Renewable Energy 103 (2017) 197e207

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Renewable Energy
journal homepage: www.elsevier.com/locate/renene

Cultivation of Chlorella vulgaris using nutrients source from domestic

wastewater for biodiesel production: Growth condition and kinetic
studies
Man Kee Lam a, *, Mohammad Iqram Yusoff a, Yoshimitsu Uemura a, Jun Wei Lim b,
Choon Gek Khoo c, Keat Teong Lee c, Hwai Chyuan Ong d
a
Chemical Engineering Department, Universiti Teknologi PETRONAS, 32610 Seri Iskandar, Perak, Malaysia
b
Fundamental and Applied Sciences Department, Universiti Teknologi PETRONAS, 32610 Seri Iskandar, Perak, Malaysia
c
School of Chemical Engineering, Universiti Sains Malaysia Engineering Campus, Seri Ampangan, 14300 Nibong Tebal, Pulau Pinang, Malaysia
d
Department of Mechanical Engineering, Faculty of Engineering, University of Malaya, 50603 Kuala Lumpur, Malaysia

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

Article history: Owning to the presence of essential mineral nutrient content in wastewater, cultivation of microalgae
Received 12 January 2016 using wastewater sources provides an alternative and sustainable solution for biodiesel production.
Received in revised form Hence, the potential of using domestic wastewater as nutrient source to cultivate Chlorella vulgaris was
11 October 2016
presently studied. It was found that the microalgae was favoured to grow in domestic wastewater under
Accepted 15 November 2016
the conditions of 0.02 v/v of wastewater, initial pH of 3, and 0.03 v/v of initial amount of microalgae seed
Available online 24 November 2016
with 24 h of continuous illumination. Under these conditions, a high lipid content of 32.7% was
embedded within the microalgae biomass. From the analysis of fatty acid methyl ester (FAME) profile, the
Keywords:
Microalgae
extracted microalgae lipid was suitable for biodiesel production. The existing growth kinetic models
Biodiesel were able to predict the growth of Chlorella vulgaris using the domestic wastewater as nutrients source.
Domestic wastewater The fair model fitting was however limited to contaminant-free conditions, where the growth decays of
Chlorella vulgaris the microalgae was negligible.
Growth kinetic © 2016 Elsevier Ltd. All rights reserved.

1. Introduction acids, which can be extracted for subsequent biodiesel production.

Biofuels are recognized as renewable energy since its precursors
are derived from biodegradable resources such as corn kernels,
sugar cane, sweet sorghum, cassava, woody biomass, vegetable oils,
microalgae, waste cooking oil and etc. [1,2]. Commercially available
bioethanol is derived from sugar cane, cassava flour, corn starch
[3e6] whereas biodiesel is produced from oil crops [3,4] or waste
cooking oil [7,8]. Due to the food for fuel dilemma, non-food crops
and aquatic microorganisms such as microalgae have been pro-
posed as an alternative feedstock for biodiesel production, whereas
lignocellulosic biomass [9] are increasingly used in the bioethanol
production [10,11]. Microalgae are photosynthetic microorganisms
that only require CO2, water and nutrients (e.g. nitrogen, phos-
phorus and potassium) for their growth. The produced microalgae
biomass contains a significant amount of lipid in the form of fatty
* Corresponding author.
E-mail address: lam.mankee@utp.edu.my (M.K. Lam).
It was reported that approximately 50% of the microalgae biomass
constituents are lipids, and sometimes may reached up to 80% by
cultivating them under heterotrophic condition [12].
Wastewater can be used as alternative nutrient source to grow
microalgae, in which the wastewater will be simultaneously bio-
remediated prior to discharge. Accordingly, the amount of nitro-
gen and phosphorus sources can be eliminated to prevent the
eutrophication phenomenon in receiving water bodies of dis-
charged wastewater. Hence, by using wastewater to cultivate
microalgae provides mutual benefit of producing biofuels while
removing nitrogen, phosphorus and organic carbon from the
wastewater [13]. However, wastewater-based microalgae cultiva-
tion is still facing uncertainties and challenges, including variation
of wastewater compositions due to various sources, contamination
by toxic compounds and presence of suspended solid particles that
may affect the light transmission efficiency in cultivating micro-
algae [14].
The aim of the present work is to evaluate the feasibility of
microalgae cultivation using unsterilized domestic wastewater. The

