Algal oil extraction from macroalgae

1. Optimization and kinetic studies on algal oil extraction from marine macroalgae
Ulva lactuca
Tamilarasan Suganya, Sahadevan Renganathan ⇑
Department of Chemical Engineering, Alagappa College of Technology, Anna University, Chennai 600025, India
a r t i c l e i n f o
Article history:
Received 12 September 2011
Received in revised form 5 November 2011
Accepted 10 December 2011
Available online 19 December 2011
Keywords:
Biodiesel
Marine macro algae
Ulva lactuca
Oil extraction
Extraction kinetics
a b s t r a c t
In this present investigation, kinetic studies on oil extraction were performed in marine macroalgae Ulva
lactuca. The algal biomass was characterized by scanning electron microscopy and Fourier Transform-
Infra Red Spectroscopy. Six different pre-treatment methods were carried out to evaluate the best
method for maximum oil extraction. Optimization of extraction parameters were performed and high
oil yield was obtained at 5% moisture content, 0.12 mm particle size, 500 rpm stirrer speed, 55 °C temper-
ature, 140 min time and solvent-to-solid ratio as 6:1 with 1% diethyl-ether and 10% methylene chloride in
n-hexane solvent mixture. After optimization, 10.88% (g/g) of oil extraction yield was achieved from 30 g
of algal biomass. The rate constant was obtained for the first order kinetic study by differential method.
The activation energy (Ea) was calculated as 63.031 kJ/mol. From the results obtained in the investiga-
tion, U. lactuca biomass was proved to be a suitable source for the biodiesel production.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Biodiesel seems to be a viable choice, but the most significant
drawback is the cost of crop oils, that accounts for 80% of total
operating cost, used for the biodiesel production (Demirbas,
2007). Biodiesel is usually produced from oleaginous crops such
as rapeseed, soybean, sunflower and palm (Gouveia and Oliveira,
2009). Moreover, the availability of the oil crops serve as the
sources for the biodiesel production are limited (Chisti, 2008).
Therefore, it is necessary to find new feedstock suitable for biodie-
sel production, which does not drain on the edible vegetable oil
supply. One alternative to the conventional oil crop is the algae be-
cause they contain oil, suitable for esterification/transesterification
reaction for the biodiesel production.
Biodiesel production from algae is widely considered as one of
the most efficient methods. It appears to represent the recent
renewable source of oil that could meet the global demand for trans-
port fuels (Miao and Wu, 2006). The occurrence of algal blooms
greatly disturbs the ecosystems by modifying food chains and faunal
community structure. The accumulation of algal biomass relocating
natural communities of sea grasses and higher plants (Taylor et al.,
1995). Similarly, macroalgal blooms cause changes in the main bio-
geochemical cycles of C, N, P and S (Viaroli et al., 2001).
As a result of this problem there is much attention needed to
clean up the macro algae biomass. Hence this biomass is utilized
for the production of biodiesel.
Only less information is available for the production of biodiesel
from marine macro algae and limited research work was observed
for the extraction of oil from marine macro algae.
Extraction is one of the fundamental processing steps used for
recovering oil from biomass for the production of biodiesel. Vari-
ous methods are available for the extraction of algal oil, such as
mechanical, enzymatic, chemical extraction through different or-
ganic solvents and supercritical extraction. Solvent extraction is a
common and an efficient technique for oil extraction. Solvent
extraction involves the transfer of a soluble fraction from a solid
material to a liquid solvent. Commercial grade n-hexane was used
as a solvent for the extraction of oil from biomass for many years
(Amin et al., 2010).
Ulva lactuca is a thin flat green algae. The margin is ruffled and
often torn. The membrane is thick, soft and translucent, and also
grows without a stipe. This species in the Chlorophyta is formed
of two layers of cells irregularly arranged.
In this investigation, oil extraction from U. lactuca was studied.
The main aim of the study was to find the efficiency of algal oil
extraction using 6 different extraction methods and 12 different
solvent systems. Optimization study for extraction was established
with various parameters, such as moisture content, particle size,
stirrer speed, extraction temperature, extraction time and sol-
vent-to-solid ratio to obtain maximum oil extraction. The rate con-
stant and activation energy was determined using the first order
rate kinetics for the extraction of oil from U. lactuca biomass.
0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.12.045
⇑ Corresponding author. Tel.: +91 9941613532; fax: +91 4422352642.
E-mail address: rengsah@rediffmail.com (S. Renganathan).
Bioresource Technology 107 (2012) 319–326
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2. 2. Methods
2.1. Materials
Organic solvents of analytical grade (Extra pure 99%) were pur-
chased from Merck Ltd., Mumbai, India. They were reused after
preliminary distillation.
2.2. Collection of algal sample
U. lactuca belongs to green algae chlorophyta family. U. lactuca
was collected from Rameswaram, Mandapam, South coast (Gulf
of Mannar), India. The macroalgae was collected by hand picking
from the intertidal and sub tidal regions. Sample collection was
carried out during low tide period.
