Nitrogen is an essential element for life. Biological nitrogen fixation is the process by which nitrogen-fixing bacteria and archaea convert atmospheric nitrogen into organic nitrogen compounds. This process occurs through a complex biochemical process involving the nitrogenase enzyme. It is an important natural process that adds nitrogen to soil and makes it available to plants. Some key points are: 1) Certain bacteria, archaea, and some plants like legumes form symbiotic relationships where the microbes in root nodules or other tissues fix nitrogen for the plant. 2) Free-living microbes like cyanobacteria also fix nitrogen, enriching soil and ecosystems. 3) The nitrogen fixed enters the nitrogen cycle and is used by other

1. B.Sc. II, Sem. III
NITROGEN METABOLISM
Dr. Seema Gaikwad
Dept. of Botany
Vidnyan Mahavidyalaya, Sangola

2. Nitrogen Metabolism
Introduction
Nitrogen is important element of life. In importance it comes
only next to carbon, hydrogen, and oxygen. The composition of
protein, nucleic acid, growth hormones, and vitamins requires
Nitrogen. Leaves consist of about 1 to 15% nitrogen of their
dry weight but lesser % in another vegetative organ.

3. Introduction---
• The N2 is present in the atmosphere, in the form of gas. It is about 78%.
• Green plants unable to use this N2 directly in their metabolism. Only some micro-organism can
convert this N2 gas directly into organic form.
• The N2 present in the soil is called soil nitrogen. The plants growing in the soil, mainly utilize
the soil N2 for their metabolic requirements.
• In the soil the nitrogen is present in the form of nitrate nitrogen (NO3, NO2), ammonia nitrogen
(ammonia, ammonium salt), organic nitrogen and molecular nitrogen (N2).
• The converging of the free nitrogen, by natural or physical process is called nitrogen fixation…
when any biological system is involved in this process, then it is called as biological nitrogen
fixation……
• In this process of biological N2 fixation, atmospheric N2 is converted into ammonia. The nitrate
N2 absorbed by the plants is converted into ammonia by a process known as nitrate
reduction…
• For the synthesis of different organic compound, both nitrates (NO3), and ammonia (NH4) are
assimilated in plants with the help of specific enzyme. The two imp enzymes in the process are
nitrate reductase and nitrogenase…
• Only some micro-organism or prokaryotic cell can carry out biological N2 fixation, because
only these cells show presence of enzyme nitrogenase, required for process. These prokaryotic
cells have specific genes in their genome, which carry out synthesis and regulation of enzyme
nitrogenase. These genes are associated with N2 fixation, are known as nif genes…..

4. Nitrogen cycle

5. Nitrogen cycle---
•When animal wastes and dead organic remains of plant
and animal are added in the soil, their decomposition
begins due to action of micro-organisms. In this process,
some micro-organisms convert organic nitrogen into
ammonia and ammonium salt, by a process called
ammonification. By this process ammonia and
ammonium salt are added in the soil and are made
available to the plants and other micro-organism.

6. Nitrogen cycle---
• Some micro-organism convert ammonia and ammonium salt into nitrates by
a process known as nitrification. Nitrates added in the soil are mainly utilize
by the plants. So the three process namely nitrogen fixation, ammonification
and nitrification together add large amount of nitrogen in the soil and made
it available to the plants..
• Nitrates present in the soil are utilized by some micro- organisms
to produce nitrogen gas by a process called denitrification. This
denitrification produce N2 gas in the soil, which is released in the
atmosphere. This process is, responsible for returning the N2 back to the
atmosphere.
• All these process indicate that the nitrogen in the atmospheric
which enters the biomass through the soil. So atmospheric N2 completes a
cycle forms atmosphere, through soil, biomass and back to the atmosphere.
This cycle is known as ‘ Nitrogen cycle’

