1 Objectives • Measure carbon dioxide evolution and uptake in plants and animals. • Study the effect of temperature on cell respiration. • compare respiration rates in germinating and non-germinating peas. Introduction Energy is required by living organisms for movement, transport, and growth. Nothing happens without energy! The Sun is the ultimate source of virtually all energy on the planet Earth. Solar energy is captured by plants through the process of photosynthesis. The glucose molecules holding this energy are broken down by metabolic processes, creating usable energy for living systems. Cellular respiration is a series of reactions in which glucose molecules are broken down, releasing stored chemical bond energy (Figure 6.1). The released energy is used to make the energy rich molecule ATP (adenosine triphosphate). Carbon dioxide is released as a by-product of the breakdown of glucose. It is a crucial by-product from the perspective of plants, because they need CO2 to perform photosynthesis. Glycolysis is the first step in cellular respiration, and it results in the net production of two ATP molecules. In glycolysis, the 6- carbon glucose molecules are “split” into two, 3-carbon pyruvate (pyruvic acid) molecules. LAB TOPIC 6: RESPIRATION Pyruvate has two potential routes – aerobic respiration or anaerobic respiration [as either lactate fermentation or alcohol fermentation] (Figure 6.1). 1 In laboratory today, you will be examining respiration in organisms that use aerobic respiration, which makes use of oxygen. In this pathway, pyruvate is broken down completely, and h igh-energy electrons are stripped away and passed through a series of electron carriers. Energy is released at each transfer, and is used to make a net 34 ATP molecules. Oxygen is the final electron acceptor in the electron transport system, hence the name aerobic cellular respiration. In lecture you will compare this process to anaerobic respiration, which occurs in the absence of oxygen or under low oxygen conditions. The equation below summarizes the process of aerobic respiration: C6H12O6  +  6  O2  à 6  CO2  +  6  H2O  +  ATP  +  Heat   Glucose      Oxygen          Carbon        Water                                                                        Dioxide     Considering the equation for aerobic respiration what variables could you measure to monitor respiration rate?   Figure  6.1     Glycolysis  and  the  potential  fates  of  pyruvate  during  cellular  respiration.   2 2 Oxygen Consumption during Aerobic Respiration Aerobic respiration uses oxygen as the terminal electron‐acceptor in the electron transport chain and produces carbon dioxide (see equation above). You can, therefore, monitor the respiration rate of an organism by measuring its uptake of oxygen or evo ...

1. 1
Objectives
• Measure carbon dioxide evolution and
uptake in plants and animals.
• Study the effect of temperature on cell
respiration.
• compare respiration rates in germinating
and non-germinating peas.
Introduction
Energy is required by living organisms for
movement, transport, and growth. Nothing
happens without energy! The Sun is the
ultimate source of virtually all energy on the
planet Earth. Solar energy is captured by
plants through the process of photosynthesis.
The glucose molecules holding this energy are
broken down by metabolic processes, creating
usable energy for living systems.
Cellular respiration is a series of reactions in
which glucose molecules are broken down,
releasing stored chemical bond energy
(Figure 6.1). The released energy is used to
make the energy rich molecule ATP
(adenosine triphosphate). Carbon dioxide is
released as a by-product of the breakdown of

2. glucose. It is a crucial by-product from the
perspective of plants, because they need CO2
to perform photosynthesis.
Glycolysis is the first step in cellular
respiration, and it results in the net production
of two ATP molecules. In glycolysis, the 6-
carbon glucose molecules are “split” into two,
3-carbon pyruvate (pyruvic acid) molecules.
LAB TOPIC 6: RESPIRATION
Pyruvate has two potential routes – aerobic
respiration or anaerobic respiration [as either
lactate fermentation or alcohol fermentation]
(Figure 6.1).
1
In laboratory today, you will be examining
respiration in organisms that use aerobic
respiration, which makes use of oxygen. In
this pathway, pyruvate is broken down
completely, and h igh-energy electrons are
stripped away and passed through a series of
electron carriers. Energy is released at each
transfer, and is used to make a net 34 ATP
molecules. Oxygen is the final electron
acceptor in the electron transport system,
hence the name aerobic cellular respiration. In
lecture you will compare this process to
anaerobic respiration, which occurs in the
absence of oxygen or under low oxygen
conditions. The equation below summarizes
the process of aerobic respiration:

3. C6H12O6
+
6
O2
CO2
+
6
H2O
+
ATP
+
Heat
Glucose
Oxygen
Carbon
Water

4. Dioxide
Considering the equation for aerobic
respiration what variables could you measure
to monitor respiration rate?

