Algal Research 15 (2016) 59–64

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Algal Research

journal homepage: www.elsevier.com/locate/algal

Use of Spirulina biomass produced from treatment of aquaculture

wastewater as agricultural fertilizers
Shy Chyi Wuang ⁎, Mar Cho Khin, Pei Qiang Danny Chua, Yanpei Darren Luo
School of Applied Science, Temasek Polytechnic, 21 Tampines Ave 1, 529757, Singapore

a r t i c l e i n f o a b s t r a c t

Article history: Microalgal research has been an area of great interest as microalgae have higher productivities than land plants
Received 4 August 2015 and can be used for the production of valuable commodities such as biofuel, animal feeds and agricultural fertil-
Received in revised form 27 October 2015 izers, among others. To enhance the economic feasibility of algal-based commodities, the growth of microalgae
Accepted 8 February 2016
can be coupled to wastewater remediation. The technical feasibility of cultivating Spirulina platensis with fish
Available online xxxx
water for production of algae fertilizers was investigated. The remediation potential of S. platensis was found to
Keywords:
be good for ammonia and nitrate removal, but inadequate for nitrite removal. Its specific growth rate of
Water remediation 0.026 h−1 and the nutrient reduction times compare well with various literature reports. This work provides
Spirulina platensis insight into the potential of algal biomass as agricultural fertilizers, when coupled with aquaculture wastewater
Agricultural fertilizers remediation. The ability of Spirulina-based fertilizers to enhance plant growth was demonstrated in leafy vegeta-
Fish water bles such as Arugula (Eruca sativa), Bayam Red (Ameranthus gangeticus) and Pak Choy (Brassica rapa ssp.
chinensis). The germination of Chinese Cabbage (B. rapa ssp. chinensis) and Kai Lan (Brassica oleracea alboglabra)
also improved significantly in terms of seedlings' dry weight.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction benefits associated with the mass-culture exploitation of microalgae.

The global seafood consumption is increasing and the new World
Bank Report 83177-GLB [1] has projected that aquaculture will produce
half of the world's supply of fish in 2030. Intensive aquaculture utilizes
large quantities of clean freshwater, and generates nutrient-rich waste-
water streams which can cause eutrophication of coastal waters and
negatively impact downstream biological communities. Sustainable
water treatment technologies are needed to reduce nutrient and chem-
ical discharge into receiving waters. Currently, chemical fertilizers are
extensively used for agricultural crops as they are inexpensive, have
low labor application, and provide immediate availability of the nutri-
ents. However, the excessive use of chemical fertilizers contribute to
greenhouse gas emission and can lead to on-site soil degradation [2],
nutrient pollution and eutrophication [3]. Organic fertilizers, such as
farmyard manure and crop residues are possible alternatives to reduce
dependency on synthetic chemical fertilizers [4], as well as to counter-
act soil degradation [5]. However, these are inadequate in meeting the
nutrient demands for high yielding crops as essential macro- and
micronutrients may not be present in desired amounts.
Microalgae research has gained interest in recent decades and is cen-
tered mainly in the exploitation of microalgae for extraction of products
of value, and the utilization of microalgae in wastewater treatment sys-
tems. A key area of interest in microalgae biotechnology is the economic
⁎ Corresponding author.
E-mail address: scwuang@tp.edu.sg (S.C. Wuang).
The use of microalgae to remove certain excess nutrients from aquacul-
ture effluents – algal bioremediation – has been widely discussed [6–9].
A significant reduction of nutrient loads in aquaculture effluents
through algae bioremediation requires the selection of appropriate spe-
cies. This selection will also impact the applicability of algal biomass for
various uses.
Microalgal biomass contains high concentrations of protein, lipids,
and vitamins. Therefore, besides being a valuable alternative to the con-
ventional purification treatments, the use of microalgae offers several
advantages [10]. Firstly, the algal biomass can be recycled or used as fer-
tilizers and livestock feeds. Furthermore, under photosynthetic condi-
tions, algal growth allows oxygen to be released, thus enhancing the
auto-depuration potential of water. In agricultural countries, algal bio-
mass could be directly employed in soil inoculation to increase crop pro-
ductivities. They offer an alternative to the vicious cycle of chemical
fertilizers, soil exhaustion and dependency on imports. Algae fertilizers
can also boost the profitability of aquaculture by generating additional
income from fertilizers' sale. The use of algal fertilizers is not well-
established, though there are numerous reports documenting its feasi-
bility in rice plants [11–13]. In India, Tripathi et al. [12] found significant
enhancement of growth in rice plants growing on amended soils
enhanced with blue-green algae biofertilizer. Similarly, Watanabe
et al. [14] used blue-green algae as a fertilizer in approximately 40 rice
varieties and noted remarkable increases in rice yields in Japan. In
Iran, Saadatnia et al. [13] have found that the germination of rice
seeds treated with cyanobacteria was faster than that of control. These

