Development of a Single Phase Nitrate Feeding
Strategy for Enhanced Lipid Productivity from
Green Microalgae for Biodiesel Production
Sashi Sonkar and Nirupama Mallick
Agricultural and Food Engineering Department, Indian Institute of Technology Kharagpur, Kharagpur 721302, India;
nm@agfe.iitkgp.ernet.in (for correspondence)
Published online 19 August 2016 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/ep.12443
Biomass productivity and cellular lipid content are two
major components that influence the ultimate lipid produc-
tivity from algal biomass. In this report, a nitrate feeding
strategy was developed taking three green microalgae, Scene-
desmus obliquus, Chlorella vulgaris, and C. minutissima, to
achieve higher lipid productivity under single phase cultiva-
tion. Four different strategies, i.e. nitrate-sufficient, nitrate-
deficient, biphasic nitrate-deficient and low-dose sequential
nitrate feeding, were examined. The biphasic nitrate-
deficient approach depicted the maximum lipid productivity.
To bring this biphasic approach to a single phase, sequential
limited nitrate feeding strategy was examined. This showed
nitrate supplementation with a 1/100th dose of standard N
11 medium at an interval of 3 days resulted in a significant
rise in lipid productivity. The biodiesel samples also demon-
strated the predominance of saturated and monounsaturated
fatty acid methyl esters. This study, therefore, suggests that
with appropriate standardization, the usual biphasic
approach can be brought down to a single phase, thereby
reducing the overall cost of microalgal biomass production
for biodiesel purpose. Moreover, this strategy is user-friendly,
and would reduce the nitrate requirements for large-scale
cultivation, thus, would result in cost-effective and eco-
friendly biodiesel production from microalgae. V C 2016 Ameri-
can Institute of Chemical Engineers Environ Prog, 36: 222–
231, 2017
Keywords: algal biomass, cost-effectiveness, fatty acid
methyl esters, nitrate-deficient, biphasic cultivation, low-dose
nitrate feeding
INTRODUCTION
Biodiesel, composed of fatty acid methyl esters (FAMEs),
obtained from vegetable oils or animal fats by transesterifica-
tion process, is considered as a renewable source of energy.
It is an eco-friendly and CO2 neutral energy source obtained
from biological origin. In comparison to the usual oil crops,
microalgae are considered as valuable feedstocks for produc-
ing biodiesel due to their rapid growth rate and higher lipid
accumulation potential [1,2].
This article was published online on 19 August 2016. An error was
subsequently identified. This notice is included in the online and
print versions to indicate that both have been corrected 29 August
2016.
VC 2016 American Institute of Chemical Engineers
222 January 2017 Environmental Progress & Sustainable Energy (Vol.36, No.1) DOI 10.1002/ep
Microalgae have the ability to modify their metabolism
under specific growth conditions to increase their lipid pool.
For example, crude lipid content in Choricystis minor, under
nitrogen and phosphorus deficiencies, was raised up to 60% of
the dry cell weight (dcw) as compared to 27% in control [3]. In
Chlorococcum infusionum lipid content of 35% (against 15%
control) was recorded, when grown in nitrogen-limited condi-
tion [4]. In Botryococcus sp. under high light intensity, nitrogen
deficiency and high level of iron, lipid content of 35.9% (dcw)
was recorded [5]. In Nannochloropsis sp. F & M-M24 lipid con-
tent of 60% (dcw) was recorded under nitrogen deficiency [6].
Nitrogen limitation/starvation also found to enhance the lipid
content in Chlorella sp. BUM11008, Nannochloropsis oculata,
Neochloris oleabundans, Scenedesmus obliquus, and Chlorella
vulgaris [7–11].
As discussed above, nitrogen-limited/starved cultivation
enhanced the lipid accumulation in algal cells profoundly.
However, the biomass concentration under such conditions
was significantly low for the practical production of biodiesel
from the cell mass. Therefore, there is a need to study and
evaluate various nitrogen feeding strategies for achieving not
only higher cellular lipid accumulation but also elevated lipid
productivity from algal biomass. The aim of this study was
thus set to compare the effects of four different nitrate feed-
ing strategies on biomass concentration, corresponding cellu-
lar lipid concentration, and variations in fatty acid profiles of
three green microalgal species, i.e., Scenedesmus obliquus,
Chlorella vulgaris, and C. minutissima for the development
of a suitable nitrate feeding strategy to obtain maximum lip-
ids from green algal biomass.
MATERIALS AND METHODS
Test Organisms and Culture Conditions
Three green microalgae, namely, Scenedesmus obliquus
(isolated indigenously from river Subarnarekha, West Midna-
pore, West Bengal, India), Chlorella vulgaris (an isolate of river
Ganges, courtesy: Prof. L. C. Rai, Banaras Hindu University,
Varanasi, India), and Chlorella minutissima (isolated indige-
nously from river Kangsavati, West Midnapore, West Bengal,
India) were used in this study. Plankton net (Ricco sampler,
Tokyo, Japan) was used to collect the planktonic samples. The
samples were made unialgal following the standard serial dilu-
tion technique and streaking. The test microalgae were identi-
fied by the Cryptogamic Unit, Botanical Survey of India Head
Quarters (BSI), Howrah, West Bengal, India. Batch cultures
were made under the sterile condition in 100 mL of N 11 medi-
um [12] contained in 250 mL Erlenmeyer flasks without aera-
