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AGM 509 - Biofertilizer Technology
Lecture 1
 Microbial Inoculants in Agriculture
 Biofertilizer definition
 Different groups of Biofertilizers
Agriculturally important beneficial microorganisms
Beneficial microorganisms present in soil or plant applied to either the
soil or the plant in order to improve productivity and crop health.
 Microbial Inoculants also known as ‘Bio inoculants’
Based on application MI
1.Crop productive - Nutrition : Biofertilizers
Plant growth promotion- PGPM- Biostimulants
Composting : Biomineralizers
Stress mitigation
2. Crop protective- Biocontrol agents
Microbial Inoculants in Agriculture
Biofertilizers:
Formulations / preparations/ product which contains
the efficient strain of beneficial microbes in live / latent
cells when applied through seed, soil / plant surfaces ,
colonizes the rhizosphere / interior plant parts and
accelerate microbial processes
 in soil and promotes the growth by increasing the
availability and uptake of nutrients to the host plants.
Biofertilizers
 Accelerate microbial processes in soil-augment
availability of nutrients in form-easily assimilated by
plants.
Eg. Nitrogen- Amide/ Nitrate /Ammonia
N- N fixation
P- Phosphates
 BF enhance crop growth , productivity and soil fertility.
 BF add nutrients through the natural processes of
nitrogen fixation, solubilization of P,K,Si, Zn and plant
growth promotion.
Biofertilizers are,
 Cost effective
 Renewable
 Eco –friendly
 Sustainable
 Supplement fertilizer
Importance/ Advantages of Biofertilizers
• Increase the fertilizer use efficiency
 Supplement fertilizers– meet the nutrient need of crops
 Add 20-200kg/N/ha(by fixation) under optimum
conditions & solubilize/mobilise 30-50 Kg P2O5/ha
 Liberate growth promoting substances & vitamins –
maintain soil fertility
 Suppress the incidence of pathogens & control diseases
 Increase crop yield – 10-20%. Nitrogen fixers reduce
depletion of soil nutrients – provide sustainability to
farming system
 Cheaper, pollution free – based on renewable energy
source
 Improve soil physical properties, & soil health
 Reduces the chemical cost by 25 %
Types / Classification of Biofertilizers
1. Based on Organism
• Bacteria- Azospirillum, Rhizobium, Azotobacter,
Phosphobacteria
• Fungi – AM fungi
• Algal- BGA, Azolla
• Actinomycetes- Frankia
2. Based on function
3. Based on nature
TYPES OF BIOFERTILIZERS
Heterotrophic bacteria Phototrophic bacteria Fungal
Nitrogen fixers
Symbiotic Associative Non symbiotic
Frankia Rhizobium Azospirillum Azotobacter
Phosphate solubilizers
Non symbiotic
Bacillus
Pseudomonas
Nitrogen
fixers
Symbiotic
Azolla BGA
Phosphate
Solubilizers and
mobilizers
Non symbiotic
Mycorrhizae
Glomus
Gigaspora
Aspergillus
Penicillium
2. Based on function
• Diazotrophs/Biological nitrogen fixers
• Phosphate solubilizing/mobilizing microorganisms
• Potassium releasing or silicate solubilizing microorganisms
• Zinc solubilizing bacteria
• Plant growth promoting rhizobacteria
• Biotic stress and abiotic stress tolerance
• Mineralization and bioremediation
• Soil aggregation
Types of Biofertilizers
I. NIRTOGEN FIXERS
Free living Azotobacter, Beijerinckia ,Clostridium,
Klebsiella, Anabaena, Nostoc
Symbiotic Rhizobium, Frankia, Anabaena azollae
Associative symbiotic Azospirillum
Endophytic Gulconacetobacter, Burkholderia
II. PHOSPHATE SOLUBILIZERS
Bacteria Bacillus megaterium var phosphaticum,
Bacillus subtilis, Pseudomonas striata
Fungi Penicillum sp, Aspergillus awamori
III. P MOBILIZERS
VAM fungi Glomus sp, Gigaspora sp, Acaulospora sp,
Scutellospora, sp & Sclerocystis sp
Ectomycorrhizal fungi Laccaria sp., Pisolithus sp., Boletus sp., Amanita
sp.,
Ericoid mycorrhizae Pezizella ericae
Orchid mycorrhizae Armillaria, Rhizoctonia sp
IV. PGPR Pseudomonas spp., etc
Types of Biofertilizers
Diazotrophs / N fixers
 Very few organisms possess the “N-fixing” enzyme complex
necessary to carry out BNF. Bacteria that use N2 as the sole
source of N are called diazotrophs.