http://dx.doi.org/10.1016/j.algal.2016.02.009
2211-9264/© 2016 Elsevier B.V. All rights reserved.
60 S.C. Wuang et al. / Algal Research 15 (2016) 59–64
reports evidenced the potential use of algae fertilizers when applied to
other agricultural crops.
Spirulina platensis, a filamentous cyanobacterium, has been widely
used as a food supplement due to its high protein content and nutrition-
al value. Spirulina thrives in alkaline environments and this preference
prevents external contamination [15], suggesting its suitability for envi-
ronmental applications. Spirulina has also been suggested as a good
alternative to chemical fertilizers as well as a good protein supplement
in livestock feeds [16]. The objectives of this work are to assess the suit-
ability of S. platensis as a bioremediation agent for aquaculture waste-
water and to examine the applicability of its biomass as agricultural
fertilizers to leafy vegetables. The water quality of the treated water
was studied and the excess algae were harvested and studied for their
usefulness as crop fertilizers with different types of leafy vegetables
such as Arugula, Bayam Red, Pak Choy, Chinese Cabbage, Kai Lan and
White Crown. The nutrient composition of the algae fertilizer was also
compared to a commercial chemical fertilizer.
2. Material and methods
2.1. Materials
S. platensis (LB 1928) was obtained from the University of Texas
(UTEX) collection and routinely cultured in the Guillard F/2 commercial
media, Micro Algae Grow, according to the manufacturer's recommend-
ed dilution (1:1000) in tap water with 30 g/L marine salt. The Nessler
reagent set, nitrite and nitrate testing reagents (Nitriver 3, Nitraver 5),
and chemical oxygen demand (COD) vials were purchased from Fluke
South East Asia Pte Ltd. Microscopy images of the algae cells were ac-
quired using an Olympus IX51 microscopy (low magnification) and a
Nikon-E upright microscope (high magnification).
2.2. Cultivation of S. platensis
Fish water was obtained during water changes for catfish (Pangasius
hypophthalmus) reared in Temasek Aquaculture Facility, Temasek
Polytechnic (TP), Singapore. The rearing of catfish was approved by TP
Institutional Animal Care and Use Committee (IACUC control number:
2012-50A). 30 g/L NaCl was subsequently added to the fish water.
Batch cultivation (2 L) of the microalgae was performed using the ob-
tained fish water in glass bottles and air-aerated with a flow rate of
10–15 mL/min. An average of 2 × 104 algae cells per milliliter of fish
water was used as the starting concentration. The growth and nutrient
profiles of the water were monitored for 3 days. Illumination was pro-
vided by fluorescent lamps with a luminance of 500–1000 lx, typical
of indoor lighting and similar to the natural light intensity on an over-
cast day. Temperature has a diurnal range of 28–30 °C. The flasks were
placed on a tray with the fluorescent light placed 8 cm away from the
side of the flasks. The various trays were covered with opaque boxes,
with artificial light as the only light source. pH was not controlled but
closely monitored during the experiments. Throughout the study, the
only form of agitation was provided by air aeration via single tubing in
each cultivation bottle.
For algal density quantification, 3 mL of algae suspension was col-
lected and the cell optical density was measured at 660 nm using a spec-
trophotometer (HACH DR2800). The relative cell density was calculated
by the following formula:
absorbanceðAt Þat time t
Relative cell density ¼ : ð1Þ
A0 at t ¼ 0
Specific growth rate (μ, in h−1) was calculated based on the equation
A two-sample t-test. The differences observed between the treatment
ln ðAy Þ
μ¼ ty −tx .
x
2.3. Harvesting and analytical methods
Stipulated amounts of algae suspension (50 mL) were withdrawn
from the cultivation flasks and subjected to centrifugation at 2650 g
for 10 min. The supernatants were withdrawn and analyzed for ammo-
nia, nitrite and nitrate. Ammonia, nitrite and nitrate concentrations
were determined using standard methods [17]. Absorbance readings
