Proc Indian Natn Sci Acad 80 No. 2 June 2014 pp.

301-316
Printed in India. DOI: 10.16943/ptinsa/2014/v80i2/55108

Review Article
Advancements in the Utilization of Azolla-Anabaena System in Relation
to Sustainable Agricultural Practices
R K YADAV1, G ABRAHAM1, Y V SINGH1 and P K SINGH*2
1Centre for Conservation and Utilization of BGA, Indian Agricultural Research Institute, New Delhi 110012,

India
2Department of Botany, Centre for Advanced Studies in Botany, Banaras Hindu University, Varanasi 221 005,

India

(Received on 26 March 2013; Revised on 21 January 2014; Accepted on 27 February 2014)

The nitrogen fixing aquatic pteridophyte Azolla has the ability to fix atmospheric nitrogen at cheaper and faster rates due
to the presence of a symbiotic cyanobacterium Anabaena azollae. Because of this property it has been exploited widely as
biofertilizer for rice plants. In addition to this it has several other uses such as food, feed, biogas producer and hyper-
accumulator of heavy metals etc. Because of the multifaceted uses the promotion and use of Azolla-Anabaena system
would be ideal and environment friendly in sustainable agriculture. This article provides a brief account of the importance
as well as developments in the utilization of Azolla-Anabaena system in agriculture and allied sectors.

Key Words: Azolla-Anabaena; Biofertilizer; Nitrogen; Multifaceted Uses; Sustainable Agriculture

Introduction body weight of the chicks upon feeding with Azolla

was observed. Azolla is a rich source of protein and
The aquatic pteridophyte Azolla is an excellent
essential amino acids and contains several vitamins
biofertilizer and green manure having global
such as vitamin-A, vitamin B-12 and beta carotene.
distribution. Ability of Azolla-Anabaena system to
It is also rich in minerals such as Calcium,
fix atmospheric nitrogen at faster rates makes it an
Phosphorous, Potassium, Magnesium, Copper and
outstanding agronomic choice for the cultivation of
Zinc etc. The protein composition of Azolla is 25-
rice under tropical conditions. Nitrogen fixation
35% on dry weight basis and is easily digested by
potential of the Azolla-Anabaena system has been
poultry (Parashuramulu et al., 2013). Azolla has many
estimated to be 1.1 kg N ha–1 day–1 and one crop of
uses such as animal feed, human food, medicine,
Azolla provided 20-40 kg N ha–1 to the rice crop in
production of biogas, hydrogen fuel, water purifier,
about 20-25 days (Watanabe et al., 1977). The ability
weed control, reduction of ammonia volatilization and
of nitrogen fixation is due to the presence of a
is aptly referred it as green gold mine (Wagner, 1997).
heterocystous cyanobacterium Anabaena azollae
which is confined to the dorsal leaf cavity of the fern Azolla plants in huge numbers sequestered significant
(Moore, 1969). This cavity is formed by the enfolding quantities of atmospheric CO 2 and converted it
of the adaxial epidermis in the dorsal leaf lobe (Peters, directly into biomass of Azolla (Speelman et al.,
1976). Azolla has multifaceted uses and has gained 2009). According to them the plants died and sank to
considerable importance in the recent times as the bottom of the Arctic Ocean and were deposited
biofertilizer, green manure and as poultry feed and as sediments that now form a layer beneath the Arctic
cattle fodder (Singh and Subudhi, 1978). Increase in Ocean. Thus these plants had a significant impact on

*Author for Correspondence: E-mail: [email protected]

302 R K Yadav et al.

Earth’s climate 50 million years ago (Bujak, 2007). attempts are required for the popularization of the
The plant has the ability to multiply fast which is a system. Therefore, the present review takes stock of
remarkable aspect in its exploitation as biofertilizer. the developments related to the successful
Agronomic potential of Azolla has been demonstrated exploitation and application of Azolla-Anabaena
and successful exploitation of this organism in the system and its multifaceted uses for sustainable
agriculture context of Asian countries has also been agricultural practices.
highlighted (Singh, 1977a; Singh, 1989). Besides this
there are several reports on its potential and Distribution, Habit and Habitat and Morphology
agronomic significance (Watanabe, 1982). Rice Azolla is found in both temperate and tropical regions.
ecosystems are characterized by the pattern of rain It grows luxuriantly in ditches, fresh water ponds and
fall, depth of flooding and drainage and by the paddy fields. The Azolla plants are delicate, small
adaptation of rice to these agro-ecological factors and and triangular or polygonal in shape (Fig. 1). It is
rice is grown in flooded conditions in more than 128 free floating and aquatic but can grow on moist soils
million hectares and is suitable for the cultivation of as long as the moisture persists in the soil. The
Azolla (IRRI, 1994, Gillar, 2002). Increase in yield sporophytic plant has a horizontal rhizome of 0.5 to
due to application of Azolla was demonstrated by 7 cm in diameter with branches having densely
several studies conducted in the past at several arranged and overlapping leaves. A leaf consists of a
locations in the country. Similar results have been thick dorsal lobe and a thin ventral lobe. The
obtained with Azolla along with the application of symbiotic Blue Green Alga is confined to the dorsal
chemical nitrogen fertilizers (Singh et al., 1992). lobe (Peters and Mayne, 1974). An epidermis covers
Highest grain yield in rice plants is observed when a the surface of the dorsal lobe and the epidermis has
comparison of Azolla application is made with other vertical rows of single celled stomata and trichomes
biofertilizers. Suppression of weeds and reduction of one or more cells. The ventral lobe which helps in
in the volatilization of ammonia in rice fields due to floating due to its convex surface touching water has
the formation of a thick mat in rice fields by Azolla a few stomata and trichomes (Eames, 1936).
is observed (Singh, 2000). Use of Azolla is very
popular in India and the most widely distributed Growth and Multiplication
species of Azolla include A. pinnata, A. fuiliculoides,
Azolla is found in both temperate and tropical regions
A. rubra, A. microphylla, A. mexicana and A.
and it grows luxuriantly in ditches, fresh water ponds
caroliniana, respectively (Hills and Gopal, 1967).
and paddy fields. The pH requirement of the soil is
There are also reports on its successful use in crops
7.2 for an ideal growth coupled with a temperature
other than rice. Trials have been conducted using
of 32oC. However, studies conducted elsewhere in
Azolla successfully in wheat also as biofertilizer to
enhance the yield (Marwaha et al., 1992). Mahapatra
and Sharma (1989) observed beneficial effects of
Azolla on subsequent wheat crop with increase in
grain yield. Application of 20 tonnes of Azolla along
with 60 Kg nitrogen recorded highest yield of wheat
(Sharma et al., 1999). The plant is also used in
phytoremediation programs to clean up the polluted
and contaminated waters. Phytoremediation is fast
emerging as an excellent option to ward off the
pollutants from aquatic ecosystems (Arora et al.,
2003).
However, the full potential of the plant is still
underutilized despite its advantages and concerted Fig. 1: Azolla plants
Advancements in the Utilization of Azolla-Anabaena System 303

