1

A Paper on

Azolla: Its Biology, Phytochemical Profile and Utilization

submitted to:

Dr. Agnes T. Aranas

Chemistry Department
Ateneo de Davao University

As a partial requirement for the subjects

Chem 661 & 652

Graduate Readings and Seminar

by

MARIVIC D. CANDARI

MS Chemistry

Summer 201
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TABLE OF CONTENTS

INTRODUCTION 3

History 4

Azolla in the Philippines 4

BIOLOGY AND PHYTOCHEMIAL PROFILE OF AZOLLA 6

Biology 6

Classifiaction 6

Characteristics 7

Chemical Composition 8

UTILIZATION OF AZOLLA 12

History of Azolla Use 12

NEW DEVELOPMENT IN AZOLLA USE 13

Azolla as Biofertilizer in Rice Cultivation 13

Azolla as Nutritional Supplement for Livestock 15

Azolla in Bioremediation 16

Azolla and Bioenergy 16

Azolla as a Human Food 17

Azolla as a Component of Space Diet 17

Azolla as a Reducing Agent in Green Synthesis

of Nanoparticles 18

CHALLENGES IN AZOLLA RESEARCH 17

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I. INTRODUCTION

Azolla is an aquatic pteridophyte widely distributed in the water bodies. It has

been traditionally used as a biofertilizer for rice paddy fields owing to its potential

to fix atmospheric nitrogen. Azolla is a unique plant that can help reduce man-

made climate change and provide biofertilizer, livestock

feed, food and renewable energy anywhere in the world [1]. The main parameter

responsible for the value of Azolla as feed or fodder of the livestock and fishes is

its amino acids content followed by its other compounds like crude protein, fibre

and digestible carbohydrates.

Azolla is a genus of six species of aquatic ferns, the only genus in the family

Azollaceae. It grows naturally in stagnant water in drains, canals, ponds, rivers

and water bodies including marshy lands with temperature range of 15-35C[1].

The floating water fern Azolla is one such potential novel crop. It can be

cultivated in closed systems on non-arable land or in natural occurring freshwater

basins.. The aquatic fern azolla is one of the fastest-growing nitrogen-fixing

plants on earth and therefore considered as a potential source of biomass for

biomass production. The unique long-chain (di) hydroxy fatty acids and fatty

alcohols that are found and separated from the azolla may provide a valuable

secondary product stream when purified into biochemical, with possible

applications to chemical industry and nutrition[2].

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Azolla also has numerous names in different countries and languages,

including the following, with the local names as: Carolina fern, Duck fern,

Duckweed fern, Fairy fern, Fairy moss, Feathered water fern, Mosquito fern*,

Pacific mosquito fern, Red water fern, Water velvet [3].

History

Azolla was believed to be found on the Arctic Ocean when its primary

opening, the Turgay Sea was blocked nearly 50 million years ago. The enclosed

body of water became the perfect habitat for a small leaved fern called azolla.

Azolla took advantage of the abundant nitrogen and carbon dioxide, two of its

favorite foods, and flourished. Large populations formed thick mats that covered

the body of the lake. When rainfall increased from the changing climate, flooding

provided a thin layer of fresh water for azolla to creep outward, over parts of the

surrounding continents. The thick blanket of sediments of azolla was found in

2004 by the Arctic Coring Expedition[4].

Azolla in the Philippine

Azolla research was introduced in the Philippines through the International

Rice Research Institute as early as 1975. Applied research on Azolla utilization in

lowland rice was initiated by the Bureau of Soils in 1979. The National Azolla

Action Program and the Unified Azolla Program were created to promote Azolla

culture and utilization nationwide. They include basic and applied research
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components and training of MAF field personnel and rice farmers. The Philippine

Government fully supports the dissemination of Azolla technology in its

Masagana 99 rice production program, particularly in irrigated areas.

The National Azolla Program

The University of the Philippines at Los Baos (UPLB) and the Ministry of

Agriculture and Food (MAF) in July 1982 launched a research and extension

Azolla Program. The program was designed primarily to promote the utilization of

Azolla as an alternative source of N for wetland irrigated rice culture. The

National Azolla Action Program (NAAP) has two broad objectives: 1. To reduce

by 50% inorganic N fertilizer use in wetland irrigated rice 2. To reduce the

dependence of rice farmers on inorganic N fertilizers.

