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Dr. Subhendu Datta

Sr. Scientist
CIFE, Kolkata Centre

Background: Why bio-fertilizers?

 With the introduction of green revolution technologies
the modern agriculture is getting more and more
dependent upon the steady supply of synthetic inputs
(mainly fertilizers) which are products of fossil fuel
(coal+ petroleum).

 Excessive dependence of modern agriculture and the

supply of these synthetic inputs and the adverse effects
being noticed due to their excessive and imbalanced
use has compelled the scientific fraternity to look for

(i). Availability and cost of commercial fertilizers

 Demand is much higher then the availability.

 It is estimated that by 2020, to achieve the targeted

production of 321 million tones of food grain, the
requirement of nutrient will be 28.8 million tones, while
their availability will be only 21.6 million tones being a
deficit of about 7.2 million tones.

 Increasing costs are getting unaffordable by small and

marginal farmers.

(ii) Effect of Chemical fertilizers in soil and


 Excessive and imbalanced use of chemical fertilizers has

adversely affected the soil causing decrease in organic
carbon, reduction in microbial flora and fauna of soil,
increasing acidity and alkalinity and hardening of soil.

 Moreover, excessive use of nitrogenous and phosphatic

fertilizers are contaminating water bodies (eutrophication)
thus affecting fish fauna and causing health hazards for
human beings and animals.

 Production of chemical fertilizers adds to the pollution.

 To overcome the deficit in nutrient supply and to
overcome the adverse effects of chemical cultivation, it
is suggested that efforts should be made to exploit all
the available resources of nutrients under the theme of
integrated nutrient management.

 Under this approach the best available option lies in the

complimentary use of Biofertilizers,
Biofertilizers, organic manures in
suitable combination of chemical fertilizers.

What are bio-fertilizers

 “Biofertilizer is a substance which contains living
microorganisms which, 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
[Vessey, J.K. (2003). Plant growth promoting rhizobacteria as
biofertilizers. Plant Soil 255: 571-586].

 This definition separates biofertilizer from organic


 The latter contains organic compounds which directly, or

by their decay, increase soil fertility.

 Likewise the term biofertilizer should not be used
interchangeably with the terms, green manure

 Not all plant growth promoting rhizobacteria

(PGPR) can be considered biofertilizers.

 Bacteria that promote plant growth by control of

deleterious organisms are biopesticides, but not

 Similarly bacteria can enhance plant growth by

producing phytohormones and are regarded as
bioenhancers, not biofertilizer.

 The importance of cyanobacterial biofertilizers

was recognized as early as 1939. Since then good
deal of literature on these aspects has appeared.

 Although biofertilizers are now being used in

agriculture fields, it can equivocally be stated that
biofertilizers may also be used in fish culture

 Field studies shown that about 30 kg N/ha can be

saved by the use of cyanobacterial biofertilizers.

 Indian Definition - Biofertilizers are ready to use live
formulates of such beneficial microorganisms which on
application to seed, root or soil mobilize the availability
of nutrients by their biological activity in particular, and
help build up the micro-
micro-flora and in turn the soil health
in general.

Leguminous oilseed Crops: Soybeans

and peanut
Leguminous pulses: Arhar, Letils,
Mung, Urad, Pea, Cowpea, Gram,

Bio-fertilizer pack

Benefits of biofertilizers
 Increase crop yield by 20-
 Replace chemical N & P by 25 %
 Stimulate plant growth
 Activate soil biologically
 Restore natural fertility
 Increases soil organic matter and maintains a good soil texture.
 Environmentally friendly - Don’t pollute the environment
 Cheaper than synthetic fertilizers
 Believed to have growth promoting substances.
 Provide protection against drought and some soil borne diseases

Types of
Most biofertilizers belong to one of
these two categories:
(a). Nitrogen fixing
(a). Phosphate solubilising.

For Nitrogen:
 Rhizobium for legume crops
 Azotobacter/Azospirillum for non legume crops
 Acetobacter for sugarcane only
 BGA and Azolla for low land paddy & aquaulture

For Phosphorous
 Phosphatika for all crops to be applied with Rhizobium,
Rhizobium, Azotobacter,
Azospirillum and Acetobacter

For enriched compost

 Cellulolytic fungal culture
 Phosphotika and Azotobacter culture

 Seed treatment :
Suspend 200 gm N biofertilizer and 200 gms Phosphotika in 300-
300-400 ml of
water and mix thoroughly. Mix this paste with 10 kg seeds & dry in shade.
Sow immediately.