tion or CO2 sparging. The N 11 medium constituents were:
KNO3, 1.0 g L21; Na2HPO4
2H2O, 0.083 g L21; KH2PO4, 0.052
g L21; MgSO4
7H2O, 0.05 g L21; CaCl2
2H2O, 0.01 g L21, and
Fe-EDTA stock (10 g chelate per liter), and 1 mL of trace metal
mixture contained: MnCl2
4H2O, 0.099 g L21; NiSO4
7H2O,
0.0236 g L21; ZnSO4
7H2O, 0.063 g L21; CuSO4
5H2O, 0.005
g L21; CoSO4
7H2O, 0.0028 g L21; NH4VO3, 0.0029 g L21; and
(NH4)6Mo7O24
4H2O, 0.0018 g L21. Light at an intensity of 75
mmol photon m22 s21 PAR was supplied for a photoperiod of
14 h. At the beginning of each experiment, the pH value of the
culture medium was adjusted at 6.8 6 0.05 by 0.1 M HCl or
0.1 M NaOH solutions and temperature was maintained at
258C 6 28C. Thus, the initial pH of the medium was found to
be 6.8 6 0.05, whereas the pH value of the cultures during har-
vesting was raised up to 8.9 6 0.23. Shaking of the culture
flasks was done three to four times a day by hand to prevent
sticking of the cultures to the bottom of the flasks. These were
the usual static cultures and were also referred as controls. The
final volume of harvest from each flask was 100 mL.
Nitrate Feeding Strategies
Four different strategies were considered for this study:
Strategy 1 involved a single phase incubation of the three
test microalgae in nitrate-sufficient medium (as per N 11
medium), and evaluation of biomass and lipid concentrations
at 3 days intervals. The second strategy included a single
phase strategy to cultivate the test microalgae in nitrate-
deprived medium and evaluate the biomass as well as lipid
concentrations.
Strategy 3 was a two-phase strategy or biphasic approach:
at first, the microalgae were grown in the nitrate-sufficient
medium as to reach the maximum biomass concentration fol-
lowed by a lipid accumulation phase, i.e. transforming to
nitrate-deficient medium for a stipulated time and evaluation
of biomass and lipid concentrations.
The fourth strategy involved a sequential low-dose nitrate
feeding approach, i.e. incubation of the three microalgae in
low concentrations of nitrate-supplemented medium (1/100th,
1/50th, and 1/10th concentrations of N 11 medium) with sub-
sequent nitrate feeding at an interval of 3 days. Biomass and
lipid concentrations were analysed at regular intervals.
Dry Weight Estimation
Dry cell weight (dcw) was estimated as per the protocol of
Rai et al. [13]. A noted quantity of microalgal culture was cen-
trifuged for 10 min at 5000 rpm, after which the harvested bio-
mass was dried at 608C till a constant weight was reached. The
biomass concentration was expressed as g L21.
Nitrate Analysis
The uptake of nitrate was determined by the brucine sul-
furic acid method [14]. A 5 mL of the culture was taken and
centrifuged for 10 min at 5000 rpm. After centrifugation,
2 mL of brucine reagent (4% (w/v) in chloroform) was added
to the supernatant and kept in the ice bath. To this, 5 mL of
concentrated H2SO4 was added slowly drop by drop and
placed in a water bath at 908C for 10 min until the chloro-
form evaporated. The resultant yellow colour was measured
spectrophotometrically at 410 nm. Nitrate present in the sam-
ple was calculated using the standard curve prepared from
KNO3 and expressed as mg L21.
Extraction and Estimation of Lipid Concentration
and Productivity
Lipid extraction was carried out according to the standard
protocol of Bligh and Dyer using chloroform and methanol
Environmental Progress & Sustainable Energy (Vol.36, No.1) DOI 10.1002/ep
[15]. The pellets obtained after centrifugation of 100 mL of
culture was dried in hot air oven. The dried algal biomass
was taken in a glass vial, 2 mL of methanol and 1 mL of
chloroform were added, and kept for 1 day at room temper-
ature. The mixture was agitated in a vortex from time to
time. After 24 h, 1 mL of chloroform was added again to the
mixture and shaken vigorously for 1 min. About 1.8 mL of
distilled water was added to the mixture and agitated in the
vortex again for 2 min. The layers were separated by centri-
fugation for 10 min at 2000 rpm. The lower organic layer
containing the lipids was filtered through Whatman No. 1 fil-
ter paper into a pre-weighed vial (W1). Evaporation was car-
ried out in a hot water bath and the residue was further
dried at 1048C for 30 min in an oven. The weight of the vial
was recorded again (W2). Lipid concentration, content and
productivity were calculated as follows:
Lipid concentration (g L21) 5 (W2 – W1) 3 10
Lipid content (% dry cell weight) 5 Lipid concentration/
Biomass concentration 3 100
Lipid productivity (mg L21 day21) 5 Lipid concentration
(mg L 2 1)/Incubation period (days)
Analysis of Acid Value of the Extracted Lipids
The acid value of the extracted lipids was analysed by the
titration method of European standard EN14104 [16]. About
0.1 g of lipid was taken into a conical flask and 50 mL of sol-
vent mixture (1:1 diethyl ether and 95% ethanol) was added
to it. A few drops of phenolphthalein indicator (1% (w/v) in
millipore water) were also added. Titration was done with
0.1 N KOH solutions till the development of pink color. The
acid value was expressed as mg KOH g21 of lipid.
Acid Value 5 (56.1 3 N 3 V)/W
Where N, V, and W denote the normality of KOH (0.1 N),
titrate (mL), and weight of lipids (g), respectively.
Transesterification and Analysis of Fatty
Acid Methyl Esters (FAMEs)
Considering the high acid values (>45 mg KOH g21) of
the test microalgal lipids, acid-catalysed transesterification of
microalgal lipids was carried out using a molar ratio of oil:
methanol: HCl:: 1: 82: 4 at a reaction temperature of 658C
