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Bioresource Technology
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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
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.
941
942
N 2k 2k 6
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
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
8
>
<
>
:
0 if Y < 1
s
YL
if L 6 Y 6 T
TL
1 if Y > T
943
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)
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)
944
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
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
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.
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.
946
Fig. 3. The individual maximum response of (A) carbohydrate, (B) biomass and (C) lipid consumption.
947
948
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
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.
949
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
Photoautotrophic
25.3
18.2
4.6
Heterotrophic
2.6
35.1
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
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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