Investigating Photosynthesis with Algae (Part 1/2)

In today's lesson students will use the data from two experiments to determine the importance of light to photosynthesis. In the first experiment, students will use colored filters to determine which color of visible light causes the highest photosynthetic activity. In the second experiment, students will use paper chromatography to separate the different colors of plant pigments. This is the first day of a two day lesson. Here is an overview of what students will learn today.  

For this lab, each student group will need the following equipment: 

• computer
• Logger Pro
• Spectrometer
• cuvettes with lids (2)
• foil covered cuvette with lid (1)
• 100 W lamp
• stopwatch
• 600 mL beaker
• 250 mL beaker
• two small test tubes
• 5 mL pipet with bulb 
• eyedroppers (2)
• 10 mL DPIP/phosphate buffer solution
• unboiled algae culture
• boiled algae culture 
• ice

Students will complete a modified lab based on Vernier photosynthesis lab in which students use a spectrometer to determine the effect of light on photosynthesis. After learning how to do the protocol, students will compare the rates of photosynthesis for an algal culture in different light conditions.  

• Students should begin by obtaining two plastic eyedroppers, two cuvettes with lids, and one aluminum foil covered cuvette with a lid. 
• Students should mark one eyedropper with a U (unboiled) and one with a B (boiled). 
• Next, they should mark the lid for the cuvette with aluminum foil with a D (dark). 
• For the remaining two cuvettes, students should mark one lid with a U (unboiled) and one with a B (boiled). 
• Next, students should prepare a blank by filling a cuvette 3/4 full with distilled water and wipe the outside of the cuvette with a Kimwipe. 
• Then, students should calibrate the spectrometer.  
• After calibrating the spectrophotometer, students should obtain a 600 mL beaker filled with water and a flood lamp for each lab table. 
• Place the beaker between the lamp and the cuvettes. The beaker will act as a heat shield, protecting the algae from warming by the flood lamp. 
• Students obtain a sample of both the unboiled and boiled algae culture, resuspend the algae, and then add the solutions to the cuvettes using the correctly marked pipettes. (Note:  this culture needs to be prepared ahead.) 
• Students should place both cuvettes on ice.  
• Next, students should add 2.5 mL of DPIP/phosphate buffer solution to each of the cuvettes (Cuvette U, D, and B).  
• Students should then immediately add 3 drops of unboiled algae  to Cuvette U and 3 drops of unboiled algae to Cuvette D. 
• Students should add 3 drops of boiled algae into Cuvette B. 
• Gently mix all cuvettes, but do not introduce bubbles into the solution.  
• Place the cuvettes in front of the lamp marking its position so it can always be returned to the same spot.  
• Using the spectrometer, students should take absorbance readings for each cuvette.  Before taking any reading, students should invert each cuvette two times to resuspend the algae. 
• Once all readings are taken, replace the cuvettes in front of the lamp and turn on the lamp. 
• Students should take additional spectrophotometer readings after 5, 10, 15, and 20 minutes have elapsed.
• Students should record their findings in the data table provided. 

Making Sense of the Data

• Students should ppen the LoggerPro file, “07 Photosynthesis” on your computer. 
• Then they should manually enter the data from Table 1 into the correct column in LoggerPro. 
• Students should calculate the rate of photosynthesis for each of the three cuvettes by selecting the Linear Fit. This will perform a linear regression.  When the dialog box appears, select the three data sets (i.e.: unboiled, dark, and boiled). A box showing line of best fit will appear for each data set.
• Finally, students should record the slope of the line (m) in the table provided.  The slope is the rate of photosynthesis for each data set. 

Once the data has been analyzed, students should consider the following questions: 

• What evidence shows that the chloroplasts in the algae were able to reduce DPIP? 
• Were chloroplasts able to reduce DPIP when kept in the dark?  Explain.
• Were boiled chloroplasts able to reduce DPIP? Explain. 
• What conclusions can be made about the photosynthetic activity of algae?

based on: Redding, Kelly and David Mastermind. 2007. Photosynthesis. Biology with Vernier.  Vernier Software and Technology: Beaverton, OR

Students will use paper chromatography to separate a mixture of algal pigments.  

For this lab, each student group will need the following equipment

• 50 mL graduated cylinder
• cork stopper
• chromatography
• Filter paper strip
• pencil
• algae mat
• scissors
• coin
• solvent
• goggles
• ruler

Directions: 

• Students should obtain a 50 mL graduated cylinder.
• Students should add 5 mL of acetone to the bottom.
• Students should cut the chromatography paper so that it is long enough to reach the solvent. Then they should cut one end of the paper into a point.
• Students should draw a pencil line 2.0 cm above the pointed end of the paper.
• Students should use the coin to extract the pigments from the algal mat. Place a small section of the algal mat on top of the pencil line. They should use the ribbed edge of the coin to push the algal cells into the chromatography paper. Then they should repeat the procedure 10 times making sure to use a different part of the mat each time.
• They should place the chromatography paper in the cylinder so the pointed end just touches the solvent.
• Students should make sure the pigment is not in the solvent.
• They should place the cork on the cylinder and wait until the solvent is approximately 1 cm from the top of the paper.
• Then they should remove the chromatography paper and mark how far the acetone has travel before it evaporates.
• They should allow the paper to dry. 

Determining the distance each pigment traveled.

• After the paper dries, they should mark the bottom of each pigment band.
• Next, they should measure the distance each pigment moved from the starting line to the bottom of the pigment band.
• Students should then record the distance that each of the pigments and the solvent moved, in millimeters in the table provided

Determining the R_f value of each pigment

• Students should use the formula, R_f = distance traveled by pigment/ distance traveled by solvent to determining how far the pigment traveled. Each molecule has its own
• Finally, they should identify each of the bands and label them on the chromatography paper:  beta carotene (yellow to yellow orange), xanthophyll (yellow), chlorophyll a (bright green to blue green), chlorophyll b (yellow green to olive green).

After they complete their analysis, students should consider the following questions:  

• What types of pigments were found in the algal mat? 
• In what type of environment does algae thrive? 
• Considering the type of environment in which algae live, why would those pigments best help make food? Explain.

based on: Redding, Kelly and David Mastermind. 2007. Photosynthesis. Biology with Vernier.  Vernier Software and Technology: Beaverton, OR. 

Bring the class back together to discuss the results of the two labs. Using the information that they learned from both labs, student groups should design an experiment to test the effect of red, blue, and green light on the photosynthetic activity of algae.  They should write a lab proposal and submit it for approval by the end of the period. (Note:  I provide my students with an electronic copy of the protocol to save them time. They may email me their final draft.)

Student groups will conduct their experiment during the next class period.