Biocombustibles (Lectura 1) PDF

Biotechnology Advances 30 (2012) 709732
Contents lists available at SciVerse ScienceDirect
Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv
Research review paper
Extraction of oil from microalgae for biodiesel production: A review

Ronald Halim , Michael K. Danquah, Paul A. Webley
Bio Engineering Laboratory (BEL), Department of Chemical Engineering, Monash University, Victoria 3800, Australia
a r t i c l e
i n f o
Article history:
Received 8 March 2011
Received in revised form 14 November 2011
Accepted 4 January 2012
Available online 11 January 2012
Keywords:
Microalgae
Biodiesel
Oil extraction
Lipid extraction
Organic solvent
Supercritical carbon dioxide
Downstream process
Pre-treatment
Direct transesterication
a b s t r a c t
The rapid increase of CO2 concentration in the atmosphere combined with depleted supplies of fossil fuels
has led to an increased commercial interest in renewable fuels. Due to their high biomass productivity,
rapid lipid accumulation, and ability to survive in saline water, microalgae have been identied as promising
feedstocks for industrial-scale production of carbon-neutral biodiesel. This study examines the principles
involved in lipid extraction from microalgal cells, a crucial downstream processing step in the production
of microalgal biodiesel. We analyze the different technological options currently available for laboratoryscale microalgal lipid extraction, with a primary focus on the prospect of organic solvent and supercritical
uid extraction. The study also provides an assessment of recent breakthroughs in this rapidly developing
eld and reports on the suitability of microalgal lipid compositions for biodiesel conversion.
2012 Elsevier Inc. All rights reserved.
Contents
1.
2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Microalgal lipid composition . . . . . . . . . . . . . . . . . . . . . . .
Overview of downstream processes . . . . . . . . . . . . . . . . . . . .
Lipid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Organic solvent extraction . . . . . . . . . . . . . . . . . . . . .
4.1.1.
Basic principles . . . . . . . . . . . . . . . . . . . . . .
4.1.2.
Solubility parameters . . . . . . . . . . . . . . . . . . .
4.1.3.
Selection of organic solvents . . . . . . . . . . . . . . . .
4.1.4.
Operating variables . . . . . . . . . . . . . . . . . . . .
4.1.5.
Modications to organic solvent extraction . . . . . . . . .
4.2.
Supercritical uid extraction . . . . . . . . . . . . . . . . . . . .
4.2.1.
Basic principles . . . . . . . . . . . . . . . . . . . . . .
4.2.2.
Operating variables . . . . . . . . . . . . . . . . . . . .
4.3.
Comparison between organic solvent extraction and SCCO2 extraction
5.
Effect of cellular pre-treatment on lipid extraction . . . . . . . . . . . . .
6.
Simultaneous extraction and transesterication of microalgal lipids . . . . .
7.
Microalgal biorenery . . . . . . . . . . . . . . . . . . . . . . . . . .
8.
OriginOil Single-Step Extraction of microalgal lipids . . . . . . . . . . . .
9.
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding author. Tel.: + 61 3 990 53420.

E-mail address: ronald.halim@monash.edu (R. Halim).
0734-9750/$ see front matter 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2012.01.001
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R. Halim et al. / Biotechnology Advances 30 (2012) 709732
1. Introduction
The search for sustainable and renewable fuels is becoming
increasingly important as a direct result of climate change and rising
fossil-fuel prices. Current commercial production of biodiesel or fatty
acid methyl ester (FAME) involves alkaline-catalyzed transesterication of triglycerides found in oleaginous food crops with methanol.
However, cultivation of these food crops for biodiesel (mainly
rapeseed in Europe and soybean in the US) is no longer sustainable
as it requires substantial arable land and consumes large amounts of
freshwater (Chisti, 2007).
Microalgae are currently considered to be one of the most
romising alternative sources for biodiesel (Sheehan et al., 1998).
Since many microalgal strains can be cultivated on non-arable land
in a saline water medium, their mass farming does not place additional strains on food production (Widjaja et al., 2009). Their high photosynthetic rates, often ascribed to their simplistic unicellular
structures, enable microalgae not only to serve as an effective carbon
sequestration platform but also to rapidly accumulate lipids in their
biomass (up to 77% of dry cell mass). Even using a conservative
scenario, microalgae are still predicted to produce about 10 times
more biodiesel per unit area of land than a typical terrestrial oleaginous crop (Chisti, 2007; Rosenberg et al., 2008; Sheehan et al.,
1998; Shenk et al., 2008).
There are, however, various technological and economic obstacles
which have to be overcome before industrial-scale production of
microalgal biodiesel can take place. The selection and successful
outdoor large-scale cultivation of a robust microalgal strain, which
has optimum neutral lipid content, possesses an elevated growth
rate, and is immune towards invasion by local microbes, remain a
major upstream challenge (Sheehan et al., 1998). On the other
hand, the development of an effective and energetically efcient

