LABORATORY 7 Aerobic and Anaerobic Cellular Respiration

Cells can obtain the energy stored in sugars by breaking the sugar molecules apart in a series of enzyme-mediated reactions. Energy is extracted most efficiently in the presence of oxygen via the process known as aerobic respiration. Under conditions where oxygen is scarce or absent, some cells are still able to split glucose to obtain energy via the process of fermentation--but far less energy per glucose is extracted, since glucose cannot be fully broken down without oxygen.

In today’s lab, you will work in teams of two and design an experiment in which you manipulate some factor affecting the rate of either aerobic or anaerobic metabolism. You and your partner will choose only one system--anaerobic or aerobic--around which to design an experiment. However, you should be aware of what is going on with teams who have chosen the type of metabolism you did not choose to manipulate, as you will be responsible for understanding both types of metabolism for exams and quizzes.

I. Fermentation: anaerobic metabolism Certain organisms can produce ATP by utilizing metabolic pathways that do not

require the participation of molecular oxygen. Usually, these are referred to as pathways of fermentation or anaerobic glycolysis. In general terms, both of these pathways involve the sequential conversion of a carbohydrate into partially oxidized end product(s). The energy released during these reactions, and stored in the bonds of ATP.

One species of free-living (i.e., non-parasitic) unicellular fungus, commonly known as Brewer’s yeast (Saccharomyces cereviseae), can "ferment" various disaccharides and monosaccharides. Yeast ferment most sugars in a manner similar to the way they ferment glucose to yield ethanol and carbon dioxide.

about 11 enzymatic reactionsC6H12O6 ---------------------------------------> 2CH3CH2OH + 2CO2

We can estimate the rate of this pathway for any given sugar by determining the yield of CO2 over time. This can be accomplished easily with fermentation tubes.

A. Experimental procedureBecause our tubes are hand-blown and unmarked, you must calibrate them before

you begin your experiment. With a syringe, introduce water into the tube, 0.5 cc at a time, and tip the tube so that the water enters the sealed end of the tube. Each time you add water, mark the meniscus with a wax pencil. Make marks as far up the neck of the tube as possible, as shown in Figure 7-1.

Available at the back table are 0.5M solutions of four different sugars and a beaker of live yeast suspended in water. If you were going to feed the yeast some sugar, you might find that about 2 ml of yeast suspension and 10 ml of sugar suspension would provide a fine feast that would last for a couple of hours. Hint hint.

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Figure 7-1. A fermentation tube. To calibrate the tube, introduce 0.5 cc (= ml) of water into the bowl and tilt so that the liquid flows into the closed neck. Mark the bottom of the meniscus with a wax pencil. Repeat this procedure until the entire neck is calibrated with 0.5 cc marks, as shown in the illustration.

And now the creative process begins. Consider where wild, free-range yeast are found in nature. (HINT: what’s that “bloom” on the grape?) Now consider where the various sugars available are found in nature. Ask questions about these things. Formulate ideas....and design an experiment. We’re not going to tell you more. What is your Ho? What is your Ha? The yeast have been incubated aerobically so that they will use up all their carbohydrate reserves. Why do you suppose this was necessary? Use the handy table on the following page to record your experimental set up quantities. You may or may not use all the lines and spaces. That’s up to you.

Table 7-1. Quantities of yeast suspension and 0.5 M sugar solutions used in testing

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tube #

ml (cc) yeast suspension

ml (cc) sugar solution

type of sugar

B. Data collectionYou may use the table provided (Table 7-2 on the following page)to record your

experimental results. Provide an appropriate legend. (NOTE: It may take about 30 minutes before you observe any generation of CO2, but the process proceeds quite rapidly once it has begun.)Table 7-2.

time(minutes)

tube 1(cc CO2)

tube 2(cc CO2)

tube 3(cc CO2)

tube 4(cc CO2)

zero102030405060708090100110120

C. Data analysis1. When you have completed the experiment, use data entered in Table 7-2 to plot

the yeast's rate of fermentation of each sugar. Use the graph paper provided at the end of this exercise, and provide an appropriate figure legend. (Which is the independent variable? The dependent variable? Be sure to label your axes correctly. If you are not sure how to do so, review Appendix IV.) Place all four rate curves on the same figure for ease of comparison.

