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Green microalga Chlorella vulgaris as a

potential feedstock for biodiesel
ARTICLE in JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY JANUARY 2012
Impact Factor: 2.35 DOI: 10.1002/jctb.2694

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Research Article
Received: 29 March 2011

Revised: 5 June 2011

Accepted: 6 June 2011

Published online in Wiley Online Library: 12 July 2011

(wileyonlinelibrary.com) DOI 10.1002/jctb.2694

Green microalga Chlorella vulgaris

as a potential feedstock for biodiesel
Nirupama Mallick, Shovon Mandal, Amit Kumar Singh, Moumita Bishai
and Archana Dash
Abstract
BACKGROUND: A major bottleneck in microalgal biodiesel production is lipid content, which is often low in microalgal
species. The present study examines Chlorella vulgaris as a potential feedstock for biodiesel by identifying and evaluating the
relationships between the critical variables that enhance the lipid yield, and characterizes the biodiesel produced for various
properties.
RESULTS: Factors affecting lipid accumulation in a green microalga, Chlorella vulgaris were examined. Multifactor optimization
raised the lipid pool to 55% dry cell weight against 9% control. When C. vulgaris cells pre-grown in glucose (0.7%)-supplemented
medium were transferred to the optimized condition at the second stage, the lipid yield was boosted to 1974 mg L1 , a value
almost 20-fold higher than for the control. The transesterified C. vulgaris oil showed the presence of 82% saturated fatty acids,
with palmitate and stearate as major components, thus highlighting the oxidative stability of C. vulgaris biodiesel. The fuel
properties (density, viscosity, acid value, iodine value, calorific value, cetane index, ash and water contents) are comparable
with the international (ASTM and EN) and Indian (IS) biodiesel standards.
CONCLUSION: C. vulgaris biomass with 55% lipid content and adequate fuel properties is potentially a renewable feedstock for
biodiesel.
c 2011 Society of Chemical Industry


Keywords: biodiesel; cetane index; Chlorella vulgaris; iodine value; RSM

INTRODUCTION

J Chem Technol Biotechnol 2012; 87: 137145

Correspondence to: Nirupama Mallick, Agricultural and Food Engineering

Department, Indian Institute of Technology Kharagpur-721302, West Bengal, India. E-mail: nm@agfe.iitkgp.ernet.in
Agricultural and Food Engineering Department, Indian Institute of Technology,
Kharagpur, West Bengal, India

www.soci.org

c 2011 Society of Chemical Industry

137

Microalgae are the fastest growing photosynthetic organisms,

and need about 2 tons of CO2 for production of 1 ton of algal
biomass. As part of photosynthesis, algae produce oil. According
to figures compiled by the Global Petroleum Club, USA, soya
typically produces 450 L of biodiesel per hectare per year, canola
1200 L, and palm 6000 L. Researchers predict that a hectare of
algae could produce 90 000 L of biodiesel, and has the potential
to go even higher.1 Oil squeezed from algae can be used as a
feedstock for biodiesel production, and the rest of the biomass
can be converted into ethanol or animal feed.
Biodiesel production from microalgae seems to be feasible,
but the lipid content in the microalga is required to be high
to satisfy the economics of the process. Table 1 summarizes a
few attempts where a significant increase in lipid content was
observed under specific growth conditions. An oil content of
86% of dry cell wt (dcw) was reported in the brown resting
state colonies of Botryococcus braunii, while the green active
state colonies were found to account for only 17% of lipid dry
weight.2 The major obstacle to using B. braunii as an industrial
organism for biodiesel production is its poor growth rate.3 Piorreck
et al.4 reported an increase in lipid content from 21 to 50%
(dcw) in Scenedesmus obliquus under nitrogen-limited conditions.
Takagi et al.5 reported a lipid content of 51% (dcw) against 31%
control in Nannochloris sp. UTEX LB1999 under modified NORO
medium in which the microalgae were grown in continuous
nitrate-limited medium with 3% CO2 purging. It was observed

by Illman et al.6 that Chlorella emersonii and C. minutissima could

accumulate lipid up to 63 and 57% (dcw), respectively, in lowN medium. Chlorella protothecoides also contained a lipid pool
of 55% (dcw) when grown heterotrophically with corn powder
hydrolysate under nitrogen limitation.7 Lipid content increased
to 33% (dcw) in 1% sucrose-supplemented culture of Chlorella
sp. against 15% control.8 Chiu et al.9 reported an accumulation
up to 50% (dcw) in Nannochloropsis oculata NCTU-3 aerated
with 2% CO2 . In Scenedemus obliquus lipid accumulation up
to 58% (dcw) was recorded when subjected to simultaneous
nitrogen and phosphorous limitation in the presence of sodium
thiosulphate against 13% control.10 Sobczuk and Chisti11 also
observed an accumulation of 59.5% (dcw) in Choricystis minor
under simultaneous nitrate and phosphate starvation, whereas
the control culture showed an accumulation of only 27%
(dcw).
Most of these reports present the effects of a single variable on
the lipid pool of the test microalga, except for S. obliquus10 and
C. minor11 , where interactive effects of more than one variable
were investigated. In this report, Chlorella vulgaris biomass was

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Table 1.

