Ecological Engineering 99 (2017) 47–53

Contents lists available at ScienceDirect

Ecological Engineering
journal homepage: www.elsevier.com/locate/ecoleng

Heterotrophic cultivation of microalgae using aquaculture

wastewater: A biorefinery concept for biomass production and
nutrient remediation
Abhishek Guldhe, Faiz A. Ansari, Poonam Singh, Faizal Bux ∗
Institute for Water and Wastewater Technology, Durban University of Technology, P.O. Box 1334, Durban, 4000, South Africa

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

Article history: Cultivation of microalgae utilizing wastewater substrate could form a sustainable biorefinery with double
Received 13 July 2016 benefit of biomass generation and nutrient remediation. In this study potential of aquaculture wastewater
Received in revised form is evaluated for cultivation of Chlorella sorokiniana in heterotrophic mode for generation of high value
21 September 2016
biomass. Nutrient removal potential is also assessed. Aquaculture wastewater with 400 mgL−1 sodium
Accepted 13 November 2016
Available online 18 November 2016
nitrate supplementation resulted in biomass productivity of 498.14 mgL−1 d−1 . The biomass generated
showed lipid productivity of 150.19 mgL−1 d−1 , carbohydrate productivity of 172.91 mgL−1 d−1 and protein
productivity of 141.57 mgL−1 d−1 . The nutrient removal efficiencies were 75.56% for ammonium, 84.51%
Keywords:
Microalgae for nitrates, 73.35% for phosphates and 71.88% for COD (chemical oxygen demand). The findings of this
Aquaculture wastewater study underline the potential of aquaculture wastewater for production of valuable microalgal biomass
Heterotrophic which can be utilized for biofuels or feed application. This biorefinery concept also polished aquaculture
Biomass wastewater which can be effectively reused.
Biorefinery © 2016 Elsevier B.V. All rights reserved.

1. Introduction of expensive inorganic chemicals (Medeiros et al., 2015; Rawat

Microalgal biomass has been proven as a sustainable feedstock
for biofuels, feed and numerous value added products pertinent to
nutraceuticals and therapeutic industry. Microalgae can be effec-
tively grown using wastewater for biomass generation and nutrient
remediation (Ma et al., 2016; Rawat et al., 2011). Heterotrophic
cultivation of microalgae is advantageous in terms of eliminating
light dependency and higher biomass yields over phototrophic cul-
tivation (Kim et al., 2013; Venkata Mohan et al., 2015). Microalgal
biomass generated is rich in lipids, carbohydrates and proteins.
The microalgal biomass can be utilized primarily for biofuels gen-
eration (biodiesel, biomethane, biohydrogen etc.) and for feed
applications (aquaculture and animal) (Singh et al., 2015a). Alter-
natively, microalgal biomass can also be utilized for value added
products such as pigments, nutraceuticals, bioplastic etc. (Suganya
et al., 2016). Use of synthetic medium makes the commercial scale
microalgal biomass generation unfeasible. Heterotrophic cultiva-
tion using different waste streams have been gaining interest from
researchers as it reduces the production cost by dropping the usage
∗ Corresponding author.
E-mail address: faizalb@dut.ac.za (F. Bux).
et al., 2011).
Biorefinery concept where different industries are integrated
together for various products and mutual benefits could prove
as a sustainable and economical approach for microalgal biomass
generation. Aquaculture is one of the fastest growing food indus-
tries. This growing industry generates wastewater rich in nutrients
such as ammonia, nitrates, phosphates and organic load (Gao et al.,
2016; Lananan et al., 2014). Wastewater generated in aquaculture
industry needs treatment prior to its reuse or release in envi-
ronment to avoid the eutrophication. This wastewater treatment
step adds to the production cost of aquaculture produce. Existing
wastewater treatment processes used in aquaculture are denitri-
fication process to release nitrogenous compounds in atmosphere
and chemical precipitation using ferrous chloride to remove phos-
phorous compounds. These processes are not only adding to the
cost but also lead to toxic by-products (Mook et al., 2012; Nasir
et al., 2015). Microalgal cultivation using aquaculture could prove
itself as a promising biorefinery for economical biomass generation
and sustainable wastewater remediation. Microalgae can utilize the
nutrients and organic load present in aquaculture wastewater to
produce valuable biomass which can be subsequently utilized for
biofuels or feed applications. Microalgae have been studied as a
feed supplement and also as a whole feed for aquaculture industry.
Microalgae contain proteins, long chain fatty acids, pigments which

