Algal Research 7 (2015) 117123

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Algal Research
journal homepage: www.elsevier.com/locate/algal

Review article

A review on the extraction of lipid from microalgae for

biodiesel production
M. Mubarak a, A. Shaija a,, T.V. Suchithra b
a
Mechanical Engineering Department, National Institute of Technology Calicut, Kerala 673 601, India
b
School of Biotechnology, National Institute of Technology Calicut, Kerala 673 601, India

a r t i c l e i n f o a b s t r a c t

Article history: Biofuels produced from algal biomass are the most suitable alternative fuels for the future, as microalgae biomass
Received 11 March 2014 can accumulate lipids within their cell similar to vegetable oils with a potential to produce 100 times more oil per
Received in revised form 18 October 2014 acrel and than any other plants. The methods used for the extraction of lipid from microalgae are either mechan-
Accepted 29 October 2014
ical or chemical method. The chemical methods of lipid extraction are Soxhlet extraction, supercritical uid ex-
Available online 17 November 2014
traction, accelerated solvent extraction and mechanical methods are oil expeller, microwave assisted
Keywords:
extraction, ultrasonic assisted extraction. This paper is a review of different methods used for extracting oils or
Biomass productivity lipids from microalgae biomass for biodiesel production. It is seen that lipid extraction yield from microalgae
Ultrasonication could be increased by using pretreatment methods such as ultrasonication and microwave-assisted techniques
Microwave assisted extraction along with solvent extraction.
Solvent extraction 2014 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
2. Lipid composition of microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
3. Lipid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
3.1. Mechanical methods of extraction of lipid or oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
3.1.1. Expeller or press . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
3.1.2. Ultrasound assisted extraction of oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
3.1.3. Microwave assisted extraction of oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
3.2. Chemical methods for extraction of oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
3.2.1. Solvent extraction method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
3.2.2. Supercritical CO2 extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
3.2.3. Ionic liquids as solvent for extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
4. Extraction of oil from wet algae biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
5. Effect of pretreatment on the lipid extraction from algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

1. Introduction
The escalating prices of the fast depleting fossil fuels and the impact
of its usage on the environment have lead to the search for alternative
fuels for diesel engines [1]. The biofuels that are produced from biomass
Corresponding author.
E-mail address: shaija@nitc.ac.in (A. Shaija).
are considered as promising alternative fuels and are classied as solid
(bio-char), liquid (ethanol, vegetable oil, and biodiesel), and gaseous
(biogas, biosyngas and biohydrogen) fuels.
The liquid biofuels are categorized into different generations of
biofuels based on the type of feedstock used. The rst-generation liquid
biofuels are derived from edible feedstock such as corn, soybean, sugar-
cane, and rapeseed while the second-generation biofuels are from non-
edible feedstock such as Jatropha, Miscanthus, and Switch grass.

http://dx.doi.org/10.1016/j.algal.2014.10.008
2211-9264/ 2014 Elsevier B.V. All rights reserved.
118 M. Mubarak et al. / Algal Research 7 (2015) 117123
However, the escalating demand for edible feedstock as a food source,
coupled with the nite availability of arable land for the cultivation of
edible and non-edible feedstock makes the rst and second-
generation biofuels unsustainable. Thus, the third generation biofuels,
which are derived from micro- and macro-algae have an edge over
the previous two categories and the fourth generation biofuels are
based on metabolic engineering of photosynthetic organism to produce
fuel [2]. Microalgae are microscopic photosynthetic organisms that are
found in marine and freshwater environment [3,4] and have a potential
of xing 1.83 tons of atmospheric CO2 when producing 1 ton of algae
biomass [5]. Microalgae are considered to be good candidates for biofuel
production because of their higher photosynthetic efciency, higher
biomass production and faster growth rate compared to other energy
crops such as rapeseed and soybean [6]. Moreover, it is easy to cultivate
microalgae with wastewater even in lands that are unsuitable for agri-
culture [7,8]. The lipid accumulation in the cells of microalgae ranges
from 2575% of its dry weight [9]. Hence, production of biodiesel from
microalgae is a promising step towards nding a suitable alternative
fuel for diesel engine. The key processes involved in the biodiesel pro-
duction from microalgae are cultivation, harvesting, biomass process-
ing, lipid extraction, and its transesterication. Among these methods,
lipid extraction is an important and costly process. The economical pro-
duction of biodiesel from microalgae mainly depends upon the energy
utilized for the processing of biomass and the type of lipid extraction
process used [10].
