Nitrogen fixation by free living organisms and symbiotic nitrogen fixation

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Nitrogen fixation is a distinctive property possessed by a select group of organisms, because of the presence of the enzyme nitrogenase in them. The process of nitrogen fixation is primarily confined to microbial cells like bacteria and cyanobacteria. These microorganisms may be independent and free living.

Some free living microbes which fix nitrogen

Image showing some free living microbes which fix nitrogen.
Image showing some free living microbes which fix nitrogen.




Anaerobic bacteria (Non-photosynthetic)


Facultative bacteria (Non-photosynthetic)


Aerobic bacteria (Non-photosynthetic)


Purple, non-sulphur bacteria (Photosynthetic)


Cyanobacteria (Photosynthetic)

Some microbes may become associated with other organisms and fix nitrogen. The host organism may be a lower plant or higher plant. The host organism and the nitrogen fixing microbes establish a special relationship called symbiosis and this result in symbiotic nitrogen fixation.

Some symbiotic nitrogen fixing organisms

Image showing some symbiotic nitrogen fixing organisms.
Image showing some symbiotic nitrogen fixing organisms.




Cyanobacteria and Fungus


Cyanobacteria and Anthoceros


Cyanobacteria and Azolla


Cyanobacteria and Cycas


Legumes and Rhizobium


Non-leguminous and actinomycetes

(such as Almus, Myrica, Purshia)


Brazilian grass (DIgitaria), Corn and Azospirillum

Mechanism of Biological fixation of Nitrogen

Nitrogen fixation requires;

-the molecule nitrogen,

-a strong reducing power to reduce nitrogen like reduced FAD (Flavin adenine dinucleotide) and reduced NAD (Nicotinamide Adenine dinucleotide),

-a source of energy (ATP) to transfer hydrogen atoms from NADH2 or FADH2 to dinitrogen,

-enzyme nitrogenase,

-compound for trapping the ammonia formed since it is toxic to cells.

The reducing agent (NADH2 and FADH2) and ATP are provided by photosynthesis and respiration.

The overall biochemical process involves stepwise reduction of nitrogen to ammonia. Biological nitrogen fixation is the process in which certain living organisms convert the free nitrogen into those compounds which can be used by plants. The organisms may be free living bacteria (e.g. Azotobacter), symbiotic bacteria like Rhizobium or cyanobacteria. The enzyme involved in the process is nitrogenase, which is a Mo-Fe protein. During this process, the atmospheric nitrogen is reduced by the addition of hydrogen. The three bonds between the two nitrogen atoms (N=N) are broken and hydrogen is progressively added to form ammonia. The fixation requires three components; ATP, to supply energy, a strong reducing agent, to transfer hydrogen atoms and the enzyme system (nitrogenase).

NH3 is not liberated by the nitrogen fixers. It is toxic to the cells and therefore these fixers combine NH3 with organic acids in the cell and form amino acids. The overall reaction is as follows:

Molecular nitrogen is a very stable molecule. Therefore, sufficient amount of cell energy in the form of ATP is required for stepwise reduction of nitrogen to ammonia.

Soil bacteria called rhizobia are symbiotically interact with legume roots to form specialized structures called nodules in which nitrogen fixation takes place. The biochemical steps for nitrogen fixation are same. Legume nodules possess a special protein called leghemoglobin, which is hemoglobin like red pigment and is essential for nitrogen fixation. Leghemoglobin color gives root nodules a pinkish cast. Leghemoglobin strongly binds oxygen to maintain bacteroid respiration without shutting down the action of nitrogenase. Leghemoglobin is a group of proteins called nodulins are also synthesized which help in establishing symbiosis and maintaining nodule functioning.

Leghemoglobin is produced as a result of interaction between the bacterium and legume roots. Apparently, Rhizobium gene codes for theme part and legume root cell gene codes for Globin moiety. Both the coded products together constitute the final protein leghemoglobin.

Image showing flowsheet of biochemical steps for nitrogen fixation.

Flowsheet of Biochemical Steps for Nitrogen Fixation

Image showing flowsheet of biochemical steps for nitrogen fixation.

Specific functions for legumes only because free living microbes do not possess nitrogen fixing leghemoglobin. However, it has also not been found in cyanobacterial symbiosis with other plants.

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