Nitrogen Fixation

Nitrogen fixation 

The process of conversion of free nitrogen of the atmosphere into utilisable compounds of nitrogen such as nitrate, ammonia, amino acids etc is termed as nitrogen fixation.  About 78% of nitrogen is present in the atmosphere which exists in the form of an inert gas.

Hence the plants cannot use molecular nitrogen. It is therefore, necessary to fix free nitrogen of the atmosphere into some elements or compounds in soil converting them into nitrogenous salts to make them available for plants as nutrients.

Plants absorb nitrogen from the soil in the form of nitrates, nitrites, ammonium salt, and organic nitrogenous compounds.  These compounds are present in the soil in the form of proteins, amino acids, urea etc.  Some plants absorb urea directly, but plants do not absorb proteins and amino acids directly.

Fixation of nitrogen into the soil takes place by the process of rainfall, electric discharge due to thunderstorm and due to the activity of microorganisms.

Following are the three methods of nitrogen fixation- physical, biological and industrial nitrogen fixation.

Physical nitrogen fixation:

During thunder and lightening, N2 and O2 of atmosphere react to form NO (nitric oxide).


NO (nitric oxide) on further oxidation forms nitrogen dioxide.


The nitrogen oxides dissolve in water to form nitric acid and nitrous acid.  They enter into the soil along with rain water and react with alkaline earth metals to form corresponding nitrate and nitrite salt.

K+HNO3→KNO3 (potassium nitrate)

Ca+HNO3→Ca (NO3)2 (calcium nitrate)

K+HNO2→KNO2 (potassium nitrite)

Biological nitrogen fixation:

Conversation of atmospheric nitrogen into inorganic or organic usable forms through the agency of living organism is termed as biological nitrogen fixation.  It is the major source of nitrogen fixation.  This process is mainly carried out by two types of nitrogen fixation depending on the microorganisms:

  1.  Asymbiotic nitrogen fixation.
  2.  Symbiotic nitrogen fixation.

Asymbiotic or non- symbiotic biological nitrogen fixation:

Nitrogen is fixed asymbiotically in the soil by free living microorganism.  Asymbiotic or free nitrogen fixers can be classified into three groups,

(a)  Aerobic bacteria, for example, Azotobacter.

(b)  Anaerobic bacteria, for example, Clostridium, Rhodospirillum and

(c)  Blue green algae, or cyanobacteria, for example, Anabaena, Nostoc, Aulosira, Cylindrospermum, TrichodesmiumAulosira is the most active nitrogen fixer in rice fields while Cylindrospermum is active in sugarcane and maize fields.

These microorganisms are abundant in the soil and contribute to the nitrogen content of the soil.  All nitrogen fixing blue green algae (Cyanobacteria) possess long, thick-walled colourless cells called heterocysts.  Heterocysts are the site of nitrogen fixation.

A proper amount of minerals like molybdenum, iron and calcium in the soil is essential for nitrogen fixation.  Moreover, all nitrogen fixing microorganisms require molybdenum for their activity.

Mechanism of asymbiotic biological nitrogen fixation:

Nitrogen fixation is a reductive process which requires the following:

  1.  Enzyme- Nitrogenase.
  2.  Reductant- Ferredoxin.
  3.  Energy-ATP.

Following is the process of asymbiotic biological nitrogen fixation :

1:  Pyruvic acid is broken down into acetyl-PO4 and hydrogen is released.

2:  Ferredoxin is reduced by H+ ion.

3:  Acetyl phosphate reacts with ADP to form ATP and acetate.

4:  In the presence of ATP and reduced ferredoxin, molecular N2 is adsorbed on the surface of enzyme nitrogenase.

5:  Transfer of H+ ion takes place to N2 and reduction of N2 takes place until it is completely reduced to NH3.

Symbiotic nitrogen fixation:

Symbiotic nitrogen fixers are those which fix nitrogen being associated symbiotically with other plants.  The symbiotic fixers include Rhizobium, Nostoc, Anabaena and the plants include legumes (root nodules), cycas (coralloid roots) etc.

The most common forms of symbiotic nitrogen fixing bacteria (e g., several species of Rhizobium) occur in the root of the members of Leguminosae.  Besides these, many other vascular plants (e g.,Casuarina, Myrica, Purshia, Cerococarpus etc) are also host to nitrogen fixing microorganisms. In some plants nodules also occur on stems (e g., Aeschynomene, Sesbania ) and leaves (for example ,Psychotria, Azolla).

Biochemistry of symbiotic nitrogen fixation:

It is similar to that of asymbiotic nitrogen fixation.  Here, an additional pigment leghemoglobin is present in the root nodule cells.  Leghemoglobin has the ability to combine rapidly with oxygen. Thus it helps to maintain the activity of enzyme nitrogenase to release oxygen.  Leghemoglobin provides oxygen to nitrogen fixing bacteroid. As a result, these produce ATP for nitrogen fixation.

Symbiotic nitrogen fixers in leguminous plants inhabit small knob-like protuberance called nodules on the roots of the plant.  These nodules vary considerably in their shape and size.  They may be spherical and may have finger-like projections.  Their size vary from pin head to 1 cm in diameter.

Formation of nodules:

At first, the root secrets certain glycoproteins, which attract specific Rhizobium species. The bacterial cells produce some extracellular hormones and enzymes that produce following two effects:

  1.  Cause the root hair to curl in a characteristic crook-shape.
  2.  Help in the partial destruction of the cell wall of root hair. This is followed by the invasion of root hairs by the threads of mucilaginous substance in which bacterial cells are imbedded.  Later, the thread moves into the cortex.

Mechanism of formation of nodules:

This mucilaginous threads or infection threads stimulate the outer cortical cells to increase the DNA content of the nuclei, as a result of which, the chromosome compliment becomes polypoid.  Repeated division of these polypoid cells form nodules.

Bacteria in the nodules secrete a growth hormone called indole acetic acid (IAA).  It stimulates the nodules to grow in size.  As a result, they become effectively supplied with vascular tissue.

Bacterial cells multiple rapidly inside infected host cell. The cells as a result are  transferred into swollen form called bacteroides.  The functional nodule contains a reddish pigment called leghemoglobin (hemoglobin of legumes). It is a oxygen scavenger and is related to blood pigment haemoglobin. Leghemoglobin protects nitrogen fixing enzyme nitrogenase from oxygen.

This pigment appears to be an oxygen carrier in the process of nitrogen fixation in nodules. Hence, it facilitates the diffusion of oxygen to the vigorously respiring bacteria within the cells of nodules, thereby stimulating the production of ATP.  Nitrogen fixation requires ATP.  Therefore, cells lacking leghemoglobin are not capable of fixing nitrogen.

Nitrogen fixation (Nodulation in legumes)

Fig: Nodulation in legumes

Industrial nitrogen fixation:

In various fertilizer industries, compression of atmospheric N2 and H2  takes place at a particular temperature and pressure to produce ammonia (NH3) by Haber’s process.  NH3 then converts into different fertilizers including urea.