You are on page 1of 6

Algal Research 15 (2016) 59–64

Contents lists available at ScienceDirect

Algal Research

journal homepage:

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
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 use of microalgae to remove certain excess nutrients from aquacul-
The global seafood consumption is increasing and the new World ture effluents – algal bioremediation – has been widely discussed [6–9].
Bank Report 83177-GLB [1] has projected that aquaculture will produce A significant reduction of nutrient loads in aquaculture effluents
half of the world's supply of fish in 2030. Intensive aquaculture utilizes through algae bioremediation requires the selection of appropriate spe-
large quantities of clean freshwater, and generates nutrient-rich waste- cies. This selection will also impact the applicability of algal biomass for
water streams which can cause eutrophication of coastal waters and various uses.
negatively impact downstream biological communities. Sustainable Microalgal biomass contains high concentrations of protein, lipids,
water treatment technologies are needed to reduce nutrient and chem- and vitamins. Therefore, besides being a valuable alternative to the con-
ical discharge into receiving waters. Currently, chemical fertilizers are ventional purification treatments, the use of microalgae offers several
extensively used for agricultural crops as they are inexpensive, have advantages [10]. Firstly, the algal biomass can be recycled or used as fer-
low labor application, and provide immediate availability of the nutri- tilizers and livestock feeds. Furthermore, under photosynthetic condi-
ents. However, the excessive use of chemical fertilizers contribute to tions, algal growth allows oxygen to be released, thus enhancing the
greenhouse gas emission and can lead to on-site soil degradation [2], auto-depuration potential of water. In agricultural countries, algal bio-
nutrient pollution and eutrophication [3]. Organic fertilizers, such as mass could be directly employed in soil inoculation to increase crop pro-
farmyard manure and crop residues are possible alternatives to reduce ductivities. They offer an alternative to the vicious cycle of chemical
dependency on synthetic chemical fertilizers [4], as well as to counter- fertilizers, soil exhaustion and dependency on imports. Algae fertilizers
act soil degradation [5]. However, these are inadequate in meeting the can also boost the profitability of aquaculture by generating additional
nutrient demands for high yielding crops as essential macro- and income from fertilizers' sale. The use of algal fertilizers is not well-
micronutrients may not be present in desired amounts. established, though there are numerous reports documenting its feasi-
Microalgae research has gained interest in recent decades and is cen- bility in rice plants [11–13]. In India, Tripathi et al. [12] found significant
tered mainly in the exploitation of microalgae for extraction of products enhancement of growth in rice plants growing on amended soils
of value, and the utilization of microalgae in wastewater treatment sys- enhanced with blue-green algae biofertilizer. Similarly, Watanabe
tems. A key area of interest in microalgae biotechnology is the economic 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
⁎ Corresponding author. Iran, Saadatnia et al. [13] have found that the germination of rice
E-mail address: (S.C. Wuang). seeds treated with cyanobacteria was faster than that of control. These
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 2.3. Harvesting and analytical methods
other agricultural crops.
Spirulina platensis, a filamentous cyanobacterium, has been widely Stipulated amounts of algae suspension (50 mL) were withdrawn
used as a food supplement due to its high protein content and nutrition- from the cultivation flasks and subjected to centrifugation at 2650 g
al value. Spirulina thrives in alkaline environments and this preference for 10 min. The supernatants were withdrawn and analyzed for ammo-
prevents external contamination [15], suggesting its suitability for envi- nia, nitrite and nitrate. Ammonia, nitrite and nitrate concentrations
ronmental applications. Spirulina has also been suggested as a good were determined using standard methods [17]. Absorbance readings
alternative to chemical fertilizers as well as a good protein supplement were obtained using the HACH DR2800 spectrophotometer. The per-
in livestock feeds [16]. The objectives of this work are to assess the suit- centage reduction in ammonia, nitrite and nitrate was calculated by
ability of S. platensis as a bioremediation agent for aquaculture waste- the following formula:
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 concentration at t ¼ 0−concentration at time t
% reduction ¼  100:
usefulness as crop fertilizers with different types of leafy vegetables concentration at t ¼ 0
such as Arugula, Bayam Red, Pak Choy, Chinese Cabbage, Kai Lan and ð2Þ
White Crown. The nutrient composition of the algae fertilizer was also
compared to a commercial chemical fertilizer.
The analysis of the constituents of S. platensis was performed by
Pacific Lab Services, Singapore. The N, P, K percentages in the chemical
2. Material and methods fertilizer (Triple Pro 15-15-15) are calculated based on its inorganic
constituents, and metal contents were determined via inductively
2.1. Materials coupled plasma spectrometry.

