Algal Research 7 (2015) 24–32

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

An advanced hybrid medium optimization strategy for the enhanced

productivity of lutein in Chlorella minutissima
R. Dineshkumar a, Gunaseelan Dhanarajan a, Sukanta Kumar Dash b, Ramkrishna Sen a,⁎
a
Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal 721302, India
b
Department of Mechanical Engineering, Indian Institute of Technology Kharagpur, West Bengal 721302, India

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

Article history: The biosynthesis of lutein in microalgae is critically influenced by the media components, which either increase
Received 28 August 2014 the key enzyme concentrations or act as cofactors for the enzymes involved in lutein metabolic pathway. In this
Received in revised form 15 November 2014 study, an advanced medium optimization strategy, involving Plackett–Burman design for screening the Bold's
Accepted 29 November 2014
Basal Medium (BBM) components, followed by artificial neural network modeling and particle swarm optimiza-
Available online 6 December 2014
tion (ANN–PSO), was implemented for improving the productivity of lutein in Chlorella minutissima. In this two-
Keywords:
step hybrid optimization endeavor, Plackett–Burman analysis helped select NaNO3, KH2PO4, MnCl2·4H2O and
Microalgae CuSO4·5H2O as critical components that were subsequently optimized by ANN–PSO technique. On experimental
Lutein validation, the optimized medium resulted in three-fold greater lutein productivity, as compared to BBM. Fur-
Plackett–Burman design thermore, the maximum lutein productivity of 3.45 ± 0.07 mg L−1 d−1 was obtained upon cultivation of
ANN modeling C. minutissima in a 2-L airlift photobioreactor using the optimized medium with appropriate process conditions.
Particle swarm optimization Thus, the hybrid optimization approach was instrumental in enhancing lutein productivity by about 187%, which
was significantly higher than the relevant values reported in literature. To our knowledge, this is the first report
on the application of an integrated optimization strategy for the enhanced lutein productivity in C. minutissima as
a model photo-autotrophic organism.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction complicated by the problems of inherently low lutein productivity.

In recent years, microalgae have emerged as attractive sources
for many value-added molecules, in addition to their capabilities to
sequester carbon dioxide and remediate waste water. Lutein, one of
the major carotenoids present in green microalgae, has been reported
to have commercial importance due to its potential applications in
nutraceuticals and also in the prevention and treatment of diseases
such as cataracts, macular degeneration, atherosclerosis and different
types of cancer [1]. Traditionally, lutein has been derived from the petals
of marigold flowers by employing labor and land intensive processes.
The ever increasing demand for lutein for various health-care applica-
tions has driven the researchers to look for more sustainable alternative
source like microalgae, thereby minimizing the dependency on agricul-
tural land and intense human labor [2]. Despite the fact that microalgae
can serve as a sustainable source for a high-value product like lutein, it
has not been possible to realize the full commercial potential, due to
the high cost involved in algal biomass cultivation and the subsequent
downstream processing steps in large scale [2]. This situation is further
⁎ Corresponding author.
E-mail address: rksen@yahoo.com (R. Sen).
Therefore, it is important to enhance the intracellular content of lutein
by optimizing the medium and process conditions.
The biological function of the carotenoids is to protect chlorophyll
and other photosynthetic components from detrimental effects of free
radicals, reactive oxygen species (ROS), metal and light-induced ROS
[3]. Generally, carotenogenesis in microalgae is induced by oxidative
stress factors such as heavy metals and by other cultural or environmen-
tal parameters [4]. So, it is believed that providing appropriate nutrients
and trace metals at their optimal levels would increase the accumula-
tion of lutein. Although there are some reports that elucidate the effect
of nutrients on lutein production via single-factor-at-a-time strategy, no
systematic study on the formulation of a production medium for lutein
production in photoautotrophic mode has been carried out. Hence,
there is a need to formulate a suitable medium for improved lutein syn-
thesis in microalgae. In order to design a production medium, it is im-
portant to select and optimize the critical nutritional factors using
appropriate statistical methodologies such as response surface method-
ology [5]. In recent years, the use of advanced mathematical modeling
and optimization techniques, such as artificial neural network (ANN)
modeling and particle swarm optimization (PSO), has been found to
be more efficient in deriving global optimal solutions for complex
non-linear bioprocesses. The principles and advantages of ANN–PSO
are discussed in previous reports [6–8].

http://dx.doi.org/10.1016/j.algal.2014.11.010
2211-9264/© 2014 Elsevier B.V. All rights reserved.
 R. Dineshkumar et al. / Algal Research 7 (2015) 24–32 25