http://dx.doi.org/10.1016/j.renene.2016.11.032
0960-1481/© 2016 Elsevier Ltd. All rights reserved.
198 M.K. Lam et al. / Renewable Energy 103 (2017) 197e207
effects of various cultivation parameters such as amount of nutri-
ents, initial pH and amount of microalgae seed were systematically
investigated. Subsequently, the growth kinetic studies were con-
ducted to illustrate the growth pattern of microalgae cultivated in
wastewater medium, rendering more information for scale-up
study.
2. Materials and methods
2.1. Cultivation of Chlorella vulgaris using Bold’s Basal Medium
(BBM)
Chlorella vulgaris was provided by Prof. Dr. Lee Keat Teong
(School of Chemical Engineering, Universiti Sains Malaysia). Prior to
the experiments, the microalgae was preserved and grown in BBM
consisting of: (1) 10 mL per liter of culture medium with the
following chemicals: NaNO3 (25 g/L), CaCl2$2H2O (2.5 g/L),
MgSO4$7H2O (7.5 g/L), K2HPO4 (7.5 g/L), KH2PO4 (17.5 g/L), NaCl
(2.5 g/L) and (2) 1 mL per liter of culture medium with the following
chemicals: EDTA anhydrous (50 g/L), KOH (31 g/L), FeSO4$7H2O
(4.98 g/L), H2SO4 (1 mL), H3BO3 (11.4 g/L), ZnSO4$7H2O (8.82 g/L),
MnCl2$4H2O (1.44), MoO3 (0.71 g/L), CuSO4$5H2O (1.57 g/L),
Co(NO3)2$6H2O (0.49 g/L) [15]. The initial pH of the medium was
adjusted to 3.5. The seed culture was grown in a 5 L bottles con-
taining 4.5 L of medium, aerated with compressed air with sur-
rounding temperature ranging from 25 to 28
C and illuminated
continuously with cool-white fluorescent light.
2.2. Cultivation of Chlorella vulgaris using domestic wastewater
The wastewater samples were collected from a domestic
wastewater treatment plant (inlet point of secondary treatment) in
Universiti Teknologi PETRONAS, Perak, Malaysia. The collected
wastewater samples were left to settle tranquilly for 24 h. The su-
pernatant of wastewater samples were then filtered using 1.2 mm-
pore size GF/C filter (Whatman co.) to avoid any false indication in
the microalgae growth study due to the presence of suspended
solid. Then, the filtrate was being analysed for total nitrogen and
total phosphorus according to the HACH standard methods. Then, a
pre-determined volume ratio of the domestic wastewater to total
working volume (v/v) and seed culture to total working volume (v/
v) were introduced into the Erlenmeyer flask with total working
volume of 1 L. The pH of the cultivation medium was adjusted ac-
cording to the intended study parameter. Subsequently, the
Erlenmeyer flasks were continuously aerated with compressed air
and illuminated with cool-white fluorescent light (light intensity of
60e70 mmol m2 s1). All experiments were carried out in triplicate
with ±5% error and average values were reported.
2.3. Measurement of microalgae growth
Dry cell weight is commonly used to determine the biomass
production in biological process and it is obtained by measuring the
total suspended solids concentration in the culture medium. A
correlation between the optical density of Chlorella vulgaris and
concentration of biomass was pre-determined. In this regard, the
optical density was determined daily at 688 nm using spectro-
photometer (Agilent UV-VIS). Then, 20 mL of sample was dried in
an oven at 100
C for 24 h and the weight of biomass was deter-
mined using weighting balance. The correlation between the
microalgae biomass concentration and optical density was estab-
lished as shown below:
Dry weightðg=LÞ ¼ 0:3793  OD688nm ; R2 ¼ 0:9674 (1)
The specific growth rate (m) and biomass productivity (P) were
determined by using Equations (2) and (3), respectively:

