Environmental Science and Pollution Research

https://doi.org/10.1007/s11356-019-04633-0

RESEARCH ARTICLE

Optimising of Scenedesmus sp. biomass production in chicken

slaughterhouse wastewater using response surface methodology
and potential utilisation as fish feeds
Maizatul Azrina Yaakob 1 & Radin Maya Saphira Radin Mohamed 1 & Adel Al-Gheethi 1 & Athirah Tiey 1 &
Amir Hashim Mohd Kassim 1

Received: 9 July 2018 / Accepted: 19 February 2019

# Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
Production of Scenedesmus sp. biomass in chicken slaughterhouse wastewater (CSWW) is a promising alternative technique for
commercial culture medium due to the high nutritional content of the generated biomass to be used as fish feeds. The current
work deals with optimising of biomass production in CSWW using response surface methodology (RSM) as a function of two
independent variables, namely temperature (10–30 °C) and photoperiod (6–24 h). The potential application of biomass yield as
fish feeds was evaluated based on carbohydrate, protein and lipid contents. The results revealed that the best operating parameters
for Scenedesmus sp. biomass production with high contents of carbohydrates, proteins and lipids were determined at 30 °C and
after 24 h. The actual and predicted values were 2.47 vs. 3.09 g, 1.44 vs. 1.27 μg/mL, 29.9 vs. 31.60% and 25.75 vs. 28.44%,
respectively. Moreover, the produced biomass has a high concentration of fatty acid methyl ester (FAME) as follows: 35.91% of
C15:1; 17.58% of C24:1 and 14.11% of C18:1N9T. The biomass yields have 7.98% of eicosapentaenoic acid (EPA, C20:5N3)
which is more appropriate as fish feeds. The Fourier transform infrared (FTIR) analysis of biomass revealed that the main
functional groups included hydroxyl (OH), aldehyde (=C–H), alkanes and acyl chain groups. Scanning electron micrograph
(SEM) and energy-dispersive X-ray spectroscopic analysis (EDS) indicated that the surface morphology and element distribution
in biomass produced in BBM and CSWW were varied. The findings have indicated that the biomass produced in CSWW has
high potential as fish feeds.

Keywords Slaughterhouse wastewater . Scenedesmus . Lipid contents . Fish meals

Introduction et al. 2017). Microalgae biomass has the potential to substitute

The potential of microalgae biomass as fish meals depends
mainly on the presence of high contents of carbohydrates,
lipids and proteins in the biomass yield. It has been demon-
strated that microalgae cells have 30–40% protein, 10–20%
lipid content and 5–15% carbohydrates (Brown 2002; Jais
Responsible editor: Philippe Garrigues
* Radin Maya Saphira Radin Mohamed
maya@uthm.edu.my
* Adel Al-Gheethi
adel@uthm.edu.my
1
Faculty of Civil and Environmental Engineering, Micro-Pollutant
Research Centre, Universiti Tun Hussein Onn Malaysia, Parit
Raja, Malaysia
fish feed due to its easy culturing methods with rapid growth
rates compared to terrestrial crops. Moreover, microalgae cells
can be easily digested by fishes due to its extremely small size
(Hemaiswarya et al. 2011; Apandi et al. 2018a). The potential
of microalgae biomass as fish feeds has been reported in the
literature. Radhakrishnan et al. (2015) replaced fish meal with
25, 50, 75 and 100% of Chlorella vulgaris biomass and inves-
tigated their effects on growth performance, energy utilisation
and digestive enzyme of freshwater prawn post-larvae
(Macrobrachium rosenbergii). The study revealed that 50%
fish meal replaced by C. vulgaris enhanced growth perfor-
mance (1.25 g/day), survival rate (93.33%) and feeding rate
efficiency (2.54%). It was concluded that C. vulgaris as a
source of protein for fish feed replacement was easily
absorbed and digested by animals. However, one of the limi-
tations for utilisation of microalgae biomass as fish feed is the
high cost of the production process in the commercial media.

Therefore, many of the researchers have shifted to use waste-
water as a production medium for microalgae biomass.
Slaughterhouse wastewater is a type of wastewater which
contains high amounts of organic matter (nitrogen, phospho-
rus and carbon) and includes proteins, blood residues, fat and
lard, which are available in the range required for microalgae
growth (Bazrafshan et al. 2012; Al-Gheethi et al. 2019a). The
microalgae require approximately 0.03–0.06% of phosphorus
in the culture medium to sustain growth (Hannon et al. 2010;
Atiku et al. 2016). Phosphorus is present in wastewater as
orthophosphate (PO43−) which is superior for microalgae bio-
mass (Solovchenko et al. 2016). However, the nutrients avail-
able in CSWW are not the sole requirement for microalga
growth. Many factors such as light, temperature and pH might
contribute to the quality and quantity of growth, and the pH of
CSWW (pH 7–9) is within the range for most algal species
(Yaakob et al. 2018).
Temperature and light play a critical role in algal growth, and
temperature affects biochemical composition. Light is an essen-
tial factor for microalgae growth, because microalgae cell con-
verts light into energy via the photosynthesis process. Both tem-
perature and light factors might be overcome by using indige-
nous microalgae strains that have been acclimated to local envi-
ronments (Pahazri et al. 2016). Moreover, light and temperature
are adjustable factors which might be optimised to be within the
optimum range of microalgae growth. Optimisation of
microalgae is performed using response surface methodology
(RSM), which is a statistical design of experimental tools that
leads to the peak of process performance. RSM put all responses
together via optimisation approaches which lead to the discovery
of ideal conditions where all specifications are met at minimal
cost (Anderson and Whitcomb 2016; Al-Gheethi et al. 2019b).
The effect of two or more factors can be screened using the
factorial design. Factorial designs have several advantages such
as efficiency and precision than one-factor-at-a-time experiment,
can avoid misleading conclusion from factorial interaction and
allow the effect factor to be estimated at several levels of other
factors, yielding conclusions that are valid over a range of exper-
imental conditions (Montgomery et al. 2015). Skorupskaite et al.
(2015) optimised Chlorella sp. biomass production in the culture
medium using RSM. The study revealed high Chlorella sp. bio-
mass concentration that reached 2.41 g/L, and it was achieved in
a growth medium containing 0.114 g/L nitrogen and 2.70 g/L
technical glycerol. CSWW has higher concentrations of these
elements, which is expected to induce the production of
microalgae biomass. Singh et al. (2015) used RSM for
optimising Ankistrodesmus falcatus biomass production to be
used as a biofuel feedstock. The study recorded high lipid content
(74.07 mg/L/day). Andrade et al. (2014) optimised psychrophilic
microalgae biomass production in a temperature between 10 and
20 °C in a Rodriguez-Lopez medium. Based on the quadratic
prediction model, high biomass production for the Rodriguez-
Lopez medium (RL) was observed as 600 mg/L at 10 and 20 °C.
Environ Sci Pollut Res
Wahidin et al. (2013) optimised light intensities and photoperiod
of Nannochloropsis sp. growth and lipid content. The study re-
ported that the maximum lipid content was recorded at
100 μmol m−2 s−1 light intensity and photoperiod of 18 h.
However, the optimisation studies performed by authors in the
literature might be insufficient to be applied for microalgae pro-
duction in the CSWW due to differences in the chemical com-
position of the production media as well as microalgae species.
Therefore, optimisation for the Scenedesmus sp. biomass produc-
tion in CSWW is required. The aim of the current study was to
optimise the Scenedesmus sp. biomass production using RSM as
well as to determine the quality of biomass yield for fish feeds in
a comparison to a commercial fish meal based on the carbohy-
drate, protein and lipid contents.
Material and methods
Chicken slaughterhouse wastewater sampling
CSWW was collected from a discharge point of a slaughter-
house located at Parit Raja, Malaysia (coordinates 1° 51′ 03 N;
103° 05′ 37 E). The sampling was performed through the grab
sampling method to ensure that all the variations in the efflu-
ent contents are covered. Five litres of CSWW was collected
using a plastic bottle of polyethylene terephthalate (PET). The
samples were transported, preserved and stored according to
standard methods (American Public Health Association,
American Water Works Association, Water Pollution Control
Federation and Water Environment Federation 1915). The
characteristics of the CSWW samples were determined as
described in a previous study (Yaakob et al. 2018)
(Appendix 1).
Scenedesmus sp. cultivation
Scenedesmus sp. was obtained from Endau-Rompin
Rainforest, Johor and identified as mentioned in a previous
work (Apandi et al. 2018a, b). Scenedesmus sp. strain was
cultivated in Bold’s basal medium (BBM) and prepared to
be used as inoculum solution as detailed by Apandi et al.
(2018a, b).
Optimisation of Scenedesmus sp. grown in CSWW
The central composite design (CCD) in Design Expert v 6.0.4
software of RSM was used for optimising the microalgae pro-
ductivity and nutritional content. The selected operating con-
ditions in the present work included temperature (10 to 30 °C)
and photoperiod (6 to 24 h). The range for each parameter was
selected according to the ability of the Scenedesmus sp. cells
to grow which is similar to their native growth conditions
(Lage et al. 2018). The optimisation experiments were carried
Environ Sci Pollut Res
out in three replicates at three centre points and making a total
of nine sets of experimental runs for small design. There were
5 times of repetition of 20 °C and 15 h conditions. Repetition
of an experiment, under the same conditions, was carried out
to estimate the variability of the results and to increase the
accuracy of the estimated data by assuming that no bias, sys-
tematic error was present. The design was composed of three
levels (low, medium and high, being coded as − 1, 0 and + 1);
thus, the combination of parameter was based on the three-
level software coded for relative significance factors of com-
plex interactions (Bai et al. 2015).
The experimental data were plotted to investigate the effect
of the factors on the growth of Scenedesmus sp. The optimum
conditions were selected according to the optimisation results.
Then, the microalgae were mass cultivated for biomass pro-
duction. The purpose of this optimisation experiment was to
determine the best microalgae biomass quality in terms of
protein, lipids and carbohydrates before it was applied as
feeds. Experimental data was obtained from microalgae
growth and was compared to the predicted data (RSM).
Validation test was carried out to predict the errors as shown
in Eq. (1). RSM models are valid if predicted error is smaller
between actual and predicted data.
Actual error−predicted error
Error ¼  100 ð1Þ
Actual error
Photobioreactor system setup
The experiment setup and design used for the production of
Scenedesmus sp. biomass is illustrated in Fig. 1. Three
(6000 mL of capacity for each) aquaria were placed on a
Fig. 1 Experiment setup of
Scenedesmus sp. biomass
production optimisation in
CSWW
polystyrene box and filled with 4500 mL CSWW in each
aquarium. Bulb, air pump and thermostat were attached to
the polystyrene box. Bulb was used to replace sunlight.
Light-emitting diode (LED) light (18 W is equivalent to
1600 lx and 250 V) was used for inducing microalgae growth
and photosynthesis (Delgadillo-Mirquez et al. 2016). Light
intensity used in this study was 21.6 μmol m−2 s−1. LED light
was used due to several advantages such as high photovoltaic
conversion efficiencies, long service lives, fixed wavelengths
and low heat production (Hsia and Yang 2015). In addition, an
aquarium air pump (220 V, 5 W, 3.5 L/min) was used in this
experimental work for agitation and ensured microalgae do
not accumulate in the bottom of the aquarium. The thermostat
was used to control the temperature and it has the ability to
control a wide temperature that ranged from − 9.9 to 99.9 °C
(Willhi Electronics Co. Ltd 2016). Scenedesmus sp. was inoc-
ulated with 1 × 108 cells/mL into CSWW. Experimental oper-
ating conditions were conducted as designed by RSM.
Microalgae cell counting was carried out every 2 days for
8 days using a haemocytometer and harvested from CSWW
for biomass production nutritional content as described in a
previous work (Apanda et al. 2018a).
Microalgae biomass nutritional analysis as potential
fish feeds
The microalgae biomass nutritional content was determined in
terms of total carbohydrates, total lipids, total protein and fatty
acids. Analysis of the total carbohydrate, total protein and total
lipids was carried out after microalgae cultivation. Cultivation of
microalgae was carried out on the 8th day and harvesting was
done using the centrifugation method. The biomass harvested