A few review papers have explained the different steps involved in
biodiesel production from algae, like cultivation, harvesting, lipid ex-
traction, and production of biodiesel without emphasizing the various
methods of extracting lipids or oils from microalgae. Hence, this paper
is an attempt to give an overview of the different techniques adopted
for the extraction of lipid from microalgae.
2. Lipid composition of microalgae
Lipids are biological molecules, which are soluble in organic sol-
vents. The fatty acids, which are the main constituents of both neutral
and polar lipid molecules, are of saturated and unsaturated types. Satu-
rated fatty acids do not have double bonds, while unsaturated fatty
acids consist of at least one double bond. The lipid content of microalgae
varies considerably for different species. The composition and fatty acid
prole of lipids extracted from a particular species is affected by the cul-
tivation conditions, such as medium composition, temperature, illumi-
nation intensity, ratio of light/dark cycle and aeration rate and ranges
from 12 to 22 carbons in length [11].
Lipid extraction methods
Mechanical methods
1. Oil expeller or press
2. Ultrasound assisted
3. Microwave assisted
Fig. 1. Flow chart of different lipid extraction methods from microalgae.
3. Lipid extraction
The methods used for the extraction of lipid from microalgae are cat-
egorized as mechanical and chemical methods [11]. The chemical
methods of lipid extraction are Soxhlet extraction, supercritical uid ex-
traction, and accelerated solvent extraction; mechanical methods are oil
expeller, microwave assisted extraction, and ultrasonic assisted extrac-
tion [12,13], as shown in Fig. 1. The chemical method uses organic sol-
vents like n-hexane that is toxic and inammable, and vacuum
distillation, which affects health and environment. The supercritical
uid extraction technology eliminates the use of toxic solvents and
uses non-toxic CO2 gas as solvent. The efciency of oil extraction can
be increased using solvents such as chloroform, methanol and n-
hexane. Both polar and non-polar solvents are used for the extraction
and its selection depends on the polarity of the target compound as it
dictates reciprocal solubility; polar compounds are better-extracted
using polar solvents and vice versa. Hexane (non-polar) has been used
extensively throughout the world as a solvent for extracting vegetable
oils [14].
The process of lipid or oil extraction from microalgae cells is an ener-
gy intensive and costly procedure because the use of solvents for the ex-
traction of lipids requires extra energy for recovering the same solvents
from lipids after the extraction [15]. In addition, the method used
should be fast and should not damage the extracted lipids.
3.1. Mechanical methods of extraction of lipid or oil
3.1.1. Expeller or press
This method uses a mechanical press to break the cells and com-
press out the oil from the dry biomass and can extract about 75% of
oil. But, this method is quite slow and requires a large quantity of
biomass [16]. Niraj et al. [17] extracted about 75% of lipid from la-
mentous algae using a screw expeller press and some amount of
lipid present with the cake formed was removed using solvent ex-
traction method.
3.1.2. Ultrasound assisted extraction of oil
The thick cell walls of microalgae blocks the release of intralipids
present inside and the use of methods like solvent extraction and me-
chanical press, yields less lipid [18]. The principle behind ultrasound
assisted extraction method is that the intense sonication of liquid gener-
ates sound waves that propagate into the liquid media and results in al-
ternate high-pressure and low-pressure cycles. During high-pressure
cycle, the small vacuum bubbles, which are produced in the low-
pressure cycle, collapse violently and result in a phenomenon called
Chemical methods
1. Solvent extraction
2. Supercritical CO2
3. Ionic liquid extraction
 M. Mubarak et al. / Algal Research 7 (2015) 117123
cavitation. The high pressure and high speed liquid jets form shearing
forces around the algae cells during cavitation and break the cell struc-
ture mechanically and improve material transfer supporting the extrac-
tion of lipids. Hence, ultrasonic assisted extraction technique with
sound waves having frequencies higher than 20 kHz is used and due
to this high intensity, small vacuum bubbles are created in the liquid
[19]. This effect supports the extraction of lipids from microalgae and
an oil yield enhancement by 50500% with 10 fold reduced extraction
time [20] is achieved. Adam et al. [21] proposed a new method as
solvent-free ultrasound assisted method to extract lipids from
Nannochloropsis oculata biomass. They used response surface methodol-
ogy (RSM) for the optimization of oil recovery using parameters like
ultrasonication power, extraction time and moisture content of biomass
and have compared it with the oil yield extracted using Bligh Dyer's
method. Widjaja et al. [22] reported no improvement in the lipid yield
and the extraction time for Chlorella vulgaris using ultrasonication
method.
