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CHAPTER 21

AZOLLA AS A POTENTIAL PARTIAL SUBSTITUTE OF

ANIMAL FODDER IN DEVELOPING COUNTRIES UNDER
CLIMATE CHANGE IMPACTS

Medical Dr. Mohmmad Mouammer HAKKI 1

Pharmacy Rajaa AL-ZAGLOL2

1
SPU University, Faculty of Pharmacy, Damascus, Syria. Researcher in Nwat Center
for Scientific Studies, Food Security Department. E-mail:
[email protected] (ORCID: https://orcid.org/0000-0002-8373-
782X)
2
Institute of Pharmacy, Shaam Academy of Medical Sciences, Damascus, Syria. Nwat
Center for Scientific Studies, Food Security Department. E-mail:
[email protected] (ORCID: https://orcid.org/0000-0002-3066-6896)
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INTRODUCTION
Azolla is known for centuries in some agricultural
communities, it has been known as a multipurpose plant in
southern China and North Vietnam (Azolla.Foundation,
Accessed on 15.10.2021), using of Azolla is known for more
than 2000 years in rice farming as written in the ancient
Chinese book Er-Ya (Qiu et al., 2003), the earliest record of its
cultivation is written in Jia Ssu Hsieh (Jia Si Xue) book in 540
A.D on The Art of Feeding the People (Chih Min Tao Shu)
(Azolla.Foundation, Accessed on 15.10.2021). They grow
Azolla as a rice biofertilizer since it can efficiently fix
atmospheric nitrogen and make a barrier for repelling
mosquitoes from laying eggs in rice ponds. The spread of
Azolla implementation in rice production was extended by the
Chinese Government which introduced a series of programs in
1962 to improve rice production (Liu, 1979). Nowadays Azolla
used as a biofertilizer has been widespread globally especially
in the far East, including Bangladesh, Brazil, Burma, China,
India, Indonesia, Korea, Nepal, Pakistan, Sri Lanka, Thailand,
Vietnam, and The Philippines. Also, it was introduced recently
20 years ago into the Mediterranean region especially in Egypt
(Zahran HH et al., 2007) and Syria too after the war and forced
migration. Farmers are using it for the previously mentioned
benefits and as a supportive fodder ingredient for livestock.
The genus name Azolla is based on Greek azo, to dry + ollyo,
to kill, an allusion to death from drought.

The Azolla Biology, Physiology, Morphology, And Distribution

Azolla is a genus of a free water fern plant that belongs to the family
Salviniaceae, also known as the mosquito fern, duckweed fern, fairy
moss, and water fern. It is a tiny, delicate annual plant. The tiny small
leaves no larger than 2 mm across, alternatively attached to the
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branching stems, often overlapping in two to three ranks. The leaves

have an upper green lobe with hydrophobic hairs and a colorless lobe
submerged underwater surface. Unbranched roots produced from the
axile of branches are hanging into the water. In optimum conditions, 2-
3 layers of Azolla could be overlap making a mat of 5 cm (Small et al.,
2011). In nature, the lower layer decays with time and is replaced by
the previous layer; with time the carbon content will increase because
of the rapid biomass production of Azolla. The most amazing part of
Azolla is its symbiosis with N-fixing bacteria cyanobacterium
Anabaena azollae which is found in pouches at the upper leaf lobes on
their lower side (El-Bassel et al., 1996) (Small et al., 2011). This
relationship is very rare in the plant kingdom, and this led to the Azolla
being called a “super-plant”. It can grow well in the range between 15-
35 degrees, and different strains perform differently according to the
habitat and species. 6 species are well known which are: A. caroliniana,
found in eastern North America, and the Caribbean, this strain is the
most cold-tolerant of Azolla species; it can survive frost and prolong ice
coverage, and can thrive in mud (Small et al., 2011), A. filiculoides,
found in southern South America through western North America and
Alaska, it is cold-tolerant and can survive under a thin layer of ice. A.
microphylla, found in tropical and subtropical America, it’s highly
adapted to high temperature ( > 35Co) (Dawar et al., 2002), A.
mexicanam, found in northern South America through western North
America, this strain is also cold tolerant, A. nilotica, found in upper
reaches of the Nile to Sudan, this specie has large leaf structure which
could extend to 32 cm long, this structure has a big advantage
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harvesting practice, we know little information about this specie until

now (Annonyomus, Accessed on 10.10.2021) (Lumpkin et al, 1982). A.
pinnata, found in most of Asia and the coast of tropical Africa. Each
species has special characteristics that researchers are trying to figure
out to be implemented in agriculture and industrial practices.

