World J Microbiol Biotechnol (2013) 29:915–922
DOI 10.1007/s11274-012-1248-2
ORIGINAL PAPER
Optimization of biomass and fatty acid productivity
of Scenedesmus obliquus as a promising microalga
for biodiesel production
Mostafa El-Sheekh • Abd El-Fatah Abomohra •
Dieter Hanelt
Received: 7 December 2012 / Accepted: 20 December 2012 / Published online: 27 December 2012
Ó Springer Science+Business Media Dordrecht 2012
Abstract Nowadays, microalgae are discussed as a
promising feedstock for biodiesel production. The present
study examines the possibility of enhancement of fatty acid
productivity of Scenedesmus obliquus by modifications of
the culture medium composition. The effect of different
concentrations of sodium bicarbonate, salinity, potassium
nitrate, glycerol and sugarcane molasses on the enhance-
ment of biomass and esterified fatty acids production was
studied. NaHCO3 caused an increase in the biomass pro-
ductivity at low concentrations (0.5 g L-1), while nega-
tively affected fatty acid productivity at all tested
concentrations. Increase of salinity enhanced both biomass
and fatty acid productivity. The optimum NaCl concen-
tration and sea water ratio were 0.94 g L-1 and 25 %
which resulted in 56 and 39 % increase in fatty acid pro-
ductivity, respectively. Nitrogen deficiency showed
increase in fatty acid content by 54 % over control but fatty
acid productivity was decreased as a result of growth
inhibition. Nitrogen-free cultures and cultures treated with
-50 % concentrations of KNO3 showed 96 and 42 %
decrease in EFA productivity, respectively, as compared
with the control. Addition of 0.05 and 0.1 M of glycerol
increased the biomass productivity by 6 and 5 %, respec-
tively but showed no significant effect on fatty acid pro-
ductivity as a result of decrease in fatty acid content.
Finally, usage of sugarcane molasses stimulated both bio-
mass and fatty acid content. The increase in fatty acid
M. El-Sheekh (&)  A. E.-F. Abomohra
Phycology Research Unit, Botany Department, Faculty
of Science, Tanta University, Tanta 31527, Egypt
e-mail: mostafaelsheekh@yahoo.com
D. Hanelt
Department of Cell Biology and Phycology, University
of Hamburg, Ohnhorststrasse 18, 22609 Hamburg, Germany
productivity was 32, 65 and 73 % above the control level at
1, 3 and 5 g L-1 of sugarcane molasses, respectively.
Keywords Biomass productivity  Fatty acid productivity 
Microalgae  Biodiesel  Optimization
Introduction
In recent years, microalgae have been investigated as a
feed stock for biofuels. Renewable, carbon–neutral fuel
applications exploiting algal components include transe-
sterification of lipids to biodiesel (Wu and Miao 2006;
Chisti 2007), saccharification of carbohydrates to ethanol
(Matsumoto et al. 2003), gasification of biomass to syngas
(Lv et al. 2007), cracking of hydrocarbons and isoprenoids
to gasoline (Milne et al. 1990; Rohmer 1999), and the
direct synthesis of hydrogen gas (Ghirardi et al. 2002;
Prince and Kheshgi 2005). Many research reports and
articles described many advantages of using microalgae for
biodiesel production in comparison with other available
feedstocks (Sheehan et al. 1998; Tsukahara and Sawayama
2005; Hossain et al. 2008; Rosenberg et al. 2008; Mata
et al. 2010).
The first step in developing an algal process is to choose
the suited algal species (Griffiths and Harrison 2009). Fast
growth promotes high biomass productivity which conse-
quently increases yield per harvest volume in a certain
period (productivity) and decreases cost. Whether condi-
tions are favorable or not, the intricate metabolic pathways
of a cell are heavily influenced by its environment. In the
case of microalgae, specialized cultivation can stimulate
changes in metabolism, thereby providing a simple method
of enriching biomass with a target metabolite (Rosenberg
et al. 2008). For microalgae that are able to survive
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heterotrophically, exogenous carbon sources offer prefab-

