Nitrogen is an essential element of many biomolecules, the most important being nucleic acids and amino acids. Although nitrogen is the most abundant gas (about 80%) in the atmosphere, neither animals nor plants can use this nitrogen to synthesize biological compounds. However, there are certain microorganisms on which the living plants (and animals) are dependent to bring nitrogen into their biological systems.The phenomenon of fixation of atmospheric nitrogen by microorganisms is known as diazotrophy and these organisms are collectively referred to as diazotrophs. Diazotrophs are biological nitrogen fixers, and are prokaryotic in nature.

1. NItrogen Metabolism
Dr. Naveen Gaurav
Associate Professor and Head
Department of Biotechnology
Shri Guru Ram Rai University
Dehradun

2. Nitrogen Fixation in Plants: Certain beneficial microorganisms, present in the soil, are
known to influence the plant growth, development and yield. These bacteria and fungi
may provide growth-promoting products to plants or inhibit the growth of soil pathogenic
microorganisms (phytopathogens), which hinder the plant growth. The former is the direct
effect while the latter is the indirect effect of growth- promoting bacteria in plants.
Biological Nitrogen Fixation: Nitrogen is an essential element of many biomolecules,
the most important being nucleic acids and amino acids. Although nitrogen is the most
abundant gas (about 80%) in the atmosphere, neither animals nor plants can use this
nitrogen to synthesize biological compounds. However, there are certain microorganisms
on which the living plants (and animals) are dependent to bring nitrogen into their
biological systems.The phenomenon of fixation of atmospheric nitrogen by microorganisms
is known as diazotrophy and these organisms are collectively referred to as diazotrophs.
Diazotrophs are biological nitrogen fixers, and are prokaryotic in nature.
Nitrogen Cycle:An outline of the nitrogen cycle is depicted in Fig. 52.1. Nitrogen enters the
soil with the deposits of dead animals and plants, and urea of urine. These waste materials
(proteins, urea) are decomposed by soil bacteria into ammonia and other products. The
ammonia is converted to nitrite (NO2) and then nitrate (NO3) by certain bacteria belonging
to the genera Nitrosomonas and Nitrobacter.

4. The nitrate is degraded by various microorganisms to release nitrogen that enters atmosphere. This
atmospheric nitrogen is taken up by the nitrogen fixing bacteria (present on the roots of leguminous
plants) and used for the synthesis of biomolecules (e.g. amino acids). As the animals consume the
leguminous plants as food, the nitrogen cycle is complete.
Nitrogen Fixing Bacteria:
It is estimated that about 50% of the nitrogen needed by the plant comes from nitrogen fixing
bacteria. These are two types of nitrogen fixing microorganisms-asymbiotic and symbiotic.
Asymbiotic nitrogen fixing microorganisms:
The gaseous nitrogen of the atmosphere is directly and independently utilized to produce nitrogen-
rich compounds. When these non- symbiotic organisms die, they enrich the soil with nitrogenous
compounds. Several species of bacteria and fungi can do this job e.g. Clostridium pasturianum,
Azatobacter chrooccum. The mechanism of nitrogen fixation by asymbiotic bacteria is not clearly
understood. It is believed that nitrogen is first converted to hydroxylamine or ammonium nitrate,
and then incorporated into biomolecules.
Symbiotic nitrogen fixing microorganisms:
These microorganisms live together with the plants in a mutually beneficial relationship,
phenomenon referred to as symbiosis. The most important microorganisms involved in symbiosis
belong to two related genera namely Rhizobium and Brady-rhizobium. These symbiotic bacteria also
referred to as nodule bacteria are Gram negative, flagellated and rod-shaped. The host plants
harbouring these bacteria are known as legumes e.g. soybean, peas, beans, alfalfa, peanuts, and
clover.
Each one of the species of Rhizobium and Bradyrizobium are specific for a limited number of plants,
which survive as the natural hosts (Table 52.1). It is now clearly known that these bacteria do not
interact with plants other than the natural hosts.