2.3. Preparation of U. lactuca algal biomass
The collected algae was brought to laboratory and it was
washed with fresh water followed by distilled water to separate
potential contaminants such as adhering impurities, sand particles,
epiphytes and animal castings. The samples were spread for shade
drying. The dried biomass was grounded and particle size distribu-
tion was determined using a sieve analyzer as per ASTM standards.
2.4. Characteristics of U. lactuca algal species
To gain further insight into the effect of the ultra sonic pre-
treatment on the algal oil extraction, the microstructure of the al-
gal biomass was analyzed with scanning electron microscopy
(SEM, JEOL Ltd, Tokyo, Japan). The analysis was carried out for
the algal biomass before and after treating with ultra sonication
to identify the changes in the surface morphology. The presence
of various functional groups in the algal biomass was analyzed
using Fourier Transform Infra Red Spectroscopy (Spectrum RX1,
US).
2.5. Sequence strategy for oil extraction from biomass
The extraction of oil from U. lactuca marine macro algal biomass
was performed based on the following sequence: (a) pre-treatment
was performed to destruct the algal cells with various methods to
increase the efficiency of the extraction, (b) after destruction of al-
gal cells and cell wall, the algal biomass was mixed along with sol-
vent mixture placed in a temperature controlled extraction unit
with a magnetic stirrer for agitation, (c) solvent systems were se-
lected for oil extraction to increase the efficiency of oil extraction,
(d) optimization study was carried out with various extraction
parameters to achieve high oil yield, (e) the extraction parameters
were optimized for the maximum oil extraction yield from algal
biomass, and (f) kinetic study was carried out for the algal oil
extraction from U. lactuca marine macro algae to determine the or-
der of the reaction, reaction rate constant and activation energy for
oil extraction.
2.6. Destruction of algal cells
Dry algal biomass of 30 g along with water (water to biomass
ratio as 3:1) was taken into a 250 ml of conical flask. In order to
compare the oil extraction yield with direct extraction using sol-
vent, the following methods for destruction of algal cells were
tested: (1) ultra sonication using ultra sonic probe at 24 kHz with
constant temperature (50 °C ± 1) for 5 min, (2) heat treatment was
performed using auto clave. The experimental conditions for auto-
clave method were maintained as temperature of 121 °C, pressure
of 15 lbs and time duration of 5 min (Kasai et al., 2003), (3) deep
freezing pre-treatment was carried out using deep freezer. The al-
gal biomass sample was placed under freezing conditions at
À20 °C, (4) microwave pre-treatment was conducted in the micro-
wave oven for 5 min time duration at 100 °C, 500 W and 2455 MHz
(Lee et al., 2010), (5) lyophilization was carried out at 4 °C under
vacuum pressure (14 Pa) using lyophilizer and (6) bead-beater
pre-treatment was performed with 1 mm glass beads at high speed
of 1500 rpm. After pre-treatment, the algal biomass was separated
from water by using filtration technique through filter paper. Then
the algal biomass was dried in hot air oven to maintain specific
moisture content (Kabutey et al., 2011). The different moisture
content % (MC%) of the algal biomass obtained were calculated
from Eq. (1) (Kabutey et al., 2011):
MC ð%Þ ¼
Mi À Mf
Mi
 100 ð1Þ
where Mi is weight of the sample in the initial state (g) and Mf is
weight of the sample after drying (g).
The dried algal biomass with specific moisture content was al-
lowed to mix with solvent mixture for the extraction of oil. After
particular time of extraction, the slurry was transferred to a sepa-
rating funnel to separate solvent–oil mixture and biomass. Solvent
was separated from oil by distillation. All experiments were con-
ducted with triplicate.
2.7. Solvent systems for oil extraction
After the destruction of algal cells by ultrasonication, 12 differ-
ent solvent systems were used for oil extraction such as n-hexane,
methyl tertbutyl ether, chloroform:methanol (1:1), n-hexane:ether
(3:1), chloroform:methanol (2:1), 1% diethyl ether and 10% meth-
ylene chloride in n-hexane, chloroform:2 propanol (2:1), hexane:2
isopropanol (3:2), dichloromethane:methanol (1:1), dichlorometh-
ane:ethanol (1:1), acetone:dichloromethane (1:1) and hex-
ane:ethyl alcohol (1:1). During the selection of solvent study, the
solvent-to-solid ratio was maintained as 5:1 for the oil extraction
from algal biomass.
2.8. Extraction experimental set up
Extraction set up mainly consists of a three necked round bot-
tom flask (250 ml). The large neck in the middle of the flask was
connected to a reflux condenser, a thermometer was placed in
one of the two side necks and the third neck was used for taking
samples during the extraction process. The flask was submerged
in a temperature controlled water bath with magnetic stirrer.
2.9. Optimization of extraction parameters to enhance the oil yield
There are many factors influencing the oil extraction yield. The
extraction parameters such as moisture content (2–6%), particle
size (0.359–0.104 mm), stirrer speed (200–600 rpm), extraction
temperature (35–65 °C), extraction time (20–160 min) and sol-
vent-to-solid ratio (3:1–7:1) were optimized to increase the oil
extraction yield.