http://dx.doi.org/10.1016/j.ecoleng.2016.11.013
0925-8574/© 2016 Elsevier B.V. All rights reserved.
48 A. Guldhe et al. / Ecological Engineering 99 (2017) 47–53
are essential for fish nutrition. Whole microalgae or lipid extracted
microalgae can thus be utilized in fish feed production (Ju et al.,
2012).
There are several studies on utilization of wastewater for
microalgal cultivation (Caporgno et al., 2015; Ramanna et al., 2014).
However, using aquaculture wastewater for this purpose is not
well explored area. There are very few studies which report the
use of aquaculture wastewater for biomass production, but most of
these studies mainly focus on phototrophic cultivation and nutri-
ent removal. Very few studies report the biomass composition,
which is important for its applications. Thus it is important to inves-
tigate the potential of aquaculture wastewater for heterotrophic
cultivation of microalgae and evaluate the biomass composition.
In this study, aquaculture wastewater has been used to cultivate
Chlorella sorokiniana heterotrophically. Microalgal growth, nutrient
removal and biomass composition were thoroughly investigated
and compared with synthetic media cultivation. Sodium nitrate
supplementation strategy has also been developed to achieve pos-
sible sealing biomass and primary metabolite productivities.
2. Materials and methods
2.1. Wastewater collection and characterization
The aquaculture wastewater was collected from aquaculture
research facility, Durban, South Africa. At this facility Nile tilapia are
reared in 5000 L tanks in controlled temperature (27–32 ◦ C) with
continuous aeration. A biofiltration system was used to remove
nutrients and organic load from recycled water at regular interval.
For this study wastewater was sourced from the collection tank
before the treatment. Aquaculture wastewater (AW) was collected
in 25 L containers and brought to laboratory immediately after
collection. The pH, electrical conductivity, temperature, salinity,
dissolve oxygen (DO), dissolve oxygen percentage were measured
at the time of sample collection by YSI MP – AES. The total solid
and total dissolve solid were calculated by standard methods of
APHA 2005 (APHA-AWA-WEF, 2005). The chemical oxygen demand
(COD) was determined by closed refluxed method. Centrifuged
sample were used to analyze ammonia (NH4 + ), nitrates (NO3 − ),
nitrites (NO2 − ) and phosphates (PO4 3− ) concentration by GalleryTM
Automated Photometric analyzer (Thermo Scientific, USA). For the
heavy metals analysis, sample were digested in microwave (Mile-
stone S.R.L., Italy, output power 1200 W) at 180 ◦ C for 20 min at
1000 W using acid mixture (15 mL HNO3 and 4 mL HClO4 ). After
cooling the solution was allowed to evaporate from digested sam-
ples until the volume reduced to 5 mL. The samples were filtered
through filter paper and further diluted using deionized water to
50 mL for heavy metals analysis using microwave plasma atomic
emission spectrometry (Agilent Technologies 4200 MP-AES). Bac-
terial count was determined by determined by heterotrophic plate
count method.
2.2. Microalgae cultivation
Chlorella sorokiniana strain was used for the heterotrophic cul-
tivation using aquaculture wastewater (AW). The AW was first
filtered using glass fiber filter papers and then autoclaved prior to
microalgal inoculation (Nasir et al., 2015). The microalgal cultures
were maintain in 1 L conical flask with 500 mL working volume. The
cultivation conditions were: temperature 25 ◦ C, shaking at 110 rpm
in complete dark phase. Microalgal culture flasks were wrapped
in aluminum foil and were kept in dark conditions. These condi-
