6CO2 + 6H2O + light C6H12O6 + 6O2 1- Planet Tenebrio
Pre-lab assignment:
Before lab you must read the lab intro-duction, perform the calculations de-scribed below and answer the questions. You will hand in a copy of this work when you come to lab, instead of the weekly quiz.
Objectives
1. Review the processes of photosynthesis, including the light reactions (non-cyclic and cyclic) and the Calvin cycle. 2. Test the reciprocal processes of gas
ex-change and the obligate symbiosis be-tween heterotrophic and autotrophic or-ganisms.
3. Separate the photosynthetic pigments by performing paper chromatography. 4. Observe a demonstration of the
fluores-cence of isolated photosynthetic pig-ments.
Introduction
The first procedure in this week’s lab is to build closed “biospheres” and test the recip-rocal gas exchange between photosynthesis and respiration. This procedure will also be the basis of your lab report.
As you know, photosynthesis consumes CO2 and produces O2, according to the
fol-lowing equation:
because each uses the gases produced by the other. As a result, autotrophic and hetero-trophic organisms (roughly, plants and ani-mals) depend on each other to maintain an atmosphere suitable for life. Of course, plants have both chloroplasts and mitochon-dria, so they are a bit less dependent on ani-mals in this respect than aniani-mals are upon plants. Chloroplasts use light energy to strip hydrogen from water, combine it with CO2 to make carbohydrate, and release the
leftover O2. Mitochondria recombine
car-bohydrate with O2, deriving useful energy,
and release CO2 and water.
The global “biosphere” is the surface, the oceans, and atmosphere of planet Earth. The global biosphere is essentially a closed system. Nothing significant enters or leaves except for the light that arrives from the sun, and the heat that is radiated to space. Ecologists have created experimental closed ecosystems in order to investigate the proc-esses that maintain balance and livable con-ditions on earth. For example, the Bio-sphere-2 facility is a large live-in research station in Arizona (Figure 1).
We will construct closed systems or “biospheres” by sealing mealworms and plants in closed containers. You can con-sider these to be like miniature worlds, with you in control of conditions. We’ll vary the
Conversely, respiration consumes O2 and
produces CO2. These two metabolic
path-ways are thus co-dependent on each other,
Figure 1. The Biosphere 2 complex near Tuc-son, Arizona is a large scale closed ecosystem operated by Columbia University. There is a link to the Biosphere 2 website on the lab page.
The instructor will provide these. There is a folded paper wick inside to provide a large surface area. Insert the capsule above the mealworms, and work the support pin down to the bottom of the tube– don’t skewer any mealworms.
Keep the tube upright from now on.
The KOH will absorb CO2. The
sys-tems that don’t have KOH have an equal volume of plain water, to keep the contents of these closed systems to test
pre-dictions about photosynthesis, respiration, and reciprocal gas exchange.
On planet Tenebrio, as on planet Earth, the survival of the inhabitants is dependent on reciprocal gas exchange. The response variable that we will measure is the survival of the mealworms during the lab period. You will predict and interpret the results based on what you know about the proc-esses of photosynthesis and respiration.
The class will prepare 6 biospheres. If there are 6 student groups in lab, each group can prepare one. All 6 are necessary, and all groups will share results. It is important to work fast and get these set up within the first 15- 20 minutes of lab, so that there will be time to see the results.
Biosphere contents A. mealworms, water B. mealworms, KOH
C. mealworms, plant, water, light D. mealworms, plant, KOH, light E. mealworms, plant, water, dark F. mealworms, plant, KOH, dark Directions for setting up the biospheres
1. Weigh out 3 grams of mealworms, as nearly as practical. Make sure that each worm is alive and active- don’t pick any that are mummified.
2. Place the worms in the test tube, and add a pinch of dry oatmeal so they’ll have something to eat.
3. Add a reservoir containing either 5% KOH solution or water, depending on which biosphere that you are preparing.
STOPPER
ELODEA (in
C, D, E, & F)
RESERVOIR and wick with KOH so-lution to absorb CO2
(B, D, & F) or just water (C, E).
SUPPORT PIN (supports the reser-voir above the crew compartment)
THE CREW (Tenebrio molitor)
Figure 2. A “biosphere” or mini-planet. You can also think of it as a space station. All are closed systems so far as matter goes.
gas volume in each system the same, and to provide a high humidity so the
Elodea won’t dry out.
4. Add a plant, if your biosphere calls for a plant. Your lab instructor will provide a 5-6 cm frond of Elodea. Shake off any excess water so it won’t drip, but do not allow it to dry out. Insert the frond “headfirst” into the tube above the reser-voir. Do not allow it to contact the wick. If its too long, trim the base.