 Seedling root dip:

For vegetables 1 kg each of two biofertilisers be mixed in sufficient
quantity of water. Dip the roots of seedlings in this suspension for 30-
min before transplanting.
For paddy make a bed in the field and fill it with water. Mix biofertilisers in
water and dip the roots of seedlings for 8-
8-10 hrs.

 Soil treatment:
Mix 4 kg each of biofertilisers in 200 kg of compost and leave it overnight.
Apply this mixture in the soil at the time of sowing or planting.
In plantation crops apply this mixture near root zone and cover with soil.

Nitrogen fixing biofertilizers
 Free-living or symbiotic bacteria and blue-green algae
(Cyanobacteria) fix atmospheric gaseous nitrogen as
ammonia and release it, increasing the fertility of soil and

 These include Rhizobium, Azotobacter, Acetobacter,

Azospirillum, Blue Green Algae (BGA) and Azolla.

 While Rhizobium, A. azollae requires symbiotic association

to fix nitrogen, others can fix nitrogen independently.

 Anabaena azollae living in leaf cavities of Azolla (aquatic

fern) are very efficient nitrogen fixers, and contribute about
500 kg N/ha/year.

Phosphate solubilising biofertilizers

 Phosphate solubilising micro-organisms (PSM)

secrete organic acids which enhance the uptake of
phosphorus by plants by dissolving rock phosphate
and tricalcium phosphates.

 PSMs (e.g. Phosphatika)

Phosphatika) are particularly valuable as
they are not crop specific and can benefit all crops.

Composting bio-fertilizers
o Compositing biofertilizers are used for
hastening the process of composting and for
enriching its nutrient value.

o Composting process is enhanced by

enzymes secreted by microorganisms
(Cellulolytic fungal culture) to hydrolyse
pectins, xylans, hemicellulose, cellulose
releasing beneficial micronutrients for the

Nitrogen fixing biofertilizers

Rhizobia (Soil bacteria of the genus Rhizobium)

produces root nodules in legumes.

Legume/Rhizobium Nodules are Red. This is due to

the production of Leghaemoglobin which sequesters
oxygen. This helps to create a low oxygen

The enzyme which fixes nitrogen (Nitrogenase) needs

an anaerobic environment.

 Another microsymbiont with nitrogen
fixing capacity the principal genus of
microbes is Frankia.

 Morphology of Frankia is similar to that

of actinomycetes and produces nodules in
woody non-legumes, like Alnus, Casuarina,
Myrica etc. Many of these are "pioneer"
species which colonize barren sites.

 These nodules fix more atmospheric Root nodules of Alnus

nitrogen than legume/Rhizobium

 Some of these are alien species like Myrica

Myrica,, grows much faster
than native competitors, and they alter the soil nitrogen levels
markedly compared to native stands.

Azolla-Anabaena azollae relationship

o An Azolla plant floating on the surface of the
water is roughly triangular or circular in shape
and rarely exceeds 3–4cm (except in the species
Azolla nilotica).

o The cyanobacterium Anabaena azollae occurs as

filaments located on the stem apexes of Azolla
(aquatic fern) and inside the leaf cavities in
Symbiotic association,
association which are inoculated
during their formation with some Anabaena from
the apex.

Reference: C. Van Hove and A. Lejeune

(2002). Biology And Environment: Proceedings of
the Royal Irish Academy, Vol. 102B (1): 23–26.

The Azolla–A.
azollae association
can develop on a
medium devoid of
nitrogen compounds
because of the ability
of A. azollae to
reduce N2 to NH3.

