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.  The growth-promoting activity of micro­organisms and the biotechnological approaches are described briefly with respect to the following aspects: 1. Biological nitrogen fixation. 2. Bio-control of phytopathogens. 3. Bio-fertilizers.

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

2. Growth-Promoting Bacteria 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.
The growth-promoting activity of microorganisms and the biotechnological approaches
are described briefly with respect to the following aspects:
1. Biological nitrogen fixation.
2. Bio-control of phytopathogens.
3. Bio-fertilizers.
1. 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. 2. Bio-control of Phytopathogens: Phytopathogens can drastically reduce the crop yield,
which may be in the range of 25-100%. Chemical agents are commonly used to control
them. This is associated with ill-health affects on humans, besides environmental pollution.
There are certain bacteria that can lessen or prevent the deleterious effects of plant
pathogens- fungi or bacteria. And thus, they promote plant growth by indirect means.
Plant growth-promoting bacteria are capable of producing a wide range of substances that
can restrict the damage caused by phytopathogens to plants. The important plant growth-
promoting substances are siderophores, antibiotics and certain enzymes.
Siderophores: There are some bacteria in the soil that can synthesize a low molecular
weight (400-1,000 Daltons) iron-binding peptides, collectively referred to as siderophores.
Siderophores have high affinity to bind to iron in the soil and transport it to the microbial
cell. This is required since iron is essential, and cannot directly enter the bacteria due to a
very low solubility.The growth-promoting bacteria, through siderophores can take up large
quantities of iron from the soil, and make it unavailable for the growth and existence of
fungal pathogens. This is possible since the siderophores produced by fungi have a very low
affinity when compared to siderophores of bacteria. There is no harm to the plants since
they can grow at a much lower concentration of iron in the soil.
Genetic manipulations for siderophores:
It is now clearly established that siderophores can prevent the proliferation of phytopathogens. It is therefore
logical to think of siderophore genes for more effective bio-control of plant pathogens. Pseudobactin is a
siderophore synthesized by plant growth-promoting bacteria Pseudomonas putida. By genetic
complementation and other techniques of molecular biology, at least five separate gene clusters involved in
pseudobactin production have been identified.

10. 3. Bio-fertilizers: It is estimated that more than 100 million tons of fixed nitrogen are
needed for global food production. The use of chemical/synthetic fertilizers is the
common practice to increase crop yields. Besides the cost factor, the use of fertilizers is
associated with environmental pollution.
Scientists are on a constant look for alternate, cheap and environmental-friendly sources
of nitrogen and other nutrients for plants. The term bio-fertilizers is used to refer to the
nutrient inputs of biological origin to support plant growth. This can be achieved by the
addition of microbial inoculants as a source of bio-fertilizers.
Bio-fertilizers broadly includes the following categories:
i. Symbiotic nitrogen fixers
ii. Asymbiotic nitrogen fixers
iii. Phosphate solubilizing bacteria
iv. Organic fertilizers.
The most important microorganisms used as bio-fertilizers along with the crops are listed
in Table 52.2.

11. Some of the important features of these bio-fertilizers are briefly described.
Symbiotic Nitrogen Fixers:
The diazotropbic microorganisms are the symbiotic nitrogen fixers that serve as bio-
fertilizers. e.g. Rhizobium sp and Brady rhizobium sp. The details on these bacteria with
special reference to nitrogen fixation must be referred now. Many attempts are being
made (although the success has been limited) to genetically modify the symbiotic
bacteria for improving nitrogen fixation.
Green manuring:
It is a farming practice wherein the leguminous plants which are benefited by the
symbiotic nitrogen fixing bacteria are ploughed into the soil and a non-leguminous crop
is grown to take benefits from the already fixed nitrogen. Green manuring has been in
practice in India for several centuries. It is a natural way of enriching the soil with
nitrogen, and minimizing the use of chemical fertilizers. Rhizobium sp can fix about 50-
150 kg nitrogen/hectare/annum.
Asymbiotic Nitrogen Fixers:
The asymbiotic nitrogen-fixing bacteria can directly convert the gaseous nitrogen to
nitrogen- rich compounds. When these asymbiotic nitrogen fixers die, they enrich the
soil with nitrogenous compounds, and thus serve as bio-fertilizers e.g. Azobacfer sp,
Azospirillum sp.
Blue-green algae (cynobacteria):
Blue-green algae multiply in water logging conditions. They can fix nitrogen in the form
of organic compounds (proteins, amino acids). The term algalization is used to the
process of cultivation of blue-green algae in the field as a source of bio-fertilizer.

