PHOTOSYNTHESIS page 51

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1 PHOTOSYNTHESIS page 51 Pre-lab assignment: Before lab you must read the lab introduction, perform the calculations described 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 exchange and the obligate symbiosis between heterotrophic and autotrophic organisms. 3. Separate the photosynthetic pigments by performing paper chromatography. 4. Observe a demonstration of the fluorescence of isolated photosynthetic pigments. 1- Planet Tenebrio Introduction The first procedure in this week s lab is to build closed biospheres and test the reciprocal gas exchange between photosynthesis and respiration. This procedure will also be the basis of your lab report. As you know, photosynthesis consumes CO 2 and produces O 2, according to the following equation: because each uses the gases produced by the other. As a result, autotrophic and heterotrophic organisms (roughly, plants and animals) depend on each other to maintain an atmosphere suitable for life. Of course, plants have both chloroplasts and mitochondria, so they are a bit less dependent on animals in this respect than animals are upon plants. Chloroplasts use light energy to strip hydrogen from water, combine it with CO 2 to make carbohydrate, and release the leftover O 2. Mitochondria recombine carbohydrate with O 2, deriving useful energy, and release CO 2 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 processes that maintain balance and livable conditions on earth. For example, the Biosphere-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 consider these to be like miniature worlds, with you in control of conditions. We ll vary the Figure 1. The Biosphere 2 complex near Tucson, Arizona is a large scale closed ecosystem operated by Columbia University. There is a link to the Biosphere 2 website on the lab page. 6CO 2 + 6H 2 O + light C 6 H 12 O 6 + 6O 2 Conversely, respiration consumes O 2 and produces CO 2. These two metabolic pathways are thus co-dependent on each other,

2 PHOTOSYNTHESIS page 52 contents of these closed systems to test predictions 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 processes of photosynthesis and respiration. Materials and Methods 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 minutes of lab, so that there will be time to see the results. 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 CO 2. The systems that don t have KOH have an equal volume of plain water, to keep the 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. STOPPER ELODEA (in C, D, E, & F) Biosphere contents A. mealworms, water B. mealworms, KOH C. water, light D. KOH, light E. water, dark F. KOH, dark RESERVOIR and wick with KOH solution to absorb CO 2 (B, D, & F) or just water (C, E). 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. SUPPORT PIN (supports the reservoir above the crew compartment) THE CREW (Tenebrio molitor)

3 PHOTOSYNTHESIS page 53 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 reservoir. 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 mealworms- 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 mealworms will signal you if they experience 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 reservoir, 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. Written assignment: Complete before this lab and hand it in at the beginning of this lab. 1. Calculate how long it will take 3 grams of mealworms to consume all the oxygen in 20 ml of air. Assume that the mealworms 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 differences. 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.

4 PHOTOSYNTHESIS page 54 Table 1. Results of the biosphere experiment Contents of the biospheres Time the hatch closed Time movement stopped Minutes of survival Number of casualties A mealworms, water B C D E F mealworms, KOH water, light KOH, light water, dark KOH, dark Lab Report due the week after lab 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 noncyclic photosynthesis. Then describe the rationale of the experiment. Methods : Briefly describe the experimental set-up in your own words. Make predictions for each of the 6 biospheres, as you did in the pre-lab assignment, based on your assumptions about the processes and conditions. Results : The results of this experiment are simple. How long did the mealworms remain 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 correspond to the observed survival time? Did the mealworms in the dark do better or worse with the plant present? In considering your results, don t forget that plants respire, too. How did removing the CO 2 affect 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 CO 2 (hypercarbia). Which biospheres would experience these conditions? Would there be any consequences of excess CO 2? 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 populations? There are many problems that result 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 ecosystem 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?

5 PHOTOSYNTHESIS page Separation of Pigments In order for photosynthesis to occur, light must be absorbed. Certain wavelengths of light are absorbed by pigments (colored protein molecules) present in the chloroplasts. This absorbed energy causes electrons to move to higher energy levels or even to leave the pigment altogether a process called photo-oxidation. The energetic electrons then drive the electron transport chain and the production of ATP, or they are transferred to NADPH for carbohydrate synthesis. There are a variety of pigments in the chloroplasts. Chlorophylls a and b, xanthophylls, and carotenes all play roles in photosynthesis. These pigments are not very soluble in water, but they are readily soluble in less polar solvents such as acetone, ethanol, and petroleum ether. Your lab instructor has extracted the photosynthetic pigments from leaves (pine needles, spinach, or grass). The leaves were placed in a blender with ethanol, and ground up to rupture the cells. The homogenate 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 pigments by light. The solution contains a mixture of several kinds of pigments. Generally, the chlorophylls are more concentrated than the carotenoids and xanthophylls. We will separate the various pigments from one another using a procedure called paper chromatography. The pigment extract is applied in a narrow band near one end of a strip of paper. Next, the lower end of the paper is immersed 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 pigment molecules, they dissolve and are carried up the strip. The rate at which they travel is slower than the movement of the solvent, because they are somewhat attracted to the cellulose fibers of the paper. The rate at which each kind of pigment travels up the paper is unique to that pigment. The mobility of the pigments is quantified as a factor called R f which is defined 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 manner so as to make the stripe narrow. Blow the stripe dry, and then make more applications. 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.

6 PHOTOSYNTHESIS page 56 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 pigment. 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 absorbed). 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 energy and transfer energetic electrons to the chlorophylls. Xanthophylls seem to have the opposite effect, perhaps dissipating excessive light energy and protecting the chlorophyll 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. Table 2. Movements of solvent and pigments in paper chromatography. State the pigment color Distance from origin Rf Probable identity Solvent front Fluorescence When the photosynthetic pigments are isolated from the membrane systems in the chloroplasts, they remain capable of absorbing 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 precisely determined by the structure of the pigment molecule. This emitted light is called fluorescence. There are other substances 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 illuminating 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.