25 Lab Protocol: Cellular Respiration and Photosynthesis

Each student group will design experiments to test the effect of the following ingredients on CO2 production by fermentation:

1. Yeast (provides the enzymes for glycolysis and ethanol fermentation, prepare by dissolving one packet of yeast in 500 mL warm water, use 5 mL)

1. 5% Glucose (see Figure 1, use 3 mL)

1. 3M Pyruvate (see Figure 1, use 3 mL)

1. 0.1M MgSO4 (an activator, use 5 mL)

1. 0.1M NaF (an inhibitor, use 5 mL)

Note: water will be used to equalize the volume of each reaction.

Note: determine the volume of liquid needed to fill the respirometer.

1. Looking at Figure 1 and the ingredient list, it appears that yeast and either glucose or pyruvate will be required for CO2 production. Explain the role of glucose, pyruvate, and yeast in CO2 production.

1. Design an experiment using experimental and control tubes to verify that these are the minimum ingredients.

1. Design an experiment with experimental and control tubes to determine if MgSO4 activates glycolysis or fermentation or both. What experimental results would convince you that fermentation but not glycolysis is activated by MgSO4?

1. Design an experiment with experimental and control tubes to determine if NaF is an inhibitor of glycolysis or fermentation or both. What experimental results would convince you that glycolysis but not fermentation is inhibited by NaF?

1. Carry out the experiments you designed in steps 2, 3, and 4. Measure CO2 production and record in a separate data table for each experiment. This table should indicate which ingredients were added as well as the CO2 yield. Also record how long you incubated the respirometers at 37 degrees Celsius.

Minimimal ingredients experiment:

Conclusions:

Measuring CO2 production in Fish by aerobic respiration

Aerobic respiration breaks down glucose to release 6 CO2 molecules. This CO2 can react with water to produce carbonic acid (H2CO3) which in turn can release bicarbonate (HCO3-) and hydrogen (H+) ions. Thus, the numerical decrease in pH of the fish water will correlate with the amount of CO2 produced and the rate of aerobic respiration.

We will use phenolphthalein as a pH indicator. It is colorless in acidic solutions and turns pink at or above a pH of 8. The lower the pH of the fish water solution, the more NaOH will be necessary to increase the pH to the point where phenolphthalein turns pink. It is important to note that phenolphthalein is very toxic to fish. Hence all glassware must be washed thoroughly to ensure the fish stays healthy and alive. Furthermore, fish do not do well in chlorinated tap water; hence, be sure to use Aqua Safe-treated water in this experiment.

1. Thoroughly clean glassware before use.

1. Measure 100 mL Aqua Safe-treated water twice and add to two washed beakers.

1. Use a net to capture a fish from the aquarium and gently place it into one of the beakers.  Cover the beakers with a petri dish. Start the timer for 30 minutes.

1. After the incubation time is complete, remove the fish and place it back into the aquarium.

1. Add 4 drops phenolphthalein to the 100 mL water in the control beaker (the one which did not have the fish).

1. Add 100 mL 2.5 mM NaOH, mix well, and look for a color change to pink.

1. Repeat step 6 as many times as needed counting the number of additions until the water stays pink after mixing. Record the amount of NaOH added in data table.

1. Add 4 drops phenolphthalein to the 100 mL water in the experimental beaker (the one which did have the fish).

1. Add 100 mL 2.5 mM NaOH, mix well, and look for a color change to pink.

1.  Repeat step 9 as many times as needed counting the number of additions until the water stays pink after mixing. Record the amount of NaOH added in data table.

Explain the results you obtained in this experiment.

Design a similar experiment in which you would test one of the following variables:

1. The effect of water temperature.

1. The effect of the type of organism: fish versus snail.

1. The effect of the length of incubation time.

You will not carry out this experiment.

Observe photosynthetic electron transport in leaves

The light reactions of photosynthesis include electron transport chains to pump hydrogen ions across the thylakoid membrane and to produce NADPH. The dye 2,6-dichlorophenol-indolephenol (DCPIP) can be used to measure the rate of electron transport.  DCPIP, in its oxidized state, is blue. After accepting electrons, it becomes colorless. When designing experiments measuring electron transport, it is important to maintain a constant pH since the rate of electron flow is pH dependent.

Assemble reaction tubes:

#Record the color of the contents of each tube before light treatment. Please also take a photograph of the 4 tubes.

*Cover tube 4 with tin foil to test for the effect of light on electron transport. Place tubes in rack equidistant from light source (~ 1 – 2 feet away, do not want tubes to get too hot). from light source to reduce the effect of high temperature.

@ Set the timer for 15 minutes. Record the color of the contents of each tube after light treatment. You may want to adjust the time depending on the rate of color change. Please also take a photograph of the 4 tubes.

Compare the results of tubes 3 and 4 to make a conclusion of the effect of light on the rate of electron transport.

Herbicides can target various aspects of photosynthesis. Suppose herbicide A inhibits the enzyme Rubisco which fixes carbon dioxide in the Calvin cycle, and herbicide B inhibits the transfer of electrons from plastoquinone A to QB during the light reactions of photosynthesis.  Plan an experiment to test the effect of each of these herbicides on electron transport using PCPIP.  Predict what you think the results of the experiment will be.  Explain your reasoning. You will not carry out this experiment.

Determine the absorption spectrum of a leaf extract

Leaves contain various pigment molecules to absorb sunlight and power photosynthesis.  Each type of pigment absorbs a specific spectrum of wavelengths. Thus, using a variety of different pigments increases the range of wavelengths that can contribute to photosynthesis. The pigment molecules are embedded in thylakoid membranes within chloroplasts; hence they are at least partially hydrophobic and will dissolve better in a nonpolar solvent such as acetone.

You will design and carry out an experiment to graph the absorption spectrum of the pigments from a particular species of plant.

1. Use a mortar and pestle to grind the leaves in acetone.

1. Filter out debris using cheese cloth.

1. Fill a test tube to take absorbance readings using a spectrophotometer. Cover top of tube with parafilm.

1. Fill a negative control tube and cover with parafilm.

1. Take readings at 20 nm increments starting at a wavelength of 380 nm and ending at 700 nm. Record readings on a data table.

1. Take another set of readings at the same wavelengths and record on the data table.

1. Calculate the average of each pair of absorbance readings and record on data table.

1. Construct a graph to visualize the absorption spectrum. Label the graph with a title and label the X and Y axes of the graph.

Questions:

1. What should be used to fill the negative control tube?

1. What is the purpose of the negative control tube?

1. How similar are the two readings at a given wavelength?

1. Which wavelength gave the highest absorption reading?

1. Which wavelength gave the lowest absorption reading?

1. Which wavelength of light contributes the most towards photosynthesis?

1. Explain the reasoning for your answer to question 6.

1. Compare your results to the results of another group.