Some of the
ammonia is supplied
to the fern, and
the fern supplies the
cyanobacterium with

Morphology of Azolla stem

(longitudinal section).
1. Stem;
2. Stem apex;
3. Apical Anabaena colony
without heterocysts;
4. Other bacteria;
5. Leaf primordium;
6. Young leaf;
7. Branched hair;
8. Single hair;
9. Upper leaf lobe;
10. Lower leaf lobe;
11. Leaf cavity (showing a
central gaseous region and a
peripheral mucilaginous
12. Involucre;
13. Indusia;
14. Microsporocarp;
15. Microsporangia;
16. Megasporocarp;
17. Megasporangium;
18. Akinetes of Anabaena;
19. Vegetative cell of Anabaena;
20. Heterocyst.

Azolla A. azollae symbiosis
Importance of Azolla–
 The Azolla–A. azollae symbiosis is has long been used by farmers, mainly in
Asia, as feed for their animals and as green manure.

 Azolla is one of the most nutritive aquatic plants, owing to its high crude
protein and carotenoid contents (anthocyanine ) and generally good amino-
acid profile.

 It can be incorporated into the feed of fish, pigs, poultry, rabbits and even

 A number of laboratory and field studies have shown beyond any doubt the
beneficial effect of Azolla as an organic nitrogen fertiliser, mainly in terms of
increasing rice grain yield.

 The presence of an Azolla mat on the surface of the water body has been
shown to significantly reduce weed development, limit evapotranspiration,
reduce volatilisation of applied N fertilisers and purify water.

 Recent research has focused on the use of Azolla in integrated farming systems, mainly rice–
fish–Azolla and pig–poultry–fish–Azolla.

Azotobacter species are free-living (mostly root

associated), aerobically nitrogen fixing bacteria.

Nostoc, Calothrix, Gloeotrichia, Stigonema, etc are free-living

aerobically nitrogen fixing Cyanobacteria.

In addition, Vesicular Arbuscular Mycorhizae (VAM

fungi) are free-living soil forms that increase nutrient
uptake (specially by converting organic phosphorus into
inorganic phosphorus), plant growth, nodulation and
nitrogen fixation in legumes.

 In coastal areas of some countries, seaweeds are also used
as biofertilizers.

 However, all these life forms may be grown artificially and

inoculated in seed, root or soil as biofertilizer.

 The nitrogen fixers releases nitrogen during their life time

and also add other elements after their death and decay,
essential for the growth of crops.

Biofertilizers generally used in


1. Azolla

2. Phosphatase bacteria

3. Phosphate solubilizing bacteria

4. Nitrogen fixing bacteria

5. Nitrogen fixing cyanobacteria

6. Enriched compost

What is Azolla?
 Azolla is a dichotomously branched (dichotomy
is a mode of branching by repeated bifurcation) free
floating aquatic fern which is naturally
available mostly on moist soils, ditches
marshy ponds and is widely distributed in
tropical belt of India.

 The shape of Indian species is typically

triangular measuring about 1.5 to 3.0 cm in
length 1 to 2 cm in breadth.

1. Azolla…
Roots emanating from growing branches remained
suspended in water. The dorsal lobe which remains exposed
to air is having a specific cavity containing its symbiotic
partner, a Blue Green Algae (BGA), the Anabaena Azolae.

The fern is capable of fixing atmospheric nitrogen in the soil

in the form of NH4+ and becomes available as a soluble
nitrogen for the wet land rice crop, which is the major cereal
for the people of the North East.

Owing to the poor economic conditions of the farmers of the

North Eastern States, rice crop is mostly grown under natural
soil fertility with minimum inputs and amelioratives.

 But for taking a good crop of rice, judicious
application of nutrients is necessary.

 Besides, this, the farmers of the state of Meghalaya

and other north eastern states have apathy in using
chemical fertilizers in crop production.

 For sustainable crop production, there is a practice

to supply some quantity of nutrients through
organic manure, viz; FYM and composted plant
residues and bio-fertilizers.

In the context of depletion of soil fertility and high prices of
chemical fertilizer, it has become imperative to use
biofertilizer which is a cheaper and renewable source of low
cost plant nutrient and playing a major role in Integrated
Plant Nutrient Supply System.

Use of Azolla fern as a bio-fertilizer is advocated to

minimize the dependency of chemical fertilizer.

Azolla supplements nitrogen to rice crop by fixing

atmospheric nitrogen in the soil for crop growth, crop
production and maintain soil fertility.

