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3. Cyanobacteria.ppt

  1. 1. 1. Habitat 2. Morphology 3. Cultivation 4. Reproduction 5. Evolution 6. Genetics 7. Photosynthesis
  2. 2. y Division Cyanophyta Cyanobacteria ‘formerly known as’ BlueGreen Algae - Cyano = blue - Bacteria – acknowledges that they are more closely related to prokaryotic bacteria than eukaryotic algae
  3. 3.  Introduction to Cyanophyceae:  It is a primitive group of algae, consists of 150 genera and about 2,500 species. In India, the division is represented by 98 genera and about 833 species. Members of the class Myxophyceae (Cyanophyceae) are commonly known as blue green algae. The name blue green algae is given because of the presence of a dominant pigment c- phycocyanin, the blue green pigment.  In addition, other pigments like chlorophyll a (green), c-phycoerythrin (red), β-carotene and different xanthophylls are also present. The members of this class are the simplest living autotrophic prokaryotes.
  4. 4.  సైనోఫిసీ పరిచయం: ఇది ఆల్గే యొక్క ఆదిమ సమూహం, ఇందులో 150 జాతులు మరియు సుమారు 2,500 జాతులు ఉన్నా యి. భారతదేశంలో, ఈ విభాగాన్నా 98 జాతులు మరియు 833 జాతులు సూచిసుున్నా యి. తరగతి మైక్సో ఫిసీ (సైనోఫిసీ) సభ్యు లను సాధారణంగా నీలం ఆకుపచచ ఆల్గే అంటారు.  నీలం ఆకుపచచ ఆల్గే అనే పేరు పెట్టబడంది ఎందుక్ంటే ఆధిపతు వరణద్రవు ం సి-ఫైక్ససైన్నన్, నీలం ఆకుపచచ వరణద్రవు ం.  అరనంగా, క్ల క్సరోఫిఫి్ ఎ (ద్ీన్), సి-ఫైక్సరిద్ిన్ (ఎరుపు), β- కెఫిటిన్ మరియు వివిధ జాంతోఫి్ో వంటి ఇతర వరణద్రవు ం కూడా ఉన్నా యి. ఈ తరగతి సభ్యు లు సరళమైన జీవన ఆటోద్టోఫిక్ ద్ొకాఫిు ట్లరో.
  5. 5.  Important Characteristics of Cyanophyceae:  1. The individual cells are prokaryotic in nature. The nucleus is incipient type and they lack membrane bound organelles.  2. Both vegetative and reproductive cells are non- flagellate.  3. Cell wall is made up of microfibrils and is differentiated into four (4) layers. The cell wall composed of mucopeptide, along with carbohydrates, amino acids and fatty acids.  4. Locomotion is generally absent, but when occurs, it is of gliding or jerky type.  సైనోఫిసీ యొక్క ముఖ్ు మైన లక్షణాలు: విభజన యొక్క ముఖ్ు మైన లక్షణాలు ద్రంది విధంగా ఉన్నా యి: 1. వు ర ుగత క్ణాలు ద్పక్ృతిలో ద్ొకాఫిు టిక్. న్యు ర రోయస్ ద్ారంభ రక్ం మరియు వాటిర ొర క్ట్లటకునా అవయవాలు ల్గవు.  2. ఏపుగా మరియు పునరుతప తిు క్ణాలు రండూ క్ల ా రో లల్గట్ కాన్నవి. 3. సె్ గోడ మైద్క్సఫైద్ి్ో తో రూొందించబడంది మరియు ఇది
  6. 6.  5. The principal pigments are chlorophylls a (green), c- phycocyanin (blue) and c-phyco- erythrin (red). In addition, other pigments like β-carotene and different xanthophylls like myxoxanthin and myxoxanthophyll are also present.  6. Membrane bound chromatophore are absent. Pigments are found embedded in thylakoids.  7. The reserve foods are cyanophycean starch and cyanophycean granules (protein).  5. ద్పధాన వరణద్రవాు లు క్ల క్సరోఫిఫి్ో ఎ (ఆకుపచచ ), సి- ఫైక్ససైన్నన్ (నీలం) మరియు సి-ఫైక్స-ఎరిద్ిన్ (ఎరుపు). అరనంగా, β- కెఫిటిన్ వంటి ఇతర వరణద్రవు ం మరియు మైకాో క్ో ంతిన్ మరియు మైకాో కాశ ంతోఫి్ వంటి విభినా శంతోఫి్ో కూడా ఉన్నా యి.  6. మంద్ేన్ బండ్ ద్క్సమాటోఫోర్ ల్గదు. వరణద్రవు ం థైలాక్సయిడ్ో లో ొందుపరచబడ ఉంటాయి.  7. రిజర్్ ఆహారాలు సైనోఫిషియన్ క్ల సా ట ర్చ మరియు
  7. 7.  8. Many filamentous members possess specialized cells of disputed function (supposed to be the centre of N2 fixation) known as heterocysts.  9. Reproduction takes place by vegetative and asexual methods. Vegetative reproduction takes place by cell division, fragmentation etc. Asexual reproduction takes place by endospores, exospores, akinetes, nannospores etc.  10. Sexual reproduction is completely absent. Genetic recombination is reported in 2 cases.  8. చాలా మంది ఫిలమంట్స్ సభ్యు లు వివాదాసప ర ఫంక్షన్ యొక్క ద్పత్యు క్ క్ణాలను క్లిగి ఉంటారు (N2 క్ల సిరీకక్రణకు కంద్రంగా భావించాలి) హెటెఫిసిక్ల స్టో అన్న పిలుసా ు రు.  9. ఏపుగా మరియు అలంగిక్ పరధతుల దా్ రా పునరుతప తిు జరుగుతుంది. వృక్షసంపర పునరుతప తిు క్ణ విభజన, విచిి నా ం మొరలన వాటి దా్ రా జరుగుతుంది.
