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Cost Effective Approach for Production of Chlorella pyrenoidosa: A RSM Based

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DOI: 10.1007/s12649-018-0330-x

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Waste and Biomass Valorization
https://doi.org/10.1007/s12649-018-0330-x

ORIGINAL PAPER

Cost Effective Approach for Production of Chlorella pyrenoidosa: A RSM

Based Study
M. Mubarak1 · A. Shaija1 · T. V. Suchithra2

Received: 16 June 2017 / Accepted: 10 May 2018

© Springer Science+Business Media B.V., part of Springer Nature 2018

Abstract
Microalgae are considered as a promising feedstock for biodiesel production due to their faster growth rate, higher biomass
productivity and lipid content which ranges from 20 to 50% of its dry weight. In this work, optimization of bold basal media
(BBM) for the production of Chlorella pyrenoidosa are studied with central composite design (CCD) using response surface
methodology. The classical optimization is used for identifying the best pH for the growth of C. pyrenoidosa and found to
be 8. The screening of significant factors for the growth was studied using Plackett–Burman design (PBD) with 7 factor
experiment. The significant factors identified were M­ gSO4⋅7H2O, ­CaCl2⋅2H2O, ­KNO3, ­Na2EDTA from PBD of experiment.
These significant factors were included in a CCD, resulting in 31 experiments, that were further performed. The chlorophyll
a, chlorophyll b, chlorophyll a, b and turbidity were measured regularly during experiments. The optimum concentration
of ­MgSO4⋅7H2O, ­CaCl2⋅2H2O, ­KNO3, and N ­ a2EDTA were found to be 0.1, 0.0375, 0.375, and 0.0625 g/L, respectively.
The turbidity, chlorophyll a, chlorophyll b and chlorophyll a, b obtained with optimum concentrations of BBM were 0.809,
5.7504, 1.6137 and 7.3642 mg/L respectively. The results showed a yield of more than 5 times higher with optimized BBM
as compared to the actual media. Moreover, the effect of light and urea on C. pyrenoidosa growth were studied and it was
found that turbidity, chlorophyll a, chlorophyll b and chlorophyll a, b were maximum when an artificial light of 3500 lx was
used. The nitrogen starvation during the growth of C. pyrenoidoa showed a maximum lipid content of 36.03% dry weight
on 16th day which is quite sufficient for the production of biodiesel. Hence, C. pyrenoidosa could be cultivated with the
optimum BBM for biodiesel production.

Keywords  C. pyrenoidosa · Bold basal media · Plackett–Burman design · Response surface methodology · Turbidity

Abbreviations CCD Central composite design

A665 Absorbance at 665 nm PBD Plackett–Burman design
A650 Absorbance at 650 nm
OD680 Optical density at 680 nm
Y1 Turbidity at 680 nm Introduction
Y2 Chlorophyll a content in mg/L
Y3 Chlorophyll b content in mg/L The irreversible depletion of fossil fuels and its rising
Y4 Chlorophyll a, b content in mg/L price, accumulation of greenhouse gases in the atmos-
DF Degrees of freedom phere due to burning of fossil fuels demands for a clean
BBM Bold basal media sustainable alternative fuel. Biodiesel from edible, non-
ANOVA Analysis of variance edible crops and microalgae is considered as a sustainable
RSM Response surface methodology alternative fuel for diesel engines due to its biodegradabil-
ity, renewability, non-toxicity, reduced emissions [1]. The
* A. Shaija biodiesel production from edible crops such as soybean,
shaija@nitc.ac.in palm, coconut, rapeseed, sunflower, peanut are limited
due to the requirement of more land for cultivation and it
1
Mechanical Engineering Department, National Institute gives rise to food versus energy competition [2]. The use
of Technology Calicut, Calicut, Kerala 673 601, India
of non-edible crops such as jatropha, pongamia, rubber
2
School of Biotechnology, National Institute of Technology seed, neem, tobacco for the biodiesel production creates
Calicut, Calicut, Kerala 673 601, India

13
Vol.:(0123456789)

scarcity of agricultural land for food production [3, 4]. The
biodiesel production from microalgae has an edge over
edible and non-edible crops with lesser requirement of
land for cultivation.
Microalgae are able to synthesize and accumulate sub-
stantially higher amount of lipids than terrestrial plant and
their biomass doubling time ranges from 3.5 to 24 h [3, 5].
The other advantage of microalgae is its ability to consume
higher amounts of ­CO2 for its production,i.e for producing
1 kg of microalgae, 1.83 kg of C ­ O2 is consumed [6]. They
can grown in land which are unsuitable for agriculture and
in wastewater. The biodiesel production from microalgae
involves stages like cultivation, harvesting, biomass pro-
cessing, lipid extraction and transesterification. The factors
such as light, temperature, nutrient concentration, pH, ­CO2
affects the growth of microalgae [7]. After the cultivation,
microalgae can be harvested using centrifugation, filtration,
flocculation, flotation and processing involves drying and
powdering [8, 9]. The extraction of lipid from this processed
microalgae can be done by mechanical or chemical meth-
ods [2]. Finally, this extracted lipid can be converted to its
biodiesel using transesterification process [10]. Among all
these steps, cultivation of microalgae is the most costly step
and economical production requires optimized conditions of
factors such as nutrients concentration, light, temperature,
pH and ­CO2.
The concentration of nutrients in the cultivation media is
one of the important factors affecting the growth of micro-
algae. The nutrients can be either macro nutrients, such as
nitrogen, magnesium, potassium, calcium or micronutrients,
such as sodium and iron which are essential for the growth
of microalgae [11]. The composition of these nutrients var-
ies depending upon the type of media. The components
of media for macronutrients are ­MgSO4⋅7H2O, ­K2HPO4,
­CaCl2⋅2H2O, ­KNO3 while for micronutrients are ­FeSO4,
NaCl, ­Na2EDTA. Optimization of these components are
essential for the efficient and economic production of micro-
algae. The classical method of optimization which uses only
one variable at a time by keeping all other variables constant
is time consuming and cannot be effective in representing
the combined effects of all the factors involved [12, 13].
Whereas, the statistical method of optimization, response
surface methodology (RSM) can use a number of variables
at a time with minimum number of experiments. RSM is a
combination of mathematical and statistical techniques used
for developing, improving and optimizing the processes and
used to evaluate the relative significance of each factors even
in the presence of complex interactions [14, 15]. The appli-
cation of RSM in combination with PBD is widely described
in many scientific fields [13, 16, 17]. Some researchers have
reported on lipid enhancement of microalgae using nitrogen
starvation for biodiesel production [18, 19]. The production
of microalgae can be estimated with the measurement of
13
Waste and Biomass Valorization
turbidity, chlorophyll a, chlorophyll b and a, b during its
growth period [20, 21].
In order to increase the lipid content of microalgae, vari-
ous cultivation conditions like nitrogen starvation, phos-
phorus and iron limitation and increased C ­ O2 supply have
been reported [22–26]. Feng et al. [25] studied the effect of
nitrogen and phosphorus on lipid accumulation and biomass
productivity of C. zofingensis and reported maximum lipid
content in nitrogen deficient media.
In this work, the best pH for the growth of C. pyrenoidosa
was identified using classical optimization and then screen-
ing of significant factors such as ­MgSO4⋅7H2O, ­K2HPO4,
­CaCl2⋅2H2O, ­KNO3, NaCl, N ­ a2EDTA and F ­ eSO4 was done
using Plackett–Burman design (PBD) in order to reduce the
number of experimental runs in RSM through the construc-
tion of CCD. Then, RSM through the construction of CCD
was used for that was further built for optimizing the above
said identified significant factors obtained from PBD for the
cultivation of C. pyrenoidosa. The growth of C. pyrenoidosa
was then compared by using actual BBM and optimized
BBM composition from RSM study considering light and
urea. The lipid enhancement of C. pyrenoidosa was also
studied using nitrogen starvation experiments.
Materials and Methods
Strain Collection and Inoculum Preparation
Chlorella pyrenoidosa, (Strain No. 2738) a species of
microalgae was procured from National Centre for Indus-
trial Microorganisms (NCIM), Pune, India. The stock
culture of C. pyrenoidosa was phototrophically grown in
250 mL flasks, in actual BBM at room temperature under
natural light. The composition of 1 L of the actual BBM
is ­KNO3-0.25 g, ­K2HPO4-0.074 g, ­MgSO4⋅7H2O-0.073 g,
­C aCl 2 .2H 2 O-0.024  g, NaCl-0.025  g, ­F eSO 4 -0.005  g,
­Na2EDTA-0.045 g. The pH was adjusted to 7. The cultur-
ing was done by adding 10% inoculum to the sterilized
BBM and incubated for 16 days. In order to avoid error and
chances of contamination, individual tubes were used to
measure turbidity and chlorophyll content of C. pyrenoidosa
in alternate days.
Determination of Turbidity and Chlorophyll Content
Turbidity and chlorophyll contents were measured on
alternate days of the cultivation. As a measure of total
cell density, the turbidity of the culture was measured at
680 nm using a UV–Visible spectrophotometer with glass
cuvettes of 1 mL sample capacity [21, 27]. Even though all
the experiments were performed in aseptic conditions, to
exclude the interference of bacterial/fungal contamination if
Waste and Biomass Valorization

