Carbon Dioxide Uptake and Production in
Photoautotrophic Elodea and Heterotrophic Goldfish
BC1001 Revolutionary Concepts in Biology
LAB 3, September 27-October 1, 2004
This week in lab you will use the scientific inquiry method to design and test a hypothesis. In lecture you have learned about autotrophic and heterotrophic organisms; in this lab you will measure aerobic respiration and photosynthesis. You will use a goldfish as an example of a heterotroph and Elodea as an example of an autotroph (the same aquatic plant whose leaf cells you examined under the microscope in lab 2). Please remember that while only the plant carries out photosynthesis, BOTH the PLANT and the ANIMAL carry out aerobic respiration in their mitochondria.
Summary equation for aerobic respiration (animals and plants)
C6H12O6 + 6 O2 à 6 CO2 + 6 H2O + ATP + Heat
Summary equation for photosynthesis (plants)
6 CO2 + 6 H2O + light energy à C6H12O6 + 6 O2 + Heat
Note that carbon dioxide is produced by aerobic respiration and consumed by photosynthesis. In this experiment, you will be indirectly measuring amounts of CO2 in an aquatic system—allowing you to monitor respiration and photosynthesis. The goldfish will be producing CO2 through aerobic respiration, but the Elodea will be both producing CO2 through aerobic respiration and consuming CO2 through photosynthesis.
CO2 reacts with water to form carbonic acid through the following reaction:
CO2 + H2O à H2CO3
In your experiments in lab this week, you will use a pH indicator (phenolphthalein) to detect changes in pH resulting from the production and consumption of carbonic acid (and thus CO2). Phenolphthalein is colorless in acidic solutions and pink in basic solutions. You will measure the volume of NaOH necessary to neutralize the carbonic acid produced by CO2; this is an indirect method that allows you to calculate the amount of CO2 produced or consumed.
Scientific Inquiry Method
Scientists use the scientific inquiry method to further scientific knowledge. The steps in this method are the following:
make observations
ask question
develop hypothesis
make prediction
design experiment
collect data
analyze data
draw conclusions
The scientific method begins with the process of making observations. From these observations, you ask a question about what you see. Next, you develop a hypothesis, often defined as an educated guess to answer that question. You can make a prediction based on whether or not your hypothesis is true, and then design and carry out an experiment to test your hypothesis and prediction. Data (remember that “data” is plural, “datum” is singular) must be collected and analyzed. From the results of your experiment, you can draw conclusions about whether your hypothesis is or is not supported. You will use all the steps in the scientific method in lab this week.
Please note that you can NEVER “prove” a hypothesis to be true, you can only have your hypothesis supported by data. NEVER, EVER use the word “prove” in a scientific setting. Really, NEVER.
Preliminary experiments
AUTOTROPHS VS. HETEROTROPHS
Living things obtain their energy in a variety of ways. Some organisms are able to produce energy from sunlight or other environmental sources. Of the organisms that produce their own energy, the autotrophs, you are probably most familiar with the green plants. Other organisms, heterotrophs, cannot produce their own energy; they must obtain it from other organisms. There are several ways of making a living as a heterotroph. You can be an herbivore and obtain nutrients by eating living plant matter. You can be a carnivore and obtain energy by eating other animals. Or, you can be a saprophyte and obtain nutrients from decaying organic matter, as mushrooms do. Other organisms are parasites, obtaining energy from a plant or animal without, at least initially, killing it.
Whether an organism is an autotroph or a heterotroph, one can get an idea of the rate at which it is carrying out metabolic processes by measuring the amount of carbon dioxide it uses or produces, respectively. Autotrophs convert carbon dioxide to oxygen and carbohydrates by photosynthesis. Both autotrophs and heterotrophs produce carbon dioxide as a result of metabolizing food in the presence of oxygen.
In today's lab you will measure carbon dioxide use by a green plant and carbon dioxide production by an aquatic animal. While doing this exercise, think about how carbon dioxide and oxygen are involved in the lives of the organisms. In particular, keep in mind that autotrophs not only produce energy, they also use it.
Textbook reading prior to coming to lab: Tobin and Dusheck 3rd edition: pp. 109-144.
