The document discusses associative nitrogen fixation (ANF), where diazotrophic bacteria live freely in soil and fix atmospheric nitrogen, making it available to plants. ANF involves various free-living bacteria, fungi, and algae that inhabit terrestrial and aquatic environments. Key factors like temperature, moisture, and presence of nutrients influence ANF. Though typically lower yield than symbiotic fixation, ANF is important for fertilizing rice and grasses without legume hosts.

2. Content
 Introduction
 Nitrogen source
 Role of nitrogen in plant
 Bio-chemistry of nitrogen fixation
 Mechanism of nitrogen fixation
 Basic requirement of nitrogen fixation
 Type of nitrogen fixation
• Physical nitrogen fixation
• Chemical nitrogen fixation
• Biological nitrogen fixation
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3.  Types of biological nitrogen fixers
• Symbiotic nitrogen fixation
• Non-symbiotic (Associative) nitrogen fixation
 Difference between associative & symbiotic Nitrogen
fixation
 Associative Nitrogen fixation (ANF)
 Importance of associative nitrogen fixation
 Factors affecting nitrogen fixation
 Summary
 References
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4. Introduction
 Nitrogen gas are found 78.09% (Wikipedia) on atmosphere of earth.
 Nitrogen are found in molecular N2 (N=N) form in soil.
 Nitrogen fixation is a process by which nitrogen of the Earth’s
atmosphere is converted into ammonia (NH3), nitrogen salts or
other molecules available to living organisms.
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5. Nitrogen sources
 Atmospheric Nitrogen
 Nitrates, Nitrites and ammonia
 Amino acids in the soil
 Organic nitrogenous compounds in insects
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6. Role of nitrogen in plant
 Major substance in plants next to water
 Building blocks
 Constituent element of
Chlorophyll
Cytochromes
Alkaloids
Many vitamins
 Plays important role in metabolism, growth reproduction and heredity.
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7. Biochemistry of nitrogen fixation
 Nitrogen fixation process is given Burris 1966.
 Nitrogen fixation in atmosphere N2 is convert into ammonia or nitrates
presence in nitrogenase enzyme.
 Nitrogen fixation in first stable compound is ammonia.
 Nitrogen convert into ammonia is a reduction.
 In this process, N2 is spilt up into free atoms by breaking the triple bond
with the help of enzyme nitrogenase.
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8.  This reaction is endergonic (energy consuming) it requires an input of
nearly 160kcal energy.
N2 + 160kcal. Nitrogenase 2N
 Free nitrogen combines with hydrogen forming NH3 presence of
hydrogenate enzyme.
 The reaction is exergonic (energy releasing) (13kcal, energy).
2N + 3H2
Hydrogenase 2NH3 + 13kcal
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9. Mechanism of nitrogen fixation
 Nitrogen fixation, natural and synthetic, is essential for all forms of
life because nitrogen is required to biosynthesize basic building
blocks of plants, animals and other life forms,
 e.g. -: nucleotides for DNA and RNA and amino acids for proteins.
 Nitrogen fixation is a process by which nitrogen in the Earth’s
atmosphere is converted into ammonia (NH3) or other molecules
available to living organisms. Atmospheric nitrogen of molecular
dinitrogen (N2) is relatively inert, it does not easily react with other
chemicals to form new compounds.
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10. Basic requirement of nitrogen fixation
 Nitrogenase and hydrogenase enzymes.
 Protective mechanism against Oxygen.
 Ferrodoxin
 Constant supply of ATP.
 Hydrogen releasing system or electron donor (pyruvic acid or glucose/
sucrose.)
 Co-enzyme and cofactors TPP, CoA, Inorganic phosphate and Mg 2+
 Cobalt and Molybdenum.
 A carbon compound
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11. Type of nitrogen fixation
Nitrogen
fixation
Physical
N2 fixation
Chemical
N2 fixation
Biological
N2 fixation
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12. Physical nitrogen fixation
 Physical nitrogen fixation are occur by the natural factor such as,
lightening, thunder.
 It occur in several steps and starts with combination atmospheric discharge
and thunder to produce nitric oxide.
N2 + O2
Lightening Nitric oxide [2NO]
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13.  The nitric oxide is then oxidized to nitrogen peroxide in presence of
oxygen.
2NO + O2
Oxidation Nitrogen peroxide [2NO2]
 During rains, the nitrogen peroxide combines with rain water to form
nitrous acid and nitric acid which come to ground along with rains.
2NO2 + rain water HNO2 + HNO3
 On ground the alkali radicals of the react with nitric acid to produce
nitrites and nitrates which are soluble in water and which can be absorbed
by the plant through roots.
Ca+2 or K+ salts + HNO3
soil Ca+2 or K+ Nitrates
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14. Chemical nitrogen fixation
 Reduction of N2 into NH3 by the chemical process is called
chemical nitrogen fixation.
 In the use of Haber’s method.
 Haber’s process.
 Haber’s method use of high pressure (nearly 200Atm) and high
temperature (nearly 200 0C).
N2 + 3H2
200 ℃/200 atm 2NH3
 The method is used in agriculture and industry.
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15. Biological nitrogen fixation
 Fixation of atmospheric nitrogen into nitrogenous salts with the help of
micro-organisms.
 Biological nitrogen fixation (BNF) occurs when atmospheric nitrogen is
converted to ammonia by an enzyme called nitrogenase.
 Biological nitrogen fixation in all micro-organisms are prokaryotes.
 Those micro-organism are called is diazotrophs.
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16. Types of biological nitrogen fixers
 Biological nitrogen fixers classified based on fixing micro-organism.
 Per year 60% part of total nitrogen fixing by biological nitrogen fixing on
Earth
 Two types
• Symbiotic
A part of biological relationship in which plants provides a
niche and fixed carbon to bacteria in exchange for fixed nitrogen.
• Non symbiotic (Associative)
A process of biological nitrogen fixation performed by a group
of autotrophic bacteria living free in the soil either aerobically or
anaerobically and not depend on plant. 16