any, chlorophyll content was also taken into consideration. Table 1  Actual values of factors at two level in Plackett–Burman
The chlorophyll content was measured using the method of design (PBD)
Fu et al. [28]. One mL of C. pyrenoidosa culture was taken Symbol Factors Low (− 1), g/L High (+ 1), g/L
in an eppendorf tube and centrifuged at 8000 rpm for 5 min
A MgSO4⋅7H2O 0.04 0.08
and the supernatant was discarded. 90% methanol was then
B K2HPO4 0.04 0.08
added to the C. pyrenoidosa pellet formed after centrifuga-
C CaCl2⋅2H2O 0.015 0.03
tion. This caused 50–100 times dilution of C. pyrenoidosa
D KNO3 0.125 0.25
depending on the cell concentration. The C. pyrenoidosa
E NaCl 0.015 0.03
with methanol in eppendorf tube was bathed at 50 °C for
F FeSO4 0.0025 0.005
50 min and centrifuged at 12,000 rpm for 5 min. The absorb-
G Na2EDTA 0.025 0.05
ance of the supernatant was measured at 650 and 665 nm
using UV–Visible spectrophotometer. The contents of chlo-
rophyll a, chlorophyll b and chlorophyll a, b was calculated
using the following equations [28, 29]. Table 2  Comparion of turbidity and chlorophyll content measured
using classical optimization
Chlorophyll a = 16.5 × A665 − 8.3 × A650 (1)
pH Turbidity Chlorophyll a Chlorophyll b Chlorophyll a, b
Chlorophyll b = 33.8 × A650 − 12.5 × A665 (2) 6 0.060 0.738 0.220 0.959
7 0.186 2.582 1.061 3.643
Chlorophyll a, b = 25.5 × A650 + 4.0 × A665 (3)
8 0.285 2.841 0.994 3.834
Classical Optimization and Purity Check

The classical optimization was performed by varying one
factor pH of actual BBM medium ranging from 6, 7 and
8. The experiments were conducted in conical flasks of
250 mL capacity. The chances of bacterial and fungal growth
in open flasks were analysed by streaking lines on nutrient
agar plates in alternative days and were kept in room tem-
perature for 2 days. The chlorophyll content and turbidity
were measured on alternate days of the total 16 days of the
culture period.
Screening of Factors Using Plackett–Burman Design
(PBD)
The screening of significant factors such as ­MgSO4⋅7H2O,
­K2HPO4, ­CaCl2⋅2H2O, ­KNO3, NaCl, ­Na2EDTA, ­FeSO4
and pH was required to reduce the number of experimen-
tal runs used in RSM. The PBD is a fraction of two level
factorial designs (− 1 and + 1) which allows investigation
of ‘n-1’ independent variables with at least ‘n’ experiments
[30]. The PBD was performed with 7 factors (all chemicals
used for BBM and pH 8) as shown in Table 1 for C. pyr-
enoidosa cultivation. The factors having significant effects
on the chlorophyll content and turbidity of C. pyrenoidosa
were identified using Minitab 17. As shown in Table 2, 12
experiments of PBD was set up in triplicates to study the
effect of seven factors. For a total 36 runs, 30 mL of media
was prepared in 36 test tubes of 50 mL capacity and 10% of
C. pyrenoidosa culture with an initial tubidity of 0.176 was
added to each test tube as inoculum. The measurements of
turbidity and chlorophyll content were done from 0th to 16th
day on alternate days.
Optimization by RSM Through the Construction
of a Central Composite Design
RSM was applied through the construction of a CCD
for optimizing the growth of C. pyrenoidosa. The PBD
experiments showed four factors which are significant for
improving the turbidity and chlorophyll content of C. pyr-
enoidosa. In order to optimize the growth of C. pyrenoi-
dosa, CCD with four factors evaluated at five levels (− 2,
− 1, 0, + 1 and + 2) were employed for the experimen-
tal design as shown in Table 4. The factorial experiment
with 31 runs (16 cube points, 7 centre points and 8 axial
points) were designed in Minitab 17. The triplicates for
each run were done for in order to minimize the error in
the experimental reading. 10% of C. pyrenoidosa inocu-
lum was used in the triplicates of 31 culture tubes con-
taining 5 mL media and was placed in an orbital shaker
with 150 rpm at room temperature under natural light.
Experimental readings were taken every 4th day from 0th
to 16th day. In order to minimize errors, tubes were kept
separately and only after proper mixing of the culture was
ensured, turbidity and chlorophyll content were measured.
The response variable (Y) representing the growth of C.
pyrenoidosa was fitted using a second order polynomial
equation Eq. (4) as,
Y = 𝛽o + 𝛽1 X1 + 𝛽2 X2 + 𝛽3 X3
+ 𝛽4 X4 + 𝛽12 X1 X2 + 𝛽13 X1 X3 + 𝛽14 X1 X4
(4)
+ 𝛽23 X2 X3 + 𝛽24 X2 X4 + 𝛽34 X3 X4 + 𝛽11 X1 2
+ 𝛽22 X2 2 + 𝛽33 X3 2 + 𝛽44 X4 2

