Bioresource Technology 200 (2016) 940950

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Multi-objective optimization of media nutrients for enhanced

production of algae biomass and fatty acid biosynthesis from Chlorella
pyrenoidosa NCIM 2738
Kamaraj Kanaga, Ashutosh Pandey, Sanjay Kumar , Geetanjali
Department of Biotechnology, Motilal Nehru National Institute of Technology Allahabad, Allahabad 211004, India

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

Medium was optimized by multi-

objective (MO) optimization using C.

pyrenoidosa.

Increase in lipid productivity by MO
optimization against control was
found 2.90-fold.

MO optimization was showed 23.6%
improvement than single-response
optimization.

a r t i c l e

i n f o

Article history:
Received 5 September 2015
Received in revised form 8 November 2015
Accepted 9 November 2015
Available online 14 November 2015
Keywords:
Microalgae
Lipid
Multi-objective optimization
C. pyrenoidosa
Biofuel

a b s t r a c t
This study aimed to optimize significant medium nutrient parameters for maximization of algal lipid and
biomass production by using multi objective optimization strategy. Nutrients (nitrate, phosphate and
carbohydrate) were investigated to improve the lipid accumulation, biomass production and carbohydrate consumption individually and cumulative manner using a central composite design for the
Chlorella pyrenoidosa NCIM 2738 cultivation. Maximum lipid, algal biomass and carbohydrate utilization
for individual response optimization were found 34.8% (w/w), 1464.3 mg L1 and 93.4%, respectively at
different optimum level of selected parameters. Whereas, maximum lipid accumulation, biomass production and glucose consumption values in multi-response optimization were observed 28.9%,
1271.2 mg L1 and 89.2%, respectively at optimum level of 16.8 mM NaNO3, 300.9 lM K2HPO4 and
2.6% (w/v) glucose. The overall enhancements in lipid productivities by single and multi-response optimization in comparison with control medium conditions were found 2.35 and 2.90-fold with productivity
level of 24.8 and 30.6 mg L1 day1, respectively.
2015 Elsevier Ltd. All rights reserved.

1. Introduction
Rising world population means growing demand for the daily
needs of human life. Apart from food another most promising need
of humans is energy. Fossil fuels are the nonrenewable energy
resources, whose overconsumption from last few decades has

Corresponding author.
http://dx.doi.org/10.1016/j.biortech.2015.11.017
0960-8524/ 2015 Elsevier Ltd. All rights reserved.

caused many serious threats to the society and environment. One

of the major environmental concerns is global warming caused
by increasing concentration of greenhouse gases (such as carbon
dioxide) in biosphere from the burning of fossil fuels that accounts
for 76.7% (v/v) (Ho et al., 2014). According to British Petroleum (BP)
Statistical Review of World Energy 2010, crude oil and natural gas
may be consumed by 45.6 and 62.8 years, respectively. The need to
develop sustainable and renewable sources of energy is driving
industry and research institutions to find a replacement for

K. Kanaga et al. / Bioresource Technology 200 (2016) 940950

fossil-derived diesel as a significant source of mobile power for

transportation (Fan et al., 2014). Biofuel derived from biomass such
as oil crops and microalgae are sustainable, renewable and ecofriendly transportation energy substitute in near future. It has also
reported that exhaust of biodiesel during combustion has lesser
carbon monoxide, particulate matter, hydrocarbon and sulfur dioxide as compared to that of petro-diesel (Nautiyal et al., 2014).
Second generation feedstocks contains non-edible seed crops
such as jatropa, jojoba, mahua and waste cooking oil, karanja
which are not sufficient for present transportation needs (Nigam
et al., 2011). The biodiesel derived from microalgae is considered
to be the third generation biofuel. Microalgae which is 75% of total
algae, contribute to 40% of the atmospheric oxygen (Ponnuswamy
et al., 2013). Microalgae are sunlight driven energy sources that
translate sunlight, water and CO2 to triacylglycerols (TAGs). Photosynthetic CO2 bio-fixation by microalgae is the most effective and
environmental friendly carbon sequestration method on earth. In
large scale microalgal cultivation, the CO2 capture efficiency can
be as high as 99%, efficiently capturing 1.8 kg of CO2 per kg of algal
biomass (Lim et al., 2012). Microalgae have the ability to uptake
inorganic salts (NO3, NH+4, PO3
4 ) as nutrient materials from waste
streams (including, flue gases and wastewaters) and produce biomass (feedstocks for biodiesel) 50 times higher than land plants
(Prajapati et al., 2014). One of the main obstacle in utilizing
microalgae as the feedstock for biodiesel is the -production cost
mainly due to our lack of knowledge for microalgae metabolism
and efficient process. Estimated current production cost of
microalgae oil ranged from 2.4 to 10.6 US $ per liter which need
to be reduced by 10 times in order to be competitive with fossil
fuel oil (Lim et al., 2012).
Chlorophyceae is the most widely utilized microalgae in
biotechnological applications including biofuel production. Chlorella sp. show more suitability for biofuel process development
due to a high fixation capacity for CO2 from flue gas generated by
combustion processes and has high pollution tolerance
(Bhatnagar et al., 2010). The genus Chlorella contains several species
that produce different amounts of lipids, e.g., Chlorella pyrenoidosa
211.9%, Chlorella ellipsoidea 449%, Chlorella protothecoides 8
55% which are considered to have great potentials for commercial
production of biodiesel (Ngangkham et al., 2012). C. pyrenoidosa is
unicellular green alga grows in freshwater which has been demonstrated for phycoremediation of industrial wastewater (Zhang et al.,
2014). However, till date there is little information available for
enhancement of lipid productivities from C. pyrenoidosa.
Process optimization efficiently enhances the yield of a desired
metabolic product in a microbial system. Response Surface
Methodology (RSM) is a mathematical approach that can be used
for process parameter optimization (Abdelaziz et al., 2014). Various individual response optimization analysis approaches were
used to maximize the production of lipid accumulation, substrate
uptake, chlorophyll level or algal biomass production for microalgae cultivation (Abdelaziz et al., 2014). However, a few studies
have been performed using multi-objective optimization to improvise the productivity in microalgae production. Multiple response
optimizations using desirability function has many advantages as
it improves economy and efficiency. To find out the optimal condition for a single response is relatively less informative than multiobjective optimization using response surface designs (Bezerra
et al., 2008).
Nutrient starvation is one of the most important lipid induction
techniques used in microalgae cultivation. In many studies, it was
found that nitrogen starvation enhanced the lipid content as well
as lipid productivity (Anand and Arumugam, 2015). Nitrogen and
phosphorous deficiency along with availability of carbon source
change cellular carbon flux from protein to fatty acid synthesis
leads to enhanced lipid content in algae (Mutlu et al., 2011).

