Cellular Respiration Lab

Photosynthesis and Respiration
Objectives:
In this activity we will review the process of photosynthesis and cellular respiration, and become familiar with the use of a pH indicator for investigating these processes. Following this activity your group will generate a new research question about photosynthesis or respiration, and utilize the pH indicator system for the experiment that your group will design, implement and interpret.
Introduction:
Photosynthesis is the process by which plants and some other organisms convert solar energy to chemical energy in the form of sugars. In this reaction, the energy from sunlight is harnessed and utilized to form sugar molecules from carbon dioxide and water, forming oxygen gas as a byproduct.
Photosynthesis: 6 CO2 + 6 H2O + sunlight ---> C6H12O6 + 6 O2
While photosynthesis creates sugar with the sun's energy, cellular respiration utilizes the chemical energy in sugar to meet the energetic needs of organisms. During respiration, sugar is combined with oxygen, resulting in energy and carbon dioxide. Note that the equation for respiration is the opposite of photosynthesis, indicating the products of photosynthesis become reactants in respiration, and vice-versa.
Respiration: 6O2 + C6H12O6 -----> energy + 6CO2 + 6H2O
While only plants and select other organisms are capable of photosynthesis, all organisms (plants included) carry out respiration to convert their food (sugar) to the energy needed to power the wide variety of biochemical reactions that are necessary to sustain life. After all, plants are not creating sugar from carbon dioxide and sunlight for you, they're making it for themselves, and you're simply taking advantage of the process by consuming the biomass the plant produces through photosynthesis. Many students incorrectly uniquely associate photosynthesis with plants, and respiration with animals. It is important that you realize that plants carry out both respiration and photosynthesis, as this information will be useful during your lab analysis.
If you think about the gases inhaled and exhaled by humans, the process of respiration should be easy to remember. We breathe in oxygen and exhale carbon dioxide because we are constantly carrying out cellular respiration. In our lungs, oxygen from the air enters your blood and is carried to cells throughout your body where it is used for cellular respiration. The waste product of respiration, carbon dioxide, is carried back to your lungs by your blood, and then exhaled into the environment.
Photosynthesis and Respiration: Bringing an abstract concept to life
So let's take a minute and recap. You know that plants carry out photosynthesis to create sugar, and that all organisms use sugar to fuel cellular biochemical reactions through the process of cellular respiration. Not all of the sugar created by the plant is used up in respiration though, as some is used to create new plant biomass like roots, leaves, stems, wood, and bark. The carbon in plant biomass is only stored temporarily, as it will return to the atmosphere when the biomass decomposes, burns, or is eaten and metabolized.
Up to this point you have read about the abstract concepts of photosynthesis and cellular respiration and, being the trusting souls that you are, accepted these ideas without ever having observed them. In today's lab you will complete exercises that will show you both of the processes in action, in under an hour's time. These exercises will help you to make an abstract concept more concrete, and aid you in understanding the role of plants and other organisms in the cycling of carbon.
Carbon dioxide and pH
In the first exercise, you will investigate photosynthesis with Elodea, an aquatic plant. In the second exercise, you will examine cellular respiration with seeds of the mung bean (Phaseolus aureus). In each of these cases, we will follow the movement of carbon dioxide between a plant or seed and the water that surrounds it. So we need to have some way of observing changes in carbon dioxide concentrations in water. One way to do it would be to put radioactive carbon into the water, add the plants/seeds, allow them to interact with water for 45 minutes or so, and then compare the amount of radioactivity in the water and in the plants/seeds. While this is the method used by scientists who study photosynthesis, the potential lawsuit implications are staggering, and this won't be mentioned again. Another way would be to use sensors or processes to directly measure carbon dioxide concentrations in the water, but this is too costly and/or complicated for our purposes. pH and gas exchange
To understand this relationship, imagine a ruler with pH on one end and carbon dioxide concentrations on the opposite end. Note that if you raise the concentration of carbon dioxide in the water (by raising that end of the ruler), pH declines. If you decrease the amount of carbon dioxide in the water, pH increases. As carbon dioxide uptake is one way to measure photosynthesis rates, and changes in carbon dioxide concentrations cause changes in pH, we can use pH changes as an indirect measure of photosynthesis rates.
If we place a plant in water with dissolved carbon dioxide and oxygen, the plant will remove carbon dioxide from the water as it photosynthesizes, reducing carbon dioxide concentrations in the water, and raising the pH of the solution. If we place a plant or seed in the water and it undergoes respiration, it will release carbon dioxide into the water, and lower the pH of the solution. So by monitoring the changes in the pH of a solution, you can determine if the plant or seed in the solution is photosynthesizing (pH goes up) or respiring (pH goes down). This relationship between pH and photosynthesis/respiration is key to today's lab, so be sure you understand it completely.
By now, you see that we will be observing the processes of photosynthesis and respiration by observing changes in the pH of solutions containing aquatic plants or seeds. So how do we measure pH? There are a variety of methods available, but we will be using a method commonly used in laboratory exercises like this one - bromothymol blue. Bromothymol blue is a pH indicator solution that is blue when its pH is basic (and carbon dioxide concentrations are low) and yellow when its pH is acidic (and carbon dioxide concentrations are high). Figure 3 shows these color changes as a function of solution pH. Note that the solution is blue when basic, greenish when neutral, and yellow when acidic.
In today's lab you will be examining changes in solution pH to indirectly measure carbon dioxide concentrations in solutions containing Elodea plants and mung bean seeds. By looking at changes in carbon dioxide concentrations over time, you will be able to observe firsthand the processes of photosynthesis and respiration.