13

where Y was the predicted response, β o a constant,
­ 1–X4 were the input variables, β1–β4 were the linear coef-
X metrically using Bligh and Dyer’s method [31] and drying
ficients, β12–β34 were the second order interactive coeffi-
cients, and β11–β44 were the quadratic coefficients.
Comparison of Growth of C. pyrenoidosa Using
Actual BBM and Optimized BBM
Six conical flasks of 1 L capacity were used for introducing
the prepared media, where three flasks were used for actual
BBM and remaining for the optimized BBM. 50 mL of C.
pyrenoidosa culture with an initial turbidity of 0.178 was
added to each flask as inoculum. The growth of C. pyrenoi-
dosa was measured and compared using actual and opti-
mized BBM composition which was obtained from RSM
study. The growth was measured based on turbidity meas-
ured at 680 nm and by measuring the chlorophyll a, chloro-
phyll b and a, b content of C. pyrenoidosa.
Comparison of Effect of Light and Urea
on the Growth of C. pyrenoidosa
Two sets of experiments were conducted under natural and
artificial light using urea as substitute for ­KNO3 in actual
BBM and optimized BBM to study the growth of C. pyr-
enoidosa. The artificial light was provided by a compact
fluorescent lamp of 14 W and the intensity was measured
as 3500 lx using a lux meter. The natural light intensity was
600 lx and experiments were conducted at room tempera-
ture. The growth of C. pyrenoidosa was measured by meas-
uring the turbidity at 680 nm, chlorophyll a, chlorophyll b
and a, b on 16th of cultivation.
Effect of Nitrogen Starvation on Lipid Enhancement
of C. pyrenoidosa
The effect of nitrogen starvation on lipid enhancement of C.
pyrenoidosa was studied using feed and starve method. In the
first day of experiment, C. pyrenoidosa was inoculated in 7
conical flasks having 2 L capacity by adding 10% inoculum
with an initial turbidity of 0.180 at 680 nm and incubated for
7 days. From these culture flasks, the biomass of the 6 flasks
were used for nitrogen starvation experiments and remaining
one was used as control using the same nitrogen concentra-
tion of optimized BBM. The C. pyrenoidosa harvested from
each flasks were inoculated to the newly prepared media on
8th day of cultivation. The optimized BBM was prepared
but with varying concentration of ­KNO3 (0, 10, 20, 40, 60,
80%) in six conical flasks. The lipid contents were measured
on 16th day of cultivation. 50 mL of C. pyrenoidosa culture
was taken in a preweighed centrifuge tube and centrifuged at
3000 rpm for 10 min. The supernatant was discarded and the
C. prenoidosa pellet was dried using hot air oven at 60 °C.
13
Waste and Biomass Valorization
The total lipid content of C. pyrenoidosa was estimated gravi-
was continued until the weight was constant.
Results and Discussion
The classical optimization is used for confirming the pH
of the actual BBM and then the screening of significant
media components for the growth of C. pyrenoidosa was
done using PBD with 7 factor experiments. The turbidity
and chlorophyll content of C. pyrenoidosa culture were
measured as response. It was applied RSM through con-
struction of a CCD was used in order to find the optimized
composition of BBM for the economical cultivation of C.
pyrenoidosa. Analysis of variance was done to investigate
the statistical significance of each media components, and
based on the application of the desirability function, the
optimum composition of BBM was obtained. The growth
of C. pyrenoidosa was compared between actual BBM and
optimized BBM composition. Also, the effect of light and
urea on the growth of C. pyrenoidosa was studied and com-
pared with to actual BBM composition. The enhancement of
lipid concentration was investigated by applying the nitrogen
starvation which trigger lipid production.
Classical Optimization and Purity Check
The classical optimization results reveals that culture at
pH 8 showed turbidity, chlorophyll a, chlorophyll b and
chlorophyll a, b were 0.285, 2.841, 0.994 and 3.834 mg/L
respectively as shown in Table 2 compared to pH 6 and 7.
The streaking of lines over the nutrient agar plate showed
contamination of fungus and bacteria for pH 6 and 7 as com-
pared to pH 8. Thus, maintaining pH at 8 was found to be
conducive to reduce bacterial contamination in open pond
set up for further studies.
Screening of Significant Factors
As shown in Table 3, the run number 2 of PBD experi-
ment with 7 factor showed maximum values of turbid-
ity, chlorophyll a, chlorophyll b and chlorophyll a, b were
0.161, 1.633, 0.653 and 2.285 mg/L for the corresponding
BBM compositions M ­ gSO4.7H2O-0.08 g, ­K2HPO4-0.08 g,
­C aCl 2 .2H 2 O-0.015  g, ­K NO 3 -0.3  g, NaCl-0.015  g,
­FeSO4-0.0025 g, ­Na2EDTA-0.025 g.
As shown in Table 4, the screening of factors using 7 fac-
tor PBD experiment showed factors such as M ­ gSO4·7H2O,
­CaCl2·2H2O, ­KNO3 and N ­ a2EDTA were found to be significant
­ 2 value
for increasing the turbidity with p value < 0.05 with R
of 97.93%. Also, the same factors were found significant for
chlorophyll a with ­R2 value of 98.71%. Hence, the maximum
Waste and Biomass Valorization

Table 3  Experimental design Run A B C D E F G Y1 Y2 Y3 Y4

and results using the PBD of
screening 7 factors 1 1 − 1 1 − 1 − 1 − 1 1 0.090 0.570 0.272 0.842
2 1 1 − 1 1 − 1 − 1 − 1 0.161 1.633 0.653 2.285
3 − 1 1 1 − 1 1 − 1 − 1 0.080 0.469 0.235 0.704
4 1 − 1 1 1 − 1 1 − 1 0.158 1.526 0.571 2.096
5 1 1 − 1 1 1 − 1 1 0.136 1.284 0.429 1.714
6 1 1 1 − 1 1 1 − 1 0.078 0.548 0.229 0.777
7 − 1 1 1 1 − 1 1 1 0.120 0.795 0.355 1.149
8 − 1 − 1 1 1 1 − 1 1 0.152 1.426 0.439 1.865
9 − 1 − 1 − 1 1 1 1 − 1 0.160 1.575 0.622 2.197
10 1 − 1 − 1 − 1 1 1 1 0.106 0.615 0.284 0.899
11 − 1 1 − 1 − 1 − 1 1 1 0.060 0.133 0.080 0.213
12 − 1 − 1 − 1 − 1 − 1 − 1 − 1 0.115 0.652 0.335 0.986

Table 4  Screening of factors using 7 factor PBD experiment As shown in Table 6, regression model terms with high
Factor Effect p value 2
R value
F value and minimum p value are highly significant for
turbidity. It was clear that regression model with F value
Turbidity ­(Y1) 10.78 and p value less than 0.01 depicts its significance for
 MgSO4⋅7H2O 0.01489 0.029 97.93% predicting the turbidity of C. pyrenoidosa. The regression
 CaCl2⋅2H2O − 0.02944 0.003 model consisted of linear ­(X1, ­X2, ­X3, ­X4), squared ­(X12,
 KNO3 0.03433 0.002 ­X22, ­X32, ­X42) and interactive terms (­ X1*X2, ­X1*X3, ­X1*X4,
 Na2EDTA 0.03300 0.002 ­X2,X3, ­X2*X4, ­X3*X4) used for the experimental study.
Chlorophyll a ­(Y2) Among the linear terms, X ­ 2, ­X3 and X­ 4 and square terms,
 MgSO4⋅7H2O 0.1876 0.029 98.71% ­X32 and ­X42 and also interactive terms, ­X2*X3 and ­X3*X4
 KNO3 0.3798 0.001 were significant with p values less than 0.05 which indicates
 CaCl2⋅2H2O − 0.3227 0.001 its significance for predicting the turbidity of C. pyrenoi-
 Na2EDTA − 0.2720 0.003 dosa. As shown in Table 5, regression model terms with F
Chlorophyll b (­ Y3) value 10.07 and p value < 0.01 depicts its significance for
 MgSO4⋅7H2O 96.77% predicting the chlorophyll a content of C. pyrenoidosa. In
 KNO3 0.2722 0.001 the regression model, among the linear terms, X ­ 2 and X
­ 4 and
 CaCl2⋅2H2O 2
the square terms, ­X1 interactive terms, ­X2*X3 and ­X3*X4
 Na2EDTA − 0.1307 0.012 were found to be significant with p values less than 0.01.
Chlorophyll a, b ­(Y4) As shown in Table 5, regression model with F value 5.74
 MgSO4⋅7H2O 0.2496 0.027 98.76% and p value < 0.01 depicts its significance for predicting the
 KNO3 1.1476 0.000 chlorophyll b content of C. pyrenoidosa. Among the diferent
 CaCl2⋅2H2O terms in the regression model, ­X3 and ­X4, ­X12, ­X1*X2 and
 Na2EDTA − 0.3939 0.006 ­X1*X3 was found to be significant with p values less than
0.05. As shown in Table 6, the regression model with higher
F value 8.78 and p value < 0.01 implies its significance for
number of significant factors shown in Table 3 obtained from predicting the chlorophyll a, b content of C. pyrenoidosa.
PBD is considered for CCD irrespective of responses. Hence, Among the different terms in regression model ­X2 and ­X4,
the factors such as M
­ gSO4⋅7H2O, ­CaCl2⋅2H2O, ­KNO3 and ­X12, ­X1*X2, ­X1*X3, ­X2*X3, ­X2*X3 and ­X3*X4 were signifi-
­Na2EDTA were considered for further study. cant with p values less than 0.01 which indicates its signifi-
cance for predicting the turbidity of C. pyrenoidosa.
Optimization of BBM Media Using RSM Through The estimated values of regression coefficients were
Construction of CCD used for forming regression model which is a second order
polynomial equations [Eqs. (5–8)] for turbidity (­ Y1), chlo-
As shown in Table 5, the best run was identified as 23 with rophyll a ­(Y2), chlorophyll b (­ Y3) and chlorophyll a, b (­ Y4)
maximum turbidity, chlorophyll a, chlorophyll b and chlo- as follows:
rophyll a, b.