and reaction time of 6.4 h, as optimized in our laboratory
[17]. The top organic phase, which contained FAMEs was
pipetted out and was taken for GC-MS analysis using Clarus
680 GC (Perkin-Elmer, Shelton, CT) equipped with an Elite-1
methylpolysiloxane capillary column (30 m 3 0.25 mm 3
0.25 lm) and MS (Clarus 600 T MS) equipped with a photo-
multiplied tube detector which has a phosphor screen. The
pressure of the carrier gas (He) was 90 Psi at the initial oven
temperature with the flow rate 20 mL min21. The samples
were injected in the split mode (50:1). The final temperature
of the injector was 2508C; the oven temperature was 508C,
rose to 2508C at a rate of 108C min21 (total run time 22 min).
The mass spectrometer was operated in electron impact (EI)
mode at 70 eV with the scan range of 50–650 m/z. The injec-
tion sample volume was 0.2 lL. Different FAMEs were identi-
fied by comparing their mass fragmentation with NIST mass
spectral reference library [17].
Statistical Analysis
MSTAT-C software (Plant and Soil Sciences Division, Mich-
igan State University, USA) was used for statistical analysis of
the results by Duncan’s new multiple range test (DMRT). To
check the reproducibility, all experiments were done in trip-
licate and repeated thrice.
January 2017 223
RESULTS AND DISCUSSION
Impact of Single Phase Cultivation in Nitrate-sufficient
and Nitrate-deprived Media on Biomass and Lipid
Concentrations
Figure 1A shows the time-course studies on the three test
microalgae under single phase cultivation in N 11 medium of
the standard composition. One phase strategy in nutrient suffi-
cient medium demonstrated a biomass concentration of 1.10 6
0.02 g L21 for S. obliquus on day 21 of incubation. Maximum
lipid concentration was 0.12 6 0.01 g L21 (Figure 1B). For C.
vulgaris, the biomass and lipid concentrations were 1.31 6 0.01
and 0.18 6 0.02 g L21, respectively. Similarly, for C. minutis-
sima, these values were 1.39 6 0.03 and 0.25 6 0.02 g L21 in
the above order (Figures 1A and 1B). The cellular lipid content
was 11.1 6 0.3, 13.8 6 0.6 18.0 6 0.9 (% dcw), respectively for
S. obliquus, C. vulgaris, and C. minutissima (Figure 1B).
Single phase cultivation of S. obliquus, C. vulgaris, and C.
minutissima in nitrate-deprived medium resulted into maxi-
mum biomass concentration of 0.19 6 0.02, 0.18 6 0.01, and
0.20 6 0.02 g L21, respectively, for an incubation period of 7
days, after which the cultures showed a declining trend (data
not shown). The lipid concentration was only 0.08 6 0.01
g L21 (41.5 6 0.9% dcw), 0.08 6 0.01 g L21 (44.8 6 0.8% dcw)
and 0.10 6 0.01 g L21 (50.9 6 1.5% dcw), respectively, in the
above order (Figures 2A and 2B). It is clearly evident from
Figure 2A that there was a profound rise in cellular lipid con-
tent in the three test microalgae under nitrogen deprivation.
However, the overall lipid concentration was significantly
lower as compared to the values of nitrate sufficient cultiva-
tion (Figure 1B). These results are consistent with several
Figure 1. (A) Growth behaviour and (B) lipid accumulation
in S. obliquus, C. vulgaris and C. minutissima grown in
nitrate-sufficient N 11 medium.
224 January 2017 Environmental Progress & Sustainable Energy (Vol.36, No.1) DOI 10.1002/ep
earlier reports as prearranged in the introduction section.
Further, in S. obliquus, under N-deficiency, cellular lipid con-
tent reached up to 32–40% (dcw) against 10% control [18].
Similarly, C. vulgaris showed a lipid content of 35.6% as
compared to 12.29% control [19]. C. minutissima MACC 360,
under nitrogen deficiency, also showed a lipid content of
40–46%, whereas the control value was 11–12% [20]. The
probable reason for such an observation may be that under
nitrogen deficiency/limitations, any carbon dioxide fixed is
stored as lipids rather than protein due to reduction/inhibi-
tion of protein synthesis [21]. Under nitrate limitation,
NADPH consumption was decreased due to unavailability of
nitrogen pool, which blocks the amino acid synthesis path-
ways, especially the reaction from a-ketoglutarate to gluta-
mate, thus resulting into accumulation of excess NADPH in
the cells [22,23]. Under such condition, acetyl-CoA could not
enter into the tricarboxylic acid (TCA) cycle as high concen-
tration of NADPH inhibit the enzyme citrate synthase, one of
the key enzymes of TCA cycle, leading to an increase in the
pool of acetyl-CoA [24]. The latter might be converted into
malonyl-CoA, catalyzed by acetyl-CoA carboxylase (ACCase),
the central carbon donor for fatty acid synthesis [25]. The first
step in the fatty acid biosynthesis is the conversion of acetyl-
CoA into malonyl-CoA by addition of CO2 in an ATP depen-
dent reaction in the presence of acetyl-CoA carboxylase
(ACCase) [26]. The gene encoding the biotin containing sub-
unit of ACCase, biotin carboxylase (BC), was extensively up-
regulated in response to nitrogen limitation/starvation [27].
To begin with the biosynthesis of fatty acids, malonly-CoA is
transferred by malonyl-CoA ACP transacylase (MAT) into
acyl-carrier protein (ACP). This step is followed by a number
of enzymes such as beta-ketoacyl-ACP synthase (KAS), beta-
ketoacyl-ACP reductase (KAR), beta-hydroxyacyl-ACP
Figure 2. (A) Maximum lipid content and (B) lipid concen-
tration under control, nitrate-deprived and biphasic cultiva-
tion of the three test microalgae.
Table 1. Maximum lipid concentration and productivity recorded for S. obliquus, C. vulgaris, and C. minutissima under
sequential low-dose nitrate feeding condition.