Thus, the current study focuses on enhancing the lutein biosynthesis Table 1
in a microalga, Chlorella minutissima, by implementing an advanced hy- Plackett–Burman design matrix for 11 medium components with lutein productivity as
process response.
brid medium optimization strategy. The strategy involves the following
steps: (1) screening of critically influencing Bold's Basal Medium (BBM) Runs Medium components (mg L−1)a Lutein
constituents for lutein production by Plackett–Burman statistical de- productivity
A B C D E F G H J K L
(mg L−1d−1)b
sign; (2) optimization of the critical medium components by ANN–
PSO and (3) experimental validation of the predicted global optimal 1 750 150 75 150 75 75 1 0.2 4 1 0.2 0.223 ± 0.008
2 250 150 225 50 75 75 10 0.2 4 0.1 2 0.122 ± 0.004
values. Further, the performance of modified BBM is assessed by culti-
3 750 75 225 150 25 75 10 2 4 0.1 0.2 0.249 ± 0.005
vating the microalga in a 2-L airlift photobioreactor under appropriate 4 250 150 75 150 75 25 10 2 16 0.1 0.2 0.113 ± 0.003
process conditions. 5 250 75 225 50 75 75 1 2 16 1 0.2 0.074 ± 0.002
6 250 75 75 150 25 75 10 0.2 16 1 2 0.194 ± 0.007
2. Materials and methods 7 750 75 75 50 75 25 10 2 4 1 2 0.383 ± 0.010
8 750 150 75 50 25 75 1 2 16 0.1 2 0.274 ± 0.011
9 750 150 225 50 25 25 10 0.2 16 1 0.2 0.167 ± 0.006
2.1. Microalgae and culture conditions 10 250 150 225 150 25 25 1 2 4 1 2 0.150 ± 0.004
11 750 75 225 150 75 25 1 0.2 16 0.1 2 0.193 ± 0.007
The microalga C. minutissima (MCC-27) was procured from Centre 12 250 75 75 50 25 25 1 0.2 4 0.1 0.2 0.079 ± 0.003
for Conservation and Utilization of Blue Green Algae, Indian Agricultural a
A — NaNO3; B — K2HPO4; C — KH2PO4; D — MgSO4·7H2O; E — NaCl; F — CaCl2·2H2O; G —
Research Institute, New Delhi. It was maintained in BBM, which has the FeSO4·7H2O; H — CuSO4·5H2O; J — ZnSO4·7H2O; K — CoCl2·6H2O; and L — MnCl2·4H2O.
b
following composition in mg L−1: NaNO3, 250; KH2PO4, 175; K2HPO4, Data are the average values of three experiments ± S.D.

75; MgSO4·7H2O, 75; NaCl, 25; CaCl2·2H2O, 25; EDTA, 63.7; KOH, 31;
H3BO3, 11.42; FeSO4·7H2O, 4.98; ZnSO4·7H2O, 8.82; CuSO4·5H2O, productivity). The effect of each variable E(xi) was calculated by Eq. (1)
1.57; MnCl2·4H2O, 1.44; Co(NO3)·6H2O, 0.49; and MoO3, 0.71. The and the significance level (p-value) of each variable was determined by
screening and optimization experiments were carried out in 150 mL student's t-test using Eq. (2) and the analysis of these statistical parame-
shake flasks using a temperature controlled shaking incubator (illumi- ters is listed in Table 2.
nation facility of 50 μmol m−2 s−1) with the following cultivation con-
ditions: working volume, 80 mL; inoculum concentration, 0.05 g L−1; hX i
X
initial pH, 8.0; temperature, 28 °C; shaking speed, 120 rpm and contin- 2 RðHÞ− R ðL Þ
uous illumination. All experiments were performed in triplicates and Effect ¼ ð1Þ
N
expressed as mean with standard deviation (S.D.).
where, R(H) = all responses when variable xi at high levels, R(L) = all
2.2. Analytical methods responses when variable xi at low levels and N = total number of runs.

2.2.1. Determination of microalgal growth and nitrate concentration

Microalgal growth was measured by taking optical density (OD750) of
samples using UV–visible spectrophotometer (Chemito Instruments-UV
2100). The dry weight of biomass was calculated based on the standard
graph plotted between OD750 and dry cell weight. Dry weight biomass
(g L−1) = 0.43 ∗ OD750 nm. Nitrate concentration was estimated according
to Ho et al. [9].
2.2.2. Determination of lutein concentration
Lutein concentration was determined using reverse phase High
Performance Liquid chromatography (Agilent, USA) [10]. For sample
preparation, a known amount of microalgal biomass (10 mg) was suit-
ably disrupted and treated with 10 mL of methanol:dichloromethane
(3:1 v/v) to extract the carotenoid lutein. This was followed by saponi-
fication by adding 1 mL of 4% KOH at 40 °C for 45 min. The carotenoids
were then extracted using dichloromethane and the lutein concentra-
tion in the extracts was analyzed using reverse phase C-18 column
(4.6 × 250 mm, 5 μm particle size) with isocratic solvent system
methanol:dichloromethane:acetonitrile:water (67.5:22.5:9.5:0.5, v/v)
at a flow rate of 1 mL min−1 at 450 nm.
2.3. Experimental design
Eðxi Þ
t ðxi Þ ¼ ð2Þ
S:E:
where, S.E. is the standard error of the concentration effect.
2.3.2. Central composite design (CCD)
A 24 full factorial CCD for four significant nutrients, namely, NaNO3;
KH2PO4; MnCl2·4H2O and CuSO4·5H2O as identified by Plackett–
Burman statistical analysis, was used for optimization by ANN–PSO.
The experimental design that consists of 30 runs showing the levels of
the selected variables along with the responses (lutein productivity) is
given in Table 3.
2.3.3. Artificial neural network modeling
The ANN topology consists of an input layer, a hidden layer and an
output layer. It was suitably trained by utilizing the CCD experimental
data with feed-forward back propagation (FFBP) algorithm. The addi-
tional data points required for training the network were generated
Table 2
Statistical analysis for the medium components included in the Plackett–Burman design.