lipid extraction process from the microalgal cells is critical for the successful upscaling of the downstream processes. Despite the routine
use of laboratory-scale extraction protocols to determine microalgal
lipid contents, the variables affecting lipid extraction from microalgal
cells are not well understood and no method for industrial-scale
extraction is currently established (Halim et al., 2011).
This paper attempts to address the knowledge gap surrounding
microalgal lipid extraction by summarizing and critiquing recent
studies in the eld. We report on the suitability of microalgal lipid
compositions for biodiesel conversion and review the different conventional downstream bioprocessing steps required for microalgal
biodiesel production. We then examine the technologies currently
available for laboratory-scale microalgal lipid extraction, paying special attention to the use of organic solvent extraction and supercritical
uid extraction. We conclude with an assessment on how different
cellular pre-treatment processes can effect microalgal lipid extraction
as well as with an update on the recent advances in the eld, such as
the development of a simultaneous microalgal lipid extractionmethylation method and the establishment of a novel single-step
microalgal lipid extraction method by OriginOil, Inc.
2. Microalgal lipid composition
A fatty acid (FA) molecule consists of a hydrophilic carboxylate
group attached to one end of a hydrophobic hydrocarbon chain
(Fig. 1). Fatty acids are constituents of lipid molecules (both neutral
and polar) and designated based on their two most important
features the total number of carbon atoms in the hydrocarbon
chain: the number of double bonds along the hydrocarbon chain.
Saturated fatty acids have no double bond, while unsaturated fatty
Fig. 1. (a) Fatty acid chains. Saturated fatty acid (C18:0 or stearic acid) on the left. Unsaturated fatty acid (C18:1 or oleic acid) on the right. Oleic acid is of cis-isomerism. (b) Lipid
molecules. Triacylglycerol (neutral lipid) on the left. Phospholipid (polar lipid) on the right. R, R, R in the triacylglycerol molecule represent fatty acid chains. Phospholipid
molecule is negatively charged.
Modied from Nelson and Cox (2000).
acids consist of at least one double bond (Nelson and Cox, 2000).
When the carboxylate end of the fatty acid molecule is bonded to
an uncharged head group (e.g. glycerol), a neutral lipid molecule is
formed (e.g. triacylglycerol). On the other hand, the association of a
fatty acid molecule to a charged head group (e.g. glycerol and phosphate complex) forms a polar lipid molecule (e.g. phospholipid).
Lipids can be dened as any biological molecule which is soluble
in an organic solvent. As mentioned above, most lipids contain fatty
acids (Fig. 1) and can generally be classied into two categories
based on the polarity of the molecular head group (Kates, 1986a):
(1) neutral lipids which comprise acylglycerols and free fatty acids
(FFA) and (2) polar lipids which can be further sub-categorized into
phospholipids (PL) and glycolipids (GL). Neutral lipids are used
primarily in the microalgal cells as energy storage, while polar lipids
pack in parallel to form bilayer cell membranes. Acylglycerol consists
of fatty acids ester-bonded to a glycerol backbone and is categorized
according to its number of fatty acids: triacylglycerols (TG), diacylglycerols (DG), monoacylglycerols (MG). FFA is a fatty acid bonded to a
hydrogen atom.
It is noted that there are also some types of neutral lipids that do
not contain fatty acids, such as hydrocarbons (HC), sterols (ST),
ketones (K), pigments (carotenes and chlorophylls). Even though
these lipid fractions are soluble in organic solvents (hence tting
the denition of lipids), they are not convertible to biodiesel. Readers
are referred to other sources for a more comprehensive description
regarding lipids, their varieties, and their molecular structures
(Kates, 1986a; Mathews and van Holde, 1996; Volkman et al.,
1989). The term oil is often used to refer to any lipid fraction that
exists as a liquid at ambient conditions. Since lipids, especially those
obtained from microalgae, are extracted as composite mixtures
consisting of various fractions, they do not always exist as liquids.
As such, the term oil is not used in any part of this study.
Microalgal lipid content varies considerably from one species to
another and could range, in terms of dry biomass, from 5 to 77 wt.%
(Brown et al., 1997; Chisti, 2007). Brown et al. (1997) studied the nutritional properties of 40 different Australian microalgal species and
concluded that they comprise, as a weight fraction of dry cell mass,
between 5 and 20% lipids. Microalgal lipid composition also varies
considerably from one species to another (Brown et al., 1997). During
their study investigating the lipid compositions of various microalgal
species, Lv et al. (2010) demonstrated that some microalgal species
are richer in neutral lipids than other species.
The composition and fatty acid prole of lipids extracted from a
particular species is further affected by the microalgal life cycle as
well as the cultivation conditions, such as medium composition,
temperature, illumination intensity, ratio of light/dark cycle, and
aeration rate (Guzman et al., 2010; Ota et al., 2009; Ramadan et al.,
2008; Rao et al., 2007). Microalgal cells harvested during the stationary phase have lower polar lipid contents than the same species
obtained during the logarithmic phase (Dunstan et al., 1993). Some
microalgal species have been known to increase their lipid contents
from ~ 10 wt.% to almost 20 wt.% during oxygen deprivation
(Dunstan et al., 1993). Microalgal cells generally respond to nutrient
starvation by intensifying the metabolic pathway which synthesizes
neutral lipids. However, this increase in cellular lipid production
usually does not result in an overall increase in oil productivity per
unit mass as it is often performed by sacricing growth and through
the cessation of cell division. Due to the aforementioned inter- and
intraspecic variations, the suitability of microalgal lipids for biodiesel production is difcult to assess and often needs to be examined
on a case-by-case basis.
Acylglycerols are desirable for commercial-scale biodiesel production for two main reasons. Firstly, industrial-scale alkaline-catalyzed
transesterication is designed to process acylglycerols (TG, DG, and
MG) and has limited efcacies on other lipid fractions, such as polar
lipids and free fatty acids (Christie, 2007; Lang et al., 2001). Secondly,
711
since acylglycerols generally have a lower degree of unsaturation