5. The relationship between the symbiotic bacteria and the legumes is well recognized. On the
roots of legumes, there are a number of nodules (swellings) in which Rhizobium sp thrive.
These bacteria trap atmospheric nitrogen and synthesize nitrogen-rich compounds (amino
acids, proteins etc.) used by the legumes. At the same time, the legumes supply important
nitrogen compounds for the metabolism of Rhizobia.
The growth of legumes has been known to enrich the soil fertility. This is due to the fact
that the concentration of nitrogen compounds in the soil increases as a result of the
presence of symbiotic bacteria. For this reason normally, nitrogen fertilizers are not
needed in the fields cultivated legumes.

6. Mechanism of Nitrogen Fixation: Inside the root nodules of leguminous plants, the bacteria
proliferate. These bacteria exist in a form that has no cell wall. The bacteria of the nodules
are capable of fixing nitrogen by means of the specific enzyme namely nitrogenase.
Nitrogenase: Nitrogenase is a complex enzyme containing two oxygen sensitive
components. Component I has two α-protein subunits and two β-protein subunits, 24
molecules of iron, two molecules of molybdenum and an iron molybdenum cofactor
(FeMoCo). Component II possesses two a-protein subunits (different from that of
component I) and a large number of iron molecules. Component I of nitrogenase catalyses
the actual conversion of N2 to ammonia while component II donates electrons to
component I (Fig. 52.2).

7. Leg-hemoglobin: A protein comparable of hemoglobin in animals has been identified in the
nodules of leguminous plants. Leg-hemoglobin (LHb) contains iron and is red in colour. It is
an oxygen binding protein. The heme part of leg-hemoglobin is synthesized by the bacterium
while the protein (globin) portion is produced by the host plant. Leg-hemoglobin is
absolutely necessary for nitrogen fixation. The nodules that lack LHb are not capable of fixing
nitrogen.
It is LHb that facilitates the appropriate transfer of oxygen (by forming oxyLHb) to the
bacteria for respiration to produce ATP. And energy in the form of ATP is absolutely required
for nitrogen fixation. Another important function of LHb is that it prevents the damaging
effects of direct exposure of O2 on nitrogenase.
In Fig. 52.2, the fixation of nitrogen by symbiotic bacteria is depicted. As the oxyLHb supplies
O2, bacterial respiration occurs. The ATP generated is used for fixing nitrogen to produce
ammonia. The complex reaction is summarized below.
N2 + 8H+ + 8e– + 16 ATP=2NH3 + H2↑ + 16 ADP + 16 Pi
Hydrogenase:During the course of nitrogen fixation by nitrogenase, an undesirable reaction
also occurs. That is reduction of H+ to H2 (hydrogen gas). For the production of hydrogen, ATP
is utilized, rather wasted. Consequently the efficiency of nitrogen fixation is drastically
lowered. It is possible theoretically to reduce the energy wastage by recycling H2 to form H+.
In fact, some strains of Brady rhizobium japonicum in soybean plants were found to use
hydrogen as the energy source. These strains were found to possess an enzyme namely
hydrogenase. Recycling of the hydrogen gas that is formed as a byproduct in nitrogen fixation
is shown in Fig. 52.3.

8. It is advantageous for nitrogen fixation if the symbiotic bacteria possess the enzyme
hydrogenase. However, the naturally occurring strains of Rhizobium and Bradhyrhizobium
do not normally possess the gene encoding hydrogenase.