To achieve the goals of the program, four major thrusts were considered 1.

establishment of a National Inoculum Center (NIC) at UPLB and subcenters in

selected agricultural institutions. The center and subcenters serve as sources of

pure inoculum of superior Azolla strains. They also conduct short-term research

in support of the Action Program; establishment of Regional Propagation Centers

(RPCs) in the 12 administrative regions of the country to serve as initial sources

of inoculum for distribution to provincial and community nurseries; 3. preparation

of an area operation plan for each region to identify, classify, and prioritize

wetland rice growing areas suitable for Azolla growth; and 4. training and the

production of information and extension materials. [5].

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II. BIOLOGY AND PHYTOCHEMICAL PROFILE OF AZOLLA

Biology

The Azolla are heterosporous free-floating freshwater ferns that live

symbiotically with Anabaena azolla, a nitrogen-fixing blue-green algae. Azolla

plants consists of a main stem growing at the surface of the water, with alternate

leaves and adventitious roots at regular intervals along the stem. Secondary

stems develop at the exit of certain leaves. The azolla fronds are triangular or

polygonal and float on the water surface individually or in mats. Plant diameter

ranges from 1-2.5 cm for small species like Azolla pinnata to 15 cm or more for

A. niloticaa [6].

Classification

The genus Azolla was established by the French naturalist Jean-Bapitiste

Lamarck in 1783 based on the specimens collected by Philibert Commerson and

his assistant Jeanne Bar from the Magellan region of South America

during Louis-Antoine de Bourgainvilles 1766-1769 expedition around the world

[7].

The genus Azolla belongs to the single genus family Azollaceae which

includes six species distributed widely throughout temperate, sub-tropical and

tropical regions of the world.

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The six recognizable species within the genus are grouped under two

subgenera: Euazolla (characterized by three megaspore floats and septate

glochidia) and Rhizo sperma (characterized by nine megaspore floats). The four

species under the sub-genus Euazolla are A. filiculoides, A. caroliniana, A.

mexicana and A. microphylla. It is thought that these four species originated from

temperate, sub-tropical and tropical regions of North and South America. The two

species under sub-genus Rhizosperma are A. nilotica and A. pinnata. A. nilotica

is a native of East Africa and can be found from Sudan to Mozambique. A.

pinnata has two different varieties that vary in their distribution patterns. A.

pinnata var. imbricata originates from subtropical and tropical Asia while A.

pinnata var. pinnata occurs in Africa and is known as African strain[6,13].

Characteristics

Because Azolla has a higher crude protein content (ranging from 19 to 30

percent) than most green forage crops and aquatic macrophytes and a rather

favourable essential amino acid (EAA) composition for animal nutrition (rich in

lysine), it has also attracted the attention of livestock, poultry and fish farmers [8].

In Asia, Azolla has been long used as green manure for crop production

and a supplement to diets for pigs and poultry. Some strains of Azolla can fix as

much as 2-3 kg of nitrogen/ha/day. Azolla doubles its biomass in 3-10 days,

depending on environmental conditions. Azolla provides a protected environment

and a fixed source of carbon to the blue-green filamentous algae Anabaena . [9].
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Chemical composition

The chemical composition of Azolla species varies with ecotypes and with

the ecological conditions and the phase of growth. The dry matter percentage of

different Azolla species varies widely and there is little agreement between the

published data on this subject: values of 5 to 7 percent can, however, be taken

as fair estimates [4] However, a higher percentage (8.7%) was also reported

[10].

The chemical compostion of Azolla pinnata is presented in Table 1 below.

Table 1. Chemical Composition of Azolla

Parameters Values in % of dry matter

Dry matter 8.0

Crude protein 24.61

Crude fibre 15.0

NDF 43.8

ADF 31.8

Lignin 11.4

Ether extract 3.8

Ash 15.9

Starch 4.1
Source: Roy et al. Advances in Life Sciences 5(7).
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Hassan and Chackrabati also presented a summary of the chemical

compostions of various Azolla species presented in Table 2. As seen in table 2,

under natural conditions, values near 20-22 percent are frequent. Also, Azolla

have high ash contents, varying between 14-20 percent. No clear interspecific

difference in the crude lipid levels of various Azolla species occurs; the value is

around 3-6 percent on a DM basis.

Table 3 showed the mineral content of Azolla as presented by Roy et

al.[11]

The amino acid composition and the phytochemical components of the

azolla are shown in Table 3 and 4 respectively.