 Some live in a symbiotic relationship with plants of the legume
family (Rhizobium)
 Some establish symbiotic relationships with plants other than
legumes (Frankia )
 Some exhibits symbiotic relationship with waterfern, Cycads
and angiosperms (Nostoc )
 Some nitrogen-fixing bacteria live freely in the soil (BGA,
Azotobacter )
 Free living Nitrogen fixers are both aerobic and anaerobic and
some of them are phototrophs and some are heterotrophs
Types of Biofertilizers in Use
Azospirillum Azotobacter Rhizobium
Cyanobacteria Azolla
Arbuscular Mycorrhiza
Phosphobacteria
Biofertilizer Consortium
Azospirillum Phosphobacteria
+ = AZOPHOS
1. AZOPHOS
2. Gluconacetobacter diazotrophicus
For sugarcane
PPFM
• 1st identified by Winogradsky (1894)
• First isolated free living bacterium- Clostridium pasturianum.
• Anaerobic, rod shaped, non spore forming , G-ve bacteria
• After 7 yrs 2 more important microbes identified by Beijerinck
(1901)- A.chroococcum , A.agile
• Majority of asymbiotic n fixers belongs to Azotobacteriaceae
and heterotrophic in nature.
• They inhabit both terrestrial and aquatic habitats
• Different gps-Aerobic, anaerobic, facultative anaerobe
• Imp.Spp: A.chroococcum, A.vinelandii A.agilis, A.paspali
• Qty of N fixed is more in Azotobacter than Clostridium
Non symbiotic / Asymbiotic/ Free living N fixation
• Free living aerobic bacteria
– Azotobacter, Azomonas, Azotococcus
– Beijerinckia
– Klebsiella
– Cyanobacteria (lichens)
• Free living anaerobic bacteria
– Clostridium
– Desulfovibrio
– Purple sulphur bacteria
– Purple non-sulphur bacteria
– Green sulphur bacteria
1.Non symbiotic / Asymbiotic/ Free living ‘N’ fixers
S.No Group Examples
1 Obligate aerobe Azotobacter, Beijerinckia,
Derxia, Bacillus polymyxa
2 Obligate anaerobe Clostridium
3 Facultative
anaerobe
Klebsiella, Aerobacter,
Pseudomonas
4 Photosynthetic Rhodospirillum, Rhodobacter
5 Methanogens Methanobacterium
6 Cyanobacteria Nostoc, Anabaena
7 Fungi Macrosporium, Cladosproium,
A.tenuiis
Groups of Free living microorganisms
Mechanism of N fixation- Indirect
Azotobacter
• Most widely investigated genera , Aerobic, heterotrophic
• Azotobacteriaceae constitutes majority of heterotrophic FL N fixers
Azotobacter, Beijerinckia , Derxia
Habitat:
• Present in neutral or alkaline soils
• > Popln. in soils rich in organic carbon
• Population rarely exceeds 10 4 to 105 /g soil
• Low in rhizosphere of crop plants & in uncultivated soils
• Rice, wheat, Maize, cotton, S.cane, veg, plantation crops.
• A.paspali- Grass
• A.chroococcum is the most commonly occurring sp. in arable soils
• Utilize atm. N for their cell protein synthesis.
• Cell protein is then mineralized in soil after the death of cells &
contributes N availability of crop plants
• Popln influenced by other microbes in soil. (Cephalosporium)
Morphology
• G-ve , free living soil bacterium polymorphic in nature
• Cells are short rod thick with round end.