Increased lipid content of microalgae under various specific growth conditions

Microalga

Growth condition

Botryococcus braunii

Brown resting state

Scenedesmus obliquus

Nitrogen limitation

Chlorella emersonii

Nitrogen limitation

C. minutissima

Nitrogen limitation

Nannochloris sp. UTEX LB1999

Nitrogen limitation

Chlorella protothecoides
Chlorella sp.

Heterotrophy with corn powder

hydrolysate under nitrogen limitation
Heterotrophy with 1% sucrose

Nannochloropsis oculata NCTU-3

2% CO2

Scenedesmus obliquus

Nitrogen and phosphate limitations in

presence of sodium thiosulphate
Nitrogen and phosphorus deficiencies

Choricystis minor

N Mallick et al.

Lipid content (% dcw)

86
17
50
21
63
29
57
31
51
31
55
15
33
15
50
31
58
13
60
27

Reference
Brown et al. (1969)2
Piorreck et al. (1984)4
Illman et al. (2000)6
Illman et al. (2000)6
Takagi et al. (2000)5
Xu et al. (2006)7
Rattanapoltee et al. (2008)8
Chiu et al. (2009)9
Mandal and Mallick (2009)10
Sobczuk and Chisti (2010)11

Lipid content of control culture.

examined as a possible feedstock for biodiesel production by

identifying the critical variables that influence lipid accumulation
in the cell. For evaluation of the interrelationship of these critical
variables, a multifactor optimization study was conducted with
the help of response surface methodology (RSM) in combination
with central composite rotary design (CCRD) to maximize lipid
accumulation. The biodiesel produced was characterized for
various properties, and was compared with the international
(ASTM and EN) and Indian (IS) biodiesel standards.

MATERIALS AND METHODS

Organism and growth conditions
Established cultures of the green microalga Chlorella vulgaris
were grown in 150 mL Erlenmeyer flasks containing 50 mL N
11 medium12 at pH 6.8 in a culture room at 25 2 C under
a photoperiod of 14 : 10 h at light intensity of 75 mol photon
m2 s1 PAR without sparging with air or CO2 . The mineral
salt medium composition per litre of distilled water was as
follows: 1.0 g KNO3 , 0.083 g Na2 HPO4 2H2 O, 0.052 g KH2 PO4 ,
0.05 g MgSO4 7H2 O, 0.01 g CaCl2 2H2 O, 1 mL Fe-EDTA solution,
and 1 mL SAZ solution. The Fe-EDTA stock solution contained
10 g chelate per litre. The SAZ solution contained (per litre
of distilled water) 0.099 g MnCl2 4H2 O, 0.0236 g NiSO4 7H2 O,
0.063 g ZnSO4 7H2 O, 0.005 g CuSO4 5H2 O, 0.0028 g CoSO4 7H2 O,
0.0029 g NH4 VO3 and 0.0018 g (NH4 )6 Mo7 O24 4H2 O. The cultures
were hand shaken two or three times daily to avoid sticking. This
was referred to as control culture.

138

Dry weight measurement

Dry cell weight (dcw) was determined gravimetrically according
to Rai et al.13 A known volume of algal culture was centrifuged at
5000 rpm for 10 min and the harvested biomass was dried at 60 C
to constant weight.

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Extraction and estimation of lipid from algal biomass

Extraction of lipid was done following the protocol of Bligh and
Dyer.14 To a 15 ml glass vial containing a known amount of algal
biomass, 2 mL methanol and 1 mL chloroform were added and
kept for 24 h at room temperature. The mixture was agitated in
a vortex for 2 min, and 1 mL of chloroform was again added and
the mixture shaken vigorously for 1 min; 1.8 mL of distilled water
was added and the mixture was agitated in a vortex again for
2 min. The layers were separated by centrifugation for 10 min at
2000 rpm. The lower layer was filtered through Whatman No. 1
filter paper into a previously weighed clean vial (W1 ). Evaporation
was carried on in a water bath and the residue was further dried at
104 C for 30 min. The weight of the vial was again recorded (W2 ).
Lipid content was calculated by subtracting W1 from W2 , and was
expressed as % dcw.
Evaluation of cultural variables for lipid accumulation
A number of variables, viz. pH (5.510.5 at an interval of 1.0),
temperature (2045 C at an interval of 5 C), culture density
(15120 mg dcw L1 ), spectral quality, various nutrient (N, P, K, Ca,
Mg, Fe, and S) deficiencies/limitations, mixotrophy (under glucose
supplementation) and stresses such as heavy metals (Cu, Ni, Pb and
Cd), heat (45 C for 30120 min) and chilling (4 C for 30120 min)
stresses were examined to increase lipid accumulation in the
cells. The variables raising the lipid pool significantly are detailed
below.
Effects of N, P and Fe limitation/deficiencies on biomass and
lipid accumulation
To study the effects of nitrate and phosphate limitation on
lipid accumulation, C. vulgaris cells were grown under different concentrations of nitrate (0.0050.1 g L1 ) and phosphate
(0.0050.1 g L1 ). N-deficiency was achieved by substituting KNO3