3.1.3. Microwave assisted extraction of oil
Microwaves are electromagnetic radiation of frequency from 0.3 to
300 GHz. The microwave assisted heating uses a non-contact heat
source, which can penetrate into the biomaterials, interact with polar
molecules like water in the biomass, and heat the whole sample uni-
formly. The higher oil yield with superior quality and reduced extraction
time are the main advantages of microwave-assisted extraction (MAE).
Iqbal and Theegala [15] investigated the solvent potential of biodiesel
(methyl soyate) containing 20% (BD20) and 40% (BD40) biodiesel
blended with ethanol at three different temperatures of 80C, 100C
and 120C for microalgae lipid extraction using MAE. They compared
the yield with MAE using chloroform methanol and with Soxhlet extrac-
tion. The results showed improved lipid extraction yield with BD40
compared to other traditional toxic solvents like n-hexane and chloro-
form. A resonant continuous microwave processing system was used
for extracting oil from microalgae, Scenedesmus obliquus with hexane
as solvent and 77% of the total lipid content was extracted at 95C in
30 min [14].
Chen et al. [23] measured neutral lipids from 13 green algae
strains namely, Nannochloris sp. LB 229 (UTEX); Desmodesmus
quadricauda; Palmellococcus miniatus; Pseudochloroccocum sp.
(ASU strain 1); Ankistrodesmus pseudobranunii 245 (UTEX);
Ankistrodesmus falcatusvar stipitatus; Scenedesmus dimorphus (ASU
strain 1); Ankistrodesmus pseudo pseudobranunii LB 1380 (UTEX);
Neochloris oleoabundans 1185 (UTEX); Chlorella zongiensis (ASU
strain 2); Ankistrodesmus braunii 750 (UTEX); Scenedesmus sp.
(ASU strain 1), and Chlorella ellipoidea (ASU strain 3) using micro-
wave assisted Nile red uorescence. They used optimized timing
of 50 and 60 s in the microwave oven for pretreatment of algae bio-
mass and staining process, respectively. This method was effective
for quantifying the lipid content of the algae.
3.2. Chemical methods for extraction of oil
3.2.1. Solvent extraction method
The chemistry concept of like dissolving like is the basic principle
behind lipid extraction from microalgae using solvents. An ideal solvent
requires high levels of specicity towards lipids especially acylglycerols
and the solvent must be volatile enough to ensure low energy distilla-
tion to separate the lipid from solvents. The extraction of lipids from
algae biomass can use non-polar solvents such as hexane, benzene,
toluene, diethyl ether, chloroform and polar solvents such as methanol,
acetone, ethyl acetate, and ethanol. The non-polar solvents disrupt the
hydrophobic interactions between non-polar and neutral lipids
available in the algae biomass. The solvents used for extracting lipid
from microalgae biomass are n-hexane, ethanol, 1-butanol, DBU (1,8-
diazabicyclo-[5.4.0]-undec-7-ene), dimethyl ether, and mixtures
of chloroform/methanol, n-hexane/ethanol, n-hexane isopropanol,
119
n-hexane/2-propanol, methanol/1-ethyl-3-methyl imidazolium methyl
sulfate, DBU/ethanol, DBU/octanol, methylene chloride/methanol, di-
chloroethane/methanol, dichloroethane/ethanol, and acetone/dichloro-
methane [18]. Among these, chloroform/methanol (1/2 v/v) is the most
widely used organic solvent system extracting lipid from biomass and
animal tissues, as the extraction time is reduced with increased yield.
