Biological Nitrogen Fixation

Biological Nitrogen Fixation:-

The conversion of atmospheric nitrogen into the nitrogenous compounds by living organisms is called biological nitrogen fixation. Only prokaryotes can fix nitrogen. Nitrogen fixation require anaerobic conditions because oxygen inactivates nitrogenase enzyme. 
Hence for obligate anaerobes nitrogen fixation is easy, but in case of facultative anaerobes the nitrogen fixation occurs only in anaerobic conditions. In case of obligate aerobes the oxygen level inside the cell must be kept low for nitrogen fixation.
1. Nitrogen Fixers (Diazotrophs):- Among the earth’s organisms, only some prokaryotes like bacteria and cyanobacteria can fix atmosphere nitrogen. They are called nitrogen fixers or diazotrophs. They fix about 95% of the total global nitrogen fixed annually by natural process.
a. Asymbionts (Free living)
b. Symbionts
a. Asymbionts (Free living):-
i. Bacteria:- They add up to 10-25 kg, of nitrogen/ha/annum.
Azotobacter (Aerobic, Saprophytic)
Beijerinckia (Aerobic, Saprophytic)
Clostridium (Anaerobic, Saprophytic)
Desulphovibrio (Chemotrophic)
Rhodopseudomonas (Photoautotrophic)
Rhodospirillum (Photoautotrophic)
Chromatium (Photoautotrophic)
ii. Blue Green Algae (Cyanobacteria):- Heterocysts are the special cells that fix nitrogen. They add 20-30 kg Nitrogen/ha/annum.
Nostoc
Anabaena
Aulosira:- A. fertilissima is the most active nitrogen fixer in Rice fields.
Cylindrospermum:- It is active in sugarcane and maize fields.
Trichodesmium
b. Symbionts:- Live in close symbiotic association with other plants.
i. Bacteria:- 
Rhizobium:- It is aerobic, gram negative nitrogen fixing bacterial symbionts of legume roots. Sesbania rostrata has Rhizobium in root nodules and Aerorhizobium in stem nodules. 
Frankia:- It is symbiont in root nodules of many non-leguminous plants like Casuarina and Alnus.
Xanthomonas and Mycobacterium:- They occur as symbiont in the leaves of some members of the families Rubiaceae and Myrsinaceae (e.g., Ardisia). 
ii. Blue Green Algae (Cyanobacteria):-
Nostoc and Anabaena:- They are common symbionts in lichens, Anthoceros, Azolla and Cycas roots. 
Anabaena azollae:- It is found in fronds of Azolla pinnata (a water fern). It is often inoculated to Rice fields for nitrogen fixation.
2. Rhizobium Nitrogen Fixation:-
Rhizobium bacteria:-
i. Free living
ii. Gram negative
iii. Aerobic
iv. Soil bacteria
> Rhizobium becomes anaerobic upon entry into roots. 
Leghaemoglobin (legHb or symbiotic Hb):- 
- It is a pink coloured pigment.
- It occurs in the root nodules of leguminous plants. 
- It acts as an oxygen scavenger. It provides anaerobic conditions for the nitrogenase enzyme and protects the enzyme from inactivation.
Two main steps:-
a. Nodule formation
b. Nitrogen fixation
a. Nodule formation:- Root nodule formation is initiated, when the soil contains a low level of nitrogen. Steps of nodulation are:
i. Aggregation:- Roots of legumes secrete flavonoids, which attracts rhizobia towards the root. Rhizobia aggregate around root hairs.
ii. Developmental changes:- Rhizobia secrete nod factors, which causes stimulate many developmental changes:
- Membrane depolarization
- Curling of root hairs 
- Cell division in the root cortex 
- Intracellular calcium movement
iii. Infection thread:- The nod factor attaches to receptors present on the plasma membrane of the root hairs, which leads to the formation of the infection thread. 
iv. Entry:- Infection thread provides the passage to bacteria to enter epidermal cells. Rhizobia then enter cortex cells, each bacterium gets surrounded by a plant-derived membrane known as symbiosome.
v. Nodulation:- Nodule formation is initiated by chemicals produced by rhizobia. It is a result of calcium dependent signal transduction pathway, which triggers biochemical changes leading to cell division and nodule formation. Cytokinin also plays an important role in nodules formation.
vi. Bacteroids:- Within nodules, bacteria get differentiated into bacteroids, which fix nitrogen. The Rhizobia stop dividing, loose cell wall and become nitrogen fixing cells as bacteroids . Vascular tissues are developed for nodules for exchange of nutrients.
b. Nitrogen fixation:-
- The nodule serves as site for N2 fixation. 
- Nodule contains nitrogenase and leghaemoglobin. 
- The nitrogenase has 2 components:
i. Molybdoferredoxin (Mo-Fe protein)
ii. Azoferredoxin (Fe-protein)
- The free di-nitrogen first bound to MoFe protein and is not released until completely reduced to ammonia. 
- In this process ferredoxin serves as an electron donor to Fe-protein (nitrogenase reductase) which in turn hydrolyzes ATP and reduce Mo-Fe protein, the Mo-Fe protein in Turn reduce the substrate N2. The electrons and ATP are provided by photosynthesis and respiration of the host cells.
- Many intermediates are formed to form ammonia (NH3).
Dinitrogen → Hydrazine → Diamine → Ammonia
- Ammonia (NH3) is immediately protonated at physiological pH to form ammonium ion (NH4+). As NH4+ is toxic to plants, it is rapidly used near the site of generation to synthesize amino acids.