Biofertilizers, merits, usefulness, methodology of preparation and application methods etc.

1. Bio-fertilizers
Dr. P. K. Mani
Bidhan Chandra Krishi Viswavidyalaya
E-mail: pabitramani@gmail.com
Website: www.bckv.edu.in

2. Bio-fertilizers or microbial inoculants are the carrier-based
preparations
containing
sufficient
number
of
microorganisms in a viable state inoculated to soil or seed
to augment the nutrient availability to plant by enhancing
the growth and proliferation of microorganisms.
A biofertilzer is an organic product containing a specific
microorganism (microbial inoculant) in concentrated form
(107 to 109 g-1), which is derived either from the nodules of
plant roots or from the soil of root zone (Rhizosphere).
Biofertilizers may be referred to as inoculants after
the name of microorganisms they contain, viz. Rhizobium
inoculant or Azospirillum inoculant

3. Energetics
Industrial fixation N≡N
Haber-Bosch (100-200 atm, 400-500°C,
8,000 kcal kg-1 N)
3CH4 + 6H2O --> 3CO2 + 12H2
4N2+12H2 --> 8NH3
Biological Nitrogen Fixation
Nitrogenase (4,000 kcal kg-1 N)

4. Fritz Haber
(1868-1934)
Carl Bosch
(1874-1940)
Haber and Bosch: Most influential persons of the 20th Century (
Nature, July 29,1999)

5. In 1828, the German chemist Friedrich Wöhler obtained urea artificially by
treating silver cyanate with ammonium chloride.[5][6][7]
AgNCO + NH4Cl → (NH2)2CO + AgCl
This was the first time an organic compound was artificially
synthesized from inorganic starting materials, without the involvement
of living organisms. The results of this experiment implicitly
discredited vitalism: the theory that the chemicals of living organisms
are fundamentally different from inanimate matter.

7. The relationship between activation energy ( ) and enthalpy of formation (ΔH) with and
without a catalyst, plotted against the reaction coordinate. The highest energy position (peak
position) represents the transition state. With the catalyst, the energy required to enter

8. In chemistry, activation energy is a term introduced in 1889 by
the Swedish scientist Svante Arrhenius that is defined as the
minimum energy that must be input to a chemical system,
containing potential reactants, in order for a chemical reaction to
occur. Activation energy may also be defined as the minimum
energy required to start a chemical reaction. The activation energy
of a reaction is usually denoted by Ea and given in units
of kilojoules per mole.
Activation energy can be thought of as the height of the potential
barrier (sometimes called the energy barrier) separating
two minima of potential energy (of the reactants and products of
a reaction). For a chemical reaction to proceed at a reasonable rate,
there should exist an appreciable number of molecules with energy
equal to or greater than the activation energy.

9. Chemical fertilizers
FEATURES
Vs
CHEMICAL FERTILIZER
Biofertilizers
BIOFERTILIZER
Raw material
Non-renewable
Renewable
Energy
Reductant
Fossil Fuel
H2
Solar
Organic
Catalyst
Al, Fe, Mo oxides
Nitrogenase enzy
Temp. & Pr.
750oF, 200-600atm
Ambient T, P
Energy reqt.
680 kJ.mol-1.NH4+
355 kJ.mol-1.NH4+
Efficiency
40-45%
Exists due to indiscriminate use
90%
Pollution free
Cost
High cost input @ Rs. 6/kgN Rs.
14/kg P2O2
Low cost input @Rs.
0.20/kg
Soil Health
Deteriorates
Improves
Pollution effect

10. Classification of Bio fertilizers
Nitrogen fixer
i)
Symbiotic
ii)
Associative
iii)
Free living
(Nonsymbiotic)
Rhizobium
(legume)
Frankia
(non-legume)
BGA: Anabaena
(Azolla)
Azospirillum
Beijerinckia,
Azotobacter ,
Clostridium,
Acetobacter
Phosphate fixers
Phosphate
solubilizers:
Organic
matter
decomposer
Phosphate a)
mobilizers: Cellulolytic:
Bacillus
VAM
Trichoderma
Pseudomonas Glomus,
Aspergillus
Gigaspora
Penicillium
b)
Lignolytic:
Agaricus,
Polyporus
Frankia-filamentous gram positive actinomycetes (vesicles for N fixation
site). Actinorhizal plants are Alnus, Myrica, Casuarina