 ln N2
m day1 ¼ N1
(2)
t2  t1
N2  N1
Pðg=L=dayÞ ¼ (3)
t2  t1
where N1 and N2 are the biomass (g/L) at time t1 (day) and t2 (day),
respectively.
2.4. Harvesting and collecting of microalgae biomass
After the Chlorella vulgaris had reached the stationary growth
phase, the aeration to the cultivation medium was stopped. The
microalgae biomass was left to settle naturally in the Erlenmeyer
flask for three days. Two distinguished layers were then formed, in
which water with suspended microalgae cells were located at the
upper layer and concentrated microalgae biomass was deposited at
the bottom layer. The upper layer was carefully decanted, whereas
the bottom layer was transferred to 500 mL beaker and dried at
100
C for 24 h. The dried microalgae biomass was scrapped from
the beaker and kept in empty container for later use.
2.5. Microalgae lipid extraction
1 g of dried Chlorella vulgaris biomass was placed in a 500 mL
conical flask containing 200 mL mixed methanolechloroform so-
lution (volume ratio of 2:1). The mixture was stirred at 400 rpm for
24 h at room temperature. After that, the biomass was filtered using
filter paper and the filtrate was evaporated in a rotary evaporator at
100
C. The leftover crude microalgae lipid was collected after all
the solvent was evaporated. The filtered microalgae biomass was
then mixed with the solvent recovered from the rotary evaporator
for second cycle of lipid extraction [16].
2.6. Transesterification reaction of crude microalgae lipid and FAME
analysis
1 g of crude lipid of Chlorella vulgaris, 15:1 of methanol to lipid
molar ratio and 3 wt% of concentrated sulphuric acid were mixed
during the transesterification process. The reaction was carried out
in a water bath shaker at 60
C for 3 h 1 mL of the reaction product
was subjected to gas chromatography-mass spectrometry (GCeMS;
PerkinElmer Clarus 600) analysis after the reaction was completed.
The gas chromatography was equipped with a flame ionization
detector (FID) and Elite 5-MS column
(30 m  0.25 mm  0.25 mm). The initial oven temperature was
65
C, held for 2 min and raised to 280
C at a ramping rate of 8
C/
min and held at 280
C for 10 min [15].
2.7. Kinetic growth model
Three non-linear mathematical models, namely Logistic, Gom-
pertz, and Richards models were used to validate the experimental
data of biomass production by Chlorella vulgaris. The experimental
data were referred to the biomass yield of Chlorella vulgaris under
the supplementation of different concentrations of wastewater in
1 L cultivation setup.
2.7.1. Logistic model
The logistic model describes the growth of microbial based on
the initial population density, time, growth rate and final
 M.K. Lam et al. / Renewable Energy 103 (2017) 197e207
population density [17]. The original logistic function model was
developed by Pearl and Reed (1977) and expressed as follow [18]:
AþC
y¼ (4)
1 þ expBðtMÞ
where A is the asymptotic of ln Xt/Xo as t decreases indefinitely, C is
the asymptotic of ln Xt/Xo as t increases indefinitely, B is the relative
growth rate at time M (day1), t is the residence time (day), M is the
time at which the maximum growth rate is reached (day), Xt is the
biomass concentration at time t (g/L) and Xo is the initial biomass
concentration (g/L).
2.7.2. Gompertz model
Gompertz model has been widely used in literature and most of
the kinetic data were described based on model shown below [19]:
y ¼ A þ C expexp½BðtMÞ (5)
where A is the asymptotic of ln Xt/Xo as t decreases indefinitely, C is
the asymptotic of ln Xt/Xo as t increases indefinitely, B is the relative
growth rate at time M (day1), t is the residence time (day), M is the
time at which the maximum growth rate is reached (day), Xt is the
biomass concentration at time t (g/L) and Xo is the initial biomass
concentration (g/L).
2.7.3. Richards model
Richards model is a four-parameter model as shown below [20]:
y ¼ Að1 þ y exp½kðt  tÞÞð1=yÞ (6)
where A is the asymptotic of ln Xt/Xo as t decreases indefinitely, t is
the residence time (day), n and k are shape parameter whereas t is
time at the inflexion point (lag phase).
2.7.4. Analysis of microalgae growth kinetic
A nonlinear regression technique was used to solve the growth
models by using POLYMATH 6.0. The algorithm for microalgae