than other lipid fractions (i.e. polar lipids), they produce FAME with
higher oxidation stability. As shown by the lipid proles of three
microalgal species (Nannochloropsis oculata, Pavlova lutheri, Isochrysis
sp.) in Fig. 2, microalgal lipids usually comprise high levels of polar
lipids and non-acylglycerol neutral lipids (HC, ST, K, FFA). As such,
they often need to be puried before they can be transesteried.
When comparing lipids obtained from logarithmic and stationary
growth phase, stationary-phased lipids, despite having an abundance
of polar lipids at 5157 wt.%, contain higher levels of TG (2041 wt.%
of total lipid) and appear more attractive for biodiesel processing
than logarithmic-phased lipids (Dunstan et al., 1993).
Microalgal fatty acids range from 12 to 22 carbons in length and
can be either saturated or unsaturated. The number of double bonds
in the fatty acid chains, however, never exceeds 6 and almost all of
the unsaturated fatty acids are cis isomers (Medina et al., 1998).
The fatty acid prole of lipid extracted from Tetraselmis suecica during
early stationary phase is shown in Fig. 3 (Volkman et al., 1989). T. suecica is a common green microalga and its fatty acid prole is used
here as an example to illustrate the suitability of microalgal lipids
for biodiesel synthesis. Having C16:0, C18:1, and C18:2 as its the
principal fatty acids, Tetraselmis lipid appears to have the required
fatty acid prole for conversion to high-quality biodiesel. Saturated
neutral lipids breakdown
Fig. 2. Compositions of crude lipids extracted from three microalgal species during
logarithmic phase and stationary phase. Top: Nannochloropsis oculata, middle: Pavlova
lutheri, bottom: Isochrysis sp. For neutral lipids, TG: triacylglycerols, HC: hydrocarbons,
ST: sterols, K: ketones, FFA: free fatty acids.
Modied from Dunstan et al. (1993).
712
30
25
20
15
10
5
14
:0
16
:0
C
18
:0
C
16
:
C 1
16
:1
C t
18
:1
C
20
:1
C
16
:2
C
18
:2
C
16
:3
C
18
:3
C
16
:4
C
18
:
C 4
20
:4
C
20
:5
0
C
wt% of total fatty acids
fatty acid chain
No. of double bonds in the fatty acid chain