Limited nitrate Incubation Lipid Lipid Lipid con-

feeding KNO3 period concentration productivity tent
Microalga (g L21) (days) (g L21) (mg L21 day21) (% dcw)
S. obliquus 0.1 27 0.25 6 0.01a 9.2 6 0.5a 22.2 6 0.1b
0.02 27 0.34 6 0.01b 12.8 6 0.5b 31.2 6 0.3d
0.01 30 0.39 6 0.02c 13.1 6 0.5b 34.5 6 0.2e
C. vulgaris 0.1 27 0.26 6 0.01a 9.6 6 0.5a 17.8 6 0.7a
0.02 30 0.36 6 0.02b 11.9 6 0.5b 26.3 6 0.2c
0.01 30 0.38 6 0.01c 12.7 6 0.4b 33.0 6 0.4b
C. minutissima 0.1 30 0.33 6 0.02b 11.1 6 0.5b 22.7 6 0.1b
0.02 30 0.38 6 0.01c 12.5 6 0.5b 30.0 6 0.1d
0.01 30 0.48 6 0.01d 16.0 6 0.4c 50.5 6 0.4f

Values are mean 6 SE, n 5 3. Values within a column superscripted by different alphabets (a–f) are significantly different from
each other (P < 0.05, DMRT). A separate analysis was done for each column. 0.1, 0.02, and 0.01 were respectively 1/10th, 1/
50th, and 1/100th doses with respect to N 11 medium (1.0 g L21 KNO3).

Table 2. Comparison of maximum biomass and lipid concentrations, and lipid productivity recorded for the three test microal-
gae under various strategies.

Incubation Biomass Lipid Lipid Lipid

period concentration concentration productivity content
Microalga Strategy Culture condition (days) (g L21) (g L21) (mg L21 day21) (% dcw)
S. obliquus 1 N 11 medium 21 1.10 6 0.02c 0.12 6 0.01a 5.8 6 0.7a 11.1 6 0.3a
2 Nitrate-starved 7 0.19 6 0.02a 0.08 6 0.01a 11.1 6 1.2c 41.5 6 0.9c
3 Biphasic nitrate-starved 21 1 7 0.83 6 0.01b 0.36 6 0.02d 13.0 6 0.6c 43.8 6 1.3c
4 Limited nitrate feeding 30 1.14 6 0.05c 0.39 6 0.02d 13.1 6 0.5c 34.5 6 0.2b
(0.01 g L21 of KNO3)
C. vulgaris 1 N 11 medium 21 1.31 6 0.01d 0.18 6 0.02b 8.7 6 0.8b 13.8 6 0.6a
2 Nitrate-starved 7 0.18 6 0.01a 0.08 6 0.01a 11.5 6 1.3c 44.8 6 0.8c
3 Biphasic nitrate-starved 21 1 7 1.07 6 0.04c 0.50 6 0.01e 17.7 6 0.5d 46.2 6 1.1c
4 Limited nitrate feeding 30 1.15 6 0.03c 0.38 6 0.01d 12.7 6 0.4c 33.0 6 0.4b
(0.01 g L21 of KNO3)
C. minutissima 1 N 11 medium 21 1.39 6 0.03d 0.25 6 0.02c 11.9 6 0.8c 18.0 6 0.9a
2 Nitrate-starved 7 0.20 6 0.02a 0.10 6 0.01a 14.5 6 1.6c 50.9 6 1.5d
3 Biphasic nitrate-starved 21 1 7 1.12 6 0.01c 0.59 6 0.01f 20.9 6 0.5e 51.9 6 1.7d
4 Limited nitrate feeding 30 0.95 6 0.05b 0.48 6 0.01e 16.0 6 0.4d 50.5 6 0.4d
(0.01 g L21 of KNO3)

Values are mean 6 SE, n 5 3. Values within a column superscripted by different alphabets (a–f) are significantly different from
each other (P < 0.05, DMRT). A separate analysis was done for each column. 0.01 g L21 was the 1/100th dose of N 11 medium
(1.0 g L21 KNO3).