11. Symbiotic Nitrogen Fixation
Microorganisms
Host plants
Location
Isolated
Plant group
Tissue
Inside or
outside
plant cell
Rhizobium,
Bacteria
Bradyrhizobium
(α-Proteobacteria)
Azorhizobium
Legumes and
Parasponia
Nodule
(induced)
Inside
Yes
Actinomycetes
Frankia
Betulaceae
and
8 family(trees)
Nodule
(induced)
Inside
Yes
Nostoc
Bryophytes
(Antheros
etc.)
Leaf
cavity
Outside
Yes
Nostoc
(Anabaena)
Pteridophyte
(Azolla)
Leaf
cavity
Outside
No
Nostoc
Cycadophyta
(Cycas,Macro
zamia etc.)
Collaroid
root
Outside
Yes
Nostoc
Angiosperm
(Gunnera)
Gland
tissue
Inside
Yes
Large group
Cyanobacteria
(BGA)
Genera

12. Types of Biological Nitrogen Fixation
Cyanobacteria
Azospirillum
Rod shaped rhizobia in the nodule of cowpea (Vigna unguiculata).

13. Frankia and
Actinorhizal Plants
Actinomycetes
(Gram
+, filamentous); septate
hyphae; spores in
sporangia; thick-walled
vesicles
Azolla

14. Ecology of nitrogen-fixing bacteria

15. Significance
of
Biofertilizer:
Biological N-fixation (BNF): 69% of global N-fixation. Legume-Rhizobium
symbiosis is the most significant as it supplies 80-90% of the total N
reqt. of legumes, increases grain yield by 10-15% (Verma and
Bhattacharyya, 1990). Rhizobium bacteria can fix 50-100 kg N/ha/yr.
Azotobacter and Azospirillum inoculation on several non-legumes
crops experienced 5-15% yield increase and N contribution about
25 kg/ha.
Use of Azospirillum as seed inoculant can save 20-30 kg N/ha in crops
like Barley, Sorghum and Millets (Subba Rao et al., 1980)
Use of Blue Green
(submerged condition)
Algae provides 25-50 kg N/ha to rice crop
The use of Phosphobacterin has been found to increase the efficiency of
ground rock phosphates and superphosphates applied in in neutral to
alkaline soils
Vesicular-Arbuscular Mycorrhizae (VAM) has prominent role in P availability.

16. Merits/ advantages/ usefulness of
Biofertilizer
use
Reqd. in smaller quantities. 1g carrier of a BF contain 10 million cells of
a specific strain (500g/ha material may be sufficient)
Tandon (1991) reported estimates of Nutrient equivalent potential as
follows:
Rhizobium: 19-22 kg N/ha
Azotobacter and Azspirillum: 20 kg N/ha
BGA: may fix 20-30 kg N/ha
Azolla : may fix 3-4 kg N/ton of Azolla
BF increases the yield : 10-30 % by supplying N in the soil, adding
organic matter to the soil.
Bio fertilizers provides residual effect on soil fertility
BF like Azospirillum and phosphobacterin produce growth promoting
substances like hormone, vitamin etc favouring root growth.
The fixed P become available by the application of phosphobacterin
and the demand of P to the plants meets accordingly.

17. Class-8

18. legume
Fixed nitrogen
(ammonia)
Fixed carbon
(malate, sucrose)
rhizobia

19. Rhizobium:
A bacteria having the capacity to form morphologically well defined
nodules on the roots of leguminous plants
Gram negative
Short rod 1.2-3.0 μm (L) x 0.5-0.9 μm (B)
Have flagella (single polar or peritrichous
Do not form endospore
Aerobic, mesophilic , Chemoorganotroph
2 types of growth habit : Fast and slow (Bradyrhizobium)
2 distinct type of colony colour: Pink and creamy white
Rhizobium has been placed in Bergey’s Manual of Systematic
Bacteriology (1984) , belongs to the family Rhizobiaceae
Rhizobium, Bradyrhizobium and Azorhizobium
Azorhizobium ( stem nodule of Sesbania rostrata) A. caulonodans

20. Buchanon (1926)
Shape: spherical, elongated, palmate
Nodule Size: max. 6 cm
Rhizobium
Colour of nodule: white, green , black, pink
Pink colour is effective strain: leghaemoglobin. 15-17kd, 1 peptide bond
epiderm
cortex
Meristem
Central zone having
bacteriods
Vascular bundle
Cross section of nodule:

21. Nodulation process:
(i) Multiplication in rhizosphere
1. Pre-infection:
(ii) Attachment of root surface
(iii) Branching of root hair
(iv) Root hair curling
2. Infection and
nodule formation
3. Nodule function
(v) Formation
of infection thread
VI. Nodule development
VII.Releasing Rhizobia from Infection
thread
VIII. Bacteriod formation
IX. Reduction of N2 to NH3
X. Complementary functions
XI. Nodule persistence