was sun dried and then nutritional content analysis was carried
out.
Total carbohydrates
The determination of carbohydrates from microalgae biomass
was conducted using the calorimetric method based on
phenol–sulphuric acid hydrolysis as described by Dubois
et al. (1956). The experiment was conducted by weighing
50 mg of dried microalgae biomass, and 1 mL water was
added to the samples. Then, 1 mL of 5% phenol solution
and 5 mL of concentrated sulphuric acid were added to the
prepared samples and were rapidly mixed together. The test
tubes were vortexed for 10 s and incubated at room tempera-
ture for 1 h. After the incubation process, the test tubes were
centrifuged at 4600 rpm, 10 min and the absorbance samples
were measured at 750 nm using a UV spectrophotometer. The
blank preparation was followed by the preparation of the sam-
ples, but the samples were replaced by deionised water. Then,
the standard curve was plotted and total carbohydrates were
determined from the standard curve.
Total protein
In this study, the Lowry method (Lowry et al. 1951) was used
to measure the protein content in microalgae biomass. Bovine
serum albumin (BSA) is the most common protein standard
used for calibration curves in a spectrophotometer (Barbarino
and Lourenço 2005). For the investigation of total protein,
50 mg of dried microalgae biomass was weighted and ground
manually using mortar into a fine powder. The process was
followed by the addition of 4 mL distilled water to the samples
and kept in a refrigerator for 15 h at 4 °C. Samples were
homogenised for 5 min using a vortex and 4 mL of distilled
water was added to the tubes before being centrifuged at 4 °C,
30 min. The supernatant was collected at the end of contact
time after which 1 mL of 0.1 N NaOH was added to the
precipitate, and they were mixed thoroughly before incubation
at room temperature for 1 h. Samples were centrifuged for
20 min, 15,000 rpm, and the supernatant collected and added
to the previous supernatant; 25% trichloroacetic acid (TCA)
was added to the algal extract and kept on ice for 30 min. The
supernatant was discarded and 2 mL NaOH (0.1 N) was added
to the precipitate; 5 mL reagent C and 0.5 mL diluted (1:2)
Folin–Ciocaltu were added to 1 mL of the sample mixture,
where reagent C was a mixture of 1 mL reagent A (2%
Na 2CO 3 and 0.1 N NaOH) and 50 mL reagent B (5%
CuSO4, 5H2O and 1% C4H4Na KO6, 4H2O), respectively.
Samples were incubated for 30 min, and then, absorbance
was measured at 750 nm using the UV spectrophotometer
(T80 Plus, PG Instrument, UK).
Environ Sci Pollut Res
Total lipids
Investigation of lipid contents in the microalgae biomass was
determined using the modified lipid extraction methods by
Folch et al. (1957) and by Bligh and Dyer (1959). The lipid
was extracted from microalgae using chloroform:methanol sol-
vent as shown in Appendix 2. In brief, 50 mg of dried microalgae
biomass was weighted and extracted with 3 mL
chloroform:methanol (2:1). Then, the weighted samples were
homogenised and centrifuged at 1500 rpm, 4 min, 4 °C. The
supernatant was transferred into a clean tube and was placed on
ice, whereas the residue was added to 3 mL of
chloroform:methanol (1:2) using vortex and homogeniser to en-
sure complete mixing. Then, the centrifugation process was con-
ducted at 1500 rpm, 4 min, and 4 °C, and the supernatant was
transferred into a previous supernatant tube.
The supernatant tubes were added with 1.5 mL saline (0.9%)
and vortexed before been incubated at 4 °C for 60 min. When
there was an observation of two layers formed, the samples were
centrifuged again (10 min, 4 °C, medium speed) until the layer
was clearly separated. The contents in the upper phase were
discarded and the lower phase was transferred into an aluminium
dish and was allowed to evaporate in the oven at 60 °C. The
aluminium dish was weighted and the total lipid concentration
was recorded according to Eq. (2) as follows:
Lc ¼ A f −Ai ð2Þ
where Af is final aluminium dish weight, Ai is initial aluminium
dish weight and Lc is the lipid concentration.
From Eq. (3), lipid percentage was determined by Eq. (3).
Lc
Lipid Percentage ð%Þ ¼  100% ð3Þ
Ws
where Lc is the lipid concentration and Ws is the sample
weight.
Fatty acid methyl ester by gas chromatography
Fatty acids in the microalgae biomass were converted into fatty
acid methyl ester (FAME) which is regarded as the volatile form
of lipids. The process was followed by quantifying the FAME
using gas chromatography–mass spectrometry (GC–MS).
Firstly, 50 mg of dried microalgae biomass was mortared and
ground into a fine powder. Then, the dried microalgae biomass
was extracted with chloroform:methanol (1:2) by using the
Soxhlet extraction method. The extraction process of the biomass
was achieved for 6 h at 140 °C, and the process was followed by
30 min of solvent rinse and 30 min of solvent evaporation. Then,
1 μL FAME samples were injected into a GC injection chamber
and carried into a separating column by helium (He) as carrier
gas with 1.3 mL/min flow rate. The GC analysis was performed
in duplicate. Then, the temperatures for the injector and detector
Environ Sci Pollut Res
were set at 250 and 280 °C, respectively. The FAME passed
through a 30-m capillary column and separated into a number
of peaks based on molecular weights and polarities, and then
FAME was quantified. Peaks were compared to a reference ma-
terial’s retention time, and in this experimental work, the refer-
ence material used was 37-FAME mix analytical standard from
Sigma Aldrich. Based on microalgae biomass FAME composi-
tion obtained, this study highly focused on docosahexaenoic acid
(DHA, C22:6 n-3) and eicosapentaenoic acid (EPA, C20:5 n-3)
composition which are essential for fish feed formulation.
FTIR and SEM-EDS analysis of Scenedesmus sp.
biomass and commercial fish feed
In order to identify the presence of the functional groups in the
Scenedesmus sp. biomass and commercial fish feed, Fourier trans-
form infrared (FTIR) spectroscopy was used. The Scenedesmus
sp. biomass and commercial fish feed were recorded on a Perkin-
Elmer spectrum 100 FTIR spectrometer. FTIR was operated be-
tween the wavelength region 400 and 4000 cm−1, resolution
4 cm−1, scan number 32 and scan speed 0.5 cm s−1. In order to
determine the surface morphology of the prepared Scenedesmus
sp. biomass and commercial fish feed, scanning electron micros-
copy (SEM) SUI510 was used according to the procedure de-
scribed by Ribeiro et al. (2016). The chemical composition of
the adsorbent materials was achieved using the energy-dispersive
analysis X-ray (EDS) unit attached with the SEM JOEL JSM-
6380LA. One gram of the Scenedesmus sp. biomass and commer-
cial fish feed was spread on a carbon tape for the analysis. The
elemental composition can be illustrated in the EDAX spectrum.
Data analysis
The data for microalgae growth, CSWW characteristics and
proximate analysis of microalgae biomass were investigated,
and the data obtained were analysed by one-way ANOVA with
95% confidence level using SPSS software. A significance level
of p value less than 0.05 was used for all tests. Finally, optimi-
sation of Scenedesmus sp. dry biomass, protein, lipid and carbo-
hydrate content was analysed using Design Expert Software ver-
sion 6.0 to achieve the optimum operating parameter. Tables,
figures, graphs and bar chart were used in illustrating the results
from this research.
Results and discussion
Optimisation of microalgae biomass production
and contents
Scenedesmus sp. biomass production in CSWW as well as
carbohydrate, protein and lipid contents was studied using
RSM as a function of two independent variables, namely
temperature (10–30 °C) and photoperiod (6–24 h). Nine runs
were carried out to cover all possible combinations of factor
levels which were then analysed using analysis of variance
(ANOVA, p < 0.05). The design arrangements and responses
are illustrated in Appendix 3. It was noted that the highest
biomass obtained in CSWW was 3.1 g at 30 °C and a photo-
period of 24 h for both observed and predicted outcomes.
Meanwhile, the minimum dry biomass obtained was 0.97 g
at 20 °C and a photoperiod of 15 h for the observed outcome
which was slightly different from the predicted results (±
0.46). The data obtained in this study was similar to a previous
study by Krzemińska et al. (2014) who revealed that
microalgae biomass had a correlation with photoperiod
length. Mantzorou et al. (2017) also reported that microalgae
biomass productivity was higher under high temperature
(25 °C). Thus, it can be concluded that microalgae dry bio-
mass production in CSWW increased as temperature and pho-
toperiod increased.
In contrast, the lowest carbohydrate content was observed
at 10 °C after a 6-h photoperiod at 0.31 μg/mL. The highest
carbohydrate content was recorded at 20 °C after a 15-h pho-
toperiod at 1.59 μg/mL. Microalgae undergo photosynthesis
under suitable environmental conditions and this process ac-
cumulates carbohydrates in the form of starch, polysaccha-
rides and also cellulose in the cell walls or chloroplast (Chen
et al. 2013). The results obtained in this study were similar to a
previous study by Juneja et al. (2013) who reported that
microalgae growth at low temperatures undergoes the photo-
inhibition process which affects microalgae growth rates and
subsequently reduces carbohydrate production. The highest
protein content was recorded at 20 °C after a 15-h photoperiod
at 33.14%, while the lowest protein content was observed at
30 °C after a 6-h photoperiod at 26.59%. The highest lipid
content of 38.4% was recorded at 20 °C after a photoperiod of
15 h. The lipid data obtained in this study was similar to a
previous study by Singh and Singh (2015) who revealed that
the optimal conditions to produce lipid from Scenedesmus
armatus were a temperature of 20 °C and a photoperiod of
14 h. Singh and Singh (2015) stated that the cultivation of
Scenedesmus armatus at 20 °C and a 14-h photoperiod for
15 days resulted in 112 and 14.7 (gP)−1 for lipid productivity
and tri-acyl glycerol, respectively.
The ANOVA of the response surface quadratic model for
Scenedesmus sp. dry biomass and carbohydrate, protein and
lipid contents is presented in Appendix 4. The ANOVA results
revealed that dry biomass production was influenced by tem-
perature and photoperiod. The effect of temperature and pho-
toperiod on microalgae biomass production was significant
(p < 0.0314) with determination coefficients (R2) equal to
0.8939 and adjusted R2 0.7877, respectively. The results
achieved in this study indicate the aptness of the model for
the prediction of the investigated factors. The interaction be-
tween temperature and photoperiod has stimulated microalgae