Folch et al. [24] used a mixture of chloroform: methanol 2:1 by volume,
for extracting lipids from animal tissues and named it the Folch method
[25]. The Bligh and Dyer method is used for total lipid extraction and pu-
rication with methanol and chloroform as solvents, and water as co-
solvent [26]. A number of works have been reported in the literature
using the Bligh and Dyer method for the extraction of lipids [22,
2730] and modied forms of this method have also been used
[3135]. Lee et al. [31] evaluated the performance of ve different sol-
vent systems such as chloroform/methanol (2:1, v/v); hexane/
isopropanol (3:2, v/v); dichloromethane/methanol (1:1, v/v); dichloro-
methane/ethanol (1:1, v/v) and acetone/dichloromethane (1:1, v/v) via
bead beating of Botrycoccus braunii biomass. The chloroform/methanol
(2:1 v/v) solvent system showed higher lipid yield of 28.6%. Cheng
et al. [30] investigated the mixed solvent extraction of ultrasonically
pretreated Pavlov species lipids with solvent systems such as ethyl ace-
tate/methanol toluene/methanol hexane/methanol and showed higher
lipid yield of 98% with ethyl acetate/methanol solvent system.
Balasubramanian et al. [36] classied the lipid into three categories
as neutral lipid, free fatty acid (FFA) and polar lipid using solid-phase
extraction column and investigated the factors like biomass drying,
moisture content and solvent systems such as chloroform:methanol
(2:1) and hexane:methanol (3:2) which affect the intracellular lipid ex-
traction from marine microalgae. They found that the FFA content of the
lipid extracted from solar dried algae biomass was three times higher
than freeze-dried algae biomass. The extraction of oil using chloro-
formmethanol solvent system showed maximum lipid extraction ef-
ciency and the moisture content of more than 5% reduces the oil
extraction yield and increases the FFA fraction of oil.
The use of toxic solvents like hexane and chloroform cause ad-
verse health and environmental hazards and is the major drawback
of solvent extraction method of algae oil [37]. Laboratory scale
work generally uses batch process for extracting oil from algae
with organic solvents. The lipid content from biomass into the sol-
vent reaches a state of equilibrium with the lipid concentration in
the solvent, which limits further transfer of lipid to the solvent in
the batch process of extraction, whereas a continuous organic sol-
vent extraction method could overcome this limitation, but is expen-
sive, as it requires a large amount of organic solvents. During the
cycles of solvent evaporation and condensation, Soxhlet apparatus
continuously replenishes biomass with fresh organic solvents and
minimizes the solvent consumption [11]. The Soxhlet apparatus con-
sists of three main compartments: a continuously heated round bot-
tom ask, Soxhlet extractor to hold the microalgae biomass and a
condenser for continuous cooling. Guckert et al. [38] used methylene
chloride and methanol solvent systems for extracting lipids from
Chlorella and recovered greater amounts of neutral lipid as a similar
modied Bligh and Dyer's method, which used phosphate buffer in
addition to the chloroform, methanol and water mixture. D'Oca
et al. [39] used different methods like Soxhlet extraction, magnetic
stirring, and ultrasonic bath with ve solvent systems such as mix-
tures of chloroform and methanol (2:1 v/v), methanol, chloroform,
ethanol, and hexane for lipid extraction from the dry biomass of
Chlorella pyrenoidosa. They reported that a mixture of chloroform/
methanol used as a solvent for extraction yields more lipids from
microalgae than other solvents.
The use of organic solvents for extracting lipids uses energy in-
tensive distillation after extraction for separating lipid from the sol-
vents [11]. Boyd et al. [40] used a switchable hydrophilic solvent N,
N-dimethylcyclohexylamine for extracting lipid from freeze- dried
samples of Botryococcus braunii. The Liquid ChromatographyMass
120 M. Mubarak et al. / Algal Research 7 (2015) 117123

Spectrometry (LCMS) analysis of the crude lipid showed high con-
centrations of long chain mono-, di- and tri-acylglycerides without
any phospholipids.
Even though the solvent extraction techniques are cheaper and easy
to execute, the use of toxic solvents and longer execution times are the
main drawbacks. In order to minimize these drawbacks, a new acceler-
ated solvent extraction technique was introduced, which uses solvents
at temperatures between 50 and 200C and pressures between 10 and
15 MPa. The high pressure ensures that the solvent always remains in
liquid state for safe and rapid extraction. Pieber et al. [41] used pressur-
ized uid extraction (accelerated solvent extraction) of polyunsaturated
fatty acid from N. oculata, taking n-hexane, a mixture of n-hexane and
propan-2-ol (2:1 vol.%) and ethanol (96 vol.%) as solvents. The elevated
temperatures increase the solubility of solvent in biomass and the
higher pressure keeps the solvent in liquid state at high temperatures
above the boiling point.
