AP Biology Lab 5
Cell Respiration Lab Activity: Poikilotherms vs. Peas
Objectives:
 Measure the amount of carbon dioxide produced by poikilotherms and peas using a LabQuest and
a CO2 sensor
 Quantify and graph the effects of temperature on the rate of respiration
 Demonstrate the relationship between dependent and independent variables
 Compare the rates of cellular respiration in varying temperatures
Background
In order for our bodies to function, we need fuel. This fuel comes from the foods we eat in the
form of proteins, carbohydrates, and fats. Of these organic compounds, it is the carbohydrate glucose that
is the most preferred energy source for our bodies. However, in order for this energy to be useful to us, it
must first be converted to ATP (adenosine triphosphate), the common carrier of chemical energy in the cell.
All body cells split glucose molecules to transfer the energy to ATP through a process called
cellular respiration. This energy transfer occurs in two stages: glycolysis and aerobic respiration. In the
first stage, a small amount of ATP is produced when glucose is broken down to pyruvate during glycolysis.
In the second stage, the pyruvate either passes through aerobic respiration (producing a large amount of
ATP) or (anaerobic) fermentation (producing a small amount of ATP). In the absence of oxygen, pyruvate
is converted to either lactate or ethanol and carbon dioxide. The difference between aerobic respiration, and
anaerobic respiration, is that in aerobic, ATP is produced by cells breaking down organic compounds with
oxygen as the final electron acceptor. With anaerobic respiration, energy is liberated that does not require
the presence of oxygen.
ATP provides the energy used for synthetic reactions, active transport, and all cell processes.
Nearly all organisms’ cells respire and in eukaryotes, this occurs in the mitochondria. However, in bacteria
and other prokaryotes, which do not have mitochondria, the conversion of NADH and pyruvate to ATP
occurs in the cell membrane. The overall equation for the breakdown of glucose can be represented in the
following way:
Glucose + Oxygen
Carbon Dioxide + Water + Energy (ATP)
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The energy-rich carbohydrates that undergo cellular respiration in plants and animals are produced
in the chloroplasts of plants and algal cells. Photosynthesis uses carbon dioxide, water, and light energy to
produce oxygen and carbohydrates. Cellular respiration uses carbohydrates and oxygen to produce carbon
dioxide, water, and energy for the activities of the cell. Cellular respiration, then, is essentially the reverse
of photosynthesis. It is the link between the energy captured by plant cells and the energy utilized by both
plant and animal cells and is the means by which energy flows from the sun through living things.
Glucose Metabolism
Most cells use glucose as an energy source for producing ATP. The first stage of glucose
metabolism is referred to as glycolysis, a process that occurs outside the mitochondria in the cytosol.
Glycolysis is anaerobic, meaning that it does not require oxygen. Glucose is converted to two molecules of
pyruvate with little breakdown; however, oxidation by removal of hydrogen atoms (e -+ H+) does occur, and
the energy of oxidation is used to generate two molecules of ATP. A small amount of NADH is also
produced.
The fate of pyruvate, the end product of glycolysis, depends on the availability of oxygen. It can
either be involved in fermentation or incorporated into the Krebs Cycle. Under anaerobic conditions,
fermentation occurs within the cytosol and pyruvate accepts the two molecules of hydrogen that were
removed from glucose during glycolysis. Because only two molecules of hydrogen are available,
fermentation yields only two ATP molecules.
Pyruvate can be reduced in one of two ways. It can become lactate in fermentation, which is used
by many bacteria and some small animal cells, or it can be reduced to alcohol with the release of carbon
dioxide, which is known as alcoholic fermentation and is utilized by yeast and plant cells.
Cellular Respiration
If oxygen is available to the cell, cellular respiration occurs. This is an aerobic process that
produces much more energy than fermentation. In this case,
pyruvate is not reduced, but instead enters the mitochondria and
is completely oxidized to carbon dioxide and water. The
removal of hydrogen atoms (e-+ H+) is accompanied by the
release of carbon dioxide, which diffuses out of the
mitochondria and the cell; the hydrogen atoms are sent to an
electron transport system located in the mitochondrial cristae,
which are folds of the inner membrane. As electrons are passed
from one carrier to the next in the cristae, energy is made
available that is eventually used to generate ATP molecules.
