BIOLOGICAL NITROGEN FIXATION

SOIL SCIENCE DISCIPLINE

KHULNA UNIVERSITY
 BIOLOGICAL NITROGEN FIXATION

Course Title: Project Thesis

Course No: SS - 4106

This project paper has been prepared for the partial fulfillment
of four years professional degree of Bachelor of Science in Soil
Science, from Soil Science Discipline, Khulna University.

SUBMITTED BY
ID No: 021334
Session: 2004 - 2005

Soil Science Discipline

Khulna University.
July - 2007
BIOLOGICAL NITROGEN FIXATION

APPROVED AS TO THE STYLE AND CONTENT BY

MD. SANAUL ISLAM

CHAIRMAN
EXAMINATION COMMITTEE
SOIL SCIENCE DISCIPLINE
KHULNA UNIVERSITY
 DECLARATION

This project paper is entirely the candidate’s own investigation and no part
of this paper has been accepted nor is it being concurrently submitted to any
other University or Institution for any degree.

SUPERVISOR

Md. Sadiqul Amin

Lecturer
Soil Science Discipline
Khulna University

ID No: 021334
(Candidate)
DEDICATED TO

MY BELOVED PARENTS

AND

MY FAMILY
 ACKNOWLEDGEMENT

It is a great pleaser and privilege for me to present this report on “Biological Nitrogen
Fixation”. So at first, I express my heartiest and deep sense of gratitude to the Lord, creator
and Almighty Allah, who gave me able to complete this study successfully.

I wish to express my deepest sense of gratitude to my supervisor Md. Sadiqul Amin, Lecturer,
Soil Science Discipline, for his incentive guidance and valuable suggestions regarding this
work. His help and inspiration was undoubtedly a matter of pride.

I pay my heartfelt thanks and respect to Professor Dr. Rakib Uddin, and Head of Soil Science
Discipline, Khulna University.

I also express my cordial thanks to all of my teachers, especially Sonia Nasrin, Soil Science
Discipline, Khulna University, for her constructive advice to make this thesis a success.

I convey my thanks to many of my friends and classmates for their invaluable suggestions.

Finally, I acknowledged to my beloved parents, husband and other family members for their
mental supportation and encouragement.

July, 2007 Author

 CONTENTS

Title Page
List
of Tables III
List of Figures IV

1. Introduction 1

2. Concept of Biological Nitrogen Fixation 3

2.1. Characteristics of Biological Nitrogen Fixation 3

2.2. Physiology of Biological Nitrogen Fixation 4

2.3. Classification of BNF 5

3. Different nitrogen fixation processes 6

3.1. Symbiotic nitrogen fixation 7

3.1.1. Major groups of organisms involved and their characteristics 7

3.1.2. Symbiotic fixation with legumes 9

3.1.3. Symbiotic fixation with nonlegumes 12

3.1.3.1. Nodule forming nonlegumes 12

3.1.3.2. Stages of nodule formation 13

3.1.3.3. Symbiotic nitrogen fixation without nodules 17

3.1.3.4. Nitrogen fixation by Symbiotic

blue green algae without nodules 18

3.1.3.5. Azolla-Anabaena Association 19

3.1.3.6. Lichens association 21

3.1.3.7. Blue-green algal association with bryophytes 22

3.2. Nonsymbiotic nitrogen fixation 22

 3.2.1. Major groups of organisms and their charectistics

involved in nonsymbiotic nitrogen fixation 23

3.2.2. Fixation with Heterotrophs 27

3.2.3. Fixation with Autotrophs 28

3.2.4. Nitrogen fixation by free living blue green algae 30

4. Factors affecting N fixation 32

5. Amount of N fixed in soil by BNF organisms 34

6. Measurement of nitrogen fixation with 15N 35

7. Importance of BNF in soil, plant, living organisms, agriculture 36

8. Future prospects in Biological Nitrogen Fixation 38

9. Conclusion 39

10. Reference 40
 Lists of Tables

Table Page
Table 3.1 Information on different systems of Biological 07
Nitrogen Fixation

Table 3.2 Representative genera of microorganisms involved in 08

Symbiotic N fixation

Table 3.3 The cross-inoculation groups and Rhizobium-Legume 12

association

Table 3.4 Number of major Actinomycete-Nodulated nonlegume 13

angiosperm

Table 3.5 Typical levels of nitrogen fixation from Symbiotic N 18

fixation without nodules

Table 3.6 Typical level of nitrogen fixation from different systems 19

Table 3.7 Representative genera of microorganisms involved in

Nonsymbiotic N fixation with Atutotrophs 28

Table 3.8 Representative genera of microorganisms involved in 30

Nonsymbiotic N fixation with Atutotrophs

Table 3.9 Nitrogen fixing genera of blue-green algae 31

Table 3.10 Typical levels of nitrogen fixation from different systems 35

Table 6.1 Economically important involved in Biological 38

Nitrogen Fixation
 List of Figures

Figure Page

Figure 2.1 The classification of BNF 06

Figure 3.1 Nodule of Alnus and nodules 09

containing Frankia

Figure 3.2 Soybean nodules and nodule section 10

which is formed by Rhizobium

Figure 3.3 Part of a clover root system bearing naturally 11

occurring nodules of Rhizobium

Figure 3.4 Steps in the formation of root nodule 15

Figure 3.5 A dorsal lobe of the water fern Azolla showing 20

the dark- green pocket filled with cells of
the microsymbiont Anabaena (Nostoc) azollae.

Figure 3.6 A filament of the cyanobacterial microsymbiont 20

(Anabaena [Nostoc] azollae

Figure 3.7 Fruticose and squamulose lichens. 21

1. Introduction

Molecular nitrogen (N 2 ) is the major component (approximately 80%) of the earth's

atmosphere. The element nitrogen is an essential part of many of the chemical
compounds, such as proteins and nucleic acids, which are the basis of all life forms.
However, N 2 cannot be used directly by biological systems to build the chemicals
required for growth and reproduction. Before its incorporation into a living system, N 2
must first be combined with the element hydrogen. This process of reduction of N 2,
commonly referred to as "nitrogen fixation" (N-fixation) may be accomplished
chemically or biologically (Brady and Weil, 2002).

The continuous drain on the nitrogen resources of the soil and the necessity for higher
crop yields have led to an ever-increasing emphasis on means of conserving the limited
supply of the element. Because only a fraction of the total agricultural need for nitrogen
comes from synthetic and natural fertilizers, the remaining portion must be satisfied from
the soil reserves and through the biological fixation of atmospheric nitrogen. A number of
free living microorganisms can assimilate molecular nitrogen, but no higher plant or
animal has the needed enzyme to catalyze the reaction. In certain instances, however, a
sysbiosis can become established in which one of the more prominent effects of the
association is the acquisition of nitrogen from the atmosphere. Two members required for
the association - a plant and microorganisms. The classical example such a symbiosis is
that between leguminous plants and bacteria of the genus Rhizobium. The seat of the
symbiosis is with in the nodules that appear on the plant roots (Alexander, 1977).
Microorganisms have a central role in almost all aspects of nitrogen availability and thus
for life support on earth (Burn and Hardy, 1975). Prokaryotes are the only organisms that
are able to tap the nitrogen reservoir of the atmosphere and fix molecular nitrogen
(Schlegel, 1993.)

Some bacteria can convert N 2 into ammonia by the process termed nitrogen fixation;
these bacteria are either free-living or form symbiotic associations with plants or other
organisms (e.g. termites, protozoa). Other bacteria bring about transformations of
ammonia to nitrate, and of nitrate to N 2 or other nitrogen gases. Many bacteria and fungi
degrade organic matter, releasing fixed nitrogen for reuse by other organisms (Burn and
Hardy, 1975).

The most important contribution to BNF comes from the symbiotic association of certain
micro-organisms with the roots of higher plants. A classic example is that of the bacteria
(Rhizobium) which characteristically infect the roots of leguminous plants (e.g., bean,
soybean, clover, and peanut) with a high degree of host specificity. Small nodules are
formed on the roots and these become filled with an altered form of the bacteria
(bacteroides) which fix appreciable amounts of nitrogen. This symbiosis alone accounts
for 20% of global biological nitrogen fixed annually. The legumes represent a major
direct source of food for man and forage for livestock and therefore represent a critical
contribution to world food production (Vincent, 1974).