3.2.2. Supercritical CO2 extraction
Supercritical uids are found to be suitable as an extraction solvent
because the solvent power of a supercritical uid, being a function of
density, can be varied by changing the extraction pressure and temper-
ature, and is capable of producing solvent free crude lipids [42]. Super-
critical CO2 (SCCO2) is the primary solvent commonly used for the
majority of supercritical uid extractions due to its moderate critical
pressure (7.4 MPa) and low critical temperature (31.1C) [43]. The op-
erating variables inuencing the performance of SCCO2 extraction of
microalgae lipids are pressure, temperature, and uid ow rate. Cram-
pon et al. [44] used SCCO2 method of extraction from N. oculata with
air ow drying and freeze drying as pretreatment methods. The airow
drying at a temperature of 35C was the most adequate pretreatment
method for extracting 90% by weight of triglycerides without any
phospholipids. Halim et al. [12] evaluated the performance of SCCO2 ex-
traction and hexane extraction based on the yield and fatty acid compo-
sition of lipid extracted from Chlorococcum sp. for biodiesel production.
The lipid yield decreased with increase in temperature and pressure
using SCCO2 extraction. They also reported that Soxhlet extraction
using hexane took 5.6 times the time required for SCCO2 extraction for
achieving a comparable lipid yield of 0.058 g lipid/g dried microalgae
and using co-solvents like ethanol with CO2 could increase the lipid ex-
traction yield in SCCO2 extraction. Liau et al. [45] used co-solvent mod-
ied SCCO2 extraction of lipids from the microalgae, N. oculata and
reported that at 35 MPa, 50C and 16.7 of weight percentage of ethanol
addition as co-solvent yielded 239.7 mg of triglycerides per gram of ex-
tracted oil.
The optimum conditions of the operating variables like pressure,
temperature, modier addition, and uid ow rate are important for re-
ducing the energy requirement and maximizing the lipid yield in SCCO2
extraction. Tang et al. [46] obtained the optimized conditions for SCCO2
extraction of lipids from microalgae, Shizochytrium limacinum as
35 MPa, 40C with 95% volume ethanol as co-solvent. Hu et al. [47] re-
ported that the highest lipid yield of C. pyrenoidosa extracts was obtain-
ed at 32C, 40 MPa, 20l/h with dosage of modier as 1 ml of ethanol per
gram sample for 3 h. Santana et al. [42] reported that the optimal condi-
tions obtained for extracting lipids from B. braunii was 2225 MPa and
50C. They concluded that as the pressure and density of CO2 increases,
the amount of unsaturated compounds present in the extracted lipid
also increases causing more unsaturated fatty acids in the triglycerides.
Noble et al. [48] extracted 33% of lipid from Nannochloropsis sp. at the
best operating conditions of 40C and 30 MPa with a CO2 ow rate of
0.62 g/min. Gouveia et al. [49] used a supercritical uid extraction
(SFE) method to study the effect of modiers (oil mixed with
microalgae and ethanol with SCCO2) for extracting lipid and pigments
from C. vulgaris at 30 MPa and 40C. They compared the lipid yield
with that obtained by using acetone and soybean oil at room and higher
temperature and concluded that the SCCO2 extraction showed superior
yield of pigments and lipid with reduced time. Table 1 shows the com-
parison of the different extraction methods based on the species of
algae, solvent used, operating conditions, and their lipid yields.
3.2.3. Ionic liquids as solvent for extraction
Ionic liquids are suitable for the extraction of lipids from algae due to
their non-volatility, thermal stability, and synthetic exibility. Kim et al.
[53] used a mixture of ionic liquids like [Bmim][CF3SO3] and methanol,
which could extract 19% of lipids from C. vulgaris using Bligh and Dyer's
method. This limited the yield to 12.5% of lipids and characterized the
fatty acid proles of extracted lipids using gas chromatograph. Kim
et al. [54] used ionic liquids like [MeSO4][Bmim] [MeSO4] with
ultrasonication and reported the total amount of lipid extracted from
C. vulgaris by Soxhlet's and Bligh and Dyer's methods are 21 and
29 mg/g dry cell weight, respectively compared to 47 mg/g dry cell
weight using ionic liquids with ultrasonication. The lipid extracted
with ionic solvents was 1.6 times higher than Soxhlet's and Bligh and
Dyer's methods. The fatty acids' proles of lipids extracted showed sim-
ilarity with those lipid obtained by Bligh and Dyer's method. The lique-
faction of microalgae, Spirulina with Fe (CO)5-S catalyst at 350C for
60 min in tetralin under 5 MPa of hydrogen showed an increased oil
yield from 52.3 to 66.9 wt.% [55].