tions were kept constant for all the experiments. Microalgae were
also cultivated in BG11 nutrient medium supplemented with glu-
cose for comparative analysis. Microalgae were also grown in BG11
medium and AW under phototrophic condition for comparative
analysis. Each set of experiment was done in duplicate. Supple-
mentation experiments were carried out by adding 200, 400, 600
and 1500 mgL−1 sodium nitrate in AW.
2.3. Analytical methods
2.3.1. Growth and biomass analysis
Microalgal growth was monitored daily by determining optical
density at 680 nm using spectrophotometric method. Biomass was
estimated gravimetrically at initial, middle and late log phases of
growth. Biomass productivity (mgL−1 d−1 ) was calculated at late log
phase gravimetrically (Singh et al., 2015b). Biomass was harvested
using centrifuge and freeze dried using lyophilizer (Mini lyotrap,
LTE scientific Ltd., United Kingdom) for further analysis.
2.3.2. Nutrient removal
The nutrients removal efficiency was determined on every alter-
nate day. For analysis 10 mL of sample were collected from culture
flask and centrifuged. The samples were then filtered using 0.45 ␮m
syringes filters. These samples were analyzed for nitrates (NO3 − ),
nitrites (NO2 − ), TON, ammonia (NH4 + ) and phosphates (PO4 3− )
using GalleryTM Automated Photometric analyzer (Thermo Scien-
tific, USA) (Gupta et al., 2016). The chemical oxygen demand (COD)
was analyzed by closed refluxed method. The removal efficiency in
percentage was determined by using following equation
Percentage removal =
Initial concentration − Final concentration
× 100 (1)
Initial concentration
2.3.3. Biochemical composition of biomass
Microalgal biomass collected from each experiment was ana-
lyzed for lipids, carbohydrates and proteins. Lipids were extracted
from the harvested biomass using microwave assisted sol-
vent extraction. Dried biomass was mixed with chloroform and
methanol (2:1 v/v) and then subjected to microwave treatment
(100 ◦ C for 10 min at 1000 W) for cell disruption (Guldhe et al.,
2014). Biomass residues were removed by filtration. The organic
layer was collected and oven dried at 70 ◦ C for lipid recovery. The
lipids obtained were measured gravimetrically and the percentage
lipid content was determined based on lipid recovered from known
weight of dry biomass. Lipids productivity was calculated according
to equation described by (Singh et al., 2015b)
lipid content
Lipid productivity = biomass productivity × (2)
100
Where, biomass productivity is in mgL−1 d−1 , lipids content is in
percentage per dry biomass weight.
Proteins extraction was done following the method given by
Lopez et al. (Lopez et al., 2010). The quantitative analysis of proteins
was done by Lowry’s method. A spectrophotometer (Spectroquant-
Pharo 300, Merck) was used to measure the absorbance of the
extraction mixture at 750 nm. Standards for calibration were pre-
pared by using bovine serum albumin (BSA) in lysis buffer. The
standard calibration curve prepared using BSA was used for pro-
teins quantification. Proteins productivity was determined using
equation 3.
protein content
Protein productivity = biomass productivity × (3)
100
Where, biomass productivity is in mgL−1 d−1 , proteins content
is in percentage per dry biomass weight.
Total carbohydrates were quantified using the phenol-sulfuric
acid method (Prajapati et al., 2013). Dried biomass was mixed with
sulfuric acid (2% v/v) and autoclaved for 30 min at 121 ◦ C for hydrol-
ysis. The mixture was then neutralized with 1 M NaOH/H2 SO4 .
Supernatant was collected by centrifugation at 1509 × g for 10 min.
 A. Guldhe et al. / Ecological Engineering 99 (2017) 47–53 49

