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Biomass Growth and Composition of Azolla (Azolla Pinnata R. BR.)

Supplemented With Inorganic Phosphorus in Outdoor Culture

Article  in  SAARC Journal of Agriculture · June 2021

DOI: 10.3329/sja.v19i1.54788

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SAARC J. Agric., 19(1): 177-184 (2021) DOI: https://doi.org/10.3329/sja.v19i1.54788

BIOMASS GROWTH AND COMPOSITION OF AZOLLA (Azolla

pinnata R. BR.) SUPPLEMENTED WITH INORGANIC
PHOSPHORUS IN OUTDOOR CULTURE
M.A. Hossain1*, S.A. Shimu1, M.S.A. Sarker3
M.E. Ahsan2 and M.R. Banu2
1
Department of Aquaculture, 2Department of Fisheries Management
Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh
3
School of Agriculture and Rural Development, Bangladesh Open University
Gazipur, Bangladesh.

ABSTRACT
An experiment was conducted to know the effect of supplemental
phosphorus on biomass growth and composition of a floating aquatic
fern, Azolla pinnata, cultured in a pit system for 21 days with 4 levels of
phosphorus (0, 5, 10, and 15 ppm) supplementation. Water quality
parameters of the pits were within a suitable range for A. pinnata culture.
It was observed that fresh and dry weights of A. pinnata increased with
phosphorus supplementation up to 10 ppm. Doubling time was the
fastest when the culture medium was supplemented with 10 ppm of
phosphorus. Phosphorus content of A. pinnata was proportional to the
phosphorus supplementation in the culture medium. Supplementation of
phosphorus also improved the protein and lipid contents of A. pinnata. It
was concluded that supplementation of 10 ppm phosphorus to water
used for culturing A. pinnata is optimum under outdoor conditions.
Keywords: Azolla, Nutrient, Growth, Phosphorus

INTRODUCTION
Azolla is a free-floating aquatic fern, which is a member of the family Salviniaceae. It
is a dichotomously branched plant and naturally available on moist soils, ditches, and
marshy ponds. This fern grows extensively in association with nitrogen-fixing
bacteria (Anabaena azollae), which allows it to thrive on waters low in nitrogen but
containing phosphorus. Azolla grows on floating water surfaces in the temperate and
subtropical regions (Katole et al., 2017). It can reproduce sexually, by forming
spores. However, it mainly reproduces vegetatively by breaking off side branches at
a rapid rate. Under ideal conditions, it grows exponentially, doubling its biomass
every 2 to 5 days (De et al., 2015; Kathirvelan et al., 2015).

*
Corresponding author: [email protected]

Received: 14.12.2020 Accepted: 08.03.2021

178 Hossain et al.

Azolla is a good source of protein. It contains almost all essential amino acids,
minerals such as iron, calcium, magnesium, potassium, phosphorus, manganese, etc.,
apart from appreciable quantities of vitamin A’s precursor, beta-carotene, and
vitamin B12. It is also found to contain probiotics and biopolymers (Bhaskaran and
Kanappan, 2015). Thus, Azolla appears to be a potential source of nutrients and has a
considerably high feeding value (Anitha et al., 2016).
Azolla is used as a feed or feed supplement for a variety of animals, including broiler
chicken (Balaji et al., 2009), laying hens (Alalade et al., 2006), black tiger Shrimp
(Sudaryono, 2006), tilapia (Das et al., 2018; Hundare et al., 2018) and buffalo calves
(Indira et al., 2009). Azolla is also used in diets for sows and for partial replacement
of protein for growing or fattening pigs (Leterme et al., 2010). Due to easy
cultivation and high biomass yield, Azolla can be an ideal feed substitute for animals.
Apart from animal feed, Azolla is also widely used as a bio-fertilizer for paddy
cultivation. It is a mosquito repellent and bio-scavenger as it takes away all heavy
metals from water (Bhuvaneshwari, 2012; Bhuvaneshwari and Singh, 2015).
Phosphorus (in the form of phosphate) is the primary limiting nutrients for Azolla
growth and yield. The importance of phosphorus on Azolla growth has been
confirmed in the Anzali wetland (Sadeghi et al., 2013). In laboratory experiments,
Janes (1998) found that increasing phosphorus supply led to increased sporulation in
Azolla. There have been few reports about the requirement of phosphorus for
sustained Azolla spp. growth (Herzalla et al., 2003; Cheng et al., 2010). Most of those
studies have been conducted with pure nutrients in laboratory conditions. However,
scientific information on phosphorus’s effect on Azolla culture in the outdoor
conditions is limited. Therefore, the present study was undertaken to know the impact
of supplemental phosphorus on biomass growth and composition of A. pinnata.