  8. 8.  Thallus Organisation in Cyanophyceae:  Plants of this group show much variation in their thallus organisation.  The thallus may be of unicellular or colonial forms:  1. Unicellular Form: In unicellular form, the cells may be oval or spherical. Common members are Gloeocapsa (Fig. 3.23A), Chroococcus and Synechococcus.  2. Colonial Form: In most of the members the cells after division remain attached by their cell wall or remain together in a common gelatinous matrix, called a colony.  The colonies may be of two types: a. Non- filamentous, and b. Filamentous
  9. 9. Growth Forms Synechococcus Crococcus Cells of Anacystis Oscillatoria Spirlina spp. Anabaena
  10. 10. Fossil Cyanobacteria
  11. 11. "Ooid" sand grain containing Endolithic Cyanobacterium (CB). Erosion caused by Endolithic Cyanobacteria
  12. 12. Structural drawing of the fine structural features of a cyanobacterial cell. (D) DNA fibrils; (G) gas vesicles; (Gl) glycogen granules; (P)plasmalemma; (PB) polyphosphate body; (Ph) polyhedral body; (Py) phycobilisomes; (R) ribosomes; (S) sheat; (SG) structured granules (cyanophycin granules); (W) wall.
  13. 13.  a. Non-Filamentous Type: The cells of this type divide either alternately or in three planes, thereby they form spherical (Gomphosphaera, Coelosphaerum), cubical (Eucapsis alpine, Fig. 3.23C), squarish (Merismopedia) or irregular (Microcystis, Fig. 3.23B) colony.  b. Filamentous Type: By the repeated cell division in one plane, single row of cells are formed, known as trichome. e.g., Oscillatoria (Fig. 3.23D), Spirulina, Arthosporia etc. The trichome when covered by mucilaginous sheath is called a filament. The filament may contain single trichome (Oscillatoria, Lyngbya) or several trichomes (Hydrocoleus, Microcoleus, Fig. 3.23E).  The trichomes may be unbranched (Oscillatoria, Lyngbya), branched (Mastigocladus limilosus, Fig. 3.23J) and falsely branched (Scytonema, Fig. 3.23K and Tolypothrix).
  14. 14.  ఒక్. న్నన్-ఫిలమంట్స్ రక్ం: ఈ రక్మైన క్ణాలు ద్పత్యు మాా యంగా ల్గదా మూడు విమాన్నలుగా విభజసా ు యి, తదా్ రా అవి గోళాకార (గోంాస్ఫే రా, క్సలోస్ఫే రం), కూు ిక్్ (యూకాపిో స్ ఆలప న్, Fig. 3.23 సి), సాక ్ రిష్ (మరిస్పో పీడయా) ల్గదా సద్క్మంగా (మైద్క్ససిసిటస్, ఫిగ్. 3.23 ి)  కాలనీ. ి. తంతు రక్ం: ఒక్ విమానంలో పునరావృతమయ్యు క్ణ విభజన దా్ రా, ఒక వరుస క్ణాలు ఏరప డత్యయి, వీటిన్న ట్రైక్సమ్ అంటారు.  ఉదా., ఓసిల్గటోరియా (Fig. 3.23D), సిప రులిన్న, ఆఫిరీస్పప రియా మొరలనవి. ముసిలాజనస్ క్సశంతో క్పప బడనపుప డు ట్రైక్సమ్ను ఫిలమంట్ అంటారు. ఫిలమంట్లో సింగి్ ట్రైక్సమ్ (ఓసిల్గటోరియా, లింగాో ు ) ల్గదా అనేక్ ట్రైక్సమ్లు (హైద్డోక్సలియస్, మైద్క్సక్సలియస్, Fig. 3.23E) ఉండవచ్చచ .  ట్రైక్సమ్లు ద్రంచ్ చేయబడన్నవి (ఓసిల్గటోరియా, లింగాో ు ), శఖ్లుగా (మాసిటగోకారో డస్ లిమిలోసస్, Fig. 3.23J) మరియు తపుప గా కొమో లుగా ఉండవచ్చచ
  15. 15.  Reproduction in Cyanophyceae:  The blue green algae (Cyanophyceae) reproduce by both vegetative and asexual means. Sexual reproduction is absent.  The vegetative reproduction performs through fission (Synechococcus), fragmentation (Oscillatoria, Cylindrospermum muscicola), hormogonia formation (Oscillatoria, Nostoc), hormospores (Westiella lanosa), planococci and Palmelloid stage.  During asexual reproduction various types of asexual spores are formed. These are akinetes (Anabaena sphaerica, Gloeotrichia natans, Calothrix fusca), endospores (Dermocarpa), exospores (Chamaesiphon) and nannocyte (Microcystis) (Fig. 3.27).