941

Phosphorus limitations improved the cellular total lipid content

mainly due to TAG accumulation during microalgae growth. Thus,
in order to achieve high growth rate as well as high lipid content, it
is necessary to optimize all these factors.
Therefore, this study is focused on significant medium parameters (nitrate, phosphate and carbohydrate source) optimization for
obtaining higher lipid, biomass production and carbohydrate consumption of C. pyrenoidosa NCIM 2738 employing multi-objective
optimization by desirability function approach using a central
composite experimental design. Assessment of individual and
multi-objective optimization analysis was also performed to compare lipid and biomass productivity.
2. Methods
2.1. Organism, growth medium and culture conditions
The culture of green unicellular algae C. pyrenoidosa NCIM 2738
was procured from National Centre for Industrial Microorganisms
(NCIM), Pune, India. C. pyrenoidosa was grown heterotrophically
using 2% inoculum (6  105 cells mL1) into 500 mL Erlenmeyer
flasks containing 200 mL BG11 medium (Cheirsilp and Torpee,
2012) {in g L1: NaNO3, 1.5 [17.6 mM]; K2HPO4, 0.04 [229.6 lM];
MgSO47H2O, 0.075; CaCl22H2O, 0.036; citric acid, 0.006; ferric
ammonium citrate, 0.006; EDTA, 0.001; Na2CO3, 0.02; 1 mL trace
elements solution [in g L1: H3BO3, 2.86; MnCl24H2O, 1.81; ZnSO4
7H2O, 0.222; NaMoO42H2O 0.39; CuSO45H2O, 0.079; Co(NO3)2
6H2O, 0.0494]; initial pH 7.0 1}. Temperature at 25 2 C under
illumination of white fluorescent lamps with a photoperiod of
16:0 h light and 8:0 h dark at light intensity of 54 lmol m2 s1
(540 Testo Ltd, Alton) was used for growth at 100 rpm.
2.2. Analytical methods
2.2.1. Cell growth measurement
The cell concentration was measured by turbidity change in the
culture medium using a UVVIS spectrophotometer (Cary 60 Agilent Technologies, CL) at 600 nm twice a day till completion of
experiment.
Dry cell weight (dcw) was calculated using a 5 mL volume of
algal culture by centrifugation at 6500g for 10 min. The algae cell
pellet was washed twice with distilled water, and centrifuged biomass was oven dried at 70 C until it reached constant weight.
2.2.2. Total lipid estimation
Total algal lipid from the samples was extracted using
methanol-chloroform (2:1) by modified Bligh and Dyer extraction
method (Ryckebosch et al., 2012). Algal culture (200 mL) was centrifuged at 6500g for 10 min. The algal biomass was suspended
after discarding the supernatant and washed with distilled water
and, centrifuged again. Then pellet was oven dried at 70 C for
overnight and weight of the biomass was recorded after total drying. Algal biomass (g), methanol (mL) and chloroform (mL) was
mixed in ratio of 1:2:1 and incubated for 18 h at 25 1 C. Then,
mixture was vortexed for 2 min, followed by the addition of 1 mL
of chloroform and vortex for 1 min. Distilled water (1 mL) was
added in the mixture and vigorously vortexed the whole content
for 2 min. Separation in different layers were done by centrifugation for 10 min at 3000g. The lower lipid layer containing chloroform was separated carefully in a clean and pre-weighed
centrifuge tube (W1). The chloroform phase was evaporated in a
water bath at 70 C for 20 min. The weight of the centrifuge tube
was again determined (W2). Lipid content was calculated by
subtracting W1 from W2, and divided by of dried algal biomass
and multiplying by 100 and finally expressed as % dcw.