5. Figure
6.1
Glycolysis
and
the
potential
fates
of
pyruvate
during
cellular
respiration.
2
2
Oxygen Consumption during Aerobic
Respiration
Aerobic respiration uses oxygen as the
terminal electron‐acceptor in the electron
transport chain and produces carbon dioxide
(see equation above). You can, therefore,
monitor the respiration rate of an organism by
measuring its uptake of oxygen or evolution of
carbon dioxide. Here, we will measure the
respiration rate of the crickets, Gryllus sp., and
English peas, Pisum sativa, using a gas‐phase

6. CO2 probe and meter. This equipment can
be used to measure the level of CO2 in the
atmosphere of a closed chamber in units of
parts per million (ppm) or mg/L.
Respiration rate is dependent on a variety of
factors including the size and the level of
activity of an organism, temperature, etc. At a
given temperature, would you expect a crickets
or peas to have a higher respiration rate on a
per mass basis?
Exercise 6.1
Evolution of Carbon Dioxide by a Plant
1
Here we will measure respiration in
germinating peas. We will also address
the questions “Do peas undergo cell
respiration before germination?” and
“What is the effect of temperature on the
cell respiration of peas?” Using your
collected data, you will be able to answer
these questions.
Hypothesis
Construct null and alternative hypotheses
(e.g. for the effect of germination and

7. temperature on respiration).
Remember, your hypotheses must be
testable.
2
Prediction
Predict the result of the experiment based on
your hypotheses. Your prediction would be
what you expect to observe as a result of this
experiment (if/then).
Plants undergo both respiration and
photosynthesis (at least during the day). As
these two processes offset each other to

8. some degree or another, the amount of CO2
evolved or consumed depends on how active
photosynthesis is relative to respiration. To
eliminate this problem from the determination
we will make today, we will use pea seeds.
Since these are not significantly
photosynthetic, changes we measure in CO2
concentration in the chamber will be due to
respiration alone. These peas undergo cell
respiration during germination as they begin
the process of seedling growth.
3
3
PROCEDURE – Part 1 Effects of
Germination (You will be using Logger Pro
as you did for the spectrophotometer)
1. Obtain 25 germinating peas and blot
them dry between two pieces of paper
towel.
2. Place the germinating peas into the
respiration chamber.
3. Place the shaft of the CO2 Gas Sensor in

9. the opening of the respiration chamber.
4. Wait one minute, then begin measuring
carbon dioxide by clicking the green arrow
button (Experiment/ Start Collection).
Collect data for 5 minutes then Stop
Collection.
5. Remove the CO2 Gas Sensor from the
respiration chamber. Remove and weight
the peas (record the weight below).
6. Place the peas on ice for Part 2 of the
experiment.
7. Use a notebook or notepad to fan air
across the openings of the probe shaft of
the CO2 Gas Sensor for 1 minute. Make
sure the CO2 readings return to 300-400
ppm.
8. Fill the respiration chamber with water,
empty, dry the inside thoroughly with a
paper towel.
9. Determine
the

10. rate
of
respiration:
a. Move
the
mouse
pointer
to
the
point
where
the
data
values
begin
to
increase.
Hold
down
the
left
mouse
button.
Drag
the
mouse
pointer
to
the

11. end
of
the
data
and
release
the
mouse
button.
1
b. Click
“Analyze”
and
the
“Linear
Fit”
button
to
perform
a
linear
regression.
A
floating
box
will
appear

12. with
the
formula
for
a
best
fit
line.
c. Record
the
slope
of
the
line,
m,
as
the
rate
of
respiration
for
germinating
peas
at
room
temperature
in
Table
6.1.

13. d. Close
the
linear
regression
box
by
clicking
on
it
and
deleting.
e. Move
your
data
to
a
stored
run
by
choosing
“Store
Latest
Run”
from
the

14. Experiment
menu.
10. Obtain
25
non-­‐germinating
peas
and
place
them
in
the
respiration
chamber
11.
Repeat
Steps
3–9
for
the
non-­‐
germinating
peas
and
record
the

15. rate
in
Table
6.1.
4
21
Part 2 – Effects of Temperature
1. Remove
the
peas
from
the
ice
and
blot
them
dry
between
two
paper
towels.

16. 2. Repeat
Steps
3–9
from
Part
1
to
collect
data
with
the
cold
germinating
peas
and
record
the
rate
in
Table.6.1.
TABLE
6.1
Peas
Temperature
(ºC) Rate
of

17. respiration
Mass-­‐Corrected
Respiration
Rate
(ppm/g/min)
Germinating,
Room
Temperature
20
Non-­‐germinating,
Room
Temperature
20
Germinating,
Cool
Temperature
4
QUESTIONS
1. Do
you
have
evidence
that

18. cell
respiration
occurred
in
peas?
Explain.
2.What
is
the
effect
of
germination
on
the
rate
of
cell
respiration
in
peas?
3.What

19. is
the
effect
of
temperature
on
the
rate
of
cell
respiration
in
peas?
4.Why
do
germinating
peas
undergo
cell
respiration?