dry biomass production with a confidence level of 95%. These
results indicated that the synergistic effects of temperature and
photoperiod were significant.
The carbohydrate content was directly influenced by
temperature. The effect of temperature on carbohydrate
content was significant (p < 0.0079) with determination
coefficients (R 2 ) equal to 0.9478 and adjusted R 2
0.8955, respectively. The interaction between temperature
and photoperiod stimulates carbohydrate content in
microalgae biomass production. These results indicated
that there is a synergistic effect of temperature and pho-
toperiod towards carbohydrate content in microalgae bio-
mass. In contrast, the ANOVA results also revealed that
protein content was not directly influenced by temperature
and photoperiod. The effect of temperature and photope-
riod on protein content was not significant (p < 0.9755)
with determination coefficients (R2) equal to 0.0933 and
adjusted R2 0.8134, respectively. The interaction between
temperature and photoperiod does not stimulate protein
content in microalgae biomass production. These results
indicated that there is no synergistic effect of temperature
and photoperiod towards protein content in microalgae
biomass. The lipid content was not directly influenced
by temperature and photoperiod. The effect of tempera-
ture and photoperiod on lipid content was not significant
(p < 0.7858) with determination coefficients (R2) equal to
0.2986 and adjusted R2 0.4029, respectively. The interac-
tion between temperature and photoperiod does not stim-
ulate lipid content in microalgae biomass production.
These results indicated that there is no synergistic effect
of temperature and photoperiod towards lipid content in
microalgae biomass.
The results of the interaction between temperature
and photoperiod and their effects on microalgae dry
biomass production were presented using a 3D graphical
representation of the system behaviour. The 3D curve
plot of temperature versus photoperiod for dry biomass
produced in CSWW is presented in Fig. 2a. As a result
of the interaction between temperature and photoperiod,
the maximum biomass production was 3.1 g. The opti-
mal carbohydrate content was 1.59 μg/L (Fig. 2b).
Based on the effect of interaction between temperature
and photoperiod, the optimal protein content was
33.14% (Fig. 2c). The interaction between temperature
and photoperiod recorded 38.4% of lipid production.
Andrade et al. (2014) had studied the best culture con-
dition for Chlorella vulgaris by using RSM at different
temperatures. The highest biomass for Chlorella
vulgaris obtained at 20 °C in 50% diluted RL medium
was 753 ± 5.8 mg/L compared to 691.4 ± 3.8 mg/L in
50% diluted RL medium at 10 °C.
The response of the quadratic equation describes the rela-
tionship between the independent factors [temperature (A);
Environ Sci Pollut Res
photoperiod (B)] on Scenedesmus sp. biomass production in
CSWW as well as carbohydrate, protein and lipid contents
with selected factors as given by Eqs. (4), (5), (6) and (7).
y1 ¼ þ1:44 A−0:062B þ 0:26A2 þ 1:10AB ð4Þ
y2 ¼ þ1:47A þ 0:31B þ 0:18A2−0:82AB þ 0:1 ð5Þ
y3 ¼ þ28:93A−0:31B þ 1:28 A2 þ 0:50AB þ 1:24 ð6Þ
y4 ¼ þ27:74A þ 1:50B−3:46A2 þ 3:80AB−1:14 ð7Þ
The best operating parameters for Scenedesmus sp.
biomass and carbohydrate, protein and lipid contents
The optimisation of the statistical experimental strategies was
carried out, in which one run of additional confirmation ex-
periments was conducted. The best operating parameters are
presented in Table 1. Dry biomass production was close to
those estimated using RSM. The actual optimum values of
temperature and photoperiod were 30 °C and 24 h, respective-
ly. The quantified microalgae biomass production in
optimised condition was 2.475 g. To confirm the optimisation
experiment on carbohydrate production, one additional con-
firmation experiment was carried out. The carbohydrate con-
tent in microalgae biomass was 1.44 μg/mL that was close to
the carbohydrate estimated, 1.27 μg/mL. Moreover, 29.9% of
protein was recorded in microalgae biomass which was close
to that estimated using RSM (31.6%); 25.75% of lipid content
was recorded in microalgae biomass which was close to that
predicted using RSM (28.44%). The percentage error of the
responses between the actual and predicted data was less than
5%. These findings clearly showed that no substantial differ-
ence was observed. This also testifies that the RSM approach
was appropriate for optimising the operational conditions of
the lipid production in treated microalgae biomass.
The perturbation plot was used to determine the effect of
each factor on the responses, based on the perturbation plot in
Appendix 5, and it can be noted that photoperiod (B) did not
show an effective production of microalgae dry biomass as the
graph is linear with p value exceeding 0.05 (p < 0.1784).
However, temperature (A) had more effect on microalgae
dry biomass production. The perturbation graphs above
showed that each of the variables used in the present study
has its individual effect on microalgae dry biomass production
from the phycoremediation of CSWW. This clearly indicates
that it is important to maintain temperature and photoperiod at
an optimum level as these factors can affect microalgae bio-
mass production. Photoperiod (B) did not show an effective
production of carbohydrate content in microalgae biomass as
the graph is almost linear. Temperature (A), however, had
more effect on carbohydrate production. The perturbation
graphs above showed that each of the variables used in the
present study has its individual effect on carbohydrate
Environ Sci Pollut Res

