Introduction

We all know how important nitrogen is to plants, but plants cannot directly take nitrogen from the atmosphere. Biological nitrogen fixation is a process by which nitrogen is fixed in plants with the help of certain anaerobic bacteria (works in the absence of oxygen). So in the following section, we will study the whole process of how these anaerobic bacteria fix nitrogen in plants and the whole process with certain conditions necessary for nitrogen fixation.

• Nitrogen is also a limiting nutrient for both natural and agricultural ecosystems because plants compete with microbes for the limited nitrogen available in the soil.

Nitrogen fixation is of two types: 

1. Abiological nitrogen fixation
2. Biological nitrogen fixation

Abiological nitrogen fixation:

This is also known as physical nitrogen fixation. The conversion of free nitrogen into nitrogenous compounds without any help of biological means is known as abiological nitrogen fixation. 

It is of two types:

1. Natural/Electrical
2. Industrial/Artificial 

Natural/Electrical:

Atmospheric N2 + Atmospheric O2 in the presence of energy from UV radiations and lightning converts to Nitrogen Oxides (NO, NO2, N2O).

Nitrogen oxides (NO, NO2, N2O) + water vapours                             

HNO3, HNO2, HNO

HNO3 NO3-+ H+; HNO NO- + H+; HNO2 NO2-+ H+

These NO2-, NO3- and NO- are uptake by plants and utilised.

Industrial/Artificial

Haber method of NH3 preparation:

N2 + 3H2→ 2NH3

This ammonia is further incorporated into urea. Urea is used as a fertiliser by farmers.

Biological nitrogen fixation

Biological nitrogen fixation is a process by which nitrogen is fixed in plants with the help of certain anaerobic bacteria. Biological nitrogen fixation was discovered by Dutch microbiologist Martinus Beijerinck in 1901. Only certain organisms like prokaryotes, such as bacteria and cyanobacteria, can fix atmospheric nitrogen. We call these bacteria and cyanobacteria nitrogen fixers or diazotrophs. They fix 95% of the total global nitrogen only by natural processes. They utilise the enzyme nitrogenase to catalyse the conversion of atmospheric nitrogen (N2) to ammonia (NH3). Plants can readily assimilate NH3 to produce the aforementioned nitrogenous biomolecules.

1. N2 in the presence of nitrogenase converts into NH3.

(a). Free-living nitrogen-fixing bacteria fix nitrogen in a free-living way.

Examples: Azotobacter, Beijernickia, Rhodospirillum, Bacillus, Clostridium, Rhodopseudomonas, etc.

(b). Symbiotic nitrogen-fixing bacteria: These nodules are small outgrowths on the root.

(c). By loose symbiotic bacteria: This is symbiosis without nodule formation, e.g., Azospirillum with maise root.

2. By Cyanobacteria or Blue-Green Algae: They fix nitrogen inside specialised cells called heterocysts. BGA fix nitrogen in two ways:

(a). Free-living nitrogen-fixing BGA: They fix nitrogen without the help of the host. E.g., Nostoc, Anabaena, Oscillatoria, Aulosira, Cylindrospermum etc. 

(b). Symbiotic nitrogen-fixing BGA: They fix nitrogen with the symbiotic association. BGA makes symbiotic association with almost every group of plants in the Plantae kingdom.

Biological Nitrogen Fixation

This process can be done in selected stages:

Stage-I: Rhizobium contacts a susceptible root hair. 

• This attraction is chemotactic (rhicadhesin protein of bacterial cells identifies host root). To attract rhizobia, the root of the legume releases amino acids, sugars, organic acids, and flavonoids.

Stage-II: Bacteria invade the root hair (infection). 

Curling of root hair is induced by specific complex polysaccharides found on the surface of rhizobia, recognised by lectins (small protein of host plant root).

The bacteria get modified into rod-shaped Bacteroides and cause inner cortical and pericycle cells to divide.

For dedifferentiation of cortical and pericycle cells, PGRs (auxin and cytokinin) are required. Auxin is provided by the host root, while bacteria provide cytokinin.

Mechanism of Biological Nitrogen Fixation

Requirements for biological nitrogen fixation:

1. Substrate – Nitrogen
2. An enzyme – Nitrogenase
3. Source of energy (ATP)

• During free-living nitrogen fixation, the prokaryote itself is a source of ATP.
• During symbiosis, ATP is obtained from the respiration of the host cells.

