~1.0 x 10¹⁰ kg/yr ~8.0 x 10¹⁰ kg/yr ~1.8 x 10¹¹ kg/yr

Nitrogen Fixation

~4% of total ~30% of total ~66% of total

Role of nitrogen in plants

• 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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Sources of nitrogen

• Atmospheric Nitrogen
• 78% of atmosphere
• Plants cannot utilize this form
• Some Bacteria, Blue Green Algae, leguminous plants
• Nitrates, Nitrites and Ammonia
• Nitrate is chief form
• Amino acids in the soil
• Many soil organisms use this form
• Higher plants can also taken by higher plants
• Organic Nitrogenous compounds in insects
• Insectivorous plants

Nitrogen fixation

• The conversion of free nitrogen into nitrogenous salts to make it available for absorption of plants

Types of nitrogen fixation

Non biological fixation

• The micro-organisms do not take place
• Found in rainy season during lightning

1. N₂ + O₂ lightning 2 NO

(Nitric oxide)

2. 2NO + O₂ oxidation 2NO₂

( Nitrogen per oxide)

3. 2NO₂ + H₂O HNO₂ + HNO₃

4. 4NO₂ + 2H₂O + O₂ 4HNO₃

(Nitric acid)

5. CaO + 2HNO₃ Ca (NO₃)₂ + H₂O

(Calcium nitrate)

6. HNO₃ + NH₃ NH₄NO₃

(Ammonium nitrate)

7. HNO₂ + NH₃ NH₄NO₂

(Ammonium nitrite)

Biological fixation

• Fixation of atmospheric Nitrogen into nitrogenous salts with the help of micro-organisms
• Two types
• Symbiotic
• Non-symbiotic

Non-symbiotic

• Fixation carried out by free living micro-organisms
• Aerobic, anaerobic and blue green algae
• Bacteria: special type (nitrogen fixing bacteria) types -
• Free living aerobic : Azotobacter, Beijerenckia
• Free living anaerobic : Clostridium
• Free living photosynthetic : Chlorobium, Rhodopseudomonas
• Free living chemosynthetic :Desulfovibro,Thiobacillus

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Contd..

• Free living fungi: yeasts and Pillularia
• Blue green algae:
• unicellular – Gloeothece, Synechococcus
• Filamentous (non heterocystous) -Oscillatoria
• Filamentous (heterocystous) – Tolypothrix, Nostoc, Anabaena

Symbiotic nitrogen fixation

• Small, knob-like protuberances-root nodules
• Size and shape varies
• Spherical, flat, finger-like or elongated
• From Pin head to one centimeter in size
• Various spp. Of Rhizobium noted
• Named after the host plant
• Pea – Rhizobium leguminosarum
• Beans – R. phaseoli
• Soyabeans – R. japonicum
• Lupins – R. lupini
• Two types of Rhizobium-
• Bradyrhizobium – slow growing spp.
• Rhizobium - fast growing spp.

Nodule formation in leguminous plants

• 2500 spp. Of family leguminosae ( Cicer arientium, Pisum, Cajanus, Arachis) produce root nodules with Rhizobium spp.
• They fix Nitrogen only inside the root nodules
• Association provides-food and shelter to bacteria

-bacteria supply fixed nitrogen to plant

• Nodules may buried in soil even after harvesting – continue nitrogen fixation

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Nodule formation in non-leguminous plants

• Some other plants also produces root nodules
• Causuarina equisetifolia – Frankia
• Alnus – Frankia
• Myrica gale – Frankia
• Parasponia – Rhizobium
• Leaf nodules are also noted
• Dioscorea, Psychotria
• Gymnosperms – root – Podocarpus,

- leaves – Pavetta zinumermanniana, Chomelia

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Non-nodulation

• Lichens - cyanobacteria
• Anthoceros - Nostoc
• Azolla – Anabaena azollae
• Cycas – Nostoc and anabaene
• Gunnera macrophylla - Nostoc
• Digitaria, Maize and Sorghum – Spirillum notatum
• Paspalum notatum – Azotobacter paspali

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rhizobium

• Gram negative
• Non spore forming
• Micro-aerobic
• Show a degree of specificity
• The two partners (Bacteria and Host) recognized by chemical substance LECTINS - phytoagglutinins (carbohydrate containing plant protein)

Formation of root nodules in legumes

• Root nodules formed due to infection of Rhizobium
• Free living bacteria growing near root of legumes unable to fix nitrogen in free condition
• Roots of the legumes secrete some growth factors helps in fast multiplication of bacteria
• (E.g.) Pisum sativum secretes homo serine also carbohydrate containing protein Lectins over their surface

Contd..

• This helps in recognition and attachment of rhizobial cells
• Rhizobial cells have carbohydrate receptor on their surface
• Lectins interact with the carbohydrate receptor of rhizobial cells
• Occur between root hairs and young root hair
• Bacteria enter the roots through soft infected root hairs
• Tips are deformed and curved
• Tubular infection thread is formed in the root hair cell and bacteria enters into it

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After entry, new cell wall is formed

Tubular infection contains mucopolysaccharides where bacteria embedded and start multiplication

It grows much and reaches the inner layers of cortex and the bacteria is released

It induces the cortical cells to multiply which result in the formation of nodule on the surface

The bacterial cells multiplies and colonize in the multiplying host cells

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Contd..

• After host cells are completely filled, bacterial cells becomes dormant-bacteroids
• Float in leghaemoglobin – reddish pigment in cytoplasm of host cells
• Efficient O₂ scavenger
• Maintains steady state of oxygen
• Stimulates ATP production
• Nitrogenous compounds synthesized is translocated through vascular tissues
• Groups of rhizobia surrounded by double membrane originated from host cell wall
• Bacteroids lack firm wall (osmotically liable)

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Biochemistry of nitrogen fixation

• Basic requirements for Nitrogen fixation
• Nitrogenase and hydrogenase enzyme
• Protective mechanism against Oxygen
• Ferrodoxin
• Hydrogen releasing system or electron donor (Pyruvic acid or glucose/sucrose)
• Constant supply of ATP
• Coenzymes and cofactors TPP, CoA, inorganic phosphate and Mg⁺²
• Cobalt and Molybdenum
• A carbon compound

Nitrogenase enzyme

• Plays key role
• Active in anaerobic condition
• Made up of two protein subunits
• Non heme iron protein ( Fe-protein or dinitrogen reductase)
• Iron molybdenum protein (Mo Fe-protein or dinitrogenase)
• Fe protein reacts with ATP and reduces second subunit which ultimately reduces N₂ into ammonia

N₂ + 6H⁺ + 6e^- 2NH₃

Two components MO-FE AND FE – 180-235 kDa ,30-72-KDa

Dinitrogenase

Reductase

Dinitrogenase

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2NH₄⁺ + H₂

N₂ + 10H⁺

8

Nitrogenase Complex

Electrons are passed through redox centers to N₂ and H⁺ (8 electrons total).

Contd..

• The reduction of N₂ into NH₃ requires 6 protons and 6 electrons
• 16 mols of ATP required
• One pair of electron requires 4 ATP
• The modified equation

N₂ + 8H⁺ + 8e^- 2NH₃ + H₂

• Hydrogen produced is catalyzed into protons and electrons by hydrogenase

hydrogenase

H₂ 2H⁺ + 2e^-

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