2. General purposes:
1- To make the students aware with the role of
microbes in maintaining environment, existing
microbial interactions and recycling of
nutrients in nature:
2- A technique for the isolation of a free living soil
bacterium Azotobacter.
3- A technique for the isolation of root nodule
Bacterium Rhizobium sp.

3. Cycling can be studied at different scales

4. Sulphur

5. What is nitrogen?
Or nitrogen cycle?

6. By traveling through one of the
four processes in the Nitrogen
Cycle!
(1) Nitrogen Fixation
(4) Denitrification
Nitrogen
Cycle
(3) Nitrification (2) Ammonification
(mineralization)

7. Nutrients- The Nitrogen Cycle
•modified from Goldman and Horne. 1994. Limnology. McGraw Hill.

8. N
N
N
Why does
N
atmospheric
nitrogen need to be
converted?
N N

9. It is one of nature’s
great ironies…
Nitrogen is an essential component of DNA, RNA,
and proteins—the building blocks of life.
why is N fixation important?
• atmospheric N2 is inert – biotically unavailable.
• availability of fixed N is often the factor most
limiting to plant growth

10. How could atmospheric nitrogen
be changed into a form that can
be used by most living
organisms?
N
N

11. There are three ways that
nitrogen could be “fixed”!
(a) Atmospheric Fixation
(b) Industrial Fixation
(c) Biological Fixation
Bacteria

12. Atmospheric Fixation Lightning “fixes” Nitrogen!
(Only 5 to 8% of the Fixation
Process)
The enormous energy of N
N O
lightning breaks nitrogen Nitrogen
combines
molecules apart and enables with Oxygen
the nitrogen atoms to combine
with oxygen forming nitrogen Nitrogen oxides forms
oxides (N2O). Nitrogen oxides (N2O)
Nitrogen
dissolve in rain, forming (NO3) oxides dissolve
nitrates. Nitrates (NO3) are in rain and
change to
carried to the ground with the nitrates
rain.
Plants use
nitrates to grow!

13. Industrial Fixation NN
H
Under great pressure, at a
temperature of 600 degrees N
Celsius, and with the use of H3
a catalyst, atmospheric
nitrogen (N2) and hydrogen Industrial Plant combines
are combined to form nitrogen and hydrogen
ammonia (NH3). Ammonia
can be used as a fertilizer. (NH3) Ammonia is formed
Ammonia is used a fertilizer in soil

16. 3. Biological Fixation:
a. Non-symbiotic bacteria) Free Living Bacteria: (“fixes” 30% of N2)
Highly specialized bacteria live in the soil and have the ability to combine
atmospheric nitrogen with hydrogen to make ammonia (NH3).
Such as Azotobacteraceae
b.Symbiotic Relationship Bacteria: (“fixes” 70% of N2)
Bacteria live in the roots of legume family plants and provide the plants with ammonia
(NH3).
Among the most beneficial microorganisms of the soil are those that are able to convert
gaseous nitrogen of the air to “fixed forms” of nitrogen that can be utilized by other
bacteria and plants. Without these nitrogen-fixers, life on this planet is probably
disappear within a relatively short period of time. The utilization of free nitrogen
gas by fixation can be accomplished by organisms that are able to produce the
essential enzyme nitrogenase. This enzyme, in the presence of traces of
molybdenum, enables the organisms to combine atmospheric nitrogen with other
elements to form organic compounds in living cells.
Such as Rhizobiaceae.
Other organisms of less importance that have this ability are a few strains of Klebsiella,
some species of Clostridium, the cyanobacteria, and photosynthetic bacteria. In
this exercise we will concern ourselves with two activities: the isolation of
Azotobacter from garden soil and the demonstration of Rhizobium in root nodules
of legumes.

17. Biological Fixation
There are two types of “Nitrogen Fixing Bacteria”
Free Living Bacteria Symbiotic Relationship Bacteria
(“fixes” 30% of N2) (“fixes” 70% of N2)

18. Free Living Bacteria
Highly specialized bacteria live in the soil and have the
ability to combine atmospheric nitrogen with hydrogen to
make ammonia (NH3).
N
N H
NH3
Free-living bacteria live
in soil and combine
atmospheric nitrogen
with hydrogen
(NH3)
Nitrogen changes
into ammonia
Bacteria

19. Symbiotic Relationship
Legume plants
Bacteria
Bacteria live in the roots of
legume family plants and
provide the plants with N
ammonia (NH3) in exchange
for the plant’s carbon and a
protected-home. NH3
N
Roots with nodules
where bacteria live
Nitrogen changes into
ammonia.