Figure 1. Approximate modern distribution of Azolla (Small et al.,

2011)
Most of Azolla spp. changes its color to reddish-brown color when
stressed by extremely high or low temperature or mechanical or feeding
damage by herbivores or by lack of enough phosphorous (Carrapiço,
2010) (Tung et al., 1983) . This color changing is due to the production
of anthocyanin pigments in the vacuoles in the epidermal cells of the
leaves, when anthocyanins levels are increased, it reduces the activity
of photosynthesis (Zimmerman et al., 1985).

Life Cycle and Reproduction

Azolla is a heterosporous aquatic floating pteridophyte plant, so it
neither produces flowers nor seeds. It’s heterosporous because it
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produces 2 kinds of spores, male and female spores structure. Sexual

reproduction is not common in the Azolla life cycle, it’s initiated due to
environmental circumstances that stress the plant to stop vegetative
proliferation and initiate sexual reproduction. We observed in our
experiments in Syria that high temperatures (in early summer) or low
temperature (in early winter) are triggers for the plant to start sexual
reproduction, (Carrapiço et al, 2000) and (Watanabe, 1982) had
confirmed that too. Sporocarps are observed in these circumstances
which are spherical structures located under the branches. When the
sporocarps are formed, the vegetative growth rate is retarded
(Watanabe, 1982).

Figure 2. A: Azolla nilotica, Photo by Ikrami Subhi, Egypt. B: Azolla

mexicana photo by Russ Kleinman., C: A. pinnata (Annonymous,
Azolla pinnata, Accessed on 10/10/2021), D: Azolla caroliniana
(Annonymous, Azolla caroliniana, Accessed on 12/10/2021)
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Sporocarps occur in pairs of either microsporocarps, the male organs,

and mega(macro)-sporocarps represent the female organs. As seen in
Figure 4 the megasporocarp is smaller than the microsporocarp which
is brown-yellow or brownish-red in color. The male sporocarp
“microsporocarp” has a structure that resemble the egg sac of an insect
or a spider, it contains a small granules-like structure called
microsporangia (or male sporangia), within the periplasmodium of
male sporangia 32 to 64 microspores which will develop and aggregate
into massulae (mucilaginous extensions of the tapetum which enclose
the microspores), from massulae glochidia develop. Microspores
germinate and release antherozoids which fertilize the oospore in the
megaspore. The female organ megasporocap is smaller than
microsporocap and produces only one megaspore. After fertilization a
zygote is produced and develops into an embryo and by further
developing a new sporophyte plant is present (Carrapiço et al, 2000)
(BECKING, 1987) (Watanabe, 1982).
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Figure 3. Key stages in the sexual reproduction of Azolla filiculoides.

(a) Sporophyte; bar, 10mm. (b) Megasporocarps (arrows) and
microsporocarp (mi) at the underside of a sporulating plant; bar, 1 mm.
(c) Detached megasporocarp; bar, 0.2 mm. (d) Microsporangium
containing four massulae; bar, 0.2 mm. (e) The massulae’s glochidia
(arrows) allow it to attach to the megasporocarp; bar, 0.2 mm. (f)
Megasporocarp floats (fl) emerge from beneath the indusium cap (ic);
bar, 0.2 mm. (g) Cotyledons (co) push away indusium cap and emerge
from the megasporocarp; bar, 0.5 mm. (h) Sporeling with root (arrow)
detaches from the megasporocarp; bar, 0.5 mm. (i) Azolla sporeling
floating; bar, 1 mm. the image from (Brouwer et al., 2014)
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Fertilization is facilitated by the close proximity of male and female

structures, glochidia have a big role in anchoring massulae to the
filamentous floats that cover megasporocarps thus fertilization occurs
(See Fig 4, 5). The subsequent growth of the embryo to be a mature
vegetative plant takes about 1, 2 months, thus using megasporocarps as
a spawn for inoculation purposes is not practical, we can use it for
research and shipping purposes. The fertilized megaspore can be used
for storage since it can withstand desiccation for more than one year
when stored in dry conditions (Watanabe, 1982), therefore this is one
of the strategies that can be used to save the germplasm collection
during extreme conditions. Usually, its asexual propagation is achieved
by the multiplication of fragmented fern leaves, which is faster than
sexual reproduction. During optimal conditions, Azolla can double its
weight every 2-3 days (Wagner, 1997). The most amazing part of
Azolla is its symbiosis relationship with cyanobacterium Anabaena
azollae which can fix atmospheric nitrogen into ammonium that
transfers to Azolla parts like amino acids (Ray et al., 1978). The bacteria
can pass itself during vegetative and sexual reproduction, the bacteria
can pass from generation to generation in a way as they can’t be
separated during all stages of reproduction. In this sense, we can
consider bacterium-Azolla as an ecosystem model where bacteria and
plants at the boundaries of leaf cavity ( where Anabaena thrive Fig. 6)
had recognized each other and exchanging benefits. The trichomes are
involved in the transfer of nutrients between the fern and bacterium
colonies (Howland et al., 1990). Azolla-Anabaena symbiosis is the only
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system that is applicable to the aquatic ecosystem ( in comparison to

legume-rhizobia systems which are terrestrail) (Qiu et al., 2003).