growth kinetic analysis are as follow: (1) the values for ln Xt/Xo was
calculated based on the experimental data, (2) the data was loaded
to the POLYMATH program, (3) the respective growth model was
then inserted to the POLYMATH program under the ‘Nonlinear’
column, (4) all the dependent, independent and model variables
are shown in the programme after inserting the respective model
and checking, (5) initial estimates of the parameters values were
required and, (6) several statistic indicators were used to assess the
quality of the estimated values and the regression models, such as
coefficient of determination (R2), variance (S2), root mean square
deviation (RMSD), graph (experiment versus predicted) and re-
sidual plot. The following guidelines were used in determining the
goodness-of-fit of the data to the kinetic models:
(a) R2: The coefficient of determination is always used as an
indicator to represent the preciseness of model in fitting the
experimental data. A coefficient of determination close to
one implies the accuracy of the model. The coefficient of
determination can be calculated based on the following
equation:
Pn  2
2 i¼1 yiobs  yicalc
R ¼1 Pn  2 (7)
i¼1 yiobs  y
199
!
1 X
n
y¼ yiobs (8)
n
i¼1
where, ‘n’ is referred to number of observation, ‘obs’ is referred to
the observed data and ‘calc’ is referred to the calculated data.
(b) S2 and RMSD: Similar to coefficient of determination, these
two indicators are frequently used for comparing the accu-
racy of different models that represent the experimental
data. A model with smaller variance and RMSD indicate the
data are more accurate than a model with larger values of
these indicators. The following equations describe the
calculation of S2 and RMSD:
Pn
ðyi  yÞ2
S2 ¼ i¼1
(9)
n1
!2
1 Xn  2
RMSD ¼ y  yicalc (10)
n i¼1 iobs
where ‘n’ is referred to number of observation, ‘obs’ is referred to
the observed data and ‘calc’ is referred to the calculated data.
3. Results and discussions
3.1. Effect of nutrients amount
Nutrient uptake by microalgae is usually affected by the overall
composition of nutrient available in the cultivation medium. In this
regard, nitrogen (N) and phosphorus (P) source are usually required
in the cultivation medium to support the growth of microalgae
biomass. Thus, the N/P ratio in the cultivation medium must be
fine-tuned properly in order to ensure the simultaneous utilization
of both nutrient sources by microalgae and attain optimal nutrient
removal efficiency [21]. Nevertheless, there are some limitations
that need to be addressed when wastewater is used as nutrient
source in cultivating microalgae, such as the resistance of micro-
algae species towards biotic pollutants (e.g. bacteria, fungi and
zooplanktons) that co-existing in wastewater [22].
In the present study, different amounts of unsterilized waste-
water were filled into Erlenmeyer flask to study the effect of
wastewater towards the growth of Chlorella vulgaris. From Fig. 1,
Chlorella vulgaris was found to have higher biomass yield when
supplied with low amount of wastewater (0.02 v/v) than high
amount of wastewater (0.15e0.2 v/v). This observation was rather
contradict to most of the previously published reports showing
high amount of wastewater tended to accelerate the growth of
microalgae due to the immense availability of nutrients in the
cultivation medium [23,24]. However, in the present study, the
wastewater source was directly utilized without sterilizing. This
led to the severe contamination by biotic pollutants from the high
amount of wastewater cultivation, in which inhibited the growth
of microalgae. In addition, the polluted microalgae cultivations
were noticed turned into light brown colour and rotifers (micro-
algae grazer) were detected under microscope observation. The
results were further supported by Fig. 2, showing the highest
microalgae biomass productivity (0.0409 g/L/day) was attained
with the minimum amount of wastewater (0.02 v/v). The cultiva-
tion of Chlorella vulgaris in the medium containing 0.02 v/v of
wastewater also recorded the highest specific growth (0.30 day1)
as compared with other cultivations loaded with higher amount of
wastewater.
200 M.K. Lam et al. / Renewable Energy 103 (2017) 197e207