Variable Variable Effect % F-value p-Value Significance

2.3.1. Plackett–Burman statistical experiments codes terms contribution
Among fifteen medium components from BBM, only eleven were se- A NaNO3 0.13 55.76 133.67 0.0003 Yes
lected as the Plackett–Burman design parameters, while the remaining B K2HPO4 −0.019 1.21 – N0.1 No
four components were maintained at optimal levels throughout the trial C KH2PO4 −0.054 9.77 23.41 0.0084 Yes
experiments. Design Expert version 7.1.3 (Stat-Ease Inc., Minneapolis, D MgSO4·7H2O 0.005 0.10 – N0.1 No
E NaCl 0.0008 0.002 – N0.1 No
USA) was used to construct the Plackett–Burman design. For all screen-
F CaCl2 0.010 0.35 – N0.1 No
ing and optimization experiments, lutein productivity (mg L−1 d−1) G FeSO4·7H2O 0.037 4.80 11.50 0.0275 Yes
was used as the objective, since it combines the effects of both lutein H CuSO4·5H2O 0.042 6.16 14.78 0.0184 Yes
content (mg g−1) and biomass productivity (g L−1 d−1) [11]. Table 1 J ZnSO4·7H2O −0.034 3.83 9.18 0.0388 Yes
presents the design matrix involving 12 independent runs at their K CoCl2·6H2O 0.029 2.77 6.64 0.0615 No
L MnCl2·4H2O 0.067 15.24 36.54 0.0038 Yes
corresponding high and low levels along with the responses (lutein
26 R. Dineshkumar et al. / Algal Research 7 (2015) 24–32

Table 3
Central composite design matrix of four critical medium components as independent process variables with lutein productivity (mg L−1 d−1) as the responses. Each experimental run was
carried out in triplicate.

Run order NaNO3 KH2PO4 MnCl2·4H2O CuSO4·5H2O Lutein productivity

(mg L−1 d−1)
Level mg L−1 Level mg L−1 Level mg L−1 Level mg L−1

1 1 1500 −1 50 1 7.5 1 6 0.330 ± 0.011

2 −1 500 1 150 −1 2.5 1 6 0.240 ± 0.008
3 1 1500 −1 50 −1 2.5 1 6 0.289 ± 0.013
4 0 1000 0 100 0 5.0 2 8 0.202 ± 0.003
5 1 1500 −1 50 1 7.5 −1 2 0.286 ± 0.007
6 −1 500 1 150 −1 2.5 −1 2 0.256 ± 0.009
7 0 1000 0 100 0 5.0 0 4 0.507 ± 0.014
8 1 1500 1 150 1 7.5 −1 2 0.244 ± 0.008
9 2 2000 0 100 0 5.0 0 4 0.270 ± 0.012
10 −2 0 0 100 0 5.0 0 4 0.090 ± 0.001
11 0 1000 0 100 0 5.0 0 4 0.503 ± 0.011
12 1 1500 1 150 −1 2.5 1 6 0.230 ± 0.006
13 0 1000 −2 0 0 5.0 0 4 0.541 ± 0.015
14 −1 500 −1 50 1 7.5 −1 2 0.310 ± 0.009
15 0 1000 2 200 0 5.0 0 4 0.430 ± 0.012
16 1 1500 1 150 1 7.5 1 6 0.264 ± 0.008
17 −1 500 1 150 1 7.5 −1 2 0.234 ± 0.004
18 1 1500 −1 50 −1 2.5 −1 2 0.262 ± 0.007
19 0 1000 0 100 −2 0 0 4 0.187 ± 0.003
20 0 1000 0 100 0 5.0 0 4 0.510 ± 0.011
21 −1 500 −1 50 −1 2.5 −1 2 0.298 ± 0.006
22 0 1000 0 100 2 10.0 0 4 0.196 ± 0.003
23 0 1000 0 100 0 5.0 −2 0 0.211 ± 0.007
24 −1 500 1 150 1 7.5 1 6 0.234 ± 0.005
25 0 1000 0 100 0 5.0 0 4 0.510 ± 0.012
26 −1 500 −1 50 −1 2.5 1 6 0.297 ± 0.008
27 1 1500 1 150 −1 2.5 −1 2 0.230 ± 0.005
28 −1 500 −1 50 1 7.5 1 6 0.314 ± 0.010
29 0 1000 0 100 0 5.0 0 4 0.500 ± 0.017
30 0 1000 0 100 0 5.0 0 4 0.510 ± 0.012