S. platensis (LB 1928) was obtained from the University of Texas

(UTEX) collection and routinely cultured in the Guillard F/2 commercial 2.4. Potted plants experiment
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 Potted plants experiments were conducted with Arugula (Eruca
reagent set, nitrite and nitrate testing reagents (Nitriver 3, Nitraver 5), sativa), Bayam Red (Ameranthus gangeticus), and Pak Choy (Brassica
and chemical oxygen demand (COD) vials were purchased from Fluke rapa ssp. chinensis) plants. For each plant, four trials were conducted —
South East Asia Pte Ltd. Microscopy images of the algae cells were ac- Control, Spirulina (5 g/pot), chemical fertilizer (Triple Pro 15-15-15,
quired using an Olympus IX51 microscopy (low magnification) and a 0.3 g/pot/week), and Spirulina (5 g/pot) plus chemical fertilizer
Nikon-E upright microscope (high magnification). (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
2.2. Cultivation of S. platensis
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-
Fish water was obtained during water changes for catfish (Pangasius
phyll content, root length, fresh weight and dry weight were deter-
hypophthalmus) reared in Temasek Aquaculture Facility, Temasek
mined. Chlorophyll content was measured using the Hansatech meter
Polytechnic (TP), Singapore. The rearing of catfish was approved by TP
(Model CL-01) which has a range of 0–2000 CCI (Chlorophyll Content
Institutional Animal Care and Use Committee (IACUC control number:
Index) units.
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 2.5. Seed germination study
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 Seed germination studies were performed for three types of
profiles of the water were monitored for 3 days. Illumination was pro- vegetable — Chinese Cabbage (B. rapa ssp. chinensis), Kai Lan (Brassica
vided by fluorescent lamps with a luminance of 500–1000 lx, typical oleracea alboglabra) and White Crown (B. rapa ssp. chinensis, F1 hybrid).
of indoor lighting and similar to the natural light intensity on an over- For each type of vegetables, Spirulina inoculation on seed germination
cast day. Temperature has a diurnal range of 28–30 °C. The flasks were trials was conducted using 100 seeds per treatment for 6 treatments;
placed on a tray with the fluorescent light placed 8 cm away from the T1 to T5, containing various concentrations of Spirulina (2 g/L, 4 g/L,
side of the flasks. The various trays were covered with opaque boxes, 6 g/L, 8 g/L and 10 g/L respectively) in tap water and control T0 (tap
with artificial light as the only light source. pH was not controlled but water only). The seeds were soaked in the respective solution overnight
closely monitored during the experiments. Throughout the study, the before germination on tissue towel. Triplicate runs of 100 seeds per run
only form of agitation was provided by air aeration via single tubing in were performed for each treatment group and the average results were
each cultivation bottle. described. Germination rate, shoot length, root length, vigor index and
For algal density quantification, 3 mL of algae suspension was col- dry weight of 100 seedlings were measured. Germination rate is the av-
lected and the cell optical density was measured at 660 nm using a spec- erage number of seeds that germinate over a 7-day period. Seed vigor
trophotometer (HACH DR2800). The relative cell density was calculated index is obtained by multiplying the germination rate with the shoot
by the following formula: length [18].

2.6. Statistical analysis

absorbanceðAt Þat time t
Relative cell density ¼ : ð1Þ
A0 at t ¼ 0
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
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 .
groups were considered significant for P b 0.05.
S.C. Wuang et al. / Algal Research 15 (2016) 59–64 61