wt% of total fatty acids
4
20.8%
0
27.6%
3
14.2%
2
16.2%
1-trans
0.8%
1
20.3%
Fig. 3. Fatty acid composition of crude lipid extracted from the species Tetraselmis
suecica at the end of logarithmic phase (the beginning of stationary phase): (a) in
terms of fatty acid chain; (b) in terms of number of double bonds in the fatty acid
chain. In (a), the letter t after the fatty acid name (C16:1t) denotes trans-isomerism.
When no letter t appears, fatty acid is of cis-isomerism. In (b), the word -trans after
the number of double bonds denotes that fatty acids are of trans-isomerism. When
no isomerism is mentioned, fatty acid is of cis-conguration.
Modied from Volkman et al. (1989).
fatty acid content (27.6 wt.%) is relatively low when compared to the
total cis-unsaturated content (71.6 wt.%). This is desirable as FAME
derived from cis-unsaturated fatty acids often has advantageous
cold ow properties (a low cloud point and a low pour point). In
contrast to saturated chains which pack rapidly upon temperature
decrease to form tight semicrystalline structures, cis-unsaturated
fatty acids are prevented from forming regular molecular packing
due to the bends imposed by the cis double bonds and consequentially freeze at a much lower temperature (Lang et al., 2001; Mathews
and van Holde, 1996). The extracted lipid contains a relatively modest
amount of polyunsaturated fatty acids (PUFA) with 4 or more double
bonds (C16:4 being the most abundant at 7.9 wt.%). This is desirable
as highly unsaturated PUFAs are known to be responsible for the
poor volatility, the low oxidation stability, and the tendency for
gum formation observed in some oilseed-derived biodiesel (Lang
et al., 2001). In terms of lipid classes, it is noted that acylglycerols
generally have a lower degree of unsaturation than polar lipids and,
as such, are more suited for biodiesel conversion.
3. Overview of downstream processes
Fig. 4 shows the downstream processing steps required to produce
biodiesel from microalgal biomass (Halim et al., 2011). Table 1 lists
the different laboratory-scale technological options currently available for each step. The table also examines the scale-up potential of
each technology. After the microalgal culture is harvested from the
bioreactor, it is concentrated in a dewatering step. The concentrated
microalgal culture is then processed in a pre-treatment step to prepare it for lipid extraction. During lipid extraction, lipids are extracted
out of the cellular matrices with an extraction solvent. The lipids are
then separated from the cellular debris, isolated from the extraction
solvent and any residual water, and nally converted to biodiesel in
the transesterication step. Each of the downstream processes is