using the regression equation (Supplementary Eq. A.1) as reported by updates its velocity and position at different time intervals according
Maji et al. [12]. The neuron connections in each layer are characterized to Eqs. (5) and (6), respectively.
by weights and bias. During training, the model updates its weights
and implements an activation function (Eq. (3)) which produces an out-
   
put accordingly. The model also minimizes the error between actual k
Vi ¼ w
k−1
þ Vi
k−1 k−1 k−1
þ C 1 R1 Li −P i
k−1 k−1
þ C 2 R2 Gi −P i ð5Þ
output and simulated output by iteratively feeding the error backwards
using FFBP algorithm. In ANN modeling, the optical neural network ar-
k k−1 k
chitecture has to be determined in order to avoid over-fitting of data Pi ¼ Pi þ Vi ð6Þ
and it was evaluated using mean squared error (MSE) as performance
index (Eq. (4)) and overall correlation coefficient (R) as precision of where, Vik and Vik − 1 are the velocities of particle i at iteration k and k − 1,
the model. respectively; wk − 1, inertia weight; C1 and C2, learning factors; R1 and R2,
uniformly distributed random variables between 0 and 1; Lki − 1, local best
solution of particle i; Gki − 1, global best solution of the group; and Pik and
X 
Yj ¼ xi wi j þ b j ð3Þ Pik − 1 are the positions of particle i at iteration k and k − 1, respectively.

3. Results and discussion

where, Yj = activation function, wij = weight connection between neu-
rons i and j, xi is the input at neuron i and bj is the bias of neuron j. 3.1. Plackett–Burman screening of critical components for lutein production