Growth Requirement
The major requirements for Azolla growing are temperature, water,
light, pH, air and inorganic nutrients. Usually, those variables are
studied individually while others are constant, but from the practical
point of view these variables are integrated in a way results from
laboratory experiments couldn’t be the same as in the fields. Due to that,
reported results are different in the literature.

Figure 4. Sporocarp of Azolla spp. photo by “Department of Botany,

Faculty of Science, Chulalongkorn University”.
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Temperature

Temperature is one of the most important environmental factors for

Azolla spp. growth and geographical distribution in nature (HEIDE et
al., 2005). For mass production for most species the temperature should
be between 18 - 28 Co, some species can be as high as 30 Co for species
such as A. pinnata, A. mexicana, and A. Carolinian (Azolla.Foundation,
Accessed on 15.10.2021). We should consider the relation between
temperature and growth rate and yield, (Peters et al., 1980) had studied
different parameters, including temperature on the growth rate,
acetylene reduction rate, C to N ratio, dry matter, and Chlorophyll
(CHl) a to b ratios of four Azolla species. Under a constant temperature
in the range between 15 - 40 Co under a light

Figure 5. Heterosporous life reproductive cycle of Azolla (Braun-

Howland, 1990).
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intensity of 16 Klx with 16/8-h day/night photoperiod, the maximum

growth rate, nitrogen content, and nitrogenase activity were at their
maximum at 30 Co for A. pinnata and A. mexicana, at 25 to 30 Co for A.
caroliniana, and at 25 Co for A. filiculoides. The strains' tolerance to
elevated temperature at 40 Co for a prolonged time were as the
following A. mexicana > A. pinnata > A. caroliniana > A. filiculoides.
(Tung et al., 1983) worked on 5 species of Azolla, it was measured for
6 weeks under a light intensity of 20klx with a 12-h photoperiod and a
thermoperiod at 37/29 Co, under these conditions A. pinnata stopped
after 4 weeks. The doubling time increased from 2.3 to 3.5 d, while A.
microphylla doubling time decreased by half. Doubling times of A.
caroliniana and A. filiculoides were increased and considered to be heat
sensitive. The strains' tolerance to an elevated temperature at 40 C o for
a prolonged time was as the following A. pinnata > A. mexicana > A.
microphylla > A. caroliniana > A. filiculoides. Table 1 summarized the
most important parameters of each strain; this table guides the farmer
which strain is better according to the local environment and time of the
year. The acclimation of species to specific temperatures could occur,
(Talley et al., 1980) reported that A. filiculoides had increased its
nitrogenase activity with increasing temperature condition stepwise up
to 40 Co and remained high even at 45 Co. Thus, even if we don’t have
all the strains that we need for mass production, we could adapt our
available strains following up the
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Figure 6. Azolla ’s dorsal leaf cavities provide a home for anabaena

from (Azolla.Foundation, Accessed on 15.10.2021), (Carrapiço,
2010).

procedures mentioned in this review to get the best yield. In general,

different reported results are found in literature, to find what is better
for the farmer one should understand the physiology, procedures, and
methods of Azolla cultivation around the world which will guide us to
find what could fulfill our conditions.
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Table 1. Azolla species’ optimal parameters, * (Ashton, 1976)

Optimal Cold
Best Parameters Heat Tolerance Ref.
Temperature tolerance
(Peters et al., 1980)
A. pinnata 30 Heat tolerant
(Bartholomew et al., 1986)
Cold
A. mexicana 30 Heat tolerant (Peters et al., 1980)
sensitive
Cold (Peters et al., 1980)
A. caroliniana 25-30 Heat tolerant
tolerant (Li et al., 1984)
Frost (Peters et al., 1980)
A. filiculoides 20, 27.5* Heat sensitive
tolerant (Talley et al., 1980)
Cold
A. microphylla 25-35 Heat tolerant (Bartholomew et al., 1986)
sensitive
Heat (Li et al., 1984) (Uheda et
A. rubra Cold tolerant
sensitive al., 1999)
Cold
A. nilotica Heat sensitive (Li et al., 1984)
sensitive

Water, Relative Humidity, and pH

Azolla contains 90-95% (Kobiler et al., 1982) water which is
responsible for all the biochemical activities inside plant tissue. Azolla
species are free-floating fern so it needs some depth to grow, shallow
water of 3-5 cm is the best, although it can grow well in greater depth
(Wagner, 1997). When the water content of Azolla decreased to more
than 80% the nitrogenase activity decreased to the fifth of its maximum
(Hechler et al., 1995). If desiccation does occur, growth and nitrogen-
fixing activity are irreversibly inhibited within 24 h. (Ray et al., 1978).
Water turbulence and winds could adversely affect Azolla growth.
Water temperature is related to air and water source temperature, the
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stability of water temperature is crucial for mass and steady production.