Numerous genera of non-leguminous angiosperms, such as Alnus, Casuarina, Coriaria,

Myrica, etc., form root nodules in response to infection by the actinomycete Frankia.
These associations may achieve fixation rates as high as 100 kg N/ha/yr and may occur as
climax vegetation or as pioneer species in adverse soil environments. Some
gymnosperms, such as Cycas, Macrozamia, and Podocarpus, are capable of forming
similar nitrogen fixing root nodule associations (Killham, 1994).

There is great diversity in the metabolic types of free-living microorganisms which are
capable of BNF. This includes about 20 genera of non-photosynthetic aerobic
(Azotobacter, Beijerinckia) and anaerobic (Clostridium) bacteria and about 15 genera of
photosynthetic cyanobacteria (blue-green algae) such as Anabaena and Nostoc. The leaf
surface (phyllosphere) of certain plants in warm, humid tropical regions may provide an
additional favorable environment for the growth and nitrogen fixing activity of such free-
living bacteria as Azotobacter, Beijerinckia and Kiebsiella (Stevenson, 1986).

The objectives of the project paper are:

1. To know about different types of biological nitrogen fixation and the activities of
microorganisms involved in the process of fixation in soil,

2. To know how important biological nitrogen fixation could be for soil, agriculture,
and economics.
2. Concept of Biological Nitrogen Fixation

The conversion of atmospheric inert dinitrogen gas (N 2 ) into nitrogen containing organic
compounds by some prokaryotic microorganisms through biological reactions that
become available to all forms of life through nitrogen cycle is known as biological
nitrogen fixation (BNF). Biological nitrogen fixation occurs at ordinary temperatures and
pressures. It is commonly carried out by certain bacteria, algae and actinomycetes which
may or may not be associated with higher plants (Brady and Weil, 2002).

Prokaryotes, either as free living organisms or in symbiosis with higher plants can carry
out reactions that result in incorporation of nitrogen into organic compounds and hence
directly or via plant substances, into the protein reservoir of the soils (Schlegel, 1993).

2.1. Characteristics of Biological Nitrogen Fixation

There are three conditions must be met to prove that biological nitrogen fixation occurs.
First, there must be a large population of these organisms that is consistent with the
observed nitrogen fixation rates. Second, there must be rapid cell formation, which
indicates that nitrogen fixation is linked to growth. Third, the nitrogen must be
atmospheric, not inorganic or organic; this is one of the most difficult conditions to meet.

Nitrogen fixation is energy intensive. Microorganisms must have a ready supply of

electrons for sufficient nitrogen fixation to occur. Electrons are necessary because
nitrogen fixation is a reductive process. Nitrogen gas gains electrons to become ammonia.
Nitrogen fixation requires an enzyme complex called nitrogenage and nitrogenage must
operate in a microenvironment protected from oxygen (Coyne, 1999).

2.2. Physiology of Biological Nitrogen Fixation

Biological nitrogen fixation is catalyzed by an enzyme association called nitrogenage.

This is strictly a prokaryotic enzyme complex. Nitrogenage is composed of two soluble
proteins: the Fe protein-dinitrogenase reductase and the MoFe protein- dinitrogenase.
MoFe stands for molybdenum-iron, an essential cofactor in dinitrogenase. The two
enzymes in the complex work are: dinitrogenase reductase reduces dinitrogenase, and
dinitrogenase reduces nitrogen. Both enzymes are required for fixation to occur. An
equation for nitrogen fixation can be written as follows; Mg2+ is a required cofactor:

N 2 + 6e- +6H++12ATP+Mg2+ 2NH 3 +12ADP+12Pi+Mg2+

Electrons come from organic and inorganic sources, flow through several electron
carries, flow through nitrogenage, and ultimately reduce nitrogen:

e- => Ferrodoxin => Flavodoxin => Fe Protein => MoFe Protein => N2

Each electron transferred requires two ATP. Unfortunately, nitrogenage is not perfectly
efficient. It also reduces other compounds such as H+, N 2 O, NO 3 -, and CN-. Hydrogen
evaluation is a general by-product of nitrogen fixation, particularly at low electron flux:

2H++ 2e-+ 4ATP H 2 + 4ADP + 4Pi

The overall equation for nitrogenase -catalyzed nitrogen fixation is therefore:

N 2 + 16ATP + 8e- + 8H+ + Mg2+ 2NH 3 + H 2 + 16ADP +16Pi +Mg2+

(Coyne, 1999).

Several salient facts about this enzyme and its function are worth noting, for nitrogenage
is unique and its role in the nitrogen cycle is of great importance to humankind.

1. The reduction of nitrogen to ammonia by nitrogenage requires a great deal of

energy to break the triple bond between the nitrogen atoms. Therefore the process
is greatly enhanced by association with higher plants, which can supply this
energy from photosynthesis.

2. Nitrogenase is destroyed by free oxygen, so organisms that fix nitrogen must

protect the enzyme from exposer to oxygen. When nitrogen fixation takes places
in root nodules, one means of protecting the enzyme from free oxygen is the
formation of leghemoglobin. This compound, which gives active nodules a red
interior color, binds oxygen in such a way as to protect the nitrogenase while
making oxygen available for respiration in other parts of the nodule tissue.
 3. The reduction reaction is end product inhibited- an accumulation of ammonia will
inhibit nitrogen fixation. Also, too much nitrate in the soil will inhibit the
formation of nodules.

4. Nitrogen fixing organisms have a relatively high requirement for molybdenum,

iron, phosphorus, and sulfur, because these nutrients are either part of the
nitrogenase molecule needed for its synthesis and use (Brady and Weil, 2002).

2.3. Classification of BNF

Biological nitrogen fixation known to occur to a varying degree in many different

environments, including soils; fresh and salt waters and sediments; on or within the roots
stems and leaves of certain higher plants; and within the digestive tracts of some animals.
The potential for nitrogen fixation exists for any environment capable of supporting
growth of microorganisms. Biological systems which are capable of fixing nitrogen are
historically classify as nonsymbiotic or symbiotic, depending on the required
involvement of one or more than one organism, respectively, in the process (Hubbell and
Kidder, 2003).

The classification of BNF is shown in figure 1-

BNF

Symbiotic fixation Symbiotic fixation Nonsymbiotic nitrogen

With legumes with non legumes fixation

Nodule forming Symbiotic fixation Fixation Fixation

Nonlegumes without nodule by Heterotroph by Autotroph

Figure 1. The classification of BNF (Stevenson, 1986).

3. Different nitrogen fixation processes

Biological nitrogen fixation occurs through a number of organisms systems with or

without direct association with higher plants. The organisms that fix nitrogen are
conveniently placed into two groups: (1) the nonsymbiotic fixers, or those that fix
molecular nitrogen apart from the specific host; and (2) the symbiotic fixers, or those that
fix nitrogen in association with higher plants, including some nonlegumes (Stevenson,
1986). Although the legume symbiotic systems have received the most attention
historically, recent findings suggest that the other systems are also very important
worldwide and many even rival the legume associated system as suppliers of biological
nitrogen to the soil (Brady, 1990).

Information on different systems of Biological Nitrogen Fixation are observed in table-1:

Table 1: Information on different systems of Biological Nitrogen Fixation (Burn and

Hardy, 1975)

N-fixing systems Organisms Plants involved Site of fixation

involved

Symbiotic Bacteria Rhizobia Legumes Root nodules

Obligatory and Bradyrhizobia
Legumes

Nonlegumes Actinomycetes Nonlegumes Root nodules

(angiosperms) (Frankia) (angiosperms)

Associative Blue-green algae, Various higher Leaf and root

Morphological bacteria plants and nodules, lichens
involvement microorganisms

Nonmorphological Blue-green algae, Various higher

involvement bacteria plants and
microorganisms

Nonsymbiotic Blue-green algae, No involved with Soil, water

bacteria plants independent of
plants
3.1. Symbiotic nitrogen fixation

Generally, the symbiosis means mutually beneficial relationship. The living together in
intimate association of two dissimilar organisms, the cohabitation being mutually
beneficial (Brady and Weil, 2002). An association between two organisms or
populations, which, in the absence of environmental change, is stable (Killham, 1994).