were obtained using the HACH DR2800 spectrophotometer. The per-
centage reduction in ammonia, nitrite and nitrate was calculated by
the following formula:
concentration at t ¼ 0−concentration at time t
% reduction ¼  100:
concentration at t ¼ 0
ð2Þ
The analysis of the constituents of S. platensis was performed by
Pacific Lab Services, Singapore. The N, P, K percentages in the chemical
fertilizer (Triple Pro 15-15-15) are calculated based on its inorganic
constituents, and metal contents were determined via inductively
coupled plasma spectrometry.
2.4. Potted plants experiment
Potted plants experiments were conducted with Arugula (Eruca
sativa), Bayam Red (Ameranthus gangeticus), and Pak Choy (Brassica
rapa ssp. chinensis) plants. For each plant, four trials were conducted —
Control, Spirulina (5 g/pot), chemical fertilizer (Triple Pro 15-15-15,
0.3 g/pot/week), and Spirulina (5 g/pot) plus chemical fertilizer
(0.3 g/pot/week). Each pot consists of 500 g of soil. The duration of ex-
periments ranged between 21 and 40 days. All trials were performed in
triplicates. Weekly measurement of plant growth was recorded in
terms of leaf number per plant and plant height. At the end of the trials,
the plants were harvested and their leaf number, plant height, chloro-
phyll content, root length, fresh weight and dry weight were deter-
mined. Chlorophyll content was measured using the Hansatech meter
(Model CL-01) which has a range of 0–2000 CCI (Chlorophyll Content
Index) units.
2.5. Seed germination study
Seed germination studies were performed for three types of
vegetable — Chinese Cabbage (B. rapa ssp. chinensis), Kai Lan (Brassica
oleracea alboglabra) and White Crown (B. rapa ssp. chinensis, F1 hybrid).
For each type of vegetables, Spirulina inoculation on seed germination
trials was conducted using 100 seeds per treatment for 6 treatments;
T1 to T5, containing various concentrations of Spirulina (2 g/L, 4 g/L,
6 g/L, 8 g/L and 10 g/L respectively) in tap water and control T0 (tap
water only). The seeds were soaked in the respective solution overnight
before germination on tissue towel. Triplicate runs of 100 seeds per run
were performed for each treatment group and the average results were
described. Germination rate, shoot length, root length, vigor index and
dry weight of 100 seedlings were measured. Germination rate is the av-
erage number of seeds that germinate over a 7-day period. Seed vigor
index is obtained by multiplying the germination rate with the shoot
length [18].
2.6. Statistical analysis
All experiments were performed in triplicate sets and the data pre-
sented are means ± standard deviation. The differences in the results
obtained with two treatment groups were analyzed statistically using
groups were considered significant for P b 0.05.
 S.C. Wuang et al. / Algal Research 15 (2016) 59–64
3. Results and discussion
3.1. Cultivation of S. platensis
S. platensis is microscopic blue-green algae and appears as spiral fil-
aments under a Nikon Ni-E upright microscope (see Fig. 1). It is charac-
terized by cylindrical, multicellular trichomes in an open helix. As the
filament length varies between different cells, counting the filaments
is not an accurate way to assess algal growth. The green pigment within
the Spirulina cells absorbs strongly at 660 nm, and this absorbance is
used as a gauge of cell growth.
Fig. 2 shows the relative algal density and pH profiles of the fish
water used for Spirulina cultivation. The alga grows well in fish water
with a specific growth rate of 0.026 h−1 (0.623 day−1) and a doubling
time of 28 h. These growth parameters compare favorably with those
reported elsewhere [19–20], indicating the suitability of cultivating
S. platensis in fish water. Throughout the experiments, the pH remained
approximately consistent between 8.23–8.46.
As cells assimilate nutrients during growth, the phytoremediation
potential of algae is closely linked to its growth. Greater algal growth
will promote better nutrient removal from the cultivation media. In