2.10. Determination of oil extraction yield
Oil extraction yield was calculated with respect to time for dif-
ferent temperatures. The oil extraction yield (% w/w) was calcu-
lated using the following Eq. (2) (Gutierrez et al., 2008):
Oil extraction yield ð%Þ ¼
Weight of oil extracted ðgÞ
Weight of algal biomass ðgÞ
 100 ð2Þ
320 T. Suganya, S. Renganathan / Bioresource Technology 107 (2012) 319–326

3. 2.11. Characterization of U. lactuca algal oil
Fatty acid composition was analyzed by Gas Chromatography–
Mass Spectrometry (GC-MS-QP 2010, Shimadzu) equipped with
VF-5 ms capillary column (length – 30 mm, diameter – 0.25 mm,
film thickness – 0.25 lm). The column temperature of each run
was started at 70 °C for 2 min, then raised to 300 °C and main-
tained at 300 °C for 10 min. GC conditions were: column oven tem-
perature – 70 °C, injector temperature – 260 °C, injection mode –
split, split ratio – 10, flow control mode – linear velocity, column
flow – 1.51 ml/min, carrier gas – helium 99.9995% purity. MS con-
ditions were: ion source temp – 200 °C, interface temp – 240 °C,
scan range – 40–1000 m/z, solvent cut time – 5 min, MS start time
– 5 (min), MS end time – 35 (min) and ionization – EI (À70 eV).
2.12. Determination of molecular weight of algal oil
Molecular weight (g/mol) of algal oil can be calculated by using
following Eq. (3) (Phan and Phan, 2008):
MWoil ¼ 3 Â
X
ðMWi  % miÞ þ 38 ð3Þ
where MWoil is average molecular weight of the oil (g), MWi is
molecular weight of fatty acid i and % mi is percentage of fatty acid
i. Chemical properties of algal oil were analyzed using titration
methods.
3. Results and discussion
3.1. Characterization of U. lactuca algal biomass
3.1.1. Scanning electron microscopy analysis
To further increase in the cell wall damage and porous surface
of the algal cell, the ultra sonic pre-treatment method was imple-
mented on the algal biomass for the oil extraction. Before and after
the pre-treatment, the microstructure of the algal biomass was
analyzed with scanning electron microscopy (SEM). From the
SEM analysis, the surface morphology and porous surface of algal
biomass which exposed for the solvent extraction was examined.
It was also observed that the cell wall breakage caused by the
ultrasonic cavitation energy and porous surface was found to be
more on the surface of the biomass after making the pre-treatment
when compared to the biomass without pre-treatment. This in-
crease in porous surface leads to the increase in the oil extraction
yield from the U. lactuca biomass due to the penetration of solvent
to the inner surface of the algal biomass. This consequence is re-
flected in the fact that the oil extraction yield increased and ob-
tained as 10.88% (g/g) from algal biomass with ultrasonic pre-
treatment.
3.1.2. Fourier Transform-Infra Red Spectroscopy analysis
The FTIR spectra of the algal biomass showed the presence of
the relevant functional groups. Alkynes bend (C–H bending) was
observed at 669.87 cmÀ1
. The presence of hydroxyl group (O–H
stretch) was characterized by the absorption peak at
3440.51 cmÀ1
. The ester group (C–O stretch or C–H bend) was
identified at 1054.93 cmÀ1
. The presence of aromatic amine (C–N
stretch) was confirmed by the absorption band at 1259.82 cmÀ1
.
The peaks corresponding to the presence of alkenes (–C@C–
stretch) were found at 1645.99 cmÀ1
. The alkane (C–H bending
(scissoring)) group was identified at 1428.07. These peaks confirm
the presence of triglyceride functional groups in the U. lactuca bio-
mass. The presence of triglyceride groups for the different types of
oils and fat were already reported by Yang et al. (2005). Similar
type of result was previously reported by Patil et al. (2011) for
the extraction of lipid from micro algae Nannochloropsis sps.
3.2. Effects of extraction methods
Oil extraction yield from U. lactuca biomass was evaluated by
six different extraction methods (Fig. 1). The highest oil extraction
yield of 8.25% (g/g) was achieved in ultra sonication method, which
was adopted for further study. The extraction efficiency of this
method was 2.25 times higher than that of direct extraction and
0.56 times higher than that of bead-beater of algal cells. The bead
beater pre-treatment method has been widely used for lipid
extraction from microalgae (Lee et al., 1998).
An autoclave method is a heat treatment technique incorpo-
rated for oil extraction from the algal biomass. The oil extraction
yield of 7.88% was obtained by this autoclave pre-treatment meth-
od. The membrane of U. lactuca is two cells thick. Hence, this heat
treatment method is one of the very efficient method to destruct
the membranes to enhance the oil yield.
Deep-freezing, lyophilization and microwave pre-treatment
methods caused partial hydrolysis and pre-esterification of the
oil. These pre-treatment methods did not show much improve-
ment in oil extraction yield when compared with direct extraction
using solvent.
