1. Presence of nitrogen-fixing bacteria
Nitrogen fixing bacteria are very important components of soil. In order to build 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 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 Nfixing 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 N-fixers
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.
The idea with this method is to put seeds into replicated combinations of potting soil with no Nfixing bacteria in it, and mixed with increasing amounts of the soil being tested for the presence
of N-fixing bacteria.
Obtain a multi-chambered planting tray (Speedwell or similar tray). Fill one row of chambers
with sterile potting soil, a second row filled with soil that is mixed 75% potting soil, 25% site
soil, a 3rd row with soil t hat is 50% potting soil, 50% site soil, and a 4th row with 100% site soil.
Plant a bean seed in each well and water appropriately. After one month, gently lift the plants
form the soil, wash roots gently, measure root length and count the visible nodules on the roots.
Nodules containing nitrogen-fixing bacteria will appear as pale or slightly pinkish balls or lumps
on the roots. Calculate the number of nodules per centimeter of root. Calculate an average
number of nodules for all plants from each percentage of site soil (0%, 25%, 50%, 100%). Root
dry weight can be assessed by drying each root system and weighing the dried root. Shoot
weight, root and shoot branching could also be assessed.
The chambers with no site soil act as a control; no nodules should grow on the roots of plants
grown in that mixture. The number of nodules per cm of root should increase as the percentage
of site soil increases. If most or all of the plants in the 25% site soil mixture have nodules on
their roots, you may infer that nitrogen-fixing bacteria are present in relatively high numbers. If
nodules are found only in the 100% site soil treatment, and on less than half the plants, you may
infer that nitrogen-fixing bacteria are present in relatively low numbers. Greater numbers of
nodules per cm of root indicate better grassland soil, with a higher capacity for nitrogen fixation.
Methods and Materials
Materials
• multi-chambered planting tray (Speedwell or similar tray)
• sterile potting soil
• site soil
• bean seeds
• rhizobial bacteria inoculant (available from garden supply store)
Nitrogen-fixing Bacteria
Procedure
1. Prepare the inoculant and coat one set of bean plants with it. Plant the bean plants
alongside the experimental plants. This will give you a known set of plants
containing nodules.
2. Fill one row of chambers of a multi-chambered planting tray with sterile potting
soil.
3. Fill the second row with soil that is mixed 75% potting soil and 25% site soil. The
third row is filled with 50% potting soil and 50% site soil, and the fourth row is
filled with 100% site soil.
4. Plant a bean seed in each well and water consistently in all rows.
5. After one month, lift the plants from the soil and gently wash the roots. Ask each
group to inspect the bean plants that grew with the inoculant to learn to identify
nodules that contain nitrogen fixing bacteria.
6. Measure each plants root length and record in a data table.
7. Count the visible nodules on each plants roots. Nodules containing nitrogenfixing bacteria will appear as pale or slightly pinkish balls or lumps on the roots.
Results
Create a data table to record root lengths and numbers of nodules for each plant. Make
sure that each plant is identified in some manner, and % potting soil vs. % site soil is
clearly labeled in your table.
Analysis
1. Calculate the number of nodules per centimeter of root for each plant and record
in a data table (add this information to the data table with results or construct a
new table). Use the following relationship:
Number of nodules/cm = # nodules on plant
length of root system
2. Calculate an average number of root nodules/cm for all plants from each
percentage of site soil (0%, 25%, 50%, 100%).
3. The number of nodules per centimeter of root should increase as the percentage of
site soil increases. If most or all of the plants in the 25% site soil mixture have
nodules on their roots, it may be inferred that nitrogen-fixing bacteria are present
in relatively high numbers. If nodules are only found in the 100% site soil, it may
be inferred that N-fixing bacteria are rare in site soil. Greater numbers of nodules
per centimeter of root indicate a higher capacity for nitrogen fixation.
Conclusions
Discuss the results of this assay in terms of the data you obtained. What can you conclude
about the soil collected from the experimental site in terms of its ability to support plant
life? Were there any sources of error that could have had an effect on your interpretation
of the results? What would you do to further analyze this soil? Are there any pieces of
information that you need to help better understand the significance of this test?
Soil Biological Assays Data Tables
Presence of Nitrogen-fixing Bacteria
25% site
soil: Plant
1
25% site
soil: Plant
2
25% site
soil: Plant
3
25% site
soil: Plant
4
25% site
soil: Plant
5
25% site
soil: Plant
6
50% site
soil: Plant
1
50% site
soil: Plant
2
50% site
soil: Plant
3
50% site
soil:
Plant 4
50% site
soil:
Plant 5
50% site
soil: Plant
6
Root
Length
(cm)
Number
Nodules
Number
Nodules / cm
Average #
nodules / cm
Root
Length
(cm)
Number
Nodules
Number
Nodules / cm
Average #
nodules / cm
100% site
soil: Plant
1
Root
Length
(cm)
Number
Nodules
Number
Nodules / cm
Average #
nodules / cm
100% site
soil : Plant
2
100% site
soil: Plant
3
100% site
soil: Plant
4
100% site
soil: Plant
5
100% site
soil: Plant
6