13
 Waste and Biomass Valorization

Table 5  Central composite Run MgSO4⋅7H2O CaCl2⋅2H2O KNO3 Na2EDTA Experimental values Predicted values
design and resulting medium
composition Y1 Y2 Y3 Y4 Y1 Y2 Y3 Y4

1 0.08 0.0300 0.150 0.0250 0.44 1.62 0.79 2.41 0.42 1.42 0.65 2.07
2 0.06 0.0225 0.225 0.0375 0.45 2.27 1.02 3.29 0.45 2.12 1.05 3.18
3 0.04 0.0300 0.300 0.0500 0.55 2.79 1.13 3.92 0.54 2.81 1.06 3.90
4 0.06 0.0075 0.225 0.0375 0.48 2.15 1.34 3.49 0.43 1.81 1.07 2.89
5 0.06 0.0225 0.225 0.0625 0.62 2.95 1.39 4.34 0.63 2.75 1.21 4.00
6 0.04 0.0150 0.150 0.0250 0.39 1.93 0.80 2.72 0.45 2.09 0.87 2.96
7 0.04 0.0300 0.150 0.0250 0.43 1.76 0.83 2.59 0.41 1.45 0.74 2.20
8 0.06 0.0225 0.075 0.0375 0.22 1.44 0.68 2.12 0.22 1.79 0.79 2.59
9 0.08 0.0300 0.300 0.0250 0.45 2.13 1.19 3.31 0.47 2.10 1.12 3.23
10 0.06 0.0225 0.225 0.0375 0.43 2.16 1.08 3.24 0.45 2.12 1.05 3.18
11 0.06 0.0225 0.225 0.0375 0.43 2.08 1.16 3.24 0.45 2.12 1.05 3.18
12 0.06 0.0225 0.225 0.0375 0.44 1.92 1.13 3.05 0.45 2.12 1.05 3.18
13 0.06 0.0375 0.225 0.0375 0.47 2.46 1.20 3.66 0.50 2.82 1.37 4.21
14 0.08 0.0150 0.150 0.0250 0.41 1.73 0.39 2.12 0.41 1.51 0.38 1.89
15 0.04 0.0150 0.300 0.0500 0.45 1.35 0.68 2.02 0.49 1.71 1.03 2.77
16 0.04 0.0150 0.150 0.0500 0.44 2.29 1.13 3.42 0.42 2.12 1.11 3.25
17 0.08 0.0300 0.150 0.0500 0.39 2.36 1.07 3.43 0.40 2.07 0.94 3.05
18 0.04 0.0300 0.150 0.0500 0.38 1.68 0.70 2.39 0.39 1.75 0.80 2.58
19 0.06 0.0225 0.225 0.0125 0.55 1.01 0.33 1.34 0.52 1.22 0.42 1.64
20 0.04 0.0300 0.300 0.0250 0.38 1.73 0.68 2.40 0.40 1.67 0.74 2.42
21 0.10 0.0225 0.225 0.0375 0.44 1.19 0.55 1.75 0.45 1.31 0.60 1.93
22 0.06 0.0225 0.225 0.0375 0.47 2.20 1.00 3.20 0.45 2.12 1.05 3.18
23 0.08 0.0300 0.300 0.0500 0.64 3.60 1.53 5.13 0.60 3.60 1.67 5.30
24 0.04 0.0150 0.300 0.0250 0.37 0.75 0.49 1.25 0.36 0.84 0.53 1.38
25 0.06 0.0225 0.375 0.0375 0.35 2.40 1.38 3.78 0.33 2.07 1.17 3.26
26 0.06 0.0225 0.225 0.0375 0.47 2.10 0.93 3.03 0.45 2.12 1.05 3.18
27 0.06 0.0225 0.225 0.0375 0.49 2.12 0.97 3.09 0.45 2.12 1.05 3.18
28 0.08 0.0150 0.300 0.0250 0.35 0.63 0.40 1.03 0.37 0.72 0.51 1.25
29 0.08 0.0150 0.300 0.0500 0.47 1.85 1.22 3.07 0.49 1.96 1.23 3.22
30 0.02 0.0225 0.225 0.0375 0.46 1.20 0.63 1.83 0.43 1.10 0.48 1.60
31 0.08 0.0150 0.150 0.0500 0.38 1.68 0.70 2.39 0.38 1.90 0.85 2.78

Y1 = 0.886 − 2.22X1 − 15.37X2 + 1.019X3 − 22.22X4
2 2 2
− 9.4X12 + 44X2 + 8.01X3 + 191.8X4
(5)
+ 91.7X1 ∗ X2 + 7.50X1 ∗ X3 − 5X1
∗ X4 + 40X2 ∗ X3 + 26.7X2 ∗ X4 + 42.67X3 ∗ X4
Y4 = 8.21 + 17.9X1 − 314.5 X2 − 30.93 X3 − 16.2 X4
− 885 X1 2 + 1643X2 2 − 11.3X3 2 − 575X4 2 + 1575X1
∗ X2 + 155 X1 ∗ X3 + 590 X1 ∗ X4 + 802 X2 ∗ X3
+ 253 X2 ∗ X4 + 291 X3 ∗ X4 (8)

Y2 = 6.39 + 19.9X1 − 233.3X2 − 23.06X3 − 42X4 where ­X 1, ­X 2, ­X 3, ­X 4 denotes the concentration of

2 2 2
− 572X1 + 868X2 − 8.43X3 − 208X4 + 908X1
∗ X2 + 77.5X1 ∗ X3 + 360X1 ∗ X4 + 653X2 ∗ X3 (6)
+ 707X2 ∗ X4 + 225.3 X3 ∗ X4
Y3 = 1.86 − 2.1X1 − 82.2X2 − 8.05X3 + 25.7X4
− 315.2X1 2 + 781X2 2 − 2.86X3 2 − 375X4 2
+ 679X1 ∗ X2 + 77.9X1 ∗ X3 + 228X1 ∗ X4
+ 152.2X2 ∗ X3 − 473X2 ∗ X4 + 67.3X3 ∗ X4
2 ­MgSO4⋅7H2O, ­CaCl2⋅2H2O, ­KNO3 and N ­ a2EDTA which
varies from − 2 to + 2 levels shown in Table 4, respectively.
From the Eq. (5), it can be seen that the concentration of
­KNO3 has positive effect on turbidity of the C. pyrenoidosa.
The correlation coefficient, R ­ 2 obtained from the statistical
analysis using Minitab-17 was 90.1% which is close to 100, it
shows that the experimental values of turbidity are very close
to the ones predicted by the regression model. It can be seen
(7)
from the Eq. (6) that the concentration of M ­ gSO4⋅7H2O has