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K. Kanaga et al. / Bioresource Technology 200 (2016) 940950

2.2.3. Carbohydrate estimation

Algae sample (1 mL) was centrifuged at 5000g for 10 min and
supernatant was used for total carbohydrate estimation. Sample
was diluted (10100 times) and analyzed for reducing sugar by
DNS standard method as described by Miller (1959). The absorbance of tested sample was analyzed at 540 nm using UVVIS
spectrophotometer.
2.2.4. Effect of light intensities on lipid and biomass production
Light intensities effect was investigated for heterotrophic cultivation of microalgae. Effect of this parameter was studied on
growth and lipid production of C. pyrenoidosa NCIM 2738 at three
different light intensities (25, 40, 54 lmol m2 s1) along with control kept at low light intensity (1 lmol m2 s1).
2.3. Fatty acid analysis
The fatty acids component of the oil from C. pyrenoidosa NCIM
2738 was analyzed by GC-linked mass spectrometry (GCMS)
using DB-5MS (5% phenyl)-methylpolysiloxane nonpolar column
(30 m  0.25 mm  0.25 lm) (Li et al., 2015). Acidic transesterification of the lipids was performed to obtain fatty acid methyl
esters as described by Miao and Wu (2006).
2.4. Cell count
Cell concentration of C. pyrenoidosa NCIM 2738 was determined
by counting duplicate samples in Naebaeur haemocytometer.
2.5. Experimental design and process variable optimization using RSM
2.5.1. Central composite design
A 2k factorial central-composite model was used as the experimental design model to investigate the key process parameters.
The design was created by statistical software Minitab 17.1, PA,
USA. The total numbers of experiments are calculated using the
Eq. (1):

N 2k 2k 6

where, N = total number of experiments, k = number of parameters

and 6 is the number of replicates at the center points to calculate
the pure error. The common form of second order quadratic model
is presented in the Eq. (2):

Y b0

n
n
n X
n
X
X
X
bi X i
bii X 2i
bij X i X j
i1

i1

i1 j1

where, Y = response, n = number of parameters, b0 = offset

term, bi = linear term coefficient, bii = quadratic term coefficient,
bij = interactive term coefficient, X = uncoded level of independent
parameters in the model.
Lipid content, carbohydrate consumption and biomass produced were studied under varied NaNO3 (1.029.0 mM), K2HPO4
(10.0450 lM) and glucose (0.54.5%) conditions with the aim to
establish the potential of the organism under heterotrophic conditions. The experimental design was applied after selecting a range
of each variable (maximum and minimum), as shown in Table 1.
2.5.2. Multi-objective optimization analysis
In this study, desirability function approach was used to analyses multi-objective optimization for maximization of cumulative
lipid, carbohydrate consumption and biomass production using
Minitab 17.1, PA, USA. The value of each response (lipid, biomass
production and carbohydrate consumption) for a given combination of controllable variables was translated to a number between
zero and one known as individual desirability. If the maximum

Table 1
Various factors and their levels in the central-composite design.
Factor

Name

Units

A
B
C

NaNO3
K2HPO4
Glucose

mM
lM
%

Range of levels
2

1

+1

+2

1
10
0.5

8
130
1.5

15
250
2.5

22
370
3.5

29
490
4.5

value of objective type was obtained, the desirability function

would be defined as mentioned in Eq. (3):

8
>
<
>
:

0 if Y < 1
s

YL
if L 6 Y 6 T
TL

1 if Y > T

where, d = desirability function, L = lower acceptable value to the

response, T = target value, Y = response and s = weight of the
response. Thus, when s = 1 (d is linear), s > 1 (high importance specified near the target value), s < 1 (low importance specified near the
target value).
2.6. Model validation
Confirmatory experiment were performed at optimum levels of
parameters for maximization of lipid, biomass and carbohydrate
consumption individually as well as cumulative manner to validate
the developed models and kept other medium components level as
mentioned in BG11 medium composition. The medium was inoculated with 2% inoculum (6  105 cells mL1) and maintained at
temperature of 25 2 C under illumination of white fluorescent
lamps with a photoperiod of 16:0 h light and 8:0 h dark at light
intensity of 54 lmol m2 s1 for growth at 100 rpm. The samples
were withdrawn at regular interval for biochemical parameters
estimation such as biomass, lipid and carbohydrate consumption.
All model validation experiments and its analysis were conducted
in duplicates and average of the result was reported.
3. Results and discussion
3.1. Growth characteristics of C. pyrenoidosa NCIM 2738
Microscopic observation of the microalgae C. pyrenoidosa NCIM
2738 under bright field with oil immersion at 100  magnification
showed unicellular and spherical shaped cells. Previous studies
reported that glucose used as most favorable monosaccharide carbon source for the growth of C. pyrenoidosa, subsequently galactose
and fructose (Zhang et al., 2014). Considering this, glucose was
used throughout the experiments as carbon source for the growth
of C. pyrenoidosa NCIM 2738. Addition of carbon source (glucose)
comparatively increased the growth of C. pyrenoidosa NCIM 2738
under photoheterotrophic cultivation than photoautotrophic condition (data not shown). The growth curves of C. pyrenoidosa NCIM
2738 in BG11 medium with 1% glucose concentration showed that
the late exponential growth phase was observed after 11 days of
cultivation. Therefore, 12 days old grown C. pyrenoidosa NCIM
2738 cultures were used further for different biochemical analysis
(lipid, biomass production and carbohydrate consumption) in CCD
experiments.
3.2. Optimization of medium components to maximize the lipid,
biomass production and carbohydrate assimilation
The objective of the CCD approach was to determine the best
levels of the selected significant parameters, viz., NaNO3 (1.0
29.0 mM), K2HPO4 (10.0490.0 lM) and glucose (0.54.5%) for

