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Nitrogen Cycle: Overview, Nitrogen Fixation (Atmospheric, Industrial, Biological), Nitrification, Assimilation, Denitrification, Ammonification, Practice Problems and FAQs

Nitrogen Cycle: Overview, Nitrogen Fixation (Atmospheric, Industrial, Biological), Nitrification, Assimilation, Denitrification, Ammonification, Practice Problems and FAQs

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Nitrogen is the fourth  most abundant element in the living system. It is a constituent of a number of organic compounds like amino acids, proteins, nucleotides etc. Its availability from soil is limited, so the plants have to compete with microbes in natural and agricultural ecosystems to obtain this. 

 Obtaining nitrogen

Nitrogen is abundant in the atmosphere, but plants cannot directly absorb it, therefore it is the most critical element.

Availability of nitrogen

Regular supply of nitrogen to the plant is maintained through the nitrogen cycle. Two atoms joined by a triple covalent bond in nitrogen. Let's understand the metabolism of nitrogen in detail in this topic.

Nitrogen bonds

Nitrogen Cycle

The nitrogen cycle is a biogeochemical process. Plants, the soil, the atmosphere and nitrogen are involved in this cycle.

Nitrogen cycle

The nitrogen cycle helps the nitrogen go from the atmosphere to the soil, to the plants and re-enter the atmosphere.

 Pathway of nitrogen

Nitrogen Fixation

It is the process of conversion of atmospheric nitrogen or dinitrogen (N2) to ammonia and other related compounds.

Conversion of nitrogen

During this process the triple covalent bond holding the nitrogen atoms is broken to give ammonia and other nitrogen compounds. Plants cannot break the covalent bond on their own. Hence, they rely on nitrogen fixation to convert atmospheric nitrogen into forms which can be absorbed easily.

 Nitrogen conversion

Modes of Nitrogen Fixation

There are three ways in which nitrogen fixation happens in nature as follows:

 Modes of nitrogen fixation

Atmospheric Nitrogen Fixation

It is the process of fixation of atmospheric nitrogen into oxides of nitrogen, nitrates, or nitrites by the processes that involve high energy circumstances like lightning, UV exposure etc. High energy of lightning breaks the triple bond between nitrogen and combines it with oxygen in air, leading to the formation of nitric oxide, nitrogen dioxide or nitrous oxide. The nitrogen oxides combine with rain water to form nitrates which seep into the soil. Hence, during atmospheric nitrogen fixation, nitrogen is converted into nitrates, rather than ammonia.

Nitrogen conversion

Industrial Nitrogen Fixation

It is the process of fixation of atmospheric nitrogen into ammonia, oxides of nitrogen, nitrites, or nitrates by industrial means under artificial reaction conditions like high temperatures, high pressures and catalysts.

Sources of industrial nitrogen fixation

Sources of industrial nitrogen fixation include the following:

  • Industrial smoke - Released from the chemical reactions in industries. 
  • Vehicular exhaust - Released from burning of fuels in vehicles
  • Forest fires - It causes the nitrogen held in the plants to combine with oxygen in the air to form nitrogen oxides.

The oxides are formed mainly by the reaction of nitrogen and oxygen in air, facilitated by the high temperatures in industrial processes and burning of fuels in vehicles. These nitrogen oxides eventually find their way into the soil as nitrates.

Sources of nitrogen

Biological Nitrogen Fixation

Certain bacteria have the capability of fixing atmospheric nitrogen into the soil. This type of conversion of atmospheric nitrogen into ammonia is known as biological nitrogen fixation. It is the major contributor to the nitrogen fixation step of the nitrogen cycle. It is mainly performed by nitrogen fixing microbes known as diazotrophs

Biological nitrogen fixation

: Nitrogen conversion

Requirements for Biological Nitrogen Fixation

The following ingredients are required for biological nitrogen fixation:

  • Strong reducing agents like NADPH2, FMNH2 etc.
  • Source of energy like ATP.
  • Compounds formed from the reduction of dinitrogen.
  • Enzymes like nitrogenase 

Nitrogenase

The nitrogen fixing bacteria contain a special enzyme called nitrogenase. It is a protein containing a Fe and a Mo-Fe component. This enzyme causes the reduction of the triple bonds in the nitrogen to ammonia.

Nitrogenase

Mechanism of Action of Nitrogenase

Nitrogenase uses atmospheric nitrogen as a substrate and reduces the triple bond between the two atoms of nitrogen. Iron and molybdenum attach to the dinitrogen molecule and weaken the bond between the two atoms. The weakened nitrogen molecule is then reduced with the help of reducing agents like NADPH2, FMNH2 etc. It results in the formation of dimide (N2H2), hydrazine (N2H4). At the end of reaction, 2 molecules of ammonia are released from a single molecule of N2. Once the reaction is completed, the enzyme is free to bind with another molecule of nitrogen.