dehydrase (HAD) and enoyl-ACP reductase (EAR), which cata-
lyse the condensation, reduction, dehydration, and further
reduction reactions, respectively. The expression of genes
responsible for encoding KAR was suppressed, whereas MAT,
KAS, HAD and EAR were up-regulated in the absence of nitro-
gen in the cells. After 6-7 cycles, the number of C atoms
became 16 (C16:0-[ACP]) or 18 (C18:0-[ACP]); as a result, the
synthesis of fatty acids hindered. The thioesterases oleoyl-ACP
hydrolase (OAH) and Acyl-ACP thioesterase A (FatA) generate
the end product of fatty acid synthesis (i.e. palmitic (C16:0)
and stearic (C18:0) acids) by removing the ACP residues [28].
Genes encode for OAH and FatA were over-expressed in the
absence of nitrogen in the cells. The over expression of OAH
encoding genes reduces the feedback inhibition that partly
controls the production rate of fatty acid biosynthesis which
results in the overproduction of fatty acids, as shown in Phaeo-
dactylum tricornutum [29]. It has been further suggested that
due to increasing in expression of FatA gene and the related
acyl-ACP hydrolysis may help in increased transportation of fat-
ty acids to the endoplasmic reticulum site from the chloroplast
where triacylglycerol (TAG) assembly occurs [30,31]. Nitrogen
limitation has also been seen to activate the enzyme diacylgly-
cerol acyltransferase, which converts diacylglycerols to triglycer-
ides [32]. Moreover, in the absence of nitrogen, pentose
phosphate pathway encoding genes were up-regulated for sup-
plying reducing equivalents via NADPH for fatty acid biosynthe-
sis. The proportion of unsaturation in fatty acids was increased
due to the changed in the level of genes expression associated
with the formation of double bonds [28].
In the single phase nitrate-starved medium the biomass
concentration was reduced severely in all the test microalgae.
Therefore, although the cellular lipid content under such
condition was raised profoundly (Figure 2A), the overall lipid
concentration was found to be significantly hampered

Environmental Progress & Sustainable Energy (Vol.36, No.1) DOI 10.1002/ep January 2017 225
Figure 3. Comparative growth behaviour of (A) S. obliquus, (B) C. vulgaris, and (C) C. minutissima grown in complete N 11, and
limited nitrate-fed media. Variations in nitrate concentrations in the experimental sets with N 11 medium, and limited nitrate feed-
ing sets of 1/10th, 1/50th, and 1/100th doses for the three test microalgae {D) S. obliquus, (E) C. vulgaris, and (F) C. minutissima.
Nitrate concentrations: 1. N 11 medium (scale down by 100 times); 2. Limited nitrate feeding sets of 1/10th dose (scale down by 10
times); 3. 1/50th dose (scale down by 10 times); 4. 1/100th dose shows the actual values.

(Figure 2B) as compared to the values recorded for cultures
grown in complete N 11 medium (Figure 1B). This confirms
the earlier notation that high lipid content and lipid yield are
reciprocally exclusive [33].
Impact of Biphasic Cultivation and Sequential Low-
dose Nitrate Feeding on Biomass and Lipid
Concentrations
In the two-phase strategy (biphasic approach), which
consisted of incubation of the test microalgae in nitrate-
sufficient medium for 21 days followed by cultivation in
nitrogen-starved medium for 1 week, a reduction in the over-
all biomass concentration was clearly evident in the second
phase (data not shown). However, the corresponding lipid
concentration was increased profoundly, i.e., 0.36 6 0.02,
0.50 6 0.01, and 0.59 6 0.01 g L21, respectively for S. obli-
quus, C. vulgaris, and C. minutissima for an incubation peri-
od of 28 (21 1 7) days (Figure 2B). Cellular lipid content also
showed significantly higher values (Figure 2A). This is well
in agreement with the report of Josephine et al. [34], where a
significant enhancement in lipid content of Chlorella vulgaris

226 January 2017 Environmental Progress & Sustainable Energy (Vol.36, No.1) DOI 10.1002/ep
Table 3. Relative % of fatty acid methyl esters (FAMEs) of S. obliquus biodiesel grown under various strategies.

Relative % of FAMEs
FAMEs Strategy 1 Strategy 2 Strategy 3 Strategy 4
Palmitic acid 43.3 6 1.1a 46.2 6 1.3b 45.9 6 0.9b 46.1 6 1.1b
Stearic acid Nil 3.5 6 0.3a 8.9 6 0.2c 5.1 6 0.8b
Oleic acid 35.0 6 1.2a 42.8 6 1.7c 40.3 6 2.2c 38.8 6 2.1b
Linoleic acid 11.2 6 0.9c 4.16 0.7a 3.2 6 0.4a 6.5 6 0.5b
Linolenic acid 10.5 6 0.8c 3.4 6 0.3b 1.7 6 0.2a 3.5 6 0.5b
% of SFA 1 MUFA 78.3 6 3.2a 92.5 6 3.3b 95.1 6 3.3b 90.0 6 4.0b

Values are mean 6 SE, n 5 3. Values within a row superscripted by different alphabets (a–c) are significantly different from
each other (P < 0.05, DMRT). A separate analysis was done for each row. MUFA—monounsaturated fatty acids, SFA—saturated
fatty acids. Peak area < 0.1% were considered to be negligible.

Table 4. Relative % of fatty acid methyl esters (FAMEs) of C. vulgaris biodiesel grown under various strategies.

