LAB #6 – Photosynthesis and Cellular Respiration Introduction In order to survive, organisms require a source of energy and molecular building blocks to construct all of their biological molecules. The ultimate source of energy for almost all of life on Earth is the light that comes from the sun (see the box on the next page for an example of organisms that do not depend on light as the ultimate source of energy). Photosynthesis and cellular respiration are two of the most important biochemical processes of life on Earth. Both are a series of reactions that are catalyzed by unique enzymes at each step. Although it is somewhat of an oversimplification to describe them as “opposite” sets of reactions, for introductory purposes let us examine them as such. Photosynthetic (“light” “forming”) organisms are those that can take simple molecules from the environment such as carbon dioxide (CO2) and water (H2O), and using the energy of the sun, create their own biological macromolecules such as carbohydrates, proteins, lipids and nucleic acids. You will note that the reactions of photosynthesis are both endothermic and anabolic, in that they require energy and use small molecules to make larger ones. These reactions take place in the chloroplasts of plant cells. We generally summarize the series of reactions of photosynthesis in terms of the initial reactants and the final products - leaving out details of all the reactions in between. In introductory biology, we simplify what is happening by showing only the monosaccharide glucose as the ultimate organic molecule that is produced.
Simplified 6 CO2 + 6 H2O carbon water dioxide
sunlight
C6H12O6 glucose
+
6 O2 oxygen
In reality, the products of photosynthesis include the formation of all of the biological macromolecules the organism requires. In addition, photosynthetic organisms must have a source of nitrogen (e.g. fertilizer) to make its proteins and nucleic acids. In this lab, we will use the simplified equation above for our discussion.
Actual sunlight 6 CO2 + 6 H2O + (N source) carbon water dioxide 1
carbohydrates, proteins, lipids, nucleic acids
+
6 O2 oxygen
You will note that one of the products of photosynthesis is oxygen. Essentially most of the oxygen in our atmosphere comes from the process of photosynthesis. Like photosynthesis, cellular respiration is also a series of chemical reactions. However, rather than requiring energy, this process involves the breakdown of molecules (e.g., glucose) releasing energy that can be used for any energy-requiring process in a cell. Thus, we call these reactions catabolic and exothermic. These reactions occur in the cytoplasm of bacteria and the mitochondria of eukaryotic cells (including plants!). Ultimately, the energy released from these reactions is used to form molecules of ATP, which can be described as the “energy currency” in the cell. Just as our country uses dollars as a means to transfer wealth, cells use molecules of ATP as a means to transfer energy from the reactions of cellular respiration to other reactions in the cell that require energy. In essence, cells convert the energy derived from a molecule like glucose (e.g., a savings bond) into more useable molecules with energy called ATP (e.g., dollar bills that can actually be spent). In addition, since larger molecules such as glucose are broken down into the smaller molecules carbon dioxide (CO2) and water (H2O), this is catabolic process. Once again, for introductory purposes, we summarize the many chemical reactions of cellular respiration in one simple, overall equation: C6H12O6 glucose
6 CO2 + ▼ carbon dioxide energy ▼ ADP + Pi ATP
+ 6 O2 oxygen
6 H2O water
Although many cells prefer to use glucose as the primary molecule for cellular respiration, they can also use other carbohydrates, fats and proteins as a source of energy production when needed. Note again that one of the products of cellular respiration is carbon dioxide.
Deep sea organisms thrive in the absence of any light source In 1984, scientists made one of the most amazing discoveries in the history of science – organisms that have evolved next to deep ocean volcanic vents that use chemical energy rather than sunlight as the basis of life. These organisms are known as chemoautotrophs (“chemical” “self” “feeding”). This discovery led to the hypothesis that such forms of life may be present on other planetary bodies in our solar system or other parts of the universe!
