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Welcome

Welcome. Biotechnology of Biological Nitrogen Fixation. Dr. P.V. Madhusoodanan Prof. & Head Department of Botany University of Calicut Email: pvmadhu@gmail.com web: http// pvmadhu.tripod.com. BIOFERTILIZERS. BIOFERTILIZERS FOR SUSTAINABLE AGRICULTURE AND CLEAN ENVIRONMENT.

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  1. Welcome

  2. Biotechnology of Biological Nitrogen Fixation Dr. P.V. Madhusoodanan Prof. & Head Department of Botany University of Calicut Email: pvmadhu@gmail.com web: http// pvmadhu.tripod.com

  3. BIOFERTILIZERS BIOFERTILIZERS FOR SUSTAINABLE AGRICULTURE AND CLEAN ENVIRONMENT

  4. Biofertilizers Fertilizers with life Microorganisms (inoculam) applied to improve the fertility of the soil N P K Nitrogen and Phosphorous Why N2 is scarce in the soil?

  5. Norman E. Borlaug E. Borlaug

  6. Bad type of storing ! ‘The Hindu’ 6th Sep 2010

  7. Relevance of Biofertilizers • HYV are fertilizer responsive- Green revolution - Grow more food • Susceptible to pests and pathogens • Demand high amount of chemical fertilizers and pesticides • Agroindustries are the major contributors of pollution in the world • Farmers apply excess amount of chemicals • Chemical fertilizers provide one nutrient but wipes out essential elements • They alter the pH of the soil • Affect the soil dynamics by the soil/microorganisms- kill them- earthworms • Deteriorate agroecoystem-’Chemophobia’ • Biofertilizers can substitute to a great extent the chemical fertilizers • Organic farming- Sustainable development • One straw revolution- Masanobu Fukuoka

  8. BIOLOGICAL NITROGEN FIXATION • NN • Diazo • 79% inert vital element • Ionization energy 336 kcal/mol • Haber-Bosch Process, 8000C, high pressure (300 Atm.) N2 NH3 • 1.5 kg fuel needed for 1 kg N fertilizer to farm. • BNF only 1/150 • N2 deficiency – no ‘nitroplasts’ – 200 mt/y by few microorganisms • Nitrogenase enzyme – BNF – nif genes • Example- Potential of enzyme –catalyzed reaction

  9. Biogeochemical Cycle

  10. Nitrogen Cycle

  11. Nitrogen cycle

  12. Drews (1928) Cyanobacteria fix N2 • De (1939) Cyanobacteria fix N2 in paddy fields- Indian • Beijerlink (1888) isolated Rhizobium • Reduction of Gaseous N2 to NH3 • N2+ 8H++ 8e- + 16ATP+16H2O 2NH3+ H2 + 16 ADP + 16 Pi • ‘Terminus Technicus’ chemically ‘impossible’ • Eukaryotic cells completely lack this ability • The intermediate steps of N2 fixation still unknown NN HN=NH H2N NH2 2NH3 DiimineHydrazine

  13. The total cost of N2 reduction is 8 electron transferred and 16 ATP hydrolyzed (physiologically 20-30 ATPs) • Nitrogenase also reduces H2 O H2 HN=NH + H2 N2 + 2H2 This happens when ATP level is low-Futile cycle

  14. Nitrogenase • 2 iron - S proteins • Individually inactive • 1. Mo-Fe protein 36 Fe and 36S & 2 Mo atoms • 220,000 MW – component I/ Nitrogen reductase • 2. Fe protein - 4Fe 4S cluster/ 60,000 MW • Component II/ Nitrogenase reductase • Both O2 sensitive • Fe protein more sensitive • Anaerobic envt. essential First isolated from Clostridium pasteurianum (1960) CRL of Dupond de Numeurs (USA)

  15. Chemistry of Nitrogen fixation Nitrogenase Glutamine synthatase Glutamate synthase (GOGAT= Glutamate oxogluterate aminotranferase)

  16. Genetics of N2 Fixation Klebsiella pneumoniae – facultative anaerobe- Fix N2 only under anaerobic condition- related to E.coli Easy to culture Nif genes are studied only in K. pneumoniae Genetic map Nif operons appx gene location Nitrogenase DG Q B A L F M V S U X N E Y K D H J 0 8 12 16 20 28 kb NR Synthesis of Fe-Mo cofactor Synthesis polypeptide for electrone transport NR

  17. The genes for histidine (DG) and nif genes are linked 15 nif genes have been identified +2 unknown function x,y Gene Coding H - Nitrogenase reductase FJ - Synth. polypeptide DK - Nitrogenase MSV - maturation of complete BQVNE - Fe Mo Co factor functional nitrogenase XY - Unknown AL - Regulate expression of nif genes Nif genes of other organisms are similar but usually the genes are scattered- not clustered as in K. When a small portion of chromosome of K. pneumoniae was transferred to E.coli, the latter started N2 fixation

  18. Major Biofertilizers Important microorganisms as Biofertilizers Microorganisms Nutrient fixed Actinorrhizae (Frankia sp) 150 kg N/ha/Y Azospirillum 10-20 kg Azotobacter > 20 kg Rhizobium 50-100 kg Azolla (Anabaena) 120-150 kg (> 900kg) Cyanobacteria > 25 kg Mycorrhizae (VAM) Solubilize P