Mechanism of action of nitrogenase

Fig: Biological nitrogen fixation

This reaction requires a lot of energy in the form of ATP (16 ATP molecules). Depending on the nitrogen fixer, the ATP molecules and reducing agents are provided by photosynthesis or respiration.

 Nitrogen fixation reaction

Types of Biological Nitrogen Fixers

There are different types of biological nitrogen fixers as follows:

Asymbiotic or Non-symbiotic Nitrogen Fixer or Free-Living Nitrogen Fixer

They are not forming any symbiotic association with plants. It include the following organisms:

  • Aerobic bacteria - Azotobacter and Beijerinckia
  • Anaerobic bacteria - Clostridium
  • Photosynthetic bacteria - Rhodospirillum
  • Chemosynthetic bacteria - Desulfovibrio
  • Cyanobacteria - Nostoc, Anabaena
  • Fungi - Yeast

Symbiotic Nitrogen Fixer

They form symbiotic association with tissues of higher plants. Examples include Rhizobium leguminosarum

Nitrogen fixing bacteria

Rhizobium

It is a rod-shaped, non-sporulating, Gram negative, motile and anaerobic microorganism.

 Rhizobium

Stages of Nodule Formation by Rhizobium

Rhizobium forms symbiotic association with the tissues of higher plants like beans, gram, groundnut etc., which belongs to the family Leguminosae. In the root, the vascular bundles are enclosed by the pericycle. This layer of cells is followed by the cortex. On the outside, the root has thin, thread-like root hairs.

T.S of root hair

Stage 1 - Multiplication of Rhizobium

Plants release certain chemical signals, such as flavonoids. In response to these signals, the Rhizobium around the root hairs start multiplying around the hairs. After multiplication, the bacteria attach and colonise on the root hairs and on the epidermis of the root hair.

HAIR

Stage 2 - Curling of Root Hair

After the colonisation of the microbes, on the root hair, the hairs start curling up. Specific chemicals released by bacteria help in curling. 

Curling of root hair

Stage 3 - Formation of the Infection Thread

An infection thread is formed from the root hair into the cortex of the root. The infection thread is formed by plasma membrane. It separates the infected tissue from the rest of the plant. This helps in the entry of microorganisms to the root hairs. 

Formation of infectious thread

Now Rhizobium enters into the cortex of the root. Cell division is stimulated in the infected tissue and more bacteria invade the newly formed tissues.

Entry of Rhizobium

Stage 4 - Formation of Nitrogen-fixing Cells

Rhizobium infects the cortex. These cells differentiate into specialised nitrogen fixing cells. Some of the bacteria enlarge to become membrane bound structures called bacteroides

Formation of nitrogen-fixing cells

Slowly, the nodules start forming at the sites of infection. Cytokinin produced by the invading bacteria and auxin produced by the plant cells helps in nodule formation by promoting cell division. 

Formation of nodule

The nodule expands into the pericycle, thus giving the nitrogen fixing cells access to the vascular bundles. The nodule also bulges outwards to form a swelling. Nitrogen fixation is controlled by nod, nif and fix genes. 

Invasion of nodule

Stage 5 - Formation of Vascular Connections

The nodule establishes a direct vascular connection with the plant host. The bacteria present in the nodule fix atmospheric nitrogen into nitrates, which are transported to the plant through the vascular connections and the xylem.

Formation of vascular connections

The roots of the legumes have many such nodules which fix atmospheric nitrogen.

Root nodules

Nitrogen Fixation in Nodules

Nitrogen fixation in the nodules happens with the help of the enzyme nitrogenase, which is present in the nodules. The enzyme nitrogenase breaks the triple covalent bond of atmospheric nitrogen to yield ammonia.

Nitrogen conversion

Leghaemoglobin

Enzyme nitrogenase is highly sensitive to oxygen. Oxygen can irreversibly inactivate the enzyme and hence, it requires anaerobic conditions. The enzyme nitrogenase has a protector, known as leghaemoglobin. It is found in the nodules of the leguminous plants, that's why it is known as leghemoglobin.

Leghaemoglobin is an oxygen scavenger, which means that it has the ability to bind to oxygen molecules. Thus, it prevents the oxidation and inactivation of nitrogenase enzymes and hence ensures that nitrogen fixation happens smoothly.