India have shown that the region specific choice of culture conditions. The normal mode of reproduction
the species for their use as biofertilizer. A study was is vegetative but sexual reproduction has also been
conducted at Centre for Conservation and Utilization noticed although this has been observed in limited
of Blue Green Algae, Indian Agricultural Research time periods of the year. This has resulted in problems
Institute, New Delhi to compare the biomass related to precise identification of the Species.
production and nitrogen fixation potential of different Therefore molecular tools have been employed for
species of Azolla (Arora and Singh, 2003). Based on precise identification of the species. RFLP and
the experiments A. microphylla was found to perform isozyme patterns to identify the Sections of Azolla
better and hence selected for mass multiplication. This are employed (Zimmerman et al., 1991a; Zimmerman
strain is maintained round the year as it is able to et al., 1991b; Coppenolle et al., 1993). Taxonomy of
withstand both high as well as low temperature the Family Azollaceae is highly controversial (Reid
conditions. et al., 2006). The uniqueness of A. nilotica has been
confirmed from the data obtained from the loci of
Taxonomic Status of Azolla plastid genome (Metzgar et al., 2007). Integration of
The Genus Azolla was established by Lamarck in the different types of data based on morphology,
year 1783 and placed it in the family Salviniaceae vegetative characters and molecular biology to
under the order Salviniales. However, Azolla is placed provide a firm footing for the taxonomy of Azolla
in the monotypic family Azollaceae and there are has been suggested (Perreira et al., 2011). Species
seven extant Species of Azolla (Hills and Gopal, 1967; specific SCAR primers (sequence characterized
Konar and Kapoor, 1972). Azolla is categorized into amplified region) for the precise identification of
two Sub-Genus viz. EuAzolla and Rhizosperma different species of Azolla is developed recently
(Svenson, 1944). The Sub-Genus EuAzolla is (Abraham et al., 2013).
characterized by the presence of three floats of
Sporulation and its Importance in Azolla
megasporocarps and consists of Species such as A.
caroliniana, A. filiculoides, A. mexicana, A. rubra Mode of reproduction in Azolla is mainly vegetative
and A. microphylla. In contrast, the Sub-Genus and because of this reason the biomass has to be
Rhizosperma consists of nine megaspore floats. A. maintained round the year. The fern is heterosporous
pinnata and A. nilotica belong to this Sub-Genus. The and produce both mega and micro sporocarps and
trichomes are important in the identification of the their germination is influenced by several factors
organism at the Species level (Lumpkin and
Plucknett, 1982; Nayak and Singh, 1988). The basic Table 1: Sporocarp germination in Azolla as influenced by
chromosome number in all Species of the section as various conditions
well as the section Rhisosperma is n = 22 except in
Treatment No. of No. of Germination
A. nilotica where n = 26. The cytological sporocarps sporocarps (%)
investigations show that the somatic chromosome incubated germinated
numbers for Species of the Section Rhizosperma are
Rhizosperma are A. pinnata R.Br. (India) 2n = 44, A. Control (distilled water) 188 31 24.4

pinnata R.Br. (Africa) 2n = 44, A. pinnata R.Br. IRRI medium (solid) 155 44 37.1
(Vietnam, Green) 2n = 66 and A. nilotica Decne.2n IRRI medium (liquid) 181 37 26.7
= 52 (Tan et al., 1986). The somatic chromosome
numbers for Species of the Section Euazolla are A. Soil solution 184 46 28.9
Mexicana Presl. 2n = 48, Azolla filiculoides Lam. 2n Phosphorous (30 ppm) 126 100 63
= 40 with the basic chromosome numbers n = 24 and
Kinetin (100 ppm) 197 132 55.2
n = 20, respectively (Nayak and Singh, 1989). The
taxonomic assignment of Azolla is difficult because Gibberellic acid (100 ppm) 192 106 48
many Accessions do not form sporocarps under Singh et al., (1990)
304 R K Yadav et al.