Figure 3: Bromothymol blue color change with pH pH 2 is yellow, pH 6 is green, pH 10 is blue
Procedures: Photosynthesis
In the first exercise, you will examine the processes of respiration and photosynthesis with the aquatic plant, Elodea. Elodea is commonly found in freshwater lakes and ponds, where it floats in the water column. Because it does not require a root system to survive, Elodea is commonly used in freshwater aquaria and can be purchased at pet stores. When exposed to sunlight, Elodea will begin photosynthesizing and will remove carbon dioxide from the solution. The plant will respire constantly regardless of light conditions, as its cells always need energy.

Photosynthesis Exercise:
1.
Place about 75 ml of bromothymol blue into an 125 ml Erlenmeyer flask (the triangular glass containers at your work bench) and return to your workstation.
2.
Observe the color of the solution. The next step is to introduce carbon dioxide into the solution. We could do this by getting a carbon dioxide tank like those used for soda dispensers, and have you carbonate your solution, but that would be overkill. Luckily, there is a ready source of cost-free carbon dioxide within you right now - your lungs. Use a straw from Station 1 to slowly blow carbon dioxide from your lungs into the solution until it just turns yellow.
3.
Carry the solution to the front board and compare its color to the printout showing the color of bromothymol blue at various pH's. Find the color on the printout that best matches the color of the solution. If your color is between the colors shown, then use the pH value that is intermediate between them. Record the color of the solution (Initial color) and estimated pH (Initial pH) for each of the tubes in Table 1. When comparing or describing colors, be sure to hold the tubes in front of a white background and be as detailed as possible when describing the color (e.g., use terms like "greenish-blue" or "yellow-green").
4.
Pour the solution into three screw cap tubes, dividing it fairly evenly. Obtain 3-inch pieces of Elodea from the open dish at Station 1, place it in one of the tubes, and cap it. Obtain two 3-inch pieces of Elodea from the covered dish at Station 1, place it in a second tube covered with contact paper (to prevent the entry of light), and cap it. In both tubes, be sure the plant is completely submerged and the tube is full of the indicator solution. Cap the test tube that has no plant in it. Place the test tubes into a test-tube rack and put the rack about 8 to 10 inches from a lamp so that all three tubes are the same distance from the lamp. While you allow the experiment time to run, go ahead and set up the respiration experiment described on the next page.
5.
Allow the plants to sit undisturbed for 50 minutes, and then compare the colors of the solutions by removing the plants and holding the tubes in front of a white background. Record the Final colors of all three tubes in Table 1, and estimate the pH for each as described above. After all measurements have been completed, return the Elodea to Station 1, putting it in the dish marked "Used Elodea". Rinse out your glassware.
Procedures: Respiration
In the second exercise, you will examine the process of respiration with seeds of the mung bean (Phaseolus aureus). One problem with studying respiration in plants is that they will begin photosynthesizing when in the presence of sunlight, and this will counteract the increase in carbon dioxide concentrations we are attempting to measure.
As you saw in the first exercise, one way to measure respiration is by placing a plant in the dark. In this exercise, we'll use a slightly different approach. Seeds are immature plants that cannot yet photosynthesize, but are capable of respiring. By using seeds we can look at respiration in this group of organisms, and avoid conflicts with photosynthesis when interpreting our results.