Biomass productivity
600

Biomass productivity (mg/l/day)

500

400

300

200

100

0
BG11 H AW H AW H200 AW H400 AW H600 BG11 P AW P

Fig. 1. Biomass productivities of C. sorokiniana in BG11 medium and aquaculture wastewater with different concentration of sodium nitrate supplementation.
BG11 H- Blue green 11 media heterotrophic, AW H- Aquaculture wastewater heterotrophic, AW H 200- Aquaculture wastewater heterotrophic supplemented with 200 mgL−1
sodium nitrate, AW H 400- Aquaculture wastewater heterotrophic supplemented with 400 mgL−1 sodium nitrate, AW H 600- Aquaculture wastewater heterotrophic
supplemented with 600 mgL−1 sodium nitrate, BG11 P- Blue green 11 media phototrophic, and AW P- Aquaculture wastewater phototrophic.

240
Lipid productivity

200
Lipid productivity (mg/l/day)

160

120

80

40

0
BG11 H AW H AW H200 AW H400 AW H600 BG11 P AW P

Fig. 2. Lipid productivities of C. sorokiniana in BG11 medium and aquaculture wastewater with different sodium nitrate supplementation.
BG11 H- Blue green 11 media heterotrophic, AW H- Aquaculture wastewater heterotrophic, AW H 200- Aquaculture wastewater heterotrophic supplemented with 200 mgL−1
sodium nitrate, AW H 400- Aquaculture wastewater heterotrophic supplemented with 400 mgL−1 sodium nitrate, AW H 600- Aquaculture wastewater heterotrophic
supplemented with 600 mgL−1 sodium nitrate, BG11 P- Blue green 11 media phototrophic, and AW P- Aquaculture wastewater phototrophic.

0.1 mL of supernatant was diluted to 1 mL, and then mixed with
1 mL of phenol (5% w/v) and 5 mL of 96% H2 SO4 . After cooling to
25–30 ◦ C, the absorbance of this solution was measured at 490 nm
using a spectrophotometer (Spectroquant Pharo 300, Merck). Total
carbohydrates were quantified from a calibration curve prepared
using glucose as a standard (Prajapati et al., 2013). Carbohydrates
productivity was determined by the Eq. (4).
Carbohydrate productivity = biomass productivity
carbohydrate content
× (4)
100
Where, biomass productivity is in mgL−1 d−1 , carbohydrate content
is in percentage per dry biomass weight.
3. Results and discussion
3.1. Wastewater characterization
Aquaculture wastewater (AW) was analyzed for presence of
nitrates, ammonia and phosphates required for microalgal culti-
vation. The physicochemical parameters for collected AWW are
depicted in Table 1. The collected wastewater from aquaculture
facility showed ammonia 5.32 mgL−1 , nitrates 40.67 mgL−1 , phos-
phate 8.82 mgL−1 and COD 96 mgL−1 (Table 1). Guo et al., 2013
used aquaculture wastewater for growth of microalgae Platymonas
subcordiformis. In their study aquaculture wastewater showed
presence of 47.8 mgL−1 nitrates and 8.87 mgL−1 phosphates, which
is similar to aquaculture used in this study. The pH of the collected
wastewater was 7.28. The bacterial load in AWW was found to be
50 A. Guldhe et al. / Ecological Engineering 99 (2017) 47–53

Carbohydrate productivity
200

Carbohydrate productivity (mg/l/day)

150

100

50

0
BG11 H AW H AW H200 AW H400 AW H600 BG11 P AW P

Fig. 3. Carbohydrate productivities of C. sorokiniana in BG11 medium and aquaculture wastewater with different sodium nitrate supplementation.
BG11 H- Blue green 11 media heterotrophic, AW H- Aquaculture wastewater heterotrophic, AW H 200- Aquaculture wastewater heterotrophic supplemented with 200 mgL−1
sodium nitrate, AW H 400- Aquaculture wastewater heterotrophic supplemented with 400 mgL−1 sodium nitrate, AW H 600- Aquaculture wastewater heterotrophic
supplemented with 600 mgL−1 sodium nitrate, BG11 P- Blue green 11 media phototrophic, and AW P- Aquaculture wastewater phototrophic.