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K. Kanaga et al. / Bioresource Technology 200 (2016) 940950

measured experimental responses (lipid, biomass production and

carbohydrate consumption) using C. pyrenoidosa NCIM 2738.
Experiments of 20 sets were performed and the statistical treatments of tested parameters (medium components) along with
the various experimental responses corresponding to each combination are summarized in Table 2. The lipid concentrations were
varied from 9.84 to 34.29 (% dcw) whereas, biomass were varied
from 479.9 to 1358.3 mg L1 in various experimental runs. However, the data of cell biomass and lipid production inferred that
the production of lipids from C. pyrenoidosa NCIM 2738 was nongrowth associated. A large variation in carbohydrate utilization
was also observed during CCD experiments from 65.8 to 92.8%.
3.2.1. Medium components optimization to maximize the individual
response
Multiple regression analysis on the CCD experimental data were
performed to determine the relationship between tested parameters (NaNO3, K2HPO4 and glucose) with measured responses (lipid,
biomass production and carbohydrate assimilation) (Table 3). The
regression equations coefficients were calculated and data were
fitted to a second-order polynomial equations for respective
response. The final equation for the lipid, biomass and carbohydrate consumption in terms of uncoded parameters value are presented below in Eqs. ((4)(6)):

Y Lipid % dcw 6:21 1:04A 0:19B 5:02C  0:05A2

 0:01B2  0:85C 2  0:01AB 0:07AC
 0:01BC

Y Biomass mg L1 217:4 70:53A 2:74B 182:71C

 1:97A2  0:01B2  34:45C 2 0:01AB
4:88AC  0:03BC

Y Glucose mg L1 43:34 2:89A 0:07B 6:90C  0:06A2

 0:01B2  0:87C 2 0:01AB  0:16AC
0:01BC

where A is NaNO3, B is K2HPO4 and C is glucose.

The model equation coefficients, t values and consequent P values were presented in Table 3. All linear and quadratic term of variables (medium components) for different responses (lipid, biomass
production and glucose assimilation) from regression analysis
were indicated their high significance on the basis of their P value
(P < 0.05). The interaction effects of K2HPO4 with NaNO3 and
K2HPO4 with glucose were highly significant for model of lipid production. The interaction of NaNO3 with glucose was observed statistical significant for biomass production model. Whereas,
interaction effects of glucose with K2HPO4 and glucose with NaNO3
were found vital for glucose utilization model. Thus, interaction of
K2HPO4 with glucose were significant to maximize the lipid production as well as glucose consumption and interaction of glucose
with NaNO3 were important to improve the biomass production as
well as glucose consumption using C. pyrenoidosa NCIM 2738.
The results of CCD experiment that was carried out to determine the best levels of the selected significant parameters for measured experimental responses were further analyzed using the
analysis of variance (ANOVA) (Table 4). According to the ANOVA
analysis, developed quadratic regression models for lipid, biomass
production and glucose assimilation were highly significant, as it
was apparent from the F value (161.0, 78.6 and 143.4 respectively)
with a lower P value 60.003 for all three models. This analysis confirmed that the collective effects of all parameters for developed
models contribute to maximize the lipid, biomass production and
glucose assimilation. The goodness of fit for the individual models
were evaluated by R2 value (>96.07%), which indicates that these
three models for measured responses (lipid, biomass production
and glucose assimilation) were attributed to the tested parameters.
The predicted and adjusted R2 value were observed in the range of
92.5697.14% that reasonable agreement, signifies the enhance fitness of model to the experimental data.
The normal plots of residuals were plotted between studentized
residuals with predicted probability (%) and which specify that
uniform variance hypothesis. The diagnostic details provided from
the analysis of all data set points of the normal probability and studentized residuals were approximately linear. This also indicates
excellent prediction of the response along with parameters value
and competence of the developed quadratic models.
The three-dimensional (3-D) surface diagram were plotted to
visualize the optimum values for all three responses (lipid, biomass

Table 2
Central composite design in real and coded values with various experimental responses for lipid, biomass production and carbohydrate utilization.

*
a

Run

NaNO3 (mM)
A

K2HPO4 (lM)
B

Glucose (%)
C

Lipida (% dcw)

Biomassa (mg L1)

Carbohydrate consumeda (%)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15*
16*
17*
18*
19*
20*

8 (1)
22 (+1)
8 (1)
22 (+1)
8 (1)
22 (+1)
8 (1)
22 (+1)
1 (2)
29 (+2)
15 (0)
15 (0)
15 (0)
15 (0)
15 (0)
15 (0)
15 (0)
15 (0)
15 (0)
15 (0)

130 (1)
130 (1)
370 (+1)
370 (+1)
130 (1)
130 (1)
370 (+1)
370 (+1)
250 (0)
250 (0)
10 (2)
490 (+2)
250 (0)
250 (0)
250 (0)
250 (0)
250 (0)
250 (0)
250 (0)
250 (0)

1.5
1.5
1.5
1.5
3.5
3.5
3.5
3.5
2.5
2.5
2.5
2.5
0.5
4.5
2.5
2.5
2.5
2.5
2.5
2.5

24.39 0.03
15.68 0.35
34.29 0.20
21.98 0.25
23.18 0.46
18.10 0.56
30.70 0.65
18.78 0.17
27.96 0.39
12.73 0.34
09.84 0.35
22.00 0.30
25.89 0.35
26.87 0.71
29.24 0.12
30.47 0.39
30.16 0.60
29.81 0.28
29.41 0.35
30.12 0.63

770.5 27.9
1050.0 28.3
793.0 12.0
1169.5 7.4
838.2 24.8
1321.0 7.1
908.8 11.5
1358.3 22.8
479.9 22.7
1135.7 25.8
816.0 3.5
1001.0 15.6
915.8 31.5
1198.0 39.6
1179.0 8.5
1184.3 18.2
1206.0 5.7
1220.0 21.2
1223.5 25.8
1151.5 3.9

75.2 0.5
87.0 0.6
79.5 0.2
92.8 0.5
76.7 0.2
84.2 0.6
78.7 0.2
87.5 0.5
65.8 0.8
83.8 0.9
78.7 0.2
85.7 0.4
86.1 0.1
81.4 0.7
87.7 0.2
87.2 0.7
87.6 0.8
87.5 0.4
87.2 0.8
87.7 0.8

(1)
(1)
(1)
(1)
(+1)
(+1)
(+1)
(+1)
(0)
(0)
(0)
(0)
(2)
(+2)
(0)
(0)
(0)
(0)
(0)
(0)

Experiments performed at the central value.

