Kitch 1 Tristan Kitch Mrs. Hayden IB Biology HL 2 8 September 2014 An Investigation of the Effect of Wavelength on Photosynthesis DESIGN: Research Question: The purpose of this lab is to determine how the wavelength of light that alfalfa sprouts are exposed to affects the rate of photosynthesis in those plants. Hypothesis: Photosynthesis will occur at the highest frequency when the alfalfa plants are exposed to blue light (475 nm) ("What Wavelength Goes With a Color?"). The second highest amount of photosynthesis will occur when the alfalfa plants are exposed to red light (650 nm) ("What Wavelength Goes With a Color?"), and the lowest amount of photosynthesis will occur when the alfalfa plants are exposed to green light (510 nm) ("What Wavelength Goes With a Color?"). This is because the pigments in the plants’ chloroplasts, such as chlorophyll a, chlorophyll b, and carotenoids, absorb the most light in the blue color range, the second most light (of the selected colors) in the red range, and the least amount of light in the green range (see Figure 1). Kitch 2 Figure 1: Amount of Light Absorbed by Chloroplast Pigments Chlorophyll a absorbs the most light in the blue to indigo and red to orange ranges, chlorophyll b absorbs the most light in the blue and orange ranges, and carotenoids absorb the most light in the blue to blue-green range (“Absorption Spectrum”). Background: Originally, an experiment was run using chloroplast solutions rather than alfalfa sprouts. However, CO₂ levels did not change significantly throughout the experiment, so the method had to be changed. Then, peas that had already germinated were used instead of chloroplast solutions. CO₂ concentrations increased throughout that experiment, indicating a large amount of Kitch 3 cellular respiration was being undergone rather than photosynthesis. Then, it was discovered that peas use all available resources before producing new resources. In other words, peas underwent cellular respiration and derived energy from sugar that was already produced rather than unnecessarily producing new sugar and energy via photosynthesis (Brennan). Finally, alfalfa sprouts were decided on as the subject of the experiment due to their durability and quick germination time (Phipps). Photosynthesis ultimately produces all energy that is consumed by organisms on Earth. Photosynthesis occurs based on the chemical reaction: 6CO₂+6H₂O⟶C₆H₁₂O₆+6O₂ and, photosynthesis is stimulated by light. Since CO₂ is consumed as photosynthesis occurs, CO₂ can be monitored to measure the amount of photosynthesis that an organism is undergoing. Given that photosynthesis must occur in the presence of light, it is possible that certain colors of light may favor higher amounts of photosynthesis. In this experiment, the color, or wavelength, of light was altered in order to understand at which wavelength is most conducive to a plant’s undergoing of photosynthesis. Variables: Independent Variable: The independent variable was the wavelength (λ, in nm ± 1 nm) of light the chloroplasts received. The independent variable was altered by exposing the alfalfa plants to different wavelengths of light, specifically blue, red, and green light, along with a control alfalfa plant that only received the extraneous light from the room in which the experiment was run (the same amount of extraneous light that the experimental groups received). Those colors of light were chosen because, generally, chloroplasts in plants absorb the largest amount of blue light, Kitch 4 the smallest amount of red light, and an amount of red light in between the other two; differences in the amount of photosynthesis should have been evident between each of the light conditions. Dependent Variable: The dependent variable was the amount of photosynthesis that occurred, made quantifiable through measurement of the concentration of CO₂ molecules that were consumed by the alfalfa in photosynthesis, logged on a Vernier LoggerPro. The dependent variable was measured using a Vernier CO₂ Gas Sensor. Kitch 5 Controlled Variables: Table 1: Controlled Variables Controlled Variables Method to Control Variable Distance of Light Bulb from Chloroplasts All chloroplast solutions were kept about 10 cm away from their respective light bulbs, helping the