20. 5
Exercise 6.2
Evolution of Carbon Dioxide by an Animal
2
Animals, like plants and other eukaryotes
obtain energy for growth and day to day
metabolism through metabolism of sugars
through the glycolytic pathway of respiration
and produce CO2 as a result of this activity.
Thus, we can measure the rates of respiration
as we have done above for peas. However,
because they do not photosynthesize, any
changes in CO2 concentration
are due to respiration alone.
Table 6.2
Hypothesis
Construct null and alternative hypotheses (e.g.
for the effect temperature on respiration and a
comparison of peas and crickets).
Remember, your hypotheses must be testable.

21. Prediction
Predict the result of the experiment based on
your hypotheses. Your prediction would be what
you expect to observe as a result of this
experiment (if/then).
PROCEDURE
1. Obtain
5
crickets
from
your
instructor.
2. Place
the
crickets
into
the
respiration
chamber.

22. 3. Start
a
new
File.
Perform
the
experiment
as
outlined
in
steps
3-­‐9
above
at
room
temperature
and
4°C
(pre-­‐
incubate
in
the
refrigerator
and
then
held
on
ice).

23. 4. Record
your
results
in
Table
6.2.
.
.
.
.
.
.
Crickets
Temperature
(ºC) Rate
of
respiration
Mass-­‐Corrected
Respiration
Rate

24. (ppm/g/min)
Room
temperature
20
Cool
temperature
4
6
Questions for
1. What is the importance of cellular
respiration to living organisms?
2. The concentration of oxygen in
an environment can affect the
respiration rate of an organism.
With this in mind, why would you
need to limit the length of time of
the experiments we ran today?

25. 3. You are designing an experiment to
examine the effect running speed
has on the respiration rate of cross‐
country runners. State null and
alternative hypotheses for this
experiment:
Vocabulary
1
Objectives
• Examine carbon fixation
• Examine electron transport in
photosynthesis.
• Examine the effects of inhibitors on
photosynthesis.
Terms
Photosynthesis
Biochemical reaction of photosynthesis
Photochemical reaction of photosynthesis
Photosynthetic electron transport chain
Chlorophyll accessory pigments
Introduction
Photosynthesis uses light energy to split H2O

26. and harvest high-energy electrons. These
energetic electrons (and accompanying H+)
are passed to CO2. In doing so, CO2 is
reduced to form energy-storing sugars.
Cellular respiration removes electrons from
(i.e. oxidizes) sugar, captures the energy in
adenosine triphosphate (ATP), and ultimately
passes the electrons to oxygen to form H2O.
Organisms use the energy stored in ATP to
conduct cellular business such as transport,
synthesis of biomolecules, reproduction, and
sometimes cellular movement.
LAB TOPIC 6: Photosynthesis
Remember that in photosynthetic eukaryotes
(plants and algae), respiration occurs as it does
in animal and fungal cells (Figure 6.1).
When in an aqueous solution, carbon dioxide
reacts with water to form carbonic acid.
This results in lowering the solution's pH. As
CO2 is consumed by aquatic plants through
photosynthesis, the level of carbonic acid in a
solution will decrease, leading to an increase in
pH. Thus, monitoring pH provides an indirect
measure of the amount of CO2 consumed in
photosynthesis. This experiment uses
bromothymol blue (BTB), a pH indicator that
turns yellow at pH < 6.0, green at pH 6.0 - 7.6,

27. and blue at pH > 7.6. We will be looking at
changes in pH of water in the light and dark and
presence and absence of the aquatic plant.
Exercise 6.1
Carbon dioxide fixation by an aquatic plant
Figure 6.1 Overviews of aerobic respiration and photosynthesis.
2
Procedure 6.1 Carbon dioxide consumption
1. Place 75 ml of BTB solution into a 100 ml
beaker. Blow exhaled air through a straw
into the BTB solution until it changes from
blue to yellow-brown.
2. Obtain two 2-cm sprigs of plant, and place
one in a tube labeled "light" and the other
"dark". The other two tubes will have
no plant in them. Use these for
comparison.
3. Fill the four test tubes 3/4 full with C02-rich
BTB solution.
4. Place the two "light" tubes directly in
front of the grow light, and the other tubes in the dark.
.
5. Allow the tubes to "incubate" for 1 hour.
Proceed to the next exercise while you wait.