8. Nitrogen cycle---
 The nitrogen cycle is a complex pattern of exchange of nitrogen, the N2 present in the environment is distributed between 3
constituents, namely surrounding atmosphere, soil and biomass. The center for exchange of atmospheric N2 within these three
constituents is the soil.
 The soil fertility is directly proportional to the amount of available N2 in the soil. In case of land under cultivation, the soil
fertility is maintained by adding fertilizers. While in case of land not utilized for cultivation (forest land) the soil fertility is
maintained by some natural process.
 Two such imp processes are physical N2 fixation and biological N2 fixation.
 The process of physical nitrogen fixation occurs during electrical discharges and thunder-storms in rainy season. During this
process initially atmospheric N2 combines with oxygen, under the influence of electric discharges and thunder to produce nitric
oxide.
 Further, oxidation of nitric oxide produces nitrogen peroxide. When this N2 peroxide mixes with rain water, forms nitrous acid
and nitric acid.
 In the form of nitric acid, the atmospheric nitrogen is added in the soil. The alkaline substances in the soil react with nitric acid
and release nitrates and nitrites. These are water soluble and can be absorbed by the plants.
 This physical nitrogen fixation process adds about 5 to 10% of nitrogen in the soil. Remaining 90% of the soil nitrogen is
added by biological nitrogen fixation process and activities of micro-organism in the soil.

9. Biological Nitrogen fixation
• The conservation of atmospheric free N2 into organic form is known as Nitrogen
Fixation and when it is carried out whith the help of a biological system(organism), it
is called biological nitrogen fixation. Biological nitrogen fixation is one of the most
important processes in nitrogen cycle, responsible for addition of nitrogen in the soil.
• The green plants, except blue green algae, are eukaryotic organism. These eukaryotic
organism, though considered as evolved, do not have the appropriate
machinery(enzymes) to fix atmospheric N2. Bacteria and blue green algae, which are
with prokaryotic, relatively primitive cells, have the machinery to fix atmospheric
nitrogen.

10. Biological Nitrogen fixation ---
• Biological N2 fixation is a complex biochemical and physiochemical process. The prokaryotic
organisms that carry out N2 fixation are called nitrogen fixers. The nitrogen fixing organism may
be free living (asymbiotic ) or symbiotic. some nitrogen fixers are anaerobic, while some are
aerobic organisms. Some N2 fixing organisms are photosynthetic (e.g.- Blue green algae) while
some are non- photosynthetic (e.g.- root nodule bacteria).
• The N2 fixers can broadly be divided into two categories- a) bacteria b) Blue green algae
(BGA). Some fungi can also carry out N2 fixation (e.g- pink yeast). These N2 fixers carry out
fixation of nitrogen either independently or with the help of some other organisms. Free living N2
fixers carry out N2 fixation by asymbiotic mechanism, while symbiotic organism carry out N2
fixation by symbiotic mechanism.

11. A. Non-symbiotic Nitrogen fixation
Nitogen fixation by free living micro-organism was identified in 19th century
by Winogardsky(1894). He isolated free-living anaerobic bacterium
Clostridium pastorianum, which is a nitrogen fixing organism, The
nonsymbiotic nitrogen fixers can broadly be classified into three categories-
1. Bacteria 2. Blue green algae 3. Fungi
1. Bacteria:- Many free living bacteria have the ability to fix molecular nitrogen.
These bacteria require ions of minerals like Mo+, Cu+2, Fe+2 for efficient N2
fixation. The free living bacteria may be aerobic, anaerobic or photosynthetic.