explained in more detail below.
Cultivation of microalgae is performed in either an indoor or an
outdoor system (Chisti, 2007). Indoor cultivation systems normally
use photobioreactors, while outdoor systems employ either raceway
ponds or photobioreactors. In an outdoor system, the microalgae are
grown in the open environment where cultivation parameters
(temperature and light intensity) are dependant on day-to-day
weather conditions. The microalgae grown in such a system often
suffer from inconsistent growth rates and are more susceptible to
local species invasion. On the other hand, the microalgae grown in
an indoor system are placed in a greenhouse-type structure where
cultivation conditions can be tightly controlled. Despite providing
better protection against local species invasion, the indoor system is
not preferred due to its high operating cost (Chisti, 2007). Throughout its cultivation, microalgal culture needs to be aerated with CO2
supply and replenished with growth medium consisting of essential
elements, such as nitrogen, phosphorous, and iron (Chisti, 2007).
Harvested microalgal culture exists as a dilute aqueous suspension
(from 0.1 to 2 g dried microalgal biomass/L culture, depending on cultivation method) and needs to be concentrated in order to reduce the
cost of downstream processing (Danquah et al., 2009; Molina Grima
et al., 2003). Solidliquid separation methods, such as centrifugation,
ltration, and occulation, are used to dewater the microalgal culture
to a concentration between 10 and 450 g dried microalgal biomass/L
culture. Concentrated microalgal culture is referred to as concentrate.
When dewatered beyond 200 g dried microalgal biomass/L culture,
the concentrate is transformed to a sludge suspension and is often
referred to as paste or pellet. Developing a cost-viable and an
energy-efcient dewatering technology is currently an active eld of
research.
Among the myriads of dewatering technologies (Table 1),
occulation appears to be the most advantageous due to its low energy requirement (Uduman et al., 2010; Wijffels and Barbosa, 2010).
During occulation, microalgal cells adhere to one another to form
heavy aggregates which then settle to become concentrate. Cationic,
anionic, and non-ionic polyelectrolytes (or polymer) are typically
used to occulate microalgal cells. More details on microalgal dewatering and occulation can be found elsewhere (Uduman et al., 2010).
Post-dewatering, the concentrate undergoes pre-treatment process aimed at enhancing the efciency of subsequent lipid extraction
(Lee et al., 1998, 2010). As shown in Fig. 4, the pre-treatment process
can take different pathways depending on the desired biomass alterations. The pre-treatment can be performed in either a single step or
multiple steps. Sometimes, no pre-treatment is performed and the
concentrate is directly processed for lipid extraction. In one option
of the pre-treatment pathways, the concentrate is exposed to a cell
disruption method (such as high-pressure homogenization) which
ruptures the cellular structures. This pre-treatment forces the release
of intracellular lipids to the surrounding medium, thus assisting the
lipid extraction process. Microalgal concentrate that has been
subjected to cell disruption process is referred to as disrupted concentrate. In a typical 2-step laboratory-scale pre-treatment pathway, the
concentrate is completely dried and then milled into ne powders.
This pre-treatment eliminates residual water known to prohibit effective mass transfer during the lipid extraction step and results in the
formation of dried powder. Detailed discussion on the types and
effects of pre-treatment are deferred to Section 5.
During lipid extraction, the pre-treated microalgal biomass
(existing as one of the following physical states: concentrate or
disrupted concentrate or dried powder) is exposed to an eluting
extraction solvent which extracts the lipids out of the cellular matrices. Concentrate or disrupted concentrate still contains a certain
level of residual water, while dried powder will be completely devoid
of residual water. The principles and processes involved in lipid extraction are discussed in detail in the following section (Section 4).
713
Fig. 4. Process ow diagram showing the downstream processing steps needed to produce biodiesel from microalgal biomass.
Microalgal lipid extraction generally uses either organic solvent or

supercritical uid (such as supercritical carbon dioxide) as an
extraction solvent.
After lipid extraction, the resulting mixture, consisting of extraction solvent, residual water (only when extraction is performed on
concentrate or disrupted concentrate), lipids, and cell debris, is

submitted to a solidliquid separation method, such as ltration, to
remove the cell debris. For organic solvent extraction, a liquidliquid
separation method, such as distillation, vacuum evaporation, or solidphase solvent adsorption, is then employed to remove the extraction
714
Table 1
Different laboratory-scale technologies available for each downstream processing
step required to produce biodiesel from microalgae. The scale-up potential of each
technology is examined. . * * *: highly scalable. *: lack scalability.
Process step
Cultivation
Dewatering
Pre-Treatment: cell
disruption
Pre-Treatment: drying
Pre-Treatment: particulate
size reduction
Lipid extraction
Removal of cell debris

from extraction solventresidual water-lipids
mixture
Removal of extraction
solvent and residual water
from lipids. This step is
not applicable to
supercritical fluid
extraction.
Lipid fractionation
Transesterification
Technologies
Scale-up potential
raceway ponds
***
photobioreactors
**
agglomeration
***
centrifugation
**
filtration
**
flocculation
***
pressure dewatering
ultrasonication
**
high-pressure homogenization
***
french pressing
**
bead miling
** *
microwave
**
chemical lysis (acids &

enzymes)
**
osmotic shock
**
oven drying
freeze drying
spray drying
milling with specific sieve
**
crushing with pestle and mortar
organic solvent extraction
**
supercritical fluid extraction
**
organic solvent extraction with