Observed values of lipid, biomass, carbohydrate were the mean values of duplicates with standard deviation (mean SD).

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K. Kanaga et al. / Bioresource Technology 200 (2016) 940950

Table 3
Model coefficient estimate by multiple regression analysis for lipid, biomass production and carbohydrate consumption.
Term

Lipid production

Biomass production

Carbohydrate consumption

Coeff

SE

t value

P value

Coeff

SE

t value

P value

Coeff

SE

t value

P value

Constant
A
B
C
A2
B2
C2
AB
AC
BC

6.21
1.04
0.19
5.02
0.05
0.01
0.85
0.01
0.07
0.01

2.81
0.16
0.01
1.12
0.01
0.01
0.15
0.01
0.04
0.01

2.21
6.59
20.64
4.48
15.55
22.85
5.59
4.86
1.87
3.73

0.035
0.012
<0.001
0.024
0.005
0.002
0.001
0.005
0.072
0.001

217.40
70.53
2.74
182.71
1.97
0.01
34.45
0.01
4.88
0.03

135.67
7.64
0.45
54.18
0.15
0.01
7.33
0.02
1.86
0.11

1.60
9.23
6.17
3.37
13.20
9.76
4.70
0.62
2.63
0.30

0.120
<0.001
0.004
0.002
<0.001
0.002
<0.001
0.538
0.014
0.767

43.34
2.89
0.07
6.90
0.06
0.01
0.87
0.00
0.16
0.01

2.75
0.16
0.01
1.10
0.01
0.01
0.15
0.01
0.04
0.01

15.78
18.70
7.25
6.29
21.01
8.51
5.87
1.33
4.21
2.25

0.001
0.002
0.012
0.002
<0.001
<0.001
0.004
0.195
0.002
0.032

A nitrate source (NaNO3), B phosphate source (K2HPO4), C glucose; Coeff coefficient; SE Standard Error; lipid production model: R2 = 98.04%; biomass production:
R2 = 96.07%; carbohydrate consumption: R2 = 97.80%.

Table 4
Analysis of variance for various responses (lipid, biomass production and carbohydrate consumption).
Source

df

Model
Linear
Square
Interaction
Residual error
Lack-of-fit
Pure error
Total

9
3
3
3
29
19
10
39

Lipid production

Biomass production

Carbohydrate consumption

SS

MS

F value

P value

SS

MS

F value

P value

SS

MS

F value

P value

1673.9
884.1
742.5
47.4
33.5
26.8
6.7
1707.5

186.0
169.9
247.5
15.8

161.0
147.0
214.2
13.7

0.003
0.012
0.003
0.025

0.003
<0.001
0.002
0.082

1.08
0.01

0.470

158.9
130.4
169.3
9.1
1.1
1.2
1.0

<0.001
0.004
<0.001
0.031

0.113

1429.7
894.5
508.0
27.2
32.1
22.3
9.8
1461.8

143.4
117.7
152.9
8.2

2.11

212,383
87,898
202,631
6657
2701
2770
2570

78.63
32.54
75.02
2.46

1.41
0.67

1,911,450
1,283,587
607,892
19,971
78,327
52,623
25,704
1,991,127

1.2

0.398

df = degree of freedom; SS = sum of squares; MS = mean square.

production and glucose assimilation) against the tested medium

components and simultaneously examined the statistical significance of interactions between the variables. The 3-D surface diagrams were plotted by keeping the response on the Z-axis
against with any two independent parameters, while maintaining
third parameters at its optimum level. The 3-D surface plot showed
that lipid (% dcw) accumulation by C. pyrenoidosa NCIM 2738 was
influenced by all three parameters (Fig. 1). The accumulation of
lipid (% dcw) was greatly influenced by interaction of K2HPO4 with
NaNO3 and glucose was found to be highly statistically significant
(P value = 0.005 & 0.001) (Fig. 1(A) and (C)). Whereas, the interactive effect between NaNO3 and glucose for lipid accumulation displayed less statistical significant (P value = 0.072) (Fig. 1(B)). Three
dimensional surface plots were showed prominent interactive
effect of NaNO3 and glucose for biomass production
(P value = 0.014) (Fig. 2(B)). While, very less influence of phosphate
source with other variables on biomass production of C. pyrenoidosa NCIM 2738 (P value = 0.538 and 0.767) was observed (Fig. 2
(A) and (C) and Table 3). Interactive effects of all variables were
also evaluated by using the perturbation graphs (Supplementary
Fig. S1) to assessment of lipid production ability of C. pyrenoidosa
NCIM 2738. Curve A represented that the decreasing effect on lipid
accumulation with the addition of NaNO3 whereas curve B showed
the increasing effect on lipid accumulation with increasing concentration of K2HPO4 in the media. Flat curves for glucose (C) indicate
that this variable have little effect as compared to other variables
on lipid accumulation by C. pyrenoidosa NCIM 2738.
The response surface plots for carbohydrate utilization were
given as Supplementary Fig. S2. Carbohydrate utilization was
mostly influenced by the concentration of glucose in the media.
There was a steep decrease in the carbohydrate utilization on
increase in glucose concentration and a maximum value of utilization was achieved when glucose was on lower concentration
(0.70 g L1). The interactive effect between glucose with NaNO3
and K2HPO4 were prominent and P value was observed <0.05.