Root nodule with leghaemoglobin

Nitrification

The process of oxidation of ammonia (formed due to nitrogen fixation) into nitrites and nitrates is known as nitrification. It is a two-step process: 

  • Nitrite formation - Primary nitrification
  • Nitrate formation - Secondary nitrification

Nitrification

Chemoautotrophs

The microorganisms catalysing this conversion are the free-living chemoautotrophs. Chemoautotrophs are the organisms that obtain their energy from the oxidation of inorganic compounds like ammonia but their source of carbon is the carbon dioxide (CO2). Only free-living microorganisms perform nitrification. 

Primary Nitrification

Nitrification is required for the conversion of the ammonia formed as a result of the biological nitrogen fixation into nitrites or nitrates so that it can be easily taken up by the plants. During primary nitrification, the ammonia is converted to nitrites through the action of microbes like Nitrosomonas, Nitrococcus etc.

Primary

Secondary Nitrification

In secondary nitrification, another set of microbes like Nitrobacter converts the nitrites to nitrates. This process is an energy generating process.

Secondary

Assimilation

It involves the uptake of nitrates, by plants from the soil through roots which is further transported to leaves. This assimilated nitrogen accumulates inside animals when they feed on plants.

Assimilation

Metabolism of Nitrates

Nitrates available in high concentration, can be toxic to plants. Nitrates are first reduced to ammonia before incorporated into the organic compounds as it cannot be used directly for the synthesis of organic compounds by plants. For example, for the formation of amino acids, the nitrates are reduced to ammonia in the leaves. Further, ammonia forms an amine group of amino acids and thus becomes a part of proteins.

Metabolism of nitrates

Reduction of Nitrates to Nitrites

Nitrate reductase helps in the reduction of nitrate to nitrite. This enzyme possesses FAD as the prosthetic group. It receives hydrogen from the reduced coenzymes like NADH or NADPH for the reduction of nitrate.

Reduction of Nitrite

The nitrite ions are reduced to ammonia with the help of the enzyme nitrite reductase. This process requires ferredoxin also. The reduced coenzymes like NADH normally serve as hydrogen donor in the process of reduction of nitrates to nitrites.

Fate of Ammonia

Ammonia is toxic to plants. At physiological pH, the ammonia is protonated to form ammonium ions. 

Formation of Amino Acids

Amino acids are the initial product of nitrogen assimilation. The formation of amino acids from the ammonium can happen through two pathways -

  •  Reductive amination 
  •  Transamination

Reductive Amination

Ammonium ions react with alpha ketoglutaric acid to form glutamate, which is an important amino acid. This reaction is catalysed by the enzyme glutamate dehydrogenase.

Metabolism of nitROGEN

Transamination

It involves the transfer of an amine group from one amino acid, which is the amino donor, to another keto acid, which is the amino acceptor. This reaction helps in production of new amino acids. It is catalysed by enzyme transaminase.

Transamination

Formation of Amides

An example of this reaction is the production of aspartic acid from glutamic acid and oxaloacetic acid. Using these amino acids, plants form amides like asparagine and glutamine, which form a part of important plant structural proteins. 

Asparagine is formed from aspartic acid. During this reaction, hydroxyl part of the acid is replaced by another NH2- radical. Glutamine is also formed by a similar reaction from glutamic acid. These reactions are catalysed by asparagine synthetase and glutamine synthetase respectively. Amides contain more nitrogen than the normal amino acids. They can be transported to other parts of the plant through xylem vessels.

Transamination

Formation of Ureides 

In some plants like soyabean, the nodules export the fixed nitrogen as ureides. These compounds normally have a particularly high nitrogen to carbon ratio. They are the degraded forms of urea. 

NITROGEN RICE PLANT

Denitrification

The process of conversion of nitrites or nitrates into atmospheric nitrogen is known as denitrification. It is carried out by denitrifying bacteria like Pseudomonas and Thiobacillus. 

Denitrification

Ammonification

Ammonification is the decomposition of organic nitrogen into ammonia in nature. The process is carried out by ammonifying bacteria such as Bacillus vulgaris and Bacillus ramosus.

Ammonification

YOUTUBE LINK:  https://www.youtube.com/watch?v=2WjvlBToyAI

Practice Problems of Nitrogen Cycle

Ques:- In the nitrogen cycle, bacteria Pseudomonas denitrificans help in ___________.