2. Calculate the fermentation rate (slope) of each sugar by yeast. Compare these numerical data in your lab report.

When discussing your results, consider the following and integrate them into your text in a logical fashion. 1. To what degree might the the yeast cell membrane permeable to each of these

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sugars?2. Even if a sugar passes through the cell membrane, it may not be fermented. Why

might this be? HINT: Consider that glycolysis is mediated by many enzymes. Also consider the evolutionary history of Saccharomyces cereviseae, a fungus which has evolved to obtain its nutrients from plant sugars. Where is each of the sugars you used found in nature?

3. Was CO2 the only component of the "gas" produced in your experiment? What else might be there? How might this affect your analysis?

4. In using the fermentation tube, did you quantify all the CO2 being generated? (HINT: one end is open! D'oh!) Does this affect your data analysis?

5. Can you state with confidence that the fermentation rates are significantly different? Why or why not? How could you determine whether the rates are significantly different? How might you repeat these experiments so that the results could be more easily analyzed with a statistical test?

II. Respiration: aerobic metabolismAlmost all eukaryotic and many prokaryotic organisms are dependent upon a

continuous supply of oxygen in order to sustain life. Ultimately, molecular oxygen taken into the organism is used as the terminal electron acceptor in the electron transport chain and appear as part of a water molecule at the end of the reaction. At the same time, just as hydrogen atoms are removed from Krebs Cycle intermediates and ultimately transferred to oxygen, carbon dioxide is removed by decarboxylation reactions of the Krebs Cycle. This carbon dioxide can be re-used in certain biochemical reactions, even in heterotrophic cells. However, the vast majority of the carbon dioxide generated in mitochondria is released as a waste product, ultimately to be expelled by the organism into its environment. As you know, the breakdown of carbohydrates to produce ATP requires a long series of enzyme mediated reactions. The overall process of aerobic metabolism, however, may be expressed as follows.

about 16 enzymatic rxnsC6H12O6 + 6O2 + 6H2O ------------------------------------> 6CO2 + 12H2O + energyWhen discussing metabolism, non-scientists often use the term "warm-blooded" to

describe mammals and birds, and "cold-blooded" to describe just about everything else. These terms are inaccurate at best, and should be avoided by the biologist. Body temperature and metabolic rate are complex phenomena. It is far more accurate to consider the source and regulation mode of body heat when studying a given organism's metabolism.

Living organisms may be endothermic or ectothermic. An endotherm obtains its body heat from its own metabolic reactions, whereas an ectotherm obtains its body heat from external sources, such as the sun or a nice, hot asphalt road (splat!). These terms should not be confused with the related phenomena of homeothermy and poikilothermy. A homeotherm is capable of regulating its own body temperature, whereas the body temperature of a poikilotherm is regulated by its external environment. In general, homeothermy and endothermy result in much greater oxygen requirements than poikilothermy or ectothermy. Why might this be so? As you analyze

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the results of your respiration experiments, consider which type of physiology (in terms of internal temperature regulation and heat source) each of your organisms has, and how this might affect your observations.

If an organism has been eating mostly carbohydrates, the amount of carbon dioxide given off will be roughly equal to the amount of oxygen taken up. Hence, if such an organism is placed into an airtight chamber which has a carbon dioxide absorbent in it, the pressure of the gas within the system will slowly drop as the organism uses up the fixed volume of oxygen present. Such a system, in which one is able to measure oxygen consumption, is called a respirometer (Figure 7-2). Study the construction of the respirometer. How might it be useful to measure the oxygen consumption of a living organism?

There are several different types of small, respiring organisms in the lab. Examine the variety of these specimens, and come up with a list of questions that you potentially could answer with a well-designed respirometer experiment.Questions:

What is your Ho?