  16. 16.  సైనోఫిసీలో పునరుతప తిు: నీలం ఆకుపచచ ఆల్గే (సైనోఫిసీ) ఏపుగా మరియు అలంగిక్ మారా ే ల దా్ రా పునరుతప తిు చేసుుంది.  లంగిక్ పునరుతప తిు ల్గదు. ఏపుగా పునరుతప క్ల తిు విచిి తిు (సైనెక్సకాక్స్), ద్ాలో ంటేషన్ (ఓసిల్గటోరియా, సిలింద్డోసెప రుో మ్ మసిో క్సలా), హాఫిో గోన్నయా న్నరాో ణం (ఓసిల్గటోరియా, నోస్ప ట క్), హాఫిో స్పప ర్ో (వెసిటఎలరో లానోసా), క్ల ా రో నోక్సర మరియు ామలా రో యిడ్ రశల దా్ రా జరుగుతుంది.  అలంగిక్ పునరుతప తిు సమయంలో వివిధ రకాల అలంగిక్ బీజాంశలు ఏరప డత్యయి. అవి అరన్నటెస్ (అన్నబెన్న స్ఫప రికా, క్ల గోరోట్రద్టిచియా న్నటాన్ో , క్లోద్ిక్ో ఫుసాక ), ఎండోస్పప ర్ో (డెఫిో కారాప ), ఎక్సో స్పప ర్ో (చమసిఫోన్) మరియు న్ననోసైట్ (మైద్క్ససిసిటస్) (Fig. 3.27).
  17. 17. • cyanobacteria can be found in almost every conceivable environment, from oceans to fresh water to bare rock to soil. • They can occur as planktonic cells or form phototrophic biofilms in fresh water and marine environments, • they occur in damp soil, or even temporarily moistened rocks indeserts. • A few are endosymbionts in lichens, plants, various protists, or sponges and provide energy for the host. • Some live in the fur of sloths, providing a form of camouflage. • Aquatic cyanobacteria are probably best known for the extensive and highly visible blooms that can form in both freshwater and the marine environment and can have the appearance of blue- green paint or scum. Habitat
  18. 18. Hot Spring at Yellowstone Park: The dark color is due to the presence of Cyanobacteria. Limestone deposit at Yellowstone Park: The localized areas of green are due to the presence of Cyanobacteria
  19. 19. A schematic outline of the acquisition, reduction, and loss of genomes and compartments during evolution. Black arrows indicate evolutionary pathways; white arrows indicate endosymbiotic events in the host cell. Endosymbiotic event 1 occurred at the origin of eukaryotes. The proteobacterial endosymbiont gave rise to mitochondria (the smaller organelles in the bottom part of the diagram). Endosymbiotic event 2 occurred at the origin of plastid-containing cells. Endosymbiotic event 3 represents the secondary and higher-order endosymbioses giving rise to numerous algal phyla, as well as apicomplexans (such as Plasmodium), which have residual plastids, and to trypanosomes, which have no plastid at all. Black, filled circles indicate nuclei or nucleomorphs; ellipses within organelles indicate bacterially derived genomes, which may be reduced or lost completely. More than one kind of host cell and of endosymbiont is involved in the secondary, and in the higher-order, symbioses. The genome of the Archaebacterium is not represented in the diagram.
  20. 20. n • Old 3.5 billion years • Dominated as biogenic reefs • During Proterozoic – Age of Bacteria (2.5 bya – 750 mya) they were wide spread • Then multicellularity took over • Cyanobacteria were first algae!
  21. 21. a Production • For BGA production dig a small pit of 6x3x9 feet size in the soil and lay down a polythene sheet in the pit to check to percolation of water. Large galvanized steel tray containing soil can also be used for this purpose. Now 10 Kg soil, 200 gm of super phosphate, 6 litre water and 100 gm BGA dry flakes containing wooden dust or mother culture of BGA are added into the prepared pit. If found any pest in the pit, spray melathion solution ( 1 ml melathion in one litre water) to destroy pests. • If green algae and diatoms are found in pit, use 0.05% CuSO4 solution which will kill the green algae. After 12-15 days, a thick layer of BGA will be found floating on the water surface of the pit. You can easily collect/harvest the BGA directly from the pit or let the pit dry after water evaporation and take out dry flakes of BGA and fill it in small polypack (100-200 gm) for sale. • By this simple method BGA culture/incoulum can be prepared for use in the paddy fields. The same methods may be used in the paddy fields for the large scale productions of BGA culture.
  22. 22. CULTIVATION STOCK CULTURE •The stock culture for maintenance of laboratory culture, 2- 3 mL of a 3 weeks old cyanobacterial stock culture was used as inoculum in 50 mL of autoclaved BG 11 medium in 150 mL Erlenmeyer flasks. •The cultivation was carried out at 20 ±2°C, under continuous illumination of 8gmol/m2 by cool fluorescence lamps. •The stock cultures were maintained for 20-30 days.