X ðExperimentalvalue−predictedvalueÞ2
MSE ¼
n essential for the growth of microalgae but also helps in the effective syn-
The developed ANN model was used as fitness function in conjunc-
tion with PSO algorithm to determine the optimal input levels required
for maximum lutein productivity. The computation of ANN–PSO was
carried out using MATLAB version 8.0 (Mathworks Inc., Natick, USA).
2.3.4. Particle swarm optimization algorithm
PSO, a population based algorithm, has the potential to evolve
during the search for optimal inputs in the developed ANN model. All
particles in the population will travel towards the global optimal so-
lution by following the position of most successful neighbors. It
The optimal combination of major and minor nutrients is not only
ð4Þ
thesis of growth associated products like carotenoids. While the major
nutrients (nitrate, phosphate, magnesium, potassium) are required for
the formation of various structural and functional components of cell,
the minor nutrients or trace elements (iron, copper, cobalt, manganese,
zinc, molybdenum) act as cofactors for enzymes involved in various
metabolic pathways including growth and carotenoid production [13].
In the current study, the initial task was to screen the nutrient variables
that influence lutein productivity in C. minutissima because one or more
nutrients and its concentration levels may affect the production signifi-
cantly. Hence, Plackett–Burman screening experiments were carried out
for 11 medium variables with lutein productivity as process response
 R. Dineshkumar et al. / Algal Research 7 (2015) 24–32
(Table 1). The ANOVA for 12 experiments is presented in Table 2. It was
observed that six out of the eleven medium components were statistically
significant (p b 0.05). The relative levels of significance of each nutrient
are indicated by Pareto-chart (Fig. 1).
3.1.1. Significant nutrient components
Among the major nutrients, nitrate and phosphate (KH2PO4) showed
significant effect on lutein productivity. Nitrate showed the highest level
of significance with an F-value of 133.67, a very low p-value of 0.0003
and % contribution of 55.76 (Table 2). Nitrogen accounts for 7–10% of
cell dry weight and it is very much essential for synthesis of fundamental
elements of cell [14]. Besides the role of promoting growth, nitrogen
source is also required for synthesis of enzymes involved in metabolic
pathways towards lutein (xanthophyll) production [11]. The positive
effect (Effect = 0.13) for nitrate suggests that the medium requires the
supplementation of high level of nitrate to improve lutein productivity.
Potassium dihydrogen phosphate, the source of K+ and PO3− 4 , showed
negative effect (Effect = −0.054) on lutein synthesis. The significance
of KH2PO4 in lutein synthesis is evident from the F-value of 23.41 with
the percentage contribution of 9.77%. Phosphorous, which accounts for
~1% of dry cell weight, plays an important role in synthesizing cellular
components required for growth and metabolism [14]. However, higher
concentration of the inorganic salt KH2PO4 affected growth rate of this
microalga and in turn lutein productivity. This observation indicates
that supplementing the medium with low level of phosphate can increase
lutein productivity in C. minutissima. This finding is in agreement with the
work of Sancho et al. [15], which reported that the excess phosphorus in
the medium (≥372 μM) reduced the growth rate of Scenedesmus obliquus.
In another work, Sánchez et al. [16] reported that high phosphate concen-
tration in the medium decreased the biomass productivity of Scenedesmus
almeriensis.
Besides the role of trace metals as key elements for inducing
carotenogenesis, they also influence the uptake of essential nutrient el-
ements such as nitrogen, phosphorous and CO2 [4,13]. Hence, it is essen-
tial to provide these trace metals at optimal levels in the medium for
concomitant microalgal growth and lutein synthesis.
In the present study, the cumulative percentage contribution of
metal ions such as Mn2+, Cu2+, Fe2+ and Zn2+ to the total effects was
found to be around 33% (Table 2), indicating their importance towards
Fig. 1. Pareto chart indicating the significance of medium components. The components A
(NaNO3), L (MnCl2·4H2O), H (CuSO4·5H2O) and G (FeSO4·7H2O) showed significant
positive effect, while C (KH2PO4) and J (ZnSO4·7H2O) showed significant negative effect
towards lutein production.
27
lutein synthesis. The percentage contribution of MnCl2·4H2O to the
total effects and the F- and p-values of this trace element were found
to be 15.24, and 36.54 and 0.0038, respectively revealing its high signif-
icance next to nitrate (Fig. 1). Hence, this study suggests that the higher
concentration of this metal favors improved productivity of carotenoid
lutein in C. minutissima. Mn2 + serves as a cofactor for phytoene
synthase in the conversion of isopentenyl pyrophosphate (IPP) to
phytoene, which is one of the critical pathways in carotenoid biosynthe-
sis [17]. It is also an essential cofactor of metallo-enzyme, which brings
oxygen evolution in Z-scheme photosynthesis [18], thus demonstrating
the importance of manganese ions towards biomass growth and lutein
synthesis. The next important trace metal influencing lutein synthesis
was Cu2+ with the F-value of 14.78 and percentage contribution of 6.16.
Cu2+ ions induce oxidative stress in cells and promote carotenogenesis
for scavenging the free radicals. This is evident from the fact that
microalgal cells, when exposed to high metal concentrations, minimize
the oxidative stress by stress-induced counteractive mechanisms such
as enzymatic (superoxide dismutase, catalase) and non-enzymatic (ca-
rotenoids, glutathione) reactions [19].
Fe2+ ion, another limiting micronutrient, has been reported to be
important for processes such as photosynthesis, respiration, nitrogen
fixation and DNA synthesis. This ion is essential for the activity of β-
carotene hydroxylase, which converts ε,β-carotene to lutein, the final
step in lutein biosynthesis pathway [17]. It is also capable of inducing
carotenogenesis via generation of hydroxyl radicals by Fenton reac-
tion [20]. Fe2 + contributes 4.80% to the total effects with the F- and
p-values of 11.50 and 0.0275, respectively showing its significance
in this process. The requirement of high concentration of ferrous ions
may be attributed to the enhanced activity of β-carotene hydroxylase
and in turn lutein productivity. Although the role of Fe2+ ion on lutein
production is not yet reported, the use of ferrous ion as an inducer for
astaxanthin and β-carotene accumulation is documented [3,20]. On the
contrary, Zn2+ showed negative effect (Effect = −0.034) with 3.83%
contribution and an F-value of 9.18. This demonstrates that −1 level
(i.e. at low concentration) of this ion in the medium favors lutein synthe-
sis in this microalga. Co2+ was found to be insignificant towards lutein
productivity in terms of % contribution (2.77) and p-value (0.0615). The
negative effect of Zn2+ and insignificant nature of Co2+ ions indicate
that these ions may not be required as cofactors for enzymes involved
in lutein synthesis.
3.1.2. Non-significant nutrient components
The major nutrients such as K2HPO4, MgSO4, NaCl and CaCl2 showed
no significance towards lutein productivity with p-value N 0.1 (Table 2).
Di-potassium hydrogen phosphate (K2HPO4) did not show any major
effect in the process, as the requirements of potassium and phosphorous
might have been met by KH2PO4. Although magnesium ions are essential
for chlorophyll synthesis and biomass growth, it showed no importance
in lutein synthesis. Salinity, an important factor to induce carotenogenesis
[21], did not show a significant effect on lutein synthesis in C. minutissima.
This observation is in agreement with that of Del campo et al. [22], who
reported that increase in salinity (NaCl) up to 100 mM showed no im-
provement in lutein yield.
3.1.3. Influence of formulated medium on lutein productivity
As per Plackett–Burman design analysis, a production medium
was formulated and the composition of the medium is as follows
(in mg L − 1 ): NaNO 3, 750; KH2 PO4 , 75; K 2 HPO4 , 75; MgSO4 ·7H2 O,
150; NaCl, 25; CaCl2·2H2O, 25; EDTA, 63.7; KOH, 31; H3BO3, 11.42;
FeSO4·7H2O, 10; ZnSO4·7H2O, 4.0; CuSO4·5H2O, 2.0; MnCl2·4H2O,
2.0; Co(NO3)·6H2O, 1.0; and MoO3, 0.71. This medium resulted in lutein
productivity of 0.403 ± 0.013 mg L−1 d−1, which was around 2-fold
higher than using BBM (Supplementary Table A.1). Thus, the Plackett–
Burman based statistical screening design helped us to screen the critical
medium components and to formulate a production medium for im-
proved lutein synthesis in C. minutissima. It was observed that the lutein
28 R. Dineshkumar et al. / Algal Research 7 (2015) 24–32

Fig. 2. Regression plot of experimental and simulated values. The overall correlation coefficient, R close to 1 indicates the accuracy of the selected neural network topology.