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Table 3. Amino Acid Composition of Azolla

Amino acids Values as % protein Values as % of dry matter

Alanine 6.4 1.52

Arginine 5.9 1.45

Aspartic acid 9.3 2.37

Cystine 1.6 0.19

Glutamic acid 12.6 2.73

Glycine 5.6 1.40

Histidine 2.1 0.47

Isoleucine 4.5 1.20

Leucine 8.4 2.15

Lysine 4.7 1.02

Methionine 1.4 0.43

Phenylalanine 5.4 1.41

Proline 4.9 1.19

Serine 4.5 1.17

Threonine 4.7 1.14

Tryptophan 1.8 0.40

Tyrosine 3.6 0.95

Valine 5.5 1.45

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Table 4. Phytochemical Components of Azolla

Source: Dr. Veerabahu et al. International Journal of Current Agricultural Research Vol. 4, No. 8

UTILIZATION OF AZOLLA

History of Azolla Use

Rice farmers used Azolla as a rice

biofertilizer 1500 years ago. The earliest

known written record of this practice is in a

book written by Jia Ssu Hsieh (Jia Si Xue)

in 540 A.D on The Art of Feeding the

People (Chih Min Tao Shu). By the end of

the Ming Dynasty in the early 17th

century, Azollas use as a green compost

was being recorded in numerous local

records.
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The use of Azolla in rice production use dates back at least a thousand

years in Vietnam. Vietnamese rice farmers had to travel each year to villages

such as Bich Du so that they

could buy new Azolla stock

called Beo Going for starter

colonies because their own

plants perished each year.

Photo shows farmers collecting

azolla from inoculum producing
pond.

Only a few families knew the secret of maintaining and multiplying Beo

Going during the hot summer months, maintaining their monopoly of

the Azolla inoculum by guarding their secrets through a system of inheritance.

This restricted Azollas use in Vietnam to provinces such as Nam-Dinh, Hai-

Duong and Hung-Yen that were close to villages with Azolla inoculum.

In 1954 the government became interested in Azollas potential to

significantly increase rice production. More than a 100 depots were established

to cultivate and distribute Azolla inoculum, gradually extending its use throughout

the country. By 1973, Vietnamese rice-Azolla cultivation had been established in

half a million hectares, mostly in the northern part of the country [4].
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In the early 1980's, the use in South Cotabato, Philippines had spread.

However, since the late 1980's, the socio-economic situation in these countries

has drastically changed with the introduction of market economy system.

Increased use of chemical fertilizers, the search for a high income crop during

azolla growth period, and the collapse of farmers' organizations who provided

inoculum for rice cultivation are factors to explain rapid decline of azolla use [12].

Azollas use as a biofertilizer is now widespread in rice cultivation

throughout the Far East, including Bangladesh, Brazil, Burma, China, India,

Indonesia, Korea, Nepal, Pakistan, Sri Lanka, Thailand, Vietnam and the

Philippines[3].

NEW DEVELOPMENT IN AZOLLA USE

Azolla as Biofertilizer in Rice Cultivation

Azolla is cultivated in the paddy field either as monocrop or as intercrop

and incorporated into the mud/soil for increasing humus and nutrient content of

the soil. This practice of Azolla cultivation is widely popular in the countries of

south-east Asia like India, China, Phillipines, Indonesia etc. [13]. When Azolla

used as a bio-fertilizer in paddy field it produces around 300 tons of green bio-
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hectare per year under normal subtropical climate which is comparable to 800 kg

(1800 kgs of urea) of nitrogen [9]. Azolla has quick decomposition rate in soil and

thus it speeds up the efficient availability of its nitrogen to rice plant. The quick

multiplication rate and rapid decomposing capacity of Azolla has become

paramount important factor to use as green manure cum bio-fertilizer in rice field

[13].

Azolla as Nutritional Supplement for Livestock

Azolla is used as food supplement for variety of animals like cattle, goat,

pigs, rabbits, chickens, ducks and fish. Out of several species of Azolla, Azolla

microphylla has been reported to be best suited for tropical climate and livestock

feeding. The addition of fresh azolla to the diets of cows also proved to increase

feed efficiency and exhibit good dairy performance [14].

Incase of Black Bengal goat, replacement of the concentrate up to 20%

with sun-dried Azolla resulted better growth with no adverse effect on its

health[15] In other experiment, broiler chicken were feed with maize and

soybean meal that was replaced by dried A. pinnata (10%) and found to be cost

effective [16]. A similar experiment with layer birds fed with fresh Azolla resulted

in a higher body weight at 8 weeks or higher egg production at 40 and 72 days

than control [13].