13
Waste and Biomass Valorization

Table 6  Analysis of variance Source Sum of squares Mean Squares F- value p value Significance
for turbidity, Chlorophyll a,
Chlorophyll b and Chlorophyll Turbidity
a, b
 Regression model 0.1730 0.01236 10.78 0.000 Significant
 Linear 0.0406 0.01015 8.85 0.001 Significant
 MgSO4⋅7H2O 0.0004 0.00041 0.36 0.555 Not significant
 CaCl2⋅2H2O 0.0060 0.00601 5.24 0.036 Significant
 KNO3 0.0181 0.01815 15.82 0.001 Significant
 Na2EDTA 0.0160 0.01601 13.96 0.002 Significant
 Square 0.0935 0.02339 20.40 0.000 Significant
 MgSO4⋅7H2O*MgSO4⋅7H2O 0.0004 0.00040 0.36 0.559 Not significant
 CaCl2⋅2H2O*CaCl2⋅2H2O 0.0001 0.00017 0.15 0.702 Not significant
 KNO3*KNO3 0.0579 0.05798 50.54 0.000 Significant
 Na2EDTA*Na2EDTA 0.0256 0.02568 22.39 0.000 Significant
 2 way interactions 0.0388 0.00647 5.65 0.003 Significant
 MgSO4⋅7H2O*CaCl2⋅2H2O 0.0030 0.00302 2.64 0.124 Not significant
 MgSO4⋅7H2O*KNO3 0.0020 0.00202 1.77 0.203 Not significant
 MgSO4⋅7H2O*Na2EDTA 0.0000 0.00002 0.02 0.884 Not significant
 CaCl2⋅2H2O*KNO3 0.0081 0.00810 7.06 0.017 Significant
 CaCl2⋅2H2O*Na2EDTA 0.0001 0.00010 0.09 0.772 Not significant
 KNO3*Na2EDTA 0.0256 0.02560 22.32 0.000 Significant
Chlorophyll a
 Regression model 10.5271 0.7519 10.07 0.000 Significant
 Linear 5.2570 1.3144 17.60 0.000 Significant
 MgSO4⋅7H2O 0.0704 0.0704 0.94 0.346 Not significant
 CaCl2⋅2H2O 1.5403 1.5403 20.63 0.000 Significant
 KNO3 0.1204 0.0120 1.61 0.222 Not significant
 Na2EDTA 3.5267 3.5267 47.23 0.000 Significant
 Square 1.6814 0.4203 5.63 0.005 Significant
 MgSO4⋅7H2O*MgSO4⋅7H2O 1.4955 1.4955 20.03 0.000 Significant
 CaCl2⋅2H2O*CaCl2⋅2H2O 0.0681 0.0681 0.91 0.354 Not significant
 KNO3*KNO3 0.0644 0.0643 0.86 0.367 Not significant
 Na2EDTA*Na2EDTA 0.0301 0.0300 0.40 0.535 Not significant
 2 way interactions 3.5880 3.5980 8.01 0.000 Significant
 MgSO4⋅7H2O*CaCl2⋅2H2O 0.2970 0.2970 3.98 0.063 Not significant
 MgSO4⋅7H2O*KNO3 0.2162 0.2162 2.90 0.108 Not significant
 MgSO4⋅7H2O*Na2EDTA 0.1296 0.1296 1.74 0.205 Not significant
 CaCl2⋅2H2O*KNO3 2.1609 2.1609 28.94 0.000 Significant
 CaCl2⋅2H2O*Na2EDTA 0.0702 0.0702 0.94 0.347 Not significant
 KNO3*Na2EDTA 0.7140 0.7140 9.56 0.007 Significant
Chlorophyll b
 Regression model 2.580 0.1842 5.74 0.001 Significant
 Linear 1.306 0.3265 10.17 0.000 Significant
 MgSO4⋅7H2O 0.019 0.0198 0.62 0.443 Not significant
 CaCl2⋅2H2O 0.139 0.1395 4.35 0.053 Not significant
 KNO3 0.222 0.2223 6.93 0.018 Significant
 Na2EDTA 0.924 0.9243 28.80 0.000 Significant
 Square 0.625 0.1562 4.87 0.009 Significant
 MgSO4⋅7H2O*MgSO4⋅7H2O 0.454 0.4546 14.16 0.002 Significant
 CaCl2⋅2H2O*CaCl2⋅2H2O 0.055 0.0551 1.72 0.208 Not significant
 KNO3*KNO3 0.007 0.0074 0.23 0.638 Not significant
 Na2EDTA*Na2EDTA 0.098 0.0981 3.06 0.099 Not significant
 2 way interactions 0.648 0.1081 3.37 0.024 Significant

13
 Waste and Biomass Valorization

Table 6  (continued) Source Sum of squares Mean Squares F- value p value Significance

 MgSO4⋅7H2O*CaCl2⋅2H2O 0.166 0.1660 5.17 0.037 Significant

 MgSO4⋅7H2O*KNO3 0.218 0.2185 6.81 0.019 Significant
 MgSO4⋅7H2O*Na2EDTA 0.051 0.0517 1.61 0.222 Not significant
 CaCl2⋅2H2O*KNO3 0.117 0.1173 3.65 0.074 Not significant
 CaCl2⋅2H2O*Na2EDTA 0.031 0.0315 0.98 0.337 Not significant
 KNO3*Na2EDTA 0.063 0.0637 1.99 0.178 Not significant
Chlorophyll a, b
 Regression model 22.317 1.5941 8.78 0.000 Significant
 Linear 11.527 2.8818 15.86 0.000 Significant
 MgSO4⋅7H2O 0.170 0.1700 0.94 0.348 Significant
 CaCl2⋅2H2O 2.600 2.6004 14.32 0.002 Significant
 KNO3 0.660 0.6600 3.63 0.075 Not significant
 Na2EDTA 8.096 8.0968 44.57 0.000 Significant
 Square 4.229 1.0573 5.82 0.004 Significant
 MgSO4⋅7H2O*MgSO4.7H2O 3.580 3.5802 19.71 0.000 Significant
 CaCl2⋅2H2O*CaCl2.2H2O 0.244 0.2442 1.34 0.263 Not significant
 KNO3*KNO3 0.116 0.1165 0.64 0.435 Not significant
 Na2EDTA*Na2EDTA 0.238 0.2385 1.31 0.269 Not significant
 2 way interactions 6.561 1.0935 6.02 0.002 Significant
 MgSO4⋅7H2O*CaCl2.2H2O 0.893 0.8930 4.92 0.041 Significant
 MgSO4⋅7H2O*KNO3 0.864 0.8649 4.76 0.044 Significant
 MgSO4⋅7H2O*Na2EDTA 0.348 0.3481 1.92 0.185 Not significant
 CaCl2⋅2H2O*KNO3 3.258 3.2580 17.94 0.001 Significant
 CaCl2⋅2H2O*Na2EDTA 0.009 0.0090 0.05 0.826 Not significant
 KNO3*Na2EDTA 1.188 1.1881 6.54 0.021 Significant

positive effect on chlorophyll a content of C. pyrenoidosa.
The correlation coefficient, R­ 2 obtained from the statistical
analysis using Minitab-17 was 90.3%, which is close to 100,
it shows that the experimental values of chlorophyll a con-
tent are very close to the ones predicted by the regression
model. It can be seen from the Eq. (7), the concentration of
­Na2EDTA has positive effect on chlorophyll b content of C.
pyrenoidosa. The correlation coefficient, R ­ 2 obtained from
the statistical analysis using Minitab-17 was 84.4% which
is above 80% reported by Daghrir et al. [32], shows that the
experimental values of chlorophyll b content are very close
to the ones predicted by the regression model. From Eq. (8),
the concentration of ­MgSO4⋅7H2O has positive effect on
chlorophyll a, b content of C. pyrenoidosa. The correlation
coefficient, ­R2 obtained from the statistical analysis using
Minitab-17 was 89.3% which is very close to 100, it shows
that the experimental values of chlorophyll a, b content are
very close to the ones predicted by the regression model.
Two-dimensional contour plots are mapped considering
two experimental factors at a time by keeping the other two
factors as hold value at its central level. Figure 1a shows
the effect of ­MgSO4⋅7H2O and ­KNO3 on turbidity (OD)
of C. pyrenoidosa culture when the other two factors such
as ­CaCl2⋅2H2O and ­Na2EDTA were 0.0225, 0.0375 g/L,
respectively as hold values. According to Song et al. [33],
circular shaped contour plot indicates that the interaction
between corresponding experimental factors are not sig-
nificant whereas elliptical shape indicates the interactions
between the corresponding experimental factors are signifi-
cant. The turbidity of the C. pyrenoidosa was increased with
increase in concentration of ­KNO3 and ­CaCl2⋅2H2O which
indicates that these nutrients were favorable for cell growth.
Figure 1b shows the effect of C ­ aCl2⋅2H2O, ­Na2EDTA on
turbidity of the C. pyrenoidosa culture when the other two
factors such as ­MgSO4⋅7H2O, ­KNO3 were 0.06, 0.225 g/L,
respectively as hold values. The turbidity of the C. pyrenoi-
dosa culture was increased with increase in concentration of
­CaCl2⋅2H2O and ­Na2EDTA.
Figure  2a shows the effect of ­M gSO 4 ⋅7H 2 O and
­Na2EDTA on chlorophyll a content of C. pyrenoidosa cul-
ture when the other two factors such as C ­ aCl2⋅2H2O, ­KNO3
were 0.0225, 0.225 g/L, respectively as hold values. It was
clear that the chlorophyll a content increased with increase
in concentration of ­MgSO4⋅7H2O and ­Na2EDTA in the cul-
tivation media. Figure 2c shows the effect of M­ gSO4⋅7H2O
and ­KNO3 on chlorophyll a content of C. pyrenoidosa by
keeping other two factors such as C ­ aCl2⋅2H2O, ­Na2EDTA
were 0.0225, 0.0375 g/L, respectively as hold value. It was