ricated chemical energy, which the cells often store as lipid
droplets (Ratledge 2004). Heterotrophically cultivated
Chlorella protothecoides has been shown to accumulate as
much as 55 % of its dry weight as oil, compared to only
14 % in cells grown photoautotrophically (Wu and Miao
2006). Another natural mechanism through which micro-
algae can alter lipid metabolism is the stress response
owing to nitrogen deficiency (Tornabene et al. 1983).
Although nitrogen deficiency appears to inhibit the cell
cycle and the production of almost all cellular components,
the rate of lipid synthesis remains higher, which leads
to the accumulation of oil in starved cells (Sheehan
et al. 1998).
Abomohra et al. (2012) screened 13 freshwater micro-
algae for fatty acids productivity. They concluded that
Scenedesmus obliquus was selected as a promising mi- Fig. 1 Kniese tube used in cultivation containing 350 mL of culture
croalga for large-scale lipid production because of its high
biomass production which resulted in high lipid and fatty
acid productivity. The present work was intended to throw measured after 12 days of incubation. Biomass productivity
some light on optimization of S. obliquus growth condi- and fatty acids productivity were calculated.
tions for high fatty acid productivity for using it as a
feedstock for biodiesel. Biomass assay

Algal growth was monitored using the optical density of

Materials and methods
Algae strain and growth conditions
Scenedesmus obliquus Kützing (Culture Collection of
Algae at Goettingen University, Germany, strain number
SAG276-10) was cultivated axenically as batch cultures in
1 L Erlenmeyer flasks with KC medium (Kessler and
Czygan 1970). Cultures were illuminated by tubular fluo-
rescent lamps (PHILIPS Master TL-D 85 W/840). The
light intensity at the surface of the culturing vessels was
100 l mol photons m-2 s-1 with a photoperiod of 16:8 h
light: dark at 25 ± 1 °C.
The effect of different nutrients namely sodium bicar-
bonate [(control (0 g L-1), 0.5, 1 and 2 g L-1)], sodium
chloride [(control (0.47 g L-1), -100 % (0 g L-1), ?50 %
(0.71 g L-1) and ?100 % (0.94 g L-1)], sea water [(0, 25,
50 and 75 %)], potassium nitrate [control (0.81 g L-1),
?100 % (1.62 g L-1), -50 % (0.41 g L-1) and -100 %
(0 g L-1)], glycerol [(control (0 M), 0.05, 0.1 and 0.2 M)]
and sugarcane molasses [(control (0 g L-1), 1, 3 and
5 g L-1)] on growth and fatty acid productivity were stud-
ied. A certain volume of exponentially growing S. obliquus
cells precultured in 1 L Erlenmeyer flasks was inoculated in
300 mL of KC medium in Kniese tubes (Fig. 1) at an initial
OD680 of 0.2. Sterile filtered air enriched with 3 % (v/v)
CO2 was continuously applied to the cultures. OD680 was
measured every other day; dry weight and EFA were
the culture at 680 nm (OD680) and by determination of
algal cellular dry weight (CDW). Biomass productivity was
calculated as according to Abomohra et al. (2012).


Biomass productivity g CDW L1 d1
¼ðCDWL  CDWE Þ  ðtL  tL Þ
with CDWE representing the CDW (g L-1) at days of early
exponential phase (tE) and CDWL at days of late expo-
nential phase (tL).
Lipid extraction
To analyse the fatty acid composition of cells, 5 mL aliquots
of each culture were collected at times specified. Lipids were
extracted following the method of Bligh and Dyer (1959).
Prior to extraction, trinonadecanoylglycerol were added to
the samples as internal standard esterified fatty acid.
Fatty acid profiles
Esterified fatty acids (EFA) from the extracts of intracel-
lular lipids were subjected to transmethylation for GC
analysis as described previously (Kaczmarzyk and Fulda
2010; Scharnewski et al. 2008). The fatty acid methyl
esters were subjected to analysis by GC and GC/MS. The
GC analysis was performed with a Varian 3900 GC-system
equipped with a capillary column (Select Fame,
50 m 9 0.25 mm; Varian).