3.3. Ultra sonication pre-treatment
Ultrasonication is an emerging powerful tool to accelerate many
physical operations (Vilkhu et al., 2008). It has been also used to in-
crease the oil extraction about three times of the conventional
method used for the oil extraction. This ultra sonication pre-treat-
ment method has many advantages over the other methods, such
as reduced extraction time, reduced solvent consumption, greater
penetration of solvent into cellular materials and improved release
of cell contents into the bulk medium. This pre-treatment method
may also provide more benefits economically and environmentally
with health and safety aspects (Vilkhu et al., 2008).
3.3.1. Optimization of ultrasonic pre-treatment time
Ultrasonic pre-treatment method improves extractions of oil
significantly with higher efficiency, reduced extraction time and
increased yield, as well as low moderate costs and negligible added
toxicity. Algal biomass and water (water to biomass ratio as 3:1)
were mixed in a flask. The ultra sonication was performed at
24 kHz with constant temperature (50 °C ± 1) and at different time
intervals ranging from 2 to 6 min. It was observed that the increase
in the ultra sonication pre-treatment time increased the oil yield
from 2 to 5 min. At 5 min the yield was found to be 8.25% and
the maximum oil extraction yield of 8.49% was obtained at 6 min
duration of pre-treatment. After 6 min, the amount of oil yield
1
2
3
4
5
6
7
8
9
10
11
1 2 3 4 5 6 7
Oilextractionyield%(g/g)
Pretreatment methods
Fig. 1. Effects of various pre-treatment methods on oil extraction. 1. Direct
extraction; 2. autoclave; 3. ultra sonication; 4. lyophilization; 5. bead-beater; 6.
micro wave; 7. deep freezing.
T. Suganya, S. Renganathan / Bioresource Technology 107 (2012) 319–326 321

4. was found to be constant. From the literature, the benefit of using
ultrasonic pre-treatment before extracting oil from the seeds of
Jatropha curcas aqueous enzymatic oil extraction (AEOE) process
was evaluated by Shah et al. (2005). Ultra sonic pre-treated J. cur-
cas seeds provided significantly higher yield with reduction in
extraction time. Thus, implementation of ultrasonic pre-treatment
method reduced extraction time that may improve through put in
commercial oil production process (Vilkhu et al., 2008).
3.4. Solvent system
The selection of the solvent system for oil extraction from algal
biomass is an important factor. Solvent selection for extraction of
oil at the initial step would allow cost-effective for fuel production
without further expense required for the purification of the prod-
uct. The solvent chosen should have good extraction capacity and
low viscosity to enhance the free circulation. An efficient extrac-
tion requires the penetration of solvent into the biomass and to
match the polarity of the targeted compounds. An organic solvent
has a higher solubility with oil, this solvent system was used to fur-
ther degrade the cell walls of the algal biomass and to dissolve the
oil to enhance the oil yield. The effect of solvent systems on algal
oil extraction yield is shown in Table 1.
3.4.1. Hexane as a solvent system for extraction of oil
Hexane is extensively used for oil extraction because of its high
stability, low greasy residual effects, boiling point and low corro-
siveness. The highest oil extraction yield of 8.75% was achieved
from U. lactuca biomass using the most effective solvent mixture
as 1% diethyl ether and 10% methylene chloride in n-hexane. This
solvent system was used for the extraction of oil from the U. lactuca
algal biomass for further studies. From the literature it was ob-
served that the same solvent mixture was utilized for the extrac-
tion of triglycerides using the bond elut procedure (Kaluzny
et al., 1985).
Approximately 8.5% of oil extraction yield was achieved using
n-hexane as a solvent for the algal oil extraction. Whereas the yield
8.16% was obtained using n-hexane–ether (1:1) as solvent mixture.
While using n-hexane–2 isopropanol (Lee et al., 1998) and n-hex-
ane–ethyl alcohol (Meziane et al., 2006) as solvent mixtures, the oil
extraction yield was found to be 7.89% and 8.09%, respectively.
Hexane is the most widely used solvent for the extraction of oil
from micro and macro algae (Miao and Wu, 2006). Many research
works were already established for the extraction of value added
products from macro algal species with the use of hexane as
extracting solvent (Aresta et al., 2003). Vijayaraghavan and Hema-
nathan (2009) used hexane as a solvent for oil recovery from fresh
water algae for the biodiesel production. Large amount of microal-
gal oil was efficiently extracted from the heterotrophic cells by
using hexane as a solvent (Miao and Wu, 2006).
3.4.2. Other solvent systems
Oil extraction yield of 8.4% was achieved using single solvent
methyl tert-butyl ether (MTBE). MTBE extraction allowed faster
oil extraction and it forms the upper layer along with oil during
phase separation, due to its low density. Approximately 7.12%
and 7.36% of oil was extracted from algal biomass using chloroform
and methanol as a solvent mixture using the method suggested by
Bligh and Dyer (1959) and Folch et al. (1957). Chloroform–2 propa-
nol mixtures achieved 7.32% of oil extraction yield. The same sol-
vent system was used to separate all neutral lipids from crude
mixture by Kaluzny et al. (1985).
Nearly 7.51% and 7% of oil extraction yield was obtained using
dichloromethane–methanol and dichloroethane–ethanol as sol-
vent system, respectively. Lee et al. (1998) conducted an experi-
ment with these solvent systems and extracted 18% of oil from
Botryococcus braunii.