3.1.1. Major groups of organisms and their charectistics involved in symbiotic

nitrogen fixation

The symbiosis of legumes and bacteria of the genera Rhizobium and Bradyrhizobium
provide the major biological source of fixed nitrogen in agricultural soils. Besides these
some others organisms are involved in fixing process. Representative genera of
microorganisms involved in Symbiotic nitrogen fixation are observed in table-2:

Table 2: Representative genera of microorganisms involved in Symbiotic nitrogen

fixation (Coyne, 1999).
Physiological Type of association Host (if any) Representative
group genera
Glycine Bradyrhizobim
Legume Rhizobiam
Heterotroph Symbiotic; aerobic Trema Rhizobiam
Alnus Frankia
Myrica Frankia
Gunnera Nostoc
Lichen
Peltigera Nostoc
Collema Nostoc
Liverwort
Anthoceros Nostoc
Autotroph Symbiotic; Biasia Nostoc
nonnodule Mosses
Sphagnum Halosiphon
Ferns
Azolla Anabaena
Intracellular
Oocystis Nostoc
Autotroph Symbiotic; nodule Gymnosperm cycad Nostoc
Frankia

1. Frankia is a genus of the bacterial group termed actinomycetes - filamentous

bacteria that are noted for their production of air-borne spores.

2. Frankia is a Gram-positive, filamentous organism characterized by sporangia and

N 2 -fixing vesicles in vitro.

3. Included in this group are the common soil-dwelling Streptomyces species which
produce many of the antibiotics used in medicine.

4. Frankia species are slow-growing in culture, and require specialized media,

suggesting that they are specialised symbionts. They form nitrogen-fixing root
nodules (sometimes called actinorhizae) with several woody plants of different
families, such as alder (Alnus species), sea buckthorn (Hippophae rhamnoides,
which is common in sand-dune environments) and Casuarina (a Mediterranean
tree genus) (Killham, 1994).

Nodule of Alnus which contains Frankia is shown in figure 2-

(a) (b)

Figure 2. Figure (a) Nodule of Alnus showing the typical multilobed structure. and figure
(b) Figure A (below) shows a young alder tree (Alnus glutinosa) growing in a plant pot,
and Figure B shows part of the root system of this tree, bearing the orange-yellow colored
nodules (arrowheads) containing Frankia. (Hubbell and Kidder, 2003).
Rhizobium and Bradyrhizobium

The symbiosis (mutually beneficial relationship) of legumes and bacteria of the general
Rhizobiurn and Bradyrhizobium provide the major biological source of fixed nitrogen in
agricultural soils.

The genus Rhizobium contains fast-growing, acid-producing bacteria while the

Bradyrhizobia are slow growers that do not produce acid. Both will be considered
together. These organisms infect the root hairs and the cortical cells, ultimate inducing
the formation of root nodules that serve as the site of nitrogen fixation (Brady and Weil,
2002).

Rhizobium which was a single genus earlier is now classified into five different
genera (Schlegel, 1993):

(a) Rhizobium,

(b) Bradyrhizobium,

(c) Azorhizobium (Rhizobium which form stem nodule),

(d) Sinorhizobium and

(e) Photorhizobium (first reported Rhizobium having photosynthetic

properties).

3.1.2. Symbiotic fixation with legumes

The most important contribution to BNF comes from the symbiotic association of certain
microorganisms with the roots of higher plants. A classic example is that of the bacteria
(Rhizobium) which characteristically infect the roots of leguminous plants (e.g., bean,
soybean, clover, and peanut) with a high degree of host specificity. Small nodules are
formed on the roots and these become filled with an altered form of the bacteria
(bacteroids) which fix appreciable amounts of nitrogen. These symbiosis alone accounts
for 20% of global biological nitrogen fixed annually (Vincent, 1974). Soyabean nodules
which is formed by Rhizobium is shown in Figure 3.
 (a) (b)

Figure 3. Soybean nodules (a) and nodule section (b) which is formed by Rhizobium.
(Hubbell and Kidder, 2003).

Apparently, plants make the heavy energy investment required for symbiotic nitrogen
fixation only when short supplies of mineral nitrogen make nitrogen fixation necessary.
Although quite variable form, amount of nitrogen biologically fixed can be quite high,
especially for those systems involving nodules, which supply energy from photosynthates
and protect the nitrogenase enzyme system (Brady and Weil, 2002). Part of a clover root
system which bears naturally occurring nodules of Rhizobium is shown in Figure 4.

Figure 4. Part of a clover root system bearing naturally occurring nodules of Rhizobium
showing two partly crushed nodules (arrowheads) with pink-colored contents (Hubbell
and Kidder, 2003).
In some leguminous plants nodules occur on the stem are known as stem nodule. These
leguminous plants, for example Sesbania rostrata, grow in damp habitats in tropical
Africa and India. The nodules appear on submerged stems and on those above soil level.
It expected that these nitrogen-fixing systems would show less oxygen sensitivity than
the other N system (Schlegel, 1993).

The cross-inoculation groups and Rhizobium-Legume association are shown in table 3.

Table 3: The cross-inoculation groups and Rhizobium-Legume association (Schlegel,

1993)

Rhizobium groups cross-inoculation group Host genera

R. leguminosarum Pea group Pisum,Vicia, Lens

R. phaseoli Bean group Phaseolus

R. trifolli Clover group Trifolium

R. meliloti Alfalfa group Melilotus, Medicago

R. lupini Lupini group Lupinus, Orinthopus

R. japonicum Soybean group Glycine

R. species Cowpea group Vigna, Arachis

3.1.3. Symbiotic fixation with nonlegumes

Symbiotic nitrogen fixation also occurs some nonlegumes. This nonlegumes can be
nodule forming or with nodules. Two types are described in below.

3.1.3.1. Nodule forming nonlegumes

Numerous genera of non-leguminous angiosperms, such as Alnus, Casuarina, Coriaria,

Myrica etc., form root nodules in response to infection by the actinomycete Frankia.
These associations may achieve fixation rates as high as 100 kg N/ha/yr and may occur as
climax vegetation or as pioneer species in adverse soil environments. Some
gymnosperms, such as Cycas, Macrozamia, and Podocarpus are capable of forming
similar nitrogen fixing root nodule associations (Hubbell and Kidder, 2003).

A variety of additional plant-symbiotic nitrogen fixing associations has been reported.

Examples include the bacterium Kiebsiella in leaf nodules of Psychotria and associations
of Cyanobacteria with fungi (lichens), liverworts (Blasia), angiosperms (Gunnera) and
the water fern Azolla (Stewart, 1973).

The tree-actinomycetete complexes are able to colonize infertile soils and newly forming
soils on disturbed lands, which may have extremely low fertility as well as other
conditions that limit the plant growth. Once nitrogen fixing plants become established
and begin to build up the soil nitrogen supply through leaf litter and root exudation, the
land become more hospitable for colonization by other species. Frankia thus play a very
important role in the nitrogen economy of areas succession, as well as in established
forest marshes (Brady and Weil, 2002).

Nearly 200 species of nonlegumes are known to develop nodules and to accommodate
symbiotic N fixation. Number of major Actinomycete-Nodulated nonlegume angiosperm
is observed in Table 4.

Table 4: Number of major Actinomycete-Nodulated nonlegume angiosperm (Brady and

Weil, 2002).

Genus Family Species nodulated

Alnus Betulaceae 33/35

Ceanothus Rhamnaceae 31/35

Myrica Myricaceae 26/35

Casurina Casurinaceae 24/25

Elaeagnus Elaeagnaceae 16/45

Coriaria Coriariaceae 13/16

3.1.3.2. Stages of nodule formation:

Nodule formation is not a single step process. Scientists have discussed few specified
stages of nodulation these take place one after another in a high ordered fashion.