22. Model developed by
Dazzo and Hubbell, 1975
Root
Lectin
R. trifolii
hair
Saccharide receptor
Lectin = glycoprotien
Root exudates like flavonoids and iso-flavonoids
stimulate ‘Nod-d’ whose product binds with ‘nod-box’
which activate other nodulating genes

24. For nodulation to take place there has to be a molecular
dialogue between the plant and the bacterial partner.
•Root hair
deformation
•Membrane
depolarization
•Induction of early
nodulin expression
Flavonoids
•Formation of
nodule primordia
nod genes
•etc
Nod factors

25. The Colonization Process
Signaling
• Rhizobia sense flavonoid compounds release by roots
• specific species sense particular flavonoids specific to a
plant
• Rhizobia move by use of flagella propelling cell through
soil water
• Rhizobia produce lipo-oligosaccharides or nod factors
• these initiate root hair deformation and curling and the
division of cortical cells in the root at very low
concentrations (< 10-9 M soil solution).

26. Flavonoids secreted by the root of their host plant help Rhizobia in
the infection stage of their symbiotic relationship with legumes like
peas, beans, clover, and soy.
Rhizobia living in soil are able to sense the flavonoids and this
triggers the secretion of Nod factors, which in turn are recognized by
the host plant and can lead to root hair deformation and several
cellular responses such as ion fluxes and the formation of a root
nodule.

27. Sequence of molecular communication
a) Plant excrete certain flavonoid compounds (differ between plants)
b) Rhizobia recognize certain flavonoids (gene nod D product is a
sensor)
c) If Nodulin protein (product of nodD genes) recognizes right
flavonoids, switch of other nod genes on, and products of nod
genes coded proteins are formed--Nod factors (oligochitin
compounds)
d) Plant, in return, recognize right Nod factors.
Early processes of nodulation is triggered by Nod factors.
e) In addition to Nod factors, extracellular polysaccharides of
bacteria may function in recognition of bacteria at later
process
of nodulation (bacteria spreading inside plant cells)
nod genes: nodulation,
nif genes: common with free-living nitrogen fixation,
fix genes; Unique to symbiotic nitrogen fixation

28. Nodule development process
1. Bacteria encounter root;
they are chemotactically attracted toward
specific plant chemicals (flavonoids) exuding from
root tissue, especially in response to nitrogen
limitation
naringenin
(a flavanone)
daidzein
(an isoflavone)

29. 2. Bacteria attracted to the root attach
themselves to the root hair surface and secrete
specific oligosaccharide signal molecules
(nod factors).
Nod factors structurally are
lipochitooligosaccharides (LCOs) that
consist of an acylated chitin oligomeric
backbone with various functional group
nodsubstitutions at the terminal or non-terminal
factor
residues.
N-acetylglucosamine

30. Nod gene expression is induced by the presence of certain
flavonoids in the soil, which are secreted by the plant to
attract the bacteria.[1]
 These chemicals induce the formation of NodD, which in turn
activates other genes involved in the expression of nod factors and
their secretion into the soil. Nod factors induce root-hair curling
such that it envelops the bacterium.
This is followed by the localized breakdown of the cell wall and the
invagination of the plant cell membrane, allowing the bacterium to
form an infection thread and enter the root hair.
The end result is the nodule, the structure in which nitrogen is
fixed. Nod factors act by inducing changes in gene expression in
the legume, most notable the nodulin genes, which are needed for
nodule organogenesis.[

31. Rhizobium
Attachment and infection
Nod factor
(specificity)
Flavonoids
(specificity)
Invasion through infection tube
Bacteroid
differentiation
Formation of
nodule primordia
Nitrogen
fixation
From Hirsch, 1992.
New Phyto. 122, 211-237

32. 3. In response to oligosaccharide signals, the root
hair becomes deformed and curls at the tip;
bacteria become enclosed in small pocket.
Cortical cell division is induced within the root.

33. 4. Bacteria then invade the root hair cell and move
along an internal, plant-derived “infection thread”,
multiplying, and secreting polysaccharides that fill
the channel.

34. Rhizobium cells
expressing GFP
(green fluorescent
protein) invade a
host root hair
infection thread

35. 5. Infection thread penetrates through several
layers of cortical cells and then ramifies within the
cortex. Cells in advance of the thread divide and
organize themselves into a nodule primordium.
6. The branched infection thread enters the nodule
primordium zone and penetrates individual
primordium cells.
7. Bacteria are released from the infection thread
into the cytoplasm of the host cells, but remain
surrounded by the peribacteroid membrane (PBM).
Failure to form the PBM results in the
activation of host defenses and/or the formation of
ineffective nodules.