MATERIALS AND METHODS

The experiment was conducted during September and October, 2018 in the Faculty of
Fisheries, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur,
Bangladesh. Twelve outdoor pits, each having a dimension of 1.5 m ×1.5 m × 0.3 m,
were made for A. pinnata culture. Silpaulin sheets of 2.0 m × 2.0 m were spread out
over each of the pits. 15 kg of sieved fertile soil (pH 6.2, organic matter 2.85%,
available phosphate 0.11 P2O5/100 g dry soil) was uniformly spread over the sheet.
Two kg of cow dung mixed with 10 liters of water was poured over it. Finally, the
water depth of the pits was raised to 20 cm with underground water.
The experiment was laid out in a complete randomized design (CRD) with 3
replications. The P was added through triple superphosphate (TSP) at 4 levels, 0, 5,
10, and 15 ppm and designated as P0, P5, P10, and P15, respectively. The required
amount of TSP was measured, dissolved in an aliquot of water and mixed well with
the respective pit's water. A. pinnata inoculum, used in this experiment, was
collected from the rice fields of Bangladesh Rice Research Institute (BRRI), Gazipur,
INORGANIC PHOSPHORUS REQUIREMENT IN AZOLLA 179

Bangladesh. The inoculum was starved by keeping it in demineralized water for 7

days before inoculating into the culture pit. Water was periodically added to maintain
20 cm water depth. A. pinnata was harvested after 21 days of inoculation.
The water temperature (°C) was recorded using a thermometer (9337U20, Thomas)
and pH by a digital pH meter (HQ11D, HACH) at the spot. Ammonia (mg/l)
determination of water sampled in leveled 250 ml black plastic bottles were done in
the laboratory with a spectrometer (DR 6000, Hach Co., Colorado, USA).
In order to monitor growth of A. pinnata, 100 plants were collected on every third
day, blotted and weighed, and the average weight was calculated. At the end of 21
days experimental period, fully grown A. pinnata was harvested from the pits,
washed, blot-dried gently and weighed for calculating fresh weight, relative growth
and doubling time. The harvested A. pinnata sample from each pi s
C for about 24 hours until constant weight, and the dry value was
calculated. The dried sample was put into a polythene bag and kept into the
refrigerator until further analysis. Dry matter, crude protein, crude lipid, and crude
ash contents were determined, following the methods described by AOAC (2007).
For P determination, the oven-dried sample was digested with a nitric acid-perchloric
acid mixture, and P in the digested sample was determined following the method
followed by Hossain and Furuichi (2000).
Growth was measured in terms of total fresh weight (kg), dry weight (kg), relative
growth rate (RGR), and doubling time (DT). In order to determine the RGR and DT,
the formulae RGR= (log Wt - logW0) /t and DT = t × log 2 [log (Wt Wo-1)]-1 were
used, respectively, where DT is the doubling time (days), RGR is the relative growth
rate expressed as g/g per day, t the experiment duration (days), Wt the final weight,
and Wo the initial weight.
Data were analyzed with one-way analysis of variance (ANOVA) to determine
whether there was any significant difference among treatments mean, while LSD test
was used to compare the treatment means (Hofmann, 2008).

