Nitrogen Fixation

By
Dr Khalil Ahmad
Assistant Professor
Emerson University Multan, Pakistan
 After Studying this topic we will be able to know about
 Nitrogen Cycle
 Nitrogen Fixation
 Nitrogen Fixation Mechanism
 Soil Nitrogen:
 Soil nitrogen exists in three general forms: organic nitrogen compounds,
ammonium (NH₄⁺) ions and nitrate (NO₃⁻) ions.
 At any given time, 95 to 99 percent of the potentially available nitrogen in the
soil is in organic forms, either in plant and animal residues, in the relatively
stable soil organic matter or in living soil organisms, mainly microbes such as
bacteria.
 This nitrogen is not directly available to plants, but some can be converted to
available forms by microorganisms. A very small amount of organic nitrogen
may exist in soluble organic compounds, such as urea, that may be slightly
available to plants.
 The majority of plant-available nitrogen is in the inorganic forms NH₄ ⁺ and NO₃ ⁻
(sometimes called mineral nitrogen). Ammonium ions bind to the soil’s negatively charged
cation exchange complex (CEC) and behave much like other cations in the soil. Nitrate ions
do not bind to the soil solids because they carry negative charges, but exist dissolved in the
soil water, or precipitated as soluble salts under dry conditions.
 Natural Sources of Soil Nitrogen:
 The nitrogen in soil that might eventually be used by plants has two sources:
nitrogen- containing minerals and the vast storehouse of nitrogen in the
atmosphere.
 The nitrogen in soil minerals is released as the mineral decomposes. This process
is generally quite slow, and contributes only slightly to nitrogen nutrition on
most soils. On soils containing large quantities of NH₄ ⁺-rich clays (either
naturally occurring or developed by fixation of NH₄ ⁺ added as fertilizer),
however, nitrogen supplied by the mineral fraction may be significant in some
years.
 Atmospheric nitrogen is a major source of nitrogen in soils. In the atmosphere, it
exists in the very inert N₂ form and must be converted before it becomes useful
in the soil. The quantity of nitrogen added to the soil in this manner is directly
related to thunderstorm activity, but most areas probably receive no more than
20 lb nitrogen/acre per year from this source .
 Natural Sources of Soil Nitrogen:
 Bacteria such as Rhizobia that infect (nodulate) the roots of, and receive much
food energy from, legume plants can fix much more nitrogen per year (some
well over 100 lb nitrogen/acre).

 When the quantity of nitrogen fixed by Rhizobia exceeds that needed by the
microbes themselves, it is released for use by the host legume plant.

 This is why well-nodulated legumes do not often respond to additions of

nitrogen fertilizer: They are already receiving enough from the bacteria .
 Plant Nitrogen Needs and Uptake:
 Plants absorb nitrogen from the soil as both NH₄ ⁺ and NO₃ ⁻ ions, but because
nitrification is so pervasive in agricultural soils, most of the nitrogen is taken up
as nitrate. Nitrate moves freely toward plant roots as they absorb water.

 Once inside the plant, NO₃⁻ is reduced to an NH₂ form and is assimilated to
produce more complex compounds. Because plants require very large quantities
of nitrogen, an extensive root system is essential to allowing unrestricted uptake.

