Photosynthesis Lab PDF

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Photosynthesis Lab from Biol 101 L (first written lab assignment)...

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I.

Introduction

Most energy on this earth results from producers and autotrophs such as plants. Plants are able to convert light energy into chemical energy through a process called photosynthesis. In order for photosynthesis to be conducted, it needs CO2, H2O, chloroplasts, and light. During photosynthesis, O2 and sugars are outputted. The balanced equation for photosynthesis is 6 CO2 + 6 H2O → C6H12O6 + 6 O2. Without photosynthesis, animals would not be able to readily get energy from plants through consumption and molecules in the air would not circulate to allow animals to conduct cellular respiration. Photosynthesis is divided into two processes inside the chloroplasts: the light reaction and the Calvin cycle. The light reaction occurs in the thylakoid in which light (photons) strike photosystems and excite electrons. These electrons move along an electron transport chain where they are eventually picked up by a transport molecule called NADP+, reducing it to NADPH. A reduction is a gain of electrons, whereas an oxidation is a loss of electrons. The movement of these electrons create energy for H+ ions to be moved up its concentration gradient through active transport. These H+ ions appear because of the splitting or reduction of water, which Robert Hill discovered in 1937 after discovering that the O2 given off during the light reaction did not come from CO2. This is called the Hill Reaction. The high concentration of H+ ions are then moved down ATP synthase through facilitated diffusion, creating ATP. ATP and NADPH are then brought to the Calvin cycle in the stroma. NADPH drops its electrons off and is oxidized back to NADP+. During the Calvin cycle CO2 is converted to sugar. (Simon et al. 108-120)

2 In order to detect if molecules are being reduced or oxidized, DPIP, a dye that intercepts electrons, replaces some of the NADP+ receptors. If electron flow is present, indicating photosynthesis is occurring, the dye will change from blue to colorless because it is accepting electrons. The loss of color will be measured by a spectrophotometer. This experiment will test the rate of photosynthesis at different temperatures. The dependent variable is the results from the spectrophotometer that is measuring transmittance, and the independent variable is the change in temperature of the ice baths. Different temperatures may factor into the percent transmittance or the rate of photosynthesis by possibly slowing it down, speeding it up, or stopping it. This test is relevant because of the threat of global warming. With rising temperatures, and changes in climate, it is important to test the effects of differing degrees. We observed that most plants start to lose their leaves in the fall and are bare in the winter, so we predicted that photosynthesis slows down during colder temperatures. This may occur because there are less leaves to conduct photosynthesis, and colder temperatures slow down chemical reactions. In warmer temperatures, such as spring and summer, more plants bloom. We predicted that photosynthesis would speed up during these seasons because of the increase in temperature and more surfaces to conduct its reaction. However, if the temperature is too hot, the plant is at risk for denaturation which would stop photosynthetic reactions. We hypothesized that higher photosynthesis rates will occur in room temperature to slightly warmer temperatures, and lower photosynthesis rates will occur in cold and hot temperatures. I.

Materials and Methods

Before we began the experiment, a chloroplast solution had to be made. This solution was made from spinach leaves. First the leaves were deveined by pulling the leafy part of the spinach away from the stem. This was done in order to obtain the highest concentration of chloroplasts.