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
Fig. 2. Relative algal density (line graph) and pH (point graph) profiles of fish water used
approximately consistent between 8.23–8.46. in cultivation of S. platensis.
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 or a combination of various species will be required to achieve adequate
fish water, the main nutrients to be removed to maintain good fish ammonia and nitrite reduction simultaneously.
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 3.2. Efficacy of S. platensis as fertilizers
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 Table 1 illustrates the efficacies of S. platensis in enhancing the
is much higher at 50 mg/L in freshwater and 100 mg/L in seawater growth of Bayam Red, Arugula and Pak Choy. For Bayam Red, the plants
[22]. The starting fish water concentrations of ammonia, nitrite and ni- cultivated in soil enriched with Spirulina showed increased plant height
trate average about 2.56 mg/L, 0.245 mg/L and 15.3 mg/L respectively. by 58.3% as well as greater fresh and dry weights, by 110.1% and 155.8%
As there were significant variations between different batches of fish respectively, when compared with the control group. There were no sig-
water, the percentage reductions of the nutrient (rather than the abso- nificant differences in chlorophyll content, root length and leaf number.
lute values) were used for comparison. Fig. 3 shows the relative ammo- These results are in agreement to previous reports [12, 13] which main-
nia, nitrite and nitrate profiles of fish water cultivated with S. platensis. ly compared the efficacy of blue-green algae to controls with no supple-
This alga assimilates ammonia and nitrate quickly, with these concen- ments added. In this study, the efficacy of S. platensis was also compared
trations rapidly decreasing with cultivation time. This is in line with a to that of a commercial fertilizer, Triple Pro 15-15-15. Bayam Red culti-
previous report [23]. However, nitrite was not assimilated and its level vated with S. platensis showed no significant differences in terms of leaf
increased with growth, possibly due to its by-production during nitrate number, plant height, root length and chlorophyll content, when com-
metabolism. This implies the necessity of a further nitrite reduction step pared to those cultivated using Triple Pro 15-15-15. However, the
to achieve the recycling of fish water, when S. platensis is used as the fresh and dry weights were significantly smaller than those achieved
phytoremediation agent. with Triple Pro 15-15-15. The combination of Spirulina and Triple Pro
In the conventional biological treatment of aquaculture wastewater, 15-15-15 resulted in greater fresh and dry weights when compared to
ammonia is first converted to nitrite and then to nitrate by different ni- the Spirulina group.
trifying bacteria which typically has doubling times of 24 h [24]. Nitrate When compared to the control, Arugula plants cultivated in soil
is a stable end product which does not harm the fish in the concentra- enriched with Spirulina showed increased plant height (by 55.3%), in-
tions typically present. In comparison, with the algal-based treatment creased chlorophyll content (by 30.2%), greater fresh and dry weights
in this work, ammonia was also efficiently reduced to non-toxic levels. (by 18.7% and 21.1% respectively). When compared to plants cultivated
However, the alga was unable to remove nitrite. It should be noted using Triple Pro 15-15-15, the plants grown in Spirulina-enriched soil
that Spirulina cultivated in marine fish wastewater exhibit similar per- showed a 71.8% increase in plant height, and comparable performance
formance (Wuang et al., unpublished work). A different algae species in all the other growth parameters. The addition of S. platensis to Triple

Fig. 1. Microscopy image of S. platensis at (a) 10× magnification and (b) 100× magnification.
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-