Soxhlet apparatus
ultrasound-assisted organic
solvent extraction
**
microwave-assisted organic
solvent extraction
**
accelerated organic solvent

extraction
**
sub-critical organic solvent

extraction
**
filtration
**
centrifugation (not applicable

to supercritical fluid extraction)
**
distillation
**
vacuum evaporation
solid-phase solvent adsorption
**
liquid chromatography
silicic acid column

chromatography
acid precipitation
**
urea crystallization
**
acid catalyst
***
alkali catalyst
***
solvent and the residual water from the lipids. When non-polar/polar
organic solvent mixture is used (refer to Section 4.1.1), residual water
is removed from the extraction solvent and the lipids via biphasic
separation and decantation. The liquidliquid separation then
removes the extraction solvent from the lipids. On the other hand,
for supercritical uid extraction, pressure decompression returns
the extraction solvent as well as the residual water to their gaseous
states and results in forced precipitation of the lipids (refer to
Section 4.2.1). As such, no extra step is needed for the removal of
extraction solvent and residual water.
The isolated lipids, referred to as crude lipids or total lipids, can
now be gravimetrically quantied. The term total lipids is primarily
used for analytical purposes. As previously mentioned in Section 2,
in addition to acylglycerols, crude lipids obtained from microalgal
biomass frequently contain polar lipids and non-acylglycerol neutral
lipids (such as free fatty acids, hydrocarbons, sterols, ketones,
carotenes, and chlorophylls). From the perspective of biodiesel
production, any non-acylglycerol biochemical fraction is a contaminant and will have to be removed from the crude lipids. As such,
crude lipids arising from microalgal biomass are often subjected to a
fractionation step before they are transesteried. Different purication methods, such as liquid chromatography, acid precipitation,
and urea crystallizations, are used for lipid fractionation (Medina
et al., 1998).
During transesterication, the fatty-acid-containing lipid fractions
in the crude lipids are reacted with alcohol (methanol, ethanol,
isopropanol, butanol) and converted to fatty acid alkyl esters. When
methanol is used, the reaction produces fatty acid methyl ester
(FAME) or biodiesel. Either an acid (such as H2SO4) or an alkali
(such as NaOH or KOH) can be used as a catalyst for transesterication (Christie, 2007; Volkman et al., 1989). Since alkali catalysts
have faster reaction rates (estimated at 4000 faster) and higher conversions than acid catalysts for the transesterication of acylglycerols,
they are commercially used in the chemical industry for conversion of
plant and animal oils to biodiesel (Huang et al., 2010). As previously
noted in Section 2, alkaline-catalyzed transesterication has limited
efcacies when applied to non-acylglycerol fatty-acid-containing
lipid fractions, such as polar lipids and free fatty acids. During alkaline
transesterication of acylglycerols, the catalyst cleaves the ester
bonds holding the fatty acids to the glycerol backbone (Fig. 5)
(Chisti, 2007). The liberated fatty acids are then reacted with
methanol to form FAME. In lab-scale experiments where only small
amounts of crude microalgal lipids are available, a large amount of
methanol (substantially in excess of stoichiometric requirement) is
often added to ensure quantitative transesterication.
Once transesterication is completed, the reaction mixture,
containing biodiesel, glycerol, reformed alkali catalyst, excess
methanol, and un-transesteried lipids, then undergoes posttransesterication purication to remove by-product contaminants
(glycerol, alkali catalyst, and excess methanol). A laboratory-scale
post-transesterication purication method typically consists of 2
steps. The reaction mixture is left to settle under gravity to induce biphasic partitioning (top biodiesel/un-transesteried lipids phase and
bottom glycerol phase). Once the biodiesel/un-transesteried lipids
phase is decanted off, it is washed repeatedly with water to eliminate
any alkali catalyst and excess methanol (Chisti, 2007; Demirbas,
2008; Demirbas and Karslioglu, 2007). More details on alkaline transesterication of acylglycerols and post-transesterication purication
can be found elsewhere (Lang et al., 2001; Wahlen et al., 2011).
Analyses of the FAME composition of the puried biodiesel/
untransesteried lipids phase are carried out using a gas chromatography (GC) system.
Variables that affect FAME conversion during alkaline transesterication include the molar ratio of acylglycerol to methanol, the molar
ratio of acylglycerol to catalyst, the reaction temperature, the reaction
time, the FFA content of the crude lipids, and the water content of the
715
Triacylglycerol
Methanol
FAME
(biodiesel)
Glycerol
Diacylglycerol
Methanol
FAME
(biodiesel)
Glycerol
Monoacylglycerol
Methanol
Free Fatty Acid
Potassium
Hydroxide
Monoacylglycerol
Water
FAME
(biodiesel)
Soap
Free Fatty Acid
Glycerol
Water
Glycerol
Fig. 5. Various reactions involving the alkali catalyst, KOH or potassium hydroxide. [1], [2], and [3] illustrate the alkaline transesterication of acylglycerol with methanol to produce
biodiesel as a main product and glycerol as a by-product. [4] illustrates the undesirable reaction between free fatty acid and KOH to form soap and water (saponication).
[5] illustrates the undesirable reaction between acylglycerol (monacylglycerol is used as a representation) and water under an alkaline condition to from free fatty acid and glycerol.
Modied from Chisti (2007) and Huang et al. (2010).
crude lipids. As illustrated in Fig. 5, FFA reacts with the alkali catalyst
to form soap and water (saponication). As such, if the crude lipids to
be reacted have a high FFA content, excess alkali catalyst must always
be added in order to compensate for the saponication loss (Huang
et al., 2010). Past works on alkaline transesterication of vegetable
oil have shown that abundant presence of water in the crude lipids
had an adverse effect on the reaction kinetics (Christie, 2007; Lang
et al., 2001). As depicted in Fig. 5, water under alkaline conditions
irreversibly reacts with acylglycerol to from free fatty acid, the
formation of which, as mentioned above, consumes the alkali catalyst
for its elimination. In a study by Lepage and Roy (1984), FAME
recoveries during the transesterication of TG standards were found
to substantially decrease once water content of the standards
exceeded 20 wt.%.
In order for microalgal biodiesel to be environmentally sustainable,
the total CO2 emitted in the downstream processing steps must be
lower than or at least equal to the total CO2 originally captured by the
microalgal cells during their cultivation. Therefore, processes selected
in each step should aim at minimizing energy consumption.
4. Lipid extraction
Depending on its pre-treatment pathway, microalgal biomass to
be submitted to lipid extraction can assume one of the following
physical states: concentrate or disrupted concentrate or dried powder. During lipid extraction, the microalgal biomass is exposed to an
eluting extraction solvent which extracts the lipids out of the cellular
matrices. Once the crude lipids are separated from the cell debris, the
extraction solvent, and water (only when extraction is performed on
concentrate or disrupted concentrate), their mass can be measured
gravimetrically. Ideally, a lipid extraction technology for microalgal
biodiesel production needs to display a high level of specicity
716
towards lipids in order to minimize the co-extraction of non-lipid