The stages in the infection and development of root nodules are discussed as follows

(a) Recognition:

There is an initial contact between the bacteria and host, which depends upon the process
“Recognition”. Through recognition, correct partner on the part of both plant and
bacterium and attachment of the bacterium to root hairs occurs at first leguminous plants
contain lectins. These are glycoproteins that bind specific polysaccharides and have
general recognition function (Dubey and Maheshwari, 1999; Schiegel, 1993).

(b) Curling and deformation of root hairs:

The first reaction of the root system to the presence of rhizobia is the curling and
deformation of root hairs. Legumes excrete a large number of substances into the
rhizosphere principally sugars, amino acids and vitamins. Entry point in root hair is
possible through stomata, a wound or abrasion on the stem. Initial attraction between
legume and Rhizobium is by flavonoids produced and secreted by the legume plants,
[luteolin and diadzein].

The bacterial wall components play a very important role in the actual entry. Four
components are:

(i) Exopolysaccharides,

(ii) B-1-2- glucans,

(iii) Lipopolysachandes and

(iv) Lectins.

The curling of root hair is attributed to Indole acetic acid (IAA) produced in root region
by Rhizobia (Sullia and Shantharam, 1998).
Figure 5. Steps in the formation of root nodule in a legume infected by Rhizobium
(Madigan et al., 2000)
Invasion of Rhziobia occurs through root hairs. Fine Studies of infected root hairs
showed the continuation of the infection thread with the cell wall of the root hair, which
lend to support to the invagination hypothesis. The physiological events leading to
infection can be summarized bellow (Dubey and Maheshwari, 1999):

Normal root hair Exudation of organic substances by root Accumulation of

rhizobia in the Rhizosphere Conversion of tryptophan to Indole acetic acid.

(c) Formation of Infection- thread and formation of nodule:

The tip of curled root hair bends and the bacteria (Rhizobia polysaccharides and DNA)
penetrate and then attached to the hair by sugar binding protein called lectins form
infection tube / thread (Lack and Evan, 2001).

There appears to be an intensive interaction between the nucleus of root hair cell and the
infection thread originating at the tip of the curled portion of the root hair. The
polysaccharides react with a component of root cell to form an’organizer’.The
‘organizer’induces the production of polygalactuonase (PG) followed by
depolymenzation of cell wall pectin. In such process, incorporation of Rhizobia into cell
wall occurs which participates in intissusception i.e. (taking in of Rhizobia by root hair
and its conversion into organic tissues). The infection tube or thread branches into the
central portions of the nodule, and the bacteria released into their symbiont’s cytoplasm
to multiply. The nucleus of the root cell guides the Rhizobia (Burn and Hardy, 1975).

(d) Development of the nodule:

Immediately, at the time of release of rhizobia into cytoplasm of the host cortical cells
rapid cell division take place in the cortical cells. Inside these cells, the bacteria alter their
morphology into larger forms called bacteroids. The root cells are stimulated by this
infection to form tumor like nodule of chromosome number of the area becomes double.
The doubling of the chromosome number occurs in the polyploids as well as diploid
legume (Dubey and Maheshwari, 1999; Sullia and Shantharam, 1998).
(e) Structure of root nodules:

The root nodules are due to tissue proliferation induced by the action of growth
promoters of rhizobial in origin, probably cytokinines. The core of a mature nodule
constitutes the ‘bacteroids zone’, which is surrounded by peribacteroid membranes;
inhabit the cytoplasm of the plant cell. The effective nodules are generally large and pink
due to presence of leghaemoglobin with well-developed and organized tissue (Burn and
Hardy, 1975; Schiegel, 1993).

(f) Function of the nodule ‘bacteroids’:

The bacteroids are the sites of nitrogen fixation. Further, incontrast to the free living
rhizobia, the bacteroids are unable to utilize sugars, and secrete ammonium ions which
are apparently incorporated into organic compounds by glutamine synthase present in the
surrounding plant cell. This process indicates the involvement of true mutual symbiosis
in which role-played by leghaemoglobin in the fixation of nitrogen is much more
significant (Dubey and Maheshwari, 1999).

3.1.3.3. Symbiotic nitrogen fixation without nodules

Among the most significant nonnodule nitrogen-fixing systems are those involving
Cyanobacteria. One system of considerable practical importance is the Azolla-Anabaena
complex, which flourishes in certain rice paddies of tropical and semitropical areas. A
more wide spread nitrogen-fixing phenomenon is that which occurs in the rhizosphere of
certain grasses and other nonlegume plants. The organisms involve are bacteria specially
those of the Spirillum and Azotobacter genera. Plant root exudates supply these
microorganisms with energy for their nitrogen fixing activities (Brady and Weil, 2002).

In both cases the bacteria grow at the expense of sugars and other nutrients that leak from
the roots. However, these bacteria can make only a small contribution to the nitrogen
nutrition of plant, because nitrogen- fixation is an expensive process, and large amount of
organic nutrients are not continuously available to microbes in the rhizosphere (Deacon,
2003).
Typical levels of nitrogen fixation from Symbiotic N fixation without nodules are
observed in Table 5.

Table 5: Typical levels of nitrogen fixation from Symbiotic N fixation without nodules
(Brady and Weil, 2002).

Crop or plant Associated organisms Typical level of

nonlegumes (non- nitrogen fixation, Kg
nodulated) N/ha/yr

Pangolan grass Bacteria 5-30

(Degeraria decumbens) (Azospirillum)

Bahia grass Bacteria 5-30

(Pasalum notatum) (Azobacter)

Azolla Cyanobacteria 150-300

3.1.3.4. Nitrogen fixation by Symbiotic blue green algae without nodules

Recent studies have called attention to several significant non legumes symbiotic
nitrogen fixation systems that do not create nodules among the most significant are those
involving blue green algae. One system of considerable practical importance is the
Azolla-Anabaena complex which flourishes in certain rice paddies of tropical and semi
tropical areas. The Anabaena blue green algae inhabit cavities in the leaves of the floating
fern Azolla and fix quantities of nitrogen comparable to those of the better Rhizobium-
legume complexes (Mishra, 2000). Typical levels of nitrogen fixation from different
systems are observed in Table 6.

]3.1.3.5. Azolla-Anabaena Association

A species of Anabaena (A.azollae) is associated with the aquatic fern Azolla occurring in
a ventral pore in the dorsal lobe of each vegetative leaf. The endophyte fixes atmospheric
nitrogen and resides inside the tissues of the water fern. Azolla is being used as green
compost for rice cultivation.
Table 6. Typical level of nitrogen fixation from different systems (Brady and Weil,
2002).

Crop or plant Associated organism Typical level of

nitrogen fixation (kg
N/ha/yr)
Symbiotic Blue-green algae 150-300
(Anabaena)
Nonlegumes
(nonnodulated) Azolla
Nonlegumes (nodulated) Blue-green algae (Nostoc) 10-20
Species of Gunnera

Nonsymbiotic Blue-green algae (various) 10-50

An added advantage is that the plant multiplies fast and provides higher yields of green
compost (200-300 t/ha/yr) than conventional green manure plants such as Sesbania,
Crotalaria and Tephrosia which are known to yield 30-50 t/ha/yr. It is also necessary to
prevent algal growth in rice fields for utilization of Azolla since algae tend to overgrow
and inhibit the proliferation of the water weed. The benefit from Azolla growth to the
associated rice crop has been variously estimated. It ranges from 95 kg N/ha/yr to 670 kg
N/ha/yr, depending on the method used in determining the amount of nitrogen fixed
(Singh, 1977).

Rice plants have been treated with pure cultures of algae in laboratory and field
conditions. In many cases the algae had a beneficial effect but the increase in yield
fluctuated greatly. In several cases there has been an appreciable increase in the rice
harvested in the third or fourth years after the introduction of nitrogen fixing blue green
algae. It has been observed that culture of Anabaena and Nostoc affects rice plants
favorably, specially when lime, super phosphate or Mo were also added (Mishra, 2000).
A dorsal lobe of the water fern Azolla which shows the dark-green pocket filled with
cells of the microsymbiont Anabaena (Nostoc) azollae is shown in Figure 6.
Figure 6. A dorsal lobe of the water fern Azolla showing the dark-green pocket filled
with cells of the microsymbiont Anabaena (Nostoc) azollae. (Lack and Evan, 2001).