Relative % of FAMEs
FAMEs Strategy 1 Strategy 2 Strategy 3 Strategy 4
Palmitic acid 43.4 6 1.4a 51.1 6 1.1b 50.2 6 1.2b 48.3 6 0.5b
Stearic acid 25.5 6 0.8a 30.9 6 0.7b 32.6 6 0.9b 27.5 6 0.7a
Oleic acid 12.3 6 0.2b 8.8 6 0.1a 7.9 6 0.2a 10.0 6 0.3a
Linoleic acid 18.8 6 0.5c 9.2 6 0.2a 7.2 6 0.4a 12.5 6 0.4b
Linolenic acid Nil Nil 2.1 6 0.1a 1.7 6 0.3a
% of SFA 1 MUFA 81.2 6 2.4a 90.8 6 1.9c 90.7 6 2.3c 85.8 6 1.5b

Values are mean 6 SE, n 5 3. Values within a row superscripted by different alphabets (a–c) are significantly different from
each other (P < 0.05, DMRT). A separate analysis was done for each row. Peak area < 0.1% were considered to be negligible.

after exposing to nitrogen starved condition for 7 days was
evident. Under the biphasic nitrate-starved approach, lipid
productivity for S. obliquus, C. vulgaris, and C. minutissima
were found to be 13.0 6 0.6, 17.7 6 0.5, and 20.9 6 0.5
mg L21 day21, respectively, as compared to 5.8 6 0.7,
8.7 6 0.8, and 11.9 6 0.8 mg L21 day21, respectively under
nitrate-sufficient cultivation (Table 2).
To make the above two-phase strategy to single phase,
sequential feeding experiments with low-dose nitrate were
conducted at various concentrations of nitrate. The time-
course analysis of the growth of S. obliquus under such condi-
tion is presented in Figure 3A. The growth of S. obliquus in
0.1 (1/10th) and 0.02 g L21 (1/50th) nitrate feeding conditions
increased steadily and was comparable with N 11 control
(nitrate concentration: 1 g L21). However, in 0.01 g L21 nitrate
feeding condition, i.e. with 1/100th dose, the growth was
affected. For C. vulgaris and C. minutissima, except for the 1/
10th dose, reduction in biomass concentration was clearly evi-
dent for 0.01 (1/100th) and 0.02 (1/50th) doses (Figures 3B
and 3C). Figures 3D–3F represent the time-course of variation
in nitrate concentrations in the culture flasks with N 11 and
low-dose nitrate-fed conditions. A detectable level of nitrate
was maintained throughout the experimental period even in
low-dose nitrate-supplemented vessels.
Table 1 presents the lipid concentrations of the three test
microalgae under sequential low-dose nitrate feeding condi-
tions. Limited feeding of nitrate in the medium led to a sig-
nificant rise in lipid concentration in the test microalgae. The
1/50th and 1/100th sequential doses led to increasing in lipid
concentration up to 0.34 6 0.01 and 0.39 6 0.02 g L21 in S.
obliquus. In the case of 1/10th dose (0.1 g L21 nitrate feed-
ing), the value was only 0.25 6 0.01 g L21. For C. vulgaris,
lipid concentrations up to 0.38 6 0.01, 0.36 6 0.02, and
0.26 6 0.01 g L21, respectively for 1/100th, 1/50th and 1/10th
doses of nitrate was recorded. These values were respective-
ly 0.48 6 0.01, 0.38 6 0.01, and 0.33 6 0.02 g L21 for C. minu-
tissima in the above order. Thus, there was two-three fold
rise in lipid concentration under the low-dose sequential
feeding as compared to cultivation in nutrient-sufficient N 11
medium (Figure 1B). The lipid productivity of S. obliquus, C.
vulgaris, and C. minutissima were 13.1 6 0.5, 12.7 6 0.4, and
16.0 6 0.4 mg L21 day21, respectively for the 1/100th dose of
nitrate (0.01 g L21 nitrate) feeding. Overall, 0.01 g L21 (1/
100th dose) sequential nitrate feeding was found to be the
best among the three concentrations studied for higher lipid
production under single phase approach. The possible rea-
son could be that the microalgae might be experiencing par-
tial nitrogen deficiency under the low nitrate-fed condition,
which stimulates lipid accumulation in microalgal cells.
Analysis of Acid Value of Algal Lipids
The acid value of oil obtained from the S. obliquus, C.
vulgaris, and C. minutissima were found to be 47.3 6 1.1,
45.1 6 2.9, and 45.2 6 3.5 mg KOH g21, respectively. Because
the extracted lipids from test microalgae have high free fatty
acid content, the choice of acid- based transesterification
over alkaline was justified.
Comparison of Biomass and Lipid Concentrations
under Various Strategies
Table 2 provides a comparative account on the biomass
as well as lipid concentration under the four cultivation strat-
egies. Lipid concentration was found to rise profoundly
under the biphasic culture condition in the three test