Oil yield of 6.55% was obtained using acetone–dichloromethane
as a solvent mixture. This solvent mixture extracted low oil yield
from algal biomass. So this solvent mixture is not used for the fur-
ther extraction experiment.
3.5. Optimization of various parameters influencing the extraction of
oil
The extraction of oil depends on the nature of the solvent, mois-
ture content, particle size, stirrer speed, extraction temperature,
time of extraction and solvent-to-solid ratio (Chaiklahan et al.,
2008; Siddiquee and Rohan, 2011).
3.5.1. Effect of moisture content
In algal oil extraction, moisture content is to be considered as
an important factor. Fig. 2a shows the effect of moisture content
on the oil extraction. Moisture content varying from 2% to 6% was
used for the oil extraction from U. lactuca algal biomass. Effect of
the moisture content was studied on oil extraction by keeping
other parameters as constant (particle size – 0.161 mm diameter,
stirrer speed 400 rpm, extraction temperature 50 °C, solvent-to-
solid ratio 1:5 and extraction time 120 min). From the Fig. 2a, it
was observed that the oil extraction was found to be increased
with increase in moisture content up to 5%. The maximum oil
extraction yield of 8.86% was achieved at 5% moisture content
of biomass. Above this 5% moisture content, the oil extraction
yield was found to be decreased with increase in moisture con-
tent. This may be attributed due to the presence of high level
moisture content as a barrier between the solvent and algal bio-
mass which may restrict the penetration of solvent into the bio-
mass. Rao and Arnold (1958) studied the extraction of oil from
oilseeds with the moisture level of 3% and maximum yield was
obtained at this 3% moisture level.
Table 1
Effects of various solvent systems on oil extraction yield (%).
S. No. Solvent/solvent mixture Ratio/percentage Oil extraction yield (%)
1 Hexane – 8.53 ± 0.04
2 Methyl tertbutyl ether – 8.42 ± 0.09
3 Chloroform:methanol 1:2 7.36 ± 0.08
4 Hexane:ether 3:1 8.16 ± 0.10
5 Chloroform:methanol 2:1 7.12 ± 0.07
6 Diethyl ether and methylene chloride in hexane 1% and 10% 8.75 ± 0.09
7 Chloroform:2 propanol 2:1 7.32 ± 0.08
8 Hexane:2 isopropanol 3:2 7.89 ± 0.06
9 Dichloromethane:methanol 1:1 7.51 ± 0.04
10 Dichloromethane:ethanol 1:1 7.00 ± 0.05
11 Acetone:dichloromethane 1:1 6.55 ± 0.04
12 Hexane:ethyl alcohol 1:1 8.09 ± 0.03
322 T. Suganya, S. Renganathan / Bioresource Technology 107 (2012) 319–326

5. 3.5.2. Effect of particle size on oil extraction
Particle size is found to be a critical parameter for the extraction
of oil from the biomass. Smaller the size of biomass leads to greater
in the interfacial area between the solid and liquid. Therefore the
increase in interfacial area increases the oil extraction yield.
The maximum oil extraction of 9.05% was obtained at 0.12 mm
diameter of particle size with 5% optimum moisture level at 50 °C
temperature with 400 rpm and solvent-to-solid ratio of 5:1 for
120 min. From the Fig. 2b, it was observed that the oil extraction
yield was found to be gradually increased from 6.57% to 9.05% with
decrease in particle size from 0.359 mm to 0.12 mm diameter. Be-
low 0.12 mm diameter of the particle, there was no any further in-
crease in oil extraction. This shows that the maximum oil
extraction yield of 9.05% was obtained with the biomass particle
size of 0.12 mm diameter. It is well recognized that the rate of sol-
vent extraction is controlled by the biomass particle size.
The lower extraction rate is attributed due to the bigger parti-
cles. This bigger particle creates difficulty for the solvent to pene-
trate into the core of the biomass to leach the oil. It is noticed
that the particle size is not only increases the extraction rate, but
also increases the oil extraction yield.
Qian et al. (2008) showed that the extraction rate of cottonseed
oil was found to increase with the reduced particle size of cotton-
seed flours. However, further decrease in the particle size did not
show much improvement in the oil extraction yield of cottonseed
oil. Particle size varying from 0.3 mm to 0.335 mm was found to be
optimum for cottonseed flour. According to Han et al. (2009), the
main reason for increasing oil yield was due to decrease in particle
size, which in turn increases the specific surface area of oilseed
interacting with the solvent.
3.5.3. Effect of stirrer speed on oil extraction
The effect of stirrer speed on oil extraction is illustrated in
Fig. 2c. Stirrer speed of the extraction also affects the oil yield. It
increases the eddy diffusion and the transfer of oil from the slurry
form of the algal biomass to the solvent mixture. The effect of stir-
rer speed on oil extraction in the range of 200–600 rpm was eval-
uated with other parameters as constant. The oil extraction yield
was found to be increased from 7.81% to 9.36% with an increase
in the stirrer speed. The maximum oil extraction yield was ob-
tained as 9.36%, at 500 rpm. However, for stirrer speed more than
500 rpm, there was no significant increase in the oil extraction
yield from U. lactuca biomass in slurry. From the experimental re-
sult, it was observed that oil extraction yield was found to be low
at low stirrer speed. In order to overcome this problem, the extrac-
tion was carried out at higher stirrer speed of 500 rpm. This is
clearly revealed that the mass transfer plays major role during
extraction with solvent system.