B
A

C
D

Fig. 2 Three-dimensional response surface plot for Scenedesmus sp. dry biomass (a), carbohydrates (b), protein (c) and lipid contents (d) in CSWW as a
response of interaction between independent factors

production of microalgae biomass from the phycoremediation and photoperiod (A and B) did not show effective production
of CSWW. This clearly indicates that keeping the temperature of protein as the graph is almost linear. Moreover, photoperiod
and the photoperiod at optimum levels affects the carbohy- (B) did not show an effective production of lipids as the graph
drate production significantly. In contrast, both temperature is almost linear. Temperature (A) was found to have more

Table 1 The best operating parameters for Scenedesmus sp. biomass production and carbohydrate, protein and lipid contents in CSWW

A B y1 y2 y3 y4

Temperature (°C) Photoperiod (h) Actual Predicted Actual Predicted Actual Predicted Actual Predicted

30 24 2.475 g 3.099 g 1.44 μg/mL 1.274 μg/mL 29.9% 31.6% 25.75% 28.44%

y1 (biomass production); y2 (carbohydrates); y3 (proteins); and y4 (lipid contents)

 Environ Sci Pollut Res

effect on lipid production. This clearly indicates that keeping Table 2 EDX spectrum of Scenedesmus sp. biomass in BBM and
CSWW
temperature and photoperiod at optimum levels affects lipid
production. Element Weight (%) Atomic (%)

Scenedesmus sp. biomass surface morphology using BBM CWW BBM CWW
a scanning electron microscope with EDX Carbon 67.26 41.25 73.70 47.25
Oxygen 30.89 21.86 25.41 18.79
SEM and EDX analysis is used in the study to establish chang-
Sodium 0.81 32.85 0.46 32.26
es in morphology and elemental composition of the
Sulphur 1.04 2.03 0.43 0.44
microalgae with a view to establish the mechanism of metal
Silicon 0.68 0.33
binding (Ajayan et al. 2015). Figure 3a, b and Table 2 show
Phosphorus 0.95 0.42
the surface morphology and elemental composition of
Nitrogen 32.85 32.26
Scenedesmus sp. biomass before and after phycoremediation
Calcium 0.64 0.22
of CSWW. Based on Fig. 3, the results of SEM micrographs
showed that the Scenedesmus sp. cell before CSWW
phycoremediation had a normal shape with smooth and trans-
parent external layer outside the cell surface, whereas after Scenedesmus sp. biomass had a protein composition of 29.9%
phycoremediation of CSWW, the cell became slightly rough which is almost similar to other commercial fish feeds in the
and had corrugated textures and some particles were found on market. FAO (2014) revealed that fish have low requirements
the surface of the cell wall. The results of the EDX spectrum for protein. In fact, the protein needed for fish growth is in-
showed that the Scenedesmus sp. cell produced in CSWW had versely proportional to fish age. Roy et al. (2011) stated that
more elements which bind on the cells’ surface compared to proteins are essential for fish growth and feeding efficiency.
the Scenedesmus sp. cell produced in BBM. These findings However, when compared to the Malaysian fish feed quality
indicate that the CSWW contributes effectively in the increas- standard, the protein composition obtained in this experiment
ing of element content of microalgae biomass. was relatively low. This can be due to environmental condi-
tions such as short microalgae cultivation period for protein
Microalgae biomass nutritional content as potential production, limited nutrient from CSWW, temperature and
fish feeds light duration (Derrien et al. 1998).
Other than that, lipid composition is an essential compo-
Commercial fish feeds in the market comprise of corn gluten nent for formulating fish feeds. This is because lipids are vital
meal, soybean meal, sunflower, palm kernel, chicken offal for metabolic functions and cellular membranes in fish. Lipids
meal, krill meal, squid meal and trash fish meal formulation. obtained from the microalgae biomass in this study were
In this study, microalgae, Scenedesmus sp., biomass proxi- 25.7% which is relatively higher compared to other commer-
mate composition was quantified as an alternative to commer- cial feeds and the Malaysian quality fish feed standards. Ji
cial fish feeds in the market. Then, proximate composition of et al. (2013) used different types of microalgae (Chlorella
Scenedesmus sp. biomass was compared to Malaysian quality vulgaris, Scenedesmus obliquus and Ourococcus multisporus)
fish feed standards as shown in Table 3. It was noted that the for domestic wastewater treatment which produced 30, 27 and

Fig. 3 a, b Surface morphology

of Scenedesmus sp. biomass
produced in BBM and CSWW
Environ Sci Pollut Res
Table 3 Proximate composition of Scenedesmus sp. biomass as fish feeds
Parameters This study Commercial fish feeds in the market
Brand A Brand B Brand C
Moisture (%) ND 11 13 13
Protein (%) 29.9 30 27 29
Lipid (%) 25.75 6 8 5 8
Fibre (%) ND 4 10 8 7
ND, non-detected; NR, not reported
31% of lipids, respectively. As stated by Griffiths et al. (2011),
microalgae have higher lipid content compared to other crops
used to formulate fish feeds. Scenedesmus sp. can produce up
to 24.66 mg/L of lipids under stress and limited nutrients (Jena
et al. 2012). This might occur due to microalgae metabolic
activity which shifts their activities from the synthesis of car-
bon dioxide to the production of lipids (Ji et al. 2013).
Chlorella vulgaris has a higher lipid content (43%) under
nutrient deficiency conditions compared to typical wastewater
nutrient conditions (26%).
It can be concluded that protein content of biomass pro-
duced in CSWW is similar to commercial fish feeds and those
found by previous studies, while lipid content is higher com-
pared to other commercial feeds and the Malaysian quality
fish feed standards. The minimum requirement of nutritional
content that is sufficient for fish growth ranged between 25
and 35% for protein and between 7 and 14% for lipids, re-
spectively (Sankian et al. 2017). An effective aquaculture
practice is comprised of complete fish feed diets which con-
tain 18 to 50% protein and 10 to 25% lipid (Craig and Helfrich
2009). However, excessive protein and lipid content in feeds
may cause problems to fish health and the environment.
Hence, it can be concluded that the protein and lipid content
in microalgae biomass was still in the suitable range needed
by fish for growth (Sankian et al. 2017). Thus, this study
demonstrated that microalgae biomass from CSWW has the
potential to be used as a fish feed alternative.
Microalgae biomass fatty acid methyl ester composition
In this study, FAME composition of Scenedesmus sp. biomass
produced in BBM and CSWW was compared to that available
in commercial fish feeds (Fig. 4a–c). The elements of FAME
in Scenedesmus sp. grown in BBM are illustrated in Appendix
6—FAME: 13:0, 15:1, 24:1 and 6:0 were detected. The
highest intensities of FAME were observed at peak numbers
5, 15, 19 and 23 at retention times of 14.997, 30.949, 32.599
and 33.513 min, respectively (Fig. 4a). The FAME profile was
used as a guideline for fish feed formulation because the fishes
have no anabolic pathway to synthesise their own FAME
(Maizatul et al. 2017). Therefore, the fishes require DHA,
Malaysian quality fish feed standards
Brand D Brand E Grade A Grade B Grade C
13 10 10 10 10
30 28 65 60 55
5 12 13 13
5 NR NR NR
EPA and arachidonic acid (ARA) for growth, disease resis-
tance and metabolic activities, and microalgae biomass is rich
in FAME, and therefore, it can be used as fish feeds (Brown
2002).
The concentration of FAME in Scenedesmus sp. biomass in
BBM is presented in Fig. 5a. The highest concentration of
FAME was 14.96% (C15:1), followed by 12.14% (C6:0)
and 11.46% (C24.1), respectively. According to Islam et al.
(2013), microalgae biomass usually contains palmitic acid
(C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid
(C18:2), linolenic acid (C18:3) and minimum quantities of
DHA and EPA. However, the FAME composition obtained
in this study was varied compared to previous studies. The
differences could be due to the microalgae species and change
in environmental factors such as temperature, nutrient in the
media, photoperiod and also pH (Derrien et al. 1998).
Fluctuating environmental conditions such as light, tempera-
ture and nutrients may alter the biology and metabolism of
microalgae cells (Stengel et al. 2011; Robertson et al. 2013). It
is thus possible to enhance and optimise lipid content and fatty
acid production by applying specific abiotic factors (Spolaore
et al. 2006; Robertson et al. 2013), but conditions optimal for
lipid production may not be optimal for growth (Stengel et al.
2011). In this research, Scenedesmus sp. was cultivated in differ-
ent environmental conditions which range between 10 and 30 °C
for temperature and 6 to 24 h for photoperiod. The optimum
condition selected for fatty acid analysis was 30 °C, 24 h condi-
tion, with 25.75% of lipid production. However, this condition
was not the optimum condition for algal growth with only 14.33
μmax/day maximum growth rates, thus producing low fatty acid
content in Scenedesmus sp. cells. According to Lu et al. (2017),
EPA content in Scenedesmus sp. increased from 5.21 to 15.89%
at 10 °C. However, the rapid increase in EPAwas not observed in
algal cells grown at 25 and 30 °C. It was confirmed that high
temperature could not be an induction factor promoting the syn-
thesis of unsaturated fatty acids with a high degree of
unsaturation (Lu et al. 2017). In addition, nutrient levels play
an essential role in the lipid synthesis and storage within
microalgae cells (Spolaore et al. 2006; Benvenuti et al. 2015).
Nutrient depletion may affect long-chain polyunsaturated fatty
acid (LC-PUFA) accumulation. Other than that, variation in