 Plants with roots restricted by compaction may show signs of nitrogen

deficiency even when adequate nitrogen is present in the soil.
 Plant Nitrogen Needs and Uptake:
 Most plants take nitrogen from the soil continuously throughout their lives, and
nitrogen demand usually increases as plant size increases.
 A plant supplied with adequate nitrogen grows rapidly and produces large
amounts of succulent, green foliage. Providing adequate nitrogen allows an
annual crop, such as corn, to grow to full maturity, rather than delaying it.
 A nitrogen-deficient plant is generally small and develops slowly because it
lacks the nitrogen necessary to manufacture adequate structural and genetic
materials. It is usually pale green or yellowish because it lacks adequate
chlorophyll. Older leaves often become necrotic and die as the plant moves
nitrogen from less important older tissues to more important younger ones.
 On the other hand, some plants may grow so rapidly when supplied with excessive nitrogen
that they develop protoplasm faster than they can build sufficient supporting material in cell
walls. Such plants are often rather weak and may be prone to mechanical injury, such as
development of weak straw and lodging of small grains.
 Nitrogen Cycle:
 Bacteria such as Rhizobia that infect (nodulate) the roots of, and receive much
food energy from, legume plants can fix much more nitrogen per year (some
well over 100 lb nitrogen/acre).
 Nitrogen can go through many transformations in the soil. These transformations
are often grouped into a system called the nitrogen cycle, which can be presented
in varying degrees of complexity.
 The nitrogen cycle is appropriate for understanding nutrient and fertilizer
management. Because microorganisms are responsible for most of these
processes, they occur very slowly, if at all, when soil temperatures are below 50°
F, but their rates increase rapidly as soils become warmer.
 The heart of the nitrogen cycle is the conversion of inorganic to organic nitrogen
and vice versa. As microorganisms grow, they remove H₄ ⁺ and NO₃ ⁻ from the
soil’s inorganic, available nitrogen pool, converting it to organic nitrogen in a
process called immobilization.
 Nitrogen Cycle:
 When these organisms die and are decomposed by others, excess NH₄ ⁺ can be
released back to the inorganic pool in a process called mineralization. Nitrogen
can also be mineralized when microorganisms decompose a material containing
more nitrogen than they can use at one time, such as legume residues or
manures. Immobilization and mineralization are conducted by most
microorganisms, and are most rapid when soils are warm and moist, but not
saturated with water.
 The quantity of inorganic nitrogen available for crop use often depends on the
amount of mineralization occurring and the balance between mineralization and
immobilization.
 Ammonium ions (NH₄⁺) not immobilized or taken up quickly by higher plants
are usually converted rapidly to NO₃⁻ ions by a process called nitrification.
 Nitrogen Cycle:
 This is a two-step process:

 Bacteria called Nitrosomonas convert NH₄⁺ to nitrite (NO₂ ⁻)

 Other bacteria, Nitrobacter, convert the NO₂⁻ to NO₃⁻

 This process requires a well-aerated soil and occurs rapidly enough that one
usually finds mostly NO₃⁻ rather than NH₄⁺ in soils during the growing season.

 The nitrogen cycle contains several routes by which plant-available nitrogen can
be lost from the soil. Nitrate-nitrogen is usually more subject to loss than is
ammonium-nitrogen. Significant loss mechanisms include leaching,
denitrification, volatilization and crop removal.
 Nitrogen Cycle:
 The nitrate form of nitrogen is so soluble that it leaches easily when excess water
percolates through the soil. This can be a major loss mechanism in coarse-
textured soils where water percolates freely, but is less of a problem in finer-
textured, more impermeable soils, where percolation is very slow.

 These latter soils tend to become saturated easily, and when microorganisms
exhaust the free oxygen supply in the wet soil, some obtain it by decomposing
NO₃⁻. In this process, called denitrification, NO₃ ⁻ is converted to gaseous
oxides of nitrogen or to N₂ gas, both unavailable to plants. Denitrification can
cause major losses of nitrogen when soils are warm and remain saturated for
more than a few days.
 Nitrogen Cycle:
 Nitrogène Fixation
 Assimilation
 Ammonification
 Nitrification
 Dénitrification
 Types of Nitrogen Fixation
 Atmospheric fixation: A natural phenomenon where the energy of lightning breaks the
nitrogen into nitrogen oxides, which are then used by plants.
 Industrial nitrogen fixation: It is a man-made alternative that aids in nitrogen fixation by
the use of ammonia. Ammonia is produced by the direct combination of nitrogen and
hydrogen. Later, it is converted into various fertilisers such as urea.
 Biological nitrogen fixation: We already know that nitrogen is not used directly from the
air by plants and animals. Bacteria like Rhizobium and blue-green algae transform the
unusable form of nitrogen into other compounds that are more readily usable. These nitrogen
compounds get fixed in the soil by these microbes.
 Nitrogen Fixation:
 We eat chocolate but not cocubines.
 We eat chapati but not wheat.
 Same is for Nitrogen.
 Present in the atmosphere in 78% but not useful for organism.
 Furthermore, nitrogen is a key nutrient element for plants. However, the
abundant nitrogen in the atmosphere cannot be used directly by plants or
animals.

 Nitrogen Cycle is a biogeochemical process which transforms the inert nitrogen

present in the atmosphere to a more usable form for living organisms.
 Nitrogen Fixation:
 Healthy plants often contain 3 to 4 percent nitrogen in their above-ground tissues. This is a
much higher concentration compared to other nutrients.
 Carbon, hydrogen and oxygen, nutrients that don’t play a significant role in most soil fertility
management programs, are the only other nutrients present in higher concentrations.
 Major component of amino acids, the building blocks of proteins. Without proteins, plants
wither and die. Some proteins act as structural units in plant cells while others act as enzymes,
making possible many of the biochemical reactions on which life is based.
 Component of energy-transfer compounds, such as ATP (adenosine triphosphate). ATP allows
cells to conserve and use the energy released in metabolism.
 Significant component of nucleic acids such as DNA, the genetic material that allows cells
(and eventually whole plants) to grow and reproduce. Without nitrogen, there would be no life
as we know it.
 Nitrogen Fixation:
 Nitrogen fixation is the essential biological process and the initial stage of the nitrogen cycle.
In this process, nitrogen in the atmosphere is converted into ammonia (another form of
nitrogen) by certain bacterial species like Rhizobium, Azotobacter, etc. and by other natural
phenomena.