• Cell size 2-10x1-2.5 m
• Young cells posses peritrichous flagella used for locomotion
• Old cells produce cyst & have enhanced resistant to heat ,
desiccation which favours the bacteria under adverse
conditions.
• Cyst germinate under favourable conditions and produce
veg.cells
• Sensitive to acidic pH, high salts and temperature >35 C
Characteristics
• Produces growth promoting substances which increase
germination, growth and yd.
• Eg. IAA, GA & B vitamins (thiamine, riboflavin) nicotin in
media
• Pigmentation & production of extracellular
slime/polysaccharides
• Also produce some anti fungal substances and check
pathogens Alternaria, Fusarium & Helminthosporium –
biocontrol agent
• 10-15 % increase in crop yield
Azotobacter spp
No Azotobacter spp. Occurrence &
Isolation
pigmentation
1 A.chroococcum Neutral & alkali
soil
Black brown, insoluble,
peritrichous
2 A.vinelandii American soil Green fluorescent ,
peritrichous, soluble pigment
3 A.beijerinckii American soil Yellow brown , no flagella
4 A.Paspali Paspalum root Green fluorescent ,
peritrichous, soluble pigment
5 A.Insignis water sample
A.Agiglis Aquatic Green fluorescent ,
peritrichous, soluble pigment
6 A.macrocytogenes soil Pink, soluble, polar flagella,
abundant slime
N fixation:
• Naturally fix N in rhizosphere soil
• Fix N only after utilizing carbon source and CO2 as byproduct.
• Also requires Ca for N fixation
• In vitro – req. org N, micronutrients for enhancing N fixing ability
• In soil an effective strain fix ~15-20kg/N /ha
• Fix 10 mg N/g carbon source under in vitro
Beijerinckia
• distribution restricted to tropical and sub-tropical regions
• Capable of growing on wide range of pH (2.9- 10)
• Higher popln in pH 4.9 – 7.4.
• Dominant in acidic soils
• 3 spp. B. Indica, B. Mobile , B.fluminensis
• Occurrence reported in rice, Sugarcane, forage grasses
(Digiteria, Panicum purpurescents, Cyanodon dactylon &
Setaria ) Plantation crops (Cocount, arecanut, chashew, cocoa
,pepper & pearl millet)
• B.indica produces peritrichous flagella, abundant slime,
Light rust brown insoluble
itrogen fixation bacteria by type of interaction with plants
Root nodule
symbiosis
bacteria
Associative
nitrogen-fixing
bacteria
Free-living
nitrogen-fixing
bacteria
Energy source high moderate moderate
Oxygen protection high moderate low
Transfer of fixed N high moderate low
Estimates of
nitrogen fixation
rates, kg N ha−1
year−1
50–465 2–170 1–80
Nitrogen fixation
2. Chemical
Industrial process
1.Natural -
Lightening
• Req high temperature for
process
• Not eco-friendly
• Make plants susceptible
• Deteriorate soil health &
plant health
• Products
Eg. Urea, DAP, MOP
• Un predictable
• Un Quantifiable
3.Biological
fixation
(Microorganisms)
• Conversion of atmospheric Nitrogen (gaseous N2) to ammonia (Usable
form)
• Inert form (NN)  Reactive Nitrogen (N)
• Reduction process ( N2 NH3)
• Eco-friendly
• Inprove soil health & fertility
• Renewable
•Sustainable
Eg. Biofertilizers
Nitrogen fixation
Symbiotic nitrogen fixation: special structure-nodule formation
Nodules: organ for storage and transfer of fixed nitrogen
Stem nodules-Sesbania
Root nodules- legumes
Endophytic
nitrogen
fixation
Sugarcane-
Gluconacetobacter
2 types of nodulation
NH3 16Pi
3.Biological Nitrogen fixation
+ 2OH- + 8 flavodoxin + 16 MgADP- + 16H2PO4
- + H2
N2 + 8 flavodoxin- + 8H+ + 16 MgATP2- + 18 H2O nitrogenase
2NH4
+

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  • 1. AGM 509 - Biofertilizer Technology Lecture 1  Microbial Inoculants in Agriculture  Biofertilizer definition  Different groups of Biofertilizers
  • 2. Agriculturally important beneficial microorganisms Beneficial microorganisms present in soil or plant applied to either the soil or the plant in order to improve productivity and crop health.  Microbial Inoculants also known as ‘Bio inoculants’ Based on application MI 1.Crop productive - Nutrition : Biofertilizers Plant growth promotion- PGPM- Biostimulants Composting : Biomineralizers Stress mitigation 2. Crop protective- Biocontrol agents
  • 3. Microbial Inoculants in Agriculture Biofertilizers: Formulations / preparations/ product which contains the efficient strain of beneficial microbes in live / latent cells when applied through seed, soil / plant surfaces , colonizes the rhizosphere / interior plant parts and accelerate microbial processes  in soil and promotes the growth by increasing the availability and uptake of nutrients to the host plants.