Table 2: Effect of Azolla and various organic treatments on rice (Pusa Basmati 1) grain yield during kharif (2003-08)

Treatment* Rice grain yield (t/ha)

2003 2004 2005 2006 2007 2008 Mean

Azolla (A) 2.87 2.54 2.43 2.36 2.29 2.06 2.43

BGA (B) 2.70 2.46 2.35 2.29 2.19 1.90 2.32
FYM (F) 2.69 2.24 2.26 2.12 2.17 2.04 2.25
Vermicompost (V) 2.90 2.66 2.53 2.60 2.39 2.18 2.54
A+B 3.35 3.25 3.03 3.16 3.02 2.68 3.08
A+F 3.70 3.42 3.38 3.29 3.18 2.57 3.26
A+V 4.08 3.85 3.67 3.57 3.43 2.64 3.54
B+F 3.33 3.26 3.43 3.48 3.37 2.60 3.25
B+V 3.91 3.50 3.47 3.52 3.48 2.82 3.45
F+V 3.75 3.58 3.64 3.56 3.61 3.02 3.53
A+B+F 4.05 3.66 3.79 3.68 3.82 3.34 3.72
A+F+V 4.08 3.70 3.81 3.89 3.93 3.45 3.81
B+F+V 4.10 3.82 3.88 3.93 3.91 3.64 3.88
A+B+F+V 4.19 4.35 4.38 4.16 4.48 3.68 4.20
N80P 40K30 4.93 4.68 4.21 4.34 4.61 3.46 4.37
N0 P0K0 (Control) 2.02 1.84 1.76 1.78 1.89 1.68 1.82
C.D (at @5 %P) 0.95 0.48 0.31 0.41 0.32 0.26 0.46
*Rate of application/ha: Azolla 1.0 t (fresh); BGA 2 kg (dry); FYM 5.0 t; Vermicompost 5.0 t.

(Table 3, Fig. 2). However, production of sporocarps Auxins in combination with Gibbrellic acid has also
is under the influence of the environment and no been reported (Kar et al., 2002). However, these
uniformity has been observed regarding their time of methods using chemicals besides being uneconomical
production by the plants. For example Azolla
microphylla sporulates throughout the year (Kar et
al., 1999). Density of population of the plants
especially high density has been found to induce the
efficiency of sporocarp production (Watanabe, 1982).
Production of sporocarps was generally favoured by
relatively short day and cool nights at Cuttack (Singh
et al., 1987). Increase in the sporocarp germination
is influenced by combined nitrogen sources. The
germination of sporocarps in Azolla caroliniana
varies in response to light, amino acids, sugars and
abscissic acid (Singh et al., 1990). Megasporocarps
of Azolla and their germination in varied paddy soils
has also been reported (Nayak et al., 2004). Enhanced
sporulation in Azolla microphylla and Azolla pinnata
due to application of Gibberellic acid has been
reported (Kar et al., 1999). Similarly high sporocarp
yield in relation to exogenous application of certain Fig. 2: Azolla microphylla plants showing sporulation
Advancements in the Utilization of Azolla-Anabaena System 305

Table 3: Chemical composition of Azolla Effects of nutrient status of the medium on the
productivity and nitrogen fixation in Azolla are
Constituents Dry matter (%)
reported (Kushari and Taheruzzaman, 1990). A
Crude protein 24-30 comparative study on the growth, pigments, nitrogen
Crude fat 3.3-3.6 fixation and nutrient status of Azolla maintained as
Nitrogen 4-5 soil and nutrient based cultures showed that the soil
Phosphorous 0.5-0.9 based cultures are as good as those raised on nutrient
Calcium 0.4-1.0
media (Dawar and Singh, 2002). Azolla production
can be carried out in nursery plots, ponds, ditches,
Potassium 2-.4.5
canals, concrete tanks and polythene lined ditches.
Magnesium 0.5-0.65
The field selected for Azolla cultivation needs to be
Manganese 0.11-0.16
thoroughly prepared and leveled uniformly. Generally
Iron 0.06-0.26
20 m x 2 m size plots are made in the field with
Soluble sugars 3.5 suitable bunds and irrigation channels with a water
Crude fire 9.1 depth of at least 10 cm and in each plot water (20
Starch 6.54 liters) is added and inoculated with Azolla (8-10 Kg).
Source: Singh and Subudhi (1978a) Single super phosphate (100 g) in 2-3 split doses is
applied at an interval of 4 days to each plot. Furadon
or carbofuran (3 % active granules) can be applied in
also lead to environmental contamination. Influence the plots (100 g plot –1) with or after a week of
of fertilizer application on the sporocarp production inoculation. 100-150 Kg fresh Azolla can be harvested
has been observed (Singh et al., 1987) and induction from each plot after 15 days from each plot. The same
of sporulation under phosphorous deficient conditions method can be used to produce Azolla in bigger plots.
was recorded (Kannaiyan et al., 1988). An increase The quantities of in-puts need to be varied as per
in the sporulation frequency and sporocarp number requirement. Depending on the availability and need
without compromising the biomass yield is observed of the inocula, Azolla can also be maintained in
due to the change in schedule of phosphorous nursery in trays or earthen or cemented pots of any
application in Azolla (Kar et al., 2001). The dimension. If the production is carried out in a pond
sporocarps germinate after 3-5 months of dormancy or canal fertilizers and insecticides are not applied.
period and percent germination is higher during Cattle slurry and animal dung for the production and
August to October (Singh et al., 1984b). Farmers have utilization of Azolla as biofertilizer for rice has been
to carry large quantity of the fresh inoculums to the reported (Singh et al., 1993). The cattle slurry and
field for application every time which involves animal dung are effective as phosphorous fertilizers
transportation and many a times and this leads to and application of Azolla is found to enhance the C,
perishing of the culture. This is also one of the reasons N and available P content of the soil.
responsible for the poor adoption of Azolla by the
Methods of Application of Azolla
farmers. Hence to make the Azolla biofertilizer
technology more effective and serious attempts have The most common mode of application of Azolla in
to be made to successfully propagate the fern through the field is as green manure or as a dual crop along
sporocarps. with rice. In case of application as green manure
Azolla collected directly from ponds/ditches is
Production Technology for Azolla Biofertilizer and
applied in the field. It may be grown in nurseries as
Modes of Application specified earlier and can also be applied in the field.
The propagation of Azolla is generally carried out in A thick mat of Azolla will be formed after application
soil-based nurseries. However, to maintain the in about 2-3 weeks time and can be incorporated in
germplasm medium based cultures is advisable. the soil. Rice can also be transplanted in the field
306 R K Yadav et al.

