GENERAL BIOLOGY LAB 1 (BSC1010L)
Lab #6: Cellular Respiration
OBJECTIVES:
• Understand the major events of glucose catabolism (cellular respiration): glycolysis, the citric acid cycle and oxidative phosphorylation.
• Compare and contrast the processes involved in aerobic and anaerobic respiration.
• Demonstrate carbon dioxide production during anaerobic respiration.
• Determine oxygen consumption during aerobic respiration.
• Measure the relative production of carbon dioxide by plants and animals.
______________________________________________________________________________
INTRODUCTION:
All living organisms have evolved mechanisms to obtain the energy needed to fuel basic 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 that is stored in the negatively charged bonds of a denosine t ri p hosphate or ATP , which is used by cells to perform essential metabolic functions.
Energy flow through biological systems occurs through oxidation-reduction or redox reactions where electrons are transferred from one molecule to another. Recall from the
Biologically Important Molecules lab (Lab 4) that reduction is defined as the gain of electrons or hydrogen atoms while oxidation involves the loss of electrons or hydrogen atoms. During cellular respiration, electrons are removed from glucose (i.e., glucose is oxidized) and some of the released energy is stored in the form of ATP. The dozens of redox reactions that take place during this process use electron acceptors for energy transfer. Two of the most important electron acceptors are nicotinamide adenine dinucleotide (NAD dinucleotide (FAD
+
) , derived from niacin (Vitamin B
3
+
) and flavin adenine
) and riboflavin (Vitamin B
2
), respectively. These molecules are reduced to NADH and FADH
2
when they acquire electrons, which they then transfer to other molecules to generate ATP. Depending on which molecule serves as the final electron acceptor, the entire process is considered 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 and sulfates or organic molecules such as ethanol or lactate (Fig. 1).
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Figure 1. Comparison of redox reactions in aerobic and anaerobic respiration
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
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Glucose catabolism begins with glycolysis in the cytoplasm (Fig. 3). Glycolysis includes a series of reactions where each entering glucose (6 carbons) molecule 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, then the processes of aerobic respiration will begin with pyruvate oxidation . During this phase, each pyruvate molecule generated from gylcolysis enters the mitochondria and is converted into carbon dioxide (CO
2
), which is released as a side-product, and acetyl, a 2 carbon 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 or tricarboxilic acid (TCA) cycle). For every turn of the Krebs cycle, one ATP molecule is produced and multiple NAD
+
and FAD
+
molecules are reduced to NADH and FADH
2
, respectively. The final products of the Krebs cycle per glucose molecule include: 2 ATP, 2
FADH
2
, 6 NADH, and 4 CO
2
.
In the final stage of cellular respiration (Fig. 3), the electrons carried by FADH
2
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
( chemiosmosis ). Each molecule of NADH yields 3 ATPs while each FADH
2
generates 2 ATPs, resulting in an overall production of 32 ATPs in this stage alone!
H
+ 2H +
H +
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 utilized differently. In the process, 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
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fermentation occurs in organisms such as yeast (Fig. 4a) which have been utilized industrially for food and alcoholic beverage production. Lactic acid fermentation, on the other hand, 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
Figure 5 summarizes the basic differences between the products of aerobic and anaerobic cellular respiration. While all three types of cellular respiration (aerobic respiration, alcohol and lactic acid fermentation) produce carbon dioxide (CO
2
), water (H
2
O), 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:
C
6
H
12
O
6
+ 6O
2
6CO
2
+ 6H
2
O +ATP +Heat
Anaerobic fermentation (plants and some microbes):
C
6
H
12
O
6
2C
2
H
5
OH + 2CO
2
+ATP + Heat
(Glucose) (Ethanol)
Anaerobic fermentation (animals and some microbes):
C
6
H
12
O
6
2CH
3
CHOHCOOH +ATP + Heat
(Lactic Acid)
Figure 5. Equations for different forms of cellular respiration
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The objective of today’s lab is to examine the chemical processes involved in aerobic and anaerobic respiration. In addition, you will compare and contrast respiratory rates of animal and plant cells.