20. Root Nodule Bacteria

22. Root nodules

23. Nitrogen Fixation
The nodules on the roots
of this bean contain
bacteria called
Rhizobium that helps by
converting nitrogen in
the soil into a form the
plant can utilize it.
14

24. Mechanism of N-fixation:
The general chemical reaction for the fixation of nitrogen (N + 3H2 + Energy -> 2NH3) is
identical for both the chemical and the biological processes. The triple bond of N
must be broken and three atoms of hydrogen must be added to each of the
nitrogen atoms. Living organisms use energy derived from the oxidation ("burning")
of carbohydrates to reduce molecular nitrogen (N2) to ammonia (NH3).
• AZOTOBACTERACEAE
Azotobacteraceae that fix nitrogen as free-living organisms under aerobic conditions:
Azotobacter and Azomonas. Both are large gram-negative motile rods that may
be ovoid or coccoidal in shape, (pleomorphic). The free-living Azotobacteraceae are
beneficial nitrogen-fixers, their contribution to nitrogen enrichment of the soil is
limited due to the fact that they would rather utilize NH3 in soil than fix nitrogen. In
other words, if ammonia is present in the soil, nitrogen fixation by these organisms
is suppressed.
• RHIZOBIACEAE
The symbiotic nitrogen-fixers of genus Rhizobium, family Rhizobiaceae, are the
principal nitrogen enrichers of soil. Three genera in family Rhizobiaceae: Rhizobium,
Bradyrhizobium, and Agrobacterium. Although the three genera are related, only
genus Rhizobium fixes nitrogen. This genus of symbiotic nitrogen-fixers contains only
three species:
• R. leguminosarum: peas, beans.
• R. meliloti: sweet clover.
• R. loti: trefoil.
• All three of these species are gram-negative pleomorphic rods (bacteroids), often X-,
Y-, star-, and clubshaped; some exhibit branching. All are aerobic and motile.

25. Examples of nitrogen-fixing bacteria (* denotes a photosynthetic bacterium)
Free living Symbiotic with plants
Anaerobic (Winogradsky
Aerobic Legumes Other plants
column)
Clostridium (some)
Azotobacter
Desulfovibrio
Beijerinckia Frankia
Purple sulphur bacteria* Rhizobium
Klebsiella (some) Azospirillum
Purple non-sulphur bacteria*
Cyanobacteria (some)*
Green sulphur bacteria*

27. Procedure for isolation of AZOTOBACTERACEAE
FIRST PERIOD (ENRICHMENT)
Proceed as follows to inoculate a bottle of the nitrogen free glucose medium with a sample of garden soil.
Materials:
• 1 bottle (50 ml) N2-free glucose medium (Thompson-Skerman) or Azotobacter agar
• rich garden soil (neutral or alkaline)
• spatula
1. with a small spatula put about 1 gm of soil into the bottle of medium. Cap the bottle and shake it sufficiently to
mix the soil and medium.
2. Loosen the cap slightly and incubate the bottle at 30° C for 4 to 7 days. Since the organisms are strict aerobes,
it is best to incubate the bottle horizontally to provide maximum surface exposure to air.
SECOND PERIOD (PLATING OUT)
During this period a slide will be made to make certain that organisms have grown on the medium. If the culture
has been successful, a streak plate will be made on nitrogen-free, iron-free agar. Proceed as follows:
Materials:
Microscope slides and cover glasses microscope, 1 agar plate of nitrogen-free, iron-free glucose medium
1. After 4 to 7 days incubation, carefully move the bottle of medium to your desktop without agitating the culture.
2. Make a wet mount slide with a few loopfuls from the surface of the medium and examine under oil immersion,
Look for large ovoid to rod-shaped organisms, singly and in pairs.
3. If the presence of azotobacter-like organisms is confirmed, streak an agar plate of nitrogen-free, iron-free
medium, using a good isolation streak pattern. Ferrous sulfate has been left out of this medium to facilitate the
detection of water-soluble pigments.
4. Incubate the plate at 30° C for 4 or 5 days.

29. Martinus Beijerinck

30. Azotobactereace on different media : a) Brown-agar medium, b)
Winogradsky solution, c) smoothed soil paste–plate surface, d)
mannitol-agar, e ,f, g, h) differential LG agar medium (different
species and components.

31. Procedure for isolation for isolation of RHIZOBIACEAE:
• Materials:
1. washed nodules from the root of a legume
2. methylene blue stain
3. microscope slides
– pink nodules were selected from the root of a legume and washed by
water, then kept in (MgCl2) for period of time, and washed again by
water
– Place a nodule on a clean microscope slide and crush it by pressing
another slide over it. Produce a thin smear by sliding the top slide over
the lower one.
– After air-drying and fixing with heat, stain the smear with methylene blue
for 30 seconds.
– Examine under oil immersion.

32. A. Questions:
1. What enzyme is responsible for nitrogen fixation? By which
mechanism level of O2 regulated to obtain maximum nitoginase
activity?
2. Why is nitrogen fixation so important?
3. from the standpoint of amount of nitrogen fixation, is this group of
nitrogen-fixers Rizobacteriaceae more or less important than the
Azotobacteraceae?
4. On your opinion does it possible to increase fixation in unamended
soil by addition of high populations of bacteria (soil inoculation)?
5. Draw some of the organisms on the Laboratory Report. Look for
typical bacteroids of various configurations.