subsequently. Single super phosphate (25-50 kg (Lumpkin and Plucknett, 1980). The rapid and
ha–1) is applied in split doses. After analyzing the substantially higher rates of nitrogen fixation coupled
soil P-status the dosage of the same can be reduced. with production of high biomass have made the
Cattle dung or slurry may also be used instead of organism an outstanding agronomic choice.
single super phosphate. In case of pest infestation or Azolla strains have been successfully exploited
attack, pest control measures have to be undertaken. as an efficient biofertilizer for rice paddy fields
Azolla application by this mode contributes around because of the nitrogen fixing potential. Dr. P K Singh
20-40 kg N ha–1. and coworkers from the Central Rice Research
In dual cropping, Azolla is grown along with Institute, Cuttack successfully popularized the use of
rice and each crop of Azolla contributes on an average Azolla as a promising biofertilizer in the Eastern parts
30 kg N ha–1. After 7-10 days of transplantation fresh of India. A. pinnata fixes 75 mg N g–1 dry weight
inoculums of Azolla is applied in the field at the rate day–1 and produces a biomass of 347 ton fresh weight
of 0.50-1.0 ton ha–1. Single super phosphate is applied ha–1 in a year. This biomass contains 868 kg N which
at the rate of 20 kg ha-1 in split doses. In about 15-20 is equivalent to 1900 Kg urea. A wide variability
days time a thick mat of Azolla is formed. Azolla thus regarding growth and nitrogen fixation among
incorporated decomposes in about 8-10 days time and different strains of Azolla is observed by a study
release the fixed nitrogen. Another crop of Azolla can conducted at Central rice Research Institute, Cuttack
be raised in a similar way during the crop cycle of (Singh, 1988). Among the several factors that
rice. Azolla production technology is simple and not influence the growth and nitrogen fixing potential of
very expensive and at the same time it is very efficient Azolla are nutrient availability, rate and the time of
in terms biomass accumulation and nitrogen fixation. inoculation etc (Kannaiyan, 1993; Singh and Singh,
The rice growing season is also conducive for the 1995). In addition to sustaining rice yields,
growth of Azolla plants. The dual application does inoculation of Azolla has been reported to enhance
not have any negative influence on the rice crop. the soil biological health. It is important to optimize
use of organic, inorganic and biological inputs in an
integrated manner taking into consideration the
Biological Nitrogen Fixation and Azolla in Relation
ecological and soil conditions to sustain crop
to Soil Fertility
productivity. Soil enzyme activity is considered as
The ability to fix atmospheric nitrogen at substantially an index of microbial activity and fertility of the soil.
higher rates has led to the exploitation of the organism Azolla decomposes rapidly in soil and supply nitrogen
as biofertilizer. Application of Azolla in rice paddy to the crop plants. It contributes significant amounts
fields has a positive role in improving the soil fertility of phosphorus, potassium, sulfur, zinc, iron and
index. The ability of nitrogen fixation is due to the molybdenum in addition to other micronutrients
presence of the symbiotic cyanobacterium Anabaena besides addition of nitrogen. The biological health
that occurs in the dorsal leaf cavities of the fronds of the soil due to application of Azolla has resulted
(Peters and Meeks, 1989). The symbiont is able to in improving mineralization and consequent increase
meet the entire nitrogen requirement of the in the microbial status of the soil. In low land rice
association. Calvin cycle operates in both the partners cultivation mineralization of organic nitrogen to
and the primary end product of photosynthesis is ammonia is an important process (Sahrawat, 1983).
sucrose (Van Hove, 1989). A strong interaction exists The rate of mineralization is influenced by factors
between nitrogen fixation and the photosynthesis and such as C: N. Azolla species with a low C: N
the source of ATP and NADPH is photosynthesis. mineralized in 2 days while the species with high
The capacity of Azolla to fix nitrogen in the field has C:N mineralized in 5 days (Wang et al., 1987). The
been estimated to be 1.1 kg N ha–1 day–1 and this decomposed organic matter plays an active role in
fixed nitrogen is sufficient to meet the entire nitrogen the development of microbial population irrespective
requirement of rice crop within a few weeks of the time taken for mineralization. Soil fertility is
Advancements in the Utilization of Azolla-Anabaena System 307