9. Nitrogen assimilation in plants
Plants absorb nitrogen from the soil in the form of nitrate (NO3
−) and ammonium (NH4
+). In aerobic
soils where nitrification can occur, nitrate is usually the predominant form of available nitrogen
that is absorbed. However this is not always the case as ammonia can predominate in
grasslands and in flooded, anaerobic soils like rice paddies. Plant roots themselves can affect the
abundance of various forms of nitrogen by changing the pH and secreting organic compounds or
oxygen. This influences microbial activities like the inter-conversion of various nitrogen species,
the release of ammonia from organic matter in the soil and the fixation of nitrogen by non-nodule-
forming bacteria.
Ammonium ions are absorbed by the plant via ammonia transporters. Nitrate is taken up by
several nitrate transporters that use a proton gradient to power the transport.Nitrogen is
transported from the root to the shoot via the xylem in the form of nitrate, dissolved ammonia
and amino acids. Usually (but not always) most of the nitrate reduction is carried out in the shoots
while the roots reduce only a small fraction of the absorbed nitrate to ammonia. Ammonia (both
absorbed and synthesized) is incorporated into amino acids via the glutamine synthetase-
glutamate synthase (GS-GOGAT) pathway. While nearly all the ammonia in the root is usually
incorporated into amino acids at the root itself, plants may transport significant amounts of
ammonium ions in the xylem to be fixed in the shoots. This may help avoid the transport of
organic compounds down to the roots just to carry the nitrogen back as amino acids.
Nitrate reduction is carried out in two steps. Nitrate is first reduced to nitrite (NO2
−) in the cytosol
by nitrate reductase using NADH or NADPH. Nitrite is then reduced to ammonia in the chloroplasts
(plastids in roots) by a ferredoxin dependent nitrite reductase. In photosynthesizing tissues, it uses
an isoform of ferredoxin (Fd1) that is reduced by PSI while in the root it uses a form of ferredoxin
(Fd3) that has a less negative midpoint potential and can be reduced easily by NADPH.[13] In non
photosynthesizing tissues, NADPH is generated by glycolysis and the pentose phosphate pathway.

10. In the chloroplasts, glutamine synthetase incorporates this ammonia as the amide group
of glutamine using glutamate as a substrate. Glutamate synthase (Fd-GOGAT and NADH-
GOGAT) transfer the amide group onto a 2-oxoglutarate molecule producing two glutamates.
Further transaminations are carried out make other amino acids (most
commonly asparagine) from glutamine. While the enzyme glutamate dehydrogenase (GDH)
does not play a direct role in the assimilation, it protects the mitochondrial functions during
periods of high nitrogen metabolism and takes part in nitrogen remobilization.
Every nitrate ion reduced to ammonia produces one OH− ion. To maintain a pH balance, the
plant must either excrete it into the surrounding medium or neutralize it with organic acids.
This results in the medium around the plants roots becoming alkaline when they take up
nitrate. To maintain ionic balance, every NO3
− taken into the root must be accompanied by
either the uptake of a cation or the excretion of an anion. Plants like tomatoes take up metal
ions like K+, Na+, Ca2+ and Mg2+ to exactly match every nitrate taken up and store these as the
salts of organic acids like malate and oxalate. Other plants like the soybean balance most of
their NO3
− intake with the excretion of OH− or HCO3
−.
Plants that reduce nitrates in the shoots and excrete alkali from their roots need to transport
the alkali in an inert form from the shoots to the roots. To achieve this they synthesize malic
acid in the leaves from neutral precursors like carbohydrates. The potassium ions brought to
the leaves along with the nitrate in the xylem are then sent along with the malate to the
roots via the phloem. In the roots, the malate is consumed. When malate is converted back
to malic acid prior to use, an OH− is released and excreted. (RCOO− + H2O -> RCOOH +OH−)
The potassium ions are then recirculated up the xylem with fresh nitrate. Thus the plants
avoid having to absorb and store excess salts and also transport the OH−.

11. Plants like castor reduce a lot of nitrate in
the root itself, and excrete the resulting
base. Some of the base produced in the
shoots is transported to the roots as salts
of organic acids while a small amount of
the carboxylates are just stored in the
shoot itself.
Nitrogen use efficiency
Nitrogen use efficiency (NUE) is the
proportion of nitrogen present that a
plant absorbs and uses. Improving
nitrogen use efficiency and thus fertilizer
efficiency is important to make
agriculture more sustainable, by reducing
pollution and production cost and
increasing yield. Worldwide, crops
generally have less than 50% NUE.Better
fertilizers, improved crop
management,and genetic engineering can
increase NUE. Nitrogen use efficiency can
be measured at the ecosystem level or at
the level of photosynthesis in leaves,
when it is termed photosynthetic
nitrogen use efficiency (PNUE).
Different plants use different pathways to different
levels. Tomatoes take in a lot of K+ and accumulate
salts in their vacuoles, castor reduces nitrate in the
roots to a large extent and excretes the resulting
alkali. Soy bean plants moves a large amount of
malate to the roots where they convert it to alkali
while the potassium is recirculated.

12. Thank you
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