200
Protein productivity
180

160
Protein productivity (mg/l/day)

140

120

100

80

60

40

20

0
BG11 H AW H AW H200 AW H400 AW H600 BG11 P AW P

Fig. 4. Protein productivities of C. sorokiniana in BG11 medium and aquaculture wastewater with different sodium nitrate supplementation.
BG11 H- Blue green 11 media heterotrophic, AW H- Aquaculture wastewater heterotrophic, AW H 200- Aquaculture wastewater heterotrophic supplemented with 200 mgL−1
sodium nitrate, AW H 400- Aquaculture wastewater heterotrophic supplemented with 400 mgL−1 sodium nitrate, AW H 600- Aquaculture wastewater heterotrophic
supplemented with 600 mgL−1 sodium nitrate, BG11 P- Blue green 11 media phototrophic, and AW P- Aquaculture wastewater phototrophic.

1.79 × 103 cfu mL−1 (Table 1). The other nutrients present in the
AWW are depicted in Table 1. AWW also showed presence of other
micronutrients such as iron, magnesium, zinc, molybdenum which
are required for various physiological activities of microalgae.
3.2. Microalgae growth and biomass production using
aquaculture wastewater
The heterotrophic cultivation of C. sorokiniana showed biomass
yields of 2.47 gL−1 in AW and 4.02 gL−1 in BG11 medium (Table 2).
Higher biomass yield in synthetic medium (BG11) is because of
the higher nutrient concentration compared to that in AW. Simi-
larly, biomass productivity of 353.36 mgL−1 d−1 in AW was found
to be lower than the BG11 medium (573.79 mgL−1 d−1 ). Li et al.
(2011) cultivated chlorella sp. in sludge centrate and observed
high biomass productivity of 920 mgL−1 d−1 . In their study sludge
centrate was composed of higher nutrient concentration (COD:
2304 mgL−1 ; ammonia: 82.5 mgL−1 ; phosphate 212 mgL−1 ) than
the aquaculture wastewater used in this study. The lower biomass
productivities suggest the need of nutrient supplement for microal-
gal cultivation in AW to achieve comparable biomass yields to that
of the synthetic medium. Phototrophic cultivation of C. sorokini-
ana using AW resulted in biomass productivity of 107.86 mgL−1 d−1
(Fig. 1). The biomass productivity in heterotrophic cultivation was 3
times higher than the phototrophic cultivation. Kim et al. (2013) in
their study found that growth rate of C. sorokiniana in heterotrophic
mode was 0.53 d−1 while it was 0.24 d−1 in autotrophic mode. This
result clearly demonstrates that the heterotrophic mode of nutri-
tion is the most suitable cultivation strategy for using aquaculture
wastewater for microalgal biomass generation.
 A. Guldhe et al. / Ecological Engineering 99 (2017) 47–53 51

Table 1 ammonium may be volatilized by increased pH and temperature.