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Azolla in Bioremediation

It was found that A. pinnata and Lamna minor removed the heavy metals

iron and copper from polluted water [17]. The pollutants at low concentration

could be treated by passing it through ponds and can be reused for Agriculture

purpose. A similar study was conducted by Cohen-Shoel et al. on the biofiltration

of toxic elements by Azolla biomass [16]. Azolla exhibits a remarkable ability to

concentrate metals Cu, Cd, Cr, Ni, Pb and nutrients directly from pollutants or

sewage water [13].

Azolla and Bioenergy

A systematic analysis of terrestrial plants for their potential to be ideal

bioenergy crops led to a number of important selection criteria which include: (1)

chemical composition and high proportion of biofuel-producing molecules; (2)

high growth rates/biomass production; (3) high harvest index/rotation period; (4)

ability to grow on marginal lands and lack of competition with agricultural crops

for arable lands; (5) high freshwater use efficiency and low growing cost; (6) low

harvest cost; and (7) production of high value co-products [18]. Azolla species

together with their evolutional cyanobacterial symbiont, A. azollae, have attracted

attention because of their potential to address all of the criteria mentioned above.
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Azolla as a human food

Azolla is widely used as a livestock feed in India and the Far East, but

its potential as a food for people is less well known.

In The Azolla Cooking and Cultivation Project artists, researchers,

farmers, gardeners, chefs and scientists experiment with cooking and cultivating

the water fern Azolla. Azolla is one of the world's fastest growing plants and a

rich source of nutrients. Yet it is virtually unexplored as a foodstuff for human

consumption[19].

Azollas protein content is close to that of soybean. It is a rich source of

minerals (10-15% of dry weight), essential amino acids (7-10% of dry weight),

vitamins and carotenoids. 20 30% of Azollas dry weight is protein, which is a

lot for a vegetable.

Azolla as a Component of Space Diet

The use of azolla is now changing and some of the impetus has come

from studies into diets that could be used for space stations, space travel, and

habitation on the Moon and Mars. Recent research by Katayama et al., 2008 in

collaboration with Space Agriculture Task Force evaluate a candidate diet and

specify its space agricultural requirements for habitation on Mars. Rice, soybean,

sweet potato and a green-yellow vegetable have been selected as the basic
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vegetarian menu. The addition of silkworm pupa, loach, and Azolla to that basic

menu was found to meet human nutritional requirements [20].

Azolla as a Reducing Agent in Green Synthesis of Nanoparticles

A study on the use of Azolla for biosynthesis of GNPs using methanol

extract of Azolla microphylla was conducted by Kunjiappan et al. [21]. In their

study, they have successfully conducted an environmentally benign synthesis of

gold nanoparticles (GNPs) using methanol extract of Azolla microphylla as the

stabilizing and reducing agent. The synthesized GNPs showed excellent

antioxidant activity.

CHALLENGES IN AZOLLA RESEARCH

A major challenge for the researchers is how to fully utilize the massive

green potential of Azolla. A group of researchers will attempt to sequence the

genomes of Azolla and its symbiotic bacteria, so that they can understand the

two-way, inter-kingdom language that codes for the molecular machinery

underlying this symbiotic partnership, and possibly tailor it to suit our needs.

In the microscale, recommendations are to have more researches of the

phytochemical and biological profile of the different species of azolla and in their
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different stages of growth to be able to maximize the use of the plant for various

applications and needs.

Much research interest is also developing toward the identification of the

medicinal values of azolla species as well as its possible uses in environmental

conservation and energy production.

REFERENCES:

1. Dr. Veerabahu, C., * Dr. Radhika, D., Mohaideen, A., Indrani, S. and
Priya, R..2015. Phytochemical and Biochemical Profiles of Azolla
microphylla Cultured with Organic Manure. International Journal of
Current Agricultural Research Vol. 4, No. 8, PP. 131-133 August 2015
http://www.wrpjournals.com/journals/V/IJCAR

2. Paul Brouwer, Adrie van der Werf, Henriette Schluepmann, Gert-Jan

Reichart, Klaas G. J. Nierop..2015. Lipid Yield and Composition of Azolla
filiculoides and the Implications for Biodiesel Production. BioEnergy
Research August 2015 DOI: 10.1007/s12155-015-9665

3. Van, H.C. 1989. Azolla and its multiple use with emphasis on Africa. Food
agricultural Organization, Rome. 21:112-116.