productivity was significantly influenced by four medium constituents
(NaNO3, KH2PO4, MnCl2·4H2O and CuSO4·5H2O) and hence, these two
major and minor nutrients were considered for subsequent optimization
studies by ANN–PSO.
3.2. Optimization of critical nutrient components using ANN–PSO
The ANN model was developed by splitting the CCD experimental
data (Table 3) as follows: 70% for training, 15% for testing and 15% for
validating. The network utilizes a maximum part of the data for training
and it also tests and validates at each run in order to avoid over-fitting of
data. The network was trained using feed-forward back propagation al-
gorithm with mean squared error (MSE) as the performance index and
overall correlation coefficient (R) as the precision of the developed
model. The learning rate and performance of the neural network will
be affected by the type of transfer function used [23]. In this study,
tangent-sigmoidal transfer function at the hidden layer node and
pure-linear transfer function at the output layer node exhibited better
performance. The determination of number of neurons in the hidden
layer is also a crucial step in developing the optimal neural network ar-
chitecture and it was evaluated as described earlier [7]. The optimum
number of neurons in the hidden layer was found to be 12 in connection
with the minimum MSE value of 0.0005 and the maximum R-value of
0.998. R-value close to 1 (Fig. 2) revealed that there is no over-fitting
of data between ANN-predicted and actual experimental outputs.
Thus, 4-12-1 neural network topology (Fig. 3) was selected and the

Fig. 3. Schematic representation of optimized ANN architecture consists of an input layer with four neurons representing four medium variables, a hidden layer (12 neurons) with tangent
sigmoidal transfer function, and an output layer (one neuron) with pure linear transfer function.
 R. Dineshkumar et al. / Algal Research 7 (2015) 24–32
Fig. 4. Best fitness plot showing the particles convergence towards the global optimum
value of 0.679 at around 60 iterations.
developed ANN model was used as fitness function in PSO algorithm for
predicting the best combination of four medium components for im-
proved lutein productivity.
The MATLAB PSO code that was suitably modified by Dhanarajan
et al. [7] was used in this study. The crucial parameters that evaluate
the performance of PSO algorithm such as population size (40), inertia
weight (0.9 to 0.4) and learning factors (C1 = C2 = 1.8) were deter-
mined as described earlier [8]. The optimal nutrient concentrations for
maximum lutein productivity were found by running the PSO algorithm
with the abovementioned parameters. Fig. 4 shows that all the particles
converged towards the global maximum solution at around 60 it-
erations. This ANN–PSO approach predicted lutein productivity of
0.679 mg L − 1 d − 1 for 1152 mg L − 1 NaNO 3 ; 34 mg L − 1 KH 2 PO 4 ;
4.57 mg L− 1 MnCl2·4H2O and 2.35 mg L− 1 CuSO4·5H2O.
3.2.1. Validation studies
The prediction ability of the developed ANN model was tested by
conducting three additional set of experiments that were different
from CCD data. The results of these experiments illustrated that the per-
centage error between predicted and actual outputs was within 3.6%
(Supplementary Table A.2). Further, the PSO-predicted optimal concen-
tration of nutrients was analyzed by performing validation experiments
(in triplicates). The experimentally tested lutein productivity was found
to be 0.655 ± 0.018 mg L−1 d−1, which was in close agreement with the
predicted value. This indicated the precision of ANN–PSO approach in
predicting the optimal nutrient concentrations for improved lutein pro-
ductivity. Moreover, the application of ANN–PSO technique significantly
enhanced the lutein productivity by around 3-fold and the cellular lu-
tein yield (YP/X) by 2-fold in comparison with the unoptimized medium
(BBM) (Table 4).
Table 4
Stage wise development of the optimized medium for enhanced lutein productivity in C. minutissima performed in shake flasks.
Media Lutein concentration
(mg L−1)
BBM 1.71 ± 0.04
Medium as per Plackett–Burman analysis 4.84 ± 0.17
Medium as per maximum response from CCD 8.12 ± 0.25
Medium optimized by using ANN–PSO 9.83 ± 0.27
Data are the average values of three experiments ± S.D.
29
3.3. Role of critical nutrient components on lutein production
In the current study, it was observed that the medium components
such as nitrate, phosphate, manganese and copper ions critically influ-
enced the accumulation of lutein in C. minutissima. Nitrate is one of
the most important limiting nutrients for cellular growth and lutein
synthesis and its requirement for maximum lutein productivity is re-
markably increased from 250 mg L−1 to 1152 mg L−1. This value was
found to be moderately in agreement with Sánchez et al. [16], who re-
ported that nitrate concentration of 1020 mg L−1 (12 mM) in the medi-
um was optimal for lutein synthesis in Scenedesmus sp. However, the
nitrate requirement for lutein production in another green microalgae
Muriellopsis sp. was found to be 1700 mg L−1 [22]. Generally, the lutein
content increases with nitrate concentration up to a critical level and
then it attains saturation or slightly decreases at higher nitrate
concentration. A similar trend was also observed in our study. It has to
be noted that a very high nitrate concentration would decrease the
productivity of both biomass and lutein [11]. Therefore, in the present
study, the optimal nitrate concentration predicted by PSO algorithm
is justified. The PSO-predicted optimum phosphate concentration
(KH2PO4) for enhanced lutein synthesis was drastically decreased
from 175 mg L−1 to 34 mg L−1. However, the concentration of phos-
phate for lutein production was found to be moderately higher in
some studies [1]. From our study, it is clear that the lower concentra-
tion of phosphate was found to be effective for lutein synthesis in
C. minutissima. The requirement of total phosphorous (P) for en-
hanced lutein productivity was observed to be 21.07 mg L− 1 (i.e.
34 mg L− 1 KH2PO4 and 75 mg L− 1 K2HPO4), which is corroborated
with the phosphorous concentration of 19.20 mg L − 1 reported by
Sánchez et al. [16].
The metal ions such as manganese and copper were found to be crit-
ically responsible for stimulating carotenogenesis in C. minutissima. The
PSO-predicted optimum concentrations of MnCl2·4H2O and CuSO4·5H2O
were found to be 4.57 mg L−1 (i.e. 1.27 mg L−1 Mn2+) and 2.35 mg L−1
(i.e. 0.60 mg L−1 Cu2+), respectively. The relatively high requirement of
Mn2+ ion might be attributed to its inevitable role (as a cofactor for
phytoene synthase which converts IPP to phytoene) in synthesizing ca-
rotenoids, particularly lutein in the test organism C. minutissima. Mei
et al. [19] demonstrated that the addition of 0.55 mg L−1 Mn2+ in the cul-
ture medium resulted in nearly two-fold increase of total carotenoid and
chlorophyll-a content in Pavlova viridis. In fact, it is apparent from our cur-
rent study that the carotenoid lutein (in particular) has been enhanced
significantly in response to Mn2+ ions. Probably, this is the first report
that elucidates the critical role of manganese ions on lutein production
and its subsequent optimization. Copper ions are an essential component
of an enzyme cytochrome oxidase that is involved in photosynthetic elec-
tron transport chain [24]. This indicates its significance in the photosyn-
thesis process and in turn microalgal biomass production. However,
some reports indicated that the copper ions even at a low concentration
(0.003–0.015 mM Cu2+) exert toxic effect to few microalgal species [25,
26]. It has to be noted that the appropriate concentration of copper ions
in the medium would improve the intracellular carotenoid accumulation
without affecting the cell growth [24]. In our study, the optimum Cu2+
ion concentration for increased lutein productivity without affecting the
Lutein productivity Biomass productivity Lutein yield Fold increase of lutein
(mg L−1 d−1) (g L−1 d−1) (mg g−1)
Concentration Productivity
0.228 ± 0.007 0.085 ± 0.002 2.67 – –
0.403 ± 0.013 0.094 ± 0.004 4.28 2.8 1.8
0.541 ± 0.015 0.108 ± 0.003 5.01 4.7 2.4
0.655 ± 0.018 0.117 ± 0.005 5.58 5.7 2.9
30 R. Dineshkumar et al. / Algal Research 7 (2015) 24–32

