National Teachers Enhancement Network: Lesson Plan

Lesson Plan: An Investigation into Effects of Rhizobium Soil Bacteria on Nitrogen Availability
Subject: Earth and Space / Soils
Classtime: 9-10 weeks
Grade Level: 7-12

Materials/Technology:

20 seeds of a leguminous plant, such as garden pea, green bean, or clover (Red clover seeds are supplied with the Nitrogen-Fixation Study from Carolina supply.) These seeds should not be pre-inoculated. (A colored coating usually indicates inoculation.)
an inoculum of Rhizobium bacteria that is host-specific to the chosen host plant. If the Nitrogen-Fixation Study Kit from Carolina Biological (see reference) is not used, then appropriate host seed and bacterial inoculum can be obtained from a seed retailer.
4 identical or closely similar pots (or other suitable containers with drainage holes) that are large enough to hold 1 pint of soil
4 pints of fine mason’s sand (sandbox sand)
1 teaspoon of a 0-20-20 fertilizer or �2 teaspoon of 0-10-10 or 0-10-5. Different ratios such as 0-12-6 can be used as long as they contain no (or almost no) nitrogen.
balance
tubs to wash soil from plants

Safety, Handling, Disposal:
Care should be used in handling the fertilizers and the bacterial inoculum.

Learner Outcomes:
Students should be able to:

Collect, record and interpret their data.
Identify the control and the variables in the experiment.
Describe the growth of plants with and without nitrogen fixing bacteria.
Name the bacterium responsible for making nitrogen available to the plant.

Problem/Purpose:
What is the effect of a nitrifying bacteria such as Rhizobium on plant growth?

Background/Inquiry:
This experiment will make use of a soil bacterium of the genus Rhizobium and a host legume to demonstrate the effects that soil microbes can have on soil fertility and nutrient availability.

Vocabulary List: Here.

Hypothesis:
Make a hypothesis after reading through and understanding the procedure. Example: 'Bacteria will have no effect on the growth of the legume.'

Procedure:

Label each container with a different label:
- no additions (sand and plants)
- inoculated -- no fertilizer (sand, bacteria and plants)
- not inoculated -- fertilized (sand, fertilizer and plants)
- inoculated -- fertilized (sand, fertilizer, bacteria and plants)
Put half of the sand (1 qt ) into a mixing bowl or container and add 1 teaspoon of the 0-20-20 fertilizer to the sand and mix thoroughly. If using a 0-10-10 grade fertilizer, or a 0-10-5 fertilizer, use 2 tsps. For other grades, adjust the amount proportionately.
Place 1 pint of the fertilized soil in each of pots #3 & #4. Place 1 pint of the unfertilized soil in each of pots #1 & #2.
Plant 5 seeds in each of the pots.
Add Rhizobium inoculum to the surface layer of the sand in pots #2 and #4.

Note: Avoid Rhizobium contamination in the not inoculated pots.
Moisten the soils of each pot until water just starts to run out of the holes in the bottom. Use tap water or distilled water. If your water is chlorinated, it may be best to let it stand in a container for a day to give the chlorine a chance to dissipate before use.
Place each of the pots in separate shallow dishes or trays. Put them together (to eliminate environmental variables) in a warm, well-illuminated location, and water as needed to maintain moisture.
If one or more of the pots has less than 5 plants after the seeds have germinated and developed into shoots several inches tall, remove the smallest plants so that each pot contains the same number of plants.
Have the students predict the results (based on the introductory remarks for this lab) and record those predictions.
Make and record measurements/observations at least 2x per week. It will take about six weeks for the plants to develop root nodules, so the experiment should run for at least 9-10 weeks to be able to observe any differences between the treatments with and without Rhizobium.
Measure and record the height of the aerial parts of the plants in each pot (cm). Determine the mean height (cm) for the plants in each pot. Record in the data table.
Observe the appearance of the plants, including color, leaf size, leaf spacing, and any other observations and record them.
At the end of the nine-week growth period harvest the plants. Harvest by immersing the container in a tub of water and carefully washing the soil off the roots. Be careful while harvesting to avoid loss of plant material.
Record the wet mass of the plants for each separate pot.
Sketch and/or describe the comparative differences between root growth, nodule formation and overall vigor of the plants in each pot.
Air dry the plants for several days in a sunny location until a constant dry weight is obtained. Record the dry weight of the entire group of plants for each separate pot.
Interpret the measured / observed results and reconcile them with your predictions as a written conclusion.

Data Table

Treatment	Pot 1 No Treatment No Fertilizer	Pot 2 Inoculated No Fertilizer	Pot 3 Not Inoculated Fertilizer	Pot 4 Inoculated Fertilizer
Date	Find individual plant height, then the average for each pot. Record the appearance, including color, leaf size, leaf spacing, and other significant observations.

Results/Analysis:
Using the data collected in the data table, create a line graph with time on the horizontal axis and plant height in cm on the vertical axis. Include all four pots on one graph for comparison. Plot the average for each pot only.

Using the data collected from the harvesting of the plants, create a bar graph with mass on the vertical axis and each pot’s label on the horizontal axis. Include the wet mass and the dry mass as two separate bars for each pot.

Write a paragraph that summarizes the differences in the four groups of plants using the data in the chart and graphs.

Conclusions:
Was your hypothesis supported? Why or why not?

What questions came up during the lab that might be answered with further investigations?

What effect do you think it would have on a crop of peas to use the Rhizobium inoculum?
How could the hypothesis that "Rhizobium bacteria adds nitrogen to the soil" be tested by further experiments with the current set-up?

Assessments:

What was the purpose of using washed sand as the "soil"?
What is nitrogen fixation?
Which plants in this experiment showed the greatest above-ground growth?
Which plants in this experiment showed the greatest nodule formation on the roots?
What is the relationship between a nitrifying bacteria and a legume such as a pea plant?

Integration: Mathematics (ratios, percent error).

Reflections:
Share your thoughts on this lesson with the NTEN team.

Please send an e-mail to Patti Harrison.

Extensions:

Use a non-legume vs a legume to study beneficial symbiotic relationships.
Examine the nodules of the roots and place on culture plates to study the bacteria present.
Examine the soil and plate for the presence of nitrifying bacteria.

References/Resources:

Print: Carolina Biological Supply’s Nitrogen-Fixation Study Kit (cat # AA-15-4743, $26.00
URL: http://www.carolina.com

Credits
Contributing Teacher: Randy Farchmin
NTEN Course: LRES 580: The Dirty Dozen: Twelve Principals of Soil Science
Instructor: Jim Bauder
Developing Team:
T.L. Buck Buchanan, Don Samuels, Patti Harrison, John Usher, Don Wilson
HTML Programmers: Luke Clemens, Ken Eklund, Ching-Kwong Chia

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