Environmental Progress & Sustainable Energy (Vol.36, No.1) DOI 10.1002/ep January 2017 227
Table 5. Relative % of fatty acid methyl esters (FAMEs) of C. minutissima biodiesel grown under various strategies.
FAMEs Strategy 1
Palmitic acid 41.9 6 1.2a
Stearic acid 14.8 6 0.8b
Oleic acid 21.0 6 0.5a
Linoleic acid 11.0 6 0.2c
Linolenic acid 11.3 6 0.4a
% of SFA 1 MUFA 77.7 6 2.5a
Values are mean 6 SE, n 5 3. Values within a row superscripted by different alphabets (a–c) are significantly different from
each other (P < 0.05, DMRT). A separate analysis was done for each row. Peak area < 0.1% were considered to be negligible.
microalgae. The cellular lipid content also showed a high
value, not varying significantly from the single phase nitrate-
starved cultures. Under limited nitrate feeding, lipid concen-
tration was found to be two-three folds higher than the cells
grown in complete N 11 medium, which was even marginal-
ly higher than the value recorded under the biphasic cultiva-
tion for S. obliquus.
Under single phase nitrate-starved condition, the lipid
productivity for S. obliquus, C. vulgaris, and C. minutissima
was recorded as 11.1 6 1.2, 11.5 6 1.3, and 14.5 6 1.6
mg L21 day21, respectively. Whereas, in biphasic nitrate-
starved approach, lipid productivity was found to be 13.0 6
0.6, 17.7 6 0.5, and 20.9 6 0.5 mg L21 day21, as compared to
5.8 6 0.7, 8.7 6 0.8, and 11.9 6 0.8 mg L21 day21 under nitrate-
sufficient cultivation (control) for S. obliquus, C. vulgaris, and
C. minutissima, respectively. The lipid productivity was also
found to be 13.1 6 0.5, 12.7 6 0.4, and 16.0 6 0.4
mg L21 day21 for S. obliquus, C. vulgaris, and C. minutissima,
respectively under 0.01 g L21 nitrate feeding condition.
Analysis of Fatty Acid Profiles of Three Test Microalgae
under Various Strategies
The FAME profile of S. obliquus biodiesel depicted the
presence of four major fatty acids, i.e., palmitic, oleic, lino-
leic, and linolenic acid methyl esters (peak area <0.1% were
considered to be negligible), when grown under control cul-
ture condition. For C. minutissima, the biodiesel was rich in
palmitic, stearic, oleic, linoleic, and linolenic acid methyl
esters. However, the latter was absent in case of C. vulgaris.
Thus, the microalgal biodiesel was found to be rich in satu-
rated and monounsaturated FAMEs with a minor proportion
of polyunsaturated FAMEs in nutrient sufficient (control) con-
dition (Tables 3–5).
The FAME profile of S. obliquus biodiesel under single
phase cultivation in nitrate-deprived medium, biphasic cultiva-
tion, and sequential low-dose nitrate feeding depicted the
appearance of stearic acid in addition to the existing four
major fatty acids (Table 3). Under the nitrate-starved condition,
S. obliquus biodiesel constituted 92.5 6 3.3% of saturated 1
monounsaturated, and only 7.5 6 1.0% polyunsaturated
FAMEs. For biphasic nitrate-starved cultures, the biodiesel was
also predominated with 95.1 6 3.3% of saturated 1 monounsa-
turated FAMEs. Biodiesel obtained from the sequential addi-
tion of 0.01 g L21 of nitrate was also found with 90.0 6 4.0% of
saturated 1 monounsaturated FAMEs as compared to 78.3 6
3.2% under control condition.
The FAME profile of C. vulgaris biodiesel from single phase
cultivation in nitrate-sufficient and nitrate-starved media
depicted the presence of four major fatty acids, i.e. palmitic,
stearic, oleic, and linoleic acid methyl esters (Table 4). For
biphasic and sequential low-dose nitrate feeding cultures a
minor quantity of linolenic acid in addition to the
228 January 2017 Environmental Progress & Sustainable Energy (Vol.36, No.1) DOI 10.1002/ep
Relative % of FAMEs
Strategy 2 Strategy 3 Strategy 4
48.4 6 0.8b 46.8 6 1.4b 47.0 6 0.9b
9.7 6 0.1a 13.7 6 0.4b 9.0 6 0.5a
24.66 0.5b 23.8 6 0.8b 26.0 6 0.6b
6.9 6 0.8b 5.5 6 0.5a 4.5 6 0.1a
10.4 6 0.6a 10.2 6 0.2a 13.5 6 0.3b
82.7 6 1.4b 84.3 6 2.6b 82.0 6 2.0b
above four fatty acids was detected. The nitrate-starved cul-
tures of C. vulgaris biodiesel constituted 90.8 6 1.9% of
saturated 1 monounsaturated FAMEs. For biphasic nitrate-
starved cultures, an equivalent quantity of saturated 1 mono-
unsaturated FAMEs was recorded. Biodiesel obtained from the
sequential addition of 0.01 g L21 of nitrate was found with
85.8 6 1.5% of saturated 1 monounsaturated FAMEs. C. minu-
tissima biodiesel, however, showed the presence of five major
fatty acids, i.e., palmitic, stearic, oleic, linoleic, and linolenic
acid methyl esters (Table 5). For nitrate-starved cultures, the
biodiesel contains 82.7 6 1.4% of saturated 1 monounsatu-
rated FAMEs. The biodiesel obtained from the biphasic nitrate-
starved cultures and cultures of sequential addition of 0.01
g L21 of nitrate were also dominated with saturated and mono-
unsaturated FAMEs. The GC-MS analysis of the biodiesel
obtained from the three test microalgae under 1/100th dose
(0.01 g L 2 1) of nitrate feeding is shown in Figure 4.
The fuel characteristics of biodiesel mainly depend on its
fatty acid profile and the levels of saturation/unsaturation.
Fatty acid profile of algal biodiesel can provide essential
information on the ecological adaptation of algal species
[35,36]. Microalgal lipids usually contain polyunsaturated fatty
acids which posses four or more double bonds. During stor-
age these bonds are likely to get oxidised, thus reducing the
suitability of microalgal oil for production of biodiesel
[10,33]. Depending upon the physiological states and culture
conditions, the fatty acid content of algae can vary both
quantitatively and qualitatively. Oxidative stability indicates
the ability of the molecule to resist oxidation by oxidizing
ions upon exposure to air. Oxidation occurs because it reacts
with the oxygen when exposed to air due to the presence of
double bonds in unsaturated fatty acids [37,38]. The GC-MS
study revealed that the biodiesel obtained from S. obliquus,
C. vulgaris and C. minutissima grown under different condi-
tions was predominated (78–95% of the total FAMEs) with a
mixture of saturated and monounsaturated fatty acid compo-
nents, which demonstrates their high oxidative stability. Gen-
erally, high amount of saturated and monounsaturated fatty
acids are ideal for high energy yield, greater oxidative stabili-
ty, and higher cetane numbers [39]. With decreased concen-
tration of nitrate under sequential addition, the rise in the
level of saturated 1 monounsaturated FAMEs were recorded
in the three test microalgae. As observed in this study, bio-
diesel obtained from the cultures of various conditions was
rich in saturated and monounsaturated fatty acid methyl
esters, thus demonstrating its aptness for use as the substitute
to diesel fuel. Thus, the test microalgae could be considered
as potential feedstocks for biodiesel production.
Cost Performance
Economic analysis can be used to calculate the costs of algal
biomass production which contribute notably to the production
Figure 4. GC-MS chromatograms of FAMEs of (A) S. obliquus, (B) C. vulgaris and (C) C. minutissima grown in limited nitrate
feeding sets of 1/100th dose showing the presence of methyl palmitate, methyl stearate, methyl oleate, methyl linoleate, and
methyl linolenate with retention time of 10.89, 11.49, 11.59, 11.70, and 11.87 min, respectively (the 1st peak was methyl penta-
decanoate, the internal standard). Peak area < 0.1% were considered to be negligible.