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Part 1: Observing and Measuring Photosynthesis We have learned that photosynthesis involves the conversion of carbon dioxide and water into organic molecules such as glucose. In doing so, oxygen is a product while carbon dioxide is a reactant that is used up during photosynthesis. In this experiment, we will be using the same plant you examined in Lab 3 called Elodea. The experimental set-up involves a qualitative measurement of the CO2 concentration in the vials. The variables to be examined in relation to carbon dioxide use are the amount of light exposure and various colors of light. The pH indicator bromothymol blue is used to estimate the amount of CO2 present in the vials. When CO2 concentrations increase in aqueous solution, it causes an increase in the concentration of H+ ions, thus decreasing the pH value. This occurs through the formation of an intermediary compound called carbonic acid, which forms by the combination of CO2 and H2O as shown here: CO2
+
H2O
< ===== >
H2CO3 carbonic acid
< ====== >
H+ +
HCO3bicarbonate
Bromothymol blue is yellow under acidic conditions, that is when the pH of the solution is less than 7 (e.g. pH = 6).This occurs when the concentration of CO2 is high. When there is little or no CO2 in the solution, the pH will be ~7.6 and the bromothymol blue will actually be blue. Picture the relationship of pH to CO2 concentration being on a meter stick. The higher the concentration of CO2 - the lower the pH. The lower the concentration of CO2 - the higher the pH. Lower pH
Higher pH
Higher CO2 Level
Lower CO2 Level
YELLOW
BLUE
Exercise 1 – Observing and Measuring Photosynthesis as CO2 is Consumed 1. Label 5 vials #1, 2, 3, 4 and 5 with a marker and line them up in order in a test tube rack. 2. Obtain 4 pieces of Elodea about 6 cm in length. 3. Obtain 2 pieces of aluminum foil and one colored plastic light filter. 4. In order to create a solution that is rich in CO2 for photosynthesis, fill a 250 ml Erlenmeyer flask to the 150 ml line with tap water. 3
5. Put 5 ml of bromothymol blue solution into the flask. 6. Take a clean straw and blow bubbles carefully into the solution until it turns a distinct yellow. Note: the yellow color demonstrates an increasing level of CO2. 7. Place a 6 cm Elodea stem with leaves in vials #1, 2, 4 and 5. Vial #4 will not have any Elodea. 8. Fill vials #1, 2, 3 and 5 with this CO2 enriched solution containing bromothymol blue. 9. Fill vial # 4 with tap water and add one dropper full of bromothymol blue. 10. Wrap a piece of aluminum foil around vials #2 and #4. 11. Select one piece of colored plastic filter and wrap it around vial #5 and secure with a rubber band. 12. Tightly screw the caps on each vial. Turn the vials upside down in the rack so the Elodea has more light exposure. This chart summarizes the contents of each vial:
CO2 Enriched Solution with b.b. H2O with b.b. Elodea stem with leaves Aluminum Foil (to block light) Light Filter (red, green or blue) b.b. = bromothymol blue
Vial 1 X
Vial 2 X
X
Vial 3 X
X X
Vial 4 X X X
Vial 5 X X X
13. Expose vials #1, 3 and 5 to the light provided. Note: vials #2 and #4 are covered with foil and therefore will not receive any light. ► Record the starting color and time in the chart on your worksheet. ► Allow the experiment to run for two hours. After that time, record the colors of the five vials on the data chart on your worksheet. ► Answer the questions related to this experiment on your worksheet.
Move on to the next experiment while this experiment continues… 4
Part 2: Chromatography - Isolation of Plant Pigments A pigment is a molecule that absorbs light. White light contains all of the different colors of the visual spectrum. This can be observed in a simple rainbow during a rain storm or by using a prism that splits white light into its various colors. Why does a shirt appear red? The red shirt has a pigment molecule that we call a dye that absorbs all of the other colors of the visible spectrum (blue, green, yellow, etc,), but reflects back the red waves of light. In plants, there are two categories of pigments used for photosynthesis: primary pigments and accessory pigments. The chlorophylls are the primary pigments of photosynthesis, with two types called chlorophyll a and chlorophyll b. The chlorophylls are green pigment molecules. What does this mean? Chlorophyll absorbs blue, red, orange, yellow, etc.…...light, but reflects green light. On the other hand, the accessory pigments are red, yellow or orange – they absorb all of the other colors. You can see these colors on trees in the northern states, and locally as well, in the fall before they drop their leaves. They serve to broaden the spectrum of light absorption in plants and they protect the plant from harmful or excessive rays of sun. Chromatograhy (“color” “measure”) is a technique that allows us to separate different molecules from a mixture based on differences in solubility. Some compounds do not like to dissolve in water. These are called hydrophobic (“water” “fearing”) compounds. On the other hand, some molecules are hydrophilic (“water” “loving”), meaning they like to dissolve in water. You should note that these properties are not absolute. For example, it is possible for one compound to be slightly hydrophobic and a different compound to be extremely hydrophilic. The golden rule for solubility is: “Like dissolves in like.” In other words, a hydrophilic compound will be more soluble in a liquid that is also hydrophilic. Likewise, a hydrophobic compound will be more soluble in a liquid that is hydrophobic. Chromatography is a method of separating and isolating molecules based on their level of hydrophobic or hydrophilic properties. In paper chromatography, we create a “molecular race track” in which molecules move through a piece of filter paper, carried along by a wave of liquid solvent. Those pigment molecules that have the highest solubility in the liquid solvent used will be “carried along” through the paper the fastest. Those pigments that are least soluble in the solvent will move more slowly or not at all. The various plant pigments have differing degrees of hydrophobicity. Therefore, if we use a liquid solvent that is hydrophobic, different plant pigments will move at differing rates through the piece of paper as the liquid solvent is absorbed upward. In this way, individual pigments can be separated into bands on the filter paper. In this experiment, you will use paper chromatography to separate the plant pigments from a plant with a green leaf (spinach) or one with a red leaf (Coleus) using a hydrophobic ether-based solvent. 5