Regression equations (Eqs. (4)(6)) were maximized by an iterative technique to acquire optimal levels of concentrations of
parameters for three individual measured responses. Uncoded concentration levels of parameters were used into the corresponding
regression equation to predict the maximum output for all three
responses (glucose assimilation, biomass and lipid production)
(Fig. 3((A)(C))). Optimize level of parameters were observed viz.,
23.06 mM NaNO3, 407.6 lM K2HPO4 and 0.7% (w/w) glucose to
maximize the glucose utilization by C. pyrenoidosa NCIM 2738
(Fig. 3(A)). The projected optimal level of NaNO3, K2HPO4 and glucose to maximize the biomass concentration identified were
23.63 mM, 286.4 lM and 4.18% (w/w), respectively (Fig. 3(B)). Similarly, optimum levels of variables for maximum lipid accumulation were 6.37 mM NaNO3, 349.4 lM K2HPO4 and 1.51% (w/w)
glucose, respectively (Fig. 3(C)). The predicted maximum individual responses were observed to be 92.59%, 1395.0 mg L1 and
33.9% (w/w) for carbohydrate utilization, algal biomass and lipid
accumulation, correspondingly. The desirability of all predicted
responses was found more than 0.97.
3.2.2. Optimization of medium components for maximization of multiresponse
Determination of optimal conditions for several responses
simultaneously is more useful and relatively difficult than single
response optimization using RSM (Cheirsilp and Torpee, 2012). In
this study, multi-objective optimization was carried out for
observing the optimal conditions for NaNO3, K2HPO4 and glucose
concentration to maximize the significant responses (lipid, biomass production and glucose assimilation) concurrently and compared with optimal variable conditions of single response
optimization. Fig. 4 showed the graphical representation of evaluated optimal conditions for multi-objective optimization by desirability function. The maximum predicted responses values for
multi-response optimization were obtained to be 89.14%,
1262.0 mg L1 and 29.12% (w/w), respectively at optimum level

K. Kanaga et al. / Bioresource Technology 200 (2016) 940950

Fig. 1. 3-D response surface plot for lipid production with varying (A) K2HPO4 (lM)
vs glucose (%), (B) NaNO3 (mM) vs glucose (%) and (C) NaNO3 (mM) vs K2HPO4 (lM);
third variable was kept at central value.

of variables 16.8 mM NaNO3, 300.9 lM K2HPO4 and glucose 2.6%

(w/v). The values obtained from multi-objective optimization were
found entirely dissimilar than single response optimized values.
The predicted value of response in single response optimization
was observed 416% higher than multi-objective optimization
responses.
The contour surfaces of different responses from important
parameters level value can be overlaid and provide the feasible
experimental region to fulfill the condition for maximizing the
responses altogether. Hence, the overlaid contour plot can be
applied to visually elucidate the relationships between the two
control factors and the three response variables, as shown in

945

Fig. 2. 3-D response surface plot for biomass production with varying (A) K2HPO4
(lM) vs glucose (%), (B) NaNO3 (mM) vs glucose (%) and (C) NaNO3 (mM) vs K2HPO4
(lM); third variable was kept at central value.

Fig. 5. Fig. 5((A)(C)) correspond to the overlaid contour diagram

for cumulative responses (lipid, biomass production and glucose
assimilation) from C. pyrenoidosa NCIM 2738 were overlapped
using Eqs. (4)(6) to represent the optimal experimental region
of selected medium components. Although the overlaid contour
plot can roughly determine the optimal region for multiple
response variables, it is limited to two experimental factors. White
region in the overlaid contour diagram indicates the feasible area
for maximum values for responses (lipid, biomass production
and glucose assimilation) under multi-objective optimized
conditions.
Fig. 6 represented the maximum lipid and biomass productivities for the different CCD experimental runs at the applicable point
in the growth phase. The productivities of biomass and lipid were
varied in the range of 40.0113.2 and 6.730.3 mg L1 day1,
respectively. The maximum lipid productivity for individual and

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K. Kanaga et al. / Bioresource Technology 200 (2016) 940950

Fig. 3. The individual maximum response of (A) carbohydrate, (B) biomass and (C) lipid consumption.

multi-response optimization were calculated 24.8 and

30.6 mg L1 day1, respectively and observed 23.6% improvement
in lipid productivity by multi-objective optimization in shake flask
studies. Improved results for maximum biomass productivity
(122.03 mg L1 day1) from C. pyrenoidosa NCIM 2738 was
observed than recent studies of Chlorella sps. cultivation
(<44 mg L1 day1) at shake flask level (Fan et al., 2014; Tale
et al., 2014; Nayak et al., 2013). Similarly, in this study, lipid content and productivity were comparable with current reports
related to lipid content and productivities at flask level are summarized in Table 5. However, the lipid content of Chlorella species was
observed in between 2553% (dcw) under various types of bioreactor level studies (Table 5). However, highest value of lipid productivity achieved was 576 mg L1 day1 from Chlorella sp. supplied
with 2% of CO2 under constant light intensity of 500 lmol photons
m2 s1 in photobioreactor (Pribyl et al., 2012). This higher lipid
productivity was explained by author due to the effect of controlled cultivation conditions like CO2 and continuous higher light
intensity used in the study.
3.3. Validation of models
To validate the developed models, experiments were performed
using modified BG11 media composition with optimal levels of