Characterization of aquaculture wastewater.
Similarly, phosphorous could get precipitated by increase in pH
Parameter Unit Value and dissolved oxygen. In heterotrophic mode for algal cultivation
pH – 7.28 the organic carbon present in the medium is the primary source
Temperature ◦
C 27.3 of energy. Initial COD observed in AW was 96 mgL−1 , which was
Total solid gL−1 0.42 reduced to 27 mgL−1 after 7 days of cultivation of C. sorokiniana in
Total dissolve solid gL−1 0.35 AW. The percentage removal efficiency of C. sorokiniana in AW for
Salinity mgL−1 0.26
COD was 71.89%. The nutrient removal efficiencies depend upon the
DO mgL−1 4.17
COD mgL−1 96 initial concentration of nutrients in the wastewater and microalgal
NH4 + -N mgL−1 5.32 strains used. In a study by Zhou et al. (2012) the initial concentration
NO2 − -N mgL−1 5.52 of ammonia was 91 mg L−1 , COD was 2324 mg L−1 and phosphates
NO3 − -N mgL−1 40.67
was 212 mg L−1 in wastewater and they cultivated Auxenchlorella
TON mgL−1 38.80
PO4 3− -P mgL−1 8.82 protothecoides which showed high removal efficiency (Table 4).
Fe mgL−1 4.05 Zhang et al. (2013) cultivated Chlorella sp. ZTY4 using domestic
Mo mgL−1 3 wastewater in heterotrophic mode. The removal efficiencies found
Zn mgL−1 0.85 in their study were 40.8% for COD, 15.8% for total nitrogen and 49.9%
Na mgL−1 66.25
for total phosphorous respectively. Microalgal cultivation thus can
Ni mgL−1 0.3
Mg mgL−1 8.45 be implemented as a polishing step for aquaculture wastewater
K mgL−1 5.9 before its reuse or release into the environment.
Bacterial density cfu mL−1 1.795 × 103

3.4. Nutrient supplementation

Table 2
Biomass, lipid, protein and carbohydrates yields in C. sorokiniana under different Biomass productivities of C. sorokiniana in AW were lower than
cultivation condition in synthetic media and aquaculture wastewater. the synthetic media. Nitrogen is major nutrient which plays role
Units Biomass Lipid content Protein Carbohydrate in algal growth, in synthetic media nitrogen is supplied in forms
gL−1 % content % content% of nitrates. Furthermore, removal rate for nitrates found in this
BG11 H 4.02 35.75 28.64 33.64 study was 84.51% which clearly demonstrates the high uptake of
AW H 2.47 39.1 24.57 36.1 this nutrient. Thus sodium nitrate supplementation in AW was
AW H200 2.9 35.3 26.38 35.97 investigated in this study. Increasing biomass productivities were
AW H400 3.49 30.15 28.42 34.71 observed with increasing concentrations of sodium nitrate sup-
AW H600 3.54 26.1 29.46 32.79
BG11 P 2.54 26 29.46 29.74
plementation. Highest biomass productivity of 505.71 mgL−1 d−1
AW P 1.51 33.45 29.46 35.43 was observed with 600 mgL−1 sodium nitrate in AW. At 400 mgL−1
sodium nitrate supplementation in AW 498.12 mgL−1 d−1 biomass
BG11 H- Blue green 11 heterotrophic, AW H- Aquaculture wastewater heterotrophic,
AW H 200-Aquaculture wastewater heterotrophic supplemented with 200 mgL−1 productivity was observed which is comparable to highest biomass
sodium nitrate, AW H 400-Aquaculture wastewater heterotrophic supplemented productivity at 600 mgL−1 sodium nitrate supplementation (Fig. 1).
with 400 mgL−1 sodium nitrate, AW H 600-Aquaculture wastewater heterotrophic It is economical to use minimum chemical supplementation while
supplemented with 600 mgL−1 sodium nitrate, BG11 P-Blue green 11 phototrophic,
using wastewater as a nutrient medium. Thus 400 mmgL−1 d−1 of
AW P-Aquaculture wastewater phototrophic.
sodium nitrate supplementation was determined to be optimum
for C. sorokiniana cultivation in AW.
3.3. Nutrient removal from aquaculture wastewater
3.5. Biochemical composition of biomass
Microalgal C. sorokiniana utilized the nutrients present in the
aquaculture wastewater for its growth. The nutrient removal effi- Lipid, carbohydrate and protein content and productivities were
ciencies were determined after the 7 day cultivation period. The determined for biomass harvested from all the experiments. Lipid
removal efficiencies were found to be 84.51% for nitrates, 96.38% content of C. sorokiniana cultivated using AW was 39.1% DCW,
for nitrites, 75.56% for ammonia, and 73.35% for phosphorus respec- while it was 35.75% DCW for synthetic medium. Similarly, carbo-
tively (Table 3). These nutrients are utilized by microalgal cells for hydrate content in C. sorokiniana grown using AW was 36.1% DCW,
various physiological processes to generate biomass rich in lipids, while it was 33.64% DCW for synthetic medium (Table 2). In pre-
carbohydrates and proteins. Nitrogen is important for protein and vious studies, Auxenchlorella protothecoides grown on wastewater
genetic material synthesis while phosphorous is major constituent showed lipid content of 33.22% (Zhou et al., 2012); Chlorococcum
of adenosine triphosphate (ATP) required for short term energy sp. RAP13 grown on dairy effluent showed lipid content of 39–42%
storage and transfer (Kim et al., 2016). Microalgae utilize inorganic (Ummalyma and Sukumaran, 2014) and Scenedesmus sp. LX1 grown
nitrogen in form of ammonium, nitrate and nitrite by assimila- using secondary effluent from domestic treatment plant showed
tion process where nitrate and nitrite undergo reduction to form lipid content of 31–33% (Xin et al., 2010) (Table 4). C. sorokini-
ammonium which is then incorporated in amino acid glutamine ana when grown in raw sewage showed lipid content of 22.74%
(Cai et al., 2013). The removal of these inorganic nutrients is not (Gupta et al., 2016). Very few studies on microalgal cultivation using
solely governed by the uptake via microalgal cells. Some amount of wastewaters report the biochemical constituents of the biomass.

