Pre-lab: Photosynthesis and Glycolysis

[Pages:6]Pre-lab: Photosynthesis and Glycolysis

After reading over the lab, complete the questions below before coming to lab!

1. State your hypothesis regarding the effect of different wavelengths on photosynthesis? 2. What are some other variables that you can think of that could affect the amount of photosynthesis

in these plants? 3. Pick two of these variables from the previous question and explain how we controlled for them.

4. This week's lab covers the concepts of photosynthesis and cellular respiration. Both of these processes are complex chains of chemical reactions that can be simplified into overall reaction summary equations. Read the lab, find the summary equations for both photosynthesis and cellular respiration, and fill them in below.

Photosynthesis summary equation:

Cellular Respiration summary equation:

5. During the first part of the lab you will be testing for starch in plant leaves. Why are we testing for starch?

6. In our first experiment, we have placed black paper on some of the leaves. Why would we put on paper that we think blocks all of the light?

7. In our second experiment, state your hypotheses regarding the effects of temperature, substrate concentration, and enzyme inhibitors on the rate of glycolysis in yeast?

Photosynthesis and Glycolysis

GOALS: After completing this lab, a student should be able to: ? List some of the factors that affect photosynthetic rate. ? Explain the connection between light and energy storage in plants. ? Be able to understand HOW and WHY temperature, substrate concentrations, and inhibitor molecules

affect the rate of glycolysis in yeast. ? Use the scientific method to investigate biological processes.

OVERVIEW:

During this lab you will perform experiments to help you understand two of the basic processes that convert energy from one form to another in biological systems. Your investigations will begin with experiments on photosynthesis. Photosynthesis is the process that plants use to convert the energy of light into the stored chemical energy of sugars. Photosynthesis can be summarized with an equation that looks like this:

6CO2 + 6H2O -------->C6H12O6 + 6O2 Light

Looking at this summary equation, you can probably guess that many factors affect the rate of photosynthesis. As with any reaction, changing the concentration of the reactants (carbon dioxide and water) or the products (glucose and oxygen) can alter the rate of the reactions. In this lab, we will investigate the influence of light on the amount of photosynthesis. As in any scientific experiment, we will try to isolate one variable, in this case the wavelengths of light, and see what effect these have on the amounts of photosynthesis in leaves.

After examining the effect of light wavelengths on photosynthesis, we will investigate the process of cellular respiration. Cellular respiration converts the stored energy from sugar molecules into a more useful form, ATP, which is used to fuel chemical reactions throughout the cell. ATP, or adenosine triphosphate, is the energy "currency" of the cell, because the cell can't "spend" sugar molecules directly to run its chemical reactions, but instead must convert sugar to a usable form of energy that the cell can use to run its chemical reactions.

Cellular respiration is a complicated process that in a simplified form looks like this:

C6H12O6 + 6O2

6CO2 + 6H2O

ADP + Pi ATP

Most organisms (plants, animals, most protists, and many fungi including the yeast we will be using today) use this process of cellular respiration. If oxygen is not available, most organisms can temporarily use anaerobic respiration to gain some ATP, but the efficiency drops dramatically. Anaerobic respiration yields only 2 ATP per glucose molecule, whereas aerobic respiration (with oxygen) can yield as much as approximately 36 ATP per glucose molecule! Remember that rapidly growing yeast will deplete oxygen and switch from aerobic respiration to anaerobic respiration over time.

Photosynthesis and light

Plant cells can synthesize carbohydrates like glucose from carbon dioxide and water in a process called photosynthesis. Recall that the equation looks like this:

6CO2 + 6H2O -------->C6H12O6 + 6O2 Light

To examine this process we need to have a way to measure the amount of glucose produced by a plant. We will use a related compound, starch, which is produced by plant leaves as an energy storage product when they have excess sugars. To make the starch visible, we will use an indicator called Lugol's iodine. This solution stains starch a dark purple/black color, but does not stain sugars such as glucose. To estimate relative amounts of photosynthesis, we will test for the presence of starch using Lugol's solution. Parts of the leaf that turn dark purple in Lugol's solution indicate the presence of starch and therefore that photosynthesis was happening at such a rate that excess sugars are produced.

Using the Lugol's solution starch test, we will test the effects of different wavelengths of light on the ability of plants to perform photosynthesis. Last week, four different colored filters were placed on the leaves of geranium plants. One filter is just black construction paper and should block all of the light to the leaf underneath it. The other three filters are transparent colored plastic films that let through only certain colors of light. For example, the red filters let only red light through to the leaf, the blue only blue light, and the green only green light. Using these filters we can alter the color (or wavelengths) of the light, and see how it affects the amount of starch produced (which is the dependent and which is the independent variable?).