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World J Microbiol Biotechnol (2013) 29:915–922 917

The total fatty acid productivity was calculated 14

2 g/L 1 g/L
according to Abomohra et al. (2012).

12 0.5 g/L 0 g/L
Fatty acid productivity mg L1 d1 ¼ðFAL  FAE Þ
10
 ðtL  tE Þ1
8

OD680
with FAE and FAL representing the total fatty acid content
(mg L-1) at days of early exponential phase (tE) and late 6
exponential phase (tL).
4

Statistical analysis 2

0
Results are presented as mean ± standard deviation (SD) 0 2 4 6 8 10 12
from three replicates. The statistical analyses were carried Age (days)
out using SAS (v 6.12). Data obtained were analyzed sta-
tistically to determine the degree of significance using one Fig. 2 Effect of different concentrations of NaHCO3 on growth of
S. obliquus for 12 days of incubation
way analysis of variance (ANOVA) at probability level
P B 0.05.
Table 1 Effect of different concentrations of NaHCO3 on the EFA
content and EFA productivity of S. obliquus after 12 days of
incubation
Results NaHCO3 Biomass EFA content EFA
(g L-1) productivity (mg g-1 CDW) productivity
(g CDW L-1 d-1) (lg mL-1 d-1)
Carbonsupply is often managed by addition of hydrogen
carbonate to the culture. Figure 2 shows the effect of dif- 0 0.191 ± 0.005 123.3 ± 2.1 25.6 ± 0.4
ferent concentrations of NaHCO3 on the growth of 0.5 0.197 ± 0.008(ns) 116.3 ± 2.7(ns) 24.8 ± 0.6*
S. obliquus for 12 days of incubation. The OD680 at 1 0.149 ± 0.006* 119.9 ± 0.8(ns) 21.8 ± 0.2*
0.5 g L-1 of NaHCO3 was 9 % higher than the control. 2 0.139 ± 0.002* 105.9 ± 1.4* 18.3 ± 0.2*
However, 1 and 2 g L-1 treated cultures showed reduction
Each value is the mean of three readings ± standard deviation
with regard to the OD680 by about 12 % after 12 days of
* Significant at P B 0.05 using one way analysis of variance
incubation. Using 0.5 g L-1 of NaHCO3 showed insignifi- (ANOVA)
cant changes on biomass productivity, while 1 and 2 g L-1 (ns)
Non significant at P B 0.05 using one way analysis of variance
of NaHCO3 significantly inhibited the biomass productivity (ANOVA)
by 22 and 27 % lower than the control (Table 1). Results in
Table 1 showed no significant effect on EFA content using
0.5 and 1 g L-1 of NaHCO3 in respect to the control. 14 + 100 % + 50 %

However, 2 g L-1 of NaHCO3 caused 14 % decrease in 12

- 100 % Control
EFA of S. obliquus lower than the control after 12 days of
incubation. On the other hand, cultures treated with 0.5, 1 10

and 2 g L-1 of NaHCO3 showed significant reduction in 8

OD680

EFA productivity by 3, 15 and 29 %, respectively, with

respect to the control after 12 days of incubation (Table 1). 6

Figure 3 shows the effect of different concentrations of 4

NaCl on the growth of S. obliquus for 12 days of incuba-
tion. Application of ?50 and ?100 % increased the OD680 2

by 15 and 29 %, respectively, with respect to the control 0

after 12 days of growth. However, the OD680 was 0 2 4 6 8 10 12
decreased by 15 % with respect to the control in the NaCl- Age (days)
free medium (Fig. 3). High salinity concentrations (?50
Fig. 3 Effect of different concentrations of NaCl on growth of
and ?100 % of NaCl) significantly stimulated the biomass S. obliquus for 12 days of incubation
productivity by 18 and 24 % higher than the control
(Table 2).Results in Table 2 showed significant increase on showed 48 % increase in EFA content of S. obliquus higher
EFA content using ?50 and ?100 % of NaCl with 19 and than the control after 12 days of incubation. On the other
27 % with respect to the control, while NaCl-free culture hand, cultures treated with ?50 and ?100 % of NaCl

