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Biofertilizers & Nitrogen fixation
 

Biofertilizers & Nitrogen fixation

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    Biofertilizers & Nitrogen fixation Biofertilizers & Nitrogen fixation Presentation Transcript

    • Biofertilizers & Nitrogen fixation (Dr.) Gunjan Mehta, Dept. of Biotechnology, Shree M. & N. Virani Science College, Rajkot Email: gjmehta@vsc.edu.in 23-04-2014 1
    • Biofertilizers • Biofertilizers are defined as “biologically active products/ microbial inoculants consisting of bacteria, algae and fungi which may help biological N2 fixation, Phosphate solubilization and phytohormone production. “ • Examples: Symbiotic N2 fixer- Rhizobium Asymbiotic N2 fixer- Azotobacter Algal Biofertilizer- BGA+ Azolla PSBs Cellulolytic bacteria/ Fungi Mychorhiza23-04-2014 2
    • 23-04-2014 3
    • Nitrogen “fixation” N2 23-04-2014 4
    • Dinitrogen energetics • Energetics • NN • Haber-Bosch (100-200 atm, 400-500°C, 8,000 kcal kg-1 N) • Nitrogenase (4,000 kcal kg-1 N) • Dinitrogen (N2) makes up 80% of the air we breathe but is essentially inert due to the triple bond (NN) • Breakdown of triple bond (NN) requires substantial amount of energy. 23-04-2014 5
    • Non Biological Nitrogen fixation Atmospheric fixation (5-8%) Industrial fixation(Variable) 23-04-2014 6
    • Biological Nitrogen fixation 23-04-2014 7
    • Some nitrogen fixers Stem nodulated leguminous plant- Sesbania spp. Only root nodulated Rhizobia symbiont- Parasponia spp. 23-04-2014 8
    • Types of nitrogen fixation Nitrogen Fixation Free living Nitrogen fixation Cyanobacteria, Azotobacter Associative Nitrogen fixation Rhizosphere–Azospirillum Lichens–cyanobacteria Leaf nodules Symbiotic Nitrogen fixation Legume-rhizobia Actinorhizal-Frankia 23-04-2014 9
    • Estimated Average Rates of Biological N2 Fixation 40-300 1-150 1-50 50-150 50 Actinorhizal plant symbioses with Frankia Alnus Hippophaë Ceanothus Coriaria Casuarina 50-100 100-600 Leguminous plant symbioses with rhizobia Grain legumes (Glycine, Vigna, Lespedeza, Phaseolus) Pasture legumes (Trifolium, Medicago, Lupinus) 10-20 300 40-80 Cyanobacterial associations Gunnera Azolla Lichens 5-25 Grass-Bacteria associative symbioses Azospirillum 25 0.3 0.1-0.5 Free-living microorganisms Cyanobacteria Azotobacter Clostridium pasteurianum N2 fixed (kg ha-1 y-1)Organism or system 23-04-2014 10
    • Free-living N2 Fixation • Energy • 20-120 g C used to fix 1 g N • Combined Nitrogen • nif genes tightly regulated • Inhibited at low NH4 + and NO3 - (1 μg g-1 soil, 300 μM) • Oxygen • Avoidance (anaerobes) • Microaerophilly • Respiratory protection • Specialized cells (heterocysts, vesicles) • Spatial/temporal separation • Conformational protection 23-04-2014 11
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    • Associative N2 Fixation • Phyllosphere or rhizosphere (tropical grasses) • Azosprillum, Acetobacter • 1 to 10% of rhizosphere population • Some establish within root • Same energy and oxygen limitations as free-living • Acetobacter diazotrophicus lives in internal tissue of sugar cane, grows in 30% sucrose, can reach populations of 106 to 107 cells g-1 tissue, and fix 100 to 150 kg N ha-1 y-1 23-04-2014 13