Table 3
Nutrients and COD removal efficiency of C. sorokiniana cultivated in aquaculture wastewater.

Units COD NO3 −1 -N NO2 −1 -N NH4 + -N PO4 3− -P

Initial mgL−1 96 40.67 5.52 5.32 8.82

Final mgL−1 27 6.3 0.2 1.3 2.35
Removal efficiency % 71.88 84.51 96.38 75.56 73.35
Removal rate mgL−1 d−1 8.63 4.3 0.67 0.5 0.8
52 A. Guldhe et al. / Ecological Engineering 99 (2017) 47–53

Microalgae are known to accumulate more lipid and carbohydrates

in stressed conditions as a defense mechanism. The nitrogen and

Sukumaran (2014)
Wang et al. (2010)

Nasir et al. (2015)

Zhou et al. (2012)

Gao et al. (2016)

phosphorous content in the AW was lower than the BG11 medium.

Ummalyma and

Xin et al. (2010)

Li et al. (2011)

Current study
Thus the cultivation of microalgae in AW is under a nutrient stress
environment which results in higher lipid and carbohydrate con-
tents. Protein content in the microalgal cells is directly related to
Refs.

the growth. Reduced growth is observed in microalgae grown on

AW compared to synthetic medium. This has been reflected in the
protein content also, the protein content for C. sorokiniana grown
removal

71.88
in AW was 24.57% DCW which was lower than the protein con-
COD

79.9
90.8
83

93
%

–
tent (28.64% DCW) of biomass generated using synthetic medium
(Table 2).
Generally, in microalgal cultivation a tradeoff is observed

63.1 − 92.2
removal
PO4 −3 -P

for biomass and lipid productivities with nitrogen stress. With

98.48

73.35
85.5

82.7
80.9

increasing concentration of nitrogen in medium biomass produc-

98
%

tivities increases while lipid content decreases. In this study with

sodium nitrate supplementation lipid productivity increased upto
400 mgL−1 supplementation after that lower lipid productivity
NO3 −1 -N
removal

was observed with 600 mgL−1 . Lipid productivity with 400 mgL−1
84.51

sodium nitrate supplementation in AW was 150.19 mgL−1 d−1 .