contaminants, such as protein and carbohydrates. To reduce downstream fractionation/purication, the lipid extraction technology
should also be more selective towards acylglycerols than other lipid
fractions that are not as readily convertible to biodiesel, i.e. polar
lipids and non-acylglycerol neutral lipids (free fatty acids,
hydrocarbons, sterols, ketones, carotenes, and chlorophylls)
(Medina et al., 1998). Additionally, the selected technology should
be efcient (both in terms of time and energy), non-reactive with
the lipids, relatively cheap (both in terms of capital cost and operating
cost), and safe (Kates, 1986b). Since dewatering the microalgal biomass beyond a paste consistency (200 g dried microalgal biomass/L
culture) is energy intensive, it will be economically benecial if the
selected lipid extraction technology is effective when directly applied
to wet feedstock, i.e. concentrate or disrupted concentrate with
concentrations between 10 and 200 g dried microalgal biomass/L
culture (Halim et al., 2011). In this section, we will review the use
of organic solvent extraction for routine lab-scale determination of
microalgal lipid contents. We will also review the application of
supercritical uid extraction, an emerging green technology that is
currently gaining considerable research attention, for microalgal
lipid quantication.
4.1. Organic solvent extraction
4.1.1. Basic principles
The principles underlying organic solvent extraction of microalgal
lipids are anchored on the basic chemistry concept of like dissolving
like. Due to the interactions between their long hydrophobic fatty
acid chains, neutral lipids participate in weak van der Waals attractions between one another and form globules in the cytoplasm
(Kates, 1986b; Medina et al., 1998). The proposed mechanism for
organic solvent extraction is shown in Fig. 6 and can be divided into
5 steps. When a microalgal cell is exposed to a non-polar organic
solvent, such as hexane or chloroform, the organic solvent penetrates