Kadi and Fellag (2001) studied the effect of stirrer speed on oil
extraction from olive foot cake using hexane as a solvent. They
have extracted oil from 6.9% to 7.7% with the use of stirrer speed
varying from 600 to 1000 rpm. Maximum yield of 7.7% was
achieved at 800 rpm.
3.5.4. Effect of temperature
The effect of temperature on the algal oil extraction yield was
examined over the range of 35–65 °C from U. lactuca biomass
(Fig. 2d). The oil yield was found to be enhanced with the rise in
the temperature. This is due to the increase in the dissolution
capacity of the solvent system. The rise in the temperature from
35 to 55 °C leads to increase in the yield from 7% to 9.75%. At
55 °C, highest oil extraction yield of 9.75% was obtained at opti-
mum conditions of 5% moisture content, 0.12 mm particle size
and 500 rpm stirrer speed for 120 min. Solvent-to-solid ratio of
5:1 was maintained. It was observed that at optimum temperature
of 55 °C, the solubility of the solvent was found to be increased
with increase in diffusion rate (Denery et al., 2004).
Solvent based extraction was developed because it allows com-
plete extraction of oil in the low temperature varying from 50 to
60 °C (Amin et al., 2010).
Karlovic et al. (1992) investigated the effect of temperature on
the kinetics of oil extraction from corn germ flakes prepared by a
dry degermination process. Increase in the temperature can en-
hance the capacity of solvents to dissolve the oil because the ther-
mal energy can overcome the cohesive and adhesive interactions
(Karlovic et al., 1992).
7
7.5
8
8.5
9
9.5
10
0 1 2 3 4 5 6 7
Oilextractionyield%(g/g)
Moisture content(%)
(a)
2
3
4
5
6
7
8
9
10
0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
Oilextractionyield%(g/g)
Particle size (mm)
(b)
5
5.5
6
6.5
7
7.5
8
8.5
9
9.5
10
100 200 300 400 500 600 700
Oilextractionyield%(g/g)
Stirrer Speed (rpm)
(c)
5
6
7
8
9
10
11
12
30 35 40 45 50 55 60 65 70
Oilextractionyield%(g/g)
Temperature (°C)
(d)
4
5
6
7
8
9
10
11
12
0 20 40 60 80 100 120 140 160 180
Oilextractionyield%(g/g)
Time (mins)
(e)
8
8.5
9
9.5
10
10.5
11
11.5
12
1 2 3 4 5 6 7 8
Oilextractionyield%(g/g)
Solvent to Solid ratio
(f)
Fig. 2. Optimization of various parameters on oil extraction from U. lactuca biomass. (a) Effect of moisture content on oil extraction [conditions: particle size: 0.161 mm
diameter; stirrer speed: 400 rpm; temperature: 50 °C; extraction time: 120 min; solvent-to-solid ratio: 5:1]. (b) Effect of particle size on oil extraction [conditions: moisture
content: 5%; stirrer speed: 400 rpm; temperature: 50 °C; extraction time: 120 min; solvent-to-solid ratio: 5:1]. (c) Effect of stirrer speed on oil extraction [conditions:
moisture content: 5%; particle size: 0.12 mm diameter; temperature: 50 °C; extraction time: 120 min, solvent-to-solid ratio:5:1]. (d) Effect of extraction temperature on oil
extraction [conditions: moisture content: 5%; particle size: 0.12 mm diameter; stirrer speed: 500 rpm; extraction time: 120 min; solvent-to-solid ratio: 5:1]. (e) Effect of
extraction time on oil extraction [conditions: moisture content: 5%; particle size: 0.12 mm diameter; stirrer speed: 500 rpm, temperature: 55 °C; solvent-to-solid ratio: 5:1].
(f) Effect of solvent-to-solid ratio on oil extraction [conditions: moisture content: 5%; particle size: 0.12 mm diameter; stirrer speed: 500 rpm; temperature: 55 °C; extraction
time: 140 min].
T. Suganya, S. Renganathan / Bioresource Technology 107 (2012) 319–326 323

6. 3.5.5. Effect of extraction time
The extraction time is an important parameter for oil yield. It
helps in deciding the optimum residence time required for the
extraction process. In this study, the effect of time on the oil
extraction was investigated with different time intervals varying
from 20 to 160 min (Fig. 2e). The results showed that oil extraction
yield increases with increase in time. The extraction was estab-
lished with the optimum condition of 5% moisture content,
0.12 mm diameter of particle size, stirrer speed of 500 rpm, tem-
perature at 55 °C and solvent-to-solid ratio of 5:1. After 120 min
of extraction, the oil extraction yield was obtained as 9.75%.
