Mechanism of Biological Nitrogen Fixation

Nitrogen Fixation

Roots of a legume secrete chemical attractants (flavonoids and betaines). Bacteria collect over the root hairs and release nod factors that cause curling of root hairs, around the bacteria, degradation of cell wall and formation of an infection thread enclosing the bacteria.

  • Infection thread grows along with the multiplication of bacteria and reaches opposite protoxylem points of the vascular strand. The infected cortical cells differentiate and start dividing to produce swellings called nodules.
  • Nodule formation is stimulated by auxin produced by cortical cells and cytokinin liberated by invading bacteria. The infected cells enlarge and bacteria stop dividing and from irregular polyhedral structures called bacteroids.
  • In an infected cell, bacteroids occur in groups surrounded by the host membrane and develop a pinkish pigment called leghaemoglobin. It is an oxygen scavenger and protects nitrogen-fixing enzyme nitrogenase from oxygen.
  • Symbiotic nitrogen fixation requires the co-operation of Nod genes of legume, ‘nod’, ‘nif’ and ‘fix’ gene clusters of bacteria.
  • Enzyme nitrogenase has iron and molybdenum which take part in the attachment of a molecule of nitrogen (N2). This attachment leads to the weakening of bonds between two atoms of nitrogen.
  • The weakened molecule of nitrogen is acted upon by hydrogen from a reduced coenzyme and hence produces diamide (N2H2), hydrazine (N2H4) and ammonia (NH3).
  • Ammonia, which is toxic even in small quantities is not liberated. It reacts with organic acids to give rise to amino acids.

Free Living Nitrogen Fixers

Free-living nitrogen fixers do not immediately enrich nitrogen. It is only after the death and decomposition of organisms, that the fixed nitrogen enters the cycling pool.

It occurs in two steps:

  1. Ammonification: It is carried out by ammonifying bacteria. They act upon nitrogenous excretions and proteins of dead bodies of organisms. Proteins are first broken up into amino acids which are deaminated to release organic acids. Organic acids are used by microorganisms for their own metabolism.
  2. Nitrification: It is the process that converts ammonia to nitrite and then nitrate.
    It is performed in two steps:
    1. Nitrite formation: in this ammonia is oxidized to nitrite by nitrosococcus, Nitrosomonas, etc.
    2. Nitrate formation: in this, nitrite (NO2–) is oxidized to nitrate (NO3–) by Nitrobacter, microcystins, etc.

Denitrification

  • Denitrification: under anaerobic conditions (e.g., waterlogging, oxygen depletion), some microorganisms use nitrate and other oxidized ions as a source of oxygen. In the process, nitrates are reduced to gaseous compounds of nitrogen. Then later escape from the soil. 
  • Common bacteria causing denitrification of soil are pseudomonas denitrificans, thiobacillus denitrificans, micrococcus denitrificans.

Nitrate Assimilation

  • Nitrate assimilation in plants: the nitrates absorbed by plant roots get converted to amino acids, and amides before incorporating into proteins and other macromolecules. The reduction of nitrate into ammonia is called nitrogen assimilation.
    It occurs in two steps:
    1. Reduction of nitrate to nitrite: it is carried out by the agency of an inducible enzyme called nitrate reductase.
    2. Reduction of nitrite: it is performed by enzyme nitrite reductase.
  • Ammonia is not liberated. It combines with some organic acids to produce amino acids. Amino acids then form various types of nitrogenous compounds.
  • Synthesis of amino acids: Amino acids are the first organic compounds of nitrogen assimilation. They are synthesized by:
    1. Reductive amination: the ammonia, formed by nitrogen assimilation (i.e., reduction of nitrates), reacts with α-ketoglutaric acid to form the amino acid- glutamic acid. The reaction occurs in presence of the enzyme glutamate dehydrogenase.
    2. Transamination: It transfers amino groups of one amino acid with the keto group of a keto acid. The enzyme required is transaminase or aminotransferase.

Ammonia Assimilation

Assimilation of ammonia in most of the higher plants involves the formation of amides.

  • Amides are derivatives of amino acids in which the –OH component of the carboxylic group is replaced by another amino group (-NH2).
  • They are generally formed by the combination of ammonia and amino acids. 
  • The two most common amides found in plants are glutamine and asparagine, composed of proteins and amino acids.
  • Amides perform two other functions- storage of excess nitrogen and transport.
  • The amino acids produced from the above-mentioned processes from amino acid chains or polypeptide over ribosomes during the process of translation of genetic code carried by mRNAs.
  • Polypeptides give rise to proteins that are utilized by the plants. 
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