  • 4. Biofertilizers  Accelerate microbial processes in soil-augment availability of nutrients in form-easily assimilated by plants. Eg. Nitrogen- Amide/ Nitrate /Ammonia N- N fixation P- Phosphates  BF enhance crop growth , productivity and soil fertility.  BF add nutrients through the natural processes of nitrogen fixation, solubilization of P,K,Si, Zn and plant growth promotion.
  • 5. Biofertilizers are,  Cost effective  Renewable  Eco –friendly  Sustainable  Supplement fertilizer
  • 6. Importance/ Advantages of Biofertilizers • Increase the fertilizer use efficiency  Supplement fertilizers– meet the nutrient need of crops  Add 20-200kg/N/ha(by fixation) under optimum conditions & solubilize/mobilise 30-50 Kg P2O5/ha  Liberate growth promoting substances & vitamins – maintain soil fertility  Suppress the incidence of pathogens & control diseases  Increase crop yield – 10-20%. Nitrogen fixers reduce depletion of soil nutrients – provide sustainability to farming system  Cheaper, pollution free – based on renewable energy source  Improve soil physical properties, & soil health  Reduces the chemical cost by 25 %
  • 7. Types / Classification of Biofertilizers 1. Based on Organism • Bacteria- Azospirillum, Rhizobium, Azotobacter, Phosphobacteria • Fungi – AM fungi • Algal- BGA, Azolla • Actinomycetes- Frankia 2. Based on function 3. Based on nature
  • 8. TYPES OF BIOFERTILIZERS Heterotrophic bacteria Phototrophic bacteria Fungal Nitrogen fixers Symbiotic Associative Non symbiotic Frankia Rhizobium Azospirillum Azotobacter Phosphate solubilizers Non symbiotic Bacillus Pseudomonas Nitrogen fixers Symbiotic Azolla BGA Phosphate Solubilizers and mobilizers Non symbiotic Mycorrhizae Glomus Gigaspora Aspergillus Penicillium
  • 9. 2. Based on function • Diazotrophs/Biological nitrogen fixers • Phosphate solubilizing/mobilizing microorganisms • Potassium releasing or silicate solubilizing microorganisms • Zinc solubilizing bacteria • Plant growth promoting rhizobacteria • Biotic stress and abiotic stress tolerance • Mineralization and bioremediation • Soil aggregation Types of Biofertilizers
  • 10. I. NIRTOGEN FIXERS Free living Azotobacter, Beijerinckia ,Clostridium, Klebsiella, Anabaena, Nostoc Symbiotic Rhizobium, Frankia, Anabaena azollae Associative symbiotic Azospirillum Endophytic Gulconacetobacter, Burkholderia II. PHOSPHATE SOLUBILIZERS Bacteria Bacillus megaterium var phosphaticum, Bacillus subtilis, Pseudomonas striata Fungi Penicillum sp, Aspergillus awamori III. P MOBILIZERS VAM fungi Glomus sp, Gigaspora sp, Acaulospora sp, Scutellospora, sp & Sclerocystis sp Ectomycorrhizal fungi Laccaria sp., Pisolithus sp., Boletus sp., Amanita sp., Ericoid mycorrhizae Pezizella ericae Orchid mycorrhizae Armillaria, Rhizoctonia sp IV. PGPR Pseudomonas spp., etc Types of Biofertilizers
  • 11. Diazotrophs / N fixers  Very few organisms possess the “N-fixing” enzyme complex necessary to carry out BNF. Bacteria that use N2 as the sole source of N are called diazotrophs.  Some live in a symbiotic relationship with plants of the legume family (Rhizobium)  Some establish symbiotic relationships with plants other than legumes (Frankia )  Some exhibits symbiotic relationship with waterfern, Cycads and angiosperms (Nostoc )  Some nitrogen-fixing bacteria live freely in the soil (BGA, Azotobacter )  Free living Nitrogen fixers are both aerobic and anaerobic and some of them are phototrophs and some are heterotrophs