Fig. 4 a Chromatogram of
Scenedesmus sp. biomass
produced in BBM. b
Chromatogram of Scenedesmus
sp. biomass produced in CSWW.
c Chromatogram of FAME in
commercial fish feeds
photoperiod length and growth temperature can also affect LC-
PUFA concentration and composition, such as EPA and DHA
formation in microalgae cells (Khoeyi et al. 2012; Robertson
et al. 2013). Previous studies have reported that under low-
temperature conditions, PUFA synthesis was increased in order
to maintain fluidity of the cell membranes (Robertson et al.
2013), while high temperatures caused a decrease in the overall
lipid content (Renaud et al. 2002).
The highest intensity of FAME in microalgae biomass gen-
erated during the phycoremediation of CSWW is presented in
Fig. 4b. It was detected at peak numbers 1, 12, 24, 34 and 49 at
retention times of 14.997, 30.956, 32.828, 36.293 and
52.733 min, respectively. The profile showed the following
FAME: 16:0, 15:1, 24:1, and 18:1N9T (Appendix 6). The
Environ Sci Pollut Res
FAME composition obtained from Scenedesmus sp. biomass
in CSWW was different compared to Scenedesmus sp. bio-
mass in BBM. The variation might be related to the wastewa-
ter composition which shifted the fatty acid formation.
F i g u r e 5 b sh o w s t h e c o n c e n t r a t i o n of FA M E in
Scenedesmus sp. biomass produced in CSWW. The highest
concentration of FAME was 35.91% (C15:1), followed by
17.58% (C24:1) and 14.11% (C18:1N9T), respectively. The
palmitic acid concentration (C16:0) obtained in this study was
4.77% (Fig. 5b). The dominant fatty acids obtained in the
current work were not similar compared to those obtained in
previous studies. In comparison to a study by Ji et al. (2013)
who used S. obliquus on municipal wastewater treatment, the
dominant fatty acids obtained were mainly composed of
Environ Sci Pollut Res
palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1),
linoleic acid (C18:2) and linolenic acid (C18:3). The FAME
profile may be different among the same microalgae species
due to types of wastewater used for cultivation and nutrient
enrichment.
According to Carta et al. (2017), palmitic acid (C16:0) was
commonly found in palm oil which is about 44% of total fats,
but it also can be significantly found in meat and dairy prod-
ucts which range between 50 and 60% of total fats. In this
research, the source of palmitic acid clearly comes from chick-
en. This statement was clearly demonstrated by Nitayapat and
Chitprasert (2014) who studied fatty acid composition on
chicken processing waste and who reported that the five most
abundant fatty acids were palmitic (C16:0), cis-9-oleic
(C18:1n9c), cis, cis-9,12-linoleic (C18:2n6), stearic (C18:0)
and palmitoleic (C16:1n7) and these are the most abundant
fatty acids found in the lipids of whole fresh chickens.
Wagenen et al. (2012) reported that high exposure of
microalgae culture to light will increase the accumulation of
triacylglycerol due to photo-oxidative stress. Thus, it can
cause a sharp increase in fatty acids such as palmitic acid
(C16:0) and palmitoleic acid (C16:1). However, low exposure
to light resulted in increases in the relative abundance of un-
saturated fatty acids, such as palmitoleic and eicosapentaenoic
acids (C20:5ω3). The amount of unsaturated fatty acids was
inversely proportional to temperature, demonstrating physio-
logical adaptations to increase membrane fluidity. Other than
that, the abundance of microalgae cell can cause shading and
reduce light penetration; thus, this condition will favour the
formation of oleic acid (C18:1ω9). Oleic acid is usually found
in aged microalgae cultures compared to new culture
(Wagenen et al. 2012). Previous data by Wagenen et al.
(2012) can justify the result reported in this research.
Scenedesmus sp. biomass contains a high amount of palmitic
acid due to prolonged exposure to light which is 24 h photo-
period. On the other hand, a high amount of oleic acid is
accumulated due to the length of the cultivation period, which
is 8 days before harvest.
The highest intensity of FAME for fish meals was observed
at peak numbers 3, 15, 19 and 20 at retention times of 15.007,
32.858, 36.2 and 36.344 min, respectively (Fig. 4c). The pro-
file showed the following FAME: 16:0 and 24:1 (Appendix
6). FAME composition obtained from commercial fish feeds
varied compared to that in Scenedesmus sp. biomass. This is
because commercial fish feeds were formulated by mixing
fish trash and several types of crop. The highest FAME con-
centration was obtained by C24:1 fatty acid at 56.64%.
Besides that, this commercial feed also comprises of small
amounts of C18 and C20 fatty acids such as methyl elaidate
(C18:1n9t), oleic acid methyl ester (C18:1n9c), linolenic acid
methyl ester (C18:3n6) and cis-5,8,11,14,17-eicosapentaenoic
acid methyl ester (C20:5n3) (Fig. 5c). Based on the overall
FAME outcomes, microalgae biomass after phycoremediation
of CSWW had higher EPA content compared to microalgae
biomass before phycoremediation and commercial fish feeds
with 7.98, 4.85 and 1.46%, respectively. Comparison among
biomass and commercial fish feed was significant for fish feed
formulation that helps fish growth.
Fourier transform infrared spectroscopy
Microalgae have the capacity to absorb nutrients from
CSWW. The sorption of nutrients from CSWW into
microalgae can occur due to the active groups and chemical
bonds which act as binding sites for nutrient and metal absorp-
tion (Mehta and Gaur 2005). Hence, this study uses FTIR for
the quantitative analysis of major functional groups present in
Scenedesmus sp. biomass. The FTIR spectra of Scenedesmus
sp. biomass before and after the phycoremediation of CSWW
were compared to the commercial fish feed spectra as shown
in Fig. 6.
Normalisation is a process to determine intensity level dis-
parity by making sure that the intensity of the same material is
as similar as possible across the spectra recorded under the
same experimental parameters but slightly different conditions
(Gautam et al. 2015). Generally, normalisation is part of the
pre-processing technique used to differentiate a spectrum of
the same material. This technique shall be applied to avoid
overlapping of spectra and avoid spectra being recorded at
different times and under different instrument conditions such
as alignment and laser power levels (Gautam et al. 2015). In
this study, samples do not undergo the normalisation method
due to different sample types and their chemical composition.
According to a previous study by Gautam et al. 2015, the
normalisation technique is not recommended when there is a
possibility of a shift in the band position across the spectra
from different samples under investigation. This can be due to
the fact that different samples have different compositions,
resulting in various infrared absorption band intensities at dif-
ferent wavenumbers, which allows for the comparison and
characterisation of samples (Chang 2012).
According to Fig. 6a, the highest transmittance intensity
was demonstrated by microalgae biomass after
phycoremediation at 747.39 cm−1. There was evidence of
the alkyl halide group between bands 800 and 600 cm−1 which
is associated with the stretching of C–Cl bonds. Generally,
CSWW contains chlorine ions from preservation and cleaning
activities. Ion exchange had occurred during CSWW
phycoremediation by microalgae. The replacement of ele-
ments with chloride ions occurred due to the strong electro-
negativity of chloride ions. FTIR spectra peaks according to
wavenumbers (cm−1) and functional groups are tabulated as
shown in Appendix 7. Ion exchange mechanisms occurred
around band 3000–2800 cm−1 due to aliphatic acid in CH,
CH2 and CH3. Meanwhile, the changes in the band between
1480 and 1350 cm−1 were due to the stretching of alkane
 c
a

b
CONCENTRATION OF FAME (%) CONCENTRATION OF FAME (%)
CONCENTRATION OF FAME (%)