c 2011 Society of Chemical Industry

J Chem Technol Biotechnol 2012; 87: 137145

Biodiesel production with Chlorella vulgaris

Table 2.

www.soci.org

Levels of the critical variables for central composite rotary design (CCRD)
Levels

Variable

Coded symbol

+2 (+)

X1
X2
X3
X4

0
0
0
4

0.025
0.025
0.001
7

0.050
0.050
0.003
10

0.100
0.100
0.005
16

L1 )

Nitrate (g
Phosphate (g L1 )
Iron (g L1 )
Incubation period (days)

of the medium with equimolar concentrations of KCl. For Pdeficiency, cultures were transferred to mineral salt medium, in
which Na2 HPO4 H2 O and KH2 PO4 were replaced by equimolar
concentrations of Na2 SO4 and KCl, respectively. To study the
effect of iron limitation, C. vulgaris culture was grown at different concentrations of iron (0.00150.006 g L1 ). Fe-deficiency
was achieved by substituting FeSO4 H2 O in the medium with
equimolar concentrations of Na2 SO4 .
Multifactor optimization study for maximization of lipid
accumulation
From the above study, the four most critical variables which
were found to have profound effects on lipid accumulation
were concentrations of nitrate (X1 ), phosphate (X2 ), iron (X3 ) and
incubation period (X4 ). Thus to examine their interactive effects
for maximization of lipid accumulation, a five-level-four-factor
CCRD15 was obtained using the commercial statistical package,
Design Expert-version 7.1.1 (Stat-Ease Inc., Minneapolis, USA).
The experimental levels of the variables and the CCRD design
matrix are given in Tables 2 and 3. Thirty experiments were
set up in N 11 medium with varying concentrations of nitrate,
phosphate and iron as per the design matrix (Table 3). Duration
of culture was employed in accordance with the experimental
design. The experimental data obtained from CCRD were analyzed
by RSM. To find the level of each variable for maximum response
(maximum lipid accumulation), a point optimization technique
was employed.
Effect of mixotrophy on biomass and lipid accumulation
The effect of mixotrophy on biomass and lipid accumulation
was studied by supplementing the N 11 medium with various
concentrations (0.31.5%) of glucose.
Interaction of mixotrophy with the optimized condition
on biomass and lipid yield
Mixotrophic growth was found stimulatory for biomass yield.
Therefore, combining both the strategies, i.e. at the first
stage C. vulgaris was cultured in N 11 medium supplemented
with glucose. The biomass was harvested by centrifugation
at 5000 rpm, washed in the deficient (optimized) medium
twice, diluted and resuspended in the optimized medium.
Biomass and lipid yield were quantified after the optimized time
period.

J Chem Technol Biotechnol 2012; 87: 137145

Response (lipid
accumulation % dcw)

Process variable
Run
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

X1

X2

X3

X4

Observed

Predicted

1
+1
1
+1
1
+1
1
+1
1
+1
1
+1
1
+1
1
+1
2
+2
0
0
0
0
0
0
0
0
0
0
0
0

1
1
+1
+1
1
1
+1
+1
1
1
+1
+1
1
1
+1
+1
0
0
2
+2
0
0
0
0
0
0
0
0
0
0

1
1
1
1
+1
+1
+1
+1
1
1
1
1
+1
+1
+1
+1
0
0
0
0
2
+2
0
0
0
0
0
0
0
0

1
1
1
1
1
1
1
1
+1
+1
+1
+1
+1
+1
+1
+1
0
0
0
0
0
0
2
+2
0
0
0
0
0
0

30.52
39.02
27.25
29.54
18.17
22.98
21.80
18.76
26.16
24.80
47.24
19.25
38.21
32.42
58.14
32.90
30.41
25.05
27.72
34.89
23.57
29.39
19.70
35.47
51.73
49.54
50.26
47.01
50.89
52.11

26.59
39.68
28.10
26.95
15.63
26.05
22.19
17.58
26.99
21.85
41.62
21.44
38.24
30.42
57.13
34.28
34.06
24.30
29.67
35.83
26.99
28.86
20.48
37.58
50.26
50.26
50.26
50.26
50.26
50.26

Model F value = 28.39, P < 0.0001.