17. Associative nitrogen fixation Symbiotic nitrogen fixation
Non-symbiotic nitrogen fixation bacteria
are free living in the soil.
Symbiotic nitrogen fixation bacteria live
in a mutualism relationship with plants.
Ammonia, nitrites and nitrates are
formed.
Ammonia, Amino acids, and ureides are
formed.
Non-symbiotic bacteria make nitrogen
available in the soil.
Symbiotic bacteria produce nitrogen for
its host.
Ex : Azotobacter spp., Beijerinkia,
Gluconobacter, etc.
Ex : Rhizobium meliloti, Rhizobium
trifolii, Rhyzobium leguminosarum.
Difference between associative & symbiotic N fixation
(Important)
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18. Associative Nitrogen Fixation [ANF]
 Associative nitrogen fixation, the process by which dinitrogen gas is
converted to ammonia by bacteria in casual association with plants has
not been well-studied in temperate ecosystem.
 Fixation carried out by free living micro-organisms.
 They inhabit both terrestrial & aquatic habitats.
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19.  Aerobic, anaerobic, and blue green algae.
 Bacteria : special type (nitrogen fixing bacteria) types,
• Free living aerobic : Azotobacter, Beijerenkia
• Free living anaerobic : Clostridium
• Free living photosynthesis : Cholorobium, Rhodopseudomonas
• Free living chemosynthetic : Desulfovibro, Thiobacillus
 Free living fungi : Yeast and Pillularia
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20.  Blue green algae;
•Unicellular- Gloethece, Synechococcus
•Filamentous;(non heterocystous)- Oscillatoria plectonema.
•Filamentous(heterocystous)- Anabeana, Nostoc
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21.  In ecosystems where legumes are sparse or absent, associative nitrogen
fixation or nitrogen fixation by free living diazotrophic bacteria
associated with the roots of non-leguminous plants, can be a mechanism
for meeting part of the N requirements of plants.
 Associative N fixation has been reported in both C3 and C4 grasses but C4
grasses may be better candidates for associative N fixation because of
their greater photosynthetic efficiency.
 Associative N fixation appears to be more common in tropical and
subtropical environments, but low rates of associative N fixation have
been reported for several C4 species including corn, sorghum and switch
grass.
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Importance of associative nitrogen fixation

22.  It has been recognized for a long time that associative N2-fixing
biological systems in wetlands enrich the soil organic N pool and supply
up to 113 kg N/ha to rice crop depending upon the ecosystem, cultural
practices and rice variety grown.
 Inhibitory effect of exogenous supply of N fertilizer indicates limited
potential of associative N2 fixation to significantly benefit agriculture.
Farmers would have to withhold N fertilizer from their rice crop in
order to increase biological N2-fixation associated with rice.
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23.  In addition, scare and the increasing cost of non-renewable chemical
fertilizers necessitates the greater use of renewable indigenous biological
N2-fixation system as source of N for the rice.
 Part of fixed N is available to the plant immediately
 Plant associated N2 fixation is less sensitive to N fertilizer application
 These associations are of great importance as a substitute for nitrogen
fertilizer.
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24. Factors affecting N2 fixation
 Presence of nitrate or ammonium.
 Presence of certain inorganic substances
 Ca, Co, Mo- influence N2 fixation along with P
 PH :
• Neutral – Azotobacter
• Acidic – Beijerinkia
 Soil moisture : adequate is good for fixation
 Temperature : mesophilic-30 0C.
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25. Summary
 In associative nitrogen fixation mainly involved free living
microorganisms as aerobically or anaerobically and not depend on plant.
Here associative nitrogen fixation microorganisms are introduced to
grasses family plants economically significant.
 Thereby nitrogen requirement will be depended on associative
microorganisms. Both free living bacteria, fungi and algae are involved
with this associative nitrogen fixation.
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26.  However this nitrogen fixation very depend on C4 or C3 variation. In
C4 plant having high level of nitrogen fixation but C3 having low level
of nitrogen fixation that C3 nitrogen fixation level can increase using
new technology such as converting C3 to C4 plant, using recombination
DNA technology.
 However that ANF is very applicable to rice plantation other than
symbiotic nitrogen fixation because of ANF bacteria do not require
special host plant. Simply, Here mechanism is free living
microorganisms are absorbed of atmospheric nitrogen to form
protoplasm, that fixed nitrogen are available for plants after the
decomposing those microorganisms.
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27. References
 Agrawal. K and Sharma. J (2008), Plant physiology & Biochemistry, 1St edition, CBH
application, Jaipur.
 Singh V, Panday PC, Jain DK. (2009-2010), Plant physiology & Biochemistry, 3rd
edition, Rastogi publication, Meerut, New Delhi.
 J. Range Manage. 471192-196, May 1994. Indications of associative nitrogen fixation
in eastern Gamagrass.
 Panday, S N and Sinha, BK. (1994), Plant physiology, 2nd edition, Vikas publication
house, New Delhi.
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