The Effects of Light Wavelength and Light Intensity on Photosynthesis by Soraya Pashaei-Marandi 28 October 2015 Bio-111-543 TA: Jian ZhouAbstract An experiment was conducted to test the effects of light intensity, light wavelength , and absorption spectrum on photosynthesis. Three different exercises were used to test each category that factors into photosynthesis. In order to test the effects of light intensity, four test tubes containing chloroplast extract, 2,6-dichlorophenolindophenol, phosphate buffer, water were set up with differing light treatments. The spectrophotometer measured the amount of absorbance for each tube and measurements were taken every five minutes for up to twenty-five minutes. The effect of the light wavelength was tested by creating an action spectrum. This is done by inferring photosynthetic activity from the production of oxygen. Spinach leaves were placed in sodium bicarbonate to test the effects of visible light blue, red, green, and white. Every five minutes the number of floating disk were recorded to indicate the photosynthetic activity. The four main pigments in spinach, carotene, chlorophyll-a, xanthophyll, and chlorophyll-b, were used to observe the absorbance spectrum. The spectrophotometer measured the wavelength starting at 400 nm and moving up 50 nm till it reached 700 nm. The absorption test is used to used to confirm which pigments carry out photosynthesis. Introduction Photosynthesis is a process that occurs in plants that converts light energy into chemical energy. This reaction occurs in the thylakoid membrane (Carter 1996). To test the effects of lightintensity a spectrophotometer is used to measure the absorbance. The absorbance takes place through the chlorophyll pigments in the thylakoid membrane. The thylakoid membrane is then split into two enzyme proteins called photosystems. When the photosystems are present, they execute the electrons and they pass from photosystem II to photosystem I. In order for the electrons to be passed is through the oxidation of NADPH to NADP+. For this experiment, the NADPH was replaced with DPIP so that the reaction could be seen. The purpose for this experiment was to show the effects of light intensity of the photosynthesis of a plant. The hypothesis for this experiment is that the tube 30 cm away will reduce DPIP the best. The pigments in plants vary in wether they absorb or reflect the colors from the action spectrum (Moore 2004). Chlorophyll-a is the pigment that allows plants to reflect green light and absorb other colors. The purpose of the floating disk experiment was to measure which wavelengths promote photosynthesis by isolating the disks in different colors of light. Different wavelengths are used in photosynthesis while the rest are reflected. The hypothesis was that white light would have the greatest effect on photosynthesis and it would have the most floating disks. Other pigments that are used during photosynthesis include carotene, chlorophyll-b, and xanthophyll. The purpose of the spectrophotometer measuring the absorbance of each of the pigments was to compare it to the given absorption spectrum. The hypothesis was that chlorophyll-a will experience the most absorbance. Method Light intensity: A spectrophotometer was set at 605 nm. There were four tubes including a blank that were each given .5 mL of chloroplasts extract and 3 ml of .1 M phosphate buffer. 400 microliters of DPIP was added to the four test tubes, but not the blank. Tube number one was wrapped in foil so there was no exposure to light. The second tube was placed in an apparatus with a light source 24 cm away. The third tube was 30 cm and the fourth tube was 49 cm away. The blank tube was only exposed to the light of the room. Each tube was measured in the spectrophotometer every five minutes for twenty-five minutes. In order to measure the dark tube,the foil was removed, but replaced immediately so no error occurred. The tube with the clearer solution is the one that reduced DPIP the best. Action Spectrum: Spinach leaf disks were placed in beakers containing sodium carbonate. A plunger was used and pressure was released from it to allow the sodium bicarbonate to fill the leaf spaces vacated by oxygen. All the pressure was released until the disks sunk to the bottom, meaning they were fully aspirated. Then 50 ml beakers were used in order to test each different light. 40 ml of sodium bicarbonate and ten aspirated spinach disks were in each beaker. The beakers were placed into light boxes with the different lights of red, white, green, and blue. The dark beaker was placed in the light-proof box. A timer was kept for every five minutes to observe the number of disk that floated to the top of the solution. The disk that float indicated that oxygen filled the intercellular spaces and a photosynthetic reaction occurred. Absorption Spectrum: There were four pigments tested in this experiment: carotene, chlorophyll-a, xanthophyll, and chlorophyll-b. A spectrophotometer was used to measure the absorbance levels of each of the pigments starting at 400 nm. The wavelength was increased in increments of 50 nm until 700 nm was reached. The pigments were tested to create an absorption spectrum that was compared to the already given absorption spectrum of those pigments. Results The dark tube have very little variation in absorbance levels while the other tube dropped in absorbance measurements (Graph 1). For the floating disk experiment, dark, green, and red experienced no floating of disk. Blue and light ended up having all disk floating at the end of the thirty minute interval (Table 2). The floating disk meant that they had photosynthetic activity as expected, however, red should have had a few floating disk by the end as well. For the absorption spectrum, the levels of absorbance of the pigments depended on the wavelength. The pigments all decreased in their amount of absorbance with some spikes to infinity at certain wavelengths (Table 3).Discussion During the light intensity experiment, the dark tube that had no exposure to light had little change in absorbance. This was due to the fact that since the chloroplasts had not seen any light then no DPIP was able to be reduced. The tube that was 30 cm away from its light source was the clearest solution. It had the highest absorbance and reduced the DPIP the best. The tube placed 24 cm away had too much light exposure which caused the stomata
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