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Table 2 Effect of different concentrations of NaCl on the EFA Table 3 Effect of different seawater ratios on the EFA content and
content and EFA productivity of S. obliquus after 12 days of EFA productivity of S. obliquus after 12 days of incubation
incubation
Percent of Biomass EFA content EFA
NaCl Biomass EFA content EFA sea water productivity (mg g-1 CDW) productivity
productivity (mg g-1 CDW) productivity (%) (g CDW L-1 d-1) (mg L-1 d-1)
(g CDW L-1 d-1) (mg L-1 d-1)
0 0.190 ± 0.007 109.1 ± 4.3 23.4 ± 0.9
Control 0.189 ± 0.006 109.1 ± 4.7 21.5 ± 0.9 25 0.219 ± 0.001* 133.5 ± 18.8 (ns)
32.5 ± 4.6*
-100 % 0.147 ± 0.002* 161.9 ± 5.8* 25.1 ± 0.9* 50 0.192 ± 0.008(ns) 153.1 ± 9.7* 29.5 ± 1.9*
?50 % 0.224 ± 0.002* 129.3 ± 13.2* 29.9 ± 3.1* 75 0.063 ± 0.000* 118.6 ± 11.7(ns) 10.4 ± 1.0*
?100 % 0.234 ± 0.008* 138.8 ± 13.1* 33.6 ± 3.2*
Each value is the mean of three readings ± standard deviation
Each value is the mean of three readings ± standard deviation * Significant at P B 0.05 using one way analysis of variance
* Significant at P B 0.05 using one way analysis of variance (ANOVA)
(ANOVA) (ns)
Non significant at P B 0.05 using one way analysis of variance
(ANOVA)
14 +75 % +50 %
+25 % Control
12
Table 3. The cultures treated with 25, 50 and 75 % of
10 seawater showed increase in EFA content by 22, 40 and
8
9 %, respectively, above the control. On the other hand,
OD680

EFA productivity of cultures treated with 25 and 50 % of

6 seawater showed increase by 39 and 26 %, respectively,
4
2 control (Table 3).
0
0 2 4 6 8 10
Age (days)
Fig. 4 Effect of different seawater ratios on growth of S. obliquus for
12 days of incubation
showed significant increase in EFA productivity by 39 and
56 %, respectively, with respect to the control after
12 days of incubation, while NaCl-free culture showed
only 17 % increase in EFA productivity after 12 days of
incubation.
As NaCl enhances the productivity the question arises if
seawater could partly replace freshwater supply of the
culture. Figure 4 clarifies the changes in the growth mea-
sured as OD680 of S. obliquus as influenced by different
seawater ratios used in the cultures during 12 days of
incubation. With respect to changes in the OD680 after
12 days of incubation, it is visible that 25 and 50 % sea-
water ratios caused an increase in the OD680 by 24 and
7 %, respectively, more than the control. However, using
75 % of seawater in the culture led to severe decrease in
growth by 91 % with respect to the control. The highest
significant biomass productivity was observed by using
seawater ratio of 25 % which showed 15 % increase in
biomass productivity over the control (Table 3). The
changes in EFA content and productivity in response to
treatment with different ratios of seawater are presented in
over the control. In contrast, 75 % of seawater ratio
induced reduction in EFA productivity by 56 % below the
Nitrogen supply is essential for the synthesis of amino
acids and is often managed by addition of nitrate. The
12
effect of different concentrations of KNO3 on the growth of
S. obliquus was recorded as OD680 at 2 days interval for
12 days of incubation. The obtained results in Fig. 5
revealed that decrease or increase of KNO3 concentration
led to reductions in growth. The most pronounced inhibi-
tion amounted to 97 % below the control at -100 % of
KNO3. It is clear from these results that cultures containing
?100 and -50 % of KNO3 resulted in decrease in the
growth by 20 and 35 %, respectively, below the control
after 12 days of incubation. The same results were
observed on biomass productivity shown in Table 4. Cul-
tures containing ?100 % and -50 % of KNO3 resulted in
decrease in biomass productivity by 50 and 59 %, respec-
tively, below the control after 12 days of incubation, while
no growth was observed in the cultures devoid of KNO3.
As apparent from Table 4, the various concentrations of
KNO3 show changes in the EFA content and productivity
of S. obliquus after 12 days of incubation. Compared to the
control, cultures treated with zero and -50 % concentra-
tions of KNO3 showed the maximum increase in EFA by
54 and 13 %, respectively. However, application of high
concentration of KNO3 (?100 %) to the culture of
S. obliquus led to reduction in EFA content by about 19 %
below the control. On the other hand, cultures treated
with zero and -50 % concentrations of KNO3 showed
96 and 42 % decrease in EFA productivity, respectively,