13
Waste and Biomass Valorization
Fig. 1  a, b Contour plot showing the effect of ­ MgSO4⋅7H2O,
­CaCl2⋅2H2O, ­KNO3 and ­Na2EDTA for turbidity of C. pyrenoidosa 
clear that the chlorophyll a content was increased to above
2.1 mg/L when the ­KNO3 and ­MgSO4⋅7H2O concentration
were ranges from 0.2 to 0.35, 0.05–0.084 g/L, respectively.
The presence of more amount of nitrogen from ­KNO3 in the
media can enhance the growth of C. pyrenoidosa.
Figure 3a shows the contour plot showing the effect of
­MgSO4⋅7H2O and N ­ a2EDTA on chlorophyll b content of
C. pyrenoidosa by keeping the other two factors such as
­CaCl2⋅2H2O, ­KNO3 were 0.0225, 0.225 g/L, respectively
as hold values. It was seen that the chlorophyll b content
was above 1.2 mg/L when the concentration of N ­ a2EDTA
and ­MgSO4⋅7H2O in the cultivation media ranges from
0.048 to 0.062, 0.065–0.095 g/L, respectively. As similar
to chlorophyll a content, the increased concentration of
­MgSO4⋅7H2O in the cultivation media increased the chlo-
rophyll b content of C. pyrenoidosa. Figure 3b shows the
effect of ­MgSO4⋅7H2O and ­KNO3 on chlorophyll b content
­ aCl2⋅2H2O, ­Na2EDTA
by keeping other two factors such as C
were 0.0225, 0.0375 g/L, respectively as hold values. It was
clear that the increase in concentration of ­MgSO4⋅7H2O
and ­KNO3 showed increased chlorophyll b content in C.
pyrenoidosa.
Fig. 2  a, b, Contour plots for showing the effect of ­MgSO4⋅7H2O,
­ a2EDTA and ­KNO3, on chlorophyll a content
N
Figure  4a shows the effect of ­M gSO 4 ⋅7H 2 O and
­ a2EDTA on chlorophyll a, b content of C. pyrenoidosa by
N
keeping the other two factors such as ­CaCl2⋅2H2O and ­KNO3
were 0.0225, 0.225 g/L, respectively as hold values. Since,
magnesium is an essential component for the production of
chlorophyll content with the increase in concentration of
­MgSO4⋅7H2O in the cultivation media increased chlorophyll
a, b content. Figure 5b shows the effect of ­MgSO4⋅7H2O
and ­KNO3 on chlorophyll a, b by keeping the other two
factors such as C ­ aCl2⋅2H2O and N ­ a2EDTA were 0.0225,
0.0375 g/L, respectively as hold value. It can be seen that
the increase in concentration of ­KNO3 and M ­ gSO4⋅7H2O
increased the chlorophyll a, b content of C. pyrenoidosa.
In general, the increase in concentration of M ­ gSO4⋅7H2O
increased the chlorophyll a, b content which is due to the
importance of magnesium for the formation of chlorophyll
content.
The optimum combination of media composition
were obtained using desirability function (d) proposed
by Derringer and Suich [34]. The d value is in the range
of 0 ≤ d ≥ 1. If d = 1, implies that the factors which influ-
ence the growth of C. pyrenoidosa are in optimum level.
The maximum condition of turbidity and chlorophyll a,
13
 Waste and Biomass Valorization

Fig. 4  a, b Contour plots for showing the effect of ­MgSO4⋅7H2O,

­ a2EDTA, ­KNO3, on chlorophyll a, b content
N
Fig. 3  a, b Contour plots for showing the effect of ­MgSO4⋅7H2O,
­ a2EDTA, ­KNO3, on chlorophyll b content
N

chlorophyll b and chlorophyll a, b condition was used

with d = 1. In this work, the media composition in g/L
such as 0.1, 0.0375, 0.375, and 0.0625 were obtained
for ­M gSO 4⋅7H 2O, ­C aCl 2⋅2H 2O, ­K NO 3 and ­Na 2EDTA,
respectively for d = 1 was obtained using Minitab 17.
The corresponding predicted values of the response val-
ues as 0.8757, 6.539, 2.584, and 9.123 mg/L were tur-
bidity, chlorophyll a, chlorophyll b and chlorophyll a, b
respectively. Experiments were conducted for checking
the adequacy and validity of optimal concentration of
media compositions showed 0.809, 5.7504, 1.6137 and Fig. 5  Comparison of yield of optimum BBM obtained from RSM
7.3642 mg/L, respectively and the values were closer to and actual BBM composition
the predicted value.

Comparison of Growth of C. pyrenoidosa with Actual
Media
The comparison of actual BBM and optimum BBM compo-
sition obtained from this study was done based on the tur-
bidity and chlorophyll content of C. pyrenoidosa. As shown
in Fig. 5, the turbidity, chlorophyll a, chloropyll b and a, b
content using actual BBM and optimum BBM were 0.144,
1.1363, 0.5682 and 1.7405 against 0.809, 5.7504, 1.6137
and 7.3642 mg/L, respectively. This indicates that with the
use of optimum BBM can give a hike of 5 times in turbidity
and chlorophyll content of C. pyrenoidosa.

13
Waste and Biomass Valorization

Comparison of Effect of Light and Urea It indicates that the optimum BBM composition obtained
for the Growth of C. pyrenoidosa from this study is capable of producing more amount of
chlorophyll content in C. pyrenoidosa under natural light.
As shown in Fig. 6, there is a significant effect in the tur- The optimum BBM with urea as substitute for ­KNO3 showed
bidity of C. pyrenoidosa with the use of aritificial, natu- chlorophyll a, chlorophyll b and chlorophyll a, b in mg/L
ral light and urea as a substitute for ­KNO3. The maximum were 1.0124, 0.3921 and 1.4045, respectively. This explores
turbidity of 0.785 was obtained with optimum BBM under the option of using urea as a substitute for ­KNO3 which
artificial light of 3500 lx followed by 0.702 for optimum further reduces the cost of media preparation for large scale
BBM with urea. From this, it is clear that urea can be used cultivation.
as a substitute for ­KNO3 which further reduces the cost of
media preparation. It can be seen that the use of natural light Effect of Nitrogen Starvation on Lipid Enhancement
of 600 lx showed lesser values of tubidity which indicates of C. pyrenoidosa
that light has major effect on the growth of C. pyrenoidosa.
Using natural light, the optimum BBM showed a turbidity The effect of nitrogen starvation on lipid content of C. pyr-
of 0.1383 which is less than 5.6 times of turbidity obtained enoidosa was shown in Table 7. It was seen that with 0%
with artificial light. nitrogen concentration in optimized BBM after 8th day of
As shown in Fig. 7, the C. pyrenoidosa cultivated using cultivation showed a lipid content of 36.03% which is 2.07
optimum BBM showed higher values of chlorophyll a, b times higher as compared to the 17.14% obtained with opti-
and a, b as compared to other media compositions. The mum BBM (100% nitrogen concentration) throughout 16
chlorophyll a, chlorophyll b and a, b of C. pyrenoidosa days of cultivation whereas a 1/6th reduction of biomass
obtained using optimum BBM were in mg/L 7.9841, 3.112
and 11.0961, respectively as compared to 4.9363, 2.3126
and 7.2489 for actual BBM. It indicates that the optimum
BBM composition obtained from this study is capable of
more amount of chlorophyll content under artificial light.
The optimum BBM with urea as substitute for ­KNO3 showed
in mg/L were 7.8897, 2.6814 and 10.5711 of chlorophyll a,
chlorophyll b and a, b, respectively. This explores the option
of using urea as a substitute for K
­ NO3 which further reduces
the cost of media preparation.
As shown in Fig. 8, the C. pyrenoidosa cultivated using
optimum BBM showed higher values of chlorophyll a, b
and a, b as compared to other media compositions. The
chlorophyll a, chlorophyll b and a, b of C. pyrenoidosa was
found to be 1.2451, 0.5261 and 1.7712, mg/L respectively
as compared to 0.8288, 0.3921 and 1.4045 for actual BBM. Fig. 7  Effect of aritifical light and urea for the chlorophyll content of
C. pyrenoidosa 