Although the time was extended up to 140 min, the oil extraction
yield was found to be improved up to 10.59%. The increase in
extraction time above 140 min did not show any further significant
improvement in the extraction. Hence, 140 min was found to be an
optimum extraction time for further studies.
The oil extraction yield was studied at different time intervals at
constant temperature. The oil extraction yield was gradually in-
creased from 4.1% to 7.89% with increase in time from 20 to
160 min at constant temperature of 35 °C. The same type of study
was conducted for different temperatures varying from 35 to 55 °C.
The oil extraction yield was found to be maximum at 55° C when
compared to all other temperatures studied for extraction with
the time duration of 140 min. From the above data the kinetic
model was analyzed for the oil extraction.
Higher oil extraction yield was obtained with much shorter time
(140 min) than with other extraction methods such as aqueous
extraction and enzyme assisted aqueous extraction (18–25 h). This
short extraction time could be due to the rapid mass transfer of oil.
From the literature, it was observed that the extraction of oil
from Cyanobacterium spirulina was carried out at different time
interval varying from 50 to 150 min. The optimum time was found
to be 120 min for the maximum production of oil (Chaiklahan
et al., 2008). The maximum yield of 26.4% oil from Cunninghamella
echinulata CCF-103 fungus species at the time duration of 180 min
was reported by Certik and Horenitzky (1999).
3.5.6. Effect of solvent-to-solid ratio
The effect of solvent-to-solid ratio on the oil extraction is shown
in Fig. 2f. The influence of solvent-to-solid ratio from 3:1 to 7:1 on
oil extraction was studied by maintaining all other parameters at
optimum conditions. As the solvent-to-solid ratio increased from
3:1 to 6:1, the oil yield was found to be increased from 9% to
10.88%. The trend was continued with increase in solvent-to-solid
ratio up to 6:1. Further increase in solvent-to-solid ratio above 6:1
did not show much improvement in the oil extraction. Therefore
the solvent-to-solid ratio of 6:1 (v/w) was found to be an optimum
ratio for the further study.
Kadi and Fellag (2001) used solvent-to-solid ratio as 4:1 for the
extraction of oil from olive foot cake using hexane as a solvent and
obtained 9.4% yield. Pokoo-Aikins et al. (2010) extracted oil from
sewage sludge by using toluene, n-hexane, ethanol and methanol
as a solvent mixture. They used solvent to sludge ratio as 5:1 for
the maximum extraction of oil.
The oil extraction yield as 10.88% obtained from the present
investigation stands in comparison to other established experi-
mental data. Hossain and Salleh (2008) extracted algal oil from
Oedogonium sp. (fresh water macro algae) for the biodiesel produc-
tion. They extracted 3 g of oil from 32.4 g of the algal biomass. The
maximum oil extraction yield was found to be 9.2% from Oedogoni-
um macroalgae biomass (Hossain and Salleh, 2008).
3.6. Composition analysis and properties of algal oil
Fatty acid composition of algal oil was analyzed by GC–MS
(Table 2). From the analysis, the saturated fatty acids (79.82%)
were found to be more when compared to unsaturated fatty
acids (20.18%). In the saturated fatty acid Palmitic acid composi-
tion was observed to be maximum of 50.16% in U. lactuca algal
oil. Properties such as molecular weight, Saponification value, Io-
dine value, Acid value and free fatty acid (FFA) were determined
(Table 3).
3.7. Extraction kinetics
A reaction rate equation for oil extraction from algal biomass
can be written as Eq. (4) (Topallar and Gecgel, 2000)
dY=dt ¼ kY
n
ð4Þ
where Y is the oil extraction yield (%), t is the time of extraction
(min), k is the extraction rate constant (minÀ1
) and n is the order
of the reaction. As the percentage of oil extraction increased in
the course of time (Table 4), the terms dY/dt have a positive sign
(Topallar and Gecgel, 2000).
Using the values in Table 4 and applying the differential meth-
od, plots of lnY versus ln(dY/dt) at different temperatures with
optimum conditions were found to be linear according to Eq. (4).
A first-order kinetic model was fitted well with average regression
coefficient (R2
) value obtained as 0.936. The reaction rate constants
and the order of the reaction were determined using the intercept
and slope of the liner plot (Fig. 3). From the analysis of the data, the
oil yield was found to be increased with increase in extraction
time. The yield was also analyzed with respect to the extraction
time at constant temperature ranging from 35 to 55 °C. The deter-
mination of reaction rate constant explains about the time re-
quired to get maximum extraction of oil from the U. lactuca
biomass. Rate constants determined from the plots were found to
be increased with increase in temperature. This may be due to
the increase in the reactivity of the solvent to enhance the rate
of extraction. Hence, reaction rates are often found to depend
strongly on temperature.
Table 2
Fatty acid profile of U. lactuca algal oil.
Name of the fatty acid No. of carbon atoms Relative %
Lauric acid 12:0 3.08
Tridecanoic acid 13:0 3.72
Myristic acid 14:0 5.85
Pentadecanoic acid 15:0 1.77
Palmitic acid 16:0 50.16
Heptadecanoic acid 17:0 0.75
Streacic acid 18:0 11.07
Nonadecanoic acid 19:0 0.02
Arachidic acid 20:0 2.05
Heneicosanoic acid 21:0 0.23
Behinic acid 22:0 1.12
Oleic acid 18:1 16.57
Linoleic acid 18:2 3.23
Linolenic acid 18:3 0.38
Table 3
Properties of algal oil extracted from U. lactuca biomass.