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-
Fig. 3. Relative concentrations of ammonia, nitrite and nitrate in fish water used in
cultivation of S. platensis. 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
Pro 15-15-15 showed no enhancement in most growth parameters, and is involved in production of new growing points and root tips. It
when compared to the trials with either Spirulina or Triple Pro alone. provides elasticity and expansion of cell walls, which keeps growing
The significant enhancements were in root length and fresh weight. points from becoming rigid and brittle. The algal biomass can supply a
For Pak Choy, there were no significant differences in between plants 3-fold higher calcium concentration and a 2-fold higher Iron concentra-
cultivated in Spirulina-enriched soil and control for all the growth pa- tion to the crops, as compared to the chemical fertilizer. Iron is a struc-
rameters except root length which is higher for the plants cultivated tural component of several essential enzymes and its shortage is a
in Spirulina-enriched soil. The plants cultivated in soil enriched with Tri- common problem in many plant species. Manganese content of the
ple Pro 15–15-15 performed better in terms of greater leaf number and algal biomass is also significantly higher than the chemical fertilizer.
29.1% increase in dry weight, when compared to those in Spirulina- This element acts as an enzyme activator for nitrogen assimilation and
enriched soil. The addition of S. platensis to Triple Pro 15–15-15 showed is essential for the synthesis of chlorophyll. Zinc, which is not detected
no enhancement in growth parameters. Prior work in the comparison in the chemical fertilizer, is involved in the synthesis of growth
study of algae-based and chemical-based fertilizers is very limited. The substances and enzyme systems. It is necessary for the production of
results of our work suggest that the efficacy of S. platensis in enhancing chlorophyll and carbohydrates and essential for promoting certain
plant growth is species-dependent and in some cases (e.g. for Arugula) metabolic reactions. Selenium, another element not detected in the
it can be a better growth supplement when compared to chemical fertil- chemical fertilizer, was found in trace amount (17.8 ppb) in the algae.
izers. Its efficacy can be explained by the presence of micro- and macro- Selenium is important, not to the crops, but more to alleviate selenium
nutrients in the microalgae. 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 Chlorella vulgaris was also found to be beneficial to the growth of lettuce
growth to similar extent as the chemical fertilizers. This is likely due (Lactuca sativa) [34]. Also, it has been shown that Spirulina, when used
to the higher amounts of other secondary and micro-nutrients which as a biofertiliser, increases the growth and yield of green gram [35].
help to moderate the amounts of primary nutrients required. From these studies, there seem to be a general consensus on the useful-
ness of algae fertilizers, though specific benefits would require compre-
3.3. Efficacy of S. platensis inoculation on seed germination hensive investigation. Our study has demonstrated that the application
of S. platensis can enhance the growth of Bayam Red, Arugula and Pak
Many studies [14, 26–27] have reported the use of dried Choy to similar extents when compared to chemical fertilizers. In addi-
cyanobacteria to inoculate soils as a means of aiding fertility in rice tion, the germination of Chinese Cabbage and Kai Lan improved signifi-
plants. On average, these results have shown increases in rice grain cantly in terms of seedlings' dry weight.
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- 4. Conclusion
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 The usefulness of S. platensis in aquaculture wastewater treatment
pre-soaking of rice in blue-green algae culture has decreased losses was studied and the subsequent application of algal biomass in fertilizer
from sulfate-reducing processes and this has been attributed to the en- studies was demonstrated. The cultivation of S. platensis was done
hancement of germination and faster seedling growth [28–29]. Further- indoors, under an illuminance of not more than 1000 lx. At these condi-
more, vigor tests are used to understand the ability of seeds to emerge tions, the algae were able to remove the ammonia and nitrate concen-
from soils under stress conditions or to maintain viability during trations in fish water, indicating its ability to treat the water despite
storage. Table 3 lists the performance of S. platensis inoculation on its inadequacy in removing nitrite. Potentially, the efficacy of water