0.6

0.5
Biomass, g/L

0.4

0.3

0.2

0.1

0
0 2 4 6 8 10 12 14
Cultivation time (day)

0.02 v/v 0.05 v/v 0.1 v/v 0.15 v/v 0.2 v/v

Fig. 1. Effect of amount of wastewater towards the growth of Chlorella vulgaris. Other cultivation condition: pH ¼ 3, amount of seed ¼ 0.1 v/v and illuminated for 24 h continuously.

0.35 0.045

0.040
0.30

Biomass productivity, g/L/day

0.035
Specific growth rate, day-1

0.25
0.030
0.20 0.025

0.15 0.020

0.015
0.10
0.010
0.05
0.005

0.00 0.000
0.02 v/v 0.05 v/v 0.1 v/v 0.15 v/v 0.2 v/v
Amount of wastewater
Specific growth Biomass productivity

Fig. 2. Specific growth rate and biomass productivity of Chlorella vulgaris cultivated in different amount of wastewater.

3.2. Effect of pH production of green microalgae such as Chlorella vulgaris in

One of the important factors in microalgae cultivation is the pH
of the culture medium. This is because variation of pH in the culture
medium can ultimately affect the cell metabolism and growth of
the microalgae biomass [25]. Suitable pH for optimum growth of
most microalgae species lies in the neutral to alkaline range.
However, there are some exceptional microalgae species (e.g.
Dunaliella) that are able to grow under very acidic condition of as
low as pH 1 [26]. According to Goldman et al. (1982), the biomass
continuous cultivation was significantly affected by the pH in the
culture medium [27]. The pH tolerance limits of the microalgae are
determined either by chemical influence on the growth medium or
metabolic effects on the cells [27]. Goldman et al. (1981) asserted
that the maximum tolerable pH is not influenced by the availability
of inorganic carbon. Nevertheless, the pH is the main determinants
of the carbon species in water that regulate the availability of
different carbon sources used for microalgae photosynthesis.
Moreover, the effect of pH level to the microalgae cultivation is
 M.K. Lam et al. / Renewable Energy 103 (2017) 197e207
crucial when flue gases are used to cultivate microalgae at indus-
trial scale. The selected microalgae species must be able to tolerate
the inconsistency of CO2 concentration in the flue gases that results
in pH variation [28].
Fig. 3 shows the effect of pH value in the culture medium to-
wards the growth of Chlorella vulgaris. From the figure, the growth
of Chlorella vulgaris cultivated at pH 3 and 5 exhibited almost a
linear growth trend with maximum biomass attained at the range
of 0.18e0.25 g/L after 12 days of cultivation. For higher pH culti-
vation medium (alkaline condition), the microalgae did not exhibit
a satisfactory growth curves. The growth of microalgae at pH 7, 9
and 12 was stagnated demonstrating insignificant increment of
biomass after 12 days of cultivation. From Fig. 4, the pH 3 cultiva-
tion medium recorded the highest specific growth and biomass
productivity at 0.1466 day1 and 0.0169 g/L/day, respectively. These
results indicated that the Chlorella vulgaris used in the present
study could adapt very well at low pH medium which was an
advantage to inhibit other biotic contaminants (e.g. fungus) that
could present in the unsterilized wastewater culture medium. Be-
sides, it was also presumed that Chlorella vulgaris in the present
study preferred to assimilate carbonic acid (H2CO3) as the carbon
source for growing. This is because at pH below 4.5, the cultivation
medium is dominated with free CO2 molecules or H2CO3 when CO2
is dissolved in water [29].
3.3. Effect of amount of seed
As the density of microalgae increases with time, the growth
will slow down due to the depletion of nutrients and decrease in
light penetration arises from increasing self-shading due to dense
microalgae population. Thus, sufficient aeration in the microalgae
cultivation system is important to agitate the microalgae cells and
to allow even distribution of light to reach the dense microalgae
population [30]. Nevertheless, introducing certain amount of
microalgae seed at the initial stage of cultivation is necessary to
initiate the fast growth rate of the microalgae (avoid long lag-
phase) and to increase the survival rate of microalgae with other
0.25
0.2
Biomass, g/L
0.15
0.1
0.05
0
0 2 4 6
Cultivation Time (day)
3 5
Fig. 3. Effect of pH value on the growth of Chlorella vulgaris. Other cultivation condition: amount of wastewater ¼ 0.02 v/v, amount of seed ¼ 0.1 v/v and illuminated for 24 h
continuously.
201
microorganisms.
Fig. 5 shows the effect of amount of seed on the growth of
Chorella vulgaris. All of the 5 experiments exhibited a similar
growth trends during the 12 days cultivation. From Fig. 6, the
highest biomass productivity was recorded with experiment con-
tained 0.2 v/v of seed which was 0.0219 g/L/day, then followed by
0.15 v/v, 0.10 v/v, 0.05 v/v and 0.03 v/v which recorded biomass
productivity of 0.0215, 0.0162, 0.0134 and 0.0086 g/L/day, respec-
tively. However, Chorella vulgaris showed the highest specific
growth rate in the culture medium with the lowest amount of seed,
which was 0.1575 day1. Based on these results, cultivation me-
dium with low amount of microalgae seed could enhance the
specific growth rate which benefiting the nutrients removal from
wastewater. Nevertheless, as for biodiesel production, higher seed
amount during the initial cultivation is necessary to boost higher
biomass and lipid productivity.
3.4. Nutrients removal
The presence of high concentration of nutrients (e.g. nitrogen
and phosphorus) in wastewater can impart eutrophication if this
wastewater is discharged directly into the water bodies. Nitrogen
can present in wastewater in the form of ammonium (NHþ 4 Þ,
organically bound nitrogen or even nitrite (NO 2 Þ and nitrate (NO 
3)
3
ions; while phosphorus commonly apprears as phosphates (PO4 Þ
ions [22]. Thus, the nutrients availability in the wastewater can be
utilized for microalgae growth. For example, Chorella sp. had been
used in numerous studies due to its effectiveness in removing ni-
trogen and phosphorus sources from different wastewater streams
[31]. In addition, microalgae cultivation could be placed as a tertiary
treatment in a typical wastewater treatment plant to further
remove the remaining NO 3 after post-denitrification process [32].
Fig. 7 depicts the result for the removal efficiency of nitrogen
and phosphorus sources in the wastewater samples by microalgae.
The concentration of total nitrogen in the wastewater sample was
reduced from 2.7 mg/L to 0.4 mg/L, or 85% of removal efficiency;
whereas, the total phosphorus was reduced from 24.19 mg/L to
8 10 12 14
7 9 12
202 M.K. Lam et al. / Renewable Energy 103 (2017) 197e207

0.16 0.0180

0.14 0.0160

0.0140

Biomass productivity, g/L/day

0.12
Specific growth rate, day-1

0.0120
0.1
0.0100
0.08
0.0080
0.06
0.0060
0.04
0.0040
0.02 0.0020

0 0.0000
3 5 7 9 12
pH
Specific growth Biomass productivity

Fig. 4. Specific growth rate and biomass productivity of Chlorella vulgaris at different pH.