Oxygen, the final acceptor for these electrons, is reduced to
water. Most of the ATP production in cells comes from this
electron transport system.
If glycolysis is followed by cellular respiration, then the
overall reaction for glucose breakdown to carbon dioxide and
water can be represented as:
C6H12O6 + 6CO2
36 ATP + 6CO2 + 6H2O
The complete oxidation of glucose by cellular respiration yields a total of 36 ATP molecules, most
of which are produced by the electron transport system. It is possible to calculate the ATP yield for the
complete breakdown of glucose to carbon dioxide and water.
Oxidative Respiration
In the presence of oxygen, the pyruvate from glycolysis enters a mitochondrion, where a series of
reactions called oxidative respiration occurs. This is the transition reaction between glycolysis and the
Krebs Cycle. The electrons made available are passed to the electron transport system by either NAD + or
FAD. The reactions of oxidative respiration drive the production of large amounts of ATP.
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In order to prepare for the ATP-producing part of oxidative respiration, the pyruvate molecules
from glycolysis are first converted from three- to two-carbon fragments. As this occurs, a carbon dioxide
molecule and a hydride ion with high-energy electrons are removed from each pyruvate molecule. The
carbon byproduct leaves the mitochondrion and then the cell. The donation of hydride ions to an NAD +
molecule forms HADH, which is later used in oxidative respiration. The remaining two-carbon fragment,
an acetyl group, is attached to coenzyme A to form a compound acetyl-CoA. Acetyl-CoA is funneled into
fat synthesis if the cell’s supply of ATP is plentiful, allowing high-energy electrons to be stored for later
needs. If the cell needs ATP immediately, however, acetyl-CoA is directed to the next phase of oxidative
respiration, which is the Krebs Cycle.
The Krebs Cycle
The Krebs Cycle is a repeating series of reactions that produces ATP, electron carriers, and carbon
dioxide. The cycle begins when the two-carbon fragment of acetyl-CoA is attached to a four-carbon
molecule found in mitochondria. This forms a six-carbon compound called citrate; it is for this reason the
Krebs Cycle is also known as the citric acid cycle. During the cycle, oxidation occurs, and most of the
hydrogen atoms are donated to NAD +, except for one instance where the hydrogen atoms are donated to
FAD, another coenzyme of oxidation and reduction in cells. Some of the energy of oxidation is used
immediately to form ATP by substrate-level phosphorylation, as in glycolysis. Since the Krebs Cycle
occurs once for acetyl-CoA, it occurs twice for each glucose molecule.
Electron Transport System
Due to the transition reaction and the Krebs Cycle, the carbons of glucose are completely oxidized
to six molecules of CO2. The hydrogen atoms (e- + H+) that were removed from the substrates of these
metabolic pathways are donated by NADH and NADH2 to the electron transport system. This system is a
series of carriers and enzymes located on the cristae of mitochondria. It is also known as the cytochrome
system, since some of the electron carriers are cytochromes. Each electron transport chain passes highenergy electrons to proton-pumping membrane channels. As electrons are passed from one carrier to the
next, oxidation occurs, and the energy released is used to form ATP molecules. (ATP leaves a
mitochondrion through other protein channels, entering the cytosol where it can be used by other reactions).
This process is sometimes known as oxidative phosphorylation because oxygen receives the energy-spent
electrons from the chain. The enzyme cytochrome oxidase splits and reduces molecular oxygen (O 2) to
water:
½ O2 + 2e- + 2H+
H 2O
The final acceptor for electrons during cellular respiration is oxygen.
Substrate-Level Phosphorylation
For every glucose molecule, four ATP are formed directly by substrate-level phosphorylation—
two during glycolysis and one during each of the two turns of the Krebs Cycle.
Oxidative Phosphorylation
Fore every glucose molecule, ten molecules of NADH and two molecules of FADH 2 take
hydrogen atoms (e- + H+) to the electron transport system. For each molecule of NADH produced inside
the mitochondria by the Krebs Cycle, three ATP are produced by the electron transport system, but for each
FADH2 there are only two ATP. This is because FADH2 delivers its hydrogen to the transport system at a
lower level, pumping only 4H + instead of 6H+ into the intermembrane space per molecule of FADH2.