  • A. nitrogen fixation
  • B. oxidising ammonia to nitrite
  • C. producing elemental nitrogen
  • D. none of these

Solution: Pseudomonas denitrificans help in the process of denitrification. Denitrification is the process by which nitrates of the soil are reduced to form gaseous nitrogen. Nitrogen fixation is the conversion of atmospheric nitrogen into usable compounds of nitrogen like nitrates and ammonia. Biological nitrogen fixation is carried out by microorganisms such as Nostoc, Anabaena, Azotobacter etc. The oxidation of ammonia into nitrite is normally carried out by 

bacteria like Nitrosomonas or Nitrococcus. Hence the correct option is c.

Ques:- Frankia produces nodules on the roots of which plant _______________.

  • A. leguminous plants like Alnus
  • B. leguminous plants like sweet pea
  • C. non-leguminous plants like sweet pea
  • D. non-leguminous plants like Alnus

Solution: Frankia is a nitrogen fixing microorganism. It forms a symbiotic relationship with the roots of trees belonging to the genus Alnus. It produces nitrogen fixing nodules on the roots of these plants. Alnus are non-leguminous plants. Leguminous plants normally form a symbiotic relationship with the nitrogen fixing bacteria like Rhizobium. Hence the correct option is d.

Ques:- Identify the gene responsible for nitrogen fixation, from the options given below.

  • A. Nif
  • B. Leg-haemoglobin
  • C. Nitrogenase
  • D. None of the above

Solution: The nif genes or the nitrogen fixation genes encode the enzymes involved in the fixation of atmospheric nitrogen. The main enzyme coded by this gene is the nitrogenase complex that converts atmospheric nitrogen to ammonia. Ammonia is used by plants for various purposes. Hence the correct option is a. 

Ques:- Which is a free-living nitrogen-fixing anaerobe, among the options given below?

  • A. Azotobacter
  • B. Rhodospirillum
  • C. Beijerinckia
  • D. All of these

Solution: Rhodospirillum is an anaerobic free living nitrogen fixing microbe. Azotobacter and Beijerinckia are aerobic nitrogen fixing microbes. Aerobic microorganisms require the presence of oxygen for their normal growth and activity while anaerobic microbes do not require the presence of oxygen. Hence the correct option is b. 

FAQ’s of Nitrogen Cycle

Ques:- What will be the effect on the nitrogen cycle, if the nitrogenase enzyme has been inactivated by radiation?

Answer: Reduction of nitrogen into ammonia by microorganisms is called biological nitrogen fixation. This reaction is catalysed by the enzyme nitrogenase. Nitrogenase is a Mo-Fe protein present exclusively in some prokaryotes. So, if nitrogenase enzyme is inactivated there will be no biological nitrogen fixation.

Ques:- The pigment that is normally present in nodulated roots of leguminous plants is __________.

Answer: Leghaemoglobin is a pink pigment present in the root nodules of leguminous plants and is essential for nitrogen fixation. The enzyme nitrogenase is sensitive to oxygen. It requires anaerobic conditions for proper functioning. Leghaemoglobin acts as an oxygen scavenger and helps in oxygen removal and thus provides anaerobic conditions for the enzyme to work.

Ques:- What is the role of Glutamate dehydrogenase?

Answer: Glutamate dehydrogenase enzyme helps in the process of reductive amination. During reductive amination, ammonia forms glutamic acid by reacting with glutamic acid. The glutamic acid helps in the formation of other amino acids by the process called transamination.

Ques:- Nodule formation of legume roots is decreased due to the deficiency of which element?

Answer: The roots of leguminous plants have symbiotic association with nitrogen fixing bacteria, Rhizobium. Rhizobium is able to infect the leguminous plants. It forms nodules in the roots of the leguminous plants to help in nitrogen fixation. 
In leguminous plants like pea plants, when there is deficiency of boron, Rhizobium bacteria infecting the host cells is reduced. Hence, under normal conditions boron deficiency can lead to a reduced number of nodules. Sulphur is considered as an essential constituent of proteins and some amino acids. It is important for development of nodules and efficient nitrogen fixation. Its deficiency leads to reduced number of nodules and reduced nodule metabolism which leads to impairment in the nitrogenase synthesis and activity.

Other Related Topics

The Living World Biological Classification Plant Kingdom
Animal Kingdom Morphology of Flowering Plants Anatomy of Flowering Plants
Structural Organization in Animals Cells: The Unit of Life Biomolecules
Cell Cycle and Division Transport in Plants Mineral Nutrition
Photosynthesis in Higher Plants Respiration in Plants Plant Growth and Development
Digestion and Absorption Breathing and Exchange of Gases Body Fluids and Circulation
Excretory Products and their Elimination Locomotion and Movement Neural Control and Coordination
Chemical Coordination and Integration

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