Different Ways of Nitrogen Fixation

 Plants are the main source of food. The nutrients obtained from plants are synthesized by
plants using various elements which they obtain from the atmosphere as well as from the soil.
This group of elements includes nitrogen as well. Plants obtain nitrogen from the soil and
utilise it in the process of protein synthesis.
 Unlike carbon dioxide and oxygen, atmospheric nitrogen cannot be obtained through the
stomata of leaves. Because the nitrogen gas present in the atmosphere can not be directly used
by plants. There are certain bacteria and some natural phenomenon that help in Nitrogen
fixation.
 Biological Nitrogen Fixation:
 Certain bacteria or prokaryotes are capable of converting atmospheric nitrogen
to ammonia. This process is called biological nitrogen fixation.
 The enzyme nitrogenase converts dinitrogen to ammonia.
 Nitrogen-fixing bacteria may be free-living or symbiotic. Some of the free-living
nitrogen fixers are Azotobacter, Beijernickia, Rhodospirillum, cyanobacteria,
etc.
 Examples of symbiotic nitrogen fixers are Rhizobium (in the root nodules of
legumes) and Frankia (in the root nodules of non-leguminous plants), etc.
 Symbiotic Nitrogen Fixation:
 A species of bacteria called Rhizobium, help in nitrogen fixation. These bacteria
live in the roots of leguminous plants (e.g., pea and beans plants) and using
certain types of enzymes, they help in fixing nitrogen in the soil.
 During this biological process, they convert the non-absorbable nitrogen form
into a usable form. This form of nitrogen gets dissolved in the soil, and plants
absorb the modified nitrogen from the soil.
 This is the reason behind farmers implementing crop rotation, where leguminous
plants help to replenish nitrogen content in the soil without the necessity of
fertilizers.
 Nitrogen fixation by bacteria is an example of the symbiotic relationship
between Rhizobium and leguminous plants. While bacteria fix nitrogen in the
soil, plants provide them food..
 Nitrogen Fixation by Lightening:
 Another process that helps in nitrogen fixation is lightning.
 It is a natural phenomenon where the energy of lightning breaks and converts the
non-absorbable form of nitrogen into a usable form.
 Even though the contribution of lightning in the nitrogen fixation is small, they
save plants from the deficiency of essential elements.
 Nitrogen oxides, e.g. NO, N2O and NO2
 are also produced in the atmosphere
 by industrial processes,
 automobile exhausts,
 power stations and forest fires
 Nitrogen Cycle:
 Assimilation

 Primary producers – plants take in the nitrogen compounds from the soil with
the help of their roots, which are available in the form of ammonia, nitrite ions,
nitrate ions or ammonium ions and are used in the formation of the plant and
animal proteins. This way, it enters the food web when the primary consumers
eat the plants.

 Ammonification

 When plants or animals die, the nitrogen present in the organic matter is released
back into the soil. The decomposers, namely bacteria or fungi present in the soil,
convert the organic matter back into ammonium. This process of decomposition
produces ammonia, which is further used for other biological processes.
 Nitrogen Cycle:
 Nitrification
 In this process, the ammonia is converted into nitrate by the presence of bacteria
in the soil. Nitrites are formed by the oxidation of ammonia with the help of
Nitrosomonas bacteria species. Later, the produced nitrites are converted into
nitrates by Nitrobacter. This conversion is very important as ammonia gas is
toxic for plants.

 The reaction involved in the process of Nitrification is as follows:

 2NH3 + 3O2 → 2NO2– + 2H+ + 2H2O

 2NO2– + O2 → 2NO3–
 Nitrogen Cycle:
 De-Nitrification

 Denitrification is the process in which the nitrogen compounds make their way
back into the atmosphere by converting nitrate (NO 3-) into gaseous nitrogen
(N). This process of the nitrogen cycle is the final stage and occurs in the
absence of oxygen. Denitrification is carried out by the denitrifying bacterial
species- Clostridium and Pseudomonas, which will process nitrate to gain
oxygen and gives out free nitrogen gas as a byproduct
 Nitrogen Cycle in Marine Ecosystem:
 The process of the nitrogen cycle occurs in the same manner in the marine
ecosystem as in the terrestrial ecosystem. The only difference is that it is carried
out by marine bacteria.