Coefficient of determination (R2 ) = 0.9636, Adjusted R2 = 0.9297.
Adequate precision = 18.68, Coefficient of variation (CV ) = 9.3%.

with the help of a gas chromatograph (Autosystem XL)

equipped with Turbomass Gold Mass Spectrometer (PerkinElmer, Shelton, CT, USA). PE-5 phenyl, methylpolysiloxane
capillary column (30 m 0.25 mm 0.25 m) was used for
the analysis. Methylpentadecanoate was used as the internal
standard.

c 2011 Society of Chemical Industry

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139

Fatty acid analysis

The acid-catalyzed transesterification of algal oil was carried
out using 90 : 1:3.8 molar ratio of methanol, oil and sulfuric acid as catalyst.16 The top organic layer was taken for
analysis by gas chromatographymass spectrometry (GCMS)

Table 3. Central composite rotary design matrix with actual and

predicted responses for lipid accumulation

Statistical analysis
All the experiments were performed in triplicate to check the
reproducibility. The results were analysed statistically by Duncans
new multiple range test, co-relation coefficient and the commercial
statistical package, Design Expert-version 7.1.1 (Stat-Ease Inc.,
Minneapolis, USA).

RESULTS
Accumulation of lipid in relation to growth
Figure 1 presents the time-course of growth and lipid accumulation in C. vulgaris in N 11 medium under batch mode study. Growth
of C. vulgaris increased steadily with a lag of 3 days followed by
the logarithmic phase, and attained the stationary phase on day
18. Maximum accumulation of lipid was observed at the stationary
phase (96.3 mg L1 , 9.2% dcw). After 24 days the lipid pool showed
a declining trend.

140

Evaluation of various cultural variables for lipid accumulation

Experiments conducted at varied pH, temperature, culture density,
spectral quality, various nutrient deficiencies/limitations and
stresses such as heavy metals, heat and chilling stresses resulted in
marginal increases/decreases in lipid yield. However, a profound
increase in lipid pool was observed under N and P deficiencies,
and iron limitation; the lipid pool reached 42.4 and 40.8% (dcw),
respectively, on day 7 of N and P deficiency, and 38.9% (dcw) when
iron concentration was reduced to 0.003 g l1 from the initial
value of 0.006 g L1 on day 12 of incubation (data not shown). On
further decreasing the iron concentration to 0.0015 g L1 , the lipid
pool dropped to 33% (dcw). In Fe-deficient cultures the lipid pool
reached only 19% (dcw). All these limited/deficient conditions had
severe inhibitory effects on the biomass yield (data not shown).

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N Mallick et al.
1.2

12

10

0.8

0.6

6
Growth

0.4

Lipid content

0.2

Lipid Content (% dcw)

Fuel properties of C. vulgaris biodiesel

The fuel properties such as density, viscosity, calorific value, iodine
value, acid value, cetane index, ash content and water content of
C. vulgaris biodiesel were determined by the following standard
techniques and were compared with American (ASTM), European
(EN) and Indian (IS) standards.17 19
The density was determined in accordance with ASTM D 4052-96
standard using relative density bottles of 10 cm3 volume (Borosil
make) and was expressed as kg m3 . The kinetic viscosity of
biodiesel was determined in accordance with the Indian standard19
using a Cannon Fensky viscometer tube in a kinetic viscometer
bath (Maharana Instruments Mfg. Co., Ajmer, India).
Calorific value is an important characteristic of fuel. It expresses
the amount of heat generated by complete combustion of a unit
weight of fuel. It was deterimined by bomb calorimeter (Maharana
Instruments Mfg. Co., Ajmer, India) according to ASTM D 240-02
standard. The iodine value was determined by titrimetry method.20
Acid value was determined following Vicente et al.21 The cetane
index was measured following Krisnangkura.22
The ash content is a measure of the amount of incombustible
material remaining after burning the fuel and is expressed in terms
of percentage of original weight of the biodiesel. The ash content
of the biodiesel was determined in accordance with the procedure
given in ASTM D 482-74. The water content in the biodiesel was
measured by Karl Fisher Titrator (TKF-55, Toshniwal Instruments
Pvt. Ltd., Ajmer, India).

Biomass Concentration (g l-1)

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0
0

12

15

18

21

24

27

30

Days of Incubation

Figure 1. Lipid accumulation in C. vulgaris with reference to growth in N11

medium.

