Investigation 5: Photosynthesis

Introduction
How does the oxygen that we breathe enter the air? And how does the carbon dioxide that we exhale get taken out of the air so that we do not suffocate on it? The answer to both of these questions is photosynthesis, a chemical process for creating energy and sugars within a plant. Photosynthesis takes place in the leaves of a plant, and within the leaves' cells, in the chloroplast. In the chloroplast are specialized molecules called chlorophyll that absorb light and this absorption excites an electron that then goes through a complex series of reactions to form NADPH, an electron carrier, and ATP, an energy source for the cell.

An Overview of Photosynthesis

However, the chlorophyll only absorb a certain frequency of light, and it only absorbs light from the visible light spectrum, so colored light. The chlorophyll do not even absorb the entire spectrum of visible light, usually only absorbing one or two colors of light between a certain wavelength very well, and reflecting the rest. The plant has developed two different types of chlorophyll to account for that, chlorophyll a and chlorophyll b, as well  separate compound called carotenoids.

Absorption Rates of Chlorophyll and Carotenoids

On Friday, November 6th, Vikram, Vinay, Shreyan and I entered the lab again to learn more about photosynthesis. We decided to test the rate of the reactions of photosynthesis under different colored light so that we could see to what affect the absorption of light by the chlorophyll and carotenoids affected the overall reaction. We hypothesized that the rate of reaction would slow when under green light because the chlorophyll and the carotenoids have very low absorption rates in green light as seen above. Also, leaves reflect green light, as seen in their green color.

Our first problem is finding a method by which we could measure the rate of photosynthesis in a plant. Because it is a minuscule process, we couldn't observe it directly through a microscope, but instead would have to find evidence that it is occurring. Here is the chemical equation for photosynthesis:

To find the rate of photosynthesis, we could track the production of one of the products above, or the diminishing of one of the products. Carbon dioxide is a possibility, but would be difficult to measure in an open environment. Water is likewise hard to track, as well as glucose, because we cannot directly see the number of molecules. This leaves us with oxygen, which is also difficult to track individual molecules. Instead of doing that, however, we decided to use the buoyant properties of oxygen in water to measure the rate of reaction. Because as oxygen is produced in leaves during photosynthesis it is kept in the center of the leaf before being expelled through the stomata, the leave actually becomes quite airy. So we could put the leaves in water and watch them float to the top. But to photosynthesize, there must be a source of light, a source of H2O and a source of CO2. If the leaves were submerged, they would not have access to CO2 and would therefore not photosynthesize. Therefore we submerged the leaves in a bicarbonate solution, which would put CO2 into the water for the plant to grab and use for photosynthesis.

The problem with this plan was that the leaves already contained oxygen, so they would float on their own. The only way around this was to evacuate the oxygen from the leave by sucking it out using pressure. At a lower pressure, the oxygen will expand and fill the space, leaving the leaf for the most part and entering the surrounding space around the leaf. Doing this evacuation on a leaf would require larger syringes and tools than we have available so instead we decided to cut the leaves into small dots so they'd fit in the syringe and it would even make the submersion in water easier because then we would not have to find a large tank to submerge full leaves in.

As you can see, a leaf consists of cells but also quite a bit of empty space to hold gases like CO2 and O2

For our experimental variable, which is the color of the light that the plant would be exposed to, we figured we could change this using different colored cellophane. The cellophane would absorb all of the light that wasn't its own color, and let that color through to hit the leaf-dots below. For our controls we performed an experiment with clear cellophane that would supposedly let all light through, but we also did an experiment without cellophane to see if the clear stuff did in fact absorb even a tiny amount of light.

Because of the time consuming nature of the reaction and its effects on the leaf-dots, and the limitations of our equipment, the lab group joined forces with a lab group consisting of Christos, Jorgos and Callen. One group would do three cups with different variables, and the other would do three different cups with different variables, and the data would be shared among us all. Vikram, Vinay, Shreyan and I did the experiments with blue cellophane, yellow cellophane and no cellophane at all.

With this plan of attack, we began our experiment.

Procedure
First, the lab group went outside to pick two fresh ivy leaves to perform our experiment with. We needed to get still living leaves so that they would perform photosynthesis. Next, using the apparatus shown below, I hole-punched 30 dots of leaf out of the leaves and split them into 3 piles. These would be later used for our experiment.

Next, the group created 300mL of a 0.2% bicarbonate solution to submerge the leaves in. Then we evenly separated the solution into three cups and cut three pieces of cellophane for the cups; one blue, one yellow and one clear. Then a drop of dilute soap was added to each cup. The soap acts as a wetting agent to force the leaf dots to allow water from the solution into the cell and sink. This also helps the leaves get their source of CO2 from the solution.