amount of light of the appropriate color received by each chloroplast solution to remain constant. Light Bulb Wattage, Voltage Each light bulb had the same wattage and voltage so each chloroplast solution received the same brightness of light. Extraneous Light All chloroplast solutions were kept in the same room, keeping the amount of white light received by each chloroplast solution constant. Volume of Gas The beakers that contained the alfalfa plants were sealed so the combined volume of O₂ and CO₂ gas remained constant. Size, Amount, and Age of Alfalfa Plants Each beaker where alfalfa plants were grown contained 150 cm³ of potting soil. The mass of alfalfa seeds planted in each beaker was 0.45 g. Each group was grown in the same lighting conditions and received the same amount of water each day. Finally, they were allowed to grow for 10 days before the trials were run. Time Each alfalfa plant remained exposed to the colored light for twenty-five minutes, which was monitored with a stopwatch ( ± 0.1 s). Distance of the alfalfa plants from the bulbs and bulb wattage and voltage all were held constant to ensure the chloroplasts received equal amounts of light and equal brightness of light, helping ensure the amount of light received by the solutions did not skew the data. The beakers Kitch 6 were sealed in order to assure that the change in CO₂ concentration was caused by photosynthesis in the alfalfa plants. All alfalfa plants were grown in the same conditions in order to ensure that each group was theoretically able to consume the same amount of CO₂ when in the same lighting conditions. This ensured that the wavelength of light to which the plants were exposed led to differences in CO₂ concentrations in the beakers. Finally, the time was held constant so that the amount of photosynthesis for each group of alfalfa plants could be fairly compared at the same point in time. Required Materials: 1 bag of potting soil (600 cm³) Deionized water 5 400 mL beakers 1 bag of alfalfa seeds (1.80 g) 1 weigh boat 1 industrial scale (g ± 0.01 g) 3 lamps 1 blue light bulb 1 red light bulb 1 green light bulb 1 stopwatch (s ± 0.1 s) 4 Vernier CO₂ Gas Sensors 4 Vernier LoggerPros 1 cardboard box Glad Press’n Seal Wrap Lights to grow the plants 1 pair of scissors 1 pen Method: 1. Approximately 100 cm³ of potting soil were poured into each beaker. 2. The weight boat was placed on the industrial scale, which was then zeroed. Kitch 7 3. Alfalfa seeds were poured onto the weigh boat until the industrial scale read 0.45 g. This mass of seeds was estimated using information given on the article written by Nikki Phipps. 4. Those seeds were poured into one of the beakers. 5. Steps 3 and 4 were repeated until there were seeds in all 4 beakers. 6. An additional 50 cm³ of potting soil was poured into each beaker (Phipps). 7. Each beaker was shaken so that the soil was evenly distributed in the beaker/ 8. All 4 beakers were placed under lights so they could grow. They were watered each day, except on weekends, for 10 days. 9. Before the experiment was run, a fifth beaker and a CO₂ sensor were used to make devices that would allow each beaker of alfalfa plants to be sealed. 10. A circle was drawn with a pen on the cardboard box using the fifth beaker as a template. This circle was then cut out. 11. Another circle was drawn in the center of that circle using the CO₂ sensor as a template. This circle was then cut out so a CO₂ sensor could fit into the larger cardboard circle. 12. Steps 10 and 11 were repeated until there was a cardboard circle with a hole in the middle for each beaker of alfalfa plants. 13. The beakers of alfalfa plants were removed from under the lights, and a cardboard circle was placed on top of each beaker. 14. Glad Press’n Seal Wrap was wrapped around the top of each beaker. This locked out outside air, while the cardboard provided support to the CO₂ sensor; the Glad Press’n Seal Wrap would not have been able to support the weight of a CO₂ sensor. Kitch 8 15. Excess Glad Press’n Seal Wrap was cut from the beakers using the scissors in order to prevent the wrap from blocking light from reaching the alfalfa plants. The wrap was pressed along the sides of the beaker to ensure that no outside air could enter the beakers during the trials. 