fish water, the main nutrients to be removed to maintain good fish
health are ammonia and nitrite. The acceptable level for total ammonia
is less than 4 mg/L while that for free ammonia should be less than
0.04 mg/L [21]. The desired level of nitrite is 0–1 mg/L and the accept-
able range is less than 4 mg/L. The acceptable concentration of nitrates
is much higher at 50 mg/L in freshwater and 100 mg/L in seawater
[22]. The starting fish water concentrations of ammonia, nitrite and ni-
trate average about 2.56 mg/L, 0.245 mg/L and 15.3 mg/L respectively.
As there were significant variations between different batches of fish
water, the percentage reductions of the nutrient (rather than the abso-
lute values) were used for comparison. Fig. 3 shows the relative ammo-
nia, nitrite and nitrate profiles of fish water cultivated with S. platensis.
This alga assimilates ammonia and nitrate quickly, with these concen-
trations rapidly decreasing with cultivation time. This is in line with a
previous report [23]. However, nitrite was not assimilated and its level
increased with growth, possibly due to its by-production during nitrate
metabolism. This implies the necessity of a further nitrite reduction step
to achieve the recycling of fish water, when S. platensis is used as the
phytoremediation agent.
In the conventional biological treatment of aquaculture wastewater,
ammonia is first converted to nitrite and then to nitrate by different ni-
trifying bacteria which typically has doubling times of 24 h [24]. Nitrate
is a stable end product which does not harm the fish in the concentra-
tions typically present. In comparison, with the algal-based treatment
in this work, ammonia was also efficiently reduced to non-toxic levels.
However, the alga was unable to remove nitrite. It should be noted
that Spirulina cultivated in marine fish wastewater exhibit similar per-
formance (Wuang et al., unpublished work). A different algae species
Fig. 1. Microscopy image of S. platensis at (a) 10× magnification and (b) 100× magnification.
61
Fig. 2. Relative algal density (line graph) and pH (point graph) profiles of fish water used
in cultivation of S. platensis.
or a combination of various species will be required to achieve adequate
ammonia and nitrite reduction simultaneously.
3.2. Efficacy of S. platensis as fertilizers
Table 1 illustrates the efficacies of S. platensis in enhancing the
growth of Bayam Red, Arugula and Pak Choy. For Bayam Red, the plants
cultivated in soil enriched with Spirulina showed increased plant height
by 58.3% as well as greater fresh and dry weights, by 110.1% and 155.8%
respectively, when compared with the control group. There were no sig-
nificant differences in chlorophyll content, root length and leaf number.
These results are in agreement to previous reports [12, 13] which main-
ly compared the efficacy of blue-green algae to controls with no supple-
ments added. In this study, the efficacy of S. platensis was also compared
to that of a commercial fertilizer, Triple Pro 15-15-15. Bayam Red culti-
vated with S. platensis showed no significant differences in terms of leaf
number, plant height, root length and chlorophyll content, when com-
pared to those cultivated using Triple Pro 15-15-15. However, the
fresh and dry weights were significantly smaller than those achieved
with Triple Pro 15-15-15. The combination of Spirulina and Triple Pro
15-15-15 resulted in greater fresh and dry weights when compared to
the Spirulina group.
When compared to the control, Arugula plants cultivated in soil
enriched with Spirulina showed increased plant height (by 55.3%), in-
creased chlorophyll content (by 30.2%), greater fresh and dry weights
(by 18.7% and 21.1% respectively). When compared to plants cultivated
using Triple Pro 15-15-15, the plants grown in Spirulina-enriched soil
showed a 71.8% increase in plant height, and comparable performance
in all the other growth parameters. The addition of S. platensis to Triple
62 S.C. Wuang et al. / Algal Research 15 (2016) 59–64