  • 12. Types of Biofertilizers in Use Azospirillum Azotobacter Rhizobium Cyanobacteria Azolla Arbuscular Mycorrhiza Phosphobacteria
  • 13. Biofertilizer Consortium Azospirillum Phosphobacteria + = AZOPHOS 1. AZOPHOS 2. Gluconacetobacter diazotrophicus For sugarcane PPFM
  • 14. • 1st identified by Winogradsky (1894) • First isolated free living bacterium- Clostridium pasturianum. • Anaerobic, rod shaped, non spore forming , G-ve bacteria • After 7 yrs 2 more important microbes identified by Beijerinck (1901)- A.chroococcum , A.agile • Majority of asymbiotic n fixers belongs to Azotobacteriaceae and heterotrophic in nature. • They inhabit both terrestrial and aquatic habitats • Different gps-Aerobic, anaerobic, facultative anaerobe • Imp.Spp: A.chroococcum, A.vinelandii A.agilis, A.paspali • Qty of N fixed is more in Azotobacter than Clostridium Non symbiotic / Asymbiotic/ Free living N fixation
  • 15. • Free living aerobic bacteria – Azotobacter, Azomonas, Azotococcus – Beijerinckia – Klebsiella – Cyanobacteria (lichens) • Free living anaerobic bacteria – Clostridium – Desulfovibrio – Purple sulphur bacteria – Purple non-sulphur bacteria – Green sulphur bacteria 1.Non symbiotic / Asymbiotic/ Free living ‘N’ fixers
  • 16. S.No Group Examples 1 Obligate aerobe Azotobacter, Beijerinckia, Derxia, Bacillus polymyxa 2 Obligate anaerobe Clostridium 3 Facultative anaerobe Klebsiella, Aerobacter, Pseudomonas 4 Photosynthetic Rhodospirillum, Rhodobacter 5 Methanogens Methanobacterium 6 Cyanobacteria Nostoc, Anabaena 7 Fungi Macrosporium, Cladosproium, A.tenuiis Groups of Free living microorganisms
  • 17. Mechanism of N fixation- Indirect
  • 18. Azotobacter • Most widely investigated genera , Aerobic, heterotrophic • Azotobacteriaceae constitutes majority of heterotrophic FL N fixers Azotobacter, Beijerinckia , Derxia Habitat: • Present in neutral or alkaline soils • > Popln. in soils rich in organic carbon • Population rarely exceeds 10 4 to 105 /g soil • Low in rhizosphere of crop plants & in uncultivated soils • Rice, wheat, Maize, cotton, S.cane, veg, plantation crops. • A.paspali- Grass • A.chroococcum is the most commonly occurring sp. in arable soils • Utilize atm. N for their cell protein synthesis. • Cell protein is then mineralized in soil after the death of cells & contributes N availability of crop plants • Popln influenced by other microbes in soil. (Cephalosporium)
  • 19. Morphology • G-ve , free living soil bacterium polymorphic in nature • Cells are short rod thick with round end. • Cell size 2-10x1-2.5 m • Young cells posses peritrichous flagella used for locomotion • Old cells produce cyst & have enhanced resistant to heat , desiccation which favours the bacteria under adverse conditions. • Cyst germinate under favourable conditions and produce veg.cells • Sensitive to acidic pH, high salts and temperature >35 C