5. Now “close the hatch” on the biosphere. Insert a rubber stopper firmly into the top of the tube to seal it off from the rest of the universe. These stoppers have been lightly coated with silicon- examine the edge to be sure that there is good contact with the glass all around. Note the time
that you closed the hatch.
6. If your biosphere calls for dark, wrap the upper part (around the plant) with aluminum foil. Do not cover the meal-worms- we need to be able to see them. 7. Place all of the sealed tubes, standing in
beakers, under the grow-lights at the side of the room.
8. After about 60 minutes, begin checking all the biospheres every 10 minutes or so. Eventually, problems will develop in those biospheres that do not have adequate reciprocal gas exchange among their inhabitants. The meal-worms will signal you if they experi-ence respiratory distress. They will stop moving! Examine the mealworms carefully for movement. They don’t exactly dance around, so you have to watch the group for a minute or two to be sure.
9. When all of the mealworms in a group have stopped moving entirely for at least 2 minutes, note the time. Then
save their small but not insignificant lives. Open the stopper to renew the atmosphere. Whew!
10. Use a forceps to carefully remove the plant, if any, and return the Elodea to
the aquarium. Then remove the
reser-voir, using the forceps to grasp it by the edge. Return it to the instructor.
11. Put the mealworms in a dish and watch
them for a few minutes. They should recover quickly. If any do not recover, bury them with honors in the trash can. Put the survivors and their oatmeal back into their cage. And don’t forget to say “thank you”.
1. Calculate how long it will take 3 grams of mealworms to consume all the oxygen in 20 ml of air. Assume that the meal-worms consume 0.02 ml min-1 g-1, and that air is 20.93% oxygen. Show your calculations.
2. Review, in your textbook or lecture notes, the difference between non-cyclic and cyclic photosynthesis, and write a brief paragraph describing these differ-ences. Focus, in particular, on what goes in and what comes out.
3. How do plants make ATP at night, when there is no light available to drive the light reactions of photosynthesis? 4. Considering the factors you have just
explained, make specific predictions for each of the six biospheres described above.
Written assignment: Complete before this lab and hand it in at the beginning of this lab.
Table 1. Results of the biosphere experiment Time the hatch
closed Time movement stopped Minutes of “survival” Number of casualties A mealworms, water B mealworms, KOH C mealworms, plant, water, light D mealworms, plant, KOH, light E mealworms, plant, water, dark F mealworms, plant, KOH, dark
Contents of the biospheres
The lab report for next week will be based on the “Planet Tenebrio” experiment.
Introduction : Briefly review the processes
of respiration and photosynthesis, including the difference between cyclic and non-cyclic photosynthesis. Then describe the rationale of the experiment.
Methods : Briefly describe the
experimen-tal set-up in your own words. Make predic-tions for each of the 6 biospheres, as you did in the pre-lab assignment, based on your assumptions about the processes and condi-tions.
Results : The results of this experiment are
simple. How long did the mealworms re-main active in each biosphere? Prepare a table that shows 1) the contents of each of the 6 systems and 2) the time that elapsed before the worms stopped moving.
Discussion: Compare the results among the
6 biospheres and explain them. How did your prediction of metabolic rate
corre-spond to the observed survival time? Did the mealworms in the dark do better or worse with the plant present? In consider-ing your results, don’t forget that plants re-spire, too. How did removing the CO2
af-fect the outcome in the light? Why?
There are two situations that can develop for the animals in an unbalanced biosphere. One is too little oxygen (hypoxia). The other is too much CO2 (hypercarbia).
Which biospheres would experience these conditions? Would there be any conse-quences of excess CO2?
Try to relate this experiment to real-world processes and problems. For example, how has the balance between carbon fixation and oxidation been upset by human popula-tions? There are many problems that re-sult– some of which were not an issue in our experiment, such as global warming (unless you left the biosphere too close to the light…) Read about the Biosphere-2 facility in Arizona and other closed ecosys-tem experiments and industries (links on the lab web page). Can you relate any of your observations to problems that have occurred in Biosphere 1 and Biosphere 2?
In order for photosynthesis to occur, light must be absorbed. Certain wavelengths of light are absorbed by pigments (colored protein molecules) present in the chloro-plasts. This absorbed energy causes elec-trons to move to higher energy levels or even to leave the pigment altogether– a process called photo-oxidation. The ener-getic electrons then drive the electron trans-port chain and the production of ATP, or they are transferred to NADPH for carbohy-drate synthesis.