Exercise 2 – Separation of Plant Pigments Using Chromatography 1. Obtain a mortar and pestle in order to grind the leaves and extract the leaf pigments (your instructor will assign spinach or Coleus to each group). 2. Place a small number of leaves in the mortar and add 2 ml of the solvent (60% isopropanol and 40% acetone) to the mortar. 3. Use the pestle to grind the leaves in the solvent. You should observe the formation of a liquid in the bottom of the mortar that contains the plant pigments. 4. Acquire a rectangular piece of filter paper. Use a pencil to very lightly draw a line 0.5 cm above the short side of the filter paper. 5. Dip a small glass capillary tube into the pigment extraction solution at the bottom of the mortar to collect some of the solution. 6. Repeatedly “dab” the capillary tube with pigment solution gently across the filter paper just above the pencil line. The intention is to form a thin line or band of pigment solution that spans the width of the paper. Allow the first line to dry and then repeat this procedure three more times to concentrate the pigments along the paper. 7. Once the pigment has been properly applied to the paper, curl the paper into a cylinder so that the line of pigment is at the bottom. Use a stapler to hold the cylinder in place. 8. Acquire a 100 ml beaker. Add 2 ml of the chromatography solvent (ether:acetone 95:1) to the beaker. Note that this solvent is volatile (will evaporate quickly) and can be caustic to the eyes and nose. (Note the picture below.) 9. Move the beaker to the ventilation hood so that the vapors can be isolated from the rest of the room. 10. Carefully set the filter paper cylinder, with the line of pigments at the bottom, into the solvent at the bottom of the beaker. Make sure that the paper sits flatly on the beaker surface and so that the solvent is below the pigment line. 11. Allow the beaker to sit in the hood until the highest band of pigments rises to within 1 cm of the top of the filter paper which should take about 15 – 30 minutes. 6
► On your worksheet, draw the lines of pigments you observe on the filter paper and label the color of each line.
Move on to the next experiment while this experiment continues…
Part 3: Observing and Measuring Fermentation Cellular respiration involves numerous chemical reactions that together break down molecules (e.g. glucose) into CO2 and H2O in order to release the energy in their chemical bonds. As a result of these reactions, the energy is “captured” by the formation of molecules of ATP. The ATP molecules are then used by the cell to facilitate any energy-requiring process. As mentioned previously, it is like cashing in a “savings bond” for “dollar bills” that can then be used to purchase goods and services. The reactions of cellular respiration are commonly grouped into three processes: 1) Glycolysis; 2) The Citric Acid Cycle; 3) Electron Transport and Chemiosmosis. Interestingly, the reactions of glycolysis do not require oxygen to proceed. We say that this is an anaerobic (“not” “oxygen”) process. Even in the absence of oxygen, these reactions can produce CO2 in some organisms and very small amounts of ATP. (Oxygen is required for the reactions of the Citric Acid Cycle, Electron Transport and Chemiosmosis to occur, thus we call them aerobic processes.) You may be more familiar with the use of the terms aerobic and anaerobic in regard to exercise. When a person is doing aerobic exercise, the lungs and circulatory system are able to supply enough oxygen to more adequately meet the ATP demands of the muscles. This is because the cells can use all three stages of cellular respiration, with oxygen present, and produce vastly more ATP per glucose molecule. This would be like a simple jog down the street. However, when a person is doing anaerobic exercise, the lungs and circulatory system are unable to meet the heavy oxygen demands of the muscles during very rigorous activity. The CO2 levels in the body rapidly increase and the muscle cells produce a compound called lactic acid which causes the muscles to fatigue more quickly. This process is called lactic acid fermentation and occurs only in animal cells. You can experience this if you try to run as fast as you can – you will tire quickly – as your body at some point will be unable to keep up with the demand for oxygen in your muscles. In other organisms such as yeast, without the presence of oxygen, the cells will also use glycolysis – but they will produce CO2 and ethanol instead. This process is known as alcohol fermentation. You may be familiar with this in the production of beer (the 7
bubbles are “carbonation” or the production of CO2) and the rising of bread (the gas causing the rising is CO2). In this experiment, we will investigate alcohol fermentation in yeast under different conditions and measure the production of CO2. Exercise 3A – Observing and Measuring Alcohol Fermentation in Yeast 1. Obtain three saccharometers (as shown on the right) which you will use to set-up three different conditions to investigate alcohol fermentation in yeast (with subsequent production of CO2). 2. Use the following table to mix the proper amounts of water, yeast solution and corn syrup in small beakers. Be sure to add the water and corn syrup to each tube first – THEN the yeast last. This will allow the reactions to begin at approximately the same time. 3. Once complete, transfer each mixture to a labeled saccharometer, gently moving it side-to-side, so that no air is trapped inside the top of the tube and begin the timing of your experiment.