  23. 23. CULTIVATION SHAKE CULTURE Aliquots of 50 mL from the stationary phase stock cultures were used to inoculate 500 mL of autoclaved BG11 medium in 1.5 liter Fehrnbach flasks. These samples were cultivated at 20 ±20°C, under continuous illumination of 8gmol/m2 by cool fluorescence lamps. The cyanobacterial cultures were harvested after 4-6 weeks. The cells were separated from the medium by centrifugation (4000 rpm/ 10 min/ 100C) followed by filtration with filter paper. The biomasses were lyophilized and stored at -20°C until use while cultivation media were concentrated to 1/10 (v/v) by rotary evaporation in vacuum at 400°C and extracted immediately with EtOAc solvent.
  24. 24. The large scale cultivation was carried out in a 45 liter- glass fermentor. The fermentor was cleaned by distilled water and 70% isopropanol before use. At the beginning, the fermentor was filled with 15 L of medium and after 1- 2 hours 1.5 L of growing culture (after 20 days of cultivation in three Fehrnbach flasks) was added. Afterwards, every day 5 L of medium were added into the fermentor until 35 L of medium were reached. The cultures were illuminated continuously with banks of cool white fluorescent tubes of 8gmol/m2 and incubated at temperature of 26°C to 28°C adjusted using a heater. The pH-value of the large scale culture was adjusted to 7.4-8.5 using CO2 supplementation. The biomass was collected by centrifugation at 6500 rpm in a refrigerated continuous-flow centrifuge and lyophilized, then stored at -20°C.
  25. 25. Composition : Magnesium Sulphate (MgSO4 .7H2O) 0.025 g Calcium chloride 0.05 g Sodium chloride 0.20 g Dipotassium hydrogen phosphate 0.35 g A5 trace elements stock solution 1.0 ml Glass-distilled water 1,000 ml General purpose media for cyanobacteria (blue green algae) : Allen and Arnon's Medium (modified): This medium is generally used for nitrogen-fixing cyanobacteria. If 0.20 g of potassium nitrate is added, the medium supports the growth of many non- nitrogen-fixing cyanobacteria.
  26. 26. Life cycle of Cyanobacteri a
  27. 27. Heterocysts are the sites for Nitrogen fixation.
  28. 28. Akinetes are asexual propagules which derived from the vegetative cells. It protects in unfavourable condition this aids in asexual reproduction
  29. 29. Provides a buffering microenvironment.
  30. 30. Pilli : secretion
  31. 31. Storage products 1. Phosphate Storage: •Polyphosphate bodies contain metals, mostly potassium, calcium, and magnesium. Polyphosphate bodies in heterocysts of Anabaena showed an increased content of S and Mo and a reduction in the Ca content. The fact that polyphosphate bodies can take up heavy metals led to the hypothesis that polyphosphate bodies may be important in heavy-metal accumulation. •The enzyme involved in the synthesis of polyphosphates in cyanobacteria is thought to be polyphosphate synthetase (polyphosphate kinase), which catalyses the formation of polyphosphate from ATP. No polyphosphate is required as a primer but the enzyme requires magnesium.Polyphosphates are broken down by alkaline polyphosphatase.
  32. 32. Storage products 2. Cyanophycin •Nitrogen can be stored in cyanobacteria in the form of electron-dense granules, called structured granules, containing cyanophycin granule polypeptide (CGP) . •CGP consists of a simple polypeptide, composed of arginine and aspartic acid in a 1 : 1 molar ratio and is called multi-L-arginil-poly(L-aspartic acid), or cyanophycin. •Cyanophycin is unique to cyanobacteria ,but is not found in all species.
  33. 33. 3. Phycobilin pigments: •Phycobilisomes are composed of light-harvesting phycobilin pigments that transfer absorbed light to photosystem II reaction centres. • Phycocyanin is sometimes regarded as a nitrogen storage compound. •It is rapidly degraded during N starvation and the apoprotein synthesis is specifically repressed. • However, kinetic analyses have shown that when N is available, phycocyanin is always synthesised after the formation of cyanophycin and when N is limiting, phycocyanin is degraded after CGP. Storage products
  34. 34. s The compartmentalization of the cyanobacterial cell:The thylakoid membrane,the internal membrane system that separates the cytoplasm from the lumen and that is present in virtually all cyanobacteria,contains both photosynthetic and respiratory electron transport chains. These electron transport chains intersect,and in part utilize the same components in the membrane. Note that oxygenic photosynthesis (conversion of CO2 and water to sugars using the energy from light) essentially is the reverse of respiration (conversion of sugars to CO2 and water releasing energy). The cytoplasmic membrane,separating the cytoplasm from the periplasm,contains a respiratory electron transport chain but not photosynthetic complexes in most cyanobacteria. Therefore,in most cyanobacteria,photosynthetic electron transport occurs solely in thylakoids, whereas respiratory electron flow takes place in both the thylakoid and cytoplasmic membrane systems.