Question :
List one advantage and one disadvantage of respiring anaerobically.
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TASK 1 - Wine Production: 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 CO
2
during fermentation. In this exercise you will demonstrate CO
2
production during anaerobic fermentation by yeast through the production of wine.
I: Preparation of Yeast-Grape Juice Mixture by TA a.
Add 2L of grape juice to an Erlenmeyer flask and allow it come to room temperature. b.
To a 20mL beaker add 4mL of tap water. Heat the beaker of water to 40-43 ° C using a hot plate. Note: Distilled or deionized water is lethal to yeast. Make sure that you only use tap water. c.
Once the water is at 40-43 ° C, move the beaker to the corner of the hot plate so that the water remains at the required temperature. d.
Add 0.4g of yeast to the heated tap water. Leave the yeast-water mixture undisturbed for 15min. You should begin to see bubbles form as the yeast re-hydrates. e.
At the end of the 15min period, mix the yeast-water solution thoroughly and then pour it into the flask containing the grape juice. To ensure that all the yeast is added to the grape juice, pour about 15mL of the grape juice into the beaker that contained the yeast-water mixture, stir gently, and then pour the contents back into the flask containing the yeast-grape juice solution. Mix well using a stir bar.
II: Experimental Setup a.
Each group should obtain the following materials:
• one 500mL Erlenmeyer flask
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• one large rubber band
• one balloon b.
Label the flask with your TA’s name, section number, group number, sucrose concentration (see step c) and today’s date. c.
Your TA will assign each group a different amount of sucrose (0, 37.5g, 56.25g, 75g,
112.5g, 150g) to add to your flask.
• Weigh out your group’s assigned amount of sucrose (using a weighing boat) and then pour it into your group’s flask using a funnel. d.
Measure 325mL of the yeast-grape juice mixture and then add it to the flask. f.
Dissolve the sucrose in the yeast-grape juice solution by whirling the flask or using a glass rod to stir. g.
Measure about 1mL of the yeast-grape juice mixture with a disposable transfer pipette and add it into a small tasting cup. Set it aside. h.
Attach a balloon to the opening of the flask and secure it with a rubber band. i.
Obtain a piece of string approximately 60cm in length. j.
Measure the circumference of the balloon by recording how much string is required to wrap around the widest section of the balloon. Place the piece of string against a ruler to get the length in cm. Mark this point on the string either by drawing a line with a Sharpie marker or using a piece of colored tape. This will serve as your day 0 measurement and should be recorded in Table 1. k.
Using the previously set aside yeast-grape juice solution, have one member of your group take a small sip and note its sweetness (See Monitoring/measuring fermentation instructions below) in Table 2. l.
Over the next two lab periods (days 7 and 14), you will monitor your group’s flask and record your results in Table 1.
• Note : Make sure to share your results with the remainder of the class in order to be able to graph the class data for each treatment.
Develop a Hypothesis:
Given what you already know about anaerobic respiration, state what you expect to happen over time in the experiment you set up above ( Hint: consider the products of anaerobic respiration).
Do you expect a difference in the volume of each balloon? If so, what is being produced
(or consumed) that changes the volume?
Write your hypotheses (H o
and H a
) in the space provided below.
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Monitoring/ measuring fermentation (will be performed on days 7 and 14):
Each group will need to measure the following variables in Tables 1 and 2:
• Table 1 o
Presence/absence of bubbles produced o
Presence/absence of sediment (deposit formed at the bottom of the flask) o
Circumference of the balloon for your group’s flask using the same string from day 0. Make sure to record the circumference for day 7 and day 14 by marking each day’s point on the string with a Sharpie marker or colored tape.