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14 -100% -50% 14 0.2 M 0.1 M

+100% Control 0.05 M Control
12 12

10 10

8 8

OD680
OD680

6 6

4 4

2 2

0 0
0 2 4 6 8 10 12
0 2 4 6 8 10 12
Age (days) Age (days)

Fig. 5 Effect of different concentrations of KNO3 on growth of Fig. 6 Effect of different concentrations of glycerol on growth of
S. obliquus for 12 days of incubation S. obliquus for 12 days of incubation

Table 5 Effect of different concentrations of glycerol on the EFA

Table 4 Effect of different concentrations of KNO3 on the EFA content and EFA productivity of S. obliquusafter 12 days of
content and EFA productivity of S. obliquusafter 12 days of incubation
incubation
Glycerol Biomass EFA content EFA
KNO3 Biomass EFA content EFA (M) productivity (mg g-1 CDW) productivity
productivity (mg g-1 CDW) productivity (g CDW L-1 d-1) (mg L-1 d-1)
(g CDW L-1 d-1) (mg L-1 d-1)
0 0.193 ± 0.006 112.5 ± 1.7 26.4 ± 0.4
Control 0.193 ± 0.000 110.12 ± 3.1 25.82 ± 0.7 0.05 0.204 ± 0.005* 106.9 ± 11.2(ns) 26.3 ± 2.8(ns)
(ns)
?100 % 0.097 ± 0.001* 89.15 ± 1.7* 12.38 ± 0.2* 0.1 0.203 ± 0.004* 100.4 ± 2.7 24.5 ± 0.7(ns)
(ns)
-50 % 0.079 ± 0.002* 124.14 ± 14.1 15.03 ± 1.7* 0.2 0.155 ± 0.004* 91.7 ± 8.7* 18.0 ± 1.7*
-100 % 0.000 ± 0.001* 169.78 ± 9.3* 0.94 ± 0.1*
Each value is the mean of three readings ± standard deviation
Each value is the mean of three readings ± standard deviation * Significant at P B 0.05 using one way analysis of variance
* Significant at P B 0.05 using one way analysis of variance (ANOVA)
(ANOVA) (ns)
Non significant at P B 0.05 using one way analysis of variance
(ns) (ANOVA)
Non significant at P B 0.05 using one way analysis of variance
(ANOVA)