cost. The media cost for the production of algal biomass in the
four different strategies is shown in Table 6. The media cost for
single phase cultivation in nitrate-sufficient medium (strategy 1)
is 1000 rupees (US $16) per 1000 L21. For biphasic cultivation
(strategy 3) and sequential low-dose nitrate feeding (strategy 4),
the values are  Rs. 1200 (US $19.25) and  Rs. 300 (US $4.81),
respectively per 1000 L21 of the medium. Thus, there is a
remarkable reduction in the capital cost on the media for strate-
gy 4 as compared to strategy 1 and strategy 3 as well, without
compromising with lipid productivity.
The four nitrate feeding strategies were analyzed thor-
oughly, and the sequential feeding strategy was found to be
superlative to others. Thus, the use of low dose sequential
nitrate feeding strategy was found to be stimulatory in
increasing the lipid productivity under a single phase
cultivation, which advocates its novelty and essentiality in
algae-biodiesel research. The sequential low-dose feeding of
nitrate also evades the two times biomass harvesting of
biphasic approach, which has been proposed by the
researchers to obtain higher lipid productivity. It is also note-
worthy here that there is no single study available which
compares different nitrogen feeding strategy.
CONCLUSION
Physiological/nutritional stresses usually applied to stimu-
late lipid accumulation in microalgal cell resulted into low
cell mass vis-a-vis reduced lipid productivity. An offset for
this intricacy is the use of biphasic cultivation strategy, dedi-
cating the first phase in the nutrient-sufficient medium for
biomass production, and the second phase, a specific phase

Environmental Progress & Sustainable Energy (Vol.36, No.1) DOI 10.1002/ep January 2017 229
Table 6. Cost calculation for nutrient medium for microalgae
cultivation under various strategies.
Cost estimate
Strategy Culture condition Rupees/1000 L
1 N 11 medium 1000.00 (16.04)
2 Nitrate-starved 210.00 (3.37)
3 Biphasic nitrate-starved 1200.00 (19.25)
4 Limited nitrate feeding 300.00 (4.81)
(1/100th dose of N 11 medium)
Values in parentheses show cost in US $.
such as physiological stresses for efficient lipid accumulation.
However, this approach comprised biomass harvesting two
times, once after the growth phase and the final harvesting
after the second phase of processing. The major hurdle here
is the lack of a cost-effective harvesting technology for sepa-
ration of the biomass. In this context, standardization of a
nutrient feeding strategy eliminating the biomass harvesting
even once holds great importance. Moreover, this will reduce
the nitrate requirements for large-scale cultivation, thus,
would result into cost-effective and eco-friendly (as nitrate is
one of the major water pollutants for eutrophication) biodie-
sel production from microalgae in the long run.
ACKNOWLEDGMENTS
Mr. Sashi Sonkar is thankful to the Indian Institute of
Technology Kharagpur, India, for providing the assistantship.
The authors thank NFBSFARA, Indian Council of Agricultural
Research, New Delhi, India, for financial support. The
authors acknowledge Prof. S. P. Adhikary, Vice-Chancellor,
Fakir Mohan University, Odisha and Dr. R. K. Gupta,
Scientist-D & Dr. Sudipta Kumar Das, BSI, India for the iden-
tification of the test microalgae.
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