How does Nitrogen get
into the Soil?
Presence of Nitrogen Fixing Bacteria
Introduction:
Nitrogen fixing bacteria are very important components of soil. In order to build soil fertility,
nitrogen content must increase. N-fixing bacteria produce enzymes that under the right
environmental conditions take nitrogen gas from the atmosphere, and using sugars obtained
from the plant, fix the nitrogen into bacterial biomass. If the bacteria satisfy their nitrogen
requirements, then nitrogen is given to the plant and elevated protein levels are seen in the
plant. This elevated nitrogen is not released to the soil until part of the plant dies, or exudates
are released into the soil in the rhizosphere.
Two major divisions of N-fixing bacteria occur: symbiotic N-fixing bacteria and associative
N-fixing bacteria. Symbiotic N-fixers, such as Rhizobium, occur on legumes. These bacteria
form nodules on the roots of the plants and these nodules are easy to count. Associative Nfixers occupy the spaces between plant root cells, and do not alter the root architecture in any
way.
In grassland soils, we can use a bioassay to determine whether the symbiotic N-fixers are
present, and how many are colonizing the roots of a bioassay plant. This can be directly
related to the productivity of grassland soils, or ecosystems where legumes are normal plant
species. Some legumes found in tallgrass prairies are purple prairie clover, white prairie
clover, lead plant, milk vetch, wild indigo, Illinois bundleflower, and partridge pea.
Your class will collect small amounts of soil from a site from at least five locations and mix
in container. You will test your site soil for the presence of nitrogen–fixing bacteria as
described in the procedure. You will grow bean seeds in five different pots each having a
different proportion of sample soil and sterile potting soil. You will then count the number of
nodules per centimeter of root. This number should increase with the increasing amounts of
site soil.
Materials:
Plant Trays Plant pots
Bean Seeds (pea) Rizobidium Innoculant (gardening supplier)
Experimental Soil Potting Soil
Grease Marking Pencil
Time: This will take 1 month (allowing time for bean seedlings to grow) with one class
session at beginning and one at end of growing period.
Break into six groups
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Procedures:
Each team will follow this procedure:
Day 1 (20 minutes + travel time):
1) As a class collect a small amount of soil from a selected site from at least five
locations
2) Mix the sampled soil together in a container.
Day 2 (50 minute class):
Prepare three pots fro each group as described below in table.
Type of
bean
Percent of
potting soil
Amount of
potting soil
Percent of
sample soil
Amount of
sample soil
Group 1
Inoculated
bean
100
Group 2
Plain bean
Group 3
Plain bean
Group 4
Plain bean
Group 5
Plain bean
100
75
50
25
Group 6
Plain
bean
0
0
0
25
50
75
100
Water consistently for one month.
Day Three (50 minute class): (Note: this next step can occur only after one month)
1) After one month, lift the plants from the soil and gently wash the roots.
2) Measure the roots of each plant in centimeters and record in a data table.
3) Count the visible nodules on each plant’s roots (see figure 1). Nodules containing
nitrogen fixing bacteria will appear as pale or slightly pinkish balls or lumps on the
root. The plant that was inoculated should have visible nodules which can be
compared to the other plants.
4) Calculate the number of nodules per centimeter for each plant.
5) Calculate the average number of root nodules per soil type.
6) Record your data in your data chart. Which plant had more as compared to the
inoculated plant? What does this say about your soil at your site? Interpret your
results.
7) Analysis and Interpretation: The following questions are designed to help you make
meaningful conclusions.
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a. What can you conclude about the soil collected in terms of its ability to support
plant life?
b. What would you do to the soil to make it more suitable for plant life?
c. What other experiments would you conduct to determine the overall health of
the soil?
Figure 1
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