Economic Value

On dry weight basis Azolla contains the following chemical


Nitrogen : 5.0 %
Phosphorous : 0.5 %
Potassium : 2.0-4.5 %
Calcium : 0.1-1.0 %
Magnesium : 0.65 %
Manganese : 0.16 %
Iron : 0.26 %
Crude Fat : 3.0-3.3 %
Sugar : 3.4-3.5 %
Starch : 6.5 %
Chlorophyll : 0.34-0.55 %
Ash : 10.0 %

Classification (Taxonomy)

 Class : Pteridophyta
 Order : Salvinales
 Family : Azollaceae/Salvinaceae
 Genus : Azolla
 Sub Genus : Eu-Azolla

Common Names:
Azolla, water velvet, mosquito fern

Different species of Azolla

A. caroliniana A. Pinnata (SE Asia)

A. mexicana A. japonica

A. Microphylla A. filiculoides


Azolla Caroliniana, is identified as a cold tolerant species

and survives well even at very low winter temperature of
5ºC during the months of December to February in mid
hills of Meghalaya.

Azolla pinnata, is a local isolate found widely in the entire

North Eastern Region, but does not survive under mid
hills of Meghalaya.

However, Azolla caroliniana, has shown its adaptability in

hills and other similar locations.

 Azolla caroliniana, can be preserved in shallow

pond having 15 cm of standing water and by
providing shade 10-15 cm above the pond water
surface through weeds or paddy straw.

 For raising Azolla inoculum a pond size of 3 m x 2

m x 1 m is most desirable.

 Under such weed or straw mulch cover, the Azolla

multiplies rapidly and inoculum will be ready
within a period of 20-25 days for further releasing
in the main multiplication ponds on the onset of
monsoon in the month of April-May.

How to grow Azolla?
 Azolla can easily be cultivated separately for periodic application in fish

 The system of cultivation involves a network of earthen raceways

(20X3.0X0.6m) with water supply and drainage facilities.

 In each raceways, 6 kg of Azolla is incorpoprated. 65grams of single super

phosphate is added.

 Water depth of 10-15 cm to be maintained. Azolla is harvested


 It has been estimated that about 1 ton of Azolla can be harvested every
week from a water area 650 m2

 It has also been reported that application of Azolla @ 20 tons/ha gives

50kg N, 13kg P, 45kg K.

How Azolla fixes atmospheric nitrogen?

 The remarkable feature of Azolla is that its symbiotic

relationship with Cyanobacterium (Anabaena azollae)
which remained on the dorsal leaf cavity of Azolla.

 The fern provides protein substances to Anabaena (BGA).

 The BGA then absorbed the atmospheric nitrogen and

decomposes it through enzymic activity and converted in to
soluble ammonia (NH4+). It can fix 3-7 kg N/ha daily.

 It contains 4 % N on a dry-weight basis and is an excellent

source of nitrogen fertilizer

Favourable condition for higher
efficacy of Azolla
1. Water
2. pH
3. Salinity
4. Light & Shade
5. Herbicide level
6. Nutrition

 Azolla must grow in water or wet mud to survive. It dies in a few
hours if it becomes dry. Water control is critical, especially for
for year
round production. A water level which allows the roots to touch the
soil surface will often cause mineral deficiencies to appear.
 10-15 cm fresh current water is necessary in multiplication

Temperature: the day/night temperatures ranging between

32ºC and 20ºC have found to be most favorable. The optimum
temperature for luxurious growth of Azolla is 25-30ºC.

 Wind and wave action can eventually fragment and kill azolla.
Maintaining low water levels and rough plowing can protect azolla
from wind. In Africa, hedges, bunds, and mixed culture (with crop
plants) are used to prevent wind damage. (Von Hove et al.,
al., 1983).

Soil pH:
Azolla grows well in slightly acidic soil having pH 5.2 to 5.8.