36. transporters
bacteroid
peribacteroid
membrane

37. The Colonization Process
• Infection Thread
– Protein called recadhesin and polysaccharides
from Rhizobia and lectins from plants interact to
adhere the bacterium to the root hair
– curling of the root hair and hydrolysis of root
epidermis
– Rhizobia move down centre of the root hair toward
the root cortex
– plant produces tube called an infection thread
– in the cortex Rhizobia enter enclosed area within a
plant-derived peribacteroid membrane.
– membrane protect the rhizobia from plant defense
responses.

38. 8. Infected root cells swell and cease dividing.
Bacteria within the swollen cells change form to
become endosymbiotic bacteroids, which begin to
fix nitrogen.
The nodule provides an oxygen-controlled environment
(leghemoglobin = pink nodule interior) structured to
facilitate transport of reduced nitrogen metabolites
from the bacteroids to the plant vascular system, and of
photosynthate from the host plant to the bacteroids.

39. Shepherd
crook
Infection thread
Nodulation
Proliferation
of IT
Bacteriod
formation

40. Rhizobium Root Nodules

42. Bio-chemical considerations

43. Enzymology of N fixation
•
•
•
•
Only occurs in certain prokaryotes
Rhizobia fix nitrogen in symbiotic association
with leguminous plants
Rhizobia fix N for the plant and plant provides
Rhizobia with carbon substrates
All nitrogen fixing systems appear to be identical
They require nitrogenase, a reductant (reduced
ferredoxin), ATP, O-free conditions and
regulatory controls (ADP inhibits and NH4+
inhibits expression of nif genes

44. Nitrogenase Complex
•
•
•
•
•
Two protein components:
nitrogenase reductase and nitrogenase
Nitrogenase reductase is a 60 kD homodimer
with a single 4Fe-4S cluster
Very oxygen-sensitive
Binds MgATP
4ATP required per pair of electrons transferred
Reduction of N2 to 2NH3 + H2 requires 4 pairs of
electrons, so 16 ATP are consumed per N2

45. Nitrogenase
A 220 kD heterotetramer
• Each molecule of enzyme contains 2 Mo, 32
Fe, 30 equivalents of acid-labile sulfide (FeS
clusters, etc)
• Four 4Fe-4S clusters plus two FeMoCo, an
iron-molybdenum cofactor
• Nitrogenase is slow - 12 e- pairs per second,
i.e., only three molecules of N2 per second

46. Why should nitrogenase need ATP ??
• N2 reduction to ammonia is
thermodynamically favorable
• However, the activation barrier for
breaking the N-N triple bond is enormous
• 16 ATP provide the
needed activation energy

47. Stereochemistry of Nitrogenase reaction
+ 8e- +8H+ + 16 Mg ATP
N≡N
2NH3+ H2 + 16 Mg ADP+ 16Pi
Nitrogenase enzyme
e
-
e-
α
β
α
Amino acid
β
α
α
ATP
N2
NH4+ produced reacts
with glutamate to
Nitrogenase
form glutamine by
(Azofermo) 260kd combined activities
of GS and GOGAT
ADP + Pi
Nitrogenase reductase
(Azofer) 60kd
H2
NH4+
2H+ +2e-
Hydrogenase
Glutamate + NH4+ +ATP
ETC
GS
Mg2+
Glutamine + ADP + Pi (H3PO4)
GOGAT
Glutamine + 2-Oxoglutarate
NADPH + H+
Amino acids are
2 Glutamate transpoted by
Xylem
NADP+

48. O2
Carbon
Photo
synth
ate
1
2
l obi n
haemog
leg
O2
TCA cycle
e-
H2O
Ubiquinone → cyt b → cyt c → cyta/a3
ADP+ Pi
Cyt 559-H2
e-
ATP
Ferredoxin
3
N≡N
Hydrogen
uptake
H+
H2
Nitrogenase
complex
Bacteroid
Nodule cytosol
2NH3
Amino
acid
Nodule amino
acid pool
4

50. (i) Glutamate +
NH4+ + ATP
GS
Mg
2+
Glutamine + 2-Oxoglutarate
Glutamine + ADP + Pi (H3PO4)
GOGAT
NADPH + H+
2 Glutamate
NADP+
Low km of GS for NH4+ (0.02 mM), high affinity to bind NH4+
GS= Glutamine synthatse,
GOGAT= glutamine Oxo-glutarate amino-transferase
GDH
(ii) 2-Oxoglutarate + NH4+ NADPH + H+
Glutamate + NADP+
+ H2 O
GDH= glutamate dehydrogenase,
km of GDH for NH4+ is very high, hence it has low affinity for NH4+

51. Km is (roughly) an inverse
measure of the affinity or strength
of binding between the enzyme
and its substrate. The lower the
Km, the greater the affinity (so the
lower the concentration of
substrate needed to achieve a
given rate).