selected parameters while other media components concentration

remains the same. Maximization of single responses for C. pyrenoidosa NCIM 2738 by optimized parameters were observed experimentally 34.8% (w/w) for lipid production (biomass 854.3 mg L1
and carbohydrate consumption 82.6%), 1464.3 mg L1 for biomass
production (lipid 19.72% and carbohydrate consumption 86.6%)
and 93.4% for glucose assimilation (lipid 23.2% and biomass
1218.3 mg L1) at different optimum levels of nitrate, phosphate
and carbon source. The fitness of single optimization models
demonstrated excellent correlation between predicted and experimental data and the values of R2 were found to be 97.4%, 95.1% and
98.9% for lipid, biomass production and glucose assimilation, correspondingly. Although, multi-response optimization to maximize
lipid accumulation, biomass production and carbohydrate consumption were observed experimentally 28.9%, 1271.2 mg L1
and 89.2%, respectively at optimum level of parameters. The verification of multi-response optimization model had shown a high
degree of precision of more than 98.5%. Assessments of lipid and
biomass productivity from individual and multi-objective optimized medium are summarized in Table 6. Improvement in lipid
productivities of single and multi-response optimization approach
on comparison with 1% glucose containing BG11 medium was
observed 2.35 and 2.90 times, correspondingly. Similarly, a comparison for biomass productivities of single and multi-response

K. Kanaga et al. / Bioresource Technology 200 (2016) 940950

947

Fig. 4. Multi objective optimization condition to maximize production of all responses.

optimization approach was found 1.6 and 1.4-fold increase. The

maximum lipid productivity for single and multi-response optimization were found 24.8 and 30.6 mg L1 day1, respectively
which showed 23.6% improvement in lipid productivity by multiresponse optimization. Maximum biomass productivity for individual and multi-response optimization obtained was 122.03 and
105.93 mg L1 day1, respectively. Maximum collective responses
values at optimal level were found somewhat lower than individual response optimization values. Though, multi-objective optimization was observed more relevant technique than single
response optimization to optimize the parameters and develop
the economic bioprocess.
rdg et al. (2013) reported that moderately nitrogen stressed
conditions (70 mg L1 N) leads to increase in the lipid accumulation and productivities in C. minutissima MACC 360 and 452. In present study, similar nitrogen stressed condition was observed at
87.5 mg L1 N (NaNO3 6.23 mM) in single response optimization
to maximize the lipid production by C. pyrenoidosa NCIM 2738.
Increase in the average lipid content by 23-folds in nitrogen
stressed condition has also been reported in microalgae Chlorophyta species at 45.7% dcw and at 41% dcw (Griffiths and
Harrison, 2009). Rodolfi et al. (2009) previously demonstrated that
there was an inverse correlation between biomass and lipid production under unfavorable microalgae growth conditions. In this
study, comparable improvements in lipid content from C. pyrenoidosa NCIM 2738 was also observed from validated model results.

3.4. Extraction of lipid and fatty acid composition analysis

The total lipid content at 28.9% was extracted by multiobjective optimized medium from C. pyrenoidosa NCIM 2738 using
modified method of Bligh and Dyer protocol. The algal biomass was
harvested for its lipid content during the early stationary phase of
growth and fatty acid composition was analyzed by GCMS. The
major fatty acids detected in the lipid fractions were C14:0, 16:0
and 17:0 and 19.0.The dominant fatty acid was 2-propenoic acid,
tridecyl ester (C16H30O2) at 34.82% followed by phenol, 2,4/6bis
(1,1dimethylethyl) (C14H22O) at 21.98%. Interestingly, hexadecanoic acid, methyl ester and pentadecanoic acid, 14-methyl,
methyl ester (C17H34O2) were accounted for equal proportions of
total lipid at 9.14%. Octadecenoic acid (C19H36O2 and its isomers
were present at a combined content at 8.67%. Cyclododecane
(C12H24) was present at 2%. All other fatty acids (C11, 12, 13,
15, 18, 20, 22, 24, 30, 32, 34, 36, 38) were each present at <2% of
the total fatty acids. Earlier studies by other researchers also
reported similar type of fatty acid profile for C. pyrenoidosa
(Rajasri et al., 2013). Studies showed that C16 and C18 were the
major fatty acids in most of the microalgae. However, the fatty
acids profile of Neochloris oleoabundans showed substantial quantities of odd numbered chain fatty acids, pentadecanoic acid (C15:0)
and margaric acid (C17:0) (Tomabene et al., 1983). In the present
study, C. pyrenoidosa NCIM 2738 contains 87.54% of saturated
and 12.15% of unsaturated fatty acids.

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K. Kanaga et al. / Bioresource Technology 200 (2016) 940950

Fig. 5. Contour plot for cumulative maximum response with varying variables (A) NaNO3 (mM) vs glucose (%), (B) K2HPO4 (lM) vs glucose (%), (C) NaNO3 (mM) vs K2HPO4
(lM) and third variable at mid value in each plot.

140

Maximum Productivity (mg L-1 day-1)

Lipid Productivity

Biomass Productivity

120
100
80
60
40
20
0
1

9 10 11 12 13 14 15 16 17 18 19 20
Run

Fig. 6. Maximum lipid and biomass productivities for the different experimental runs at the appropriate point in the growth phase.