Fig. 5. Time-course profiles of biomass, lutein concentration and sodium nitrate utilization of Chlorella minutissima grown in a 2-L airlift photobioreactor using (a) BBM and (b) optimized
BBM (operating conditions: light intensity, 150 μmol m−2 s−1; CO2, 2.5%; flow rate, 0.45 vvm).

microalgal growth rate was found to be 0.009 mM (i.e. 2.35 mg L−1 of
CuSO4·5H2O). This observation is evident from the study of Vaquero
et al. [24], which reported that the lutein productivity of Coccomyxa
onubensis was increased by 20% while supplementing the medium with
Cu2+ from 0.06 mM to 0.2 mM. In another study, the carotenoid content
of C. vulgaris was found to be increased by around 2.5-fold, while increas-
ing Cu2+ ion concentration from 0.25 to 3 mg L−1 [27]. These studies sig-
nify the role of copper ions in biomass growth and lutein synthesis.
Thus, the application of Plackett–Burman design followed by ANN–
PSO technique increased the lutein productivity substantially from
0.228 ± 0.007 mg L−1 d−1 to 0.655 ± 0.018 mg L−1 d−1 and the cellular
lutein content from 2.67 mg g− 1 to 5.58 mg g− 1 in C. minutissima
(Table 4). It is important to note that there is no literature data available
on lutein production by C. minutissima for comparison purpose. However,
the optimized intracellular lutein yield and concentration obtained in the
present shake flask studies were found to be comparable with those
obtained in small scale photobioreactor studies using S. almeriensis and
Desmodesmus sp., respectively [11,16]. But the lutein productivities
could not be compared due to disparity in scale of operation. Therefore,
it would be justified if the comparison was drawn between the relevant
values obtained in reactor studies, wherein appropriately higher light in-
tensity and CO2 were provided.
3.4. Comparison of lutein and biomass production profiles of C. minutissima
cultivated using BBM and optimized BBM in a photobioreactor
The microalga C. minutissima was cultivated in a 2-L airlift
photobioreactor with the optimized production medium and appro-
priate process conditions as follows: 150 μmol m− 2 s− 1 light inten-
sity; 2.5% CO2 and 0.45 vvm flow rate. Fig. 5(a) and (b) shows the
time-course profiles of lutein, biomass concentration and nitrate uti-
lization of C. minutissima cultivated using both the unoptimized and
 R. Dineshkumar et al. / Algal Research 7 (2015) 24–32 31

Table 5
Comparison of lutein concentration, productivity and yield of Chlorella minutissima MCC-27 with those reported in the medium optimization related studies in photoautotrophic modea.

