot plate and add 5 drops of pH 8.3 buffer to one well. Add pH 10 buffer
to another well. Add one drop of concentrated Thymol blue solution to each well
and note the colors.
3.
Put 25 ml of dilute Thymol blue solution into each beaker. Slowly add 1 M
NaOH solution one drop at a time just until the solution matches the color of
thymol blue at a pH of 10 then stop immediately. It may help to place each beaker
on a white sheet of paper.
4.
Four different organisms should be available for you to put into beakers 1 - 4.
First weigh each organism then place it into a beaker. Beaker 5 will be a control
with no organisms in it. If you choose to use a plant, put the same amount of
plant material into two beakers.
5.
Cover the top of each beaker with aluminum foil. If you used a plant in two of
your beakers, completely cover one of the plant containing beakers with
aluminum foil so that no light can get in.
6.
Check the beakers every 10 min. for 30 min. If none of the solutions have turned
the color of thymol blue at a pH of 8.3, you may need to let this experiment go
longer.
7.
Once the solutions have turned colorless, with the possible exception of the
control, remove the organisms and place them back in their fish tank or where
ever you obtained them.
8.
Carbon dioxide that was released by the organisms should have formed carbonic
acid when it reacted with water as the following formula shows:
-
CO2 + H 2 O_ H 2 CO3 _ HCO3 + H
+
By raising the pH back to pH 10 (based on the color of thymol blue) with a known
amount of NaOH, it should be possible to determine the quantity of CO2 released
and consequently the rate of metabolism for each organism. This process is called
titration.
Obtain a pipette and slowly add, one drop at a time, 0.0025 M NaOH
solution until the solution turns the appropriate color. Be very careful not to over
shoot. Record the volume of NaOH solution used for each beaker. From this you
should be able to calculate the number of OH- molecules added to the solution.
As the number of OH- molecules should equal the number of H+ ions which
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should equal the number of CO2 molecules released, as indicated in the above
equation, this should tell you the number of CO2 molecules released by the
organism that was in the beaker in the time given.
If your control changed color, there will be one further calculation that you
will have to do. This indicates that CO2 from the atmosphere has dissolved into
your solutions and thus must be accounted for. Use titration to calculate the
quantity of CO2 in the control and subtract it from the total CO2 in the other
beakers.
8.
Standardize the metabolic rate for each organism by dividing the number of CO2
molecules per hour by the mass of each organism in grams. This should allow a
realistic comparison of metabolic rates between organisms. If you are a little
confused at this stage, consult the following equation, it shows mathematically
what you need to do:
(Nml - Cml)x
1l
0.0025M 106 M 1
1
x
x
x
x
1,000ml
1l
1M
Og THr
Where N is the volume of 0.0025 NaOH used in each beaker, C is the volume of
0.0025 NaOH used in the control, O is the mass of the organism in grams, and T
is the time in hours that the organism was in the beaker.
Exercise 3: Oxygen consumption during respiration
As the formula for aerobic respiration indicates, for every O2 molecule consumed,
a CO2 molecule is produced. In this exercise, CO2 produced by a small organism in a
closed container will be removed so that O2 consumption between the time the organism
was placed in the container and the time it was removed can be determined. Students are
encouraged to provide their own organisms for this laboratory.
1.
A number of respirometers are available at the front of the class room. Each is
different in shape and size so that different organisms can be accommodated, but
each operates on the same principle. As CO2 is produced by aerobic respiration, it
is absorbed by KOH or soda lime at the bottom of the container. A mechanism
for measuring the difference in volume as O2 is depleted in the containers
atmosphere is also present. If you have difficulty figuring out how to operate your
respirometer, get help from your laboratory instructor. Once you have obtained
the respirometer you intend to use and figured out how to use it, proceed to step 2.
2.
Allow the atmosphere to equilibrate inside the respirometer for 5 min., while you
are doing this weigh the organism that you intend to use.
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NOTE: Any cruelty or abject irresponsibility with the organisms used in this laboratory
will result in loss of credit for this weeks lab. If an organism is suffering respiratory
distress, release it immediately. As soda lime is very caustic care must be taken to avoid
contact between it and organisms. If your organism gets soda lime on it, immediately
remove it from the container and get help from your lab instructor.
3.
Place the organism in the respirometer and make sure that there are no air leaks.
As organisms and respirometers will vary, the length of time the organism is left
in the respirometer will have to be left to your judgement. Once a volume of O2
that can be accurately measured has been used, release the organism from the
container.
4.
Calculate the O2 consumption per gram of tissue for your organism and record it
on the board. Record the results of everyone else in this laboratory. Can you see
any patterns here?
Exercise 4: Heat produced during respiration
Newtons second law of thermodynamics tells us that as energy is changed from
one form to another, some of the energy is lost in the form of entropy or waste energy.
During metabolism this waste energy is heat, a form of energy that cannot be harnessed
by the organism. In this very simple exercise, the amount of heat being released by
germinating beans will be measured. Most of the work has already been done by the
laboratory instructors.
Forty eight hours before the start of this lab, 0.5 Kg of dry beans was soaked over
night in water. Twenty four hours before the start of the laboratory, about 4 cm of wet
cotton was placed into the bottom of a thermos flask, the beans were loaded in on top of
the wet cotton, and a thermometer was inserted into the beans. The top was then closed
so that heat would be trapped inside the thermos, but could be read from the thermometer.
All you have to do is read the temperature on the thermometer and compare it with the
ambient temperature.
Exercise 5: For A students only
Design your own experiment dealing with respiration. Here are a few
suggestions:
1.
See what happens to the pH of a solution containing yeast and sugar over time.
2.
Check the gas released in Exercise 1 to make sure it is CO2. Can you think of a
good way to do this?
3.
Try putting yeast solution in a respirometer to see if any aerobic respiration is
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going on along with fermentation.
4.
See if different seeds produce different amounts of heat when germinated
compared to the beans used in Experiment 4.
Materials
Equipment
Beakers, 150 ml 5/student
Pipettes, 1 ml 6/student
Respirometers
Test tubes, 10 ml 6/student
Thermometer
Thermos flask, 1 l
Chemicals
Concentrated Thymol Blue Solution
Dilute Thymol Blue Solution
Potassium Hydroxide, KOH
Sodium Hydroxide solution, 1 M and 0.0025 M NaOH
5% Galactose
5% Glucose
5% Fructose
5% Sucrose
Supplies
Beans, 0.5 Kg
Cotton
Critters, as many as you can lay your hands on
Elodea
Brewers yeast
Tape
Parafilm