Parameters Extracted algal oil Units
Average molecular weight 780 g/mol
Saponification value 212.9 ± 1.86 mg of KOH/g
Iodine value 97.22 ± 1.33 –
Acid value 14.27 ± 1.12 mg of KOH/g
FFA 6.6 ± 0.35 wt%
324 T. Suganya, S. Renganathan / Bioresource Technology 107 (2012) 319–326

7. Table 4
The oil extraction yield % from algal biomass at various extraction temperatures with respect to extraction time.
Temp (°C) Reaction rate constants (k) (minÀ1
) Oil extraction yield (%)
20 min 40 min 60 min 80 min 100 min 120 min 140 min 160 min
35 5.27 Â 10À4
4.15 ± 0.08 4.80 ± 0.09 5.13 ± 0.11 5.60 ± 0.10 6.30 ± 0.09 7.00 ± 0.08 7.80 ± 0.07 7.89 ± 0.06
40 2.33 Â 10À3
4.25 ± 0.09 4.80 ± 0.10 5.36 ± 0.10 6.10 ± 0.11 6.90 ± 0.12 8.12 ± 0.11 8.88 ± 0.09 8.90 ± 0.08
45 2.88 Â 10À3
4.48 ± 0.07 4.95 ± 0.08 5.60 ± 0.09 6.38 ± 0.09 7.41 ± 0.08 8.59 ± 0.10 9.79 ± 0.09 9.60 ± 0.08
50 5.53 Â 10À3
4.68 ± 0.05 5.30 ± 0.09 6.11 ± 0.07 7.05 ± 0.12 8.16 ± 0.11 9.36 ± 0.11 10.26 ± 0.09 10.20 ± 0.07
55 8.14 Â 10À3
5.31 ± 0.07 6.68 ± 0.08 7.36 ± 0.09 8.08 ± 0.11 8.88 ± 0.12 9.75 ± 0.12 10.59 ± 0.09 10.59 ± 0.09
T = 328 K y = 0.717x -4.811 R² = 0.956
T = 308 K y = 1.521x -6.261 R² = 0.904
T = 318 K y = 1.523x -6.063 R² = 0.937
T = 313 K y = 1.384x -5.851 R² = 0.933
T = 323 K y = 1.161x -5.197 R² = 0.948
-5.0
-4.6
-4.2
-3.8
-3.4
-3.0
-2.6
-2.2
-1.8
-1.4
-1.0
1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5
ln(dY/dt)
ln Y
Fig. 3. A plot of [ln(dY/dt)] versus [lnY] at different temperatures ranged from 35 to 55 °C for extraction of oil from U. lactuca biomass.
y = -7581.x + 18.21 R² = 0.943
-8
-7.5
-7
-6.5
-6
-5.5
-5
-4.5
-4
-3.5
-3
0.003 0.00305 0.0031 0.00315 0.0032 0.00325 0.0033
lnK
1 / T (K-1
)
Fig. 4. Activation energy calculation from the plot of lnk versus 1/T (KÀ1
).
T. Suganya, S. Renganathan / Bioresource Technology 107 (2012) 319–326 325

8. 3.8. Calculation of activation energy
The relation between rate constant versus extraction tempera-
ture can be described by the Arrhenius Equation (Eq. (5)) (Topallar
and Gecgel, 2000):
k ¼ AeÀEa=RT
ð5Þ
where k is the reaction rate constant (minÀ1
), A is the Arrhenius
constant or frequency factor (sÀ1
), Ea is the activation energy (kJ/
mol), R is the universal gas constant (J/mol K) and T is the absolute
temperature in K. A plot of lnk versus 1/T gives a straight line whose
slope represents the activation energy of extraction – Ea/R (Fig. 4)
(Topallar and Gecgel, 2000). Thus, the activation energy (Ea) was
calculated as 63.031 kJ/mol. The same type of result was previously
reported by Amin et al. (2010) for the extraction oil from J. curcas.
4. Conclusion
Extraction of oil from U. lactuca algal biomass using ultrasound
pre-treatment method showed better results when compared with
other methods studied. Diethyl ether of 1% and 10% of methylene
chloride in n-hexane achieved high oil yield. The maximum oil
yield of 10.88% was obtained with optimum conditions of 5% mois-
ture content, 0.12 mm of particle size, 55 °C temperature, 500 rpm
stirrer speed, solvent-to-solid ratio as 6:1 and 140 min extraction
time. Kinetic studies revealed that this extraction followed first or-
der. The oil extracted from U. lactuca was found to be one among
the suitable source for biodiesel production.
Acknowledgements
The authors gratefully acknowledge Department of Science and
Technology (DST), New Delhi for providing financial support to car-
ry out this research work under PURSE scheme. One of the authors
Ms. T. Suganya is grateful to DST, New Delhi for the award of DST-
PURSE fellowship.
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