52. Bacteroid
Contains less amount of ribosomes, mesosomes than free living
rhizobial cell
Presence of large quantities of PHB (Poly β-hydroxy butyrate) in
conspicous amount, nearly 50% of bacteroid consists of PHB
Absence of cytochrome-a in N-fixing bacteroid
(about 20-30% dry wt of nodule is due to bacteroid)
Soybean nodule:
A nodule has 3.5 X 104 plant cells
1 plant cell has about 105 bacteroids
So, Number of Bacteroid/ nodule =3.5 x 104 x105 = 3.5 x 109
Again, 1 g root has about 106 cells, So, 1 kg root has about 109 cells
1 kg root-mass may contain bacterial population equivalent to
1 medium sized nodule of Soybean

53. Class-9

54. Examples of using the cross-inoculation groups for selecting the
proper rhizobial inoculant for the legume host. The proper combination
of rhizobia and legume will
result in the best nodulation
and most nitrogen fixation.
We see that using soybean
rhizobia with soybean forms
an effective symbiosis, while
soybean rhizobia on leucaena
does not. Using information
from Table shows that cowpea
rhizobia nodulates both
mungbean and peanut.

55. Cross-inoculation group:
Rhizobium sp.
Host Legumes
1. Rhizobium leguminosarum biovar. trifolii
Rhizobium leguminosarum biovar. phaseoli
Rhizobium leguminosarum biovar. viceae
Trifolium
Phaseolus
Vicia
2. Rhizobium meliloti
Medicago
3. Rhizobium loti
Lotus
4. Rhizobium friedii
Glycine
5. Rhizobium galegae
Galega orientalis
6. Rhizobium huakii
Astragalus sinicus
7. Rhizobium tropici
Phaseolus vulgaris
Bradyrhizobium japonicum
Nodulating soybean
Bradyrhizobium sp.
Vigna sinensis
A cross inoculation group refers to a collection of
leguminous species that are capable of developing
nodules when exposed to bacteria obtained from the
nodules of any member of that particular plant group

56. Azotobacte
r
Azotobacter is a free living, aerobic, chemoheterotrophic N fixing bacteria
Important spp: Azotobacter beijerinckii. , A. chroococcum, A.vinelandii
Azotobacter paspalum is closely associated with Paspalum notatum cv batatis.
Producing 107 cfu / g of root (colony forming unit)), by ARA, it has been found
that it can fix 15-93 kg N/ha/Yr
Azotabacter has protective mechanism to safeguard the nitrogenase enzyme
from oxygen.
 (i) Respiratory protection in Azotobacter (specialised respiratory system
 (ii) conformational protection in Azotobacter
(a phenomena whereby nitrogenase activity becomes reversibly “switched on”
or “off” in response to decreased or increased pO2 is known as conformational
protection)
Azotobacter inoculants commercially known as Azotobacterin
Slurry of the carrier-based inoculant is made with minimum amount of water and
seeds are mixed with the slurry, dried in shade and sown. Seedling dip
(10-13
min) in slurry is done for transplanted crops and planted immediately.

58. Azotobacter have a full range of enzymes needed to perform the nitrogen
fixation: ferredoxin, hydrogenase and an important enzyme nitrogenase.
The process of nitrogen fixation requires an influx of energy in the form of
adenosine triphosphate (ATP).
Nitrogen fixation is highly sensitive to the presence of oxygen, and therefore
Azotobacter developed a special defensive mechanism against oxygen,
namely a significant intensification of metabolism that reduces the
concentration of oxygen in the cells.[40]
There is also a special nitrogenase-protective protein called Shethna, which
protects nitrogenase and is involved in protecting the cells from oxygen.
Mutants not producing this protein, are killed by oxygen during nitrogen
fixation in the absence of a nitrogen source in the medium.[41]
Homocitrate ions play a certain role in the processes of nitrogen fixation by
Azotobacter

59. Azospirillu

m Azospirillum could be isolated from the root of tropical grass
Digitaria decumbens (Dobereiner and Day, 1976)
Ubiquitious in nature, capable of forming colony in roots and stems
Gram negative, motile, vibroid in shape, contain Poly β-hydroxy
butyrate granules, mesophilic but can tolerate 30-400C
 Azospirillum lipeoferum (C4- Maize, sorghum, tropical forages D d
are host plant) can fix 30 kg N / ha / yr
 Azospirillum brasilense (C3- Rice/ wheat )
Azospirillum culture known to increase root biomass of rice and
wheat (Dewan and Subba Rao, 1979)
Produce growth hormones in pure culture (Tien et al, 1982).
Seed inoculation with VAM together with Azospirillum brasilense
increase the yield and P content of barley and pearl millet
(Subba Rao et al., 1985)
Carrier: FYM,
FYM + soil,
FYM + Charcoal