12. Non-symbiotic Nitrogen fixation
The aerobic bacteria belongs to genera Azotobactor, Derxia, Mycobacterium,
Azomonas,Arthbacter etc. They are widely distributed in the soil and water. These bacteria show
rapid growth and high rate of N2 fixation. Azotobactor under fevourable situation may add on an
average 10 kg of N2/ hectare/ year to soil.
The anaerobic facultative bacteria belongs to genera Aerobactor,
Klesiella,Chlorobium,Chromatium,Rhodomicrobium, Metanbacterium. They are present in the
soil and are highly resistant to advers conditions. They grow rapidly with moisture and nutrients
and add more N2 to the soil.
The photosynthetic N2 fixing bacteria are the species of sulphur bacteria.
Rhodomicrobium, Rhodopsuedomonas, They fix N2 in presence of sulphur as oxidant. They are
abundant in soil and warer but their growth is limited due to lack of light.
Among the above mentioned different N2 fixing genera Clorobium, Azotobacter
are more important. These bacteria prefer different habitats with varying nutrient level, pH,
oxygen, temprature , soil depth, season, water etc. For the optimum growth. The bacteria reduce
nitrogen to ammonia by producing different intremidiate compounds as shown by following
reaction.
Dinitrogen 2H+ Diamide 2H+ Hydrezin 2H+ Ammonia
2e- 2e- 2e-

13. 2) Yeast:-
The role of yeast in nitrogen fixation is least known however pink yeast Rhodotorula sp. isolated from
soil shows N2 fixing ability.
3) Blue Green Algae (BGA):-
BGA constitute an important group of the micro-organism capable
of fixing aerial N2. The algal members may be unicellular or filamentous in nature . The blue
green algal genera like Anabaena, Anabaenopsis, Aulosira, Nostoc, Haplosiphon, Stigonema,
Chlorogloea and Mastigocladus fix biological nitrogen.
BGA appears to be main, dominant group that fixes nitrogen in rice fields. In fresh
water lakes BGA may fix 2 to 5 kg N2/ hectare/ year.
The N2 fixation in blue green algae is associated with a special cell i.e. heterocyst.
The heterocyst ate larg, thick walled, apparently empty cells. The N2 fixing genera are localized
in the heterocyst and the activity of enzyme nitrogenase is observed in these cells.
Similarly non – heterocystous, filamentous blue green algae like Plectonema, Lyngbya
, Oscillatoria and Trichodesmium can fix atmospheric nitrogen.

14. B. Symbiotic Nitrogen Fixation
These micro-organism establish symbiotic relationship with different plants. The
symbiotic relationship between higher plant root and soil bacteria is quiet known. The
leguminous plant should be sown not only for their yield but for the benefit of next
grown crops, because , the residue of leguminous plant enriches the soil by adding
N2.The leguminous plant get N2 due to bacteria present in the root nodules. In this
symbiosis, bacteria provide fixed N2 to the host plant and receive soluble
carbohydrate from host. The symbiotic N2 fixing organisms are mainly root nodule
bacteria of leguminous plant, bacteria with non-leguminous plant and blue green
algae.

15. Leguminous plant with root nodule bacteria
• Leguminous crops fix atmospheric N2 with the help of nodule bacteria and they do not have self
ability to fix N2. i.e in association with viable bacterial strains only they can fix nitrogen . In this
symbiotic association between legume host and the bacterium both the partners are mutually
benefited, through formation of nodules on root. There are only 10 to 12 % plants of Leguminosae
shows presence of root nodules.
• The root nodule bacterium was isolated by Beijrinck (1888) and named it as Rhizobium. The bacteria
belonging to this genus are free living in the soil. Rhizobium is gram –ve , non-sporulating, aerobic,
motile, rod shaped, nodule forming bacterium. It is very specific in infecting the host i.e legume plant.
There are seven different groups recorded for species of Rhizobium and their host. Rhizobia can
grown well with supply of carbohydrates, vitamins, calcium and neutral pH under the laboratory
conditions.
• The root nodule bacteria can fix 100 to 200 times more N2 than free living organisms. The Rhizobia
are very specific in infecting the host. Establishment of Rhizobium inside the specific legume root and
development of the nodule is a complex process, but essential for N2 fixation.