Optimization of lipid accumulation

CCRD experiments with 16 cubic points, eight axial points and six
central points for studying the interactive effects of four critical
variables, i.e. concentration of nitrate (X1 ), phosphate (X2 ), iron
(X3 ) and days of incubation (X4 ) on lipid accumulation of C.
vulgaris are presented in Table 3. Different combinations of nitrate
(00.1 g L1 ), phosphate (00.1 g L1 ), iron (00.005 g L1 ) and
incubation period (416 days) resulted in lipid accumulation
varying from 18.1758.14% (dcw). The predicted values calculated
using the model were in the range 15.6357.13 (Table 3).
Regression analysis of the experimental results demonstrated
that the interactive model terms, i.e. nitrate and phosphate (X1 X2 ),
nitrate and days of incubation (X1 X4 ), phosphate and days of
incubation (X2 X4 ) and iron and days of incubation (X3 X4 ) were
significant (P < 0.05). However, the interactive model terms X1 X3
and X2 X3 , i.e. interaction of iron with nitrate and phosphate were
found to be insignificant (P > 0.05). Applying multiple regression
analysis, the results were fitted to a second-order polynomial
equation in which the insignificant model terms were omitted.
Thus, the mathematical regression model for lipid yield fitted in
terms of coded factors was as follows:
Y = 50.26 2.44X1 + 1.54X2 + 0.47X3
+ 4.27X4 3.76X1 X2 4.56X1 X4
+ 3.08X2 X4 + 5.55X3 X4 5.27X 1 2
4.37X 2 2 5.58X 3 2 5.30X 4 2

(1)

where Y represents lipid accumulation on percentage dry cell

weight basis.
Analysis of variance (ANOVA) was used to determine the
adequacy and significance of the quadratic model for lipid
accumulation and is presented in Table 3 footnote. The P-value
of the model was <0.0001, demonstrating that the model was
highly significant. Adequate precision measures the signal to noise
ratio. A ratio greater than 4 is desirable. Here, the ratio was 18.68,
indicating a highly adequate signal. The mathematical model was
found to be reliable, with an R2 value of 0.9636 (the closer R2 to 1,
the better the model fit to the experimental data). The adjusted R2
of 0.9297 also confirmed that the model was highly significant. A
very low value of coefficient of variation (Cv : 9.3%) clearly indicated
a very high degree of precision and reliability of the experimental
values.

c 2011 Society of Chemical Industry

J Chem Technol Biotechnol 2012; 87: 137145

Biodiesel production with Chlorella vulgaris

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(B)

(A)

(D)

(C)

Figure 2. 3D response surface: interactive effects of (A) varied nitrate and phosphate concentrations at zero level of iron and culture period, (B) varied
nitrate concentration and culture period at zero level of phosphate and iron, (C) varied phosphate concentration and culture period at zero level of
nitrate and iron, (D) varied iron concentration and culture period at zero level of nitrate and phosphate.

J Chem Technol Biotechnol 2012; 87: 137145

Table 4. Optimum condition of the critical variables for maximum

lipid accumulation
Lipid accumulation (% dcw)
Variable
Nitrate (g L1 )
Phosphate (g L1 )
Iron (g L1 )
Incubation period (days)

Optimum
value
0.025
0.075
0.003
13

Predicted

Experimental

57.6

55.3 1.03

lipid accumulation was increased with increasing incubation

period up to certain point but at the zero level of iron.
The interactive model terms X1 X3 (nitrate and iron) and X2 X3
(phosphate and iron) were found to be insignificant (data not
shown).
Having established the possible directions for maximization
of lipid accumulation, optimization was done using a point
optimization technique. The optimum conditions of the variables
for maximum lipid accumulation are presented in Table 4.
A maximum lipid content of 57.6% (dcw) was predicted at
0.025 g L1 nitrate, 0.075 g L1 phosphate, and 0.003 g L1 iron
for a culture period of 13 days.

c 2011 Society of Chemical Industry

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141

Localization of the optimized conditions

The interactive effects of the above four variables on lipid
accumulation were studied using three-dimensional response
graphs. The shape of the corresponding contour plots indicates
whether the mutual interactions between the independent
variables are significant or not. Each figure represents an infinite
number of combinations (within the experimental range) of two
test variables with the other two maintained at their respective
zero levels (Table 2). Figure 2(A) explains the interaction of nitrate
with phosphate at zero levels of iron and days of incubation.
In this figure, lipid accumulation increased with decrease in the
level of nitrate and increase in the level of phosphate above
the zero level up to certain point. The interaction of nitrate and
days of incubation at zero levels of phosphate and iron was
significant, as visualized in Fig. 2(B), where with decreasing nitrate
concentration and increasing incubation period, an increase in
lipid accumulation was evident up to certain point. Figure 2(C)
also explains the positive interaction between phosphate and
incubation period at zero levels of nitrate and iron, where
increase in the concentration of phosphate and days of incubation
contributes to increase lipid accumulation above the zero level
up to certain point, as depicted from convergence of the curve at
the mid-point of the surface. The 3D response surface based
on the interaction of iron and incubation period is shown
in Fig. 2(D), with the other two independent variables, nitrate
and phosphate, kept at constant (zero) level. In this figure,

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N Mallick et al.

