BIO101_Lab08_Photosynthesis

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Apr 3, 2024

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BIO 101 Lab 08: Photosynthesis v.2021 1 BIO 101 Lab 08: Photosynthesis Objectives:________________________________________________________________ • Determine the effects of light on the rate of photosynthesis • Determine the absorption spectrum of leaf pigments Background:_______________________________________________________________ Sunlight provides the majority of energy for organisms living in most ecosystems, however only a subset of organisms are capable of harvesting this energy. Plants use their chloroplasts to absorb the energy from sunlight. This energy is then stored in the covalent bonds of glucose, a simple sugar, and can be used by the plant for structural purposes (cellulose), as usable energy (ATP generation), or for energy storage (starch). Animals can eat plants to obtain glucose and produce energy through a process called cellular respiration. The overall reaction for photosynthesis is represented by the chemical equation: 6 CO 2 + 6 H 2 O + sunlight → C 6 H 12 O 6 + 6 O 2 The entire process is complex and involves many enzymatic reactions. You may notice that the photosynthesis reaction is nearly the exact reverse of cellular respiration. From the equation above the three key elements for photosynthesis to occur are carbon dioxide (CO 2 ), water (H 2 O), and light. If any of the three are missing from the system then photosynthesis will not occur, and glucose production in the plant will be negatively affected. There are two phases of photosynthesis: 1) In the light-dependent phase chlorophyll molecules located in the thylakoid membrane stacks of the chloroplasts absorb the energy from light resulting in the production of ATP and NADPH. 2) In the light-independent reactions (the Calvin Cycle ) the energy stored in ATP and NADPH is used to ultimately convert carbon dioxide to sugar. The process of taking carbon dioxide from the air to build carbohydrates is called carbon fixation .

BIO 101 Lab 08: Photosynthesis v.2021 2 In solution, CO 2 can be converted to carbonic acid (H 2 CO 3 ) when dissolved in water. The carbonic acid will then release hydrogen ions (H + ), causing the pH of the solution to decrease. CO 2 + H 2 O  H 2 CO 3  HCO 3 - + H + Bromothymol blue is a pH indicator that changes color based on the pH of a solution. Bromothymol blue turns yellow at lower pH and blue as the pH is increased. This indicator can be used to track respiration (turns yellow as CO 2 is added to the system) or photosynthesis (turns blue as CO 2 is consumed). Different colors of Bromothymol blue at the indicated pH conditions Light energy is a small part of the electromagnetic spectrum which is visible to the eye. The wavelength of visible light lies between 380 nm and 760 nm. In order to obtain the energy from light, plants must absorb light energy using pigments, namely chlorophyll a, chlorophyll b, carotene and xanthophyll. These pigments show characteristic colors because they do not absorb all light equally. By measuring the absorbance at different wavelengths, the absorption spectrum of the leaf pigments can be obtained.

BIO 101 Lab 08: Photosynthesis v.2021 3 Materials:__________________________________________________________________ Experiment 1 • Full spectrum lamp • Large beaker • Elodea • Test tubes (3) • Bromothymol blue solution • Straw • Green film Experiment 2 • Spectrophotometer • Cuvettes • Leaf extract Safety: Follow all standard laboratory safety procedures. Do not inhale or ingest water or bromothymol blue solutions. Lab coats are not to be worn outside of the lab. Procedure:________________________________________________________________ Experiment 1. Observing photosynthesis (work in groups) 1. Add about 60 ml of tap water in a beaker and add 2 dropperful of bromothymol blue. Record the color in the “before exhaling” column of Table 1 of the Data Analysis section. 2. With a clean straw, gently blow into the solution until the color just turns yellow. Record the color in the “after exhaling” column of Table 1. 3. Fill three test tubes 2/3 full with this solution. 4. Place two 3-inch sprigs of Elodea into tubes 2 and 3. Be sure that the sprigs are completely immersed in the solution. Wrap tube 2 with green film. 5. Place the test tubes in front of the light. Allow the tubes to be exposed to the light for 1 hour. 6. Record the color of the solution in each test tube in Table 2 of the Lab Worksheet 7. Make a hypothesis about how the color of bromothymol blue solution in each test tube will change and record the color you expect for each tube in Table 2 in the “Expected Color after 1 hour of light exposure” column.

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BIO 101 Lab 08: Photosynthesis v.2021 4 When making a hypothesis, consider why the pH-indicating solution turned color in the first place and what might happen if the substance that caused the color change was removed by a plant performing photosynthesis. 8. Record the color you observe for each test tube in Table 2 in the “Observed color” column. 9. Recycle the elodea sprigs and wash the test tubes. Experiment 2. Absorption spectrum of leaf extract (work in groups) 1. Turn on the spectrophotometer and allow it to warm up for 15 min. 2. Insert blank cuvette in sample holder B and the cuvette containing leaf extract in sample holder “1”. Make sure the orientation of the cuvettes is right. 3. Press “nm” button and enter “400”, press “nm” button again to confirm wavelength selection. 4. Press button “B” to rotate and then “measure blank” button to calibrate the spectrophotometer. You should see the absorbance is set to 0. 5. Press button “1” and record the absorbance in Table 3. 6. Increase the wavelength by 25 nm and repeat steps 4 and 5. Keep doing this until the wavelength reaches 750 nm. Record absorbance readings in Table 3. 7. Complete Table 3 by filling in the color of light that corresponds to the grouped wavelengths. 8. Graph your data from Table 3 using a Line Graph. Be sure to include x-axis and y-axis labels and a chart title.

BIO 101 Lab 08: Photosynthesis v.2021 5 BIO 101 Lab 08: Photosynthesis Worksheet Name: __________________________ Section: ______________________ Data Analysis and Synthesis Questions: Table 1. pH-indicating dye color Color before exhaling into beaker Color after exhaling into beaker 1. Why did the solution in the beaker change color after you exhaled? Table 2. Observing Photosynthesis Tube Color before 1 hour of light exposure Expected Color after 1 hour of light exposure Observed color after 1 hour of light exposure 1 ( Elodea + green film) 2 ( Elodea ) 3 (no plant) 2. In this experiment , what is the purpose of the tube without the plant?

BIO 101 Lab 08: Photosynthesis v.2021 6 3. Explain the color change or lack of color change in the three experimental tubes: a. Tube 1: b. Tube 2: c. Tube 3:. 4. Table 3. Absorbance of Different Wavelengths of Light Color of Light Wavelength (nm) Absorbance Color of Light Wavelength (nm) Absorbance 400 0.58 575 0.09 425 0.82 600 0.12 450 0.61 625 0.16 475 0.44 650 0.23 500 0.22 675 0.53 525 0.08 700 0.04 550 0.06 725 0.05

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BIO 101 Lab 08: Photosynthesis v.2021 7 4. Make a Line Graph of the absorbance readings versus the wavelength of light data from Table 3. Be sure to include x-axis and y-axis labels and a chart title. Graph 1. Line Graph of Absorbance versus wavelength

BIO 101 Lab 08: Photosynthesis v.2021 8 5. Which wavelength(s) and colors of light are most effectively absorbed by leaf pigments (where are the peaks in your Line Graph)? 6. Which wavelength(s) and colors of light are poorly absorbed (where are the valleys in your Line Graph)? 7. Draw a general conclusion about which color(s) of light are best for plant growth based on your data from both the measurement of absorbance of light in leaf extracts and the observations of CO 2 use by Elodea cuttings in different light conditions. Instructor signature __________________________________

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