What is your Ha? Consider whether your organisms are immature (undergoing relatively rapid mitosis) or mature (sedentary and sluggish, like your biology lecturer). What is each type of organism using for a nutrient/energy source? Carbohydrate? Protein? Fat? How might this affect its rate of oxygen consumption?

Consider metabolic modes. Is your organism endothermic or ectothermic? Homeothermic or poikilothermic? Is your organism undergoing any other metabolic reactions that might affect the amount of oxygen or carbon dioxide in your experimental system? (Consider these questions carefully, but don't assume anything. If you're not sure, ask your lab instructor or use published sources of sound information.)

A. Experimental procedureStudy Figure 7-2 to familiarize yourself with the respirometer assembly. Note the

presence of a small jar of ascarite crystals, covered with gauze. This substance, which contains corrosive sodium hydroxide, absorbs CO2.

It is essential to the success of your experiment that the ascarite be kept completely dry. Wet ascarite forms a solid mass that will not absorb CO2.

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Before placing study organisms into the jar, you must be sure they are absolutely dry, and that you do not drip any water onto the ascarite!

Before you begin, check the cover on your vial. If it is wet or stiff with dried ascarite, it must be replaced. Bring such non-functional vials to your TA, who will replace it. Place one good ascarite vial in each of your respirometer jars.

If one is to compare the rate of oxygen consumption of two different organisms, data are meaningful only when expressed as the amount of oxygen used per unit mass of living organism per unit time. (Why is this so?) Hence, you must weigh your experimental organisms to the nearest tenth of a gram on the triple beam balance provided at the center of your lab table before you begin your experiment. Use the grid below (properly labeled by you) to record these data.

Once you have placed your study organism(s) into the respirometer chamber, gently but firmly attach the chamber to the rubber stopper on the underside of the water jacket lid. First moisten the sides of the stopper with water to ensure a gas-tight seal. Gently screw the water jacket lid (with the attached respirometer chamber) onto the water jacket, which should be filled with room temperature water. The glass components of the assembly are easily broken! Be very careful as you assemble your respirometer!

Note that about half of the available respirometer jars are painted black. Do you suppose certain organisms must be kept in the dark in order to give accurate readings of oxygen consumption? Which ones, and why?

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Figure 7-2. A respirometer assembly. Organisms whose oxygen consumption is to be measured are placed inside the respirometer chamber with a vial of dry ascarite, a CO2 absorbant.

B. Data collectionAfter you have allowed the respirometers with organisms to equilibrate for about

fifteen minutes, record the temperature of your respirometer system. From the barometer at the front of the lab, obtain the following standard conditions:Room pressure: mm Hg Room temperature: oC1. Thoroughly familiarize yourself with the graduations on the pipet (it’s a manometer!). 2. Using the small dropper bottle provided, place a drop of ethylene glycol in the open

end of the respirometer pipet. If there is already a bead in the tube, you should clean it out with a pipe cleaner, or you will not get good results. In fact, it is best to thoroughly wash the pipette with detergent and hot water before you begin.

3. By very slowly withdrawing air from the apparatus with the syringe, move the drop to within five 0.01 ml divisions of the first 0.1 ml graduation on the pipet .

4. When the leading meniscus of the drop touches the 0.1 ml graduation, record the time to the second. This will be your zero time.

5. Record the position of the meniscus every 10 minutes until the meniscus reaches

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the last 0.1 ml graduation before the bend in the pipette or until the two-hour experimental time is up, whichever comes first.

6. If the meniscus reaches the last 0.1 ml graduation before the two-hour period is up, slowly push air into the system in order to back up the meniscus two or three 0.1 ml graduations, and continue recording. Do not count the time used to withdraw air.

7. If your organism seems distressed due to lack of O2, allow O2 to enter and resume readings..

8. You also should record the activity of the organism (although beans are not generally very frisky) over each time interval. Is it moving about or resting quietly? Does it appear stressed? Are its little legs pointing skyward? If so, allow in some air.