Interaction of mixotrophy with optimized condition

Under optimized conditions, lipid accumulation was found to
increase to 55.3% (dcw, Table 4), which was 6-fold higher than in
the control. However, the total lipid yield was found to increase
only to 171.4 mg l1 because of severe retardation of growth
under the optimized conditions (Table 6). It is interesting to note
here that although supplementation of glucose was not found
stimulatory for lipid accumulation, a profound rise in biomass yield
to 4.19 g L1 in 0.7% glucose-supplemented culture was evident
(Table 5). Therefore, combining both strategies, i.e. growing C.
vulgaris in N 11 medium supplemented with 0.7% glucose during
the first stage for 10 days, then transferring to the optimized
condition at the second stage, the lipid pool was boosted to
1973.9 mg L1 (Table 6), almost 20-fold higher than the control
culture.

Figure 3. Plot of internally studentized residuals vs. normal percentage

probability.

Table 5. Effect of exogenous glucose supplementation on biomass

and lipid accumulation potential of C. vulgaris for a culture period of
10 days
Glucose
concentration
(% w/v)

Biomass
concentration
(g L1 )

Lipid accumulation
mg L1

% dcw

0.0
0.3
0.5
0.7
1.0
1.5

0.33 0.02a
2.38 0.22c
2.59 0.29c
4.19 0.10d
2.14 0.08bc
1.75 0.14b

29.4 0.43a
252.3 2.1c
259.0 2.4c
441.1 3.4d
211.9 1.8b
204.8 1.9b

8.9 0.64a
10.5 0.87b
10.0 0.58b
10.6 0.70b
9.9 0.70b
11.7 0.87c

All values are mean SE, n = 3. Values in a column superscripted

by different alphabets are significantly (P < 0.05) different from each
other (Duncans new multiple range test). Separate analysis was done
for each column.

Validation of the model

Experiments were performed in triplicate and repeated three times
under the optimized conditions to verify the model. A lipid pool
of 55.3% (dcw) was achieved (Table 4). By constructing a normal
probability plot of residuals, a check was made of the normality
assumption. The normality assumption was satisfied as the residual
plot approximated a straight line (Fig. 3).

142

Effect of mixotrophy
The most significant increase in biomass yield, to 4.19 g L1 , was
obtained in 0.7% glucose-supplemented medium after 10 days
(Table 5). No further rise in biomass yield was observed with
increasing concentrations of glucose or by increasing the culture
period. Although supplementation of glucose was not found
stimulatory for lipid accumulation on the basis of percentage dry
weight, the total lipid yield was increased to 441.1 mg L1 in 0.7%
glucose-supplemented culture, which was 4.6-fold higher than in
the control.

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Analysis of C. vulgaris biodiesel

Table 7 presents the fatty acid content of C. vulgaris oil after
transesterification. Major constituents were palmitic followed by
stearic, linolenic and oleic acid methyl esters. The saturated fatty
acids, i.e. palmitic and stearic, constitute almost 82% of biodiesel,
while esters of long chain unsaturated fatty acid such as linoleic
acid were present in low quantities.
Various fuel properties of C. vulgaris biodiesel obtained under
the optimized conditions are compiled in Table 8. The density and
viscosity values were found within the range specified for biodiesel
standards. Although the acid value was marginally higher than the
EN standard, it was within the range of ASTM and IS standards. The
calorific value was 38.4 MJ kg1 , which was marginally lower than
that for petroleum diesel. The iodine value was within the limits
specified in biodiesel standards, i.e. EN 14 214 (<120 g I2 /100 g)
and IS 15 607 (115 g I2 /100 g). The cetane index was found to
be 54.7. The ash content and water content were 0.01 and 0.03%,
respectively.

DISCUSSION
Experimenting with C. vulgaris demonstrated maximum lipid
accumulation at the stationary phase when grown in complete N
11 medium (Fig. 1), which showed a declining trend after day 24.
This decrease could be due to mobilization and degradation of
lipids to maintain cellular metabolism during the stationary and
declining phases of growth. A profound rise in lipid content under
nitrate, phosphate and iron-limited conditions was observed,
which are very similar for other microalgal species.5,9,11,23 25 The
possible reason could be that under nitrate limitation, NADPH
consumption was decreased due to unavailability of a nitrogen
pool, which blocks the amino acid synthesis pathways, especially
the reaction from -ketoglutarate to glutamate, thus resulting
in the accumulation of excess NADPH in the cell.26 Under such
conditions, acetyl-CoA could not enter the tricarboxylic acid (TCA)
cycle as high concentrations of NADPH inhibit the enzyme citrate
synthase, one of the key enzymes of the TCA cycle, leading to
an increase in the pool of acetyl-CoA.27 The latter might be
converted into malonyl-CoA, catalyzed by acetyl-CoA carboxylase
(ACCase), the central carbon donor for fatty acid synthesis.28
Phosphate limitation also enhanced the accumulation of reducing
power,29 and consequently an increase in lipid pool in C. vulgaris.
Moreover, some metabolic pathways related to lipid accumulation
in C. vulgaris might be modified when iron concentration in the
medium is changed,30 which needs further exploration. With