60. Blue Green Algae:
Ubiquitious in distribution
Singled cell or consists of branched/ unbranched filaments
 possesses specialised type of cell- Heterocyst (where N fixation occurs)
 Important species are Anabaena,
Aulosira , Nostoc
In submerged rice fields bnf is essentially an algal process,
contributing 30 kg N/ha
Symbiotic association between floating aquatic fern, Azolla and its partner
Anabaena azollae (BGA) forms Azolla-Anabaena complex
Anabaena is contained endophytically in ellipsoidal cavities of aerial
dorsal lobes of the fern.
Mature heterocysts have an almost normal content of Chlorophyll a, but are
devoid of phycobiliprotein, the principal antenna pigment of Photosystem II
Due to lack of PS-II and ribulose-bis phosphate Carboxylase; they can neither
fix CO2 nor produce O2 in light
The lack of photosynthetic O2 generation coupled with H dependent
respiration, pO2 becomes very low

62. Electron
donor
Oxygenic
photosynthesis
Amino
acids
Keto
acids
Oxidised
α
pdt. gln
KG
gln
N2
Fd red
Oxidised
FdOxid
pdt.
Electron
donor
PS1 ATP
ADP
glu
glu
glu
NH3
Heterocyst
Vegetative cell
Microplasmadesmata channel
Biochemical pathway of N fixation in BGA
gln = glutamine; glu = glutamate

64. A profile of different biofertilizers
Biofertilizer
Function/Contribution
Limitation
Target Crops
Rhizobium
Fixation of 50-100 kg N/ha
10-35% increase in yield,
leaves residual N
Fixation only with
legumes, Visible effect
not reflected in
traditional area,
Needs optimum P ,
Mo
Pulse Legumes
Oilseed Legumes
Forage legumes
Tree legumes
Azotobacter
Fixation of 20-25 kg N/ha
10-15% increase in yield,
Production of growth
Promoting substances
Demands high organic
matter
Wheat, maize, cotton,
mustard,
vegetable crops
Azospirillum
Same
Poor performance in
winter crops
Sorghum. Pearl millet
minor millets, maize,
rice sugarcane
Blue Green
Algae
and
Azolla
Fixation of 20-30 kg N/ha
(BGA):30-100kg N/ha F (Azolla)
Effective only in
submerged rice
Demands bright
sunlight Survival
difficult at high temp.
Flooded rice
Phosphate
5-50% increase in yield
-
All crops
10-15% increase in yield

65. Bio-super
It is a granular material containing raw rock phosphate and
finely ground elemental sulfur. The product is inoculated with
sulfur-oxidizing bacteria Thiobacillus thiooxidans to ensure
the conversion of the sulfur to sulfuric acid. The acid in turn
reacts with the phosphate rock and making the contained
phosphorus more available to plants.
IARI-Microphos Culture
Gaur and Gaind (1984) developed improved techniques for
isolation of rock phosphate solubilizing microorganisms and by
systematic investigation new efficient bacteria such as
Pseudomonas striata, Bacillus polymixa and fungi like
Aspergillus awamori have been selected for preparation of
carrier based inoculation known as IARI-microphos culture
(Tilak, 1991).

66. Phosphate Solubilising Micro-organisms :
Bacillus, Pseuduomonas, Brevibacterium,
Corynebacterium, Flavobacterium, Micrococcus,
Sarcina, Achromobacter, Streptomyces,
Schwanniomyces, Aspergillus, Penicillum etc.
Phosphobacterin: Bacillus megatherium var phosphaticum
Release about 10-25 kg P2O5/ha/season
 Around 1350 t/a currently used in India

67. Biofertilizers
General Dosages :
 For Paddy - 3 kg/ha
 For pulses, oilseeds, vegetables etc. - 2 kg/ha
 For fruit and plantation crops - 8-10 kg/ha

68. Biofertilizers(BF)
Applications Methods :
 Seed Treatment (200 g(BF) in 400 ml water, make
slurry, mix with seed, dry in shade and sown to the
field)
 Seedling Root Dipping (200 g(BF) in 1-1.5 L water,
dipping root seedling for 30 min to 1 hr.)
 Veg. Propagule treatment (200 g(BF) in 4-5 L water ,
spray it) (Potato, sugarcane,ginger-100 no. propagule)
 Soil Treatment (2 kg Bf are mixed in 100 kg Compost,
keep it overnight mixture is incorporated in the soil at
the time of sowing or planting)

69.  Wheat, Maize, Cotton, Mustard etc. Azotobacter +
PSM at 200 g each per 10 kg of seed as seed treatment
For transplanted rice, the recommendation is to dip the roots
of seedlings for 8 to 10 hours(whole night) in a soln of
Azospirillum + PSM at 1kg each in 40L water
 Jute, Azospirillum+PSM 200g each as seed treatment
 Vegetables like Tomato, Brinjal, Chilli, Cabbage, Cauliflower
etc., Mustard, Sunflower, Cotton use Azotobacter/
Azospirillum + Phosphobacterin 1 kg each as seedling root dip.