A filament of the cyanobacterial microsymbiont (Anabaena [Nostoc] azollae) which

shows both enlarged heterocysts and normal cells of the micro-symbiont is shown in
Figure 7.

Figure 7. A filament of the cyanobacterial microsymbiont (Anabaena [Nostoc] azollae)

showing both enlarged heterocysts and normal cells of the micro-symbiont. Heterocyst
frequency is greater than 20% (Deacon, 2003).

Blue-green algae is found to grow in a paddy has some species such as Anabaena and
Nostoc etc,they fix nitrogen from air in presence of sunlight and add 30 kg nitrogen per
hectare in transplanted aman, saves the application of nitrogenous fertilizer. Besides this,
blue-green algae have been following advantages:

1. It increases the organic matter content.

2. It releases the amino-acid in the soil.

3. It brings the insoluble phosphate to the available from the plant (Das, 1987).

3.1.3.6. Lichens association

Unusually symbiotic algae (either blue green bacteria or green algae) associate with one
of several fungi in forms called lichens. They fix dinitrogen and the fungi attached to a
surface and form a protective mat that tenaciously holds water and supplies other
nutrients to the algae (Hill, 1994).

Lichen grows extremely slowly (0-30 mm per year), they help to weather rock surfaces
and they can growth on bark and other surfaces. Both the algae and the fungi can live
separately if conditions of nutrition are adequate for growth algae are found in the
cavities of liverworts where they are endophytes and also in ferns and species of Cycas
(Miller and Donahue, 1995). Different kinds of lichens such as Usnea comosa, Cladonia
pyxidata, Cladonia coccifera, are shown in Figure 8.

Figure 8. Fruticose and squamulose lichens. (C) Usnea comosa, growing on a wooden
post. This lichen has a branched, filamentous thallus which hangs down from the point of
attachment. (D) Cladonia pyxidata, growing on peaty soil. This lichen has a scale-like
squamulose structure (arrowhead) but also produces erect stalked cups termed podetia.
(E) Cladonia coccifera, similar to C. pyxidata but the rims of the podetia bear many
conspicuous red ascocarps (Hubbell and Kidder, 2003).

Most lichens are terrestrial. The algae fungus combination allows lichens to grow under
conditions where neither component could separately and leads to the production of
organic chemicals unique to the association. Although Lichens grow very slowly, they
survive in harsh environment. Lichens commonly colonize exposed rock surfaces. Lichen
distribution is sometimes limited by their sensitivity to air pollution (Sze, 1998).

3.1.3.7. Blue-green algal association with bryophytes

Certain mosses and liverworts are also known to be inhabited by blue-green algae. The
lower surface of the thallus of Anthoceros contains a species of Nostoc which stimulates
the formation of papillae within the cavities of the host. The papillae provide space for
the formation of compact colonies of the algae, which is turn, help in the nitrogen
nutrition of the host plant. Nostoc Sphaericum inhabits the cavities occurring on
outgrowths near the edges of the lower surface of Blasia and Cavicularia.The
dependence of the host on the lower symbiont for its nitrogen nutrition has been
established in the case of Blasia 15N (Subba Rao, 2001).

3.2. Nonsymbiotic nitrogen fixation

Certain free-living microorganisms present in soils and water are able to fix nitrogen.
Because these organisms are not directly associated with higher plants, the transformation
is referred to as nonsymbiotic or free-living. Several different groups of bacteria and
cyanobacteria are able to fix nitrogen nonsymbiotically. In upland mineral soils, the
major fixation is brought about by species of two genera of heterotrophic aerobic
bacteria, Azotobacter (in temperate zones) and Beijerinckia (in tropical soils). Certain
aerobic bacteria of the genus Clostridium are also able to fix nitrogen. Because pockets of
low oxygen supply exist in soils even when they are in good tilth, aerobic and anaerobic
bacteria probably work side by side in many well- drained soils (Brady and Weil, 2002).
3.2.1. Major groups of organisms and their charectistics involved in nonsymbiotic
nitrogen fixation

The organisms which are related with the nonsymbiotic fixation mostly are

1. Aerobic,

2. Facultative,

3. Anaerobic and

4. Autotrophs.

The best-characterized organism in aerobic conditions is Azotobacter. In anaerobic

conditions the best-characterized organism is Clostridium. Several cyanobacteria have
also been well characterized. In addition, some work has been done on organisms asso-
ciated with the rhizosphere of grasses; Azospirillum is an example of this type of asso-
ciative N fixer (Coyne, 1999).

Azotobacter

1. Azotobacter is a gram-negative, motile soil organism.

2. Since it is heterotrophic, mannitol is used in the isolation method to culture it

from soil.

3. It is big compared to other prokaryotes (4 to 7 μm in diameter) and yeastlike in

appearance.

4. It forms cysts that serve as resting bodies.

5. Azotobacter grows best in neutral to alkaline, mesophylic soils.

6. It does not grow when the pH is below 6 and it is not present in acidic soils.

7. Large populations are uncommon (0 to l0-3 per g is the typical

range).Consequently it does not con tribute much fixed N to soil (Coyne, 2001).

8. All Azotobacters are strict aerobes, and they apparently possess the highest known
respiratory rate.

9. These bacteria utilize few nitrogenous compounds—N ammonium, nitrate, nitrite,

urea, and an occasional organic nitrogen-containing molecule.
 10. Members of the genus are mesophilic, and their optimum temperature is near
30°C (Alexander, 1977).

Beijerincia

1. Beijerincia species also are aerobic N fixers,

2. They grow well in acid conditions and sometimes develop at a reaction as low as
pH 3.

3. This species are commonly found in tropical soils, and strains have been isolated
in South America, Asia, northern Australia, and tropical areas of Africa,

4. Their numbers ranging from a few to several thousand per gram.

5. Bnjerinckia is rarely detected in soils of the temperate zone (Alexander, 1977).

Dencia

1. Dencia is similarly an aerobic,

2. Acid- tolerant genus and it will grow in media from pH 5 to 9.

3. Strains Dencia of have been detected in soils of Asia and Brazil, but the group has
still received scant attention (Alexander, 1977).

Clostridium

1. The dominant anaerobes using N are members of the genus Clostridium.

2. The population of N clostridia in arable land may be as small as 102 or as large as

l0 cells per gram. Under some conditions, more than 10 clostridia per gram have
been detected.

3. The dominant species appear to be C. pasteurthnum, and C. acetobutylicum, but

other have been found to carry out the same biochemical process.
 4. Clostridia proliferate when organic matter is added, and they often are numerous
around plant roots.

5. In contrast with the azotobacters, which they usually outnumber, the clostridia are
found at sites of pH 5.0, and they are still capable of growth at pH 9.0 (Alexander,
1977).

Paspalum notatuin

1. Nitrogen fixation is associated with grasses such as Paspalum notatuin. Other

associations have been found in salt-marsh grass, sugar cane, rice, sorghum,
maize, millet, and wheat.

2. Associative fixation is negligible to 20 kg/ ha/ yr so grain crops still need to be

fertilized to get decent yield.

3. Inoculation of plant roots with asymbiotic N fixing organisms has given

ambiguous results. Positive results may be due to enhanced N, fixation, or may
simply be because of the production of growth stimulants and exclusion of
pathogens (Coyne, 1999).

Cyanobacteria

1. Cyanobacteria are usually free-living organisms that fix only a few kg of N/

ha/ yr. they are not of great significance compared to symbiotic fixation in
terrestrial systems, with one exception. In tropical or aquatic regions,
cyanobacteria are hugely significant.

2. Cyanobacteria are most common in aquatic and freshwater environments.

They take three basic forms:

(a) Unicellular (Chroococcus, Gloeocapsa, and Microcxstis)

(b) Nonfilamentous (Plectonema)

(c) Filamentous (Anabaena, Nostoc).

 3. Cyanobacteria can be found in soils, sometimes just below the soil surface.

4. Some can survive extreme desiccation and form dry crusts in grasslands and
deserts that begin N, fixation when moistened.