According to both air temperature and water temperature the relative
humidity (RH) could be changed accordingly, it should be between 60-
75%, less than 60% the fern dries and become fragile (Bocchi et al.,
2010). The optimal RH for good growth should be between 70-75%
(Costa et al., 1999). The best pH of water of Azolla optimal growth
should be around neutral value (Pasvisheh et al., 2013) and to some
extent, acidic condition (Wagner, 1997). (Serag et al., 2000) concluded
that Azolla can grow in culture solution at pH between 4.5 to 7 and can
survive between 3.5 to 10 provided with the needed nutrients. In Egypt
conditions (Mousa et al., 1994) reported that Azolla can grow well
between 7.1 to 9.0. As a result, the best pH value for optimal Azolla
depends on other environmental conditions that affect nutrient
solubility in water.

Nitrogen
Azolla can grow in natural or artificial conditions without nitrogen
sources, thanks to Azolla- Anabaena symbiosis (Watanabe, 1982). Even
if it can fix atmospheric nitrogen, it needs a level of nitrogen to facilitate
mass growth and nitrogenase activity. It’s a species-specific
requirement, (Carry et al., 1992) reported N-requirement (in the form
of NH4NO3) for maximum biomass production under a controlled
environment for A. pinnata is l mg L−1 of N and 10 mg L−1 of N for A.
filiculoides. More levels of medium nitrate will suppress growth rate
and nitrogenase activity as well. On the other hand, using urea 10 mg
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L−1 had increased nitrogenase activity by 50% (Wagner, 1997) (Singh

et al., 1988).

Phosphorus
This element is the limiting factor for Azolla growth (Lumpkin et al.,
1985), P-deficiency results in a low growth rate, reddish-brown small
leaves, fronds become fragile, roots will be elongated and easily
detached (Watanabe, 1982). In addition, phosphorus deficiency causes
a reduction in the number of heterocysts (Sandrah et al., 1990). In the
laboratory, 0.06 ppm of P in the form of P2O5 is required for normal
growth (Watanabe et al., 1980). However, in the field, 0.3 to 1 ppm is
required to get a good yield (Sandrah et al., 1990) (Carrapiço, 2010).
The best chemical form of P for fertilization is soluble superphosphate
P2O5, citrate soluble phosphate, calcium magnesium phosphate (Tuan et
al., 1979) (Sandrah et al., 1990). It has been estimated that using 1kg of
P2O5 results in 1.9 kg additional nitrogen in Azolla biomass (Watanabe
et al., 1980). The way we add P is very important, it has been found that
split application of a total 15 kg P2O5/ha could give the same fresh
weight and nitrogen yield as the basal application of 30 kg P 2O5. Also,
spit application of 2.5 kg P2O5/ha as superphosphate ( 2 days interval, 6
times, total 15 kg ) had increased the Azolla yield in comparison with
control. Notably, it resembled the application of 30 kg P 2O5/ha basal
using the split method of fertilization (Watanabe et al., 1980). Some
farmers use manure or urine as a source of P, in our experiments in
Syria we found the best application for A. filiculoides is 3g of soluble
super-phosphate/m2. It’s advisable to add P fertilizer in cold weather
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since P water availability is low, that is because of low water

temperature. Although, we can increase sporulation by adding more P
(JANES, 2008).

Salinity
Various studies reveal the negative effect of salinity and Azolla growth
(Sadeghi et al., 2013). High salinity is accompanied by a decrease in
growth rate and the ability of Azolla to absorb heavy metals (Arora et
al., 2003).

Iron
Iron is another limiting factor for Azolla growth since it’s an essential
element in nitrogenase enzyme activity (Watanabe, 1982). Iron-
deficient Azolla appears yellow due to depletion of chlorophyll, roots
become thin and whitish (Wagner, 1997). Iron availability is pH-
dependent. It’s decreased in neutral and alkaline conditions (Watanabe,
1982).

Light intensity
Light affects photosynthesis and nitrogenase activity in Azolla and
Anabaena. It grows well under a shaded area, less than full sunlight
except in high altitudes in spring and fall semesters (Plucknett et al.,
1980).