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Table 6. A comparative account on biomass and lipid accumulation potential of mixotrophically-grown C. vulgaris following optimized condition
with control, optimized and mixotrophy
Lipid accumulation
Culture condition

Biomass concentration (g L1 )

(mg L1 )

(% dcw)

1.05 0.13b
0.31 0.08a
4.19 0.10c
4.07 0.29c

96.3 1.72a
171.4 3.02b
441.1 3.40c
1973.9 8.16d

9.2 0.74a
55.3 1.01c
10.6 0.70a
48.5 1.19b

N 11 control
Optimized condition
Glucose (0.7%)
Glucose (0.7%) + Optimized condition

All values are mean SE, n = 3.

Values in a column superscripted by different alphabets are significantly (P < 0.05) different from each other (Duncans new multiple range test).
Separate analysis was done for each column.

Table 7. Composition and relative percentage of fatty acid methyl

esters in C. vulgaris oil
Fatty acid methyl ester
Hexadecenoic acid
methyl ester
(palmitic acid)
Octadecanoic acid
methyl ester
(stearic acid)
9,12Octadecadienoic
acid methyl ester
(linoleic acid)
9-Octadecenoic acid
methyl ester (oleic
acid)

Molecular
formula

Retention time
(min)

Relative
%

C17 H34 O2

10.85

62.4

C19 H38 O2

11.58

19.5

C19 H34 O2

11.61

9.8

C19 H36 O2

11.69

8.3

The common names of the fatty acids are given in parentheses.

Values are means of three independent observations.

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143

changing medium composition, lipid accumulation also showed

significant variation with respect to the culture period.
It is important to optimize the critical variables affecting
lipid accumulation to determine the accumulation potential
of an organism for industrial application. Using the traditional
one-factor-at-a-time optimization the concentrations of nitrate,
phosphate, iron and culture period were established as the critical
variables for increasing the lipid pool in C. vulgaris. Therefore, a
regression equation was developed using the above variables
(Equation (1)). The contour plots of the 3D response surface
(Fig. 2) show the variation in lipid accumulation as a function
of the interaction of variables. The elliptical contour plots of
the 3D response surfaces (Fig. 2(A), (B), (C) and (D)) demonstrate
that the interactions between the variables had a significant
impact on product accumulation. In order to maximize lipid
accumulation, the optimum variables were estimated using the
point optimization method in the direction of steepest ascent. The
steepest ascent procedure is a method in which the experimenter
proceeds sequentially along the path of steepest ascent, i.e. along
the path of maximum response. Thus, the optimum condition and
the predicted lipid accumulation of 57.6% (dcw) did not differ
significantly from the experimental value of 55.3% (Table 4).
Results from this study demonstrated a 6-fold rise in lipid pool
in C. vulgaris on a percentage dry cell wt basis by manipulating the
nutrient status (Table 4). The total lipid yield in these vessels was,

however, increased only to 171.4 mg L1 against 96.3 mg L1 in

the control vessels (Table 4). This is due to the fact that growth of
the test microalga was severely affected under nitrate, phosphate
and iron limitations. This necessitates a two-stage cultivation
practice, where in the first stage C. vulgaris must be cultured under
appropriate conditions to reach a high cell density, followed by
a second stage, as optimized in this study, for efficient lipid
accumulation. This hypothesis was verified experimentally.
Mixotrophic growth was found to surpass the sum of autotrophic and heterotrophic growth, as reported for Chlorella
sp. VJ79,31 Scenedesmus falcatus,32 Scenedesmus obliquus33 and
Chlorella prothecoides.5 When C. vulgaris was cultured in glucosesupplemented medium a boost in biomass yield was evident
(Table 5). Thus, when the biomass obtained after growing C.
vulgaris in glucose (0.7%)-supplemented N 11 medium was transferred to the optimized conditions in the second stage, the lipid
pool was boosted to 1973.9 mg L1 , almost 20-fold higher than
the control culture (Table 6).
The properties of biodiesel are mainly determined by its fatty
acid esters.34 Poly-unsaturated fatty acids with four or more
double bonds are quite common in microalgal oil. These bonds
are susceptible to oxidation during storage, thus reduce the
acceptability of microalgal oil for the production of biodiesel.35
GC-MS analysis (Table 7) demonstrated that the biodiesel from
C. vulgaris contains mainly saturated fatty acids (82% of the
total fatty acyl methyl esters), which highlights its high oxidative
stability. Iodine value is also a measure of total unsaturation within
a mixture of fatty acids. In C. vulgaris biodiesel it is found to be
suitably low and within the limits of European and Indian standards
(Table 8).
Density is an important parameter in airless combustion systems
because it influences the efficiency of atomization of the fuel.36
Even more than density, viscosity controls fuel atomization
and distribution. Too high a viscosity can cause excessive heat
generation in the injection equipment owing to viscous shear in
the clearance between pump plunger and cylinders. These values
in C. vulgaris biodiesel are within the prescribed limits laid down
by the ASTM, EN 14 214 and IS 15 607 standards (Table 8).
Cetane index is also a measure of diesel fuel quality related to
ignition delay time and combustion quality. High cetane index
ensures good cold start and minimizes the formation of white
smoke. The cetane index of C. vulgaris biodiesel was 54.7, which is
also suitable according to the limits established by American (47)
and European and Indian (51) standards. According to Knothe
et al.,37 high cetane index was observed for esters of saturated
fatty acids such as palmitic and stearic acids. Chlorella biodiesel,