9. Record your data in Table 7-3 and provide an appropriate table legend.Table 7-3.

time interval (min) ml O2 consumed - jar 1 ml O2 consumed - jar 20 10

10 - 2020 - 3030 - 4040 - 5050 - 6060 - 7070 - 8080 - 9090 - 100

100 - 110110 - 120

C. Data analysisIn order to calculate a standardized figure for the average amount of oxygen

consumed by the experimental organisms per unit time, you must convert the volumes of oxygen they used to standard temperature and pressure values. Use the following formula to calculate V2, the corrected value of oxygen consumed by your organisms over the period of the experiment.

V2 = P1V1T2

P2T1

P1 = atmospheric pressure of the lab (in mm Hg)

V1 = total oxygen consumed during the experiment (in ml)

T1 = temperature of the respirometer (in 0Kelvin) (0K = 0C + 273)

P2 = 760 mm Hg (standard pressure)

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T2 = 273oK (standard temperature)

Note that the units of V2 will be expressed in ml O2. As stated at the beginning of this section, your data will be meaningful only if expressed as a value of ml O2/organisms' mass/time (and you need to understand why!). Once you have calculated V2, convert it to ml oxygen consumed/g/hour by dividing V2 by the weight of the organism and then dividing this value by the number of hours over which the readings were taken.

Compare your oxygen consumption data with values obtained by other teams, for other organisms or the same as yours. Are your values different? What might account for differences in data among teams?

Do you think that in the case of an animal (such as the cricket), that oxygen consumption over time will form a constant linear relationship? What effect might the animal's activity levels have on oxygen consumed. Refer to Appendix IV to decide which type of figure is most appropriate for your type of data.

In your laboratory report, consider the questions posed throughout the lab chapter when you discuss your results. Also consider the following factors, and integrate them into your text in a logical fashion:1. Do the fluctuations in oxygen consumption correspond to organism activity level?2. Oxygen requirement of homeotherms is greater per gram of metabolizing tissue

than that of poikilotherms, and increases as the absolute size of the animal decreases. What might account for this?

3. Is either of the organisms you used homeothermic? Endothermic?4. Although you may consider the weight of your beans and crickets to be the total

weight of living, metabolizing tissue, this may not be the case. What might you be weighing-- besides metabolizing tissue--in each organism? How might this affect your results?

5. Can you state with confidence that the oxygen consumption of these two different species is significantly different? Why or why not?

Table 7-4. Oxygen consumption of various species at rest (in ml O2/g/hour). Data from Florey: An Introduction to General and Comparative Physiology, W.B. Saunders Company. Philadelphia, 1967.

Phylum/Division common name genus ml O2/g/hourCiliophora paramecium Paramecium 1.00Rhizopoda amoeba Amoeba 0.20Porifera sponge Suberites 0.24Cnidaria sea anemone Anemonia 0.013

jellyfish Aurelia 0.0034Nematoda hookworm Ascaris 0.50

roundworm Rotylenchus 0.66Mollusca clam Anodonta 0.002

mussel Mytilus 0.055octopus Octopus 0.09

Annelida lugworm Arenicola 0.031tube worm Chaetopterus 0.008

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Arthropoda crab Uca 0.005lobster Homarus 0.50crayfish Cambarus 0.10butterfly Vanessa 0.60

Echinodermata sea cucumber Holothuria 0.01sea star Asterias 0.03

Chordata goldfish Carrasius auratus 0.07trout Salmo trutta 0.22rat Rattus norvegicus 0.95cat Felis cattus 0.44human Homo sapiens 0.20

KINGDOM PLANTAE, Division Anthophyta (flowering plants)Plant name type of structure condition ml O2/g/houralfalfa seed dormant 0.38

seed germinating 1.06barley seed germinating 0.715

endosperm nutrient supply 0.076carrot root mature 0.025

leaf mature 0.440spinach leaf mature 0.515sweet potato root mature 0.96ash tree cambium mitotic 0.233

phloem mature 0.154sapwood conducting 0.047

red maple cambium mitotic 0.220phloem mature 0.170

apple flesh ripe 0.030skin ripe 0.095

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