Respiration Exercise:
1.
From Station 2, obtain three jars and three jar covers. One jar should be empty, one partially filled with germinating seeds, and the other should contain an equal number of non-germinating seeds. (Our germinating seeds have been soaked in water for about 48 hours or so).
2.
Rinse out all jars (without pouring out the seeds) to ensure that each has a fresh supply of air, then partially fill each equally with bromothymol blue. The liquid level should be just above the top of the seeds. Record the initial color and estimated pH of the solutions in Table 2.
3.
Cover the jars and set aside in the same place for 45 minutes, making sure that the two jars are in similar conditions.
4.
Filter the seeds out of the jars by pouring the solution through the provided strainer into a separate container. To control all your variables, do the same for the solution with no seeds (even though there is nothing to filter out). Compare the color of the solution in each of the jars against a white background. Record the final color and estimated pH of the solutions in Table 2.
5.
Return the seeds to Station 2 in a jar.
Case Study: Deforestation
Well, here we are. You're sitting at the table waiting for your experiments to conclude and you've got some time to kill. Let's use this time to investigate the intricate relationship between vegetation and climate change through an online case study.
By now you've seen that vegetation can have a profound influence on the cycling of carbon, and thereby on the regulation of global climate. The current increases in atmospheric carbon dioxide concentrations are thought to be caused by the combustion of fossil fuels and by the rapid rates of deforestation currently occurring in tropical forests. By adding carbon dioxide to the atmosphere and destroying the ability to remove it through photosynthesis, this combination of fossil fuel combustion and deforestation may be drastically impacting the systems that affect planetary climate.
So why are tropical forests declining? These forests are often cleared by peasant farmers with a technique called "slash and burn agriculture" in which the farmers cut all of the vegetation on a plot of land, then burn it to clear the vegetation and fertilize the soil. This process not only removes vegetation that could reduce atmospheric carbon dioxide levels through photosynthesis, but also adds carbon dioxide to the atmosphere when the vegetation is burned.
Land area occupied by tropical forests has been declining since 1800, and deforestation rates have accelerated in recent decades. In this section, you will examine a case study on tropical deforestation that illustrates the complex nature of this issue. Deforestation is influenced by a variety of factors, particularly the elevated rates of poverty in the developing world, and the case study will help you to understand the process.
The case study was created by Phil Camill of Carleton College in Minnesota for the National Center for Case Study Teaching in Science. Read sections I and II, and then complete the questions on the assignment sheet. If you are unable to load or complete the case study while in lab, you should complete it at a later time during the week.
Case study URL: http://ublib.buffalo.edu/libraries/projects/cases/amazon.html
Lab developed by Matt Laposata, SCI 1101: Interdisciplinary Science course materials, Kennesaw State University.

Student Activity Sheet
Name:_________________________________

Photosynthesis:

What is your hypothesis for the photosynthesis experiment? Remember that you are investigating the effects of light (presence or absence) on pH change in solutions with Elodea.

Data Table 1:

Tube
Initial color
Initial pH
Final color
Final pH

Elodea - light

Elodea - dark

No Elodea

Interpret your results. Evaluate your hypothesis citing data as appropriate. Be sure to explain the results for each of the three tubes, and relate them to CO2 exchanges between the Elodea plant and the indicator solution.

Respiration:

What is your hypothesis for the respiration experiment? Remember that you are investigating the effects of germination (presence or absence) on pH change in solutions with seeds.

Data Table 2:

Tube
Initial color
Initial pH
Final color
Final pH

Germinating seeds

Non-germinating seeds

No seeds

Interpret your results. Evaluate your hypothesis citing data as appropriate. Be sure to explain the results for each of the three containers, and relate them to CO2 exchanges between the seeds and the indicator solution.

On-line Case Study

Answer the questions below completely and thoughtfully, citing information from the case study for each. Brief, incomplete answers are not acceptable, so state your opinions and their justifications fully.

(a) Is it hypocritical for developed countries like the United States to criticize the exploitation of natural resources in third world countries when our country did exactly the same thing (westward expansion, Oklahoma land rush) to fuel our country’s economic growth in years past?

(b) Some economists have argued that given the current poor financial status of many third world countries, the only way to protect tropical forests is for developed countries to pay countries like Brazil not to destroy their forests. Given the global environmental benefits provided by these ecosystems, would you support the U.S. government paying countries to preserve their forests? Given that these funds would come from tax dollars, how much would you be willing to pay on an annual basis to conserve tropical forests? Ask this question of five family members, friends, or acquaintances. What were their answers? Were you surprised by their answers?

Design a new experiment:

Now that you are familiar with the Bromothymol Blue, Elodea and seeds experimental system, you are able to generate new questions that allow you to experiment, gather evidence, and make sense of your results.

What are some different aspects of photosynthesis and cellular respiration that we might explore? Develop some focus questions and hypotheses for both processes. As a class, we will develop one or two photosynthesis questions to investigate, and one or two respiration questions to investigate. Two or three lab groups (of 2-4 student each) will conduct the same experiment, allowing for comparison of results to confirm any possible patterns in the data. Once we generate our experimental questions and hypotheses as a class, groups will design the experiments (with teacher guidance), interpret the results, and explain findings to the class.