28. In Table 6.1, record any color changes that
have occurred by marking an X in the
appropriate space.
Hypothesis
Construct null and alternative hypotheses for
the effect of light and dark and presence and
absence of a plant on pH. Remember, your
hypotheses must be testable.
Prediction
Predict the results of the experiment based
on your hypotheses. Your prediction would
be what you expect to observe as a result of
this experiment (if/then).
Yellow
pH < 6.0
Green
pH 6.0 – 7.6
Blue

29. pH > 7.6
plant, light
plant, dark
NO plant, light
NO plant, dark
Table 6.1 Carbon dioxide consumption by an aquatic plant
3
Questions
Do you accept or reject your null hypotheses?
Why did the BTB turn yellow as you blew
through the straw?
What is responsible for the color change of BTB
in the tube with the plant placed in front of the light?
Why might the tube with the plant placed in the
dark exhibit an increase in pH?
Why might the tube with the plant placed in the
dark exhibit a decrease in pH?
Exercise 6.2

30. Effects of herbicides on electron
transport in
An overview of the light-dependent reactions
of photosynthesis
In the late 1930’s, Robert Hill and colleagues
observed that under proper conditions, isolated
thylakoids retained their capacity to evolve
oxygen. This phenomenon is now known as the
Hill Reaction. The Hill Reaction is part of what
are known as the photochemical reactions of
photosynthesis. This activity is associated with
Photosystem II (Figure 6.2), in which the
electrons that originate with splitting of water are
used to reduce electron acceptors. There is a
simultaneous release of oxygen. In the intact
living organism, these electrons ultimately
reduce NADP+ to form NADPH. During
photosynthetic electron transport, hydrogen ions
are moved across the thylakoid membrane as
plastoquinone shuttles electrons between PS II
and the cytochrome B6/f complex. The resulting
hydrogen ion gradient is used to produce
ATP. The ATP and NADPH then are used
in the biochemical reactions to produce
sugars, thus trapping light energy in the
chemical bonds of carbohydrates.
Hill used artificial electron acceptors,
including 2,6-dichloroindolphenol (DCPIP),
to trap electrons passed through the
electron transport chain from photosystem
II when isolated chloroplasts are exposed
to light. As the blue, oxidized form of

31. DCPlP becomes reduced it becomes
colorless. Thus the progress of the reaction
can be monitored by the change in
absorbance at 600 nm of the DCPIP
solution.
DCPIP + 2H+ + 2e- → DCPIP-H2
(blue) (colorless)
(Remember that chlorophyll is green and so only the blue
color will disappear entirely.)
The rate of the Hill Reaction is then
dependent on light intensity and can be
measured either as oxygen produced or
reduction of electron acceptors.
First we will look at electron transport in
thylakoids, then we will look at the effects
of herbicides.
Figure 6.2 Photosynthetic electron transport
4
Procedure 6.2.1 Electron Transport
1. Prepare test tubes according to Table 6.2.
Metabolically active thylakoids will be
provided to you. Add the DCPIP (blue dye)

32. last.
2. Mix the contents of each tube by inverting
each tube several times. Place tubes 2 and 3
in front of the light source for 2 minutes. Do
not position tubes behind one another or in front
of tubes from other groups. Place tube 4 in the
dark.
3. Observe color changes in the tubes and
record your observations.
What were the initial and final colors of each
tube?
Of what importance is each tube (1-4)
in this exercise? Which tubes were
CONTROLS?
Table 6.2
Solution
s for comparison of photosynthetic reaction rates

33. Tube Thylakoids 0.1 M phosphate
buffer pH 6.5
Water 0.2 M DCPIP
1 0.5 ml 3 ml 1.5 ml 0
2-LIGHT 0.5 ml 3 ml 0.5 ml 1 ml
3 0 3 ml 1.0 ml 1 ml
4-DARK 0.5 ml 3 ml 0.5 ml 1 ml
Procedure 6.2. Effects of Herbicides on
Electron Transport
Next we will monitor the effect of a photosynthetic
herbicide on photosynthesis. We will quantify the
electron transport (DCPIP reduction) using a
spectrophotometer. Monitor absorbance at
600 nm. USE WATER AS A BLANK.
Hypothesis

34. Construct null and alternative hypotheses for the
effects of herbicides on electron transport in active
thylakoids. Remember, your hypotheses must
be testable.
Prediction
Predict the result of the experiment based
on your hypotheses. Your prediction would
be what you expect to observe as a result of
this experiment (if/then).
5
1
1. Using water as a blank, calibrate your
spectrophotometer.

35. 2. Set up tube #1 as shown in Table 6.3.
3. Fill your cuvette with