16. Three stations were set up so that a different-colored lamp faced each beaker. Each beaker was approximately 10 cm away from the lamp from which the beaker was receiving light. Each lamp contained one of the following bulbs: blue, red, and green. The lamps were set up far enough from each other that the light of one lamp did not interfere with the light of another lamp. 17. The fourth beaker of alfalfa seeds was set up apart from the other three beakers and did not receive light from any lamps. This beaker acted as the control beaker. 18. The 4 Vernier O₂ Gas Sensors were connected to 4 different Vernier LoggerPros. 19. The scissors were used to poke holes in the Glad Press’n Seal Wrap on each beaker. These holes were located over the holes in the cardboard circles so CO₂ sensors could be placed through the cardboard circle and detect the levels of carbon dioxide within the beaker. 20. A Vernier O₂ Gas Sensor was placed on each beaker. Each sensor’s fit through the hole in the cardboard was snug, ensuring that air would not exit or enter the beaker while trials were being run. 21. The 3 lights were turned on simultaneously, while data collection was initiated on all 4 beakers simultaneously. Kitch 9 22. The concentration of carbon dioxide in each tube was recorded after each five-minute-long increment, beginning at 0 minutes, to check for patterns or irregularities in oxygen gas production. 23. Data collection was halted after 25 minutes. 24. The CO₂ sensors were removed from the beakers, and the positions of the beakers (i.e. the light or group to which a beaker was assigned) were rotated. 25. The next trial commenced after the readings of the CO₂ sensors were at the same level as before the previous trial was run. 26. Steps 21 through 25 were repeated two more times so three total trials were run. Kitch 10 DATA COLLECTION AND PROCESSING: Data Collection: Table 1: Carbon Dioxide Concentration Time (minutes) Carbon Dioxide Carbon Dioxide Carbon Dioxide Carbon Dioxide Concentration Concentration Concentration Concentration in When Exposed to When Exposed to When Exposed the Control Green Light Blue Light (ppm to Red Light Group (ppm ± 25 (ppm ± 25 ppm) ± 25 ppm) (ppm ± 25 ppm) ppm) Trial 1 0 820 627 415 525 5 860 686 428 509 10 867 721 456 542 15 926 763 426 565 20 930 761 523 584 25 949 781 499 612 0 687 529 357 332 5 786 672 438 464 10 791 713 490 509 15 800 736 511 542 20 800 745 480 553 25 822 767 601 568 0 676 541 352 165 5 794 700 490 283 10 769 714 410 288 15 788 730 516 313 20 793 751 505 329 25 800 751 545 347 Trial 2 Trial 3 Kitch 11 Levels of carbon dioxide increased in each trial relative to the original amount of carbon dioxide in each beaker. CO2 sensors were used to determine the concentration of carbon dioxide in each beaker. Processed Data: Table 2: Percent Change in Carbon Dioxide Concentration Trial Number Green (%) Blue (%) Red (%) Control (%) 1 15.7 24.6 20.2 16.6 2 19.6 55.0 68.3 71.1 3 18.3 38.8 54.8 110. Average 17.9 39.5 47.8 65.9 The positive changes in carbon dioxide concentration for each trial and each color group indicated an increase in the amount of carbon dioxide in each set of plants. The amounts of increase in carbon dioxide concentration for all trials of a group in certain light conditions were averaged in order to discover the increase in carbon dioxide concentration for the average set of plants. Then, these averages could be compared to discover if the increase in carbon dioxide concentration was significant between different groups. The control group had the largest increase of carbon dioxide concentration, while the group of alfalfa plants exposed to green light had the smallest increase in carbon dioxide concentration. Kitch 12 Table 3: T-Test Results for Each Set of Trials Green Green Blue Blue Red Control 2.44 2.08 1.77 0.494 0.927 Red 0.591 The average increase in concentration of carbon dioxide for each set of trials and the standard deviation for each set of trials was used to compare each set of results with each other to if there were significant differences between any of the trials. For example, the results of “green” trials were compared with the results of the “blue” trials to determine if the results were significantly different, and then the “green” results