www.soci.org

Table 8.

N Mallick et al.

Comparison of C. vulgaris biodiesel with petroleum diesel and various biodiesel standards

Property

m3 )

Density at 15 C (kg
Viscosity at 40 C (mm2 s1 )
Calorific value (MJ kg1 )
Iodine value (g I2 /100 g)
Acid value (mg KOH g1 )
Cetane index
Ash content (%)
Water content (%)

Biodiesel standards

Biodiesel from

Petroleum

C. vulgaris

diesel

ASTM

EN 14214

IS 15607

881
4.5
38.4
56.2
0.6
54.7
0.01
0.03

850
2.6
42.2

0.4
4955
0.01
0.02

1.96.0

<0.8
47
<0.02
<0.03

860900
3.55.0

<120
<0.5
51
<0.02
<0.05

870900
3.55.0

115
0.8
51
<0.02
0.05

C. vulgaris biodiesel was obtained from mixotrophically-grown cultures following optimized conditions at the second stage.
Values are means of three independent observations.

rich in these two fatty acids, might result in a high cetane index.
The report of Van Gerpen38 also supports the above view, where
an increase in cetane index with increasing methyl palmitate in a
blend was recorded.
Acid value is a measure of total free fatty acids in the biodiesel.
The presence of free fatty acids in the biodiesel leads to corrosion,
besides sludge and gum formation. The acid value of C. vulgaris
biodiesel, although found marginally higher than that specified
in biodiesel specification EN 14 214, the value was within the
limits of ASTM and IS 15 607. Acid value is mainly dependent on
the transesterification conditions and the purification steps. Thus,
the biodiesel needs to be purified further to meet the European
specification.
The calorific value of C. vulgaris biodiesel was found to be
lower than that of petroleum diesel. This could be due to the
difference in their chemical composition and the presence of
oxygen molecule (10% of weight) in the molecular structure
of biodiesel.39 The oxygen molecules present unite with the
hydrogen of the biodiesel to form water vapour even before air
or oxygen supplied for combustion reaches the hydrogen. This
results in a decrease in the available hydrogen, thus decreasing the
calorific value. However, the calorific value of C. vulgaris biodiesel
is comparable with that of Jatropha (37.2 MJ kg1 ) and karanja
(36.1 MJ kg1 ) biodiesels.40,41
The level of ash content represents the magnitude of inorganic
contaminants, such as abrasive solids and catalyst residues, and
the amount of soluble metals contained in a fuel sample.42 It is the
incombustible materials remaining after burning and correlates
with the amount of deposits formed in the combustion chamber.
Therefore, fuels having less ash content are preferred for better
engine operation and maintenance. The C. vulgaris biodiesel was
characterized by the same ash content as that of petroleum
diesel and was within the prescribed limits of biodiesel standards.
Water present in biodiesel interferes with its smooth flow through
lines and combustion inside the cylinder. The water content of
C. vulgaris biodiesel was found to be 0.03%, which complies
with various standards. All these properties thus, make C. vulgaris
biomass a potential feedstock for biodiesel production.

CONCLUSION

144

In this study, lipid content of C. vulgaris approached 55% (dcw),

which was 6-fold higher than in the control, obtained by
manipulating the nutrient status. However, biomass productivity

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was severely affected by N, P, and Fe limitation. This necessitates a

two-stage cultivation practice, such that during the first stage the
microalgae are cultured under suitable conditions to reach a high
cell density, followed by a second stage, as optimized in this study,
for efficient lipid accumulation. This is reported in Table 6, where
C. vulgaris cultures pre-grown in 0.7% glucose-supplemented N
11 medium followed by a second stage at optimized conditions
boosted the lipid pool to 2 g L1 , almost 20-fold higher than in
the control cultures. However, the addition of organic carbon
such as glucose increases feedstock cost, which warrants further
research on inexpensive carbon sources in order to reduce the
cost of algal biomass production. Therefore, our focus is to couple
algal biodiesel production with waste utilization and recycling
by taking using various carbon-rich wastes such as poultry litter,
piggery wastes, municipal and aqua discharges for algal mass
cultivation for biodiesel production.

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