NADH generated outside the mitochondria during glycolysis requires a shuttle mechanism to
transport the electrons that yield ATP, because NADH cannot cross the mitochondrial membrane. This
shuttle, which consists of an organic molecule that can cross the outer membrane, allows electrons to be
delivered to the electron transport system inside the mitochondria. The shuttle molecule crosses the outer
membrane, accepts the electrons, and delivers them to the FAD molecule in the inner membrane. At this
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point, the FAD can produce two ATP molecules; therefore, the NADH produced in the cytosol results in
the production of only two ATP rather than three.
Respiration applies to two distinct but interrelated processes: the active acquisition of oxygen by
an organism and the release of energy through the breakdown of organic compounds by metabolic chemical
oxidation. Various factors, such as temperature change, will affect respiration, as well as the chemical
oxidation of glucose. Oxygen consumption during respiration can then be measured as a change in gas
volume by using a respirometer.
In cold-blooded organisms, (poikilotherms) there is a link between the temperature of the
environment and the organism’s metabolic rate. Reptiles are a common example of a cold-blooded
organism with which most people are familiar. If you have ever seen a lizard or snake in the early morning
when the air and ground are cool, you may have noticed how slowly they move. They move slowly when
the environment is cold because they require heat from their surrounding to increase their internal
temperature and metabolism. Once their internal body temperature has warmed, they can metabolize foods
more quickly and produce the energy they need. Oxidative respiration is the process of metabolism where
sugars are broken down. Under aerobic conditions, respiration yields ATP, carbon dioxide, and water.
Part A of this lab is a cook-book lab. You will be analyzing cellular respiration in germinating
and non-germinating peas. Part B of this lab is inquiry. You will have a choice of organisms to be used to
study the effect of temperature on the metabolism of poikilothermic organisms. You will determine how
temperature affects the respiration rate of your organism by monitoring carbon dioxide production with a
CO2 gas sensor. If you want to study an organism that isn’t available, you may do so, however, you must
provide it yourself. You must remember to only bring invertebrates, and to make sure they are comfortably
accommodated throughout the experiment. Ethically, the hottest you will be allowed to go is 40ºC.
Lab Part A: Respiration in Germinating Peas
Objectives:
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Measure the amount of carbon dioxide produced by germinating peas using a LabQuest and a CO 2 sensor.
Quantify and graph the effects of temperature on the rate of respiration in plants.
Demonstrate the relationship between dependent and independent variables.
Compare the rates of cellular respiration in germinating and non-germinating peas.
Distinguish between substrate-level phosphorylation and oxidative phosphorylation and determine the energy yields of
each.
Materials List:
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25 germinating peas
25 non-germinating peas
1 CO2 sensor, with respiration chamber
LabQuest
Computer with Logger Pro Installed
200 mL beaker
1 L beaker
Ice
Paper towels
Thermometer (or temperature probe)
Procedure:
1.
2.
3.
4.
5.
6.
7.
Plug the CO2 Gas Sensor into Channel 1 of the LabQuest
Prepare the computer for data collection by performing the following steps: Click the file on the toolbar. Click
on OPEN. Open the folder Biology with Computers (not Advanced Biology). Open the folder “Experiment
11B”. The y-axis has carbon dioxide concentration scaled from 0 to 5,000 ppm. The x-axis has time scaled
from 0 to 5 minutes. The data rate is set to 6 samples/minute.
Obtain 25 germinating peas and blot them dry between two pieces of paper towel. Use the thermometer to
measure the room temperature. Record the temperature in a data table resembling data table 1 below in your
quadrille.
Place the germinating peas into the respiration chamber.
Place the shaft of the CO2 gas sensor in the opening of the respiration chamber.
Wait one minute, then begin measuring carbon dioxide concentration by clicking COLLECT on the toolbar.
Data will be collected for 5 minutes.
Remove the CO2 gas sensor from the respiration chamber. Place the peas in a 200 mL beaker filled with cold
water and ice.
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8.
Determine the rate of respiration by performing a linear regression.