 The nitrogen-containing compounds fall into the ocean as sediments get

compressed over long periods and form sedimentary rock. Due to the geological
uplift, these sedimentary rocks move to land. Initially, it was not known that
these nitrogen-containing sedimentary rocks are an essential source of nitrogen.
But, recent researches have proved that the nitrogen from these rocks is released
into the plants due to the weathering of rocks.
 Nitrogen Cycle:
 Nitrogen fertilizer rates are determined by the crop to be grown, yield goal and
quantity of nitrogen that might be provided by the soil. Rates needed to achieve
different yields with different crops vary by region, and such decisions are
usually based on local recommendations and experience.

 FACTORS THAT DETERMINE THE QUANTITY OF NITROGEN

SUPPLIED BY THE SOIL

 The quantity of nitrogen released from the soil organic matter

 The quantity of nitrogen released by decomposition of residues of the previous
crop
 Any nitrogen supplied by previous applications of organic waste
 Any nitrogen carried over from previous fertilizer applications.
 Conclusion:
 Nitrogen is abundant in the atmosphere, but it is unusable to plants or animals
unless it is converted into nitrogen compounds.
 Nitrogen-fixing bacteria play a crucial role in fixing atmospheric nitrogen into
nitrogen compounds that can be used by plants.
 The plants absorb the usable nitrogen compounds from the soil through their roots. Then,
these nitrogen compounds are used for the production of proteins and other compounds in the
plant cell.
 Animals assimilate nitrogen by consuming these plants or other animals that contain nitrogen.
Humans consume proteins from these plants and animals. The nitrogen then assimilates into
our body system.
 During the final stages of the nitrogen cycle, bacteria and fungi help decompose organic
matter, where the nitrogenous compounds get dissolved into the soil which is again used by
the plants.
 Some bacteria then convert these nitrogenous compounds in the soil and turn it into nitrogen
gas. Eventually, it goes back to the atmosphere.
 These sets of processes repeat
 Nitrogen Fixation and Metabolism:

 According to NASA, the total composition of air in the earth’s atmosphere

consists of 78% nitrogen gas, 21% of oxygen, 0.93% argon, 0.04% carbon
dioxide, and small amounts of other gases. Many cellular components are also
made up of nitrogen. Despite all of it, the concentration of a usable form of
nitrogen is very less. Nitrogen gas is vital but a limiting element. All organisms
including humans, plants, and other organisms, demand nitrogen for their daily
cellular activities.

 There is a huge demand for nitrogen gas. Now the question is, how does the
environment meet all these demands?
 Nitrogen Metabolism:
 Metabolism is a set of chemical processes, which is carried out to convert
substances into usable energy forms.
 Nitrogen metabolism is mainly based on the recycling of ammonia (NH3) into
the neutral or charged form ammonium ion (NH4+).
 The main part of nitrogen metabolism is the Nitrogen Cycle.
 Nitrogen Metabolism:
 Did you know that if you cut through a nodule, its central portion is red or pink?
What gives it this colour? It is the presence of leguminous haemoglobin or leg-
haemoglobin. Let’s learn a little more about these nodules.
Nodule Formation
 Another form of nitrogen metabolism is nodule formation. Nodule formation involves several
interactions between the roots of the host plant and Rhizobium. They are:
 The Rhizobia multiply, colonise and attach themselves to the epidermal and root-hair cells of
legumes.
 The root-hair curls allowing the bacteria to invade, create an infection thread and reach the
cortex of the root. Here the bacteria initiate nodule formation.
 The infection thread then releases the bacteria into the cells. This leads to the differentiation
of special nitrogen-fixing cells. In this way, the nodule establishes a direct connection for
exchange of nutrients with the host.
 The nodule contains the enzyme nitrogenase which converts atmospheric nitrogen to the first
stable product of nitrogen fixation – ammonia. The reaction is:
 N2 + 8e– + 8H+ + 16ATP —→ 2NH3 + H2 + 16ADP + 16Pi.
 Nodule Formation:
 The energy required for the above reaction (8ATP for each NH3 produced)
comes from the respiration of host cells. Also, the enzyme nitrogenase is very
sensitive to molecular oxygen and therefore needs anaerobic conditions. The
oxygen-scavenger called leg-haemoglobin protects nitrogenase from oxygen in
the nodules.

