NITROGEN NITROGEN METABOLISM METABOLISM

FIXATIONFIXATION

Submited by-Submited by- Anjali RaiAnjali Rai

MSc (P) BiotechnologyMSc (P) Biotechnology

NITROGEN METABOLISM

Nitrogen Fixation can be defined as the conversion of molecular nitrogen into fixed form of nitrogen to make it available for absorption by plants.

It is the third most important process after respiration and photosynthesis.

It is essential for all living organisms.

CLASSIFICATIONNITROGEN FIXATION

NON-BIOLOGICAL BIOLOGICAL

SYMBIOTIC NON-SYMBIOTIC

BIOLOGICAL N2 FIXATION

Conversion of elemental nitrogen or gaseous nitrogen into nitrogenous compounds or salts by certain microorganisms like bacteria ,blue green algae, fungi etc. is called biological nitrogen fixation.

It is carried by two types of micro- organisms. Eg Symbiotic and non-symbiotic.

NON-SYMBIOTIC N2 FIXATION

The Fixation of free nitrogen of the soil by all those micro-organisms living freely or outside the cell is called as non-symbiotic biological N2 fixation.

NON-SYMBIOTIC N2 FIXATION

SYMBIOTIC N2 FIXATION

The Fixation of free nitrogen of the soil by microorganisms living symbiotically inside the plant, is called as symbiotic biological N2-fixation.

The term “symbiosis” is coined by De bary in 1879.

CLASSIFICATION1. Nitrogen Fixation Through Nodule

Formation in Leguminous plants.2. N2 Fixation through Nodule

formation in Non-leguminous Plants.

3. N2-Fixation through Non-Nodulation.

The Nodulation process

The Nodulation process

The Nodulation process

The Nodulation process

The Nodulation process

N2-FIXATION IN NON- LEGUMINACEOUS PLANTS

In addition to legumes, there are many plants specially trees and shrubs belonging to families other than Leguminosae which produce root-nodules.

Eg- Casuarina- Frankia Alnus- Frankia Myrica- Frankia Parasponia- Rhizobium

N2 Fixation through Non-nodulation

It includes those plants where root nodules are not formed but symbiotic N2-fixation takes place.

Examples- Lichens - associated with fungi and algae Azolla- Anabaena azollae. Cycas –Anabaena or Nostoc Gunnera macrophylla- Nostoc

Associative Symbiotic N2-Fixation

When the bacteria live in close association with the roots of cereals and grasses and fix nitrogen than the association is of loose mutualism type and is called associative symbiosis whereas this nitrogen Fixation is called associative symbiotic nitrogen fixation.

Examples- Azotobactor paspali – Associated with Paspalum

notatum Azospirillum brasilense- Cereal roots Beijerinckia- Sugarcane roots

Cross-inoculation group

LEGHEMOGLOBIN The red pigment of the nodules is called

leg-hemoglobin and appears to be a product of the Rhizoboium-legume complex.

The pigment is not present in either organism grown alone.

It is a reddish pigment found in the cytoplasm of host cells.

It is an oxygen carrier & an efficient O2

scavenger

NON BIOLOGICAL N2 FIXATION

Non biological N2 fixation is usually found in rainy seasons during lightening, thunder, storms and atmoshpheric pollution.

It is of two types-1. Physical2. Chemical

NON- BIOLOGICAL n2 fIXATION (PHYSICAL )

(PHYSICAL)- 1. N2 + O2 Lightening 2NO

Thunder (Nitric Oxide) 2. 2NO + O2 Oxidation 2No2

(Nitrogen Peroxide)3. 2No2 + Rain water HNO2 + Hno3

NON- BIOLOGICAL n2 fIXATION ( CHEMICAL)

(CHEMICAL)-

N2 (g) + 3H2 (g) 2NH3 (g) (g) H = -22 Kcal

By using a temperature of about 500 C and a pressure of about 1000 atm, there is about 50% conversion of N2 to NH3

Haber’s Process

BIOCHEMISTRY OF NITROGEN FIXATION

Nitrogenase Complex- Key Enzyme In N2 – Fixation.

N2 + 8H+ + 8e- + 16ATP 2NH4+

+ 16ADP + 16Pi + H2

Overall Reaction

STRUCTURE OF NITROGENASE Fe Protein

Assimilatory nitrate reduction

Schematic diagram of nitrogenase complex

Electrons fromReduced ferredoxin

Dinitrogenase Reductase(Fe-Protein) Dinitrogenase Reductase

( MO -Fe-Protein)

ATP ADP N2NH4+

Reduction

8 Ferridoxin or 8 Flavodoxin (oxidized)

8 Ferridoxin or 8 Flavodoxin (reduced)

8 Dinitrogenase reductase(reduced)

8 Dinitrogenase reductase(oxidised)

8 Dinitrogenase reductase(reduced) + 16 ATP

8 Dinitrogenase reductase(oxidised)

8 Dinitrogenase(oxidized)

8 Dinitrogenase(reduced)

8 e-

8 e-

8 e-

8 e-

16 ATP 16 ADP+ 16Pi

2NH4+ N2

MECHANISM OF NITROGENASE REDUCTION

NITROGENASE COMPLEX ACTIVITY

1. Nitrogenase the cardinal enzyme process.2. A strong reductant such as, Ferredoxin or

flavodoxin or NADPH etc.3. ATP4. Presence of hydrogenase enzyme.5. A system for regulation of the rate of the NH3

production and one for assimilation since biosynthesis of the nitrogenase complex ceases when ammonia accumulate.

6. Protection of the nitrogen fixation system from molecular oxygen which inactivates nitrogenase and competes for reductant.

7. Coenzymes and co-factor like TPP Co-A inorganic phosphate Pi, and Mg+2.

8. The hydrogen releasing system or electron donor which is usually pyruvic acid but may also be glucose or sucrose in some instances.

NITROGEN CYCLE

Genetics of N2 Fixing Microorganisms

Genes responsible for N2 Fixation are known as nif-genes while for nodulation are called nod genes.

Nif genes have been isolated from symbiotic bacteria Rhizobium leguminosarium & free living N2

Fixing bacteria Klebsiella pneumoniae

Nif GENES In Klebsiella pneumoniae Nif genes

are present which consists of 17 genes.

Distributed into 7 or 8 oparons. Nif genes is regulated by NifA,NifL

genes. In cyanobacteria there are other

Nif genes ie,Nif H,Dand K and Nif S and Nif B.

NOD GENES Nod genes are present on sym-

plasmid. These genes are host specific Regulation of nod genes is controlled

by Nod D genes. The common Nod A,B,C genes are

conserved among rhizobium and inactivation of these genes completely depends on root hair infection and nodule formation.

CONCLUSION N2 Fixing microorganisms (free-

living and symbiotic) are known as diazotrophs.

Biological and non-biological N2 fixation.

Genetics of nitrogenase enzyme. Presence of leghemoglobin. Regulation of nitrogenase enzyme.