Table 2
Constituents of S. platensis and chemical fertilizer.

Constituents S. platensis Chemical fertilizer

(Triple Pro 15–15-15)

Carbohydrate (%) 16.8 -

Protein (%) 48.5 -
Fat (%) 4.7 -
Nitrogen (%) 7.8 12.4
Phosphorus (%) 0.8 6.6
Potassium (%) 1.6 12.5
Calcium (%) 0.4 0.1
Iron (ppm) 1057 455
Manganese (ppm) 41.9 26.1
Zinc (ppm) 155.4 -
Selenium (ppb) 17.8 -

Table 2 shows the constituents of the microalgae and chemical fertil-

Fig. 3. Relative concentrations of ammonia, nitrite and nitrate in fish water used in
cultivation of S. platensis.
Pro 15-15-15 showed no enhancement in most growth parameters,
when compared to the trials with either Spirulina or Triple Pro alone.
The significant enhancements were in root length and fresh weight.
For Pak Choy, there were no significant differences in between plants
cultivated in Spirulina-enriched soil and control for all the growth pa-
rameters except root length which is higher for the plants cultivated
in Spirulina-enriched soil. The plants cultivated in soil enriched with Tri-
ple Pro 15–15-15 performed better in terms of greater leaf number and
29.1% increase in dry weight, when compared to those in Spirulina-
enriched soil. The addition of S. platensis to Triple Pro 15–15-15 showed
no enhancement in growth parameters. Prior work in the comparison
study of algae-based and chemical-based fertilizers is very limited. The
results of our work suggest that the efficacy of S. platensis in enhancing
plant growth is species-dependent and in some cases (e.g. for Arugula)
it can be a better growth supplement when compared to chemical fertil-
izers. Its efficacy can be explained by the presence of micro- and macro-
nutrients in the microalgae.
izers. Comparatively, the algal biomass contain less nitrogen, phospho-
rus and potassium (N–P–K) which are the primary nutrients found in
fertilizers. Primary nutrients are usually applied at higher rates as they
are utilized in the largest amounts by crops, followed by secondary nu-
trients and micronutrients. Other than N–P–K, all the analyzed nutrients
(calcium, iron, manganese, zinc and selenium) were found in greater
quantities in the algal biomass. Calcium is a constituent of cell walls
and is involved in production of new growing points and root tips. It
provides elasticity and expansion of cell walls, which keeps growing
points from becoming rigid and brittle. The algal biomass can supply a
3-fold higher calcium concentration and a 2-fold higher Iron concentra-
tion to the crops, as compared to the chemical fertilizer. Iron is a struc-
tural component of several essential enzymes and its shortage is a
common problem in many plant species. Manganese content of the
algal biomass is also significantly higher than the chemical fertilizer.
This element acts as an enzyme activator for nitrogen assimilation and
is essential for the synthesis of chlorophyll. Zinc, which is not detected
in the chemical fertilizer, is involved in the synthesis of growth
substances and enzyme systems. It is necessary for the production of
chlorophyll and carbohydrates and essential for promoting certain
metabolic reactions. Selenium, another element not detected in the
chemical fertilizer, was found in trace amount (17.8 ppb) in the algae.
Selenium is important, not to the crops, but more to alleviate selenium
deficiency in the animals and humans further up the food chain [25].

Table 1
Efficacy of Spirulina in enhancing Bayam Red, Arugula and Pak Choy growth.

Plant parameter Control Spirulina Chemical fertilizer Spirulina + chemical fertilizer

Bayam Red (21 days at harvest)

Number of leaf 9.33 ± 2.08 11.00 ± 1.00 12.33 ± 1.53 13.33 ± 0.58
Plant height (cm) 16.03 ± 3.05 25.37 ± 5.90** 29.50 ± 4.71** 30.90 ± 4.11**
Root length (cm) 22.57 ± 0.50 26.00 ± 5.57 22.87 ± 3.67 19.77 ± 4.25
Chlorophyll content 5.72 ± 2.48 7.65 ± 0.85 8.98 ± 2.22 8.80 ± 2.16
Fresh weight (g) 12.52 ± 0.82 26.31 ± 4.65**,*,# 38.11 ± 5.16**,* 36.33 ± 1.74**,#
Dry weight (g) 4.28 ± 0.40 10.95 ± 1.07**,*,# 21.16 ± 3.11**,* 22.74 ± 1.05**,#

Arugula (30 days at harvest)

Number of leaf 7.67 ± 0.67 9.00 ± 0.58 9.67 ± 0.67 9.67 ± 0.33
Plant height (cm) 31.33 ± 5.93 48.67 ± 3.67**,*,# 28.33 ± 3.33* 32.00 ± 8.00#
Root length (cm) 6.00 ± 1.73 6.17 ± 0.60* 7.33 ± 0.33# 9.67 ± 0.33*,#
Chlorophyll content 13.67 ± 0.28 17.80 ± 1.87 18.60 ± 0.32 18.70 ± 0.87
Fresh weight (g) 10.67 ± 0.67 12.67 ± 0.33**,* 13.67 ± 1.45 14.67 ± 0.33**,*
Dry weight (g) 7.48 ± 0.38 9.06 ± 0.36** 9.52 ± 1.05 10.00 ± 0.05**

Pak Choy (40 days at harvest)