also influenced by the humic substances formed released into the soil (Bhuvaneshwari and Kumar,
during the decomposition of Azolla (Bhardwaj and 2013).
Gaur, 1970). For wetland rice cultivation Azolla and
Sesbania are used as green manure (Ventura and Improvement in Crop Productivity Due to
Watanabe, 1993). Continuous application increased Application of Azolla
the organic nitrogen content of the soil significantly. Productivity of rice is related to the availability of
Increased celluloytic and urea hydrolyzing activities nitrogenous fertilizers. Nitrogen can also be
in addition to significant increase in the population supplemented to the rice crops through biofertilizers
of heterotrophic bacteria were recorded (Kannaiyan such as Azolla and cyanobacteria. Application of
and Subramani, 1992; Kannaiyan and Kalidurai, Azolla to rice plants is ideal as the growth requirement
1995). Increased soil urease and phosphatase activity for both the organisms are similar. The organism is
has also been observed due to incorporation of Azolla able to multiply fast which is a remarkable aspect in
(Thanikachalam et al., 1984; Thangaraju and its exploitation as biofertilizer. Successful
Kannaiyan, 1989). exploitation of this organism in the Asian context has
Combined incorporation of nitrogen fixing also been highlighted (Singh, 1989). Field
green manures such as Sesbania and Azolla shows experiments conducted at Central Rice research
significant enhancement in the activity of soil Institute, Cuttack using high yielding varieties of rice
enzymes such as dehydrogenase, phosphatase, showed that application of 10 tons ha–1 of fresh Azolla
cellulose and amylase (Kumar and Kannaiyan, 1992). is as efficient as basal application of 30 kg of N
Similar enhancement in the microbial population, (Singh, 1977; Singh, 1978). Their experiments
total bacterial, cellulolytic, phosphate solubilising and resulted in increase of height, tiller, dry matter,
urea hydrolysing bacteria was observed number and weight of panicles, grain and straw yield
(Gopalaswamy and Kannaiyan, 2000c). Azolla helps etc. Increase in yield due to application of Azolla was
to sustain soil nitrogen supply by returning N to the demonstrated by several studies conducted at several
soil in quantities roughly equal to those extracted locations. A single crop of Azolla provides 20-40 kg
from soil by the rice plants (Cisse and Vlek, 2003). N ha–1 (Singh, 1977b). Similar results have been
Maximum population of bacteria, fungi and obtained with integrated application of Azolla and
actinomycetes and high urease and dehydrogenase chemical nitrogen fertilizers. When a comparison of
activities due to organic farming using Azolla as one Azolla is made with other biofertilizers highest grain
of the components was reported (Krishnakumar et yield in rice is observed (Singh et al., 1992). After
al., 2005). Field experiment studies have been the decomposition of the organism nitrogen content
conducted at the Centre for Conservation and is made available to the rice plants. It was found that
Utilization of Blue Green Algae, Indian Agricultural the plant decomposed 8-10 days after incorporation
Research Institute, New Delhi. In these studies into the soil and rice plants are benefitted after 20-30
optimum yield in organic Basmati rice was recorded days (Singh, 1977d). The release of N by Azolla is
and improvement in grain and soil quality noticed. slow and its availability to the first crop of rice is
The inputs used in this study included Azolla, blue- about 70% to that of ammonium sulphate. Moreover,
green algae, farmyard manure and vermicompost as the release of nitrogen is faster as compared to
organic amendments (Singh et al., 2007). nitrogen fixing cyanobacteria (Saha et al., 1982).
Experiments conducted at Banaras Hindu University, Fresh Azolla releases its N faster as compared to dried
Varanasi showed beneficial effects of Azolla in the Azolla due to rapid mineralization and mineralization
cultivation of rice (Bhuvaneshwari, 2012). of N is faster at room temperatures (Singh, 1979a;
Application of Azolla has been found to significantly Singh, 1979b). A comparative study was conducted
improve the physical and chemical properties of the at Cuttack on the N release efficiency of chemical N
soil especially nitrogen, organic matter and other fertilizers, Azolla and BGA and other organic
cations such as Magnesium, Calcium and Sodium manures. It was found that the release of N by Azolla
308 R K Yadav et al.