12. Blue-green algae, besides fixing nitrogen, accumulate biomass, which improves the physical
properties of the soil. This is useful for reclamation of alkaline soils besides providing partial
tolerance to pesticides. Cynobacteria are particularly useful for paddy fields. The most
common blue-green algae are Azobacter sp and Azospirillum sp.
Azolla: Azolla is an aquatic fern, which contains an endophytic cynobacterium Anabaena
azollae in the leaf cavities providing a symbiotic relationship. Azolla with Anaebaena is
useful as biofertilizer.
But due to certain limitation (listed below), the use of Azolla has not become popular:
i. Azolla plant requires adequate supply of water.
ii. It can be easily damaged by pest diseases.
iii. Azolla cultivation is labour intensive.
Phosphate Solubilizing Bacteria: Certain bacteria (e.g. Thiobacillus, Bacillus) are capable of
converting non-available inorganic phosphorus present in the soil to utilizable (organic or
inorganic) form of phosphate. These bacteria can also produce siderophores, which chelates
with iron, and makes it unavailable to pathogenic bacteria. Thus, besides making phosphate
available, the plants are protected from disease – causing microorganisms.
Mycorrhizas: Mycorrhizas are the fungus roots (e.g. Glomus sp) with distinct morphological
structure. They are developed as a result of mutual symbiosis between certain root-
inhabiting fungi and plant roots. Mycorrhizas are formed in plants, which are limited with
nutrient supply. These plants may be herbs, shrubs and trees. For the development of
mycorrhizas, the fungus may be located on the root surface (ectomycorrhizas) or inside the
root (endomycorrhizas).

13. In recent years, an artificially produced inoculum of mycorrhizal fungi is used in crop fields. This
practice improves plant growth and yield, besides providing resistance against biotic (pathogens)
and abiotic (climatic changes) stress. Mycorrhizas also produce plant growth-promoting
substances.
Organic Fertilizers:
There are several organic wastes, which are useful as fertilizers. These include animal dung, urine,
urban garbage, sewage, crop residues and oil cakes. A majority of these wastes remain unutilized
as organic fertilizers. There exists a good potential for the development of organic manures from
these wastes.
Benefits of Bio-fertilizers:
i. Low cost and easy to produce. Small farmers are immensely benefited.
ii. Fertility of the soil is increased year after year.
iii. Free from environmental pollution.
iv. Besides nutrient supply, some other compounds, which promote plant growth, are also
produced e.g. plant growth hormones, antibiotics.
v. Bio-fertilizers increase physicochemical properties of the soil, soil texture, and water holding
capacity.
vi. Reclaimation of saline or alkaline soil is possible by using bio-fertilizers.
vii. Bio-fertilizers improve the tolerance of plants against toxic heavy metals.
viii. Plants can better withstand biotic and abiotic stresses and improve in product yield.
Limitations of Bio-fertilizers:
i. Bio-fertilizers cannot meet the total needs of the plants for nutrient supply.
ii. They cannot produce spectacular results, as is the case with synthetic fertilizers.
Considering the advantages and disadvantages of bio-fertilizers, a realistic and pragmatic
approach is to use combination of bio-fertilizers and synthetic fertilizers for optimum crop yield.

14. 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.

15. 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−.

16. 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.

17. Thank you
References: Online notes, notes from research papers and Books by google search Engine