were compared with the “red” results, et cetera. The results of the green trials had the largest differences between the trials for other light conditions. Table 4: T-Test Critical Value Critical Value 2.78 The degree of freedom for each t-test was 4, as two sets of three data points were compared (6 total data points minus the 2 groups). The p value was 0.05 in order to determine that there was a 5% chance that the results of the experiment occurred by chance. These degrees of freedom and this p value gave 2.78 as the critical value (Caprette). Each t-test result was less than the t-test critical value. Kitch 13 Sample Calculations: Data from the group of alfalfa plants that were exposed to green light were used in all sample calculations except for the t-test; data from the “green” group and the “blue” group were used in the t-test calculation. Percent Increase in Carbon Dioxide Concentration: final concentration −original concentration original concentration − 820 ppm (100%)= 949 ppm (100%)= 820 ppm 15.7% The change in carbon dioxide concentration, the final concentration (carbon dioxide concentration at 25 minutes) minus the original concentration (carbon dioxide concentration at 0 minutes), was divided by the original concentration to determine the fractional change of carbon dioxide concentration over the 25 minutes in which each trial was run. That quotient was multiplied by 100 to determine the percent change in carbon dioxide concentration. Average Increase in Carbon Dioxide Concentration: x= x1 +x2 +x3 +···+xn n = 15.7%+19.6%+18.3% = 3 17.9% The percent change in carbon dioxide concentration for each trial in a set of trials in the same light conditions were added together. That sum was divided by the total number of trials in that set of light conditions to discover the average increase in carbon dioxide concentration associated with certain light conditions. Kitch 14 Standard Deviation: Sx = √ n ∑ (xi−x)2 x=i n −1 = √ (15.7 −17.9)2 +(19.6 −17.9)2 +(18.3 −17.9)2 3 −1 = 1.99 The magnitude of the increase in carbon dioxide concentration for each trial in a set of trials under the same conditions and the average increase in carbon dioxide concentration for that set of trials were plugged into the standard deviation formula in order to approximate the range of the set of data in that light condition. T-test: T= ∣x1 −x2 ∣ √ S2 S2 1 2 n + n = ∣39.5%−217.9%2 ∣ = √ 15.2 3 + 1.99 3 2.44 The average increase in carbon dioxide concentration and the standard deviations for the two sets of trials being compared were plugged into the t-test formula to see if the difference between the results of those sets of trials was significant. Kitch 15 Graph: Graph 1: Average Percent Increase in Carbon Dioxide Concentration of Each Light Condition The standard deviation (shown in the error bars) of each data set increased as the percent increase in carbon dioxide concentration became larger. The control group had the largest increase in carbon dioxide concentration, while the group of alfalfa plants exposed to green light had the smallest increase in carbon dioxide concentration. CONCLUSION AND EVALUATION: Conclusion: The hypothesis of the lab was that the most photosynthesis and therefore the most carbon dioxide would be consumed in the alfalfa plants exposed to blue light, with the second-most carbon dioxide being consumed in the red group, and the third-most in the green group. However, a net amount of carbon dioxide was produced, not consumed; the amount of carbon dioxide increased in each trial for each set of light conditions. This indicated that the alfalfa Kitch 16 plants underwent more cellular respiration than they did photosynthesis, as the equation for cellular respiration is the reverse of photosynthesis, C₆H₁₂O₆+6O₂⟶6CO₂+6H₂O. Also, none of the t-test results for the comparison of any two groups were above the critical t-value given the p value and degrees of freedom of the experiment. This indicated that there was no significant difference between the amount of carbon dioxide produced by any two groups, and the null hypothesis had to be accepted. The data indicated that there was no significant difference between the amount of photosynthesis undergone in alfalfa plants exposed to different wavelengths of light. However, the