Table 1
Comparison of extraction methods based on their lipid yields.

Extraction method Solvent used Operating conditions Algae used Lipid yield (%) References
(temperature, pressure, time)

Supercritical CO2 and ethanol 40C, 35 MPa, 30 min Shizochytrium limacinum 33.9 [46]
CO2 Pavlova sp. 34 [30]
Soxhlet n-Hexane 40C, 0.1 MPa, 18 h Shizochytrium limacinum 45 [46]
Dichloromethane Nannochloropsis oculata 9 [45]
n-Hexane Nannochloropsis oculata 5.79 [45]
Ethanol Nannochloropsis oculata 40.90 [45]
n-Hexane Pavlova sp. 45.2 [30]
Petroleum ether Nannochloropsis oculata 8.2 [50]
Ethanol Synechocystis PCC 6803 48 [51]
Mixed solvent extraction Hexane/ethanol 200C, 0.1 MPa, 2 h Synechocystis PCC 6803 52 [51]
Hexane/isopropanol Synechocystis PCC 6803 36 [51]
Chloroform/methanol Synechocystis PCC 6803 50 [51]
Chloroform/methanol/water Synechocystis PCC 6803 42 [51]
Pressurized uid extraction n-hexane 60C, 1012 MPa, 10 min Nannochloropsis oculata 6.1 [41]
N-hexane/propan-2-ol (2:1 vol.%) Nannochloropsis oculata 20 [41]
Ethanol 96 vol.% Nannochloropsis oculata 36 [41]
Ultrasonic assisted extraction Petroleum ether Frequency 40 kHz, 1 h Nannochloropsis oculata 3.3 [50]
Wet extraction Hexane 90C, 0.1 MPa Chlorella and Scenedesmus sp. 59.3 [52]
 M. Mubarak et al. / Algal Research 7 (2015) 117123
4. Extraction of oil from wet algae biomass
Drying and extracting oil from microalgae accounts for 90% of the
total process energy in dry extraction of lipid for algal biodiesel produc-
tion [56]. The drying of algae biomass is energy intensive and 25% reduc-
tion in energy for drying process can be attained by using wet algae
biomass [57], in which no drying is required. A few authors have report-
ed extraction of oil from wet microalgae. Kanda et al. [58] used liquid di-
methyl ether (DME) as a solvent for extracting hydrocarbons and lipids
from wet B. braunii showing the same yield as obtained by Soxhlet ex-
traction using hexane as solvent with dried algae biomass. They
concluded that with DME, the extraction process could save the energy
required for drying and cell disruption of algae biomass. Yoo et al. [57]
used osmotic treatment, which is a novel method with polar and non-
polar solvents to extract lipid from wet Chlamydomonas reinhardtii and
reported two times increased lipid recovery. A single step supercritical
process for simultaneous extraction and transesterication of
wet algae, Nannochloropsis sp. with 90% water content was reported
by Patil et al. [59]. They studied the inuence of three variables like
wet algae to methanol in weight by volume ratio, reaction temperature
and the reaction time on the conversion of Nannochloropsis sp., biomass
to fatty acid methyl esters using response surface methodology (RSM).
They obtained the optimum values of the variables, wet algae to meth-
anol (weight by volume) ratio, reaction temperature, and time as 9:1,
255C and 25 min respectively. Sathish and Sims [52] developed a
lipid extraction procedure that was capable of extracting 79% of
transesteriable lipids using acid and base hydrolyses of wet mixed cul-
tures of Chlorella and Scenedesmus sp. containing 84% moisture. Liu et al.
[60] used a water miscible solvent of 1,2-dimethoxyethane for the ex-
traction of algal oil from wet cells of B. braunii and found that the
water content of algae biomass signicantly affected the lipid extraction
efciency.
5. Effect of pretreatment on the lipid extraction from algae
Depending on the type of biomass, it is desirable to pretreat the bio-
mass it before the extraction of lipid. The pretreatment or disruption of
microalgae cells induces lipid extraction, liberating lipids from the lipid
encapsulated cell structures. The pretreatment methods can be either
mechanical or non-mechanical. Mechanical methods include high-
pressure homogenization, ultrasonication, bead milling, autoclaving, ly-
ophilization, and microwaving. The non-mechanical methods involve
treating the cells with acids, alkalis, enzymes or osmotic shocks [61,
62]. The ultrasonication method of cell disruption of microalgae,
Schizochitrium limacinum and C. reinhardtii can produce high quality
lipids with higher yields [63].
