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IIT Kharagpur
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Archana Dash
IIT Kharagpur
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Research Article
Received: 29 March 2011
INTRODUCTION
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137
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Table 1.
Microalga
Growth condition
Botryococcus braunii
Scenedesmus obliquus
Nitrogen limitation
Chlorella emersonii
Nitrogen limitation
C. minutissima
Nitrogen limitation
Nitrogen limitation
Chlorella protothecoides
Chlorella sp.
2% CO2
Scenedesmus obliquus
Choricystis minor
N Mallick et al.
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
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Table 2.
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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.
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
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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.
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N Mallick et al.
1.2
12
10
0.8
0.6
6
Growth
0.4
Lipid content
0.2
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0
0
12
15
18
21
24
27
30
Days of Incubation
(1)
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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.
Optimum
value
0.025
0.075
0.003
13
Predicted
Experimental
57.6
55.3 1.03
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N Mallick et al.
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
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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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
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
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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
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REFERENCES
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