 Interestingly, this microbe lives as aerobic, free-living organism where

nitrogenase is not functional. But during nitrogen-fixation, they become
anaerobic, thus protecting the enzyme from oxygen..
 Fate of Ammonia:
 Fate of Ammonia
 What happens to the ammonia generated after nitrogen-fixation? It is protonated
to form ammonium ion (NH4+) at physiological pH. Although plants can
accumulate nitrate and NH4+ ions, NH4+ ions are toxic to them. Thus, it is in
turn, used to synthesize amino acids in plants as follows:
 (i) Reductive amination: Here, ammonia reacts with α-ketoglutaric acid to
form glutamic acid in the presence of the enzyme – glutamate dehydrogenase.
 α-ketoglutaric acid + NH4+ + NADPH → glutamate + H2O + NADP
 (ii) Transamination: Here, the amino group of one amino acid is transferred
to the keto group of a keto acid in the presence of the enzyme – transaminase.
Asparagine and glutamine – the two most important amides in plants arise from
two amino acids – aspartic acid and glutamic acid, respectively. Another NH2–
radicle replaces the hydroxyl group of the acid to give an amide. The xylem then
transports these amides that contain more nitrogen to different parts of the plant.
 Nitrogen Fixation Mechanism:
 Biological nitrogen fixation (BNF) occurs when atmospheric nitrogen is
converted to ammonia by an enzyme called nitrogenase. The reaction for BNF
is: N2+8H++8e−→2NH3+H2.(5.15C.1)
 This type of reaction results in N2 gaining electrons (see above equation) and is
thus termed a reduction reaction.
 The exact mechanism of catalysis is unknown due to the technical difficulties
biochemists have in actually visualizing this reaction in vitro, so the exact
sequence of the steps of this reaction are not completely understood. Despite
this, a great deal is known of the process. While the equilibrium formation of
ammonia from molecular hydrogen and nitrogen has an overall negative
enthalpy of reaction (i.e. it gives off energy), the energy barrier to activation is
very high without the assistance of catalysis, which is done by nitrogenases.The
enzymatic reduction of N2 to ammonia therefore requires an input of chemical
energy, released from ATP hydrolysis, to overcome the activation energy barrier..
 Nitrogen Fixation Mechanism:
 A General Catalytic Mechanism Scheme for Nitrogenase:
 A) Components I and II are dissociated; II is ready for reduction.
 B) ATP binds to component II, which receives electrons from an electron donor
(ferredoxin or flavodoxin); binding of ATP induces an allosteric conformational
change which allows association of the two proteins. Electrons flow from the
[4Fe-4S] cluster on II to the P cluster on I.
 C) Electrons are further shuttled to the iron-molybdenum cofactor (FeMoco),
and ATP is hydrolised to ADP. This step is repeated several times before a
molecule of N2 can bind to FeMoco.
 D) The protein complex dissociates, and nitrogenase reduces dinitrogen to
ammonia and dihydrogen. Legend:I: component I (dinitrogenase; MoFe protein);
II: component II (dinitrogen reductase; Fe protein); ATP: adenosine triphosphate;
ADP: adenosine diphosphate; Fdx: ferredoxin; Fld: flavodoxin.
 Nitrogen Fixation Mechanism:
 Nitrogenase is made up of two soluble proteins: component I and II. Component
I known as MoFe protein or nitrogenase contains 2 Mo atoms, 28 to 34 Fe
atoms, and 26 to 28 acid-labile sulfides, also known as a iron-molybdenum
cofactor (FeMoco).
 Component I is composed of two copies each of two subunits (α and β); each
subunit’s stability depends on the other in vivo.
 Component II known as Fe protein or nitrogenase reductase is composed of two
copies of a single subunit. This protein has four non-heme Fe atoms and four
acid-labile sulfides (4Fe-4S). Substrate binding and reduction takes place on
component I, which binds to ATP and ferredoxin or flavodoxin proteins (Fdx or
Fld) (see step B).
 Nitrogen Fixation Mechanism:
 The hydrolysis of ATP supplies the energy for the reaction while the Fdx/Fld
proteins supply the electrons.
 Note this is a reduction reaction which means that electrons must be added to the
N2 to reduce it to NH4. Thus, the role of component II is to supply electrons,
one at a time to component I. ATP is not hydrolyzed to ADP until component II
transfers an electron to component I (see step C and D). 21-25 ATPs are required
for each N2 fixed.
 The association of nitrogenase component I and II and later dissociation occurs
several times to allow the fixation of one N2 molecule (see step B and D).

 Nitrogenase ultimately bonds each atom of nitrogen to three hydrogen atoms to

form ammonia (NH3). The nitrogenase reaction additionally produces molecular
hydrogen as a side product, which is of special interest for people trying to
produce H2 as an alternative energy source to fossil fuels.