70. For Potato, Ginger, Colocasia, Turmeric, Paddy -use
Azospiillum/ Azotobacter +PSM @ 4 kg each/acre
mixed with compost and applied as soil treatment.
Sugarcane use Acetobacter + Phosphobacterin
4 kg each/ acre as seed sett dipping.
Plantation crops use Azotobacter+phosphobacterin
4 kg each/ acre with compost & applied in soil in
two splits per year.

71. Chickpea seeds before (left) and
after (right)
treatment with biofertilizer

72. Mother culture
Carrier Powder
Flask culture
Neutralization (lime)
Bottle culture
Sterilisation (γ-irradiation)
Fermentor broth
Prepared carrier
Mixing @3:7 , moisture 40-50% (in trays)
Curing (2-7 sprays at 28-30°C)
Mass production
of biofertilizer
Packing
Product for despatch (storing at 15-30°C)

73. What precautions one should take before using
biofertilizers?
•Biofertilizer packets need to be stored in cool and dry place
away from direct sunlight and heat.
•Right combinations of biofertilizers have to be used.
•Other chemicals(Fertilizers and pesticides) should not be
mixed with the biofertilizers.
• Seed treatent chemicals like Bavistine etc. should mix 3 days
prior to mix with biofertilizer treatment.
•Sow the treated seeds(with Bio fertilizer) immediately
preferably in the morning or afternoon avoiding
scorching sunlight
•The packet has to be used before its expiry, only for the
specified crop and by the recommended method of application.

74. Biofertilizer – BCKV
PSB
Trichoderma

75. Phosphate Solubilizing Microorganisms
(PSM)
Several soil bacteria & fungi
secrete organic acids & lower
the pH in their vicinity to
bring about dissolution of
bound phosphate in soil.
e.g.- Bacillus polymyxa
Pseudomonas striata
Aspergillus awamori

76. PGPR (Plant Growth promoting Rhizobacteria)
Genera : Bacillus, Pseudomonas, Actinoplanes,
Alcaligenes, Arthrobacter, Enterobacter,
Azotobacter, Azosprillum, Clostridium etc.
Activities :
 Enhanced nutrient uptake
 Hormone production
 Vitamin production
 Enzyme production
 Biocontrol

77. Why biofertilizers are not so popular?
Inspite of several long lasting benefits the bio-inoculants are not
very popular among the farming community. There may be
several reasons for this, however some of the important ones are
as given below:
•
•
•
•
•
•
•
•
•
•
Nutrient contribution is dependent on survival of organisms.
Soil with high nutrient status do not show instant visible benefits.
Low carbon content of soils- low proliferation.
Water scarcity -possibility of desiccation.
Fluctuating soil pH- variable microflora.
Extreme temperature -in summer months.
Shelf life of organisms.
Poor storage and transportation
Lack of awareness.
Eagerness to look for instance effects.

78. The Dream…..
If
a way could be found to mimic nitrogenase catalysis
(a reaction conducted at 0.78 atmospheres N2 pressure
and ambient temperatures), huge amounts of energy
(and money) could be saved in industrial ammonia production.
If a way could be found to
transfer the capacity to form N-fixing symbioses
from a typical legume host
to an important non-host crop species such as corn or wheat,
far less fertilizer
would be needed to be produced and applied
in order to sustain crop yields

79. Azolla - the untold story

80. • Signals early in infection
– Complex handshaking between legume
root and rhizobium
Correct
signal
Incorrect
signal

81. Genetics of Nitrogenase
Gene
Properties and function
nifH
nifDK
nifA
nifB
nifEN
nifS
fixABCX
fixK
fixLJ
fixNOQP
fixGHIS
Dinitrogenase reductase
Dinitrogenase
Regulatory, activator of most nif and fix genes
FeMo cofactor biosynthesis
FeMo cofactor biosynthesis
Unknown
Electron transfer
Regulatory
Regulatory, two-component sensor/effector
Electron transfer
Transmembrane complex

