1. NITROGEN METABOLISM FIXATION Submited by- Anjali Rai MSc (P) Biotechnology MSc (P) Biotechnology

2. 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.

3. CLASSIFICATION NITROGEN FIXATION NON-BIOLOGICALBIOLOGICAL SYMBIOTIC NON-SYMBIOTIC

4. BIOLOGICAL N 2 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.

5. NON-SYMBIOTIC N 2 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 N 2 fixation.

6. NON-SYMBIOTIC N 2 FIXATION

7. SYMBIOTIC N 2 FIXATION  The Fixation of free nitrogen of the soil by microorganisms living symbiotically inside the plant, is called as symbiotic biological N 2 -fixation.  The term “symbiosis” is coined by De bary in 1879.

8. CLASSIFICATION 1. Nitrogen Fixation Through Nodule Formation in Leguminous plants. 2. N 2 Fixation through Nodule formation in Non-leguminous Plants. 3. N 2 -Fixation through Non- Nodulation.

9. The Nodulation process

15. N 2 -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

16. N 2 Fixation through Non- nodulation  It includes those plants where root nodules are not formed but symbiotic N 2 - fixation takes place.  Examples-  Lichens - associated with fungi and algae  Azolla- Anabaena azollae.  Cycas –Anabaena or Nostoc  Gunnera macrophylla- Nostoc

17. Associative Symbiotic N 2 - 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

18. Cross-inoculation group

19. 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 O 2 scavenger

20. NON BIOLOGICAL N 2 FIXATION  Non biological N 2 fixation is usually found in rainy seasons during lightening, thunder, storms and atmoshpheric pollution.  It is of two types- 1. Physical 2. Chemical

21. NON- BIOLOGICAL n 2 fIXATION (PHYSICAL )  (PHYSICAL)- 1. N 2 + O 2 Lightening 2NO Thunder (Nitric Oxide) 2. 2NO + O 2 Oxidation 2No 2 (Nitrogen Peroxide) 3. 2No 2 + Rain water HNO 2 + Hno 3

22. NON- BIOLOGICAL n 2 fIXATION ( CHEMICAL)  (CHEMICAL)-  N 2 (g) + 3H 2 (g) 2NH 3 (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 N 2 to NH 3 Haber’s Process

23. BIOCHEMISTRY OF NITROGEN FIXATION  Nitrogenase Complex- Key Enzyme In N 2 – Fixation.  N 2 + 8H + + 8e - + 16ATP 2NH 4 + + 16ADP + 16Pi + H 2 Overall Reaction

24. STRUCTURE OF NITROGENASE Fe Protein

25. Assimilatory nitrate reduction

26. Schematic diagram of nitrogenase complex Electrons from Reduced ferredoxin Dinitrogenase Reductase (Fe-Protein) Dinitrogenase Reductase ( MO -Fe-Protein) ATPADP N2N2 NH 4 + Reduction

27. 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- 16 ATP 16 ADP+ 16Pi 2NH 4 + N2N2 MECHANISM OF NITROGENASE REDUCTION

28. NITROGENASE COMPLEX ACTIVITY 1. Nitrogenase the cardinal enzyme process. 2. A strong reductant such as, Ferredoxin or flavodoxin or NADPH etc. 3. ATP 4. Presence of hydrogenase enzyme. 5. A system for regulation of the rate of the NH 3 production and one for assimilation since biosynthesis of the nitrogenase complex ceases when ammonia accumulate.

29. 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.

30. NITROGEN CYCLE

31. Genetics of N 2 Fixing Microorganisms  Genes responsible for N 2 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 N 2 Fixing bacteria Klebsiella pneumoniae

32. 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.

33. 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.

34. 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.