as compared with the control. Also, application of high
concentration of KNO3 (?100 %) to the culture of S.
obliquus led to reduction in EFA productivity by about
52 % below the control.
Glycerol is a residue of the biodiesel production from
EFAs and might be re-used as carbon source for mixo-
trophic algae. Figure 6 shows the effect of different con-
centrations of glycerol on growth of S. obliquus during
12 days of incubation. Compared to the control, cultures
treated with 0.05 and 0.1 M of glycerol showed increase in
OD680 by 7 and 18 %, respectively. While culture treated
with 0.2 M of glycerol showed inhibition of growth by
17 % below the control. The same trend was observed for
biomass productivity. Table 5 shows the changes in EFA
content and productivity of S. obliquus as influenced by
different concentrations of glycerol after 12 days of incu-
bation. It can be concluded that glycerol led to progressive
decrease in EFA content. Application of 0.05, 0.1 and
0.2 M of glycerol resulted in decrease in EFA content by 5,
11 and 18 %, respectively, with respect to the control. The
effect of 0.05 and 0.1 M of glycerol showed insignificant
decrease on EFA productivity by 1 and 7 %, respectively,
lower than the control. While 0.02 M of glycerol caused
significant decrease in EFA productivity of S. obliquus by
32 % lower than the control.
Molasses is fermented to produce ethanol as e.g. for use
as an alternative fuel in motor vehicles, but may also
directly act as carbon and energy source for mixotrophic
algae. Application of different concentrations of sugarcane
molasses as a carbon source resulted in progressive
increase in growth (Fig. 7). It can be observed that, 1, 3 and
5 g L-1 of sugarcane molasses stimulated the OD680 of the
cultures by 36, 53 and 62 %, respectively, with respect to
the control. 1, 3 and 5 g L-1 of sugarcane molasses stim-
ulated also the biomass productivity by 23, 40 and 48 %,
respectively; (Table 6). Increase in the concentration of

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14 5 g/L 3 g/L production. The obtained results suggested that 0.5 g L-1
1 g/L Control of NaHCO3 caused an increase in growth of S. obliquus,
12
while higher concentrations of NaHCO3 caused decrease in
10 growth (Fig. 2). However, this was the case when carbon is
already gaseous supplied by additional 3 % CO2. These
8
OD680