5. In rice paddies, Anabaena and Nostoc are critical components of the

production system.

6. Long-term sustainable rice production without N fertilization is possible

because N, fixation by cyanobacteria in paddies produces 30 to 70 kg N/ha/y
and meets the needs of rice growth (Coyne, 1999).

Not all blue greens and representatives of none of the other algal classes can utilize N
however. Nitrogen fixation by the active strains is stimulated by increasing light intensi-
ties, but excessive light is inhibitory. The transformation is invariably slow, and gains of
30 to 115 μg of nitrogen per milliliter of solution require periods of l 1/ 2 to 2 months for
most isolates of Aulosira, Anabaenopsic, Anabaena, Cylindrospermum, Nostoc, and
Tolypothñx. The fixation, therefore, is much less rapid than in the Azotobacters or
Clostridia. In addition to vegetative cells, certain filamentous blue-green algae—such as
Anabaena, Cylindrospermum, and Mastigocladus—possess structures known as
heterocysts. Under the light microscope, the thick-walled heterocyst is distinctive because
it gives the illusion of being an empty cell (Alexander, 1977).

Lichens

1. Lichens—which are classified as if they were separate organisms—such as

Collema, Licltina, Pettigera, and Stereocaulon thus assimilate N a process
that may aid in their growth in nitrogen-poor coastal habitats, the subarctic,
arid regions, and on glacial drifts.

2. The blue-greens are similarly able to assimilate N when living in symbiosis

with some liverworts. a preridophyte (Azolla). An angiosperm (Gunnera) and
plants classified among the Cycadaceae (Alexander, 1977).
Certain free living microorganisms present in soil and are able to fix nitrogen. Because
these organisms are not directly associated with higher plants, the transformation is
referred to as nonsymbiotic or free-living. (Brady and Weil, 2002)

There exist in soil and water, certain free-living microorganisms that are able to fix
nitrogen. Because these organisms are not directly associated with higher plants, the
transformation is referred to as nonsymbiotic or free living. There is great diversity in the
metabolic types of free-living microorganisms, which are capable of Biological nitrogen
fixation. This includes about 20 genera of non-photosynthetic aerobic (Azotobacter,
Beijerincia) and anaerobic (Clostridium) bacteria and about 15 genera of photosynthetic
Cyanobacteria (blue-green algae) such as Anabaena and Nostoc (Hubbell and Kidder,
2003).

3.2.2. Fixation with Heterotrophs

Free-living, non-photosynthetic bacteria heterotrophs depend on soil organic matter as a

food source. The nitrogen fixing activity of free-living, non- photosynthetic, aerobic
bacteria is strongly dependent on favorable moisture conditions, oxygen, and an organic
food source. Anaerobic representatives (Clostridium) predominate in grassland and
waterlogged soils and soil aggregates where moisture conditions and organic substrates
are available but oxygen supply to the micro-environment of the bacteria is severely
restricted (Hubbell and Kidder, 2003). The amounts of nitrogen fixed by free-living non-
photosynthetic bacteria in the soil may achieve an approximate maximum of 15 kg/ha/yr.
This relatively low estimated contribution is the result of limited availability of suitable
organic substrates (energy sources) and low bacterial populations in the soil environment.
Nitrogen fixation is characteristically higher in environments such as tropical soils, where
such factors as substrate availability, temperature and moisture are more favorable to the
maintenance and activity of a high bacterial population (Murder, 1975).

Representative genera of microorganisms involved in Nonsymbiotic N fixation with

Heterotrophs are observed in Table 8:
Table 7. Representative genera of microorganisms involved in Nonsymbiotic N fixation
with Heterotrophs (Coyne, 1999).

Physiological Representative
Type of association Host (if any)
group genera
Azoyobacter
Azoyomonus
Azotococcus
Beijerincia
Free living; aerobic Digitaria Derxia
Pseudomonus
Digitaria Xanthobacter
Facultative Azospirillum
Heterotroph
anaerobe Bacillus
Anaerobe Klebsiella
Thiobacillus
Associative; aerobic Digitaria Clostridium
Desulfovibrio
Desulfotomaculum
Methanobacillus
Agrobacterium

3.2.3. Fixation with Autotrophs

Among the autotrophs able to fix nitrogen are certain photosynthetic bacteria and
Cyanobacteria. In the presence of light, these organisms are able to fix carbon dioxide
and nitrogen simultaneously (Brady and Weil, 2002).

Very little information is available concerning the possible contribution of the free-living,
photosynthetic Cyanobacteria to the nitrogen economy of soils but maximum gains of 50
Kg/ha/yr have been reported. Nitrogen-fixing activity of these organisms is, of course
strongly dependent on adequate sunlight in addition to favorable moisture conditions
(Stewart, 1973).

Living plant roots release a wide variety of simple organic compounds, which may be
used as food by free-living soil bacteria. This continuous supply of food supports a higher
microbial population in the soil immediately surrounding the plant root (rhizosphere).
Evidence indicates that native nitrogen-fixing bacteria are common in the rhizosphere of
certain plants and that they may fix significant amounts of nitrogen in some cases
(Anderson et al., 1985).

Food material released from the roots would be available in greater concentration to those
microorganisms more closely associated with the root surface (Azospirillum). A striking
example is seen in certain combinations of bacteria with some tropical grasses, which
have a high photosynthetic efficiency and grow under environmental conditions favoring
high photosynthetic activity. The roots of such plants may supply the nitrogen-fixing-
microorganisms with a relatively high and sustained supply of food (photosynthates),
which is available in limited supply in the Rhizosphere of most plants (Millbank, 1974).

Under appropriate environmental conditions, aerobic Cyanobacteria may contribute

significantly to nitrogen gains in fresh water environments and anaerobic Clostridium
may play a similar important role in fresh water. Most of the nitrogen fixation in marine
environments (about 20% of the total amount of annual biological fixation) is attributed
to the Cyanobacteria but many other kinds of nitrogen fixing microorganisms have also
been found in such environments. The significant contribution of photosynthetic
(Cyanobacteria) and non-photosynthetic (Clostridium) microorganisms to nitrogen
fixation in the rhizosphere of rice is well recognized. In addition, nitrogenase activity has
been detected in the soil surrounding the mycorrhizal roots of several conifers and in the
rhizosphere of marine (Thalassia) and freshwater (Glyceria) angiosperms (Torrey, 1975).

The leaf surface (phyllosphere) of certain plants in warm, humid tropical regions may
provide an additional favorable environment for the growth and nitrogen fixing activity
of such free-living bacteria as Azotobacter, Beijerinckia and Kiebsiella (Torrey, 1975).

Representative genera of microorganisms involved in Nonsymbiotic N fixation with

Autotrophs are observed in Table 8.
Table 8. Representative genera of microorganisms involved in Nonsymbiotic N fixation
with Autotrophs (Coyne, 1999).

Physiological group Type of association Representative genera

Free living; unicellular Gloeocapsa

Filamentous; Trichodesmium
nonheterocystous

Filamentous; heterocystous Anabaena

Autotroph Calothrix
Nostoc
Anaerobic; purple nonsulfer Rhodospirillum
Rhodopseudomonas
purple nonsulfer Chromatium
Green sulfer Chlorobium

Some of the important nitrogen fixing species are: Amorphonostoc, Anabaena,

Anabaenopsis, Aulosira, Chlorogloea, Cylindrospermurn, Fischerella, Gleotrichia,
Hapalosiphon, Mastigocladus. In pure cultures, blue green algae fix varying amounts of
nitrogen ranging from 5.2 to 14.48 mg/100 ml of the medium, depending upon the
incubation time (Subba Rao, 2001). Nitrogen fixing genera of blue-green algae are
observed in Table 9.

Table 9. Nitrogen fixing genera of blue-green algae (Subba Rao, 2001).