Applications
We consider Azolla as a super-tiny plant from many biological and
economical aspects, it gives us many opportunities to find some
solutions to our Earth’s ongoing and future problems. The human
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population expansion would be about 9 billion by 2050, as a result,

large amounts of food, water, energy, and agricultural lands are required
to satisfy human nutrition needs. Although climate change is becoming
more noticeable year by year, as an example; nowadays the
Mediterranean region is facing an increased frequency and severity of
droughts, these changes will have economic and social consequences.
Rain-fed agriculture is crucial for the economy of Mediterranean
countries, accounting for more than 90% of cultivated area in Algeria,
Morocco, and Tunisia, 57% in Turkey, 64% Italy, and 56% in Portugal
(Tramblay et al., 2020) and 67% in Syria (Sagardoy et al., 2003).
Drought events are the main driver of crop failure in the world, with a
significant impact on the economy and food security. Azolla farming
could give potential solutions for new countries – as it is in some
societies, Syria is an example. What could Azolla give us in this
context? Azolla can be grown using a hydroponic technique which can
save up to 90% of water in comparison with a typical agricultural
irrigation system. Azolla mutualistic symbiosis with cyanobacterium
Anabaena azollae is unique in nature, this bioreactor can fix
atmospheric nitrogen and provide the required N fertilizer for Azolla to
grow and after it dies it releases N into the soil, thus it is called a
biofertilizer. Azolla can fix in average about 1.0 to 2.6 kg N/ha/day
(Watanabe, 1982), (Schluepmann et al., 2018) calculated N-fixation of
Azolla as 0.15–0.17 mg N/ha per gram on a dry basis (DM), which is
high compared to 0.08 mg N/ha per gram of dry biomass than N-
fixation by Rhizobia residing in the root nodules of soybean (Glycine
max), it can fix nitrogen between 400-1200 kg N/ha/year (Schluepmann
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et al., 2018), thus Azolla can be used as bio-fertilizer (green manure). It

has another advantage, it can double in weight during 2-5 days, with
protein content ranging from 200-400 g/kg of dry weight (DW). The
rough protein in soybean is around 40%, while in Azolla is between 20-
35% (Liua et al., 2008), it’s of interest to be in the formulae of fodder
for livestock (Schluepmann et al., 2018). Azolla can produce on average
56 kg of DM/ha/day, about 20 t/ha/year (Watanabe, 1982), it’s four
times the DM of wheat at its optimal yield. We hope in this section to
revise the importance of Azolla from an application point of view to
sustain food security.

As animal feed
Azolla is rich in proteins, it contains between 20-30% crude protein, 8-
12% fibers, and 10% of carbohydrate (Becerra et al., 1995) (Prajapat et
al., 2018). This high N content makes it attractive to be used as animal
feed for cows, poultry, fish, cattle, sheep, ducks, and buffalos (Sandrah
et al., 1990). The livestock sector is facing an economical challenge in
the Mediterranean region because of COVID-19 consequences on the
economy, supply chains, and climate change. Fodder prices have
increased more than 30% since 2020, soybean which is the main N
source increased by 35% globally, barely grain has doubled its prices.
Livestock and dairy producers facing a hard time, especially in
countries that rely on rain-fed agriculture.
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Figure 7. An integrated farming system of rice, fish, Azolla, and duck

(P refers to production) modified from (Cagauan et al., 2000).

The first use of Azolla as fodder came from an integrated aquaponic

cultivation system in the far east, which grows rice, Azolla, fish, and
ducks. Each part of this system has inputs and outputs, as a result, there
will be a circular advantage for all, Figure 7 illustrates this ecosystem.
(Das et al., 2018) found that substitution 25% of commercial fish feed
with A. pinnata DM will give the same product quality and contribute
to a higher profit margin for rearing Thai silver barb Barbonymus
gonionotus fishes. Other reports have similar results for tilapia ( 15%
A. microphylla ) and carp rearing (7.5 % A. filiculoides) (Magouz et
al., 2020) (Fiogbé et al., 2004) (Namiq et al., 2021). Not all fish kinds
benefit the same, (EL-SAYED, 1992) reported that feeding fingerlings
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and adults of Nile tilapia, Oreochromis niloticus (L.). with A. pinnate

(Fresh and dried Azolla) had negative growth rate and fodder utilization
efficiencies. According to an experiment we conducted in Syria rearing
broiler quails using 0%, 5%, 7%, 10%, 13% of A. filiculoides DM, we
found that substitution 13% of commercial fodder with A. filiculoides
powder gives the same body weight in comparison to control during 60
days of feeding, (MABBAYAD, 1985) found similar result by 15%
feed substitution for broiler chickens. In addition, he found that 20%
substitution has no significant negative effects on egg production and
egg size when fresh Azolla was fed to mallard ducks. (Boitai et al.,
2018) found in his experiments that 10% incorporation of Azolla for
laying hens fodder has no negative effect on egg production or egg
quality. We observed in north Syria increasing in milk production of
rearing dairy cows when we feed daily 2 kg of fresh A. filiculoides (after
washing with fresh water and filtering) mixed with an appropriate
amount of wheat bran and straw with 3k g of concentrate, 2 meals per
day, it raised milk production from 14 L to 16 L per day and lowered
concentrate consumption by 40%, see Figure 8. (Jain et al., 2019)
reported similarly increase 10% in milk production by 25% replacement
of concentrate with 2 kg fresh Azolla per day. It had reported the
digestibility of Azolla is related to its age, 77% digestible during
exponential growth and 49% digestible after it has aged (Eskew, 1985).
The increased production of milk, eggs, or meat exceeds the nutritional
value of introduced Azolla, some reported it is the rule of other
components; like carotenoids, probiotics, fibers, and bio-polymers that
contribute to better biological conversion efficiency in animal rearing
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(Shreedhar et al., 2016) (Oktavianawati et al., 2016) (Shambhvi et al.,