4. Source of reduction power (NADH)

• During free-living nitrogen fixation, prokaryote itself is the source of NADH.
• During symbiosis, NADH is provided by the respiration of host cells.

5. Oxygen-free environment

• The enzyme nitrogenase is highly sensitive to molecular oxygen; thus, its operation/activation requires anaerobic conditions. To protect this enzyme, the nodule contains an oxygen scavenger called leg-haemoglobin.
• Leg-haemoglobin (holoprotein) is a joint product of both plants and the bacterium, in which the globin (apoprotein) is produced by the plant (legume), and the heme (an iron atom bound in a porphyrin ring) is produced by the bacterium (Rhizobium).

6. Genes (nod, nif, fix): Nod genes present in both plant and bacterium while nif (nitrogen introducing factor) and fix present only in bacteria.

The fate of ammonia

At physiological pH, the ammonia is protonated to form NH4+(ammonium) ions. There are two main ways NH4+ synthesises amino acids in plants.

1. Reductive amination: In this process, the ammonia formed by nitrogen assimilation reacts with -ketoglutaric acid to form the amino acid-glutamic acid. The reaction occurs in the presence of glutamate dehydrogenase.
2. Transamination: It is the transfer of an amino group of one amino acid with the keto group of keto acid
3. Ammonification: In this process, proteins are first broken up into amino acids, then deaminated to release organic acids. Microorganisms use organic acids for their metabolism.
4. Nitrification: It is performed in two steps:

• Nitrite formation: In this step, ammonia is oxidised to nitrite by Nitrosococcus, Nitrosomonas, etc.
• Nitrate formation: In this step, nitrite is oxidised to nitrate by Nitrobacter, nitro cystitis, etc.

5. Denitrification: Nitrate present in the soil is also reduced to nitrogen. This process is called denitrification. Denitrification is carried out by bacteria Pseudomonas, Thiobacillus, and high-energy radiations.

Symbiotic nitrogen fixation

Symbiotic nitrogen fixation is the exchange between plants and bacteria, in which one provides a niche and one provides fixed nitrogen. For example, Symbiotic biological nitrogen fixation can be categorised into the following groups:

1. Nitrogen Fixation in leguminous plants: 

Symbiotic nitrogen fixers in several legume plants include the genus Rhizobium mainly. They established themselves inside specialised structures on the roots called root nodules. The bacteria fix nitrogen only when they are present inside the nodules. The association is regarded as symbiotic because the nodule bacteria is supplied by the host plant with the organic carbon (carbohydrates). The microorganisms then supply fixed nitrogen to the host plant. 

2. Nitrogen Fixation via nodule formation in the non-leguminous plants: 

Many plants belonging to families other than Leguminosae are known to produce root nodules. The important among them are primarily trees and shrubs. 

The key examples of non-leguminous plants that fix nitrogen and produce root nodules are: 

1. Genus Frankia produces root nodules associated with Alnus, Myrica gale, Casuarina equisetifolia, etc. 
2. Rhizobium also has root nodules in the genus Parasponia. 
3. Leaf nodules are formed by bacteria Klebsiella in the genus Psychotria and by bacteria Burkholderia in genus Pavetta zimermanniana.
4. Nitrogen Fixation through Non-nodulation: In some plants, symbiotic nitrogen fixation occurs, but nodules are not formed. Such associations are Pseudo symbiotic (Pseudo Symbiosis). Some of the examples are: Anthoceros, associated with Nostoc; Azolla, living in association with Anabaena.

Conclusion

 The process of converting atmospheric nitrogen into nitrogenous compounds by living organisms is called biological nitrogen fixation.Nitrogen is necessary for plant growth and development, but it is in short supply in its most common form, atmospheric nitrogen. Plants, on the other hand, rely on compounded or fixed nitrogen sources like ammonia and nitrate. Much of this Nitrogen is supplied to cropping systems in the form of nitrogen fertilisers manufactured in factories. The use of these fertilisers has resulted in a slew of global environmental issues, including the establishment of coastal dead zones. On the other hand, biological nitrogen fixation provides plants with a natural source of nitrogen. It’s an important part of many aquatic and terrestrial ecosystems all around the world.