2.1 3.71 2.72

7.05 4.35 12.14

0.09 1.11
0.44

0.97 0.45
35.91

3.62 0.79
4.77
14.96
7.5
1.55
3.06
0.29
1.33
0.61
0.34
14.11
0.51
FAME COMPOSITION

0.88

FAME COMPOSITION
FAME COMPOSITION
5.13
0.56
9.87
0.29
0.87
0.12
1.78
4.85
8.05
7.98
0.49
1.46
0.72 1.33
0.45 56.64
17.58 11.46
Environ Sci Pollut Res
 Environ Sci Pollut Res
ƒFig. 5 a FAME profile of Scenedesmus sp. biomass produced in BBM. b
FAME profile of Scenedesmus sp. biomass produced in CSWW. c FAME
profile of commercial fish feeds
groups (C=C) and CH. Besides that, at band 1360–1000 cm−1,
the highest intensity of transmittance was recorded by
Scenedesmus sp. biomass after treatment of CSWW with the
band measuring 1024.61 cm−1. This wavelength was assigned
according to the stretching of amine groups (–CN) and ether
(C–O) which is classified as the weak chemical bond of ali-
phatic amines.
Based on the FTIR spectrum shown in Fig. 6, the lowest
transmittance intensity was recorded by microalgae biomass
produced in BBM with a wavelength of 3351.74 cm−1. The
functional groups that lie within the band 3600–3200 cm−1
contain the highest bonded hydroxyl groups (–OH) and amine
groups (–NH). The abundance of this functional amine group
can be due to the nitrogen level in wastewater which has been
absorbed by microalgae cells. Sulaymon et al. (2013) stated
that nitrogen uptake by microalgae biomass has a correlation
with carboxyl, sulfonate and hydroxyl functional groups.
Besides that, the hydroxyl group is also associated with
microalgae cell walls (Sulaymon et al. 2013).
Other than nutrient removal from CSWW, the functional
groups identified using FTIR also quantified the nutritional
content of microalgae biomass. Varied environmental condi-
tions may change microalgae biomass composition in terms of
Fig. 6 Comparison of FTIR spectra of Scenedesmus sp. biomass produced in BBM (a) and CCSW (b) and compared to commercial fish feeds (c)
protein, lipids and carbohydrates (Meng et al. 2014). Based on
the data presented in Appendix 7, wavelength ranging be-
tween 3000 and 2800 cm−1 was associated with the acyl chain
stretch of C–H that was used to quantify lipids. In this study,
there is a slight difference between the transmittance intensity
of microalgae biomass produced in BBM and CSWW com-
pared to commercial fish feeds. This statement can be justified
by the experimental results which were 9.2, 25.75 and 18% of
lipids in microalgae biomass before treatment, microalgae bio-
mass after treatment and commercial fish feeds, respectively.
Besides that, the protein content in microalgae biomass can be
determined by the shift in stretching vibration observed in amide
groups and bending in NH functional groups that usually occur at
the 1650–1540-cm−1 spectra. However, no wavenumber was
recorded on the 1650–1540-cm−1 wavelength for microalgae
biomass. Thus, this FTIR spectrum for microalgae biomass be-
fore and after CSWW treatment could not be used to determine
protein content. On the other hand, commercial fish feeds also do
not show any wavenumber assigned for protein determination.
However, by using the extraction method, the protein content in
microalgae biomass and commercial fish feeds could be record-
ed. Thus, it can be justified by the wavelength shifting which
occurred due to nitrogen-limited culture conditions (Meng et al.
2014). According to this study, the carbon allocation in
microalgae biomass varied from protein to carbohydrate and
neutral lipids which changed wavenumbers (cm−1) due to
nutrient-limited culture conditions.

Other than that, total carbohydrates can be quantified based on
the wavelength band 1200–950 cm−1 with ether functional group
(C–O–C) absorption (Meng et al. 2014). In this study, microalgae
biomass after treatment (749.39 cm−1) had a higher transmittance
intensity compared to the transmittance intensity of biomass be-
fore treatment (752.01 cm−1). However, commercial fish feeds
have a higher transmittance intensity (747.63 cm−1) compared to
the transmittance intensity of microalgae biomass after treatment
(749.39 cm−1). This statement can be justified by the experimen-
tal results which obtained 14.9, 18.9 and 28% of carbohydrates in
microalgae biomass produced in BBM, CSWW and commercial
fish feeds, respectively.
Conclusion
In conclusion, Scenedesmus sp. has the ability to grow in CSWW
and produce a high-quality biomass rich in nutritional content for
fish feed potential. The findings in the current work revealed that
the best operating parameters for Scenedesmus sp. biomass pro-
duction and carbohydrate, protein and lipid contents were deter-
mined at 30 °C and after 24 h. The biomass yield has 7.98% of
Appendix 1
Fig. 7 Flow diagram of chicken
slaughterhouse activity
Environ Sci Pollut Res
EPA (C20:5N3) which is more appropriate as fish feeds.
Author contribution RM, AA and AHMK conceived and supervised the
research; MAY and AT performed the research; all authors wrote the
manuscript and contributed to the discussion; and all authors have ap-
proved the final article.
Funding information The authors gratefully acknowledge the Ministry
of Science, Technology and Innovation Malaysia (MOSTI) for the re-
search project financial support under E-Science Fund research grant
VOT NO S029 (03-01-SF0097). They are also thankful to the Ministry
of Higher Education of Malaysia by FRGS 1476 research grant that
supported microalgae collection from Endau-Rompin National Park.
Thanks are also extended to the University Tun Hussein Onn Malaysia
and Office for Research, Innovation, Commercialization, and
Consultancy Management (ORICC) for research grant IGSP VOT NO
U682 for prioritising the necessary infrastructure to carry out the research
work.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Discharge point
Fig. 8 Chicken slaughterhouse sampling area
Environ Sci Pollut Res

Appendix 2

Fig. 9 Lipid extraction procedure by using chloroform:methanol

Appendix 3

Table 4 Central composite design arrangement and responses for Scenedesmus sp. biomass production in CSWW as well as carbohydrate, protein and
lipid contents

Run A B y1 y2 y3 y4

Observed Predicted Observed Predicted Observed Predicted Observed Predicted

1 10 6 2.7 2.7 0.31 0.31 29.7 29.7 32.36 32.36

2 30 6 1.85 1.85 0.64 0.64 26.59 26.59 37.64 37.64
3 10 24 2.5 2.5 0.39 0.39 29.78 29.78 27.72 27.72
4 30 24 3.1 3.1 1.27 1.27 31.64 31.64 28.44 28.44
5 20 15 1.45 1.43 1.59 1.47 33.14 28.93 27.64 27.74
6 20 15 1.88 1.43 1.32 1.47 22.62 28.93 17.36 27.74
7 20 15 0.97 1.43 1.26 1.47 22.59 28.93 38.4 27.74
8 20 15 1.45 1.43 1.59 1.47 33.14 28.93 27.64 27.74
9 20 15 1.45 1.43 1.59 1.47 33.14 28.93 27.64 27.74

A (temperature, °C); B (photoperiod, h); y1 (dry biomass); y2 (carbohydrates); y3 (protein); y4 (lipids)

Appendix 4
Table 5 Analysis of variance (ANOVA) of the response surface quadratic model for Scenedesmus sp. dry biomass production

Source model DF Sum of squares Mean square F value Prob > F

y1 y2 y3 y4 y1 y2 y3 y4 y1 y2 y3 y4 y1 y2 y3 y4

Quadratic 4 3.49 2.06 13.7 94.24 0.87 0.51 3.43 23.56 8.42 18.13 0.1 0.43 0.0314 0.0079 0.9755 0.7858
(significant) (significant) (not significant) (not significant)
A 1 0.016 0.37 0.39 9 0.016 0.37 0.39 9 0.15 13.16 0.012 0.16 0.7177 0.0222 0.919 0.7074
B 1 0.28 0.13 6.58 47.89 0.28 0.13 6.58 47.89 2.66 4.47 0.2 0.87 0.1784 0.1020 0.6797 0.405
A2 1 2.68 1.48 0.56 32.16 2.68 1.48 0.56 32.16 25.81 52.24 0.017 0.58 0.0071 0.0019 0.9032 0.4884
AB 1 0.53 0.075 6.18 5.2 0.53 0.075 6.18 5.2 5.07 2.65 0.19 0.094 0.0875 0.1790 0.6889 0.7745
Pure error 4 0.41 0.11 133.18 221.41 0.1 0.028 33.3 55.35
Cor total 8 3.91 2.17 146.89 315.65

y1 (dry biomass); y2 (carbohydrates); y3 (protein); y4 (lipids)