Halim et al. [61] employed different pretreatment methods such as
high-pressure homogenization, ultrasonication, bead beating, and sul-
furic acid treatment for extraction of lipid from Chlorococcum sp. They
evaluated the performances based on the reduction in the intact cell
count and the average colony diameter and concluded that high-
pressure homogenization followed by sulfuric acid treatment can be
the most effective method of cell disruption. Enzymatic hydrolysis is a
recently developed method with an advantage of saving energy due to
its lower temperature and higher selectivity of components present in
the microalgae. The rst work on this was reported by Fue et al. [64],
in which the cell walls of microalgal cellulose was hydrolyzed using
immobilized cellulose into an electrospun polyacrylonitrile (PAN)
nanobrous membrane followed by lipid extraction. The microalgae
lipid extraction efciency increased to 56% from 40%. Cho et al. [65] ex-
amined the effect of enzymatic hydrolysis for disrupting the cell wall of
C. vulgaris and extracted oil using organic solvents such as chloroform,
methanol and hexane. A maximum hydrolysis yield of 85.3% and an in-
creased lipid extraction yield of 1.291.73 times higher than untreated
C. vulgaris were achieved at an optimum pH of 4.8 and a temperature
of 50C. Sander and Murthy [66] used enzymes for the degradation of
121
C. vulgaris. Horst et al. [67] reported that the use of crude papain and
bromelain was more effective in releasing tryacylglycerides with hep-
tanes/isopropyl alcohol from Phaeodactylum tricornutum. Fue et al.
[64] considered the effect of hydrolysis of cell walls of microalgae
using the enzyme cellulase and reported that lipid extraction efciency
was improved from 32% to 56%. The work reported by McMillan et al.
[68] analyzed different pretreatment methods such as microwave,
water bath, blender, ultrasonic and laser for disrupting the cell wall of
N. oculata.
The requirement of relatively low temperature makes the
ultrasonication method of cell disruption better compared to micro-
wave and autoclave methods of pretreatment. The conditions required
for sonication were important for reducing energy input and improving
the quality of lipid extracted from algae biomass. Gerde et al. [63] iden-
tied the best conditions to maximize the oil yield using sonication
method for disrupting the cells of two microalgae, Schizochytrium
limacinum and C. reinhardtii. They reported that 800J/10 ml was the en-
ergy required for maximizing cell disruption using ultrasonication.
Table 2 shows the advantages and disadvantages of different methods
of oil extraction.
6. Conclusions
As microalgae biomass can accumulate lipids within their cell with a
potential to produce 100 times more oil per acre than terrestrial plants,
the biodiesel produced from algal biomass is one of the most suitable al-
ternative fuels for the future. The chemical methods of lipid extraction
are Soxhlet extraction, supercritical uid extraction, and accelerated
solvent extraction; and mechanical methods are oil expeller, microwave
assisted extraction, and ultrasonic assisted extraction. This paper re-
views these different methods used for extracting lipid from microalgae
biomass for biodiesel production and the following conclusions are
drawn.
Most of the researchers have used organic solvents such as n-hexane,
chloroform, methanol, ethanol, petroleum ether, dichloromethane,
isopropanol for extracting oil from microalgae biomass due to lower
initial capital equipment investment.
Finding the most efcient method and optimized conditions for the
extraction of lipid are important for reducing the cost of biodiesel pro-
duction from microalgae.
Adopting a single method of extraction may not be sufcient to get the
maximum yield from algae. Hence, pretreatment methods such as
ultrasonication and microwave-assisted methods combined with sol-
vent extraction and mechanical press at the end can extract maximum
amount of algae lipids.
The supercritical uid extraction technique uses less toxic solvents for
extracting lipids, but the initial capital equipment investment is high.
The scaling up of extraction methods was important for producing
large quantities of algae oil for biodiesel production. So far, the studies
based on the extraction of oil are limited to laboratory scale and fur-
ther investigations are required for extracting large quantity of algae
oil or lipids with reduced cost.
Acknowledgement
We thank the Department of Mechanical Engineering and School of
Biotechnology National Institute of Technology Calicut, Kerala, India for
supporting the research work.
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