reliability of this result is questionable, as there were few trials run in total and the conclusion contradicts the scientific consensus on the issue (“Absorption Spectrum”). Evaluation: As previously stated, given that carbon dioxide levels increased, the plants underwent more cellular respiration than photosynthesis. This may have been because there was already glucose in the plants that could be utilized; there would have been no sense in the plants producing sugar they did not need. Cellular respiration may have allowed the plants to be efficient with their resources. The plants were in the stage I maturation phase of their lives given that they were 10 days old, meaning their production of starch and therefore amount of photosynthesis should have been high (Lai and McKersie). However, just because glucose polymerization rates and the amount of photosynthesis was high does not mean that the plants did not undergo more cellular respiration than photosynthesis. This stage of life indicated that the plants were growing rapidly, and would therefore require large amounts of energy (Lai and Kitch 17 McKersie). Plants underwent both photosynthesis and cellular respiration to maximize energy production. However, given that carbon dioxide levels increased in each trial, the plants underwent more cellular respiration than photosynthesis, which at first seemed unlikely given that photosynthesis produces the glucose required for cellular respiration. This indicated that there must have been stores of sugar already in the alfalfa plants. In repetitions of this experiment, trials should be run when the alfalfa plants are fully grown, or when they are taller than 30 cm (“Irrigated Alfalfa Management for Mediterranean and Desert Zones”). Given that there was no significant difference between any of the results, extraneous light from the lab in which the experiment was run may have contributed enough light for the alfalfa plants to undergo photosynthesis. Future experiments should be run in complete darkness to ensure that the only light the plants can derive energy from is the color that is being tested. Ideally, more trials would be run in the experiment was well, as a larger number of trials makes it much easier to discover if the data are significantly different. A larger number of trials would solidify the results of the experiment. Finally, the highest percent increases in carbon dioxide concentration often came from trials where the initial carbon dioxide concentration was low. To ensure those large percent increases did not occur just because initial carbon dioxide concentrations were low in the beginning, in future experiments, the initial carbon dioxide concentration for each trial and light condition should be about the same. Another possible experiment would be to investigate the effect of wavelength on photosynthesis in other plants, especially plants that are not green. Plants that are not green would absorb different wavelengths of light more effectively simply because of the different Kitch 18 color. For example, it is likely that a red fern would absorb blue light and green light but not red light effectively. Kitch 19 Works Cited "Absorption Spectrum." University of Illinois at Chicago, n.d. Web. 25 Aug. 2014. <http://www.uic.edu/classes/bios/bios100/lecturesf04am/absorption-spectrum.jpg>. Brennan, John. "Why Do Germinating Peas Undergo Cellular Respiration?"EHow. Demand Media, 22 Aug. 2010. Web. 13 Oct. 2014."What Wavelength Goes With a Color?" What Caprette, David R. "Selected Critical Values of the T-Distribution." Selected Critical Values of the T-Distribution. Rice University, n.d. Web. 13 Oct. 2014. "Irrigated Alfalfa Management for Mediterranean and Desert Zones." (n.d.): n. pag. Alfalfa.ucdavis.edu. UC Davis. Web. 13 Oct. 2014. Lai, Fang-Ming, and Bryan D. McKersie. "Regulation of Starch and Protein Accumulation in Alfalfa (Medicago Sativa L.) Somatic Embryos."ScienceDirect. Elsevier B. V., n.d. Web. 13 Oct. 2014. Phipps, Nikki. "Growing Alfalfa – How To Plant Alfalfa." Gardening Know How. N.p., n.d. Web. 13 Oct. 2014. Wavelength Goes With a Color? NASA, n.d. Web. 25 Aug. 2014.<http://science-edu.larc.nasa.gov/EDDOCS/Wavelengths_for_Colors. html>.
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