2020). It is advisable to vaccinate the livestock against Enterotoximea
before Azolla feeding (Van Metre et al., 2008).

Human food or supplement

Many different crops that were used before as animal feed it has become
now as precious food, e.g., potatoes, beans. Azolla's nutritional content
makes it suitable for human consumption, it could be used in salad,
soup, or burgers. Erik Sjödin a Swedish artist fascinated by Azolla
properties conducted experimental preparation of food based on this
fern, he introduced his recipe on his website (http://www.eriksjodin.
net). However, no long-term studies were conducted for the safety of
Azolla as human food.

Soil fertilization
As we mentioned before the rate of N-fixation is high in Azolla, thus it
can be used as green manure, this practice came from the co-farming of
rice and Azolla, the latter can supply about 25-30 kg N/ha per
incorporation (KANNAIYAN, 1985). It has positive effects on the
physio-chemical properties of soil when it is applied as green manure,
it decreases the specific gravity, increases the porosity and organic
matter in the soil (Lumpkins, 1980).
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Figure 8. Feeding dairy cow with Azolla-supported feed, Syria Aleppo,

Ershaf town. A: washing fresh collected Azolla at afternoon, B,C:
mixing azolla with wheat and straw, D: dairy cow eating the Azolla-
supported meal.
Other Applications

Azolla has many applications, it can be used in sewage treatment

because of its ability to absorb efficiently P, N, K, and heavy metals,
therefore it can be used in phytoremediation of heavy metals in
environmental refinement projects (Naghipour et al., 2018). Inhibiting
mosquito proliferation, because of that it’s called mosquito fern. It’s
studied for astronomical applications since it can absorb urine, NH4-N
compounds, and concentrated ions, it has strong O2-releasing capacity
and fixes atmospheric nitrogen, rapid growth rate and it’s eatable. So,
it could be a solution for astronomers as a life-supporting system (Liua
et al., 2008). Due to mass production, it can be used in biofuel
production as well.
 SUSTAINABLE AGRICULTURE AND LIVESTOCK FOR FOOD SECURITY UNDER THE
CHANGING CLIMATE | 485

Ecological and environmental precautions

Azolla farming should be under governmental control, in some
countries, it’s considered as a water weed plant. Azolla now had
introduced into many countries because of its benefits. Syria is an
example; it is considered as an introduced species that could harm the
ecosystem. It has been reported to disrupt fishing, access to water by
livestock, clog pipes, pumps, and floodgates and interfere with
watercress cultivation (Plucknett et al., 1980). Efforts of legislation and
field control should be considered in places that had been introduced.

Growing techniques and practices for Azolla production as animal

fodder-Syrian Model
We found in order to supply two cows daily with 4-8 kg of Azolla, or
38 chickens, or 190 quails (for small business) the farmer needs a
shaded pond or shaded area, it could be beside a wall or between large
branches trees (Olive tree farms are very common in Syria). An area of
20 m2 (4m*5m) should be leveled and covered with a plastic silpaulin
sheet with raised edges using a cob building system (see Figure 9), or it
could be in a pitch, 20 cm height. The leveling of the basin and its edges
is important to get a uniform water level. Sieved fertile-rich soil must
be spread on the silpaulin sheet 2-3 cm thick. After filling the basin with
water we recommend using the following fertilizer formulae for every
1 m2 (or 100 L of water); 3g superphosphate, 0.3g ferric chelators, 5g
20-20-20 NPK powder, and 0.2 kg of dry composted cow or sheep
manure, the manure should be put inside a gunny sack and soaked under
the water level. To keep Azolla production to its maximum (between
486 | SUSTAINABLE AGRICULTURE AND LIVESTOCK FOR FOOD SECURITY UNDER
THE CHANGING CLIMATE

150-200g daily/m2) the farmer should consider the following: Fertilizer

formulae application should be repeated once every month. Unless
composted manure should be applied every week. Water should be
replaced 20-30% with new freshwater every 7-10 days, to prevent
building up of nitrogen, micro-nutrient deficiency, preventing
anaerobic bacterial sediment and pest emerging. pH should be kept
between 5.5-7 Soil sediment should be replaced at least once every
three months with new sieved fertile-rich soil.