4. Jennifer Huisen. 2014. Can The Fern That Cooled The Planet Do It
Again? Scientific American. E&E News. Published on July 15, 2014 in
Climatewire with permission from Environment and Energy Publishing
LLC. www.eenews.net,202-628-6500.

5. AZOLLA UTILIZATION Proceedings of the Workshop on AzoIla Use

Fuzhou, Fujian, China 31 March-5 April 1985
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6. Ferentinos L, Smith J, Valenzuela H. 2002. Azolla. University of Hawaii.

3 p. (Sustainable Agriculture; SA-GM-2).

7. Louis Antoine de Bougainville: "Voyage around the world 1766-1769",

London 1772 (A transcription of the translation of "Le voyage autour du
monde, par la frgate La Boudeuse, et la flte L'toile" into English by
John Reinhold Forster)

8. Cagauan, A. G. ; Pullin, R. S. V., 1991. Azolla in aquaculture: Past,

present and future. In J. Muir & R.J. Roberts, eds. Recent Advances in
Aquaculture. Oxford, Blackwell Science, pp. 104-130

9. Wagner, G.M. 1997. Azolla: A Review of Its Biology and Utilization. The
Botanical Review 63(1): 1-26.

10. A. Chatterjee, Puneet Sharma, M.K. Ghosh, M. Mandal and P.K. Roy
Utilization of Azolla Microphylla as Feed Supplement for Crossbred
Cattle. 2013. International Journal of Agriculture and Food Science
Technology. ISSN 2249-3050, Volume 4, Number 3 pp. 207-214
Research India Publications http://www.ripublication.com/ ijafst.htm

11. D. C. ROY, M.C.PAKHIRA AND M.ROY. 2016. Estimation of Amino

Acids, Minerals and Other Chemical Compositions of Azolla Advances in
Life Sciences 5(7), Print : ISSN 2278-3849, 2692-2696

12. Hussner, A. (2010): NOBANIS Invasive Alien Species Fact Sheet

Azolla filiculoides. From: Online Database of the European Network on
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x/x/201x.

13. D. C. Roy, M.C.Pakhira And S.Bera. 2016. A Review on Biology,

Cultivation and Utilization of Azolla. Advances in Life Sciences 5(1), Print
: ISSN 2278-3849, 11-15, 2016

14. Tamang, Y. and Samanta, G., 1993. Feeding value of Azolla (Azolla
pinnata) an aquatic fern in Black Bengal goats. Indian J. Anim. Sci., 63
(2) : 188-191

15. Ali, M. A. and Leeson, S. 1995. The nutritive value of some indigenous
Asian poultry feed ingredients. Anim. Feed Sci. Technol., 55 (3-4): 227-
237
16. Cohen-Shoel, N.; Barkay, Z.; Ilzycer, D.; Gilath, I. and Tel, O.R. 2002.
Bio-filtration of toxic elements by Azolla biomass, Water, Air and Soil
Pollution., 135: 93-104.
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17. Jain, S.K.; Vasudevan, P. and Jha, N.K. 1989. Removal of some heavy
metals from polluted water by aquatic plants: studies on duckweed and
water valvet. Biol. Wastes., 28:115-126

18. Henry RJ. Evaluation of plant biomass resources available for

replacement of fossil oil. Plant Biotechnol J. 2010;8(3):28893.

19. Erik Sjdin.2012.The Azolla Cooking and Cultivation Project. Retrieved

from: http://eriksjodin.net/projects/theazollacookingandcultivationproject/
on May 26,2017.

20. Katayama, N.; Masamichi, Y.; Yoshiro, K.; Chung, C.L.; Watanabe, I.
and Hidenori, W. 2008. Space Ag. Task Force-Azolla as a component of
the space diet during habitation on Mars, Acta Astronautica., 63:1093-
1099

21. Selvaraj Kunjiappan, Ranjana Chowdhury , And Chiranjib Bhattacharjee.

2014. A Green Chemistry Approach For The Synthesis And
Characterization Of Bioactive Gold Nanoparticles Using Azolla
Microphylla Methanol Extract Front. Mater. Sci. 2014, 8(2): 123135 DOI
10.1007/s11706-014-0246-8

22. FAO 2015. Feedipedia: An online encyclopedia of animal feed, retrieved

from www.feedipedia.org on 27.05.2015

23. Dr. Iwao Watanabe. ABC of Azolla. http://www.asahi-net.or.jp/~it6i-

wtnb/AzollaN/azollaE.html#ch2.1 (Made on 1997.6.25, Revised on
2016.9).