Fig. 8  Effect of natural light and urea for the chlorophyll content of

Fig. 6  Effect of light and urea for turbidity of C. pyrenoidosa  C. pyrenoidosa 

13
 Waste and Biomass Valorization

Table 7  Effect of nitrogen starvation on lipid enhancement of C. pyr- • By using the optimized composition of BBM for C. pyr-
enoidosa  enoidosa cultivation can increase the chlorophyll content
S.No KNO3 in % Biomass concen- Lipid content, % and turbidity.
tration, g/L • The turbidity and chlorophyll content of C. pyrenoidosa
cultivated with optimum BBM showed 5 times higher
1 100 (Control) 0.1551 17.40
yield (turbidity and chlorophyll content) as compared to
2 0 0.0247 36.03
the actual BBM.
3 10 0.0398 29.14
• Hence, using this optimum BBM composition the cultiva-
4 20 0.0743 27.18
tion of C. pyrenoidosa can be performed with higher yield
5 40 0.0955 24.61
for biodiesel production.
6 60 0.1046 22.94
• A 5.6 times hike in turbidity of C. pyrenoidosa was
7 80 0.1151 19.20
obtained with optimum BBM using artificial light of
3500 lx as compared to actual BBM composition with
natural light.
concentration was observed with former one. Also, the • The chlorophyll a, chlorophyll b and a, b of C. pyrenoidosa
growth of C. pyrenoidosa is much depended on nitrogen obtained using optimum BBM were in mg/L 7.9841, 3.112
availability in the media, the biomass concentration was and 11.0961, respectively as compared to 4.9363, 2.3126
found minimum whereas the lipid content was increased and 7.2489 for actual BBM. It indicates that the optimum
maximum which is due to the transformations of protein BBM composition obtained from this study is capable of
or peptides present in the C. pyrenoidosa into lipids. As more amount of chlorophyll content under artificial light.
the nitrogen concentration in the media is increased the • Using natural light of 600 lx, chlorophyll a, chlorophyll b
enhancement of biomass concentration was found. Accord- and a, b of C.pyrenoidosa obtained using optimum BBM
ing to Nigam et al. [35] as the nitrogen concentration in the were in mg/L 1.2451, 0.5261 and 1.7712, respectively as
media decreased the biomass concentration also decreased compared to 0.8288, 0.3921 and 1.4045 for actual BBM.
with increase in lipid content. • The nitrogen starvation for lipid enhancement of C. pyr-
enoidosa showed a lipid content of 36.03% dryweight as
compared to 17.14% with optimum BBM.
Conclusions

The optimization of the BBM was done applying RSM
through the construction of a CCD. The classical opti-
mization is used for identifying the best pH of the actual
BBM and found to be 8. The screening of significant fac-
tors was done using PBD with 7 factor experiment. The
significant factors such as ­MgSO4⋅7H2O, ­CaCl2⋅2H2O,
­KNO3, ­Na2EDTA were identified from PBD experiment.
The RSM applied through the construction of CCD taking
in to account the significant factors identified in the previ-
ous experimental phase. The following conclusions could be
drawn from this study:
• RSM applied through the construction of CCD with four
significant factors gave an optimum composition of BBM
based on higher turbidity and chlorophyll content of C.
pyrenoidosa.
• The growth of C. pyrenoidosa requires higher level
of nutrients. The optimal BBM composition achieved
from the RSM applied through the construction of CCD
experiments were 0.1  g/L ­MgSO4⋅7H2O, 0.0375  g/L
­CaCl2⋅2H2O, 0.375 g/L K ­ NO3 and 0.0625 g/L N
­ a2EDTA.
• Under optimal conditions, the turbidity, chlorophyll a,
chlorophyll b and chlorophyll a, b was 0.809, 5.7504,
1.6137 and 7.3642 mg/L, respectively.
Acknowledgements  The authors would like to thank Mechanical Engi-
neering Department and School of Biotechnology, National Institute of
Technology Calicut, and India for providing necessary lab facility for
doing this work. The authors are thankful to Kerala State Council for
Science, Technology and Environment (KSCSTE), Kerala, India for
financially supporting (Order No. 1237/2015/KSCSTE) this investiga-
tion. A funding agency was involved in this work and facilities given
by institute were acknowledged separately. This manuscript has not
submitted elsewhere for the consideration of other journals and the
manuscript has not been published partly/ fully. The results and conclu-
sions given in the manuscript are original. No data, text, or theories by
others are presented without mentioning the reference and as if they
were the author’s own (“plagiarism”).
Compliance with Ethical Standards 
Conflict of interest  The authors declare that they have no conflict of
interest.
Ethical Approval  This manuscript was prepared according to the Com-
mittee on Publication Ethics (COPE) standard guidelines.
References
1. Abbaszaadeh, A., Ghobadian, B., Omidkhah, M.R., Najafi,
G.: Current biodiesel production technologies: a comparative