    • Legume-Rhizobium Symbiosis • The subfamilies of legumes (Caesalpinioideae, Mimosoideae, Papilionoideae), 700 genera, and 19,700 species of legumes • Only about 15% of the species have been evaluated for nodulation • Most known are: Rhizobium, Bradyrhizobium, Azorhizobium • Rhizobium o Gram -, rod o Most studied symbiotic N2-fixing bacteria o Now subdivided into several genera o Many genes known that are involved in nodulation (nod, nol, noe genes) legume rhizobia Fixed nitrogen (ammonia) Fixed carbon (malate, sucros 23-04-2014 14
    • Nitrogen fixation by diazotrophs 23-04-2014 15
    • Rank of Biological Nitrogen Fixation 0.1 - 25Free- living 5 - 25Rhizosphere associations 10 - 300Cyanobacteria- moss 50 - 600Rhizobium-legume Nitrogen Fixation (kg N/ha/year) N2 fixing system 23-04-2014 16
    • Nitrogen fixation by diazotrophs • Reductive process • Reduction of N2 to NH3 is 6 electron process • In Biosynthetic process 1 mol H2 also gets synthesized aslong with 2 mol of NH3, which additionally requires 2 electrons. • 16 ATPs are required for each N2 reduced, two for each electron transferred. N2 + 8 flavodoxin- + 8H+ + 16 MgATP2- + 18 H2O + 2OH- + 8 flavodoxin + 16 MgADP- + 16H2PO4 - + H2 nitrogenase 2NH4 + 23-04-2014 17
    • The “Nitrogenase complex” • All nitrogen fixing bacteria use highly conserved enzyme complex called Nitrogenase. 14- 24 kDa 30 kDa 30 kDa Site of N2 Fixation 220 kDa 23-04-2014 18
    • Nitrogenase- molecular mechanism FeMo Cofactor N2 + 8H+ 2NH3 + H2 8e- 4C2H2 + 8H+ 4C2H2 Dinitrogenase (Molybdoferredoxin) Dinitrogenase Reductase (Azoferredoxin) Fd(red) Fd(ox) nMgATP nMgADP + nPi N2 + 8H+ + 8e- + 16 MgATP  2NH3 + H2 + 16MgADP23-04-2014 19
    • Nitrogenase catalytic site- essential metallic component 1. Molybdanum 2. Vanadium 3. Iron23-04-2014 20
    • Genetics of Nitrogenase 23-04-2014 21
    • Oxygen insensitivity approaches PS1 absent 23-04-2014 22
    • Nitrogenase prerequisites and inhibitors Prerequisites Suitable electron donor ATPs Enzyme for assimilation Inhibitors H2 Competitive inhibitor CONon- Competitive inhibitor CS2 Rapid equilibrium inhibitor23-04-2014 23
    • Dinitrogen Diamide Hydrazine Ammonia 23-04-2014 24
    • Some symbiotic nitrogen fixers 23-04-2014 25
    • Infection and nodulation process Chemical recognition of roots and Rhizobium Root hair curling Formation of infection thread Invasion of roots by Rhizobia Cortical cell divisions and formation of nodule tissue (Crown Gall disease) Bacteria fix nitrogen which is transferred to plant cells in exchange for fixed carbon23-04-2014 26
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    • Cytokinin synthesis provokes extensive cell division High % of vacuoles 23-04-2014 29
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    • Bacteroids • Polymorphic rhizobia in microaerophilic environment. 23-04-2014 32
    • Ammonia assimilatory cycle: How nitrogen enters biological pathways NH4 + Amino acids proteins Amino acids proteins purines pyrimidines -ketoglutarate glutamate GDH + glutamate glutamine+ ATP + ADP + Pi GS + NH4 + glutamineglutamate -ketoglutarate GOGAT + Pathway 1 Pathway 2 23-04-2014 33
    • Role of route exudates • General • Amino sugars, sugars • Specific • Flavones (luteolin), isoflavones (genistein), flavanones, chalcones • Inducers/repressors of nod genes • Vary by plant species • Responsiveness varies by rhizobia species 23-04-2014 34
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