Photosynthesis is one of the most important anabolic chemical reactions that allows life to exist on Earth. With water, light energy from the sun, and carbon dioxide from the air, photosynthetic organisms are able to build simple sugars. Organisms that can make their own food are called autotrophs, and are at the base of the food chain. The basic reaction is:

Photosynthesis has two stages. Stage 1 requires light. Stage 2 can work in the light or in the dark. The energy accumulated in Stage 1 is used to drive Stage 2.

1. The light reaction is used to convert sunlight into chemical energy stored in ATP and another energy storage molecule called NADP.
2. The light-independent reaction or Calvin Cycle takes carbon dioxide and fixes it in three-carbon molecules which will eventually be synthesized into glucose.

Experiment: We will conduct a simple experiment using spinach leaves to demonstrate that, in the presence of light and carbon dioxide, leaf tissues produce gas bubbles. While we cannot prove in this experiment that the bubbles are oxygen without a gas probe, we can demonstrate, by use of a control, that the bubbles only form when the leaves are submerged in a sodium bicarbonate solution (which releases CO₂) and not when they are submerged in pure water. We can also demonstrate that the bubbles only form in the presence of strong light, by moving the experiment into the dark and making further observations. Finally, we could experimentally vary the light intensity to demonstrate the effect of light intensity on the process.

When we dissolve baking soda (NaHCO₃) in water, carbonic acid (H₂CO₃) and sodium hydroxide (NaOH) are formed. The carbonic acid then breaks down into water and carbon dioxide gas, which is why dissolving baking soda in water causes it to fizz.

Materials:

• Fresh spinach leaves
• Metal paper hole punch
• 10 mL or larger plastic syringe (without needle) - get one from your local pharmacy
• Baking soda solution (dissolve some baking soda powder in water)
• Liquid dish soap solution (dissolve 5 mL in 250 mL of water)
• 3 clear plastic cups or beakers (250 mL to 500 mL)
  • Cup 1: Detergent solution
  • Cup 2: Baking soda solution (treatment)
  • Cup 3: Water (control)
• Light source (fluorescent is good because it produces light without much heat)

Methods:

1. Use the metal hole punch to cut out 20 circular disks from the fresh spinach leaves, 10 for a control and 10 for a treatment.
2. Separate the two parts of the syringe, drop 10 of the spinach disks inside, reassemble the syringe.
3. Push the plunger almost to the bottom but don't crush the disks.
4. Control or treatment
• For the treatment, draw up a small amount ~1 mL of detergent solution, and then draw the baking soda solution up to ~3-5 mL
• For the control, draw up a small amount ~1 mL of detergent solution, and then draw the water up to ~3-5 mL
5. Point the syringe upward, tapping the sides, so that any air bubbles rise, and gently squeeze the syringe until liquid begins to come out.
6. Put a finger on the end of the syringe, and draw the plunger back slightly, creating a partial vaccum.
7. Repeat until the leaf disks are suspended in the solution. This action forces the liquid into the interior of the leaf.
8. Watch this video of the process to make sure you're doing it right.
9. Pour the contents of control and treatment syringes into two labelled clear plastic cups.
10. Swirl the liquid to try to keep the disks from sticking to each other or the sides of the cups and then let them sit.
11. Turn on a bright light, and monitor the disks every minute. Count how many disks are floating during each of the next 15 minutes.
12. After all (or most) of the disks are floating, put the cups in the dark (a shoebox or a closet) and monitor for the next 15 minutes.
13. Record how many disks remain floating after each minute until all (or most) of them have sunk.

Results:

In the light, you should expect to see the disks in the control solution (water) stay on the bottom, but the disks in the treatment solution (baking soda) should begin to rise as they use the CO₂ to undergo photosynthesis and produce oxygen bubbles. The bubbles should cause the disks to float. After you remove the light and place the cups in the dark, the treatment disks should stop undergoing photosynthesis and the disks should begin to sink.

For comparison purposes, each lab group that does this procedure should report the time at which half (5) of the disks is floating. In the example below, that time would be about 11.5 minutes. You can use this Excel spreadsheet to record your data and it will auto-generate a graph for you.

Questions:

1. How does the suction help the leaf disks to sink?
2. How does the detergent help the leaf disks to sink?
3. Why don't the leaf disks soaking in the water (control) float?
4. What is the purpose of the baking soda solution?
5. What is the purpose of the light reaction?
6. Why do the leaf disks in the baking soda solution (treatment) begin to float?
7. Why do the leaves begin to sink again in the dark?
8. Why don't the leaves in the baking soda solution continue to produce oxygen in the dark?
9. Why do we use the half-way mark as a point of comparison rather than the point at which all the disks are floating?
10. If the light-independent reaction can run without light, why does oxygen production (and presumably glucose production) stop?

References:

http://media.collegeboard.com/digitalServices/pdf/ap/bio-manual/Bio_Lab5-Photosynthesis.pdf

http://www.biologyjunction.com/5b-photoinleafdiskslesson.pdf

https://www.youtube.com/watch?v=XV9FOWleErA

http://www.berwicksclasses.org/AP%20Biology/Biology%20Assignments/AP%20BIOLOGY%20Lab%204.htm

http://www.kabt.org/2008/09/29/video-on-sinking-disks-for-the-floating-leaf-disk-lab/