3 The leaves were then placed under a lamp to expose it to light since they had been bagged previously. After exposure to light, the spinach leaves were placed in a chilled blender in order to keep them from denaturing during the blending process. Half a molar of sucrose was added to the blender for the spinach to maintain an isotonic solution. If the spinach were placed in a hypotonic the cells would burst, and if they were placed in a hypertonic solution the cells would shrink. The leaves were blended in three 10 second spurts to not create too much heat and maintain as many chloroplasts as possible. After it was blended, the chloroplast solution was drained through three layers of cheese cloth and a funnel to remove big chunks. The drained chloroplast solution was then poured into a container for use in the experiment and chilled in an ice-box as to keep it away from light and control its temperature before the experiment was conducted. To set up the experiment, we separated and labeled five test tubes on a rack. The test tubes were labeled with sharpie according to the temperature in which they were to be placed. We tested 4°C (test tub #1) , 21 °C or room temperature (test tube #2) , 29°C (test tube #3), and 45°C (test tube #4) by setting up controlled ice-boxes for each temperature. These four test tubes were all filled with 3 mL of distilled water, 1 mL phosphate buffer, and 1 mL DPIP with transfer pipettes each labeled according to their solution in order to keep liquid controlled throughout the experiment. The fifth tube, known as the calibration tube, was filled with 4 mL of distilled water, and 1 mL buffer with pipettes which can be observed in the table of Substance in Test Tubes (T.2) in the appendix. The reason buffer was added to all of the tubes was to maintain the pH since free hydrogens would make it acidic. The liquids were kept in an ice-box before placing them in their tubes in order to keep the temperature controlled. The fifth tube was the calibration tube and was only used to scale the spectrophotometer before measuring the rest of the tubes.

4 The tubes were then placed in their respected ice baths, and the calibration tube was added to the room temperature ice bath. The control was the test tube placed in room temperature that included the DPIP, because it provides a middle ground to compare the effects of warmer and colder temperatures. Before adding the chloroplast solution, we waited five minutes for the test tubes to reach the desired temperature of their ice bath. Once the temperature was reached three drops of chloroplast solution was added to every test tube. We had to wait until the tubes reached the correct temperature before including the chloroplasts in order to maintain the structure of the chloroplasts. Goose- neck lamps were placed over all of the ice baths to expose the chloroplasts to the same amount of light. The spectrophotometer was then turned on and set to 605 nm which relays orange pigment. When setting the spectrophotometer we chose a setting in which the wavelength difference was the greatest so changes in transmittance by DPIP would be easily seen. Before placing the calibration tube inside the spectrophotometer, we covered the top with Parafilm and then we inverted the tube in order to mix the solution. After it was inverted, Kim wipes were used to clean the surface of the tube from any finger prints to limit any confounding variables that would mess up the spectrophotometer’s reading. The calibration tube was then set inside the spectrophotometer and measured. Once the calibration tube read 100% the rest of the tubes could be measured. In order from coldest to hottest, the tubes were removed from their icebaths, inverted, cleaned, read, and returned to their ice bath just as the calibration tube. The data for each tube was recorded at time 0. A reading occurred by repeating the same process above at times 5, 10, and 15 (minutes). The calibration tube was read slightly before the five minute period to ensure that the

5 spectrophotometer was set correctly. This was to ensure that the transmittance read for each of the following tubes were controlled and accurate. The experiment was then repeated to ensure accuracy. II.

Results

After analyzing the data, we observed that temperature did have an effect on photosynthesis. Room temperature had the highest percentage of transmittance, followed by 29°C, 5°C, and finally 45°C as seen in the table of Transmittance with Reference to Time (T.1) and the graph Transmittance over Time (F.1) in the appendix. The controlled temperature was where the highest photosynthetic rates were seen. A rate is calculated to find specific differences in the activity level of chloroplasts in relation to the change in transmittance and time. As transmittance percentages rose, the chloroplasts conducted higher rates of photosynthesis as seen in the graph of Temperance over Time in the appendix (T.3). Chloroplasts at or below room temperature conducted photosynthesis at higher rates than that of temperatures above room temperature. III.