  19. N2- Fixing Organisms Free Living N2- Fixers • Obligate Aerobes • Azotobacter • Beijerenkia • Azotcoccus • 2.Obligate aerobes that fix N2 at low O2 • Azospirillum • Thiobacillus • Rhizobium • 3.Facultative anaerobes- fix N2 under O2 free conditions • Klebsiella pneumoniae • Bacillus polymax • Escherichia intermedia

  20. 4. Obligate anaerobes • Clostridium • Desulfovibrio • 5. Phototropic bacteria • Rhodospirillum • Chromatium • Chlorobium • 6. BGA/ Cyanobacteria • a. Unicellular aerobic • Gloethece • Aphanothece • b. Filamentous heterocystous - aerobic/ anaerobic • Nostoc, Anabaena, Cylindrospermum • c. Filamentous non-heterocystous • Oscillatria, Lyngbya, Plectonema

  21. Symbiotic N2- Fixers Rhizobium- Leguminosae (12,000 spp.) Ulmaceae Parasponia Non-Rhizobium N2 Fixers Frankia (Actinorrhizae) Alnus (Betulaceae) Elaegnus (Elaegnaceae) Coenothus (Rhamnaceae) Dryas (Rosaceae) Coriaria (Coriariaceae) Casuarina (Casuarinaceae) Lichens Collema- Nostoc Dendriscocaulon- Scytonema Water fern Azolla- Anabaena

  22. Cycads Cycas- Nostoc, Anabaena Higher Plants Haloragaceae- Gunnera- Nostoc Associative Symbiosis and casual Assn. Phyllophore- Azotobacter sp. Roots of grasses- Azotobacter Azospirillum

  23. Frankia nodules and Frankia cells

  24. Azospirillum Azotobacter

  25. Root nodules: Rhizobium

  26. Steps in the formation of root nodule infected by Rhizobium Root nodule bacteria and symbiosis with legumes

  27. Stem nodule: Sesbania rostrata Section of root nodule: Coronilla varia

  28. Azolla

  29. Cyanobacteria

  30. Scytonemaguyanense Scytonema simplex

  31. Westiellopsis prolifica Calothrix marchica

  32. Dichothrix sp. Stigonema dendroideum Calothrix brevissima

  33. Azolla rubra Anabaena azollae

  34. Azolla filiculoides Azolla pinnata

  35. VAMF

  36. Ectomycorrhizae Endomycorrhizae

  37. Effect of biofertilizers on Casuarina equisetifolia saplings 1. Control (sterilized soil) 2. Plant inoculated with Rhizobium sp. 3. Plant inoculated with Azotobacter sp. 4. Plant inoculated with Azospirillum sp. 5. Plant inoculated with Phosphobacteria 6. Plant inoculated with microbial consortium

  38. Effect of biofertilizers on Casuarina equisetifolia saplings 1. Control (sterilized soil) 2. Plant grown in unsterilized soil 3. Plant inoculated with microbial consortium and farm yard manure (FYM)

  39. Advantages of Azolla application • Contribute up to 40% N2 • Accommodate P and other nutrients and releases slowly • Can be applied as a dual crop in paddy fields • Suppresses weeds • Use as mulch, compost, fish feed, cattle feed, etc. • Edible as vegetable- Azolla thoran, Azolla vada, etc.- rich in protein • Helps in detoxify Heavy metals and many other pollutants • It also reduces nematode population • Aquatic Fern • Maintenance during summer Disadvantages

  40. Economic Importance of Cyanobacteria • N2 fixers- biofertilizers • Chinese use Nostoc commune as a delicacy • Japanese use Nostoc commune, N. verrucosum, Aphanothece sacrum and Brachytrichia quoi as side dishes • 1521- Barnal Diaz accompanied Cortez recorded Mexico city (now) ooze from Lakes having a flavour like cheese – Spirulina platensis • Chad (Africa) people collect Spirulina cakes from lakes and consume.

  41. Anabaena cylindrica- one of the richest known sources of Vit. B12 • Amino acid production- Oscillatoria, Westiellopsis • H2 (fuel) production- Phormidium, Calothrix • Antibiotics production- Phormidium, Dichothrix, Lyngbya • Phormidium valderianum shows anti-hepatitis-B • Single cell protein- Spirulina • Cosmetics and beauty products- anti wrinkle creams, skin ointments, pimple lotion, biolipstics, etc. • Effluent treatment- bioflocculation- Phormidium, Oscillatoria • Aquaculture- fish feed supplement

  42. Spirulina (CFTRI) • L.V.Venkataraman • Tablets and powder (Ambadi Gr., Madras) • Cattle and fish feed supplement • β-carotene as food colour and pro- vitamin-A • Phycocyanin-water soluble pigment (Lina Blue) • used in eye shadow, eye-liner & biolipstic (Sigam Algae Co., Bangkok)

  43. Spirulina: major constituents Proteins 65.71% Fat 6.7% β Carotene 320, 000 u/100g Calcium 658 mg P 977 mg Fe 47.7 mg Sodium 796 mg Potassium 1.14 mg Lysine 2.99% Cystein 0.47% Methionine 1.38% Phenyl alanine 2.87% Theonine 3.04%

  44. Spirulina capsules

  45. Spirulina capsules

  46. Negative: • Poisonous - Microcystis spp. give a bad smell on decaying • Decrease fish and other aquatic fauna • When ‘blooms’, biofouling “Biological softwares” - M.S. Swaminathan

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