was comparatively slower and 87% of N in chemical enhancement in grain yield of rice over absolute
fertilizer is released within 10 days. Hence the control due to the application of organic amendments
application of Azolla in combination with chemical like Azolla applied alone or in combination (Table
fertilizers offers an excellent choice for rice 2). Optimum yield of Basmati rice (cv. Pusa Basmati
cultivation (Singh et al., 1981). Increase in grain 1) can be obtained in all the years with the application
yield with application of Azolla alone and chemical of four amendments (Azolla, BGA, vermicompost and
nitrogen fertilizer (150 kg N ha-1) is reported (Singh FYM) together. Besides, enhancing and sustaining
et al., 1992). At Central Rice Research Institute, the productivity of organic rice-wheat system, higher
Cuttack, field trials demonstrated that use of Azolla productivity of vegetables like cauliflower, broccoli,
enhanced crop yield and crop N uptake significantly cabbage and carrot grown after organic rice under
as compared to treatments without Azolla (Manna and organic nutrition were recorded (Singh et al., 2012).
Singh, 1989). Utilization of Azolla pinnata as There was no serious incidence of any insect pest or
biofertilizer in acidic soils of Kerala has also been disease in organically grown rice crop during these
reported (Sevichan and Madhusoodanan, 1998). years. Microbial populations (Actinomycetes,
Suppression of weeds and reduction in the Bacteria, Fungi and BGA) were found to enhance
volatilization of ammonia in rice fields due to the over the years due to the application of organic
formation of a thick mat in rice fields by Azolla is amendments in comparison to total control and
also observed (Singh et al., 1981). Comparison of recommended fertilizer application that accordingly
Phosphorous enriched and un-enriched A. caroliniana result in a notable enhancement in dehydrogenase
on the performance of Azolla dual cropping with rice enzyme activity. The treatments have led to changes
showed higher biomass and N yield by applying in the composition of microbial communities due to
phosphorous to P-enriched inocula (Singh and Singh, fertilization treatment and application of organic
1995). Potential exploitation of the system as matter (Irisarri et al., 2001; Jha et al., 2004; Singh
phosphorous biofertilizer to improve the nitrogen and and Dhar, 2011; Singh et al., 2012). Rice grain
phosphorous balance in polluted environments was analysis for Iron, Zinc, Manganese and Copper
attempted (Singh et al., 2010). contents showed a significant increase in these
essential ion contents in the treatments having two
Azolla as an Input in Organic Agriculture or more organic amendment added altogether over
Recently there is a surge in the interest in organic control. Similar results have been obtained by
farming and Azolla is used as an important and Bhattacharya and Chakraborty (2005). Organic
potential component in the organic farming of rice. nutrient management including Azolla inoculation
Positive effect on the BGA and Azolla on rice yield showed considerable built up in soil organic carbon
has been reported earlier (Singh and Mandal, 1997; content. The values of soil physical parameters like
Singh and Mandal, 2000). Azolla has been used available water content (AWC) and water retention
successfully as a component of organic cultivation capacity (WRC) are higher under organic
in rice based cropping system. Field experiments have management compared to INM and chemical
been conducted at Indian Agricultural Research fertilization. The increase in AWC was related to the
Institute, New Delhi during 2003-2009 to find out increase in micro-and macro-porosity. Lower bulk
suitable organic amendments for sustainable density (BD) was observed in organic treatment as
productivity of Basmati rice-wheat-green gram compared to INM and chemical fertilizer treatments
cropping system (Singh et al., 2011). Different (Singh et al., 2012). Highest net return is also
treatment combinations comprising of organic recorded with integrated nutrient management (INM)
amendments such as Azolla @1.0 ton ha–1, Blue of rice that included use of Azolla in addition of
Green Algae @ 2.0 kg ha–1, vermicompost and farm chemical fertilizer and it is followed by organic
yard manure @ 5.0 ton ha–1 applied alone or in management with four inoculants including Azolla.
combination were tested. Results revealed significant These results clearly show how Azolla could be
Advancements in the Utilization of Azolla-Anabaena System 309

successfully integrated with other bio-inoculants in experiments is found to enhance the weight of the
rice as well as vegetable based organic cultivation. carp Osteobrama belangeri and resulted in better feed
conversion efficiency and protein efficiency ratio
Use of Azolla as Livestock Feed (Basudha and Vishwanath, 1997). Use of Azolla
The importance of Azolla as a sustainable feed for protein supplement for the fish Tilapia mossambica
livestock and poultry is reviewed recently (Gouri et and observed increase in feeding, absorption and
al., 2012). According to them Azolla can be used as growth rate (Sithara and Kamalaveni, 2008). They
an ideal source of feed for cattle, sheep, goats, pigs, further observed that mixing of the Azolla biomass
rabbit and fish (Fig. 3). Because of the high nutrient with other agricultural byproducts such as wheat and
content Azolla can be used as fodder for cattle and rice bran will lead to improvement in digestibility
fish as well as poultry feed. It has Proteins, Vitamins, and protein quality.
Calcium, Phosphorus, Iron, Copper, Magnesium, Beta Feeding experiments conducted on buffalo
carotene and Amino acids (Table 3). Azolla can be calves suggest that Azolla meal is a potential and
maintained throughout the year due to proper unconventional source of protein (Indira et al., 2012).
planning and cultivation practices and this in turn Although most of these studies advocate the use of
will ensure round the year availability of the organism fresh biomass, studies show that heat treated Azolla
for fodder purposes. Azolla in dried form is not meal is partially substituted for fish meal resulted in
preferred by the birds and hence the use of fresh significant growth of Labeo rohita (Maity and Patra,
biomass is an excellent poultry feed with no side 2008). An experiment was carried out to investigate
effects (Singh and Subudhi, 1978a). About 20-25% the effect of dietary inclusion of sun dried and ground
of the commercial feed can be replaced by the Azolla (Azolla pinnata) on production performance
incorporation of fresh Azolla biomass (Subudhi and of broiler chicken (Balaji et al., 2009). The per cent
Singh, 1978b). Utilization of Azolla as animal feed giblet yield of birds fed with 4.5% Azolla is found to
is suggested to be advantageous for the better be significantly higher than control and other
productivity of livestock which comprise an integral treatments. Based on the experiments it is concluded
part of the agriculture (Banerjee and Matai, 1990). that dietary inclusion of dried Azolla up to 4.5% levels
Studies conducted on Azolla pinnata indicate that the did not have any adverse effect on production
level of Amino acids in the leaf protein compare performance of broiler chicken. It is observed that
favorably with the standards of FAO reference pattern aquatic plant species especially Azolla offer a great
and chick requirements (Dewanji, 1993). Integration potential as a source of protein for animals due to
of Azolla powder in the fish meal and feeding ease of cultivation, productivity and nutritive value
(Prabha and Kumar, 2010). Therefore, keeping in
view of the importance of organic aquaculture these
days use of Azolla is found to be one of the cheapest
ways to increase the fish production in organic fish
farming. Increase in fish production was observed
when Azolla is used in organic aquaculture and it is
strongly suggested that this practice can be an
effective tool to increase the farm income and living
standards of low income farmers from the Meghalaya
region (Majhi et al., 2006). Poultry fed on Azolla
show significant increase in the body weights and
consequently have resulted in an increase in the net
return (Rai et al., 2012). These studies in general
indicate the tremendous possibility of using the Azolla
Fig. 3: Azolla used as cattle feed by farmers
310 R K Yadav et al.