Next we evacuated the leaf dots of all air. To do this, we put 10 dots at a time into a syringe with a stopper and sucked up 10 mL of the bicarbonate solution into the syringe. Then we upended the syringe and squeezed the stopper so all of the air in the solution was evacuated. Once this was done, I put my finger on the opening of the syringe and pulled back on the stopper, effectively creating a vacuum in the syringe chamber, for 10 seconds. I then released the stopper and poured out the 10 leaf dots into one of the cups. This was repeated for each separate group of leaf dots.

Once the first set of leaf dots was evacuated, we immediately started our experiment so as to avoid sunlight causing the leaves to begin their photosynthesis while we evacuated the rest of the dots. This would've caused a skew in our data, but luckily we removed that confounding variable. The experiment consisted of a piece of colored (or clear) cellophane covering the cup and the cup was then placed under a lamp. The light from the lamp then caused the cells in the leaf dots to photosynthesize. We also started a separate timer for each experiment group so we could accurately keep time.

After all of the dots were in their cups and the cups were placed under the lamp, we observed the number of leaf dots that had risen completely to the top and recorded this data. After all the dots had risen, or 30 minutes, the experiment was stopped, and the dots were poured out.

Data/Tables
Here are the results of trials with the blue, yellow and lack of cellophane.

Time (min)	# of dots risen (clear)	# of dots risen (blue)	# of dots risen (yellow)
1	0	0	0
2	0	0	0
3	0	0	0
4	0	0	0
5	0	0	0
6	0	0	0
7	0	0	0
8	0	0	0
9	0	0	0
10	0	0	0
11	0	0	0
12	0	0	0
13	0	0	0
14	0	0	1
15	0	0	2
16	0	0	4
17	0	0	5
18	2	0	5
19	3	0	7
20	6	0	7
21	8	0	7
22	10	0	7
23	-	0	8
24	-	0	8
25	-	0	9
26	-	1	9
27	-	1	9
28	-	1	10
29	-	2	-
30	-	3	-

And a picture of the experiment in action:

Here is the data received from our lab group partners Jorgos, Christos and Callen, who experimented with green cellophane and a control cup of no cellophane.

Time (min)	# of dots risen (no cellophane)	# of dots risen (green)
1	0	0
2	0	0
3	0	0
4	0	0
5	0	0
6	0	0
7	0	0
8	0	0
9	0	0
10	0	0
11	0	0
12	0	0
13	0	0
14	0	0
15	0	0
16	0	0
17	1	0
18	1	0
19	1	0
20	1	0
21	1	0
22	1	0
23	1	0
24	1	0
25	1	0
26	1	0
27	2	0
28	2	0
29	2	0
30	2	0

Here is a graph of the results of both experiments combined:

Conclusion
Looking at the results of our experiment, I'd say that the lab was a relative success. As expected, when the plant was exposed to all types of light while under the clear cellophane, the rate of photosynthesis was fastest because all 10 dots floated to the top in the shortest amount of time when compared to single-color lights. However, a confusing piece of data is the extremely low number of dots, only 2, that floated to the top without any cellophane in Christos, Jorgos and Callen's experiment. Plants do not need cellophane to photosynthesize because cellophane is not found in nature, so that cannot be the reason why the dots didn't rise. I believe that there could have been some error with the evacuation of the cells because if one evacuates the cell too hard or for too long, the cell's chloroplasts will die and it will not photosynthesize. This could have happened for our compatriots. Other points of data, however, are quite normal. We did not expect the dots to photosynthesize much while under green light because leaves do not absorb much green light, if any. I was somewhat surprised at our data for blue light because I had thought that the rate of absorption hit a peak for chlorophyll when under blue light, but only 3 dots rose to the top of the water in that group. The results of the yellow group are also predictable because some of the peak absorption of leaves happens near red light, which is right next to yellow light on the visual light spectrum. Therefore, plants should also be able to absorb a lot of yellow light.

Due to the evacuation error mentioned above, as well as other fixes for the lab, the experiment could be improved in a variety of ways. First of all, all of the leaf dots should be evacuated together so that they all have the same level of evacuation when starting the experiment. This would also make sure that different groups using different syringes and applying different pressures wouldn't skew the data as it did for Jorgos' group. Another way in which the lab could be improved is, if the lab group had a long time to spend doing the experiment, we could use the same lamp and place the cup in the same spot each time so that we can ensure that the dots in the cup are receiving the same amount of light from the lamp and that they aren't missing out on light due to position.

I believe that this can be considered a success because we got data that we expected to get, our hypothesis was proved true, but also because we made mistakes. These mistakes helped us to learn ways in which we could improve our lab for next time as well as how to look out for confounding variables in coming labs we have not performed yet.