Discussion

The data provided support for half of our hypothesis. It supported that room temperature would conduct a higher rate of photosynthesis and that hot temperatures would conduct a lower rate of photosynthesis. It did not support that cold temperatures would conduct a low rate of photosynthesis and slightly warmer temperature than room temperature would conduct higher rate of photosynthesis. We interpreted our findings as temperatures above room temperature begin to slow or stop the rate of photosynthesis. The hotter the temperature the more likely the rate of photosynthesis will decrease and possibly stop. Temperatures surrounding room temperature are the most beneficial for plants conducting photosynthesis. Temperatures colder

6 than room temperature continue to conduct photosynthesis just at a slower rate. Our controls helped us interpret our experimental results by being able to compare the warmer and colder temperatures to a temperature in which photosynthesis occurred relatively normal. Our other controlled variables such as amount of liquid and light that was shown on the solutions helped eliminate other factors that may have varied the results and allowed for the testing of one independent variable. Possible confounding variables could have been the slight change in temperature of the ice baths in which the test tubes were placed, the time it took to carry tubes to the spectrophotometer, the loss of liquid when the tubes were inverted, and smudges from finger prints on the test tubes before measuring. Some temperatures began to increase because of melting ice or because of the unstable mechanics. Although the water temperature was measured before each reading, the increases in temperature could have affected the chloroplast solution before the temperature was reduced back to its initial state. The time that it took to collect the tubes and measure them in the spectrophotometer could have reduced or increased their temperatures. Since the tube in 21°C had the same relative temperature in the air, there was minimal change in temperature, and could have been a factor in the increased rates of photosynthesis while other temperatures taken out of the ice baths were affected by the change in temperature from the environment. Some liquid was loss when the tubes were inverted. This may have had an effect on the results since some of the added solutions may have been lost, and all the tubes may not have had an equal amount of liquid for each reading. Before placing the tubes inside the spectrophotometer, the Kim wipes may not have eliminated all finger prints and residue that was on the test tubes. This may have affected the data because transmittance may have been disrupted or blocked.

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Appendix

Transmittance over Time Rate of Photosynthesis in Percent

90.00% 80.00%

F.1

70.00%

The

60.00% 5 °C 21°C 29°C 46°C

50.00% 40.00%

Rate of

30.00% 20.00% 10.00% 0.00% Time 0

Time 5

Time 10

Time 15

Time in minutes

Photosynthesis over time describes the chemical reactions of chloroplasts in different temperatures in fixed periods of times.

Percentage Transmittance Table with Reference to Time Time 0 Time 5 Time 10 Time 15

5°C 22.2% 44.3% 65.4% 81.1%

21°C 24.3% 44.3% 65.4% 81.1%

29°C 21.4% 26.6% 26.5% 32.7%

45°C 21.3% 19.1% 18.2% 16.7%

T.1 The data shows the recorded transmittance percentages of the tubes at differing times after read by the spectrophotometer.

Substances in Test Tubes 8 Calibration 1 mL buffer 4 mL distilled

5°C 1 mL buffer 3 mL distilled

21°C 1 mL buffer 3 mL distilled

29°C 1 mL buffer 3 mL distilled

45°C 1 mL buffer 3 mL distilled

water

water 1 mL DPIP 3 drops

water 1 mL DPIP 3 drops

water 1 mL DPIP 3 drops

water 1 mL DPIP 3 drops

3 drops

chloroplasts chloroplasts chloroplasts chloroplasts chloroplasts T.2 The table shows the contents that were added to the tubes in order to conduct the experiment, including the calibration tube which lacked DPIP. 5°C 21°C 29°C 45°C Calculated Photosynthetic Rates (Change in T%/Change in Time) 10 Minutes 1.74 4.11 .51 -.31 15 Minutes 1.54 3.78 .75 -.31 T.3 The rates of photosynthesis were calculated by dividing the change in percent transmittance divided by the change in time. This showed the specific changes between each time period. References Stegenga, Barabara, ed. "Photosynthesis." Laboratory Exercises for Biology 101. Chapel Hill: U of North Carolina at Chapel Hill, 2015. 24-30. Print. Simon, Eric J., Jean L. Dickey, and Kelly Hogan. "Photosynthesis." Campbell Biology: Concepts Et Connections. By Martha R. Taylor and Jane B. Reece. 8th ed. Harlow: Pearson, 2014. 108-20. Print....