biomass effectively in feed and also to bring down employed in the phytoremediation of toxic heavy
the cost involved in feeding. However, there is a need metals. Recently the phytoremediation potential of
to address the protein quality as well the Amino acid three different species of Azolla such as A.
and nutrient composition of different species of these microphylla, A. pinnata and A. filiculoides is studied
plants. This will help in the selection of promising and it is found that A. microphylla accumulated more
species with superior protein quality, amino acid and metal as compared to the other species (Arora et al.,
nutrient composition that can be used as efficient 2006). The biomass of Azolla (both live and dead)
dietary supplement. has also been used in the bio-sorption of several heavy
metals (Umali et al., 2006; Mashkani and Ghazvini,
Bioremediation Potential of Azolla 2009). Extensive work has been done using Azolla
Another interesting and applied aspect of utilization in the removal of heavy metals from aquatic
of Azolla which is gaining popularity in the recent environments (Rai 2008; Rai and Tripathi, 2009; Rai
times is its application in the process of 2010a; Rai, 2010b). However, this is an area where
bioremediation. Increase in population and rapid rates no serious efforts have been attempted despite the
of industrialization has resulted in the release of huge potential of the organism for bioremediation.
quantities of pollutants into water resources. The Capability of the organism to reduce these ions in to
pollutants released are toxic in nature and pose great metallic particles is possible since the plant itself
concern due to their adverse impact on plant and could act as a strong reducing agent.
animal health. Therefore phytoremediation offer an A hydroponic system is developed to
excellent option to ward off the pollutants from decontaminate the water containing Cadmium and
aquatic ecosystems. Azolla may be used to clean up Copper was developed recently (Valderrama et al.,
the polluted and contaminated waters (Table 4). 2012). Phytoremediation potential of Azolla was
Efficient removal of gold from waste water solutions reviewed recently (Sood et al., 2011). All these results
using the fern Azolla filiculoides is reported (Antunes show that Azolla may be successfully employed in
et al., 2001). Similarly Elmachliy et al., (2010) phytoremedation of polluted water bodies. The
demonstrated removal of Silver and Lead ions from municipal sewage water waste contains heavy load
waste waters using Azolla filiculoides. Successful of Phosphorous and Nitrogen and bioremediation
cultivation of A. microphylla biomass in secondary programs involving Azolla may be planned to
treated Municipal waste water of Delhi is attempted decontaminate them. The used biomass may be used
by Arora and Saxena (2005). Azolla biomass is also as green manure in case it is not having any heavy
metal load in it. However, when used for
Table 4: Bioaccumulation potential of heavy metals by various bioremediation of waste water having heavy metal
Azolla spp. load the biomass may be dried and extracted for the
recovery of the metal or incinerated to prevent the
Azolla spp. Heavy metal Concentration of heavy
metal accumulated
recycling of the heavy metal in the environment.
(µg metal g–1)
Research Perspectives for Future Research
A. pinnata Cadmium 2759
Mercury 450
In view of the declining soil health and productivity
due to increased anthropogenic activities maintaining
A. caroliniana Lead 416 the sustainability is a challenging task ahead. The
Chromium 964
Azolla-Anabaena system is an excellent biofertlizer
A. filiculoides Nickel 28443 for rice crop and it also has several other uses. In
Chromium 12383
order to improve its utility in agriculture and allied
A. microphylla Nickel 21785 sectors focused attention is required. Thus, there is
Cadmium 1805 an urgent need to address certain key issues in Azolla
Source: Sood et al., (2011) for its exploitation and better utilization. These
Advancements in the Utilization of Azolla-Anabaena System 311