Water Yeast Corn syrup
Tube #1 12 ml 0 ml 6 ml
Tube #2 9 ml 3 ml 6 ml
Tube #3 6 ml 6 ml 6 ml
► Write the start time on your worksheet. 4. As the reactions proceed, use a small metric ruler to measure the amount of carbon dioxide gas that is produced and collecting at the top of the tube (in mm). ► Make measurements every four minutes for twenty minutes (or shorter if it begins to overflow) and record your data on your worksheet. ► Draw a graph using your data. ► Answer the questions on your worksheet.
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Recall that carbon dioxide, when dissolved in water, causes the H+ concentration to increase, thus causing the pH value to decrease. As mentioned previously, this is due to the formation of carbonic acid. In this exercise, we will use a technique employed in an earlier lab to investigate the concentration of carbon dioxide as a person exhales at rest and then after moderate-torigorous exercise. A solution with a pH indicator called phenol red is placed in an Erlenmeyer flask. Phenol red solution is reddish-pinkish under neutral to basic conditions. As the level of CO2 increases and the H+ concentration subsequently increases, the solution will turn yellow. When ready, the subject will exhale through a straw into the solution, introducing carbon dioxide, as done in a previous activity in this lab. When enough carbon dioxide is delivered, the phenol red solution will become sufficiently acidic to change color from red-pink to yellow. Thus, phenol red will gradually turn yellow as more CO2 is introduced. Read all of the instructions below before beginning this exercise.
Exercise 3B – Examining Carbon Dioxide Before and After Exercise
Exhaling at Rest 1. Split 40 ml of diluted phenol red solution equally into two 50 ml Erlenmeyer flasks. There should be 20 ml of phenol red solution in each. 2. While at rest, have a student blow through a straw into one of the phenol red solutions. Use a clock or a stop watch to measure the amount of time required for the solution to turn completely yellow. ► Record the amount of time on your worksheet.
Exhaling after Exercise 1. Have the same student from above perform three minutes of moderate-to-heavy exercise. 9
2. Immediately upon completion of the exercise, have the student blow through a straw into the other phenol red solution. Use a clock or a stop watch to measure the amount of time required for the solution to turn completely yellow. ► Record the amount of time on your worksheet and answer the related questions.
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Laboratory 6: Photosynthesis and Cellular Respiration Name: ____________________________ Group: ___________
Date:_______________
Exercise 1 Results of the Photosynthesis Experiment Start time: ________________
End Time: _______________
Vial 1
Vial 2
Vial 3
Vial 4
Vial 5
Color at Start of Experiment Color at the End of the Experiment Was there a color change? Did Photosynthesis take place? If you need to do so, reread the introduction to this experiment before answering the following questions. What does a color change from blue to yellow indicate about a change pH and carbon dioxide level in the vial?
For the following questions, refer to the chart in the lab manual showing the contents in each vial. What happened with the color in vial #3? Explain these results based on its contents and light exposure.
What happened with the color in vials #2 and #4? Explain these results based on its contents and light exposure.
What happened with the color in vial #1? Explain these results based on its contents and light exposure.
What happened with the color in vial #5? Explain these results based on its contents and light exposure.
Exercise 2 Use a ruler to measure the distance the pigment lines moved from the starting line. Draw a picture of your chromatography paper in the box below
Exercise 3A Results of the Fermentation Experiment Start time: ________________
End Time: _______________
Measurement of CO2 Production (mm of gas at the top of the saccharometer)
0 minutes
Tube 1 Tube 2 Tube 3
4 minutes
8 minutes
12 minutes
16 minutes
20 minutes
On the grid provided below, graph the results of CO2 production from the table by plotting the amount of gas on the y-axis (mm) and time (minutes) on the x-axis. Use different colors for each of the different tubes (or dotted lines, etc.). Label both axes appropriately including the proper units.
If you need to do so, reread the introduction to this experiment before answering the following questions. Also, refer to the chart in the lab manual showing the contents in each vial. Was there a difference in the production of CO2 in the test tubes? Explain.
Exercise 3B Time required for phenol red to turn yellow
Minutes to color change Before Exercise After Exercise Was there a difference in time? Explain.