%

–
–
–

Lipid productivity with 600 mgL−1 supplementation was found

to be 131.98 mgL−1 d−1 (Fig. 2). Similar trend was observed for
removal
NH4 + -N

carbohydrate productivity; highest carbohydrate productivity of

75.56
78.3
93.9

172.91 mgL−1 d−1 was observed in C. sorokiniana grown in AW

100
%

supplemented with 400 mgL−1 sodium nitrate (Fig. 3). Protein pro-
ductivity was found to be gradually increasing with the increasing
C = 37.1P = 131.1
d−1 , C=Content

concentration of sodium nitrate in AW. Highest protein produc-

Carbohydrate

tivity mg L−1

tivity of 148.98 mgL−1 d−1 was observed with 600 mgL−1 sodium
P = Produc-

nitrate in AW. The 400 mgL−1 supplementation selected as opti-

in%

mum for biomass production resulted in protein productivity of

–
–
–

141.57 mgL−1 d−1 which is slightly lower than the highest (Fig. 4).
These results highlight the promising potential of cultivation of
C = 24.87P = 87.86

microalgae in AW for production of high quality biomass. Supple-

d−1 , C=Content
tivity mg L−1

mentation strategy has been found to be very effective to enhance

P = Produc-

the biomass productivity with adequate primary metabolites com-

Protein

position. The lipid and carbohydrates in microalgal biomass can be

in%
–
–
–

used for various biofuels synthesis such as biodiesel, biomethane

and bioethanol. Protein component of biomass makes it suitable for
C = 39.1P = 138.17

aquaculture or animal feed application.

tivity mg L−1 d−1 ,
C=Content in%

There are very few studies which report the heterotrophic culti-
P = Produc-

C = 39–42

C = 31–33

vation of microalgae in wastewater. Table 4 depicts the comparison

C = 33.22

of biomass yields, biochemical composition and nutrient removal

Lipid

efficiencies observed in this study to the previous reports on het-

–
–

erotrophic microalgal cultivation in different wastewaters. The

Previous studies reporting microalgae cultivation in different wastewaters.

biomass productivity for C. sorokiniana observed in this study using

B = 2.47 P = 353.36

AW was higher compared to the previous reported studies of het-

production g L−1

B = 1.48–1.94
P = mgL−1 d−1

erotrophic and phototrophic cultivation of microalgae in different

productivity

B = Biomass

wastewater streams (Table 4). The nutrient removal efficiencies for

Biomass

B = 1.16

P = 42.6
P = 0.11
P = 920

COD, ammonium, nitrate, nitrite and phosphates were comparable

to the previous reports of heterotrophic cultivation of microalgae
–

in various wastewater substrates. Most of the previous studies only

focus on lipid productivities of microalgae for biodiesel synthesis.
Aquaculture wastewater

Aquaculture wastewater

Aquaculture wastewater
Secondary effluent from

In this study biomass was also evaluated for protein and carbohy-
Wastewater centrate
Autoclaved centrate

domestic treatment
Type of wastewater

drate productivities for its application in other biofuels and feed

Dairy effluent

applications.
Wastewater

plant

4. Conclusion

The findings of this study have clearly highlighted the potential

Scenedesmus sp. LX1

of aquaculture wastewater as a nutrient substrate for cultivation

(Phototrophic)

(Phototrophic)
Chlorococcum sp.
protothecoides

of microalgae. Heterotrophic mode of cultivation of microalgae

Auxenchlorella

C. sorokiniana
Chlorella sp.
Chlorella sp.

Chlorella sp.

had shown better biomass and metabolites productivities than the

Microalgae

C. vulgaris
RAP13

phototrophic mode. Nutrient supplementation strategy improves

Table 4

the biomass growth as well as lipid, carbohydrate and protein

productivities. Microalgal biomass generated using aquaculture
 A. Guldhe et al. / Ecological Engineering 99 (2017) 47–53
wastewater shown high lipid, carbohydrate and protein yields
which can be used for biofuels and feed application. This biorefinery
concept forms the basis for sustainable and economic integration
of aquaculture and microalgae industry.
Acknowledgements
Authors hereby acknowledge the Durban University of Tech-
nology South Africa, National Research Foundation (South Africa)
(NRF), for financial contribution. eThekwini municipality, Durban
for the support.
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