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.

Cycling can be studied at different scales

Sulphur

What is nitrogen?

Or nitrogen cycle?

By traveling through one of the

four processes in the Nitrogen

Cycle!

(1) Nitrogen Fixation

(3) Nitrification (2) Ammonification

(mineralization)

(4) Denitrification

Nitrogen

Cycle

•modified from Goldman and Horne. 1994. Limnology. McGraw Hill.

Nutrients- The Nitrogen Cycle

Why does

atmospheric

nitrogen need to be

converted?

N

N

N

N

N

N

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

How could atmospheric nitrogen

be changed into a form that can

be used by most living

organisms?

N

N

There are three ways that

nitrogen could be “fixed”!

(a) Atmospheric Fixation

(b) Industrial Fixation

(c) Biological Fixation

Bacteria

Atmospheric Fixation

(Only 5 to 8% of the Fixation

Process)

The enormous energy of

lightning breaks nitrogen

molecules apart and enables

the nitrogen atoms to combine

with oxygen forming nitrogen

oxides (N2O). Nitrogen oxides

dissolve in rain, forming

nitrates. Nitrates (NO3) are

carried to the ground with the

rain.

Lightning “fixes” Nitrogen!

Nitrogen combines with Oxygen

Nitrogen oxides forms

Nitrogen oxides dissolve in rain and change to nitrates

Plants use nitrates to grow!

(NO3)

N N O

(N2O)

Industrial Fixation

, at a great pressureUnder

degrees 600 of temperature

Celsius, and with the use of

a catalyst, atmospheric

nitrogen (N2) and hydrogen

are combined to form

ammonia (NH3). Ammonia

can be used as a fertilizer.

Industrial Plant combines nitrogen and hydrogen

Ammonia is formed

Ammonia is used a fertilizer in soil

(NH3)

N N H

N H3

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.

Biological Fixation

of “Nitrogen Fixing Bacteria” two typesThere are

Free Living Bacteria (“fixes” 30% of N2)

Symbiotic Relationship Bacteria (“fixes” 70% of N2)

Free Living Bacteria

Highly specialized bacteria live in the soil and have the

ability to combine atmospheric nitrogen with hydrogen to

make ammonia (NH3).

Free-living bacteria live in soil and combine atmospheric nitrogen with hydrogen

Nitrogen changes into ammonia

N N

H

N H3

(NH3)

Bacteria

Symbiotic Relationship

Bacteria

Bacteria live in the roots of

legume family plants and

provide the plants with

ammonia (NH3) in exchange

for the plant’s carbon and a

protected-home.

Legume plants

Roots with nodules where bacteria live

Nitrogen changes into ammonia.

NH3

N

N

Root Nodule Bacteria

Root nodules

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

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.

Examples of nitrogen-fixing bacteria (* denotes a photosynthetic bacterium)

Symbiotic with plants Free living

Other plants Legumes Anaerobic (Winogradsky

column) Aerobic

Frankia

Azospirillum Rhizobium

Clostridium (some)

Desulfovibrio

Purple sulphur bacteria* Purple non-sulphur bacteria*

Green sulphur bacteria*

Azotobacter

Beijerinckia

Klebsiella (some)

Cyanobacteria (some)*

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.

Martinus Beijerinck

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.

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.

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.