Water pH:
 Since azolla lives in water the following refers to the pH of the water only.
Azolla can survive within a pH range of 3.5 to 10, but optimum growth is in the
range of 5 to 7.
 The relative growth rate is influenced by a direct relationship between light
intensity and pH with the highest growth rates achieved at high pH (9- (9-10) and
high light intensity and low pH (5-
(5-6) and low light.
 Nitrogen fixation is optimal at pH 6 and 200C.
 Deficiency problems can be caused in neutral to alkaline water because
because ferric
ions precipitate.
 There can also be competition between ferrous and manganous ions in water
with a neutral pH and reduction in absorption of both iron and managanese with
high calcium concentrations.
 At pH 4, ferric ions are so readily available that a high concentration
concentration of calcium
is required to balance the increased absorption of iorn,
iorn, otherwise azolla suffers
from iron toxicity. (Lumpkin and Plucknett,
Plucknett, 1980).

Salinity Tolerance
 The growth rate of azolla gradually declines as salinity

 At about 1.3% salt (33% of sea water) the growth of

azolla stops and higher concentrations will kill it.

 In rice fields where salt concentration reaches 1480-

1872 mg/l during the dry season azolla wilts.

 Salinity is a factor which should be looked wherever

azolla is being considered (Lumpkin and Plucknett,
Plucknett, 1980).

Shade Tolerance & Herbicide Sensitivity
Shade Tolerance:

Azolla prefers to grow well under partial shade. As dual cropping Azolla
gets partial shade from rice plant and therefore as dual cropping with rice
is most successful.
 Relative growth and nitrogenase activity is at a maximum at 50% of full
sunlight although the difference between growth at 50% and 100%
sunlight is not that great.
 Heavy shading is known to decrease azolla growth to almost zero
(Lumpkin and Plucknett,
Plucknett, 1980).

Herbicide Sensitivity:

 Most rice herbicides kill or inhibit azolla growth. Differences in

sensitivity are specific to the different azolla species (Moody and
Janiya, 1992).

Being an N fixing fern Azolla does not require nitrogenous
fertilizer for its growth.

Phosphorus is the most common limiting factor in the

growth of azolla. Fronds placed in P deficient solution
decrease or stop growth, become red, and develop curled
roots. The minimum P requirement is not known but it
thrives on as little as 1.1 mg P/liter.

Problems due to iron deficiency or toxicity are fairly

frequent. Azolla fronds turn yellow when iron is lacking.
Rapid growth is achieved with 1 ppm iron.

 Yield: Azolla produces around 300 tons of green

biomaas/ha/year under normal sub tropical climate which
is comparable to 800 kg of N (1800 kg of urea).

Contribution of Azolla

Basal application on green Azolla manure @ 10-12 t/ha

increases soil nitrogen by 50-60 kg/ha and reduces 30-35 kg
of nitrogenous fertilizer requirement of rice crop.

 Release of green Azolla twice as dual cropping in rice crop

@ 500 kg/ha enriches soil nitrogen by 50 kg/ha and reduces
N requirement by 20-30 kg/ha.

Use of Azolla increases rice yield by 20 to 30 %.

Rice varieties like DR-92, RCPL-1-87-8, Mendri, H-2850 and

Manipuri produced more than 30 q/ha rice when grown
with Azolla as dual cropping under natural soil fertility.

Under low land condition a thick Azolla mat does

not allow the weeds to grow in rice filed thus, Azolla
suppresses the weed growth and creates congenial
condition for rice production.

Azolla reduces evaporation from water surface and

increases water use efficiency in rice.

Dry Azolla flakes can be used as poultry feed and

green Azolla is also a good feed for fishes.

Azolla is used in carp ponds at @ 40 ton/ha/yr

proving the full complement of nutrients.

Ridged-field rice-azolla-fish model
 This design was originally developed for swampy areas with the objectives
of improving soil properties and increasing rice yield. Later it was,
stepwise, integrated with azolla and fish.

 Rice is planted on the ridge, azolla as a feed for fish as well as a

biofertilizer, and green manure and fish are stocked in the trenches (Pl. see
the diagram in the next slide).

 In tropical Asia azolla is traditionally cultivated as a green manure for rice

in two ways.

 One way is to set aside 5-10% of the crop area for year-round production.
The cultivated azolla is later added to crop fields as compost.

 In the second way, azolla is cultivated in the rice fields and incorporated
before and/or after the rice crop and between crops. Ideally azolla is grown
several times before rice transplanting.