13
Waste and Biomass Valorization
review. Energy Convers. Manag. 63, 138–148 (2012). https​://doi.
org/10.1016/j.encon​man.2012.02.027
2. Mubarak, M., Shaija, A., Suchithra, T.V.: A review on the extrac-
tion of lipid from microalgae for biodiesel production. Algal Res. 7,
117–123 (2015). https​://doi.org/10.1016/j.algal​.2014.10.008
3. Rawat, I., Ranjith Kumar, R., Mutanda, T., Bux, F.: Biodiesel
from microalgae: a critical evaluation from laboratory to large
scale production. Appl. Energy. 103, 444–467 (2013). https​://doi.
org/10.1016/j.apene​rgy.2012.10.004
4. Atabani, A.E., Silitonga, A.S., Ong, H.C., Mahlia, T.M.I., Masjuki,
H.H., Badruddin, I.A., Fayaz, H.: Non-edible vegetable oils: a criti-
cal evaluation of oil extraction, fatty acid compositions, biodiesel
production, characteristics, engine performance and emissions pro-
duction. Renew. Sustain. Energy Rev. 18, 211–245 (2013). https​://
doi.org/10.1016/j.rser.2012.10.013
5. Laurent Lardon, A.H., Sailve, B., Steyer, J.-P.: Olivier bernard life-
cycle assessment of biodiesel production from microalgae. Environ.
Sci. Technol. 43(17), 6475–6481 (2009)
6. Chisti, Y.: Biodiesel from microalgae. Biotechnol. Adv. 25(3), 294–
306 (2007). https​://doi.org/10.1016/j.biote​chadv​.2007.02.001
7. Mata, T.M., Martins, A.A., Caetano, N.S.: Microalgae for bio-
diesel production and other applications: a review. Renew. Sus-
tain. Energy Rev. 14(1), 217–232 (2010). https​://doi.org/10.1016/j.
rser.2009.07.020
8. Suali, E., Sarbatly, R.: Conversion of microalgae to biofuel.
Renew. Sustain. Energy Rev. 16(6), 4316–4342 (2012). https​://doi.
org/10.1016/j.rser.2012.03.047
9. Demirbas, A.: Use of algae as biofuel sources. Energy Convers.
Manag. 51(12), 2738–2749 (2010). https​://doi.org/10.1016/j.encon​
man.2010.06.010
10. Daroch, M., Geng, S., Wang, G.: Recent advances in liquid biofuel
production from algal feedstocks. Appl. Energy. 102, 1371–1381
(2013). https​://doi.org/10.1016/j.apene​rgy.2012.07.031
11. Lam, M.K., Lee, K.T.: Effect of carbon source towards the growth
of Chlorella vulgaris for C­ O2 bio-mitigation and biodiesel produc-
tion. Int. J. Greenhouse Gas Control. 14, 169–176 (2013). https​://
doi.org/10.1016/j.ijggc​.2013.01.016
12. Bilanovic, D., Andargatchew, A., Kroeger, T., Shelef, G.: Freshwa-
ter and marine microalgae sequestering of ­CO2 at different C and
N concentrations—response surface methodology analysis. Energy
Convers. Manag. 50(2), 262–267 (2009). https​://doi.org/10.1016/j.
encon​man.2008.09.024
13. Chen, T., Wang, Y.: Optimized astaxanthin production in Chlorella
zofingiensis under dark condition by response surface methodology.
Food Sci. Biotechnol. 22(5), 1–8 (2013). https​://doi.org/10.1007/
s1006​8-013-0221-7
14. Ravikumar, K., Pakshirajan, K., Swaminathan, T., Balu, K.: Optimi-
zation of batch process parameters using response surface method-
ology for dye removal by a novel adsorbent. Chem. Eng. J. 105(3),
131–138 (2005). https​://doi.org/10.1016/j.cej.2004.10.008
15. Gonen, F., Aksu, Z.: Use of response surface methodology (RSM) in
the evaluation of growth and copper(II) bioaccumulation properties
of Candida utilis in molasses medium. J. Hazard. Mater. 154(1–3),
731–738 (2008). https​://doi.org/10.1016/j.jhazm​at.2007.10.086
16. Deepak, V., Kalishwaralal, K., Ramkumarpandian, S., Babu, S.V.,
Senthilkumar, S.R., Sangiliyandi, G.: Optimization of media compo-
sition for Nattokinase production by Bacillus subtilis using response
surface methodology. Bioresour. Technol. 99(17), 8170–8174
(2008). https​://doi.org/10.1016/j.biort​ech.2008.03.018
17. Karthikeyan, K., Shanthi, N.K., Lakshmanaperumalsamy, K.P.:
Response surface methodology for optimization of culture condi-
tions for dye decolorization by a fungus, Aspergillus niger HM11
isolated from dye affected soil. Iranian J. Microbiol. 2(4), 213–222
(2010)
18. Ruangsomboon, S.: Effects of different media and nitrogen sources
and levels on growth and lipid of green microalga Botryococcus
View publication stats
braunii KMITL and its biodiesel properties based on fatty acid com-
position. Bioresour. Technol. (2015). https​://doi.org/10.1016/j.biort​
ech.2015.01.091
19. Lv, J.M., Cheng, L.H., Xu, X.H., Zhang, L., Chen, H.L.: Enhanced
lipid production of Chlorella vulgaris by adjustment of cultivation
conditions. Bioresour. Technol. 101(17), 6797–6804 (2010). https​
://doi.org/10.1016/j.biort​ech.2010.03.120
20. Cheirsilp, B., Torpee, S.: Enhanced growth and lipid production
of microalgae under mixotrophic culture condition: effect of light
intensity, glucose concentration and fed-batch cultivation. Biore-
sour. Technol. 110, 510–516 (2012). https​://doi.org/10.1016/j.biort​
ech.2012.01.125
21. Wang, H., Xiong, H., Hui, Z., Zeng, X.: Mixotrophic cultivation of
Chlorella pyrenoidosa with diluted primary piggery wastewater to
produce lipids. Bioresour. Technol. 104, 215–220 (2012). https​://
doi.org/10.1016/j.biort​ech.2011.11.020
22. Griffiths, M.J., Harrison, S.T.L.: Lipid productivity as a key charac-
teristic for choosing algal species for biodiesel production. J. Appl.
Phycol. 21(5), 493–507 (2009). https​://doi.org/10.1007/s1081​
1-008-9392-7
23. Ota, M., Kato, Y., Watanabe, M., Sato, Y., Smith, R.L. Jr., Rosello-
Sastre, R., Posten, C., Inomata, H.: Effects of nitrate and oxygen
on photoautotrophic lipid production from Chlorococcum litto-
rale. Bioresour. Technol. 102(3), 3286–3292 (2011). https​://doi.
org/10.1016/j.biort​ech.2010.10.024
24. Liu, Z.Y., Wang, G.C., Zhou, B.C.: Effect of iron on growth and lipid
accumulation in Chlorella vulgaris. Bioresour. Technol. 99(11),
4717–4722 (2008). https​://doi.org/10.1016/j.biort​ech.2007.09.073
25. Feng, P., Deng, Z., Fan, L., Hu, Z.: Lipid accumulation and growth
characteristics of Chlorella zofingiensis under different nitrate
and phosphate concentrations. J. Biosci. Bioeng. 114(4), 405–410
(2012). https​://doi.org/10.1016/j.jbios​c.2012.05.007
26. Baky, A.E., El-Baroty, H.H., Bouaid, G.S., Martinez, A., Aracil,
M.: Enhancement of lipid accumulation in Scenedesmus obliquus
by optimizing C ­ O2 and Fe3+ levels for biodiesel production. Biore-
sour. Technol. 119, 429–432 (2012). https​://doi.org/10.1016/j.biort​
ech.2012.05.104
27. Mouedhen, G., Feki, M., Wery, M.D., Ayedi, H.F.: Behavior of
aluminum electrodes in electrocoagulation process. J. Hazard.
Mater. 150(1), 124–135 (2008). https​://doi.org/10.1016/j.jhazm​
at.2007.04.090
28. Fu, W., Gudmundsson, O., Feist, A.M., Herjolfsson, G., Brynjolfs-
son, S., Palsson, B.O.: Maximizing biomass productivity and cell
density of Chlorella vulgaris by using light-emitting diode-based
photobioreactor. J. Biotechnol. 161(3), 242–249 (2012). https​://doi.
org/10.1016/j.jbiot​ec.2012.07.004
29. Becker, E.W.: Microalgae: Biotechnology and Microbiology. Cam-
bridge University press, Cambridge, pp. 56–62 (1994)
30. Stowe, R.A., Mayer, R.P.: Efficient screening of process variables.
Ind. Eng. Chem. Res. 56, 36–40 (1996)
31. Bligh, E.G., Dyer, W.J.: A rapid method for total lipid extraction and
purification. Can. J. Biochem. Physiol. 37, 911–917 (1959)
32. Daghrir, R., Igounet, L., Brar, S.-K., Drogui, P.: Novel electrochemi-
cal method for the recovery of lipids from microalgae for biodiesel
production. J. Taiwan Inst. Chem. Eng. 45(1), 153–162 (2014). https​
://doi.org/10.1016/j.jtice​.2013.04.013
33. Song, X., Zhang, X., Kuang, C., Zhu, L., Guo, N.: Optimization of
fermentation parameters for the biomass and DHA production of
Schizochytrium limacinum OUC88 using response surface meth-
odology. Process Biochem. 42(10), 1391–1397 (2007). https​://doi.
org/10.1016/j.procb​io.2007.07.014
34. Derringer, G., Suich, R.: Simultaneous optimization of several
response variables. J. Qual. Technol. 12, 214–219 (1980)
35. Nigam, S., Rai, M.P., Sharma, R.: Effect of nitrogen on growth
and lipid content of Chlorella pyrenoidosa. Am. J. Biochem. Bio-
technol. 7, 124–129 (2011)
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