  • 20. Characteristics • Produces growth promoting substances which increase germination, growth and yd. • Eg. IAA, GA & B vitamins (thiamine, riboflavin) nicotin in media • Pigmentation & production of extracellular slime/polysaccharides • Also produce some anti fungal substances and check pathogens Alternaria, Fusarium & Helminthosporium – biocontrol agent • 10-15 % increase in crop yield
  • 21. Azotobacter spp No Azotobacter spp. Occurrence & Isolation pigmentation 1 A.chroococcum Neutral & alkali soil Black brown, insoluble, peritrichous 2 A.vinelandii American soil Green fluorescent , peritrichous, soluble pigment 3 A.beijerinckii American soil Yellow brown , no flagella 4 A.Paspali Paspalum root Green fluorescent , peritrichous, soluble pigment 5 A.Insignis water sample A.Agiglis Aquatic Green fluorescent , peritrichous, soluble pigment 6 A.macrocytogenes soil Pink, soluble, polar flagella, abundant slime
  • 22. N fixation: • Naturally fix N in rhizosphere soil • Fix N only after utilizing carbon source and CO2 as byproduct. • Also requires Ca for N fixation • In vitro – req. org N, micronutrients for enhancing N fixing ability • In soil an effective strain fix ~15-20kg/N /ha • Fix 10 mg N/g carbon source under in vitro
  • 23. Beijerinckia • distribution restricted to tropical and sub-tropical regions • Capable of growing on wide range of pH (2.9- 10) • Higher popln in pH 4.9 – 7.4. • Dominant in acidic soils • 3 spp. B. Indica, B. Mobile , B.fluminensis • Occurrence reported in rice, Sugarcane, forage grasses (Digiteria, Panicum purpurescents, Cyanodon dactylon & Setaria ) Plantation crops (Cocount, arecanut, chashew, cocoa ,pepper & pearl millet) • B.indica produces peritrichous flagella, abundant slime, Light rust brown insoluble
  • 24.
  • 25.
  • 26. itrogen fixation bacteria by type of interaction with plants Root nodule symbiosis bacteria Associative nitrogen-fixing bacteria Free-living nitrogen-fixing bacteria Energy source high moderate moderate Oxygen protection high moderate low Transfer of fixed N high moderate low Estimates of nitrogen fixation rates, kg N ha−1 year−1 50–465 2–170 1–80
  • 27. Nitrogen fixation 2. Chemical Industrial process 1.Natural - Lightening • Req high temperature for process • Not eco-friendly • Make plants susceptible • Deteriorate soil health & plant health • Products Eg. Urea, DAP, MOP • Un predictable • Un Quantifiable 3.Biological fixation (Microorganisms) • Conversion of atmospheric Nitrogen (gaseous N2) to ammonia (Usable form) • Inert form (NN)  Reactive Nitrogen (N) • Reduction process ( N2 NH3) • Eco-friendly • Inprove soil health & fertility • Renewable •Sustainable Eg. Biofertilizers
  • 28. Nitrogen fixation Symbiotic nitrogen fixation: special structure-nodule formation Nodules: organ for storage and transfer of fixed nitrogen Stem nodules-Sesbania Root nodules- legumes Endophytic nitrogen fixation Sugarcane- Gluconacetobacter 2 types of nodulation
  • 29. NH3 16Pi 3.Biological Nitrogen fixation + 2OH- + 8 flavodoxin + 16 MgADP- + 16H2PO4 - + H2 N2 + 8 flavodoxin- + 8H+ + 16 MgATP2- + 18 H2O nitrogenase 2NH4 +