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Move the mouse pointer to the point on the graph where the data values began to increase. Hold
down the left mouse button. Drag the mouse pointer to the end of the data and release the mouse
button.
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Click the Linear Fit button on the toolbar, to perform a linear regression.
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Record the slope of the line, m, as the rate of respiration for germinating peas at room temperature in
a table that looks similar to data table two below in your quadrille.
Table Two
Table One
Environment
Room
Cold Water
Temperature
(°C)
Peas
Germinating, Room Temperature
Non-Germinating, Room Temperature
Germinating, Cold
Rate of Respiration
9. Obtain 25 non-germinating peas and place them in the respiration chamber.
10. Repeat steps 5-8for the non-germinating peas. Click SAVE last graph.
11. Place the respiration chamber in a 1 L beaker. Pour ice water into the beaker until the water level reaches the
neck of the chamber.
12. Use the thermometer, or temperature probe to measure the ice water temperature in the 200 mL beaker
containing the cold germinating peas. Record the water temperature in table 1.
13. Remove the peas from the cold water and blot them dry between two paper towels.
14. Repeat steps 5-8 to collect data with the germinating peas at a cold temperature.
15. Click SAVE last graph.
16. To Print a graph showing all three data runs:

Label all three curves by choosing Make Annotation from the Analyze menu, and typing “Room
Temp Germinated” (or “Room Temp Non-Germinated”, or “Cold Germinated”) in the edit box.
Then drag each box to a position near its respective curve.

Print a copy of the Graph window, with all three data sets and the regression lines displayed. Enter
your name(s) and the number of copies of the graph you want. Attach the copy of the graph to your
quadrille.
Part B: Inquiry Lab: Temperature and Respiration Rates in Poikilotherms
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The question to be studied is: How does temperature affect respiration rate?
Develop a hypothesis
Make your materials list
List your procedures
Develop any data tables you may need.
Things you will absolutely need:
 Computers
 Electronic balance (to mass the organisms)
 Probeware (CO2 sensors; thermometers)
 Thermometers (manual back-up)
 To perform a linear regression to calculate the rate of respiration (slope)
Processing Data:
 For each temperature, you must divide the slope of the regression line by the mass of the crickets.
 Record this value as the rate of respiration
 Record the temperature and respiration rates in a data table
 Create a graph with temperature plotted along the x-axis, and rates of respiration along the y-axis.
Possible Organisms: Crickets; Earthworms; Giant Cockroaches; Millipedes; Giant
Millipedes; any other invertebrate you may wish to supply for yourselves.
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Analysis Questions:
1.
2.
3.
4.
5.
At what temperature was the rate of carbon dioxide production highest in part A? How does this
relate to the internal body temperature of warm-blooded organisms?
Predict the rate of respiration for crickets at 50ºC. Explain.
During aerobic respiration, glucose is broken down to form several end products. Which end
products contain the carbon atoms from glucose? The hydrogen atoms from glucose? The oxygen
atoms from glucose? The energy stored in the glucose molecules?
What is fermentation? What are the two types of fermentation? What organisms use
fermentation?
Matching: Write the letter of the pathway that best fits each of the following processes.
Pathway
a.
Glycolysis
b.
Krebs Cycle
c.
Electron Transport System
Process
________1. Carbon dioxide is given off
________2. Water is formed
________3. PGAL
________4. NADH becomes NAD+
________5. Oxidative phosphorylation
________6. Cytochrome carriers
________7. Pyruvate
________8. FAD becomes FADH2
6.
Name some other biological processes that are affected by temperature. (Remember, not all
biological processes are about humans, or even animals)
7. According to your graph, what happens to the rate of carbon dioxide production by germinating
peas over time? What does this indicate to you?
8. Which of the two pea types, germinating or non-germinating, consumes the most oxygen? Why?
9. What was the effect of temperature on pea respiration? Was that different from the crickets? Was
this what you expected?
10. At this point you have observed cellular respiration in cold blooded organisms at three
temperatures, and cellular respiration on plants at cold and room temperatures. Given your
observations, predict what might happen if you were to carry out the above procedure on
germinating, and non-germinating peas in a 40° C environment.
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