Number of leaf 10.33 ± 0.88 13.00 ± 0.00* 14.00 ± 0.00**,* 13.50 ± 0.50
Plant height (cm) 15.83 ± 1.59 17.5 ± 2.50 19.17 ± 0.73 21.75 ± 0.75
Root length (cm) 2.33 ± 0.33 7.00 ± 1.00** 6.17 ± 1.59** 7.00 ± 1.00**
Chlorophyll content 13.17 ± 1.37 14.25 ± 2.85 12.77 ± 0.79 11.10 ± 0.80
Fresh weight (g) 12.67 ± 1.20 14.00 ± 3.00 17.33 ± 1.76 17.50 ± 0.50
Dry weight (g) 3.71 ± 0.60 4.79 ± 0.26* 5.91 ± 0.22**,* 5.30 ± 0.23**

* and # denote statistical significance between test groups.

** denotes statistical significance with control group.
 S.C. Wuang et al. / Algal Research 15 (2016) 59–64 63

Despite a lower N–P–K content, the algae fertilizers enhanced plant
growth to similar extent as the chemical fertilizers. This is likely due
to the higher amounts of other secondary and micro-nutrients which
help to moderate the amounts of primary nutrients required.
3.3. Efficacy of S. platensis inoculation on seed germination
Many studies [14, 26–27] have reported the use of dried
cyanobacteria to inoculate soils as a means of aiding fertility in rice
plants. On average, these results have shown increases in rice grain
yield of 15–20% in field experiments. While these prior studies have
concentrated on rice plants, the effects of Spirulina inoculation on seed
germination of leafy vegetables were studied in this work. A germina-
tion test determines the maximum seed viability. The germination
rate of a seed lot is a key indicator to its performance in the field. The
pre-soaking of rice in blue-green algae culture has decreased losses
from sulfate-reducing processes and this has been attributed to the en-
hancement of germination and faster seedling growth [28–29]. Further-
more, vigor tests are used to understand the ability of seeds to emerge
from soils under stress conditions or to maintain viability during
storage. Table 3 lists the performance of S. platensis inoculation on
seed germination of Chinese Cabbage, Kai Lan and White Crown.
For Chinese Cabbage, no improvement to germination rate was ob-
served at all the inoculated Spirulina concentrations. The only significant
enhancement was observed with an inoculation of 8 g/L Spirulina,
where the dry weight of the germinated seedlings increased by more
than 3-fold that of the control. In the case of Kai Lan, significant in-
creases in dry seedling weight were observed with 8 g/L and 10 g/L
Spirulina inoculations. Similarly, there were no significant improve-
ments for the other plant growth parameters. Spirulina inoculations at
all concentrations did not show significant effects on the germination
of White Crown. These suggest that Spirulina may not be a universal
plant growth and germination enhancer, as its effects varies consider-
ably between different plant species.
Previous studies on bio-fertilizers have reported enhanced germina-
tion rates for sunflower (Helianthus annuus L.) [30], Dill (Anethum
graveolens) [31], Cumin (Cuminum cyminum), Marigold (Calendula
officinalis) and Roma tomato (Solanum lycopersicum var. Roma) [32].
In the application of algae fertilizers, Safinaz and Ragaa [33] have report-
ed the enhancement of growth to varying extents of maize (Zea mays L.)
plants with three different types of red marine algae. The application of
Chlorella vulgaris was also found to be beneficial to the growth of lettuce
(Lactuca sativa) [34]. Also, it has been shown that Spirulina, when used
as a biofertiliser, increases the growth and yield of green gram [35].
From these studies, there seem to be a general consensus on the useful-
ness of algae fertilizers, though specific benefits would require compre-
hensive investigation. Our study has demonstrated that the application
of S. platensis can enhance the growth of Bayam Red, Arugula and Pak
Choy to similar extents when compared to chemical fertilizers. In addi-
tion, the germination of Chinese Cabbage and Kai Lan improved signifi-
cantly in terms of seedlings' dry weight.
4. Conclusion
The usefulness of S. platensis in aquaculture wastewater treatment
was studied and the subsequent application of algal biomass in fertilizer
studies was demonstrated. The cultivation of S. platensis was done
indoors, under an illuminance of not more than 1000 lx. At these condi-
tions, the algae were able to remove the ammonia and nitrate concen-
trations in fish water, indicating its ability to treat the water despite
its inadequacy in removing nitrite. Potentially, the efficacy of water
treatment can be much higher under sunlight where illuminance is
typically about 100,000 lx. The supplementation of S. platensis for leafy
vegetables found enhanced plant growth in all tested vegetables,
when compared to the controls. When compared to the performance
of chemical fertilizer, the Spirulina-based fertilizer performed compara-
bly in most plant growth parameters, and favorably for one tested
species — Arugula. Seed germination (when measured by seedling's
dry weight) also improved for all tested vegetables except White
Crown. This work has evidenced the usefulness of S. platensis in fish
water treatment and its applicability as agricultural fertilizers.
Authors' contributions and competing interests
DPQ Chua, DYP Luo and MC Khin conducted the experiments and
participated in the collection and assembly of data. SC Wuang and MC
Khin were involved in the study design and the analysis and interpreta-
tion of data. SC Wuang also conceived the study, wrote and revised the
manuscript. All authors read and approved the final manuscript. The
authors declare that they have no competing interests.