0.4

0.35

0.3

0.25
Biomass, g/L

0.2

0.15

0.1

0.05

0
0 2 4 6 8 10 12 14
Cultivation time (day)

0.03 v/v 0.05 v/v 0.1 v/v 0.15 v/v 0.2 v/v

Fig. 5. Effects of amount of seeds on the growth of Chlorella vulgaris. Other culture conditions: Amount of wastewater ¼ 0.02 v/v, pH ¼ 3, and illuminated for 24 h continuously.

15.67 mg/L, or 35% removal efficiency. The removal efficiencies of 3.5. Lipid extraction and biodiesel production from Chlorella
nitrogen and phosphorus sources in this study were also corre- vulgaris
sponding with the findings from the literature, in which Chorella
vulgaris recorded the average removal efficiency of nitrogen and Various microalgae species have been identified to have high
phosphorus of 72% and 28%, respectively, from wastewater sample lipid content which could be converted into biodiesel and thus,
[33]. In addition, the results also suggested that the relatively low providing an alternative source to petroleum based diesel [12].
yield of microalgae biomass obtained in Section 3.1e3.3 could be Generally, there are two methods to extract lipid from microalgae
due to the initial low nitrogen concentration in the wastewater. In biomass, namely chemical solvent method and mechanical press.
fact, the recommended nitrogen and phosphorus ratio for optimum One of the main advantages of using mechanical press is this
microalgae cultivation was 16:1 [32,34], whereas in the present method is conventionally suitable for most of the microalgae spe-
study, the ratio was only 0.11:1. Nevertheless, the shortfall of ni- cies. Nevertheless, it requires a thorough evaluation for large-scale
trogen concentration in the wastewater could stimulate higher extraction due to energy efficiency issue [35]. On the other hand,
lipid yield from microalgae biomass, which will be discussed after lipid extraction using chemical solvent is identified as the most
this section. reliable method to determine the overall microalgae lipid content
 M.K. Lam et al. / Renewable Energy 103 (2017) 197e207 203

0.18 0.025
0.16

Biomass productivity, g/L/day

0.14 0.020

Specific growth rate, day-1

0.12
0.015
0.1
0.08
0.010
0.06
0.04 0.005
0.02
0 0.000
0.03 v/v 0.05 v/v 0.1 v/v 0.15 v/v 0.2 v/v
Amound of seed

Specific growth Biomass productivity

Fig. 6. The specific growth and biomass productivity of Chlorella vulgaris at different amount of seed.

30

25
Amount of nutrients , mg/L

20

15

10

0
Before cultivation After cultivation

Total Nitrogen Total Phosphorus

Fig. 7. Nutrients concentrations before and after microalgae cultivation.

due to the high polarity of fatty acids towards the chemical solvent microalgae cells. Under low nitrogen concentration, starch syn-
[15]. thesis pathway of microalgae cells is blocked and photosyntheti-
In the present study, microalgae lipid extraction was conducted cally fixed carbon is redirected into fatty acids production [36].
by using a mixture of methanol and chloroform with volume ratio Hence, higher accumulation of lipid within the microalgae cells can
of 2:1. The total microalgae lipid extracted was 32.7 wt%, which was
higher than our previous study (18.1 wt%) when the microalgae was Table 2
cultivated using organic fertilizer as nutrients source [15]. This was The R2, RMSD and variance values of different growth models for Chlorella vulgaris
probably due to the low nitrogen concentration in the wastewater cultivated with different wastewater concentrations.
used as cultivation medium, which induced stress condition on the Model 0.02 v/v 0.05 v/v 0.1 v/v 0.15 v/v 0.2 v/v