Table 1:
Dissolved
Mass of sucrose
(g)
0 presence of sediment/ bubbles
Day 0
CO
2
levels =
Balloon circumference
Day 7 Day 14 presence of sediment/ bubbles
CO
2
levels =
Balloon circumference presence of sediment/ bubbles
CO
2
levels =
Balloon circumference
37.5
56.25
75
112.5
150
• Table 2 o
Taste the wine on days 0 and 14. On a scale of 0-5, with 5 being very sweet and 0, not sweet at all; compare the sweetness of the solutions.
Table 2:
Dissolved Mass of Sucrose (g) Day 0
Taste Observation (0-5)
Day 14
0
37.5
56.25
75
112.5
150
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Questions:
1.
Was there a noticeable increase in the number of bubbles and/or presence of sediment for all treatments over time?
2.
What happened to the balloon circumference of each flask as the fermentation process progressed?
3.
How does balloon circumference relate to the amount of carbon dioxide produced during fermentation?
4.
Using the graph paper provided, plot the change in CO
2
over time for each treatment.
5.
What happens to CO
2
levels with increasing sugar concentration? Is this pattern observed for all sucrose treatments?
6.
Based on your results, can you explain which ingredients are essential for fermentation to occur and why?
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7.
Overall, what can you conclude about your hypotheses? Explain.
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TASK 2 - Demonstration of Aerobic Respiration: Consumption of O
2
In this task you will examine aerobic respiration by measuring the amount of oxygen consumed by germinating and dormant (heat-killed) peas. In addition, you will assess the effect of temperature on the rate of aerobic respiration by performing the experiment at two different temperatures.
Develop a Hypothesis:
Given your knowledge of aerobic respiration, what can you predict about respiration rates in germinating vs. dormant peas? Write your hypotheses (H o
and H a
) below.
State your hypotheses (H o
and H a
) regarding the effects of temperature on respiratory rate in germinating vs. dormant peas below.
Procedure:
1.
Your TA will assign half of the class to perform the experiment in freezing conditions and the other half at room temperature. Depending on your assignment, your group will need to either setup an ice water bath or a room temperature water bath. Once this has been done, record the temperature of your group’s water bath in the space provided below.
Water Bath Temp: _____°C
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2.
Set up three respirometers as shown in Figure 6.
• Tube 1 : Place a small amount of absorbent cotton at the bottom of the tube followed by 5g germinating peas, another small plug of cotton, and then approximately 1cm of *KOH pellets. Place a rubber stopper with a glass pipette in the top of the tube and then completely cover with foil.
• Tube 2 : Place a small amount of cotton at the bottom of the tube followed by
5g dormant (heat-killed) peas, another small plug of cotton, and then approximately 1cm of *KOH pellets. Place a rubber stopper with a glass pipette in the top of the tube and then completely cover with foil.
• Tube 3 : Place a small amount of cotton at the bottom of the tube followed by
5g beads , another small plug of cotton, and then approximately 1cm of *KOH pellets. Place a rubber stopper with a glass pipette in the top of the tube and then completely cover with foil.
*KOH (potassium hydroxide) absorbs the CO
2
produced during aerobic respiration.
1 2 3
Figure 6. Respirometer Setup: 1 - germinating peas, 2 - heat killed peas, 3 - beads.
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3.
Place all three tubes in the water bath (See Fig 7). The tubes should be submerged in water/ice at least ¾ of the way up to the stopper. Allow the tubes to equilibrate at this temperature for 10 min.
Figure 7. Water bath setup showing respirometers (view from the top)
4.
Add 1 drop of colored dye into the tip of each of the three pipettes making sure that you don’t lift the tubes out of the water bath.
5.
Allow the tubes to equilibrate for 5 min and then record the temperature of the water bath as well as the initial location of the dye in the corresponding table below.
6.
Record the water bath temperature as well as dye location for each tube every 10 min
(Total time = 30 min). Make sure to also record the results obtained by a neighboring group for a different temperature treatment.