  35. 35. C-Phycocyanins Absorb Green-Yellow Light (615-620A). Allophycocyanins Absorb Orange-Red (650- 670A) C- Phycoerythrin Absorbs Green Light (495-570)
  36. 36. (PS II) uses light energy to split water and to reduce the PQ pool. Electrons are transported from the PQ pool to the cytochrome b6f complex and from there to a soluble electron carrier on the luminal side of the thylakoid membrane. In cyanobacteria this soluble carrier may be plastocyanin or cytochrome c553,depending on the species and on the availability of copper (plastocyanin is a copper containing enzyme). Either of these soluble one-electron carriers can reduce the oxidized PS I reaction centre chlorophyll,P700 . This oxidized form of the reaction centre chlorophyll is formed by a light-induced transfer of an electron from PS I to ferredoxin (Fd) and eventually to NADP. Reduced NADP can be used for CO2 fixation. Photosynthetic electron transfer leads to a proton gradient across the thylakoid membrane. In PS II,protons are released into the lumen upon water splitting,and protons formed upon plastoquinol oxidation by the cytochrome b6f complex are released into the lumen as well. The proton gradient across the thylakoid membrane is used for ATP synthesis by the ATP synthase in the thylakoid; this ATP may be applied for CO2 fixation and for other cell processes.
  37. 37. REGULATION OF PHOTOSYNTHESIS • If light is abundant,the photosynthetic electron transport chain has a much higher capacity of electron flow than has the respiratory chain, • but at very low light intensity or in darkness respiratory rates are higher than those of photosynthesis. Phycobilisomes which are attached to the Surface of the Thylakoids . Phycobilisomes contain Accessory Pigments for Photosynthesis . These are Water Soluble and are stabilized by bonds to Proteins.
  38. 38. PS I activity is abundant relative to that of the cytochrome b6f complex
  39. 39. n
  40. 40. Conditions affect on blooms A juicy, yummy microbial mat, full of cyanobacterial goodness from Yellowstone Park.
  41. 41. Nutrient Availability: Nutrients are a limiting factor for cyanobacteria populations. As long as the correct nutrients are in excess, they can grow until some other factor, often light or temperature, becomes limiting. Competition: Ability to adapt to the environment is a big factors determining whether a bloom will form. Many blue-greens are less edible, have gas vacuoles that help them float, can sequester nutrients at the sediment water interface, or can fix dissolved nitrogen, any of which can give them a competitive advantage over other algae and lead to bloom formation. Light Intensity: Since cyanobacteria are phytoplankton, light is important and different species thrive under different light intensities. If light is not extinguished by particles or color in the water, a bloom is more likely. Many blue-greens thrive under low light, and so may be favored unless light is nearly absent (such as in some high particulate reservoir systems). Mixing: Mixing allows nutrients to be more evenly distributed and affects other aspects of water quality that in turn affect algal abundance and composition. Mixing can also move algae to depths with less light, limiting growth and survival. In general, blue-greens do better wtih less mixing (Cylindrospermopsis is one taxon that seems to do well in mixed systems, though). Temperature: Surface water temperatures consistently above 28 degrees Celsius (82 degrees Fahrenheit) encourage blue-green blooms, although blooms may still occur in late fall (October, November) in the Northern U.S. Species: The above factors influence different species very differently, because each species or taxon has a unique way of dealing with their environment. There are generalizations that apply to blooms and blue-green dominance, but ther are exceptions in most cases. Algal bloom formation is a complicated ecological process. Toxicity: Not all blue-greens are toxic, so while risk may be higher during a bloom, high biomass does not necessarily result in toxicity. Also, although many toxin producing algae produce taste and odor compounds, the presence or absence of geosmin or MIB is not a predictor of the presence of toxins.
  42. 42. The pH and moisture of soil and population of cyanobacteria in four seasons of the year
  43. 43. Effects of cyanobacteria on plant and soil. Analysis was performed with independent Samples t-test
  44. 44. Algalization in paddy field (1) Increase in soil pores with having filamentous structure and production of adhesive substances. (2) Excretion of growth-promoting substances such as hormones (auxin, gibberellin), vitamins, amino acids (Roger and Reynaud 1982, Rodriguez et al. 2006). (3) Increase in water- holding capacity through their jelly structure (Roger and Reynaud 1982). (4) Increase in soil biomass after their death and decomposition. (5) Decrease in soil salinity. (6) Preventing weeds growth. (7) Increase in soil phosphate by excretion of organic acids (Wilson 2006)
  45. 45. Rice growth with Cyanobacteria
  46. 46. Mishra and Pabbi (2004) Effect of cyanobacterial biofertilizer inoculation on rice grain yield at a farmer’s field
  47. 47. for production at farmers’ level is not popular among the farming community. The main limitations of this technology are: •due to open air nature of production it can be produced for only a limited period in a year (3-4 months in summer; Production has to be stopped during rainy and winter season), •high level of contamination due to open type of production, •slow production rate, • low population density and hence need for heavy inoculum per hectare.