Rice ridge and fish ditch farming system in China

Li Kangmin (1992). Rice-fish farming in China: past, present and future, p.
17-26. In C.R. de la Cruz, C. Lightfoot, B.A. Costa-Pierce, V.R. Carangal and
M.P. Bimbao (eds.) Rice fish research and development in Asia. ICLARM
Conf. Proc. 24, 457 p.

2. Phosphatase activity with reference to bacteria
and phosphorus in tropical freshwater
aquaculture pond systems

 The bio-geochemical cycle of phosphorus is significantly influenced

by microbes in the aquatic environment.

 Phosphorus compounds are decomposed and mineralized by

enzymatic complexes such as phosphatases produced by microbes.

 Enzymatic catalysis results in the production of orthophosphate,

which can be used readily by primary producers.

 Even the smallest concentration of phosphate in water has an

influence over the production process in aquaculture systems.

Phosphatase activity…
Extracellular alkaline phosphatase activity was
observed in water and sediment media of
aquaculture ponds with different management

Heterotrophic bacterial populations as well as

phosphatase-producing bacterial populations
were higher in sediments compared with water.

Phosphatase activity…
 In the freshwater fish ponds, Bacillus spp. were
the dominant forms of bacteria producing

 The alkaline phosphatase activity of sediment was

always higher than that of water.

 The partitioning of extracellular alkaline

phosphatase in pond water by a 0.22-µm
membrane filter revealed that a proportion was
often free rather than cell associated and might
have originated as free enzymes released by
enriched sediments or by fish or microbes.

Phosphatase activity…
In the case of water, although the dissolved
alkaline phosphatase activity was lower than the
total alkaline phosphatase activity, the former was
nevertheless unimportant, as it constitute about
20% of the 'total' activity.
Free alkaline phosphatase activity shared a
negative correlation with the orthophosphate
concentration of water, whereas gross alkaline
phosphatase activity was positively correlated with
the total phosphorus and bacterial population of

Seventeen rhizobacteria isolated from different ecological
regions, i.e. Brazil, Indonesia, Mongolia and Pakistan were
studied to develop inoculants for wheat, maize and rice.

Almost all the bacterial isolates were Gram ‘-’ve, fast-

growing motile rods and utilized a wide range of carbon

These isolates produced indole-3-acetic acid at

concentrations ranging from 0.8-42.1 µg/mL, irrespective of
the region.

Isolate 8N-4 from Mongolia produced the highest amount of

indole-3-acetic acid (42.1 µg/mL), produced siderophores
(0.3 mg/mL) and was the only isolate that solubilize
phosphate (188.7 µg P/mL).

3. Nitrogen fixing cyanobacteria

o Nostoc, Calothrix, Gloeotrichia, Stigonema, etc are free-living

aerobically nitrogen fixing Cyanobacteria.

o In addition, Vesicular Arbuscular Mycorhizae (VAM fungi) are

free-living soil forms that increase nutrient uptake (specially by
converting organic phosphorus into inorganic phosphorus), plant
growth, nodulation and nitrogen fixation in legumes.

o Rhizobium producing root nodules in legumes and Anabaena

azollae living in leaf cavities of Azolla (aquatic fern) are very
efficient nitrogen fixers, and contribute about 500 kg N/ha/year.

Nitrogen fixing cyanobacteria…
Technology for production of BGA biofertiliser with simple
nutrient medium in polyhouse has been standardized.
A thick mat was found to develop within 5 days.
A suitable carrier material has been developed where survival
percentage of BGA strains was 85% even after two and half years
of storage.
The carrier material, Montmorillonite Clay has been found to be
very promising for BGA biofertilizers where it is essential to store
the inoculums for a longer period.
Technology for large scale production of BGA inoculum
biofertilizers in Flexi bioreactors was developed at Madurai
Kamraj University, Tamilnadu.

4. Nitrogen fixing bacteria

 Azotobacter species are free-living (mostly root associated),

aerobically nitrogen fixing bacteria.

 Rhizobium producing root nodules in legumes and thereby fix


5. Enriched compost
 They are used in cellulolytic fungal culture -Phosphotika
and Azotobacter culture.

 In fish culture they not yet reported.