There are a variety of pigments in the chloroplasts. Chlorophylls a and b, xantho-phylls, and carotenes all play roles in photo-synthesis. These pigments are not very soluble in water, but they are readily soluble in less polar solvents such as acetone, etha-nol, and petroleum ether.
Your lab instructor has extracted the photo-synthetic pigments from leaves (pine nee-dles, spinach, or grass). The leaves were placed in a blender with ethanol, and ground up to rupture the cells. The ho-mogenate was then filtered to remove the cellular debris. The resulting solution of pigments in ethanol was stored in an opaque bottle to prevent degradation of the pig-ments by light.
The solution contains a mixture of several kinds of pigments. Generally, the chloro-phylls are more concentrated than the caro-tenoids and xanthophylls. We will separate the various pigments from one another us-ing a procedure called paper chromatogra-phy. The pigment extract is applied in a narrow band near one end of a strip of pa-per. Next, the lower end of the paper is im-mersed in a solvent. The solvent travels up the paper, drawn upward by its attraction to the fibers of the cellulose.
As the solvent flows past the band of pig-ment molecules, they dissolve and are
car-ried up the strip. The rate at which they travel is slower than the movement of the solvent, because they are somewhat at-tracted to the cellulose fibers of the paper. The rate at which each kind of pigment travels up the paper is unique to that pig-ment. The mobility of the pigments is quantified as a factor called Rf which is
de-fined as the distance moved by the pigment divided by the moved by the solvent. (Both distances are measured from the point where the pigment was placed originally).
Materials & Methods:
Your lab instructor will supply strips of chromatography paper. Two notches are cut near one end. Use a capillary pipette like a paintbrush to apply a narrow stripe of leaf extract between the two notches. Move the capillary in a continuous sweeping man-ner so as to make the stripe narrow. Blow the stripe dry, and then make more applica-tions. About 10 applications are necessary to build up a good dark streak of pigment. Fasten the paper strip to the cork using a thumbtack. Then insert the paper and the cork into a test tube containing about 2 ml of solvent (a 9:1 mixture of petroleum ether and acetone). The tip of the paper should be immersed in the solvent, which will then wick its way up the strip. Keep track of its progress.
When the solvent front has just reached the crease at the top of the strip, remove the cork and allow the excess solvent on the strip to evaporate. Then unpin the strip and replace the cork in the solvent tube.
Results
Caution: The ether-acetone mixture is volatile and highly flammable. It should not be used near open flames and it should not be inhaled.
Table 2. Movements of solvent and pigments in paper chromatography.
State the pigment color Distance from origin Rf Probable identity
Solvent front ————————– ————————– 1. 2. 3. 4. 5.
Examine the strip. Place a pencil mark in the center of each colored band– there should be at least 4 separate bands or spots, each representing a different class of pig-ment. Measure the distance from the notches to the center of each band and from the notches to the crease at the top of the paper. Record these measurements and the color of each band in Table 2.
The pigments, in order of Rf from low to high, are chlorophyll a (closest to origin), chorophyll b, xanthophylls, and carotenes (closest to solvent front). Which pigment do you suppose is the least soluble in the non-polar solvent? Most soluble?
The colors of the leaf pigments are the light that is reflected and transmitted (not
ab-sorbed). What colors are not reflected or transmitted by these pigments, and therefore must be absorbed by them?
The xanthophylls and carotenes are called “accessory pigments”. There are several kinds of each. Carotenes absorb light en-ergy and transfer energetic electrons to the chlorophylls. Xanthophylls seem to have the opposite effect, perhaps dissipating ex-cessive light energy and protecting the chlo-rophyll from damage in bright light.
The accessory pigments are more persistent in leaves than the chlorophylls are during the fall, and they lend their colors to the fall foliage as the chlorophylls break down.
When the photosynthetic pigments are iso-lated from the membrane systems in the chloroplasts, they remain capable of absorb-ing light energy, but they are unable to pass their energetic electrons on to the normal acceptor molecules. Instead, the electrons simply drop back to their former energy level, and the absorbed energy is emitted as light.
The wavelength of the light emitted is pre-cisely determined by the structure of the
pigment molecule. This emitted light is called fluorescence. There are other sub-stances that also fluoresce– some of these are used in watch dials that glow in the dark, or in novelty items such as “moon glow” frisbees.
Your trusty lab instructor will demonstrate the fluorescence of chlorophyll by illumi-nating pigment extract with a white light. If the white light is shone from below, and the flask is viewed from the side, most of the light seen is the fluoresced wavelength, which is dark blood-red.