  48. 48. 1. The individual unit in the polyhouses can be of either RCC, brick and mortar, or even polythene lined pits in the ground. The algae are grown individually as species, by inoculating separate tanks with laboratory grown pure cultures, so as to ensure the presence of each required strain in the final product. 2. Once fully grown, the culture is harvested, mixed with the carrier material, presoaked overnight in water and multani mitti (in 1:1 ratio) and sun dried. The dried material is ground and packed in suitable size polythene bags, sealed and stored for future use. 3. The final product contains 10,000 to 1,00,000 units or propagules per gm of carrier material and, therefore, 500 g material is sufficient to inoculate one acre of rice growing area. Mishra and Pabbi (2004)
  49. 49. Luxuriant growth of Cyanobacteria in a paddy field
  50. 50. Types of Photobioreactor • Tubular Reactors –Horizontal –Vertical • Flat panel reactors • Vertical column reactors • Bubble column reactors • Air lift reactors • Stirred tank photobioreactors • Immobilized bioreactors
  51. 51. Among the proposed photobioreactors, tubular photobioreactor is one of the most suitable types for outdoor mass cultures. Most outdoor tubular photobioreactors are usually constructed with either glass or plastic tube and their cultures are re-circulated either with pump or preferably with airlift system. They can be in form of horizontal / serpentine, vertical near horizontal, conical, inclined photobioreactor. Aeration and mixing of the cultures in tubular photobioreactors are usually
  52. 52. Tubular photobioreactor are very suitable for outdoor mass cultures of algae since they have large illumination surface area. Tubular photobioreactors consist of straight, or looped arranged in coiled various ways for transparent maximizing designed capture. Properly photobioreactors completely isolate the tubing sunlight tubular culture from potentially contaminating environments, hence, allowing extended external duration monoalgal culture. photoinhibition is very common in outdoor tubular photobioreactors .When a tubular photobioreactor is scaled up by increasing the diameter of the illumination surface to volume ratio tubes, would decrease. On the other hand, the length of the tube can be kept as short as possible while a tubular photobioreactor is scaled up by increasing the diameter of the tubes. In this case, the cells at the lower part of the tube will not receive enough light for cell growth (due to light shading effect) unless there is a good mixing system.
  53. 53. • Prospects Large illumination surface area, suitable for outdoor cultures, fairly good biomass productivities, relatively cheap. • Limitations Gradients of pH, dissolved oxygen and CO2 along the tubes, fouling, some degree of wall growth, requires large land space.
  54. 54. Vertical Bioreactor Bioreactor
  55. 55. Industrial size Photobioreacto r
  56. 56. Seaweed cultivation
  57. 57.  Seaweeds are macrophytic algae, a primitive type of plants lacking true roots, stems and leaves.  Most seaweeds belong to one of three divisions - the Chlorophyta (green algae), the Phaeophyta (brown algae) and the Rhodophyta (red algae).  There are about 900 species of green seaweed, 4000 red species and 1500 brown species found in nature.  The green seaweeds Enteromorpha, Ulva, Caulerpa and Codium are utilized exclusively as source of food. Khan and Satam(2003)
  58. 58. 1) the Single Rope Floating Raft (SRFR) technique developed by CSMCRI is suitable for culturing seaweeds in wide area and greater depth. 2) A long polypropylene rope of 10 mm diameter is attached to 2 wooden stakes with 2 synthetic fiber anchor cables and kept afloat with synthetic floats. 3) The length of the cable is twice the depth of the sea (3 to 4 m). Each raft is kept afloat by means of 25-30 floats. The cultivation rope (1 m long x 6 m diameter polypropylene) is hung with the floating rope. 4) A stone is attached to the lower end of the cultivation rope to keep it in a vertical position. 5) Generally 10 fragments of Gracilaria edulis are inserted on each rope. The distance between two rafts is kept at 2
  59. 59. Chinese people provide fertilizer to seaweeds Hungry fish and animals cause damage to seaweeds
  60. 60. INTRODUTION A biofertilizer is a substance which contains living microorganisms, when applied to seed, plant surfaces, or soil, colonizes the rhizosphere or the interior of the plant and promotes growth by increasing the supply or availability of primary nutrients to the host plant. Bio-fertilizers add nutrients processes of nitrogen through the natural fixation, solubilizing phosphorus, and stimulating plant growth through the synthesis of growth-promoting substances.
  61. 61. What is Bio fertilizer? Biofertilizers are natural fertilizers that are microbial inoculants of bacteria, algae and fungi (separately or in combination). which may help biological nitrogen fixation for the benefit of plants. They help build up the soil micro-flora and there by the soil health. Biofertilizer also include organic fertilizers(manure, etc.) Use of bio-fertilizer is recommended for improving the soil fertility in organic farming
  62. 62. TYPES OF BIOFERTILIZERS Bacterial Fungal Algal Aquatic fern Earthworms VAM fungi
  63. 63. Bacteria: Symbiotic nitrogen fixers. Rhizobium, Azospirillum spp Free living nitrogen fixers. Azotobacter, Klebsiella etc., Algal biofertilizers: BGA in association with Azolla Anabena, Nostoc, Ocillatoria Phosphate solubilising bacteria: Pseudomonas, Bacillus megaterium Fungal biofertilizer VAM Earthworms
  64. 64. Bacterial biofertilizers The live cells of bacteria used as a biofertilizers These microbes contains unique gene called as Nif-Gene which make them capable of fixing nitrogen. The nitrogen fixing bacteria work under two conditions, Symbiotically Free living bacteria (non-symbiotic). The symbiotic bacteria make an association with crop plants through forming nodules in their roots. The free living bacteria do not form any association but live freely and fix atmospheric nitrogen.
  65. 65. Symbiotic nitrogen fixers. Most important symbiotic Nitrogen fixing bacteria is Rhizobium and Azospirillum. Rhizobium: Rhizobium lives in the root hairs of the legumes by forming nodules Plant root supply essential minerals and newly synthesized substance to the bacteria The name Rhizobium was established by Frank in 1889. This genus has seven distinct species based on "Cross Inoculation Group Concept".