82. Fe Protein
Fe-Mo Protein
a
b
a
b
Regulation

83. Fe Protein
Fe-Mo Protein
a
b
Regulation
Activation
of NifA
a
b
NtrC-RNA
polymerase

84. Electron Transport
Fe Protein
Fe-Mo Protein
a
ATP
Ferredoxin
b
a
b
Regulation

85. Electron Transport
Reduced
Fe Protein
Fe-Mo Protein
a
ATP
Ferredoxin
b
a
b
Regulation

86. Electron Transport
Reduced
Fe Protein
Reduction of
Fe-Mo Protein
a
ATP
Ferredoxin
b
a
b
Regulation

87. Electrons Donated to
N2
Electron Transport
Reduced
Fe Protein
Reduction of
Fe-Mo Protein
a
ATP
Ferredoxin
b
a
b
Regulation

88. Electrons Donated to
N2
Formation of NH3
Electron Transport
Reduced
Fe Protein
Reduction of
Fe-Mo Protein
a
ATP
Ferredoxin
b
a
b
Regulation

89. Genetics of Nitrogenase
Summary
NITROGENASE
acetyl-CoA + CO
ATP
2
N≡N
NifH
NifJ
NifF
NifH
pyruvate + CoA
ADP
r educing equivalents
NifD NifK
NifK NifD
2NH 3
+H2

91. Bio-technological considerations

93. Nitrogenase enzyme complex
Nitrogenase
Electron transport
MoFe protein
Fe protein
Assembling
β α
γγ α β
Fe-Mo-Cofactor
Regulator
J
H D K T Y E NX U SVWZM F L A
BQ
Physical association of nif genes in Klebsiella pneumoniae
Redrawn from www.asahi-net.or.jp/~it6i-wtnb/BNF.html

94. Rhizobia and the cross-inoculation groups of legumes they nodulate.

95. Soybean nodules
Stem nodules of
Aeschynomene afraspera

98. Short Test on 30th July, 2012
at 1.30 p.m.
Total Marks: 20
Time: 30
No. of questions: 40

99. VAM (Endomycorrhiza):
The hyphae often form swellings (vesicle) and minute branches
(arbuscles) within the cell of the host
Genera :Glomus, Gigaspora, Acaulospora, Sclerocystis
Activities : (Hacskaylo, 1972)
 P - Uptake (15-30 kg/ha/season)
 Availability of K, Mg, S, Fe, Mn, Zn, Cu, etc.
 Tolerance to adverse env. Stresses (draught
resistance)
 Tolerance to disease and Nematodes
 Produce plantVAM is mixed with Rhizobium to inoculate legume
Dual inoculation: when growth hormones
plant, it is known as Dual inoculation------better (i) nodulation, (ii) N-fixation
and
(iii) P-uptake

100. Vesicular Arbuscular Mycorrhiza
Inside root
• Intercellular mycelium
• Intracellular arbuscule
• tree-like haustorium
• Vesicle with reserves
Outside root
• Spores (multinucleate)
• Hyphae
•thick runners
•filamentous hyphae
Form extensive network of hyphae
even connecting different plants

101. GS
+
(i) Glutamate + NH4 + ATP
Glutamine + ADP + Pi (H3PO4)
Mg2+
GOGAT
Glutamine + 2-Oxoglutarate
NADPH + H+
2 Glutamate
NADP+
Low km of GS for NH4+ (0.02 mM), high affinity to bind NH4+
GS= Glutamine synthatse,
GOGAT= glutamine Oxo-glutarate amino-transferase
(ii) 2-Oxoglutarate + NH4
+
NADPH + H
GDH
+
Glutamate + NADP+
+ H 2O
GDH= glutamate dehydrogenase,
km of GDH for NH4+ is very high, hence it has low affinity for NH4+

102. GS
+
(i) Glutamate + NH4 + ATP
Glutamine + ADP + Pi (H3PO4)
Mg2+
GOGAT
Glutamine + 2-Oxoglutarate
NADPH + H+
2 Glutamate
NADP+
Low km of GS for NH4+ (0.02 mM), high affinity to bind NH4+
GS= Glutamine synthatse,
GOGAT= glutamine Oxo-glutarate amino-transferase
(ii) 2-Oxoglutarate + NH4
+
NADPH + H
GDH
+
Glutamate + NADP+
+ H 2O
GDH= glutamate dehydrogenase,
km of GDH for NH4+ is very high, hence it has low affinity for NH4+

103. Sinorhizobium meliloti
Bacteroids

105. Glutamine and glutamate are usually at much higher
concentrations than other amino acids in cells.
This is due to their role as nitrogen carriers.

106. Glutamine can combine with a-ketoglutarate to yield a pair of
glutamates. This is a reduction, catalyzed by glutamate synthase.
Glutamate synthase (bacteria and plants)