Unicellular Synechococcus,Gloeocapsa, Aphanothece,Dermocarpa

Xenococcus,Myxosarcina,Chroococcidiopsis, Pleurocapsa group

Filamentous, Plectonema Boryanum,Lyngbya,trichodesmiu, LPPgrpup,Oscillatoria

nonheterocystous ,Pseudoanabaena, Microcoleus, Schizothrix

Filamentous, Anabaena,Nostoc,Nodularia,Cylindrospermum,Scytonema,
heterocystous Fischerella,Tolypothrix,Aulosira,stigonrma,Hapalosiphon,Chloroglo
eopsis,Camptylonema,Gloeotrichia,Ostochopsis,Rivularia,Scytonema
topsis,Westiella,Westiellopsis,Wollea,Chlorogloea.
Most blue-green algae develop best in neutral or weakly alkaline media. Normally below
pH 6.5 there is adversely affected. The optimum temperature for blue-green algae is close
to 28ºC-30ºC. Sunlight is another very important factor for their development. Most of
these microorganisms seem to be obligate prototroph (Mishra, 2000).

Nitrogen assimilation is quite widespread among the blue-green algae. An ecological role
of the soil algae results from the ability of some algae to carry out non-symbiotic and
symbiotic nitrogen fixation using the enzyme nitrogenase. These algae therefore are not
only independent of preformed organic carbon, but also of fixed nitrogen. Nonsymbiotic
nitrogen fixation by blue green algae is not only of ecological importance in natural
ecosystems but may also be of considerable agronomic significance as a major source of
nitrogen to the rice plants under paddy cultivation (Killham, 1994).

Blue-green algae also prevent the soil erosion. This is an additional advantage besides
their role in accumulation of nitrogen in the soil. Singh (1961) noted the considerable
importance of algae in the improvement of alkaline soil. When rain falls on alkaline soils,
algae begin to develop. As a result of their activity the surface layer of the soil is enriched
with organic matter, the physical properties of soil improve, the pH is lowered and in the
absorbing complex, sodium is replaced by calcium, with an increased in the store of P 2 O 5
and nitrogen. Autotrophic blue green algae are the pioneer species in the reclamation of
rock. Thus the group as whole plays as an important role as pioneers in the formation soil
(Mishra, 2000).
4. Factors affecting N fixation

The most important factors influencing the quantity of N fixation by Rhizobia are soil
pH, mineral nutrient status, photosynthetic activity, climate, and legume management.
Any stress in the legume plant by these factors can severely reduce the yield and N
availability to subsequent crops (Tisdale et al., 2002). Again various environmental
factors like temperature, water stress, water logging, salinity and combined nitrogen
affect nitrogen fixation. These factors also affect the nodule growth, often differentially
affecting the process of cell division and enlargement (Mishra, 2000).

Soil pH

Soil acidity is a major factor restricting the survival and growth of Rhizobia in soil and
can severely affect nodulation and nitrogen fixing processes. Rhizobia and roots of the
host legume plants can be injured by soil acidity related to Al3+, Mn2+, and H+ toxicity, as
well as low levels of available Ca2+ and H 2 PO 4 -. There are significant differences in the
sensitivity of the various rhizobial species to soil acidity. Soil pH value below 6.0
drastically reduce the number of Rhizobiam meliloti in the root zone of alfalfa, degree of
nodulation, and yields of host alfalfa plants, whereas soil pH values between 5.0 and 7.0
have little influence on R. trifoli and the host red clover crop (Tisdale et al., 2002).

Mineral nutrient status

Except in acid soils, where Ca2+ and H 2 PO 4 - deficiencies can limit the growth of
Rhizobia, mineral deficiencies seldom reduce N fixation. N fixation in the nodules
requires more Mo than the host plant; thus Mo deficiency is the most important
micronutrient deficiency. Initiation and development of nodules can be affected by Co, B,
Fe, and Cu deficiency. Excess NO 3 - concentration in the soil can reduce nitrogenase
activity and thus N fixation. Nodules loss their pink color in a high NO 3 - soil. The
reduction in N fixation is related to the competition for photosynthate between NO 3 -
reduction and N fixation reactions (Tisdale et al., 2002).
Photosynthesis and climate

A high rate of photosynthate production is strongly related to increased N fixation by

Rhizobia .Factors that reduces the rate of Photosynthesis will reduce N fixation. These
include reduce light intensity, moisture stress, and low temperature (Tisdale et al., 2002).

Legume management

In general, any management practice that results in reduced legume stands or yield will
reduce the quantity of N fixed by legumes. These include water and nutrient stress,
excessive weed and insect pressure, and improper harvest management. Harvest practices
vary greatly with location, but excessive cutting frequency, premature harvest, and
delayed harvest, especially in the fall, can reduce legume stands and the quantity of N
fixed (Tisdale et al., 2002).

Temperature

Generally for any one host-rhizobial combination, maximum rates of activity are
obtained over a temperature range of 10º C.The actual values vary widely, usually around
15º C-25º C for tropical species. Nitrogen fixation and assimilation, gaseous diffusion
and solubility and respiratory activity are affected by temperature (Mishra, 2000).

Water stress

The effects of water stress like temperature are complex. Under conditions of
photosynthetic limitation, nitrogen fixation may fall rapidly following stomatal closure.
However, by maintaining a water supply to nodulated roots at the same time as exposing
the shoot system to a very low relative humidity, it has been possible to reduce
photosynthesis in soybean to a very low level without affecting nodule activity. Under
field conditions water stress may seriously reduce nitrogen fixation (Mishra, 2000).

Water logging

Intermittent water logging reduces the oxygen supply to the nodules and is therefore
inhibitory to the nitrogen fixation. Prolonged exposure to wet soils may result in the
formation of nodules with more air spaces and thus improved tolerance to water logging
(Mishra, 2000).
Salinity

Generally, nodulated crop plants do not like saline conditions, although at low salt levels
they may show compensation by producing larger nodules. Different legumes respond
differently to salinity. Plants grown on combined nitrogen are more tolerant than those
dependent on fixed nitrogen (Mishra, 2000).

Combined nitrogen

The reasons why combined nitrogen reduces nodulation as well as activity of performed
nodules have been studied extensively. There have been two schools of thought that
photosynthate is diverted away from nodules towards areas where combined nitrogen is
being assimilated, and that in the case of nitrate, bacteroid nitrate reductase produces
nitrite in concentrations high enough to inhibit nitroginase activity (Mishra, 2000).
5. Amount of N fixed in soil by BNF organisms

Although quite variable from site to site, the amount of nitrogen biologically fixed can be
quite high, especially for those systems involving nodules, which supply energy from
photosynthates and protect the nitrogenase enzyme system. Nonnodulating or
nonsymbiotic systems generally fix relatively small amounts of nitrogen. Many natural
plants communities and agricultural systems (generally involving legumes) drive the bulk
of their nitrogen needs from biological fixation (Brady and Weil, 2002).

Estimates of total annual biological nitrogen fixation range from 100 to 175x106 metric
tons, with about 90x106 metric tons of N per year fixed by Rhizobia. The quantity of
nitrogen fixed by properly nodulated legumes averages about 75% of the total N used for
plant growth. N fixation by most perennial legumes ranges from 100 to 200 lb/a/yr,
although under optimum conditions, N fixation can reach two to three times these values.
Short season annual legumes will often fix between50 and 100 lb N/a/yr (Tisdale et al.,
2002).

The typical levels of nitrogen fixation from different systems in soil by BNF organisms
are observed in Table 10.

Table 10: Typical levels of nitrogen fixation from different systems (Brady and Weil,
2002).