include the difficulties encountered in identification, and development of plants in general. However,
observation of sporulation, storage of sporocarps and works conducted elsewhere showed varied response
abiotic stress tolerance etc. These issues are needed by Azolla to increase in salinity. Thus, the studies
to be addressed on priority basis to further enhance conducted on Azolla regarding salinity tolerance show
the utility of this organism in agriculture and allied the potential of salinity tolerance in different species
sectors. In view of the progresses made using but no further advancement has been made in this
morphological and molecular approach in direction using advanced molecular biology tools.
taxonomical research on Azolla an integrative Since in the environment the organism is exposed to
approach is needed to solve the problems related to high and low temperature conditions, it is worth
classification using several accessions as per the list understanding the tolerance mechanisms using
of International Rice Research Institute, Philippines. advanced techniques. Such an approach will lead to
Therefore, an integrative approach involving understanding of the common proteins involved in
morphological and molecular characters must be stress tolerance. It is imperative to identify novel
taken in to consideration to assign species and create proteins, genes and their pattern of expression and
molecular finger prints. Contamination is a frequently functions in stress adaptation in order to improve the
encountered problem in many germplasm collections. stress tolerance. This could help in better
The contamination can lead to the development of understanding the abiotic stress tolerance mechanism
hybrids and we do not have any concrete tools to in Azolla. With the imminent threat of climate change
detect such a situation other than the conventional scenario and increasing contamination due to
morphological identification. Hence reliable and pesticides and heavy metals such approaches could
reproducible fingerprints must be developed for the be helpful in deciphering the mechanism of tolerance.
precise identification of the plants. In this regard, proteomics and genomics play an
important role.
The mode of reproduction in Azolla is mainly
vegetative and because of this reason the biomass Azolla may be successfully employed in
has to be maintained round the year. The farmers have phytoremedation of polluted water bodies. However,
to carry large quantity of the fresh biomass to the this is an area where no serious efforts have been
field for application every time. At times it also attempted despite the potential of the organism for
involves transportation to long distances leading to bioremediation. The municipal sewage water waste
perishing of the cultures. This is also one of the contains heavy load of phosphorous and nitrogen and
reasons responsible for the poor adoption of Azolla bioremediation programs involving Azolla may be
by farmers. Hence to make Azolla biofertilizer planned to decontaminate them. The used biomass
technology more effective, attempts must be made may be applied as green manure in case it is not
to successfully propagate the organism through having heavy metal load. However, when used for
sporocarps. Hence an important pre-requisite in the bioremediation of waste water having heavy metal
sporocarp technology is to have an understanding load the biomass may be dried and extracted for the
regarding the time of sporulation in different species recovery of the metal or incinerated to prevent the
of Azolla in relation to the factors controlling it in an recycling of the heavy metal in the environment.
ecological niche. Once we understand the pattern of
The incidence of insects and pests is a very
sporulation and conditions specific to it, then
common problem encountered in the outdoor mass
strategies could be developed for induction of
multiplication tanks of Azolla during the humid
sporulation and its storage.
seasons. These insect pests belong to Lepidopterous
Screening of the plants against abiotic stress and Dipterous orders and can damage the plants in
conditions such as salinity, high temperature, 2-3 days completely (Singh, 1977). At the Centre for
pesticide, UV-B and heavy metals is essential. Conservation and Utilization of Blue Green Algae,
Increasing soil salinity is a major deterrent for growth Indian Agricultural Research Institute, New Delhi of
312 R K Yadav et al.

late the incidence of pests is also observed during heavy metals and UV-B need to be evaluated. Strain
the months of October and November which are improvement must be attempted using innovations
otherwise months that support good Azolla growth in molecular biology. Soil fertility index studies using
(Unpublished observations). Changes in the climate soil enzymes need to be conducted employing Azolla
scenario may be responsible for such attacks and may alone or as a consortium with other micro-organisms.
be investigated in detail. At present there are chemical Sound extension strategies are also the need of the
as well as biological controls to check the incidence hour to promote the use of this biofertilizer with other
of pests and insects. Hence species which can repel benefits. The organism has tremendous potential and
the insect attack could be screened. Determination its real potential to enrich soil organic matter, soil
of phytochemical composition of different strains is enzymes and the soil microbial population has not
also important especially when Azolla is used as an yet been fully exploited. Concerted efforts are
important input in organic rice farming and poultry/ required from the part of policy makers, scientists
cattle feeding. and farmers to promote Azolla as a viable bio-
inoculant for sustainable crop production and
Conclusions development.
The fundamental to sustainable intensification of
agriculture is effective soil health management and Acknowledgements
Azolla has been known to influence the dynamics of
Financial support from the Council of Scientific
the total soil and microbial population of nitrogen
Industrial Research, New Delhi (No. 38(1319)/12/
fixing bacteria. The soil fertility index will improve
EMR-II) is gratefully acknowledged. Facilities and
due to the accumulation soil enzymes. Therefore we
support provided by IARI, New Delhi is also
need to have efficient strains of Azolla to maintain
acknowledged. The authors also thank Shri. Tirtha
the soil fertility. Efficient strains of Azolla in terms
Dasgupta (ARPS, APSA, EFIAP), Photo Officer In-
of nitrogen fixation from different agro-ecological
Charge, Central Photo Laboratory, IARI, New Delhi
zones are to be screened and their performance under
for help in the photographic work.
various abiotic stress conditions such as salinity,

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