Studying Living Organisms

Nitrogen Fixation, OR What a Gas!

Barbara J. Errington
Woodrow Wilson Biology Institute

Target age or
ability group:
Biology I and Biology II.
Class time
One fifty-minute class period for discussion and setup. Six to eight weeks for completion of project.
Materials and equipment: 1. Two 12.5 cm plastic pots (If the pots are not new, wash used ones with a 10% bleach solution)
2. Masking tape or permanent marker for labeling pots
3. Vermiculite, enough to fill the pots
4. Six legume seeds (a variety of legumes may be used: soybeans, bush-type lima, or peas) 5. Commercial Rhizobium (Check seed and feed stores or garden centers for a product called Inoculum. If it is not available in your area, Rhizobium may be ordered from any biological supply house.)
6. Plastic wrap
7. Nitrogen-free nutrients (see next page)
8. Distilled water (may be supplied by students)
Summary of activity: This is a long-term project designed to illustrate the adaptation and coevolution of organisms. Students gain skill in the design, implementation, and reporting of a scientific research project using the scientific method. Specifically, they will set up an experiment to determine the effect of adding the Rhizobium bacteria to one group of legume seeds.
Students will plant seeds in containers containing vermiculite and after the seeds germinate, they will take the pots home to continue the project. The plants are then brought back to the classroom for analysis.
Prior knowledge, concepts or vocabulary necessary to complete activity:
Students should be familiar with the concepts of coevolution and symbiosis, including mutualism, commensalism, and parasitism. They should also have been introduced to the nitrogen cycle.

Teacher Instructions

1. Start with a brief discussion on the types of symbiosis by showing visuals such as transparencies or slides depicting the various relationships. Ask students to identify the type of symbiosis and the adaptations that the participating organisms make to allow the relationship to develop.

2. Inform students thatRhizobium is a bacterium that is not harmful to humans or pets, but will cause legume roots to form nodules. Tell the students: "The purpose of our investigation will be to find out what effect bacterial interactions have on plants of the bean family."

3. Ask students: "How can we see if a plant is affected by the nodules?" Answers will vary, but get to factors such as height and weight of a plant as it grows compared to the growth of plants that do not have the nodules.

4. Elicit from students a probable hypothesis as to the effect of the nodules on plant growth.

5. After the lab setup, students are to state their hypothesis, procedures, data, table, etc.


1. Place average data from each lab team on board and determine overall class results. Discuss significant deviations, review scientific error, use of larger sample size, and other important scientific aspects of the experiment.

2. Have students suggest answers to the questions and discuss.

Optional follow up:

Remove the nodules from the plant roots and prepare a Gram stain on the bacteria.

Nitrogen-free nutrients

0.2g dihydrogen phosphate
0.8g potassium monohydrogen phosphate
0.2g magnesium sulfate
0.1g calcium sulfate
0.01g ferric sulfate

Combine the above minerals and store in a covered container. Instruct students to add 1.3 grams to one liter of distilled water.

Student Instructions

Nitrogen Fixation, OR What a Gas!


The environment of plant roots is composed of many types of organisms including bacteria, fungi, and invertebrates. To survive, plants have evolved ways to interact with these organisms ranging from defense mechanisms against pathogens to symbiotic relationships that are mutually beneficial.


To describe and explain the importance of nitrogen fixation
To investigate the symbiotic relationship between bacteria and plant roots
To gain skill in using the scientific method

Materials and Equipment:

1. Two 12.5 cm plastic pots
2. Vermiculite, enough to fill the pots
3. Six legume seeds
4. CommercialRhizobium
5. Nitrogen-free nutrients
6. Masking tape or permanent marker for labeling pots
7. Distilled water (8-12 liters)
8. Plastic wrap


1. Label each of your pots "with" or "without."
2. Fill each pot with equal amounts of vermiculite.
3. Plant three seeds 2 cm deep in "without" pot.
4. Sprinkle a small amount of Rhizobium on three seeds and plant 2 cm deep in the "with"pot.
5. Water both pots with distilled water and cover with plastic wrap.
Leave covered and do not remove plastic unit seeds germinate.
6. After the first leaves appear, uncover and water with a solution containing 1.3 grams of nitrogen-free nutrients to one liter of distilled water.
7. After six to eight weeks, remove plants from pots, taking care not to disturb nodules, and measure both the height and weight of each plant.


Alberts, Bruce, et al. Molecular Biology of the Cell. 2d ed. New York: Garland Publishing, 1989.

Nitrogen Fixation Data Sheet





What is the effect of nitrogen fixation via a symbiotic relationship with bacteria on the length and weight of a selected legume plant?

Background Information (from pre-lab and notes)



Data Table


Questions(answer on separate sheet of paper):

1. What possible error sources were there in this experiment?

2. How does Rhizobium enter the root system?

3. How much nitrogen-based fertilizer would be needed to equal the effect of the bacterium?

4. Do certain species of bean plant require a specific nitrogen-fixing bacterium?

On to Selecting Soil Organisms in Compost
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