R2
Richard 0.9942 0.8905 0.8053 0.8655 0.9146
Table 1 Gompertz 0.9935 0.8897 0.8027 0.8655 0.9140
Fatty acid composition. Logistic 0.9880 0.8900 0.8050 0.8612 0.9096
RMSD
Constituents Fatty acid methyl ester Amount (%)
Richard 0.0183 0.0528 0.0758 0.0507 0.0474
C10:0 Methyl Dodecanoate 25.57 Gompertz 0.0193 0.0530 0.0763 0.0507 0.0475
C16:1 Methyl Palmitoleate 30.54 Logistic 0.0264 0.0529 0.0759 0.0515 0.0487
C17:0 Methyl Heptadecanoate 17.17 Variance
C18:0 Methyl Stearate 13.65 Richard 0.0061 0.0501 0.1034 0.0463 0.0404
C18:1 Methyl Oleate 10.25 Gompertz 0.0060 0.0449 0.0932 0.0412 0.0362
C18:2 Methyl Linoleate 2.82 Logistic 0.0125 0.0504 0.1036 0.0478 0.0428
204 M.K. Lam et al. / Renewable Energy 103 (2017) 197e207

be attained [36,37]. Nevertheless, starch is the carbon and energy
source for microalgae; reducing the starch content in the micro-
algae cells can retard their photosynthesis efficiency and biomass
productivity [38].
Fatty acid methyl ester (FAME) is the main component of bio-
diesel and therefore, the chemical composition of distinctive FAME
profile plays a critical role in determining the properties of bio-
diesel produced. It is important to determine the microalgae lipid
profile as different cultivation conditions will induce significant
changes in the biosynthesis of the fatty acids [39]. Table 1 shows the
fatty acid composition of Chlorella vulgaris in the present study.
The overall FAME composition of Chlorella vulgaris was mostly
consisted of C10:0 (Methyl Dodecanoate), C16:1 (Methyl Palmito-
leate) C17:0 (Methyl Heptadecanoate), C18:0 (Methyl Stearate),
C18:1 (Methyl Oleate) and C18:2 (Methyl Linoleate). As depicted in
Table 1, C16:1, C17:0, C18:0, C18:1 and C18:2 encompassed the
major percentages of the FAME Chlorella vulgaris with the sum of
74.43%. These fatty acids are usually found in other oil bearing crops
such as soybean, sunflower and palm oil which have been
confirmed to be suitable for biodiesel production [15,40].

3 2.5

2.5 2
Biomass, g/L

Biomass, g/L
1.5
Y-exp
1.5
Y-cal
1
1 Y-exp
0.5 Y-cal
0.5

0 0
0 5 10 15 0 2 4 6 8 10 12 14
Cultivation time, day Cultivation time, day
(a) (b)
3 2.5

2.5
2

2
Biomass, g/L
Biomass, g/L

1.5
Y-exp
1.5
Y-cal
1
1 Y-exp
0.5 Y-cal
0.5

0 0
0 5 10 15 0 2 4 6 8 10 12 14
Cultivation time, day Cultivation time, day
(c) (d)
3 2.5

2.5 2

2
Biomass, g/L

Y-exp 1.5
Biomass, g/L

1.5 Y-cal
1
1 Y-exp
0.5 Y-cal
0.5

0 0
0 5 10 15 0 5 10 15
Cultivation time, day Cultivation time, day
(e) (f)
Fig. 8. Experiment (exp) versus calculated (cal) values. (a) Richards model with 0.02 v/v wastewater, (b) Richards model with 0.1 v/v wastewater, (c) Gompertz model with 0.02 v/v
wastewater, (d) Gompertz model with 0.1 v/v wastewater, (e) Logistic model with 0.02 v/v wastewater, and (f) Logistic model with 0.1 v/v wastewater.
 M.K. Lam et al. / Renewable Energy 103 (2017) 197e207 205

3.6. Growth kinetic study
Mathematical model has been widely used to predict the trend
of cell growth through the estimations of maximum specific growth
rate, lag phase and maximum cell concentration, which are
essentially required in the study of microbial growth and for the use
in industrial microbiology [17,41]. In the present study, three
kinetic models were used to predict the growth of Chlorella vulgaris,
namely Richards, Gompertz and Logistic models. Table 2 shows the
R2 values for the three mathematical growth models in describing
the growth kinetic of Chlorella vulgaris at different wastewater
concentrations. At 0.02 v/v, all models provided a good fit
(R2 > 0.98). However, continuous adding the wastewater to the
microalgae cultivation had resulted in unsatisfactory R2 values

0.5 0.6
0.4
yexp-ycal yexp-ycal
0.3 0.4
0.2
0.2
0.1
0 0
-0.1
-0.2 -0.2
-0.3
-0.4
-0.4
-0.5 -0.6
0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5
y y

(a) (b)
0.5 0.6
yexp-ycal yexp-ycal
0.4
0.4
0.3
0.2 0.2
0.1
0 0
-0.1
-0.2
-0.2
-0.3
-0.4
-0.4
-0.5 -0.6
0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5
y y