Crop or plant Associated organisms Typical

levels of
nitrogen
fixation, kg
N/ha/yr
Symbiotic
Legumes (nodulated)
Ipil Ipil tree (Leucena Bacteria (Rhizobium) 100-500
leucocephala)
Locust tree (Robina spp.) 75-200
Alfalfa (Medicago sativa) 150-250
Vetch (Vicia vilbosa) 50-100
Cowpea (Vigna unguiculata) 50-150
 Peanut (Arachis) 30-50
Soybean (Glycine max L.) 50-100
Pigeon pea (Cajanus) 40-80
Kudzu (Pueraria) 50-150
Nonleguminus(nodulated)
Alders (Alnus) Bacteria (Bradyrhizobium) 150-280
Species of Gunnera
Nonleguminus Actinomycetes (Frankia) 100-140
(nonnodulated) Cyanobacteria (Nostoc) 50-150
Pangola grass (Degetaria 10-20
decumbens)
Azolla Cyanobacteria (Anabena) 150-300

Nonsymbiotic Bacteria,(Azobacter,Clostridium) 5-20

Cyanobacteria (Various) 10-50

5.1 Measurement of nitrogen fixation with 15N

Qualification of nitrogen fixation rates in legumes and other nitrogen fixing plant/
15
microbial symbiosis can be achieved by N by two broad approaches. Once involves
15
growing the plant in an atmosphere containing N 2 and then at the end of the
experiment, digesting the plant material prior to mass spectrometric determination of the
15
fixed N. Although most satisfactory, this approach is expensive and so a second
approach using a 15NH 4 addition to soil has sometimes been adopted. Here uptake of 15N
by a nitrogen fixing plant is compared with a non inoculated, nonfixing plant of similar
growth stage. The difference in 15N between the two plant/ soil systems should provide
a reasonable measure of the fixation rate (Killham, 1994).
15
Fixation of nitrogen can be estimated by determining the natural abundance of N in
15
plant tissues because the fixation process leads to a natural depletion of N. Because of
variability in natural abundance in soil and plants, large numbers of samples are needed
and inter specific comparisons are difficult (Killham, 1994).
6. Importance and future prospects of BNF

Nitrogen is an essential plant nutrient. The most available from of nitrogen is gaseous N 2 ,
which constitute about 80% of the atmosphere. Nitrogen in this form is chemically
unreactive and thus unavailable to most plant and bacteria. this unavailability is a limiting
factor for plant growth. To be incorporated in organic matter nitrogen from the
atmosphere must be reduce the NH 3 , through the process called Biological Nitrogen
Fixation (Anderson et al., 1985). Biological fixed nitrogen is the most important
biochemical reaction for life on earth. Through this certain organisms convert the inert
dinitrogen gas of atmosphere (N 2 ) to nitrogen containing organic compounds that
become available to all forms of life. Because of the inertness of nitrogen gas a very
specific and sophisticated enzyme, nitrogenase is required to catalyze the reaction (Brady
and Weil, 2002). Biologically fixed nitrogen forms an important N source for uptake by
plants. In the case of the legume or other nitrogen fixing plant or microbial symbiosis, the
nitrogen transferred from the microbes to the host through translocation. The fixed
nitrogen is translocated in the legume in the form of amides where the nitrogen is
intimately bound to organic carbon. Plants other than legumes can only gain access to
this nitrogen when the nitrogen fixing organisms dies and is broken down by
microorganisms in soil (Killham, 1994).

The growth of all organisms depends on the availability of mineral nutrients, none is
more important than nitrogen, which is required in large amounts as an essential
component of proteins, nucleic acids and other cellular constituents. However nitrogen is
unavailable for use by most organisms because there is a triple bond between the two
nitrogen atoms, making the molecule almost inert. In order for nitrogen to be used for
growth and reproduction, it must be fixed in the form of NH 4 + to NO 3 -, which can be
accomplished biological nitrogen fixation (Hubbell and Kidder, 2003).

Some species of microorganisms have the ability to convert atmospheric nitrogen into
forms that are usable by plants and animals. The system most important for agriculture is
the legume rhizobia symbiosis.The fixation of atmospheric nitrogenoccurs within root
nodules after rhizobial penetration of the root. Thus, many legumes can grow vigorously
and yield well under nitrogen occurs within root nodules after rhizobial penetration of the
root. Thus many legumes can grow vigorously and yield well under nitrogen deficient
conditions, and many contribute nitrogen to the farming system in the vegetative residues
after grain harvest, or more significantly as green manure in corporate in the soil (Peoples
et al., 1989 and Takishima et al., 1989). Legumes are important source of protein, mainly
for feed in the developed world and for food in the developing world. They have been
exploited as source of nitrogen most notably in the agricultural systems of Australia and
Newziland. The successful introduction of exotic legume crops, such as alfalfa and
soybean in to the United States, necessitated the simultaneous introduction of compatible
rhizobia bacteria; such inuculant, in various forms, have been in use for about 100 years
(Ngambeki, 1985). Improvement in agriculture sustainability will require the exploitation
of BNF as a major source of nitrogen for plants. Mixed cropping and crop rotation of
nonlegumes with legumes were employed to capitalize on BNF. Nonlegumes deplete the
soil nitrogen, whereas leguminous crops can restore nitrogen as organic forms that are
easily leachable (Ngambeki, 1985). Farmers are most likely to adopt Biological nitrogen
fixation because it reduces production cost and increases yields, and also it reduces risk.
Reduced production cost or higher yields of food and feed lead to increased production
and ultimately, to lower food price (Ngambeki, 1985).

The need for food, feed, fuel and building materials has made deforestation an
increasingly processing problem in the developing world; legumes and other nitrogen
fixing trees offer a means of reversing this trend, especially the use of fast growing
nitrogen fixing trees (Ngambeki, 1985). Many organisms have the unique ability to fix
atmospheric nitrogen.

6.1 Future prospects in Biological Nitrogen Fixation

Biological nitrogen fixation is a source of combined nitrogen for plants. Some goals of
research on Biological nitrogen fixation are as follows:

1. Transfer N 2 fixing genes from bacteria to higher plants cells, thus endowing the
plant with the capability for utilizing molecular N 2 .

2. Transfer N 2 fixing genes into a beneficial bacterium capable of invading plant

cells and establishing an effective N 2 fixing system, such as nodule.
 3. Use protoplast fusion methods to create new symbiotic associations between
microorganisms and higher plants.

4. Select or develop by genetic means N 2 fixing bacteria capable of living on the

roots of such cereal crops as corn and wheat and providing adequate fixed
nitrogen for optimum plant growth.

5. Develop by genetic manipulation Rhizobiam strains that are intensive to soil NO 3 -

and NH 4 + that normally inhibit nodulation and N 2 fixation.

Developed by use of plant-breeding methods legumes that have increased photosynthetic

capabilities and, therefore, great capabilities for providing energy to N 2 fixing bacteria in
the nodules (Stevenson, 1986).

The most common economically important microorganisms which are involved in

Biological nitrogen fixation observed in Table 11.

Table 11. Economically important microorganisms involved in Biological Nitrogen

Fixation (Tisdale et al., 2002).

Organisms General properties Use in agriculture

Azotobacter Aerobic, free fixers, live in soil Proposed benefit to crops has not
water, rhizosphere, and leaf been confirmed; hormonal effect
surfaces. on root and plant growth

Azospirillum Microaerobic, free fixers or in Potential use in increasing yields

association with roots of of grasses; inoculation benefits
grasses. Inside root symbiosis crops. Hormonal effect on root
and plant growth.

Rhizobium Fix N in legume- Rhizobiam Legume crops are benefited by

symbiosis inoculation with proper strains.

Actinomycetes, Fix N in symbiosis with Potentially important in

Frankia nonlegume wood trees-alder, reforestation, wood production.
Myrica, casuarinas.

Blue-green Contain chlorophyll, as in Enhance rice in paddy soils;

algae, Anabaena higher plants, aquatic and Azolla (a water fern) - Anabaena
terrestrial azolla symbiosis; is used as
green manure.
7. Conclusion

Nitrogen (N) is the nutrient element most frequently found limiting to the growth of
green plants. This results from the continual loss of nitrogen from the reserve of
combined or fixed nitrogen, which is present in soil and available for use by plants. It is
continually depleted by such processes as microbial denitrification, soil erosion, leaching,
chemical volatilization, and perhaps most important, removal of nitrogen-containing crop
residues from the land. The nitrogen reserve of agricultural soils must therefore be
replenished periodically in order to maintain an adequate (non-growth limiting) level for
crop production. This replacement of soil nitrogen is generally accomplished by the
addition of chemically fixed nitrogen in the form of commercial inorganic fertilizers or
by the activity of biological nitrogen fixation (BNF) systems. The significance of BNF as
the major mechanism of recycling of nitrogen from the unavailable atmospheric form to
available forms in the biosphere cannot be overemphasized.
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