Microalgal strains Medium used Operating conditions Lutein Lutein Lutein yield Biomass Specific References
concentration productivity (mg g−1) productivity growth rate
(mg L−1) (mg L−1 d−1) (g L−1 d−1) (d−1)

Desmodesmus sp. F51 Modified Bristol medium Batch, 1-L; 150 μmol m−2 s−1; – 2.31 4.69 0.494 1.99 [11]
2.5% CO2
Desmodesmus sp. F51 Modified Bristol medium Batch, 1-L; 600 μmol m−2 s−1; 7.6 3.05 3.97 0.767 2.50 [11]
2.5% CO2
Scenedesmus sp. Modified Basal medium Batch, 0.5-L; 160 μmol m−2 s−1; 5.6 0.51 2.67 0.190 0.66 [28]
10% CO2
Coccomyxaacidophila Urea supplemented basal Batch, 2-L; 150 μmol m−2 s−1; – 2.0 6.1 0.25 0.34 [29]
medium 5% CO2
Chlorella sorokiniana Modified Arnon medium Batch, 1-L; 690 μmol m−2 s−1; 25 2.5 3.0 0.84 2.64 [30]
(wild type) 1% CO2
Coccomyxa onubensis K9 medium 0.2 mM Cu2+ culture in – 2.12 6.20 0.42 0.51 [24]
semi-continuous mode
Chlorella minutissima Optimized BBM Batch, 2-L; 150 μmol m−2 s−1; 17.28 ± 0.50 3.45 ± 0.07 6.05 ± 0.12 0.57 ± 0.011 1.92 ± 0.02 This study
MCC-27 2.5% CO2
a
Studies related to heterotrophic cultivation and process optimization are not considered.

optimized BBM, respectively. It was found that the maximum lutein con-
centration of 17.28 ± 0.5 mg L−1 was obtained when almost 95% of ni-
trate was consumed by C. minutissima. This finding is in agreement with
the study of Ho et al. [9]. It was also observed that the increased light
intensity and CO2 supply improved both the microalgal growth rate and
lutein productivity significantly in photobioreactors (Fig. 5a and b), irre-
spective of the medium, when compared with those in shake flask
studies. However, the maximum lutein productivity in the optimized
BBM was 3.45 ± 0.07 mg L−1 d−1, which was nearly three-fold higher
than that obtained in BBM (1.20 ± 0.02 mg L−1 d−1) under the same
conditions. The biomass productivity was consequently increased from
0.37 ± 0.009 g L−1 d−1 to 0.57 ± 0.011 g L−1 d−1. These results demon-
strate the usefulness of an efficient optimization strategy. A much clear
picture emerges by referring to Table 5 that provides a comparative esti-
mate of the lutein concentration, productivity and yield as obtained from
the reported medium optimization related studies vis-à-vis the present
study. While the optimized lutein yield and concentration values were
comparable with those of the reported values, the lutein productivity
was found to be much better than the relevant values reported in litera-
ture, irrespective of the microalgal strain or medium (Table 5). Therefore,
the most interesting finding is that the application of the advanced hybrid
optimization technique of ANN–PSO resulted in significant enhancement
in product concentration, yield and productivity.
4. Conclusion
This study stands out in its approach towards (1) identifying the
most critical components of Bold's Basal Medium for biosynthesis of lu-
tein, a pigment with versatile application potential, in C. minutissima by
employing Plackett–Burman analysis, and (2) optimizing the critical
nutrients, nitrate; phosphate; manganese and copper for the enhanced
lutein productivity by applying ANN–PSO, an advanced hybrid optimi-
zation strategy. While phosphate and nitrate are essential for growth,
nitrate enhances lutein pathway flux, manganese is a co-factor for the
pathway enzyme, phytoene synthase and copper induces lutein synthe-
sis via oxidative stress. The present study with the application of hybrid
optimization strategy is much superior to all other studies on medium
optimization for microalgal lutein production reported in literature.
This work adds value and contributes significantly to the scientific
knowledge on lutein production.
Acknowledgments
RD acknowledges the Department of Science & Technology (DST) —
INSPIRE, Government of India for his fellowship. GD acknowledges IIT
Kharagpur for his fellowship. The authors gratefully acknowledge
West Bengal Government — Department of Science & Technology
(project grant no. 560 (SANC.)/ST/P/S&T/SG-5/2011; date: 21-11-11) for
the financial support. RD is very much grateful to Mr. Vivek Rangarajan
for his valuable technical inputs on the design of the experiments.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.algal.2014.11.010.
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