  66. 66.  More than twenty cross-inoculations groups have been established.  A new classification has been established for Rhizobium.  That is 'slow growing rhizobia' known as Bradyrhizobium and the other group is 'fast growing rhizobia' called Rhizobium.  Rhizobium can fix 50-300 kg/ha Rhizobium
  67. 67. Azospirillum: It mainly present in cereal plants. inhabits both root cells as well as surrounding of roots forming symbiotic relation and increasing nitrogen fixing potential of the cereal plant. Azospirillum is recognized as a dominant soil microbe nitrogen in the range of 20- 40 kg/ha in the rhizosphere in non-leguminous plants such as cereals, millets, Oilseeds, cotton etc. Considerable quantity of nitrogen fertilizer up to 25-30 % can be saved by the use of Azospirillum inoculant. These species have been commercially exploited for the use as nitrogen supplying Bio-Fertilizers.
  68. 68. Free living bacteria Large number of free living or non -symbiotic bacteria (does not form nodules but makes association by living in the rhizosphere) present in soil. Commonly used free living bacteria are Azotobacter Klebsiella it will not associated with plant. Azotobacter is a biofertilizer which provides the required amount of nitrogen to the plant from the soil.
  69. 69. Azotobactor Azotobactor is a heterotrophic free living nitrogen fixing bacteria present in alkaline and neutral soils. Azotobactor is the most commonly occurring species in arable soils of India. Apart from its ability to fix atmospheric nitrogen in soils, it can also synthesize growth promoting substances such as auxins and gibberellins and also to some extent the vitamins.
  70. 70. Many strains of Azotobactor also exhibit fungicidal properties against certain species of fungus. Response of Azotobactor has been seen in rice, maize, cotton, sugarcane, pearl millet, vegetable and some plantation crops. It improves seed germination and plant growth. Azotobacter is heaviest breathing organism and requires a large amount of organic carbon for its growth.
  71. 71. Mass production isolated bacterial cultures were subculture in to nutrient broth The cultures were grown under shaking condition at 30±2°C The culture incubated until it reaches maximum cell population of 10¹º to 10¹¹ Under optimum condition this population level could be attained within 4-5 days for Rhizobium 5-7 days for Azospirillum and 6-7 days for Azotobacter. The culture obtained in the flask is called Starter culture For large scale production , inoculum from starter culture is transferred in to large flasks / fermentor and grown until required level of cell count is reached
  72. 72. prepare appropriate media for specific to bacterial inoculant in required quantity Inoculated with specific bacterial strain for aseptic condition Incubated at 30±2ºC for 5-7 days in rotary shaker Observe growth of the culture and estimate the population ( starter culture) The above the media is prepared in large quantities in
  73. 73. Sterilized and cooled well Media in a fermentor is inoculated with the log phase of culture grown in large flask (usually 1-2 % of inoculum is sufficient) cells are grown in fermentor by providing aeration & continuous stirring Broth is checked for the population of inoculated organisms Cells are harvested with the population load of 109 cells/ml
  74. 74. Carrier material the use of ideal carrier material is necessary for the production of god quality of biofertilizer Peat soil, lignite, vermiculture, charcoal, press mud, farmyard manure and soil mixture are used as a carrier materials Neutralized peat soil/lignite are found to be better carrier materials Ideal carrier material should be Cheaper in cost Locally available High organic matter content No toxic chemical Water holding capacity of more than 50% Easy to process
  75. 75. Preparation of inoculants packet Neutralized and sterilized carrier material is spread in a clean, dry, sterile metallic or plastic Bacterial culture drawn from the fermentor is added to the sterilized carrier and mixed well by manual or mechanical mixer Inoculants are packed in a polythene bags sealed with electric sealer
  76. 76. Specification of the polythene bags  Polythene bags should be of low density grade  Thickness of bag should be around 50-75 micron  Packet should be marked with the  Name of the manufacture  Name of the product  Strain number  The crops to which recommended  Method of inoculation  Date of manufacture  Batch number  Date of expiry  Price  Full address  storage instruction
  77. 77. Vesicular Arbuscular Mycorrhiza (VAM) The term mycorrhiza was taken from Greek language meaning 'fungus root'.term was coined by Frank in 1885 The mycorrhiza is a mutualistic association between fungal mycelia and plant roots. VAM is an endotrophic (live inside) mycorrhiza formed by aseptated phycomycetous fungi. VAM help in nutrient transfer mainly of phosphorus, zinc and sulfur.
  78. 78. Mycorrhizae is the symbiotic association between plant roots and soil fungus of the 7 types of mycorrhizae, VAM plays a great role in inducing plant growth. VAM are symbiotic entophytic soil fungi, which colonize the roots of approximately 80% plants. The VAM hyphae also help is retaining moisture around the root zone of plants It increases the resistance to root borne or soil borne pathogens and Nematodes.