results suggest that bicarbonate is also an effective carbon

6 source for microalgal growth, as optimum bicarbonate
concentration is beneficial for highest biomass production.
4 At high concentrations of NaHCO3, the pH of the medium
2
may increase due to accumulation of HCO3- ions which
can become toxic to the organism or can make other
0 chemical constituents such as ammonia toxic. This finding
0 2 4 6 8 10 12
is in accordance with Devgoswami et al. (2011) who
Age (days) mentioned that addition of bicarbonate to Chlorella,
Fig. 7 Effect of different concentrations of sugarcane molasses on Haematococcus and Scenedesmus showed high growth
growth of S. obliquus for 12 days of incubation comparing to the control. In addition, Velea et al. (2011)
studied the effect of NaHCO3 on Porphyridium purpureum
and reported that maximum growth rate was recorded at
Table 6 Effect of different concentrations of sugarcane molasses on
2 g L-1 of NaHCO3. With regard to the effect of NaHCO3
the EFA content and EFA productivity of S. obliquusafter 12 days of
incubation on EFA content and EFA productivity, the results indicated
that use of NaHCO3 in the culture caused a decrease in the
Sugarcane Biomass EFA content EFA
molasses productivity (mg g-1 CDW) productivity EFA content (Table 1) which may be explained by stim-
(g L-1) (g CDW L-1 d-1) (mg L-1 d-1) ulation of carbohydrates biosynthesis in disfavor of lipids.
These results disagree with finding of Devgoswami et al.
0 0.197 ± 0.009 109.4 ± 0.8 26.1 ± 0.2
(2011) who reported that addition of bicarbonate to
1 0.241 ± 0.002* 121.5 ± 4.6* 34.3 ± 1.3*
Scenedesmus showed high lipid content. In addition, EFA
3 0.275 ± 0.010* 135.8 ± 7.4* 42.9 ± 2.4* productivity showed continuous decrease by increasing the
5 0.292 ± 0.003* 135.2 ± 3.5* 45.1 ± 1.2* NaHCO3 concentration due to decrease in growth and EFA
Each value is the mean of three readings ± standard deviation content.
* Significant at P B 0.05 using one way analysis of variance As indicated from the present results, the increase in
(ANOVA) NaCl concentration caused an increase in growth (Fig. 3)
and EFA content of S. obliquus (Table 2). This observation
sugarcane molasses induced the increase of EFA content. is in agreement with the results obtained by Kirroliaa et al.
1 g L-1 of sugarcane molasses caused induction in EFA (2011) who mentioned that the algal biomass yield of
content by 11 % with respect to the control. While 3 and S. quadricauda increased by 7.5 % at 0.2 mM NaCl con-
5 g L-1 of sugarcane molasses showed the same percent of centration as compared to control. Salinity changes nor-
increase in EFA content by 24 % over the control mally affect phytoplankton in three ways (Moheimani
(Table 6). As a result of stimulation of growth and EFA 2005): (1) osmotic stress (2) ion (salt) stress; and (3)
production, the EFA productivity increased by increasing changes of the cellular ionic ratios due to the membrane
the concentration of sugarcane molasses. The increase was selective ion permeability. In addition, Dujjanutat and
recorded by 32, 65 and 73 % above the control level at 1, 3 Kaewkannetra (2011) reported that the lipid content of
and 5 g L-1 of sugarcane molasses, respectively (Table 6). S. obliquus under NaCl stress (1.0 M) gave 0.38 fold
higher lipid content than the control. The increase in lipid
content at higher NaCl concentration may be due to
Discussion adaptation under stress conditions which helps in accu-
mulation of lipid (Takagi et al. 2006; Kirroliaa et al. 2011).
For biodiesel production, biomass production and lipid As a result of increasing the growth and EFA content by
content play an important role from the economic point of increase of NaCl concentrations, the EFA productivity
view. Francisco et al. (2010) and Abomohra et al. (2012) increased as well. The most suitable concentration for
reported that growth and fatty acid content are inversely highest EFA productivity was identified at 0.94 g L-1.
related. So that, fatty acid productivity is more efficient In order to study the possibility of using brackish water
than fatty acid content for screening of different organisms or marine water for cultivation of S. obliquus to save the
or to choose the optimum growth conditions for biodiesel freshwater for human usage and because of the positive