seed germination of Chinese Cabbage, Kai Lan and White Crown. treatment can be much higher under sunlight where illuminance is
For Chinese Cabbage, no improvement to germination rate was ob- typically about 100,000 lx. The supplementation of S. platensis for leafy
served at all the inoculated Spirulina concentrations. The only significant vegetables found enhanced plant growth in all tested vegetables,
enhancement was observed with an inoculation of 8 g/L Spirulina, when compared to the controls. When compared to the performance
where the dry weight of the germinated seedlings increased by more of chemical fertilizer, the Spirulina-based fertilizer performed compara-
than 3-fold that of the control. In the case of Kai Lan, significant in- bly in most plant growth parameters, and favorably for one tested
creases in dry seedling weight were observed with 8 g/L and 10 g/L species — Arugula. Seed germination (when measured by seedling's
Spirulina inoculations. Similarly, there were no significant improve- dry weight) also improved for all tested vegetables except White
ments for the other plant growth parameters. Spirulina inoculations at Crown. This work has evidenced the usefulness of S. platensis in fish
all concentrations did not show significant effects on the germination water treatment and its applicability as agricultural fertilizers.
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. Authors' contributions and competing interests
Previous studies on bio-fertilizers have reported enhanced germina-
tion rates for sunflower (Helianthus annuus L.) [30], Dill (Anethum DPQ Chua, DYP Luo and MC Khin conducted the experiments and
graveolens) [31], Cumin (Cuminum cyminum), Marigold (Calendula participated in the collection and assembly of data. SC Wuang and MC
officinalis) and Roma tomato (Solanum lycopersicum var. Roma) [32]. Khin were involved in the study design and the analysis and interpreta-
In the application of algae fertilizers, Safinaz and Ragaa [33] have report- tion of data. SC Wuang also conceived the study, wrote and revised the
ed the enhancement of growth to varying extents of maize (Zea mays L.) manuscript. All authors read and approved the final manuscript. The
plants with three different types of red marine algae. The application of 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 [16] M.A.B. Habib, M. Parvin, T.C. Huntington, M.R. Hasan, A review on culture, produc-
tion and use of Spirulina as food for humans and feeds for domestic animals and
fish, FAO Fish. Aquac. Circ. (2008) (No. 1034).
The work was supported financially by the Social and Innovation [17] Standard Methods for the Examination of Water and Wastewater 4500-NH3 B & C,
Research Fund (M54), awarded by the TOTE Board, Singapore (Project 20th ed. American Public Health Association (APHA), 2000.
[18] P.C. Gupta, Germination testing, in: P.K. Agrawal (Ed.), Handbook of Seed Testing,
M54). High magnification microscopy images for the study were ac- Seednet, India 2000, pp. 242–243.
quired in the Singapore Bioimaging Consortium (SBIC)-Nikon Imaging [19] T. Göksan, A. Zekeriyaoğlu, İ. Ak, The growth of Spirulina platensis in different culture
Centre at Biopolis (Singapore) using a Nikon Ni-E upright microscope. systems under greenhouse condition, Turk. J. Biol. 31 (2007) 47–52.
[20] F.F. Madkour, A.E.-W. Kamil, H.S. Nasr, Production and nutritive value of Spirulina
platensis in reduced cost media, Egypt. J. Aquat. Res. 38 (2012) 51–57.
References [21] N.M. Stone, H.K. Thomforde, Understanding Your Fish Pond Water Analysis Report,
Cooperative Extension Program, University of Arkansas at Pine Bluff Aquaculture/
[1] World Bank, Fish to 2030: prospects for fisheries and aquaculture, Agriculture and Fisheries, 2004.
Environmental Services Discussion Paper No. 3, World Bank Group, Washington [22] Ornamental Aquatic Trade Association (OATA), Water Quality Criteria—Ornamental
DC, 2013. Fish, 2008 (Wiltshire, BA13 3JN, UK).
[2] J.H. Guo, X.J. Liu, Y. Zhang, J.L. Shen, W.X. Han, W.F. Zhang, P. Christie, K.W.T. [23] G. Xu, X. Fan, A.J. Miller, Plant nitrogen assimilation and use efficiency, Annu. Rev.
Goulding, P.M. Vitousek, F.S. Zhang, Significant acidification in major Chinese crop- Plant Biol. 63 (2012) 153–182.
lands, Science 327 (2010) 1008–1010. [24] W.J. Golz, Biological treatment in recirculating aquaculture systems, Recirculating
[3] R. Howarth, F. Chan, D.J. Conley, J. Garnier, S.C. Doney, R. Marino, G. Billen, Coupled Aquaculture in the Classroom: a Training Workshop for Agricultural Science
biogeochemical cycles: eutrophication and hypoxia in temperate estuaries and Teachers, a Proceedings of a Workshop Sponsored by the Louisiana Sea Grant Col-