RESULTS AND DISCUSSION

There was no variation in water quality parameters during the culture of A. pinnata
with different levels of P. Temperature, pH, and ammonia varied from 24.3-25.7 °C,
6.85-7.46, and 0.02-0.32 mg/l, respectively in all the P levels and did not differ
significantly as a result of P supplementation. These three parameters were within
suitable range for growth of A. pinnata. It grows relatively well in a temperature
range of 20-30°C (Cheng et al., 2010; De et al., 2015) and pH 5-8 (Sadeghi et al.,
2013). Maejima et al. (2001) reported that the lower levels of total ammonia in water
are better for Azolla.
The dry weight of A. pinnata (mg/plant) is shown in Table 1. The weight did not vary
much with P supplementation up to the 6th day as compared to the control but was
180 Hossain et al.

significantly more later. The maximum weight (4.65 mg/plant) was observed on 21st
day in P10. Sadeghi et al. (2013) observed that deficiency of P affects the growth of
A pinnata.

Table 1. Growth of Azolla pinnata cultured under different P supplementation (dry

weight mg/plant)
Days Dry weight mg/plant*
P0 P5 P10 P15
0 3.51 ± 0.15 3.51 ± 0.28 3.51 ± 0.28 3.51 ± 0.28
3 3.74 ± 0.25 3.69 ± 0.28 3.64 ± 0.33 3.64 ± 0.29
6 3.68 ± 0.42 3.69 ± 0.46 3.72 ± 0.44 3.65 ± 0.41
b a a
9 3.81 ± 0.34 3.95 ± 0.39 3.97 ± 0.33 3.93 ± 0.39a
12 3.85 ± 0.43b 4.11 ± 0.35a 4.15 ± 0.22a 4.14 ± 0.37a
15 3.76 ± 0.21b 4.21 ± 0.26a 4.20 ± 0.35a 4.12 ± 0.12a
18 3.62 ± 0.22b 4.37 ± 0.31a 4.55 ± 0.42a 4.14 ± 0.13a
21 3.42 ± 0.21c 4.25± 0.292b 4.65 ± 0.32a 4.44 ± 0.29c
*Mean ± SD. Data in the same row bearing different letters are significantly different (p<0.05).

The doubling time was calculated, which was the minimum (3.71 days) when the
culture medium was supplied with 10 ppm P (Table 2). However, further increase in
P supplementation level could not decrease the DT. DT was the highest (4.08 days)
without P supplementation to the water. Fresh weight, RGR and dry weight of A.
pinnata were enhanced by the supplementation of P and were maximum (5.01 kg/m2,
0.82 g/g per day and 0.25 kg/m2, respectively) with 10 ppm P. De et al. (2015)
observed that under P depletion, A. pinnata had recorded a significant restriction of
growth by dry matter loss. Temmink et al. (2018) in a laboratory experiment
observed significant increase in RGR in A. filiculoides, when culture medium was
supplied with 0.3 ppm P and further increase in P up to 10 ppm could not improve
the RGR. Gerek (2001) reported that 1122 g/m2 of fresh Azolla mexicana can be
harvested after15 days with the initial fresh weight of 300 g/m2 of Azolla. The final
fresh weight at harvest was more in the present study due to the difference in Azolla
species and relatively longer culture period (21 days).
INORGANIC PHOSPHORUS REQUIREMENT IN AZOLLA 181

Table 2. Effect of P supplementation on different growth parameters of A. pinnata

P in medium Doubling time RGR Fresh weight Dry weight
(mg/L) (g/g) (kg/m2) (kg/m2)
P0 8.39 ± 0.12a 0.36 ± 0.02c 3.90 ± 0.21c 0.27 ± 0.05b
P5 5.47 ± 0.21b 0.42 ± 0.06b 4.51 ± 0.18b 0.32 ± 0.03b
P10 3.74 ± 0.31c 0.54 ± 0.05a 5.92 ± 0.17a 0.41 ± 0.07a
P15 3.83 ± 0.18c 0.43 ± 0.04b 4.60 ± 0.15a 0.39 ± 0.05a
*Mean ± SD. Data in the same row bearing different letters are significantly different (P<0.05).