Environ Sci Pollut Res
Environ Sci Pollut Res

Appendix 5

Fig. 10 Factor plot representing the individual variable effect on Scenedesmus sp. biomass production and carbohydrate, protein and lipid contents

Appendix 6
Table 6 Elements of FAME in
Scenedesmus sp. biomass Peak Retention time Area (%) Height (%) Chemical formula Compound name
produced in BBM
5 14.997 30.47 23.98 C13:0 Methyl tridecanoate
15 30.949 3.58 4.61 C15:1 Methyl 10-pentadecenoate
19 32.599 7.62 9.96 C24:1 Nervonic acid methyl ester
23 33.513 9.12 9.22 C6:0 Methyl hexanoate
 Environ Sci Pollut Res

Table 7 Elements of FAME in

Scenedesmus sp. biomass Peak Retention time Area (%) Height (%) Chemical formula Compound name
produced in CSWW
1 14.997 4.77 4.59 C16:0 Palmitic acid methyl ester
12 30.956 5.17 5.96 C15:1 Methyl 10-pentadecenoate
24 32.828 23.69 25.45 C15:1 Methyl 10-pentadecenoate
34 36.293 14.11 11.76 C18:1N9T Methyl elaidate
49 52.733 5.23 3.98 C24:1 Nervonic acid methyl ester

Table 8 Elements of FAME in

commercial fish feeds Peak Retention time Area (%) Height (%) Chemical formula Compound name

3 15.007 7.5 6.5 C16:0 Palmitic acid methyl ester

15 32.838 13.65 17.24 C24:1 Nervonic acid methyl ester
19 36.2 18.58 21.04 C24:1 Methyl hexanoate
20 36.344 20.19 23.16 C24:1 Nervonic acid methyl ester

Appendix 7

Table 9 The FTIR spectral characteristics of Scenedesmus sp. and commercial fish feeds

Sources Wavelength Wavenumber (cm−1) Identified groups

range (cm−1)
Biomass Biomass produced in Commercial
produced in CSWW/difference feeds/difference
BBM

Scenedesmus sp. 3600–3200 3351.74 3344.22/− 7.52 3335.97/− 15.77 Bonded hydroxyl groups (–OH) and (–NH)
biomass (this study) groups
3100–3010 3018.6 3017.39/− 1.21 3016.82/− 1.78 Stretching of alkane groups (=C–H)
3000–2800 2945.32 2945.31/− 0.01 2944.97/− 0.35 C–H stretching, –CH, –CH2, –CH3
2833.14 2833.29/+ 0.15 2833.14/± 0 Stretching aldehyde group (=C–H)
1480–1350 1451.8 1449.89/− 1.91 1458.5/+ 6.7 Stretching of aromatic groups (C=C) and
1415.2 1408.6/− 6.6 1415.2/± 0 alkane –CH
1360–1000 1216.60 1215.80/− 0.8 1215.93/− 0.67 Stretching of amine group (–CN) and ether
1025.03 1024.61/− 0.42 1024.56/− 0.47 (C–O)
600–800 752.01 749.39/− 2.62 747.63/− 4.38 Stretching of alkyl halide (C–Cl) and
bending of alkene group (=C–H)

Publisher’s note Springer Nature remains neutral with regard to jurisdic-
tional claims in published maps and institutional affiliations.
References
Ajayan KV, Selvaraju M, Unnikannan P, Sruthi P (2015)
Phycoremediation of tannery wastewater using microalgae
Scenedesmus species. Int J Phytoremediation 17(10):907–916
Al-Gheethi AAS, Noman EA, Mohamed RMSR, Apandi NM, Yaakob
MA, Pahazri F, Kassim AHM (2019a) Recycle of greywater for
microalgae biomass production. In: Management of greywater in
developing countries. Springer, Cham, pp 205–226
Al-Gheethi A, Noman E, Mohamed RMSR, Ismail N, Abdullah AHB,
Kassim AHM (2019b) Optimizing of pharmaceutical active com-
pounds biodegradability in secondary effluents by β-lactamase from
Bacillus subtilis using central composite design. J Hazard Mater
365:883–894
American Public Health Association, American Water Works Association,
Water Pollution Control Federation and Water Environment Federation
(1915) Standard methods for the examination of water and wastewater
(Vol. 2). American Public Health Association
Anderson MJ, Whitcomb PJ (2016) RSM simplified: optimizing process-
es using response surface methods for design of experiments.
Productivity Press. Published July 25, 2016.Reference - 297 Pages
- 141 B / W Illustrations
Environ Sci Pollut Res
Andrade L, González-López J, Fenice M, Martínez-Toledo MV,
Pesciaroli C, Maza-Márquez P, Juárez-Jiménez B (2014)
Application of response surface methodology (RSM) for culture
conditions and biomass production of psychrophilic microalgae iso-
lated from High Mountains Lake during the ice-free season. Int J
Environ Res 8(3):799–812
Apandi NM, Mohamed RMSR, Al-Gheethi A, Kassim AHM (2018a)
Microalgal biomass production through phycoremediation of fresh
market wastewater and potential applications as aquaculture feeds.
Environ Sci Pollut Res:1–17. In press
Apandi N, Mohamed RMSR, Al-Gheethi A, Gani P, Ibrahim A, Kassim
AHM (2018b) Scenedesmus biomass productivity and nutrient re-
moval from wet market wastewater, a bio-kinetic study. Waste
Biomass Valoriz:1–18. In press
Atiku H, Mohamed RMSR, Al-Gheethi AA, Wurochekke AA, Kassim
AHM (2016) Harvesting of microalgae biomass from the
phycoremediation process of greywater. Environ Sci Pollut Res
23(24):24624–24641
Bai Y, Saren G, Huo W (2015) Response surface methodology (RSM) in
evaluation of the vitamin C concentrations in microwave treated
milk. J Food Sci Technol 52(7):4647–4651
Barbarino E, Lourenço SO (2005) An evaluation of methods for extrac-
tion and quantification of protein from marine macro-and
microalgae. J Appl Phycol 17(5):447–460
Bazrafshan E, Mostafapour FK, Farzadkia M, Ownagh KA, Mahvi AH
(2012) Slaughterhouse wastewater treatment by combined chemical
coagulation and electrocoagulation process. PLoS One 7(6):e40108
Benvenuti G, Bosma R, Cuaresma M, Janssen M, Barbosa MJ, Wijffels
RH (2015) Selecting microalgae with high lipid productivity and
photosynthetic activity under nitrogen starvation. J Appl Phycol
27:1425–1431
Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and
purification. Can J Biochem Physiol 37(8):911–917
Brown M.R. (2002) Nutritional value of microalgae for aquaculture. In:
Cruz-Suarez, L. E., Ricque-Marie, D., Tapia-Salazar, M., Gaxiola-
Cortés, M. G., Simoes, N. (Eds.). Advances in Aquaculture
Nutrition VI. Proceedings of the VI International Symposium on
Nutrition Aquaculture. Cancun, Quintana Roo, Mexico
Carta G, Murru E, Banni S, Manca C (2017) Palmitic acid: physiological
role, metabolism and nutritional implications. Front Physiol 8
Article 902, 14pp
Chang S (2012) Analysis of polymer standards by Fourier transform
infrared spectroscopy-attenuated total reflectance and pyrolysis gas
chromatography/mass spectroscopy and the creation of searchable
libraries. Marshall University Forensic Science Program, Atlanta
Chen CY, Zhao XQ, Yen HW, Ho SH, Cheng CL, Lee DJ, Bai FW,
Changa JS (2013) Microalgae-based carbohydrates for biofuel pro-
duction. Biochem Eng J 78:1–10
Craig S, Helfrich LA (2009) Understanding fish nutrition, feeds, and
feeding Virginia cooperativeextension, Communications and
Marketing, College of Agriculture and Life Sciences, Virginia
PolytechnicInstitute and State University. Publication, 420, 256
Delgadillo-Mirquez L, Lopes F, Taidi B, Pareau D (2016) Nitrogen and
phosphate removal from wastewater with a mixed microalgae and
bacteria culture. Biotechnol Rep 11:18–26
Derrien A, Coiffard LJ, Coiffard C, De Roeck-Holtzhauer Y (1998) Free
amino acid analysis of five microalgae. J Appl Phycol 10(2):131–
134
Dubois M, Gilles KA, Hamilton JK, Rebers PT, Smith F (1956)
Colorimetric method for determination of sugars and related sub-
stances. Anal Chem 28(3):350–356
FAO (2014) Food and agriculture organisation of United Nations. http://
www.fao.org/docrep/003/w3732e/w3732e06.htm#2.3.%20Algal%
20production. Assessed on 20 May 2018
Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the
isolation and purification of total lipids from animal tissues. J Biol
Chem 226(1):497–509
Gautam R, Vanga S, Ariese F, Umapathy S (2015) Review of multidi-
mensional data processing approaches for Raman and infrared spec-
troscopy. EPJ Tech Instrum 2(8):8
Griffiths MJ, Dicks RG, Richardson C, Harrison ST (2011) Advantages
and challenges of microalgae as a source of oil for biodiesel. In:
Biodiesel-feedstocks and processing technologies. InTechOpen
Hannon M, Gimpel J, Tran M, Rasala B, Mayfield S (2010) Biofuels
from algae: challenges and potential. Biofuels. 1(5):763–784
Hemaiswarya S, Raja R, Kumar RR, Ganesan V, Anbazhagan C (2011)
Microalgae: a sustainable feed source for aquaculture. World J
Microbiol Biotechnol 27(8):1737–1746
Hsia SY, Yang SK (2015) Enhancing algal growth by stimulation with
LED lighting and ultrasound. J Nanomater, 16(1):222
Islam M, Magnusson M, Brown R, Ayoko G, Nabi M, Heimann K (2013)
Microalgal species selection for biodiesel production based on fuel
properties derived from fatty acid profiles. Energies, 6(11):5676–
5702
Jais NM, Mohamed RMSR, Al-Gheethi AA, Hashim MA (2017) The
dual roles of phycoremediation of wet market wastewater for nutri-
ents and heavy metals removal and microalgae biomass production.
Clean Techn Environ Policy 19(1):37–52
Jena J, Nayak M, Panda HS, Pradhan N, Sarika C, Panda PK, Rao BVK,
Prasad RB, Sukla LB (2012) Microalgae of Odisha coast as a po-
tential source for biodiesel production. World Environ 2(1):11–16
Ji MK, Kim HC, Sapireddy VR, Yun HS, Abou-Shanab RA, Choi J, Lee
W, Timmes TC, Jeon BH (2013) Simultaneous nutrient removal and
lipid production from pretreated piggery wastewater by Chlorella
vulgaris YSW-04. Appl Microbiol Biotechnol 97(6):2701–2710
Juneja A, Ceballos RM, Murthy GS (2013) Effects of environmental
factors and nutrient availability on the biochemical composition of
algae for biofuels production: a review. Energies 6:4607–4638
Khoeyi ZA, Seyfabadi J, Ramezanpour Z (2012) Effect of light intensity
and photoperiod on biomass and fatty acid composition of the
microalgae, Chlorella vulgaris. Aquac Int 20:41–49
Krzemińska I, Pawlik-Skowrońska B, Trzcińska M, Tys J (2014)
Influence of photoperiods on the growth rate and biomass produc-
tivity of green microalgae. Bioprocess Biosyst Eng 37(4):735–741
Lage S, Gojkovic Z, Funk C, Gentili FG (2018) Algal biomass from
wastewater and flue gases as a source of bioenergy. Energies
11(3):664
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein mea-
surement with the Folin phenol reagent. J Biol Chem 193(1):265–
275
Lu Q, Li J, Wang J, Li K, Li J, Han P, Zhou W (2017) Exploration of a
mechanism for the production of highly unsaturated fatty acids in
Scenedesmus sp. at low temperature grown on oil crop residue based
medium. Bioresour Technol 244:542–551
Maizatul AY, Mohamed RMSR, Al-Gheethi AA, Hashim MA (2017) An
overview of the utilisation of microalgae biomass derived from nu-
trient recycling of wet market wastewater and slaughterhouse waste-
water. Int Aquat Res 9(3):177–193
Mantzorou A, Ververidis F (2018) Microalgal biofilms: A further step
over current microalgal cultivation techniques. Sci Total Environ.
651:3187-3201
Mehta SK, Gaur JP (2005) Use of algae for removing heavy metal ions
from wastewater: progress and prospects. Crit Rev Biotechnol
25(3):113–152
Meng Y, Yao C, Xue S, Yang H (2014) Application of Fourier transform
infrared (FT-IR) spectroscopy in determination of microalgal com-
positions. Bioresour Technol 151:347–354
Montgomery DC, Jennings CL, Kulahci M (2015) Introduction to time
series analysis and forecasting. John Wiley & Sons