Table 3: Ice water bath Results
Time
(min)
0
10
Temp of water bath ( o
C)
20
30
Tube 1 pipette reading (mL)
Tube 2 pipette reading (mL)
Tube 3 pipette reading (mL)
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Table 4: Room temperature water bath Results
Time
(min)
0
10
Temp of water bath ( o
C)
Tube 1 pipette reading (mL)
Tube 2 pipette reading (mL)
Tube 3 pipette reading (mL)
20
30
Questions :
1.
Using the graph paper provided, plot 2 graphs: one for the results of each of the three tubes in the ice water bath (Table 3) and the other for each of the three tubes in the room temperature water bath (Table 4).
2.
What was the purpose of using KOH pellets? What would have happened to our results had we not used them?
3.
Based on your results (look at your graphs), which was relatively faster, the germinating peas or heat-killed peas? Why? a.
How did temperature affect respiratory rate? Why do you think temperature affected or did not affect this rate?
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5.
Explain how this experiment demonstrated oxygen consumption as a part of cellular respiration.
6.
Overall, what can you conclude about your hypotheses? Explain.
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TASK 3 - Demonstration of Aerobic Respiration: Production of CO
2
When eukaryotic organisms respire they release CO
2
which can combine with H
2
0 to form carbonic acid (H
2
CO
3
). In general, acidic solutions such as H
2
CO
3
(pH less than 7) have a larger concentration of H
+
ions while basic ones (pH greater than 7) contain more OH
-
ions (Fig. 3). An indicator, such as phenolphthalein i can be used to detect changes in pH resulting from CO
2 production during cellular respiration. In an acidic solution, phenolphthalein is clear, but in basic solutions it turns pink. During this task you will examine aerobic respiration by measuring the volume of NaOH required to neutralize the acid produced by actively respiring animal (snails) and plant ( Elodea ) cells.
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Figure 3. 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?
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Develop a Hypothesis:
Considering the set-up for this procedure (Table 5), state what you expect to occur when comparing respiratory rates in animals vs. plants. Write your hypotheses (H o
and H a
) in the space provided below.
Procedure:
1.
Place 75mL pond water into each of 4 labeled beakers (see Table 5).
2.
Obtain the organisms listed in Table 3.
3.
Determine the volume of each by water displacement (refer to Lab 1 for instructions) using a 100mL graduated cylinder.
4.
Add each organism to the corresponding beaker and then cover each with parafilm. For the Elodea in the dark setup, place the covered beaker underneath a coffee can.
5.
Allow the organisms to respire for 15 min and then return them to their original culture bowls.
6.
Add 4 drops of phenolphthalein to each beaker. The contents should remain clear because the solutions are acidic
7.
Add NaOH drop by drop to the contents of the control beaker to determine the number of drops it takes to turn the solution pink (20 drops = 1mL). After each drop thoroughly mix the contents of the beaker.
8.
Record your results in Table 3.
9.
Repeat steps 6 and 7 for each of the remaining beakers.
Table 5:
Beaker
Snails –
TA setup
Total Volume of Organisms
(mL)
Milliliters (mL) of NaOH to reach endpoint
Relative Respiration
Rate of Organisms
(mL NaOH)
Respiration rate per milliliter (mL) of organism
Elodea
Elodea
(dark)
Control
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10.
To calculate the relative respiration rate of each organism, subtracting mL NaOH added to the control beaker from the milliliters added to each beaker. Record your results in
Table 5.
11.
To determine the respiration rate per milliliter of organism, divide the relative respiration rate by the volume of each organism. Record your results in Table 5.
Questions :
1.
What was the purpose of including the 4 th
setup (control)?
2.
What was the purpose of including the two different plant/light setups?
3.
Using the data collected from this experiment, what can you conclude about respiratory rates in animals vs. plants?
4.
Do you think the rates of respiration might differ for other plant or animal species?
Explain.
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5.
What possible sources of error are associated with using pond water for this procedure?
6.
Based on your results, what can you conclude about your hypotheses? Explain.
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LOOK AHEAD:
• Before coming to lab next week, make sure to read the Cell Cycle, Mitosis and Meiosis
Task sheet as well as Exercises 14 & 15 in your lab manual.
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