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results obtained by using of NaCl for cultivation of
S. obliquus, the effect of sea water on growth and EFA
content of S. obliquus was studied. Using of sea water ratio
of 25 and 50 % in the culture medium increased the growth
and EFA content, while 75 % of sea water in the medium
of S. obliquus caused sharp decrease in growth and insig-
nificant increase in EFA content with respect to control
(Fig. 4; Table 3). The salinity-induced growth reduction
may be attributed to the accumulation of reactive oxygen
species or may be attributed to the inhibition of photo-
synthesis as a result of reduction in chlorophyll. Moradi
and Ismail (2007) mentioned that reduced chlorophyll
contents at higher salinities are due to decrease in photo-
synthetic rate because of salt osmotic and toxic ionic stress.
With respect to the effect of nitrogen starvation on growth
of S. obliquus, the obtained results showed reduction in
growth and increase in EFA content. The reduction in growth
by nitrogen deficiency may be explained by the observations
of Turpin (1991) and Gordillo et al. (1999) who found that
the decrease in pigment contents and photosynthesis are a
typical response in nitrogen-limited algae. Piorreck et al.
(1984) made comparative study between chlorophyll content
of groups of green algae and cyanobacteria with different
nitrogen concentrations; they showed that chlorophyll con-
tents decreased with decreasing nitrogen concentrations.
They also concluded that, with decreasing nitrogen levels the
chlorophyll contents of the cells dropped indicating a rapid
reduction or even breakdown of the whole chloroplast
apparatus and consequently decreasing of growth. The
increase in EFA content observed in the present study as a
result of nitrogen deficiency is in agreement with the report
of Shiflin and Chisholm (1981). They reported that, accu-
mulation of fatty acid derivatives in 30 micro-algal species
belonging to chlorophyceae and diatoms under nitrogen
starvation. The possible reason could be that under nitrogen
deficiency the rest of available nitrogen is utilized for syn-
thesis of enzymes and essential cell structures. Any carbon
dioxide subsequently fixed is therefore converted into car-
bohydrate or lipid rather than protein (Richardson et al.
1969). Although the present results showed that, nitrogen
deficiency led to increasing of EFA content, the EFA pro-
ductivity was decreased as a result of inhibition of growth by
nitrogen deficiency.
The idea for using biodiesel-derived crude glycerol as a
carbon source for cultivation of oil producing microalgae
was to provide biodiesel producers with a method of disposal
for their waste glycerol. Results indicated that 0.05 and
0.1 M of glycerol increased the growth, while higher con-
centration of glycerol, 0.2 M, decreased growth. This
observation is in agreement with the results obtained by
Garcı́a et al. (2000) who mentioned that the optimal glycerol
concentration for the growth of Phaeodactylum tricornutum
was 0.1 M. On the other hand, no significant effect was
921
observed on EFA content at 0.05 and 0.1 M of glycerol while
significant decrease in EFA content was observed at 0.2 M of
glycerol (Table 4). Ivanova et al. (1999) reported that the
addition of glycerol in the culture medium of the red alga
Gratelupia doryphora caused substantial increase of the total
lipids and glycolipids while the amount of polar lipids
remained constant. Therefore, no significant changes were
observed in EFA productivity at 0.05 and 0.1 M of glycerol
and significant decrease was observed at 0.2 M of glycerol.
Our results showed that it is not profitable to use glycerol in
S. obliquus cultures for crude glycerol disposal and to
increase the EFA productivity.
The effect of different concentrations of sugarcane
molasses on growth and EFA content of S. obliquus was also
studied. The objective of this study is to investigate the
growth performance and EFA content of S. obliquus using
sugarcane molasses, a by-product of sugar refinery, as a
cheap carbon source in mixotrophic cultures. Results of this
work showed that, different concentrations of sugarcane
molasses stimulated both growth and EFA content and
consequently the EFA productivity of S. obliquus (Fig. 7;
Table 6). Leesing et al. (2011) reported that, cultivation of
Chlorella sp. KKU-S2 on BG-11 medium supplemented
with 25 g L-1 sugarcane molasses at 30 °C for 8 days caused
stimulation of growth and lipid content. The obtained results
suggested that EFA productivity from S. obliquus can be
performed with lower cost production process especially
using sugarcane molasses as a carbon source.
The present study suggests that the most effective
approach to enhance fatty acid productivity in S. obliquus
is to cultivate it in brackish water medium with 25 % sea
water, this gives 29 % increase in EFA productivity over
control. Furthermore, using 5 g L-1 of sugarcane molasses
stimulate the EFA productivity by 73 % over control. On
the other hand, nitrogen deficiency, as a common param-
eter to increase the lipid content, resulted in decrease of
EFA productivity.
Acknowledgments We would like to thank Dr. Martin Wagner
(Biocenter Klein Flottbek, Hamburg Uni, Hamburg, Germany) for his
assistance with the GC-analysis and Sigrid Mörke for her excellent
expert technical assistance. Thanks also for Dr. Dirk Warnecke (Bio-
center Klein Flottbek, Hamburg Uni, Hamburg, Germany) and
Dr. Martin Kerner (SSC GmbH) for their help and fruitful discussions
during this research. This work was supported by joint grants from
Egyptian Ministry of Higher education & Scientific Research
(MHESR) and Deutscher Akademischer Austauschdienst (DAAD) in
a German Egyptian Research Long-Term Scholarship (GERLS)
program.
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