Table 3
Efficacy of Spirulina inoculation on seed germination of Chinese Cabbage, Kai Lan and White Crown.

Treatment group Germination rate (%) Shoot Length (cm) Root Length (cm) Dry weight of 100 seedlings (g) Vigor Index

Chinese Cabbage
T0 (control) 93.3 ± 5.8 1.07 ± 0.19 1.82 ± 0.24 3.45 ± 0.59 99.8 ± 12.5
T1 (2 g/L) 88.3 ± 12.6 1.02 ± 0.08 2.01 ± 0.15 3.30 ± 0.3 90.1 ± 6.4
T2 (4 g/L) 98.3 ± 2.9 1.22 ± 0.05 2.28 ± 0.31 3.85 ± 0.44 119.9 ± 8.0
T3 (6 g/L) 100.0 ± 0.0 0.92 ± 0.14 2.71 ± 0.35 2.75 ± 0.26 92.0 ± 13.9
T4 (8 g/L) 100.0 ± 0.0 1.08 ± 0.19 2.05 ± 0.40 11.15 ± 1.38** 108.0 ± 18.6
T5 (10 g/L) 100.0 ± 0.0 0.95 ± 0.13 2.31 ± 0.17 3.09 ± 0.59 95.0 ± 12.5

Kai Lan
T0 70.0 ± 8.7 1.88 ± 0.15 1.51 ± 0.15 2.60 ± 0.13 131.6 ± 25.6
T1 56.7 ± 5.8 2.21 ± 0.17 1.56 ± 0.11 3.93 ± 1.48 125.3 ± 17.3
T2 63.3 ± 10.4 2.02 ± 0.17 2.08 ± 0.40 5.00 ± 1.20 127.9 ± 22.4
T3 75.0 ± 5.0 2.02 ± 0.23 1.65 ± 0.06 6.53 ± 2.68 151.5 ± 6.9
T4 66.7 ± 16.1 1.83 ± 0.12 1.70 ± 0.12 10.22 ± 2.78** 122.1 ± 20.8
T5 81.7 ± 17.6 1.96 ± 0.14 1.83 ± 0.13 7.15 ± 2.41** 160.1 ± 26.0

White Crown
T0 98.3 ± 2.9 2.18 ± 0.05 1.71 ± 0.21 4.80 ± 0.06 214.3 ± 7.6
T1 100.0 ± 0.0 1.98 ± 0.16 1.98 ± 0.11 4.67 ± 0.93 198.0 ± 15.3
T2 100.0 ± 0.0 2.07 ± 0.11 2.07 ± 0.31 4.32 ± 0.35 207.0 ± 10.9
T3 98.3 ± 2.9 2.05 ± 0.11 2.05 ± 0.03 4.64 ± 0.40 201.5 ± 15.3
T4 98.3 ± 2.9 1.97 ± 0.17 1.97 ± 0.16 4.90 ± 0.33 193.7 ± 11.3
T5 96.7 ± 2.9 1.91 ± 0.22 1.91 ± 0.02 4.22 ± 0.43 184.7 ± 20.4

** denotes statistical significance with control group.

64 S.C. Wuang et al. / Algal Research 15 (2016) 59–64
Acknowledgments
The work was supported financially by the Social and Innovation
Research Fund (M54), awarded by the TOTE Board, Singapore (Project
M54). High magnification microscopy images for the study were ac-
quired in the Singapore Bioimaging Consortium (SBIC)-Nikon Imaging
Centre at Biopolis (Singapore) using a Nikon Ni-E upright microscope.
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