3.5. Effect of light intensity on biomass, lipid production and glucose

assimilation
Effect of different intensities of light (25, 40 and 54 lmol m2 s1)
was analyzed for the three responses (cell biomass production, lipid

production and glucose assimilation) of C. pyrenoidosa NCIM 2738

when grown on multi-objective optimized medium. Maximum of
lipid accumulation (29.35%), and carbohydrate consumption
(90.4%) were observed at high light intensity (54 lmol m2 s1) and
maximum cell biomass production was observed at intermediate

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K. Kanaga et al. / Bioresource Technology 200 (2016) 940950

Table 5
Comparison of lipid and biomass productivity from current Chlorella species cultivation.
Chlorella sp.

Level of studies

Cultivation
strategies

Cell density
(g L1)

Biomass productivity
(mg L1 d1)

Lipid content
(% dcw)

Lipid Productivity
(mg L1 d1)

References

C. pyrenoidosa
NCIM 2738 .
Chlorella sp. Y81
C. pyrenoidosa
FACHB 9
Chlorella sp.
IMMTCC-16
Chlorella sp.

Shake flask

Heterotrophic

1.3

105.9

28.9

30.6

Present study

Mixotrophic
Heterotrophic

0.9

35.5
30.9

10.0
34.42

Lin and Wu (2015)

Fan et al. (2014)

Photoautotrophic

25.3

18.2

4.6

Nayak et al. (2013)

Heterotrophic

2.6

35.1

rdg et al. (2013)

C. vulgaris

Different type of
bioreactor

Heterotrophic

1.9

158.0

25.0

38.5

C. sorokiniana
C. pyrenoidosa
C. vulgaris
Chlorella sp.

Mixotrophic
Heterotrophic
Heterotrophic
Mixotrophic

2.7
1.8
3.8
4.5

456.0
417.0
1010.0

33.4
34.7
40.0
25.4

112.0
145.0
150.0
112.4

C. vulgaris ESP-31

Heterotrophic

2.05.0

40.053.0

67.0144.0

Najafabadi et al.
(2015)
Kumar et al. (2014)
Wen et al. (2014)
Zheng et al. (2012)
Cheirsilp and
Torpee (2012)
Yeh and Chang
(2012)

Table 6
Comparison of lipid and biomass productivity of initial, individual and multi objective optimized medium.

Medium condition

NaNO3
(mM)

K2HPO4
(lM)

Glucose
(%)

Lipida (%
dcw)

Biomassa
(mg L1)

Glucose consumptiona
(%)

Control (BG-11)

17.64

229.6

1.00

13.72

923.7

92.3

Individual response optimization

Lipid production
6.37
Biomass production
23.63
Glucose consumption
23.06
Multi objective
16.84
optimization

349.4
286.4
407.6
300.9

1.51
4.18
0.70
2.65

34.8
19.72
23.2
28.9

854.3
1464.3
1218.3
1271.2

82.6
86.6
93.4
89.2

Productivitya
(mg L1 day1)

Improvement
in
productivities
(fold)

Lipid

Biomass

Lipid

Biomass

10.56

76.98

24.77
24.06
23.55
30.61

71.19
122.03
101.53
105.93

2.35
2.28
2.23
2.90

0.92
1.59
1.32
1.38

All observed values of responses were mean values of duplicates and standard deviation less than 3%.

light intensity (40 lmol m2 s1) (Supplementary Fig. S3). The values
obtained for three responses were least found at low light intensity
(25 lmol m2 s1). Effect of light intensities (25, 40 and
54 lmol m2 s1) was not significantly different for cell biomass production and carbohydrate consumption. However, increasing trend
was observed in the lipid accumulation by C. pyrenoidosa NCIM
2738 cultivation as light intensity increased from 25 to 54 lmol m2
s1. Previous studies had also reported that a high light irradiance was
preferred for more lipid content accumulation rather than more biomass (Tansakul et al., 2005). Cheirsilp and Torpee (2012) had
observed that the growth and lipid concentration of marine Chlorella
sp. continuously improved up to the maximum level by increasing
light intensity to 112 lmol m2 s1. In this same report, marine Chlorella sp. showed improved growth and cell biomass at a light intensity
of <70 lmol m2 s1. Light saturation constant for growth of
C. pyrenoidosa was reported 35 lmol m2 s1 in continuous light
mode by Martnez et al. (1997). In the present study also not much
variation in cell biomass was observed at three different light intensities except control. It was also observed that the optimal levels of light
intensity for obtaining cell growth and lipid accumulation were
dissimilar.
4. Conclusion
In conclusion, biomass yield and lipid production along with
substrate utilization should be considered simultaneously for
optimal algal biofuel production by heterotrophic method.
Multi-objective analysis strategy gives much better performance
for optimum cell biomass and lipid production than single

response optimization method. The enhancement in lipid productivity was 23.6% higher from optimal level of multi-response
optimization analysis as compared to single response optimization
methods. In addition, fatty acid composition from C. pyrenoidosa
NCIM 2738 point towards the potential application of produced
lipid for biodiesel. Light intensity effect results also indicated its
vital role for optimal algal biofuel production.
Acknowledgements
K.K. and A.P. wish to express their thanks to Ministry of Human
Resource Development (MHRD), Government of India for M. Tech.
fellowship and Institute Ph.D. fellowship, respectively. This work
was supported by funds from Plan Grant of the Institute, MNNIT
Allahabad to S.K.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2015.11.
017.
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