  79. 79. They also mobilize different nutrients like Cu(copper), K(potassium), Al(aluminum), Mn(manganese), Fe (iron)and Mg (magnesium) from the soil to the plant roots. They posses vesicles (sac like structure) for storage of nutrients and arbuscular for funneling them into root system. Morphology External hyphae Arbuscles Vesicles External hyphae vesicles Arbuscles
  80. 80. Mechanism of Action The VAM forms an association with plant roots. It penetrates in the root cortex and spreads aroundthe roots of the plant. As the name indicates, they posses sac like structure called vesicules which stores phosphorus as phospholipids. The other structure called arbuscule helps bringing the distant nutrients to the vesicules and root.
  81. 81. Mass production VAM spores isolated Spores mixed with sterilized soil Soil filled in pots Host plant transplanted in pots Kept 3-4 months in green house
  82. 82. Soil in the pot along with roots of host plant is macerated Dried till it attains 5% moisture Dried soil inoculants used for field application
  83. 83. Uses of V AM Enhances the feeding areas of the plant root is as the hyphae spreads around the roots. Mobilizes the nutrients from distantance to root. Stores the nutrients (sp. phosphorus). Removes the toxic chemicals (example : phenolics) which otherwise hinder nutrient availability. Provide protection against other fungi and nematodes It increase growth rate in plants (citrus, maize, wheat, etc.) It reduces sensitivity of crop towards high level of salts and heavy metals
  84. 84. Algae as a biofertilizer Another group of free living nitrogen fixers are cyanobacteria. Commonly called as Blue green algae. More than 100 species of BGA can fix nitrogen. Nitrogen fixation takes place in specialized cells called ‘Heterocyst’ BGA very common in rice field. Unlike Azotobacter BGA are not inhibited by the presence of chemical fertilizers. No chemical fertilizers added, inoculation of the algae can result in 10-14% increase in crop yields.
  85. 85. They are easy to produce Usually they are mass produced in cement tanks filled with fresh water. Not require any processing Quite and cheap Cost of 10kg may be Rs.30-40 only Beneficial in certain crops like vegetables, cotton, sugarcane. Eg. of some algal biofertilizers are Anabena Nostoc Oscillatoria
  86. 86. Azolla is a tiny fresh water fern common in ponds, ditches and rice fields. It has been used as a biofertilizer for a rice in all major rice growing countries including India, Thailand, Korea, Philippines, Brazil and West Africa. The nitrogen fixing work is accomplished by the symbiotic relationship between the fern and BGA, Anabena azollae. In addition to nitrogen the decomposed Azolla also provides K, P, Zn and Fe to the crop Azolla as a bio fertilizer
  87. 87. Azolla biomass gets doubled within 5-7 days by vegetative methods. fix 40-80 kg nitrogen / ha / year. good manure for flooded rice. Increase of crop yield up to 15-20% has been observed while fertilizing the rice with Azolla Hybrids are growing faster Tolerant to heat and cold Fix 4- 5% more nitrogen
  88. 88. Bio - fertilizers application methods There are three ways of using these N-fixing/P.S.M. bacteria. Seed treatment Root dipping Soil applications
  89. 89. Seed Treatment Seed treatment is a most common method adopted for all types of inoculant. The seed treatment is effective and economic. Seed treatment with Rhizobium, Azotobacter, Azospirillum along with P.S.M. seed treatment can be done with any of two or more bacteria. no side effect. important things has the seeds must be coated first with Rhizobium or Azotobacter or Azospirillum when each seeds get a layer of above bacteria then the P.S.M. inoculant has to be treated on outer layer of the seeds.
  90. 90. This method will provide maximum number of population of each bacteria required for better results. Mixing the any of two bacteria and the treatment of seed will not provide maximum number of bacteria of individuals.
  91. 91. Root dipping Application of Azospirillum with the paddy/vegetable plants this method is needed. The required quantity of Azospirillum has to be mixed with 5-10 ltr of water at one corner of the field and all the plants have to kept for minimum ½ an hour before sowing .
  92. 92. Soil application P.S.M. has to be used as a soil application use 2 kgs of P.S.M. per acre. Mix P.S.M. with 400 to 600 kgs of Cowdung along with ½ bag of rock phosphate if available. The mixture of P.S.M., Cowdung and rock phosphate have to be kept under any tree shade or celling for over night and maintain 50% moisture. Use the mixture as a soil application in rows or during leveling of soil.
  93. 93. Precautions Store biofertilizer packets in cool and dry place away from direct sunlight and heat. Use right combination of biofertilizers Rhizobium is crop specific, so use in specified crop Do not mix with chemicals Use the packet before expiry, only on the specified crop, by the recommended method.
  94. 94. Advantage of biofertilizers  Renewable source of nutrients Sustain soil health  Supplement chemical fertilizers. Replace 25-30% chemical fertilizers Increase the grain yields by 10- 40%.  Decompose plant residues, and stabilize C:N ratio of soil Improve texture, structure and water holding capacity of soil No adverse effect on plant growth and soil fertility.
  95. 95. Stimulates plant growth by secreting growth hormones. Secrete fungistatic and antibiotic like substances Solubilize and mobilize nutrients Eco-friendly, non-pollutants and cost effective method
  96. 96. Disadvantages Biofertilizers require special care for long-term storage because they are alive. must be used before their expiry date. If other microorganisms contaminate the carrier medium or if growers use the wrong strain, they are not as effective. Biofertilizers lose their effectiveness if the soil is too hot or dry.

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