coastal marine ecosystems, Front. Ecol. Environ. 9 (2011) 18–26. lege Program, Louisiana State University, and the Louisiana Department of Educa-
[4] B. Yang, Z. Xiong, J. Wang, X. Xu, Q. Huang, Q. Shen, Mitigating net global warming tion, 6–7 December 1995, Louisiana State University, Baton Rouge, Louisiana, 1995.
potential and greenhouse gas intensities by substituting chemical nitrogen fertil- [25] R.V.S. Lavu, V. De Schepper, K. Steppe, P.N.V. Majeti, F. Tack, G.D. Laing, Use of sele-
izers with organic fertilization strategies in rice–wheat annual rotation systems in nium fertilizers for production of Se-enriched Kenaf (Hibiscus cannabinus): effect on
China: a 3-year field experiment, Ecol. Eng. 81 (2015) 289–297. Se concentration and plant productivity, J. Plant Nutr. Soil Sci. 176 (2013) 634–639.
[5] J.J.O. Odhiambo, V.N. Magandini, An assessment of the use of mineral and organic [26] G.S. Venkataraman, S. Neelakantan, Effect of the cellular constituents of the nitrogen
fertilizers by small holder farmers in Vhembe district, Limpopo province, South fixing bluegreen algae Cylindrospermum muscicola on the root growth of rice seed-
Africa, Afr. J. Agric. Res. 3 (2008) 357–362. lings, J. Gen. Appl. Microbiol. 13 (1967) 53–61.
[6] J.J. Bolton, D.V. Robertson-Andersson, D. Shuuluka, L. Kandjengo, Growing Ulva [27] U. Mishra, S. Pabbi, Cyanobacteria: a potential biofertilizer for rice, Resonance
(Chlorophyta) in integrated systems as a commercial crop for abalone feed in (2004) 6–10.
South Africa: a SWOT analysis, J. Appl. Phycol. 21 (2009) 575–583. [28] B. Nanda, S.K. Tripathy, S. Padhi, Effect of algalization on seed germination of vege-
[7] M.H. Abreu, R. Pereira, C. Yarish, A.H. Buschmann, I. Sousa-Pinto, IMTA with table crops, World J. Microbiol. Biotechnol. 7 (1991) 622–623.
Gracilaria vermiculophylla: productivity and nutrient removal performance of the [29] Z. Shariatmadari, H. Riahi, S. Shokravi, Study of soil blue-green algae and their effect
seaweed in a land-based pilot scale system, Aquaculture 312 (2011) 77–87. on seed germination and plant growth of vegetable crops, Bot. J. Iran 12 (2011)
[8] P.H. de Paula Silva, S. McBride, R. de Nys, N.A. Paul, Integrating filamentous ‘green 101–110.
tide’ algae into tropical pond-based aquaculture, Aquaculture 284 (2008) 74–80. [30] M. Jalaluddin, M. Hamid, Effect of adding inorganic, organic and microbial fertilizers
[9] R.J. Lawton, L. Mata, R. de Nys, N.A. Paul, Algal bioremediation of waste waters from on seed germination and seedling growth of sunflower, Pak. J. Bot. 43 (2011)
land-based aquaculture using ulva: selecting target species and strains, PLoS One 8 2807–2809.
(2013), e77344. [31] Y. Miri, S.B. Kochebagh, B. Mirshekari, Effect of inoculation with bio-fertilizers on
[10] G. Markou, D. Georgakakis, Cultivation of filamentous cyanobacteria (blue-green germination and early growth, Dill (Anethum graveolens), Fennel (Foeniculum
algae) in agro-industrial wastes and wastewaters: a review, Appl. Energy 88 vulgare), Cumin (Cuminum cyminum) and Marigold (Calendula officinalis), Int. J
(2011) 3389–3401. Agron. Plant Prod. 4 (2013) 104–108.
[11] R. Wang, B. Peng, K. Huang, The Research Progress of CO2 Sequestration by Algal [32] J. Garcia-Gonzalez, M. Sommerfeld, Biofertilizer and biostimulant properties of the
Bio-Fertilizer in China, J. CO2 Util, 2015, microalga Acutodesmus dimorphus, J. Appl. Phycol. (2015),
007. 1007/s10811-015-0625-2.
[12] R.D. Tripathi, S. Dwivedi, M.K. Shukla, S. Mishra, S. Srivastava, R. Singh, U.N. Rai, D.K. [33] A.F. Safinaz, A.H. Ragaa, Effect of some red marine algae as biofertilizers on growth
Gupta, Role of blue green algae biofertilizer in ameliorating the nitrogen demand of maize (Zea mayz L.) plant, Int. Food Res. J. 20 (2013) 1629–1632.
and fly-ash stress to the growth and yield of rice (Oryza sativa L.) plants, [34] F.A. Faheed, A.A.-E. Fattah, Effect of Chlorella vulgaris as bio-fertilizer on growth pa-
Chemosphere 70 (2008) 1919–1929. rameters and metabolic aspects of lettuce plant, J. Agric. Soc. Sci. 4 (2008) 165–169.
[13] H. Saadatnia, H. Riahi, Cyanobacteria from paddy fields in Iran as a biofertilizer in [35] K.L.N. Aung, Effect of Spirulina biofertilizer suspension on growth yield of Vigna
rice plants, Plant Soil Environ. 55 (2009) 207–212. radiata (L.) Wilczek, Univ. Res. J. 4 (2011) 351–363.
[14] A. Watanabe, R. Ito, C. Konishi, Effect of nitrogen-fixing blue-green algae on the
growth of rice plants, Nature 168 (1951) 748–749.
[15] E.J. Olguín, S. Galicia, R. Camacho, G. Mercado, T. Peŕez, Production of Spirulina sp. in
sea water supplemented with anaerobic effluents in outdoor raceways under tem-
perate climatic conditions, Appl. Microbiol. Biotechnol. 48 (1997) 242–247.