The following is a brief outline of the steps you will use to calculate carbon dioxide production/consumption by a heterotroph and an autotroph:
H2CO3 CO2 H2CO3 CO2
Collect data
Analyze data
Etc.
A. CO2 UPTAKE AND PRODUCTION BY AN AQUATIC PLANT
Natural waters such as ponds, lakes, and rivers are generally rich in bicarbonates [usually Ca(HCO3)2] that serve as a source of carbon dioxide for photosynthesizing aquatic plants. Using a relatively simple quantitative procedure, we can determine the uptake and production of CO2 by aquatic plants.
Here, we will use Elodea, an aquatic plant commonly found in ponds and small lakes (remember from last week that Elodea is NOT an alga). Because plants both consume and produce carbon dioxide, you can only measure the NET carbon dioxide change.
If there is an overall DECREASE in carbon dioxide, you know that more photosynthesis has occurred than aerobic respiration. If there is an overall INCREASE in carbon dioxide, you know that more aerobic respiration has occurred than photosynthesis.
PROCEDURE: The italicized questions are for you to think about as your prepare for and carry out lab. Some of these might show up on your pre-lab quiz or on your exam.
1. Add 150 ml of spring water to a clean beaker. Using a clean straw, blow bubbles into the water for one minute total (this can be done over several shorter time periods) so that the water will be saturated with CO2. Why is it important that you blow bubbles into the total 150 mL of water instead of blowing separately into each 50 mL aliquot of water in the three beakers?
2. Divide the 150 mL of CO2-saturated water into three equal portions in three beakers. Label one beaker “Elodea, Light”, one beaker “Elodea, Dark”, and one beaker “No Elodea.” Why are you using a beaker with no Elodea? Think back to the first week and what you learned about the importance of controls in designing a good experiment.
3. Using forceps, obtain a piece of Elodea that is approximately 2 g in weight. Touch the plant briefly to a piece of paper towel to remove excess water (you do not need to dry it completely, just remove the biggest drops of water) and weigh it in a dish on the balance. Submerge the piece of Elodea in one of the beakers. Obtain a second specimen of the same weight for the other beaker (if your second piece is not the same weight, remove small pieces from the cut end until it is the same weight). The third beaker will have no plant sample. Why is it important to have the same amount of plant tissue in each of the experimental beakers?
4. Place the “Elodea, Light” beaker 12 inches away from a lamp with a 100watt bulb. Place the “Elodea, Dark” beaker in the dark. Record the time you begin the experiment on Data Sheet I. Should you place your “No Elodea” beaker in the light or the dark? If it doesn’t matter, why doesn’t it matter?
5. At the end of 60 minutes, remove both pieces of plant and place them in the tank marked “Used Elodea.” Record the ending time on Data Sheet I. Save the water.
Yes, this is a long time to wait.
During the wait, you should begin your goldfish experiment and design your own experiment.
6. Add three drops of phenolphthalein to each beaker. [Your “Elodea, light” beaker may already look pink after the addition of the phenolphthalein. This is OK, just add enough NaOH to the other two beakers to get the SAME shade of pink.]
Phenolphthalein is a pH indicator that changes from colorless to pink as the solution changes from acidic to basic. The number of drops of NaOH needed to neutralize the solution so that phenolphthalein turns pink (basic) is a direct function of the acidity of the water. The more acidic (more carbonic acid / CO2) the solution is, the greater the number of drops of NaOH needed to obtain a color change.
7. Place your beakers on a sheet of blank white paper to help you see the colors more easily. With a dropper (the dropper delivers 0.05 ml per drop), slowly add 0.02 N NaOH to the “Elodea, Light” beaker, counting the number of drops until a faint but permanent pink color is obtained (if your beaker was already a faint, permanent pink color, do not add additional NaOH). Thoroughly stir the solution with a glass stirring-rod after adding each drop.
8. Count the number of drops of NaOH you need to add to the “Elodea, Dark” beaker to achieve the SAME pink color as in the “Elodea, Light” beaker. Record your results on Data Sheet I.
