Nitrogen fixation

nitrogen fixation microbiology rhizobium

1.   Nitrogen fixation Presented by S .Palaniananth Submitted to

2. : Introduction: • Nitrogen is an essential constituent of all biomolecules both in plants and in animals Most of the plants obtain nitrogen from soil in the form of nitrate or ammonium ion , but it is limited Atmosphere consists 78% of molecular nitrogen but plants unable to convert this molecular nitrogen into a useful form because the lack the enzyme nitrogenase Only prokaryote species posses this enzyme 2 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

3. :  Nitrogen fixation is the first step of nitrogen cycle where molecular nitrogen is reduced by nitrogen fixing bacteria to yield ammonia Nitrogen cycle has 3 process Ammonification Nitrification Denitrification 3 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

4. 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 4 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

5. SOURCE 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 5 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

6. NITROGEN FIXATION: The conversion of free nitrogen into nitrogenous salts to make it available for absorption of plants TYPES: 6 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

7. 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) 7 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

8. BIOLOGICAL FIXATION: Fixation of atmospheric Nitrogen into nitrogenous salts with the help of micro-organisms Two types Symbiotic Non-symbiotic 8 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

9. 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 9 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

10. Non-symbiotic Free living fungi: yeasts and Pillularia Blue green algae: unicellular – Gloeothece , Synechococcus Filamentous (non heterocystous ) - Oscillatoria Filamentous ( heterocystous ) – Tolypothrix , Nostoc , Anabaena 10 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

11. Non-symbiotic   Fixation of free nitrogen by micro-organisms in soil living symbiotically inside the plants ‘ Symbiosis’ – coined by DeBary Three categories Nodule formation in leguminous plants Nodule formation in non-leguminous plants Non nodulation 11 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

12. NODULE FORMATION IN LEGUMINOUS PLANT 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 12 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

13. 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 13 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

14. 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 14 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

15. 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. 15 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

16. 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 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 16 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

17. FORMATION OF ROOT NODULES IN LEGUMES: 17 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

18. FORMATION OF ROOT NODULES IN LEGUMES: 18 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

19. FORMATION OF ROOT NODULES IN LEGUMES: 19 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

20. FORMATION OF ROOT NODULES IN LEGUMES: 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 20 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

21. FORMATION OF ROOT NODULES IN LEGUMES: 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 Present studies indicates that leghaemoglobin is not essential 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) 21 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

22. FORMATION OF ROOT NODULES IN LEGUMES: 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 22 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

23. 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 23 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

24. NITROGENASE ENZYME: The reduction of N2 into NH3 requires 6 protons and 6 electrons 12 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 - 24 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

25. PATHWAY OF NITROGEN FIXATION IN ROOT NODULES Glucose-6-phosphate acts as a electron donor Sucrose ( synthesized in leaves) , Sucrose ( in roots ) , Glucose and fructose Glucose-6- phosphate Glucose-6-phosphate is converted to phosphogluconic acid Glucose-6-phosphate + NADP + + H2O 6- phosphogluconic acid + NADPH + H+ NADPH donates electrons to ferrodoxin . Protons released and ferrodoxin is reduced Reduced ferrodoxin acts as electron carrier. Donate electron to Fe-protein to reduce it. Electrons released from ferrodoxin thus oxidized 25 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

26. PATHWAY OF NITROGEN FIXATION IN ROOT NODULES Reduced Fe-protein combines with ATP in the presence of Mg +2 Second sub unit is activated and reduced It donates electrons to N2 to NH3 Enzyme set free after complete reduction of N2 to NH3 26 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

27. Conclusion : • Nitrogen is a limiting nutrient for plants, even though molecular nitrogen is readily available in atmosphere • Plants do not have the nitrogenase enzyme thus have to depend on prokaryotes to absorb nitrogen • Nitrogen fixation is the first step of nitrogen cycle and it is of two types biological and non biological nitrogen fixation • Biological nitrogen fixation occurs in two ways i . e symbiotic , where it involves the prokaryotic interaction with plants via root nodules and asymbiotic w here free living micro organisms fix the atmospheric nitrogen into the soil • Non biological nitrogen fixation involves the lighting process to fix atmospheric nitrogen into the soil • Because of high demand of energy this nitrogen fixation is tightly regulated by various methods 27 3/13/2021 SOIL AND AGRICULTURE MICROBIOLOGY

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