16. Mechanism of Biological Nitrogen fixation
• Healthy root nodules in leguminous plants are pink in colour due to red pigment. This pigment is
being red due to iron and it is called leghaemoglobin. The healthy nodules with leghaemoglobin can
fix N2. Leghaemoglobin is present out side the bacterial cell but close to membrane and acts as oxygen
remover Leghaemoglobin regulatates oxygen concentration and favours nitrogenase activity in
swollen form of bacterium ( essential for N2 fixation) called bacteroid. Leghaemoglobin combines
with O2 to form oxyhaemoglobin and makes low concentration of oxygen in nodules as essential for
activity of nitrogenase. It is supposed that Leghaemoglobin takes part in nitrogen fixation, indirectly
by protecting nitrogenase from adverse effects of oxygen.
• The nitrogenase activity is directly associated with metaloproteins, nitrogenase Mo- Fe protein and
nitrogenase Fe- protein. The reduction of nitrogen is found associated with electron and hydrogen
donor through ferredoxin or other reducing agent. ATP is also essential for the process. Nitrogen
reduced to diamide, hydrizine and finally to ammonia. Later on ammonia is used for the synthesis of
amino acids which are utilised by plants.

17. Non – leguminous plant with nodule
• There are many non-legume plants which also fix N2 symbioticlly. These non-legume,
root nodule N2 fixing angiosperms belong to 15 genera of woody and herbaceous
nature. These genera are Coriaria, Alnus, Myrica, Casurina, Hippophae, Elaeagnus,
Dryas, Purshia, Shephardia, Coenothus, Cerocarpus, and Arctostaphylos.
• Genus Frankia of actinomycetes fix large amount of nitrogen in root nodules of forest
plant like Alnus and Casuarina. It adds about 61.5 to 157 kg. nitrogen/ hectare/ year
and 60 kg. nitrogen / hectare / year repectively.
• Leaf nodules which are confined to members of Rubiaceae (e.g. Pavetta) and
Myrsinaceae,( e.g. Ardisia) are due to Mycobacterium and Flavobacterium, growing
in these nodules.

18. Blue green algae in symbiosis
• BGA members show symbiotic association with fungi, liverworts, mosses, ferns,
gymnosperm and angiosperms where the nitrogen fixed by algae is provided to hosts.
• Algae – fungal assosiation occurs in nature in the form of Lichens. The algal genera like
Nostoc, Calotrix.fix N2 in Lichen genera like Collema, Leptogium, Lobaria, Pannaria,
Parmelia, shows nitrogenase activity. Algal members provide N2 in the form of amino acids
to fungal partner for its growth.
• The fern Azolla shows association with algae Anabaena. Algae reside in the cavities present
on the dorsal lobe of Azolla leaf. This endophytic algae fix atmospheric nitrogen and provides
to fern.
• The species of Nostoc and Anabaena are present in coralloid root of gymnosperm genera like
Cycas, Zamia, Macro zamia, Ceratozamlia and Stangeria.

19. Significance of biological Nitrogen fixation
1. In most undisturbed non-agricultural and some agricultural ecosystems– soluble nitrogen (ammonium) is made available to
plants via a process known as biological Nitrogen fixation, in which bacteria that contain the enzyme complex called
Nitrogenase ( termed ‘Dizotrophs’) can fix atmospheric N2 into ammonia (NH3).
2. This ammonia which is potentially toxic to the organism, is usually then immediately converted into amino acids or amodes
for use by the dizotrophic bacterium in the production of proteins & peptides to facilitate its growth.
3. The fixed N incorporated into dizotrophic bacteria is then released into the environment when they die, usually in the form of
amino acids that then become mineralized & available for uptake by other bacteria & by plants.
4. Many legumes supportive of bacterial N2 fixation through the development of a specialized structure called the nodule.
5. The reactions of Nitrogen fixation are limited to the prokaryotic microorganisms as nitrogenase genes have only, been found
there. These genes which are associated with nitrogen fixation, are known as nif genes.
6. Microorganisms have a central role in almost all aspects of nitrogen availability and thus for life support on earth. Some
bacteria can convert N2 into ammonia by the process nitrogen fixation, some other bring about transformations of ammonia to
nitrate while many bacteria & fungi degrade organic matter, releasing fixed nitrogen for reuse by other organisms.
7. Some of the blue green algae increase the fertility of soil by fixing atmospheric nitrogen.