There have been variations in the levels of phosphorus reported by researchers for
sustained growth of Azolla. In laboratory experiments, a concentration of around 0.06
ppm was reported to be adequate to sustain Azolla growth. However, a range between
0.3 and 10 ppm was suggested from field surveys (Cheng et al., 2010). The response
of Azolla to different concentrations of phosphorus for optimum growth varies with
the species (Herzalla et al., 2003). Field surveys of Azolla in the Philippines showed
that P concentrations of A. microphylla were higher than those of A. pinnata var.
imbricata, and the available P contents of the soils where this species was growing
were higher (Sadeghi et al., 2013). In a pot experiment, Cheng et al. (2010) observed
that maximum biomass occurred when A. pinnata received 1 ppm of N and 5 ppm of
P, whereas A. filiculoides required 10 ppm N and up to 20 ppm P.
Supplementation of P influenced the dry matter, protein and lipid contents of A.
pinnata (Table 3).
Dry matter (4.65%), protein (28.52%) and lipid (4.15%) were the highest with 10
ppm P at the end of 21 days experimental period. Dry matter was the lowest in
control group. De et al. (2015) reported that the limitation of P resulted in a
significantly lower dry matter and protein contents of A. pinnata.

Table 3. Proximate composition of A. pinnata cultured in water with different levels

of supplemental P.
Parameters Supplemental P level
P0 P5 P10 P15
a b b
Dry matter (%) 4.05 ± 0.48 4.90 ± 0.29 4.65 ± 0.69 4.85 ± 0.28ab
Protein (% dm) 26.84 ± 0.70ab 26.02 ± 0.63b 28.52 ± 0.51a 27.23 ± 0.19b
Lipid (% dm) 5.55 ± 0.70b 4.15 ± 0.58bc 7.77 ± 0.71a 2.65 ± 0.13c
b b a
Ash (% dm) 18.37 ± 0.10 17.37 ± 0.82 21.36 ± 0.99 14.65 ± 0.21c
*dm= dry matter basis. *Mean ± SD. Data in the same row bearing different letters are significantly
different (P<0.05).
182 Hossain et al.

Oyange et al. (2019) attributed variations in the nutrient composition of Azolla to

differences in the response of Azolla strains to environmental conditions such as
temperature, light intensity and soil nutrient which consequently affect their growth,
morphology and composition. Cheng et al. (2010) concluded that the N accumulation
potential of A. filiculoides under future climate warming depends primarily on the
temperature change and P availability. The average crude protein content of A.
pinnata estimated in the present study matched with the previous findings. The P
supplementation also affected tissue P content of A. pinnata (Figure 1) and it was
maximum in P15 treatment. Tissue P content in Azolla was positively correlated with
the concentration of P in water used for culturing (Oyange et al., 2019).

0.9 a
0.8
Tissue P content (% dry

b
0.7
0.6 c
matter)

0.5
d
0.4
0.3
0.2
0.1
0
P0 P5 P10 P15
Supplemental P level

Figure 1. Effect of P supplementation in culture water on the tissue P content of A.

pinnata (Different letters indicate significant difference). The different
alphabets on each bar show level of significance (P<0.05).

CONCLUSION
Among the many inorganic nutrients required by plants, P is one of the most
important elements that significantly affect plant growth and metabolism. In the
present study, P supplementation @10 ppm to the water enhanced the growth,
decreased the doubling time and increased nutrient content of A. pinnata suggesting
that it is the optimum dose for the outdoor cultivation of A. pinnata in the ponds.
INORGANIC PHOSPHORUS REQUIREMENT IN AZOLLA 183

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