Nitayapat N, Chitprasert P (2014) Characterisation of FOGs in grease trap
waste from the processingof chickens in Thailand. Waste Manag 34:
1012–1017
Pahazri NF, Mohamed RMSR, Al-Gheethi AA, Kassim AHM (2016)
Production and harvesting of microalgae biomass from wastewater:
a critical review. Environ Technol Rev 5(1):39–56
Radhakrishnan S, Seenivasan C, Muralisankar T (2015) Effect of dietary
replacement of fishmeal with Chlorella vulgaris on growth perfor-
mance, energy utilization and digestive enzymes in Macrobrachium
rosenbergii postlarvae. Int J Fish Aquac 7(5):62–70
Renaud SM, Thinh L-V, Lambrinidis G, Parry DL (2002) Effect of tem-
perature on growth, chemical composition and fatty acid composi-
tion of tropical Australian microalgae grown in batch cultures.
Aquaculture 211:195–214
Ribeiro FM, Faria ER, Verly RM, Franco DV, Da Silva LM (2016)
Fabrication and characterisation of mixed oxide-covered mesh elec-
trodes of nominal composition Ni(x)Co(1-x)Oy supported on stain-
less-steel prepared by thermal decomposition using the slow cooling
rate method. Electrochim Acta 194:127–135
Robertson R, Guihéneuf F, Schmid M, Stengel DB, Fitzgerald G, Ross P,
Stanton C (2013) Algae-derived polyunsaturated fatty acids: impli-
cations for human health. In: Catalá A (ed) Polyunsaturated fatty
acids sources, antioxidant properties and health benefits. Nova
Sciences Publishers, Inc., Hauppauge, pp 45–99
Roy SS, Chaudhuri A, Mukherjee S, HomeChauduri S, Pal R (2011)
Composite algal supplementation in nutrition of Oreochromis
mossambicus. J Algal Biomass Util 2(1):10–20
Sankian Z, Khosravi S, Kim YO, Lee SM (2017) Effect of dietary protein
and lipid level on growth, feed utilization, and muscle composition
in golden mandarin fish Siniperca scherzeri. Fish Aquat Sci 20(1):7
Singh SP, Singh P (2015) Effect of temperature and light on the growth of
algae species: a review. Renew Sustain Energy Rev, 50:431–444
Singh P, Guldhe A, Kumari S, Rawat I, Bux F (2015) Investigation of
combined effect of nitrogen, phosphorus and iron on lipid
Environ Sci Pollut Res
productivity of microalgae Ankistrodesmus falcatus KJ671624
using response surface methodology. Biochem Eng J 94:22–29
Skorupskaite V, Makareviciene V, Levisauskas D (2015) Optimization of
mixotrophic cultivation of microalgae Chlorella sp. for biofuel pro-
duction using response surface methodology. Algal Research, 7:45–
50
Solovchenko A, Verschoor AM, Jablonowski ND, Nedbal L (2016)
Phosphorus from wastewater to crops: an alternative path involving
microalgae. Biotechnol Adv 34(5):550–564
Spolaore P, Joannis-Cassan C, Duran E, Isambert A (2006) Commercial
applications of microalgae. J Biosci Bioeng 101:87–96
Stengel DB, Connan S, Popper ZA (2011) Algal chemodiversity and
bioactivity: sources of natural variability and implications for com-
mercial application. Biotechnol Adv 29:483–501
Sulaymon AH, Mohammed AA, Al-Musawi TJ (2013) Competitive
biosorption of lead, cadmium, copper, and arsenic ions using algae.
Environ Sci Pollut Res 20(5):3011–3023
Wagenen JV, Miller TW, Hobbs S, Hook P, Crowe B, Huesemann M
(2012) Effects of light and temperature on fatty acid production in
Nannochloropsis salina. Energies 5:731–740
Wahidin S, Idris A, Shaleh SRM (2013) The influence of light intensity
and photoperiod on the growth and lipid content of microalgae
Nannochloropsis sp. Bioresour Technol 129:7–11
Willhi Electronics Co. Ltd (2016) Thermostat product manual. Shenzen,
China. 6pages
Yaakob MA, Mohamed RMSR, Al-Gheethi AA, Amir HMK (2018)
Characteristics of chicken slaughterhouse wastewater. Chem Eng
63:637–642