(c) (d)
0.5 0.6
0.4 yexp-ycal yexp-ycal
0.3 0.4
0.2
0.2
0.1
0 0
-0.1
-0.2 -0.2
-0.3
-0.4
-0.4
-0.5 -0.6
0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5
y y

(e) (f)
Fig. 9. Residual plots. (a) Richards model with 0.02 v/v wastewater, (b) Richards model with 0.1 v/v wastewater, (c) Gompertz model with 0.02 v/v wastewater, (d) Gompertz model
with 0.1 v/v wastewater, (e) Logistic model with 0.02 v/v wastewater, and (f) Logistic model with 0.1 v/v wastewater.
206 M.K. Lam et al. / Renewable Energy 103 (2017) 197e207
(R2 < 0.98) for all the three kinetic models. This was probably due to
the inaccuracy of the kinetic models to deduce the decay growth of
microalgae. As can be seen from Fig. 8, the predicted growths of
Chlorella vulgaris by the kinetics models were only coordinated well
with experiment carried out with 0.02 v/v of wastewater. However,
for experiment with 0.1 v/v of wastewater, the decay growth of
microalgae was observed due to contamination. The models were
only able to predict stagnant growth of Chlorella vulgaris during the
decay growth phase (day 9e12) and thus, resulting in poor R2
values attained. The same trends were also observed for RMSD and
variance values (Table 2), in which low RMSD and variance values
could only be attained with experiment that carried out with
0.02 v/v of wastewater.
Although growth kinetic models for Gompertz and Logistic
displayed high R2 (R2 > 0.98) and low RMSD values at 0.02 v/v of
wastewater, the growth trends predicted by these two models were
relatively more scattered as compared with the values predicted
Richards model (Fig. 8). This observation could be further clarified
by the residual plots as shown in Fig. 9. Residual plot indicates the
difference between the predicted and experimental values. A good
residual plot should not display random distribution pattern and
the values must be closed to the X-axis. From Fig. 9(c) and (e),
Gompertz and Logistic models showed a higher degree of sigmoidal
curves distribution along the X-axis, which indicated the inaccu-
racy of the models in fitting the experimental data. Although the
Richards model also showed a sigmoidal curve (Fig. 9(a) and (b)),
however the plot was more converged to the X-axis as compared
with the other models. This observation reflected the errors pre-
dicted by the Richard model were smaller than the other growth
models and therefore, acceptable to represent the growth of
Chlorella vulgaris, but only limited to low concentration of waste-
water without severe contamination. At higher concentration of
wastewater (e.g. 0.1 v/v), the residual plots as shown in Fig. 9 (b),
(d) and (f), were seen fluctuating and deviating from the X-axis for
the three kinetic models. This further explained the errors pre-
dicted by the kinetic models and incompetency of the models to
describe the decay growth of the microalgae.
4. Conclusion
The depletion of oil and gas resources and negative impacts of
burning fossil fuels to the environment have instigated the effort to
discover alternative sources of renewable energy. The production of
biodiesel using microalgae biomass is not only providing plausible
solution to diversify the current renewable biomass feedstock but
also substantially improving the life cycle of biodiesel. In addition,
by utilizing wastewater as nutrient source to cultivate microalgae
could further enhance the sustainability of this renewable feed-
stock. In the present study, Chlorella vulgaris was able to grow
under the following conditions: 0.02 v/v of domestic wastewater,
initial pH of 3 and 0.03 v/v initial amount of microalgae seed.
Although diluted wastewater was used in cultivation (low con-
centration of nutrients), stress condition had induced microalgae
cells to increase the lipid yield to 32.7%. Nevertheless, the decay
growth of microalgae was also observed when high amount of
wastewater was used in the cultivation medium due to the pres-
ence of biotic contaminants. Hence, the existing kinetic growth
models were not able to completely predict the growth of micro-
algae and a more robust kinetic growth model should be developed
to include the effects of biotic contaminants and decay growth.
Otherwise, the wastewater should be pre-treated through sterili-
zation, ultrasonic cavitation, ozone bubble or ultraviolet light prior
to microalgae cultivation to avoid any unnecessary contamination.
Nevertheless, care should be taken as this could results in addi-
tional cost to produce microalgae biodiesel using wastewater.
Acknowledgement
The authors would like to acknowledge the financial support
received from Ministry of Higher Education (MOHE) Malaysia
(Fundamental Research Grant Scheme (FRGS) with cost center
0153AB-L25) Technical support from Green Technology Mission
Oriented Research and Center for Biofuel and Biochemical Research
(CBBR) of Universiti Teknologi PETRONAS is highly appreciated.
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