Black construction paper absorbs all of the light and therefore appears black. Your jeans appear blue because they absorb all light except for blue. Your jeans reflect blue light. The plastic filters reflect and transmit the colors they appear but they absorb all the other wavelengths. These wavelengths of light do have energy, which is why the filters eventually disintegrate and dyed fabrics fade over time.

Part I Photosynthesis

This procedure involves boiling a very flammable liquid. You need to be extremely careful and follow instructions explicitly!

PROCEDURE FOR STAINING STARCH IN LEAVES:

1. Set up a beaker of boiling water for your alcohol bath. To do this, add 200 ml of tap water to a 600 ml beaker and place it on your hot plate as shown in Diagram A. Turn the hot plate to high. The hot plate will get HOT - be careful!

2. Now place approximately 100 ml of 80% ethanol in a 250-ml beaker and carefully set this beaker into the water beaker before the water boils (see Diagram B). When the water begins to boil, turn down the hot plate until the water maintains a slight boil.

3. While the alcohol is warming up, remove the leaf (or leaves) assigned to your group and take them back to your desk. Do not remove the filters!

4. Before you remove any of the filters, sketch your leaf face up so that after you boil the leaf, you will know where the filters were.

5. After sketching your leaf, carefully remove the filters and place the leaf in the boiling ethanol.

6. After the leaf is a very pale green or white color, remove it from the ethanol bath and place it in a Petri dish. Make sure you turn off your HOT plate!

7. Rinse the leaf with tap water and spread it out flat, face up, in the Petri dish. Pour off the water and add 4-5 droppers full of Lugol's iodine solution to cover the leaf.

8. After your leaf has sat in the iodine for 5-10 minutes, the leaf should darken where starch is present. [Note: Sometimes the Lugol's iodine solution is not good, since iodine is light sensitive. If your leaf does not darken, you may want to repeat steps 7-8 using a new bottle of Lugol's iodine solution.]

9. After the leaf is fully "developed," remove the leaf from the stain and place it in a clean Petri dish. Dispose of the iodine in the proper waste bottle. You may need to add a little water to get your leaf to spread out fully!

10. Now sketch your stained leaf in the table above, next to your sketch of the leaf before it was boiled for comparison.

11. After your double boiler has cooled, dispose of the ethanol in the labeled waste jar! You can put the water down the drain.

Part II Energy Metabolism in Yeast

In the second part of today's lab we will examine one of the energy yielding reactions of the normal brewing and bakers yeast Saccharomyces cerevisiae. Work in groups of four on setting up an experiment to test your hypotheses regarding the effects of substrate concentrations, temperature, and enzyme inhibitors on glycolysis. After deciding upon the experimental set up present your proposal to Mike or Ben before you begin your experiment.

You will have the following solutions and equipment available for your experiment:

10% sucrose 5% sucrose 2.5% sucrose 10% sucrose with different concentrations of EDTA (EDTA binds Mg++ which is an important cofactor for enzymes used in phosphate transfer reactions during glycolysis) An incubator at 37 degrees Ice buckets

General directions for glycolysis reaction setup:

1. Use small lumps of floral clay to seal the tips of the 10 mL pipettes.

2. Add approximately 6 mL of yeast solution to the open end of each of the 10 mL pipettes (up to the number 4). Be sure to insert the tip of the Pasteur pipette below the constriction.

3. Add enough of the appropriate test solution to fill each pyrex pipette to the top.

4. Cover the open end of each pipette securely with Parafilm and invert several times to mix the yeast and test solutions.

5. Remove the Parafilm. Invert one of the test tubes over the open end of the pipette and quickly invert both. Your instructor will demonstrate this step. A bubble of air will move into the pipette when you do this.

6. Place your "respirometer" (this is what you just made) in the test tube rack. Repeat for the other test solutions.

7. Allow 10 minutes for the respirometers to equilibrate. Take a reading from each pipette at this time (Time 0) and about every 5 minutes for 35 to 45 minutes. Remember that each space on the pipette equals 0.1 mL. You want to take the DIFFERENCE between the initial reading (Time 0) and each subsequent reading (mL at Time0 - mL at Time1; mL at Time0 - mL at Time2, etc.).

8. Record your data in a table that you have created in your lab notebook.

LAB WRITEUP

After completing your experiment on photosynthesis, answer the following questions in your lab notebook:

Was your hypothesis supported or rejected? Explain which wavelengths of light were most effective in photosynthesis. Land plants primarily use a pigment called chlorophyll to absorb light energy to power photosynthesis. What is the color of the pigment chlorophyll? Why does it appear this color?

After completing your experiment on yeast glycolysis, answer the following questions in your lab notebook:

Include the graph of volume of gas produced over time. Describe the shape of the curve observed. Was rate of production constant? When was the rate fastest? Slowest? What were the conditions in your experiment that allowed the maximum and minimum amount of glycolysis to occur? Were your hypotheses supported or rejected?

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