9. Count the number of drops of NaOH you need to add to the “No Elodea” beaker to achieve the SAME pink color as in the “Elodea, Light” and “Elodea, Dark” beakers. Record your results on Data Sheet I.
10. Use Table 3-1 to calculate the amount of CO2 consumed or produced in each of the beakers during the experiment. Record these amounts on your data sheet. Be sure to specify whether there was a net increase or net decrease in the amount of CO2 (in other words, did the use more CO2 in photosynthesis than it produced by respiration or did the plant give off more CO2 from respiration than it used in photosynthesis?).
Lab 3-18
B. CO2 PRODUCTION BY AN AQUATIC ANIMAL
To calculate the oxygen consumption of an aquatic animal, we can measure the amount of CO2 it produces in the water in a given period of time. We will use a method similar to that used in determining the CO2 uptake and production by Elodea. Note that the results of this experiment will be easier to understand because animals do not carry out both photosynthesis and respiration, only respiration.
PROCEDURE:
1. Add 200 mL spring water to each of two clean beakers. Label one beaker “Fish” and the other beaker “No fish”.
2. Use the net to carefully and respectfully move a goldfish into the “Fish” beaker. The “No fish” beaker serves as the control. Again, why do you need to use a control in this experiment?
3. Note the time on Data Sheet I and leave the beakers on the table for 30 minutes. Every 5 minutes, make note of the fish's behavior. Is it quiet or does it swim around a lot?
4. At the end of 30 minutes, carefully remove the animal and return it to the aquarium. Record the ending time on Data Sheet II.
5. Add five drops of phenolphthalein solution to each beaker of spring water.
6. Place your beakers on a sheet of clean white paper to help you see the colors more easily. With a dropper (the droppers deliver 0.05 ml per drop), slowly add 0.02 N NaOH to the solution in the “No fish” control beaker until a faint, but permanent, pink color is obtained. Thoroughly stir the solution with a glass stirring-rod after adding each drop. Record your results in Data Sheet II.
7. Count the number of drops of NaOH needed to achieve the SAME permanent pink color in the “Fish” experimental beaker.
8. Using Table 3-1, calculate the amount of CO2 neutralized by the NaOH. The difference in number of drops of NaOH is an indirect measurement of the amount of CO2 produced by the goldfish.
Table 3-1. Conversion Table of Drops of 0.02N NaOH to Volume of CO2 Values
Difference in Number of0.02N NaOH Drops / Volume of CO2 (mL)
1 / 0.05 mL
2 / 0.09 mL
3 / 0.14 mL
4 / 0.18 mL
5 / 0.23 mL
6 / 0.27 mL
7 / 0.32 mL
8 / 0.36 mL
9 / 0.41 mL
10 / 0.45 mL
Lab 3-18
What happens if there is more than a 10-drop difference in the amount of NaOH used? You’ll need to come up with a mathematical formula, which shouldn’t be difficult to do.
This lab was modified by Margaret Olney for BC1001 Revolutionary Concepts in Biology Lab, Barnard College, September 2003 and 2004.
BIOLOGY 1001 NAME______
Lab day and time______
Lab instructor______
LABORATORY 3: DATA SHEET 1
to be handed in at the beginning of next week’s lab
Be sure to write out your answers to all questions (including the italicized ones) on the data sheets.
DATA SHEET FOR PRELIMINARY EXPERIMENTS
I. NET CO2 EXCHANGE BY AN AQUATIC PLANT
A. Time experiment began: ______
Time experiment ended: ______
Length of experiment (in minutes): ______
B. Number of drops of NaOH added to “Elodea, light” beaker = ______
Number of drops of NaOH for “Elodea, dark” beaker = ______
Number of drops of NaOH for “No Elodea” = _____
C. Difference in number of drops for beaker in dark (compared to empty beaker) =
______drops
Difference in number of drops for beaker in light (compared to empty beaker) =
______drops
D. Amount of CO2 taken up or given off by plant in dark (from conversion Table 3-1) =
_____ ml
Here’s the hard part, you’ll have to think about whether this represents a net increase or net decrease in the amount of CO2—was more CO2 taken up than given off or more CO2 given off than taken up?