Figure 9. Azolla basins constructed using cob system of building

using clay, straw, stones and water to raise the edges. 13/10/2021,
Aleppo, Afrin, Syria.
Water temperature should be between 25-30Co, if it goes up, light
shading of 50% should be used (shade net or silpaulin sheets, Figure
10). Fresh healthy Azolla inoculum free of pests or diseases should be
soaked with suitable insecticide and fungicide for previous 24h,
inoculum introduced as 0.5 kg/ m2, once Azolla has fully covered all the
area ( about seven days) we can take half the grown area and inoculate
a new one, and so forth. This is called the half-saturation technique.
 SUSTAINABLE AGRICULTURE AND LIVESTOCK FOR FOOD SECURITY UNDER THE
CHANGING CLIMATE | 487

Fresh harvested Azolla should be washed with clean water before use
to get rid of any bad smell. Harvesting should be afternoon because
Azolla grows most notably during the morning hour, this practice will
help Azolla grow exponentially; when crowding occurs, the growth rate
goes down. During hot climates and high humidity, the fungal disease
could spread, shading is the cheapest procedure to do plus fungicide
e.g., Carbendazim 50ppm. Making separate basins is a good practice,
so diseases could spread in one but not in all of them. Accordingly,
treatment will be different and can be managed differently. When the
growth rate becomes low in optimal conditions, the farmer should seek
what the causes are; pests and fungal diseases are the first things to be
noticed. Quick treatment is helpful, otherwise, the crop will decline
rapidly. The previous considerations are based on the NARDEP method
of cultivation (Pillai et al., 2002) with some modifications based on our
experiments.

Figure 10. Azolla basin shaded silpaulin sheets, supply 2 cows and 15
laying hens, Aleppo, Ershaf, Syria. Photo taken by Nwat center team,
01/09/2021.
488 | SUSTAINABLE AGRICULTURE AND LIVESTOCK FOR FOOD SECURITY UNDER
THE CHANGING CLIMATE

How to Protect Azolla from High or Low Temperature

When air and water temperature go under 15 Co Azolla growth rate will
be too low, during this time farmers should protect what is left for the
next semester. Practically we found the following procedure benefits
the most. Making a small Azolla basin covered with a light-transparent
plastic sheet (used for plant nurseries) is good, or we can put them
inside a plant nursery. The amount should be enough for inoculating all
the basins in the next semester.

Figure. 11. Azolla spores collected in early winter for storage, photo
taken from Nwat center laboratory.

Growing Azolla to be thick layer 3-4 cm is helpful, that will protect it

from cold (0.7-1 kg per 1 m2, it’s called over-wintering a technique used
in China (Sandrah et al., 1990). Choosing the right species or strain
from research centers is valuable. Putting more P fertilizer in the basin
is good practice, that will encourage sporulation as well. Adding
fungicide (e.g. Carbendazim 50ppm) and insecticide 50 (e.g., 50 ppm
of Phenthoate + MIPC 3 + 3% G and Propaphos 5% G, or Cartap 4%
G, or Ethoprop 10% G ; Carbofuran 3% G and Diazinon 5%G, or
 SUSTAINABLE AGRICULTURE AND LIVESTOCK FOR FOOD SECURITY UNDER THE
CHANGING CLIMATE | 489

Benfuracarb 5% G ) is helpful to prevent losing the seeds. Spraying of

soluble silicate at 10mM enhances cold tolerance in crops via silica
deposition-induced strengthening of cell walls (Hashem et al., 2021)
(no reports are available regarding Azolla cultivation), it is useful as
well to enhance hot tolerance. In hot conditions, a farmer should
monitor air temperature, water temperature, pH, humidity, and pests
with caution. Temperatures are of main concern, shading and water
replacement are among the best procedure to prevent crop decline.
When pests are not under control, replacing the basin with a fresh one
is the best choice. In some circumstances, we can use spores as seeds,
but this is not practical. Little is known about spores’ germination
procedure what is known that light and plant density, temperature,
photoperiod are the triggers responsible for spores’ germination
(Sandrah et al., 1990). It takes about 1 to 2 months for a fertilized
megaspore to become a new sporophyte plant (Figure .11).

Harvesting and Storing

After harvesting, Azolla leaves are washed with fresh tap water to
remove debrides, bad smell (manure smell). Then we spread it on a
well-ventilated area under shade. After a while (when it loses some of
its humidity) we mix it with fodder to feed animals or leave it to dry
completely. Each 100 kg of fresh Azolla gives 10-12 kg dry matter.
Then it can be milled to be incorporated into animal fodder formulae.
Milled Azolla powder should be stored in a plastic bag in a dry place.
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THE CHANGING CLIMATE

Integrated Farming System for Local Recycling of Nutrients

Using: Azolla, BSF, Poultry
For the last six years, we have studied many solutions under the concept
of “Local Recycling of Nutrients Ecosystem (LRN Ecosystem)”, this
concept relies on circulating nutrients using biological and
biotechnological techniques. Farmers by using such an approach reduce
their consumption of fodder remarkably and maximize profits. The
coming years are challenging for humanity to satisfy food demands,
such a concept will help many countries to increase their local
production, support food security, hire more people, and recycle local
nutrients more efficiently. An application of this concept is using
Azolla, Black Solider Fly Hermetia illucens (BSF), and poultry
integrated into one place. Poultry gives manure which can be mixed
with Azolla to feed BSF larvae, BSF larvae plus fodder, and Azolla used
to feed poultry, the larvae-residue from BSF used to fertilize Azolla
with nutrients (Kawasaki et al., 2020), herein cycle closes, Figure 12.
This model is promising and is under evaluation in Syria.
 SUSTAINABLE AGRICULTURE AND LIVESTOCK FOR FOOD SECURITY UNDER THE
CHANGING CLIMATE | 491

Figure 12. Azolla, BSF and Poultry integrated farming system as an

application of local recycling of nutrients ecosystem concept (LRN
Ecosystem).

Economics of Azolla Cultivation

Few reports consider the feasibility of using Azolla as a partial
substitute for animal feed. In general, Azolla production has good
potential profits in countries the labor cost is lower than 12$/day. Azolla
farms need laborers to get mass production efficiently; the labor cost
depends on management and production goals. We calculated the cost
construction of the Azolla pond of 20 m2 as the following (based on
Syria’ costs):
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THE CHANGING CLIMATE

Table 2. Economics of Azolla Cultivation

# Items Units Unit Cost $ Total $

2
1 plastic silpaulin sheet 6*8 m 24 m 0.55 13.2
2 Labor cost for leveling and raising one time 25 25
edges
3 Shade net 30 m2 0.50 15
4 Azolla spawn 2 kg 12 24
5 Composted manure once/week for a 208 kg 12.5 12.5
year
6 Fertilizer 166g / month 1.992 g 6 6
7 Insecticide & Pesticide once / year q.s. 5 5
100.7 $

If we supposed water source is available for free and the labor is the
farmer, this infrastructure should work for nine months on average with
a minimum supposed yield at 100g/m2; accordingly, it will produce 540
kg of fresh Azolla. Therefore, the cost of production of fresh Azolla in
Syria is less than 0.18$/kg. It will replace 4 kg of daily concentrate for
a dairy cow, which means saving 1.6$/day (0.4$ concentrate/kg).
During nine months the farmer will save 432$ with better milk
production as we mentioned before. Similarly, we can calculate for
poultry, fish,.. etc.

Conclusions and Future Research

Azolla has potential applications to be used as an ingredient in animals
fodder formulae, its nutritional profile, and mass productivity in
comparison with other crops make it a “super-plant”. More research
 SUSTAINABLE AGRICULTURE AND LIVESTOCK FOR FOOD SECURITY UNDER THE
CHANGING CLIMATE | 493

should be done in understanding the adaptation of Azolla to new

conditions, to know how it will adapt to climate changes. Crossbreeding
of different Azolla species to develop better strains that could stand
thermal extremes, salinity, phosphorus deficiency, better growth rate,
and nitrogen fixation rate. Defining new strains with well-known
properties to be used commercially for different purposes. More
research in finding the best fertilizing regimen and best formulae with
better yield. Extending research about using different organic fertilizers
produced by livestock as a main source of fertilizer is very important in
developing countries. Further studies on large-scale production
management and its economy on local markets. Additionally,
developing growing procedures by which we can guide Azolla to
produce more crude protein or more carbohydrate as we need, such
progress will have valuable applications in biofuel production. Building
an open-source forum community from all over the world to share
knowledge and ideas about Azolla will have a big impact.

Acknowledgments
We acknowledge Dr. Tefide Kızıldeniz for her support. Grateful thanks
to our inspiring team at Nwat Center for Scientific Studies. We
gratefully recognize the help of Ghiras Al-Nahda organization and
NuDay Syria organization for their support. The assistance and fund
provided by Mr. Mahmoud Koubais to conduct experiments in his
Grand Farms company were greatly appreciated. We would like to
thank Sara Al Sbei for her contribution and support, without her this
chapter couldn’t be written. Special thanks to Dr. Mohamad Houssam
494 | SUSTAINABLE AGRICULTURE AND LIVESTOCK FOR FOOD SECURITY UNDER
THE CHANGING CLIMATE

Al balkhi and Dr. Mohammad Amin Mohammad whose sincerity and

encouragement we will never forget.
 SUSTAINABLE AGRICULTURE AND LIVESTOCK FOR FOOD SECURITY UNDER THE
CHANGING CLIMATE | 495

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