through the cell membrane into the cytoplasm (step 1) and interacts
with the neutral lipids using similar van der Waals forces (step 2) to
form an organic solvent-lipids complex (step 3). This organic
solventlipids complex, driven by a concentration gradient, diffuses
across the cell membrane (step 4) and the static organic solvent
lm surrounding the cell (step 5) into the bulk organic solvent. As a
result, the neutral lipids are extracted out of the cells and remain
dissolved in the non-polar organic solvent. A static organic solvent
lm is formed due to the interaction between organic solvent and
cell wall. This lm surrounds the microalgal cell and remains
undisturbed by any solvent ow or agitation.
Some neutral lipids are, however, found in the cytoplasm as a
complex with polar lipids. This complex is strongly linked via
hydrogen bonds to proteins in the cell membrane. The van der
Waals interactions formed between non-polar organic solvent and
neutral lipids in the complex are inadequate to disrupt these
membrane-based lipidprotein associations. On the other hand,
polar organic solvent (such as methanol or isopropanol) is able to
disrupt the lipidprotein associations by forming hydrogen bonds
with the polar lipids in the complex (Kates, 1986b; Medina et al.,
1998). The mechanism in which the non-polar/polar organic solvent
mixture extracts membrane-associated lipid complexes is also proposed in lower half of Fig. 6 and can be divided into 5 steps. The organic solvent (both non-polar and polar) penetrates through the
cell membrane into the cytoplasm (step 1) and interacts with the
lipid complex (step 2). During this interaction, the non-polar organic
solvent surrounds the lipid complex and forms van der Waals associations with the neutral lipids in the complex, while the polar organic
solvent also surrounds the lipid complex and forms hydrogen bonds
with the polar lipids in the complex. The hydrogen bonds are strong
enough to displace the lipidprotein associations binding the lipid
complex to the cell membrane. An organic solventlipids complex is
formed and dissociates away from the cell membrane (step 3). The
static organic
solvent film
bulk organic
solvent
cell membrane
and cell wall
1
3
2
cytoplasm
nucleus
5
Fig. 6. Schematic diagram of the proposed organic solvent extraction mechanisms. Pathway shown at the top of the cell: mechanism for non-polar organic solvent. Pathway shown
at the bottom of the cell: mechanism for non-polar/polar organic solvent mixture. lipids, non-polar organic solvent, polar organic solvent. Both mechanisms can be described
in 5 steps. Step 1: penetration of organic solvent through the cell membrane. Step 2: interaction of organic solvent with the lipids. Step 3: formation of organic solventlipids complex. Step 4: diffusion of organic solventlipids complex across the cell membrane. Step 5: diffusion of organic solventlipids complex across the static organic solvent lm into the
bulk organic solvent.
717
hexane system = 0.015 g lipid/g dried microalgal biomass, nal total

lipid yield of hexane/isopropanol system (3/2 v/v) = 0.068 g lipid/g
dried microalgal biomass).
When a non-polar/polar organic solvent mixture (such as hexane/
isopropanol or chloroform/methanol) is used, both solvents are
added simultaneously to the microalgal biomass (existing as one of
the following physical states: concentrate or disrupted concentrate
or dried powder) in the desired volumetric ratio. Once cell debris is
removed using a solidliquid separation method (such as ltration),
biphasic separation of the initially single-phase organic solvent
mixture is induced by roughly equivolume addition of the non-polar
organic solvent (hexane for hexane/isopropanol mixture and chloroform for chloroform/methanol mixture) and water. Upon complete
biphasic separation, neutral and polar lipids will mainly partition in
organic solventlipids complex then diffuses across the cell membrane (step 4) and the static organic solvent lm surrounding the
cell (step 5) into the bulk organic solvent. As such, the addition of a
polar organic solvent to a non-polar organic solvent facilitates the extraction of membrane-associated neutral lipid complexes. However,
the process also inevitably leads to the co-extraction of polar lipids.
In most laboratory practices, both non-polar organic solvent and
polar organic solvent are added to the microalgal cells to ensure the
complete extraction of all neutral lipids, both in the form of freestanding globules and in the form of membrane-associated complexes. During our previous study investigating lipid extraction from
Chlorococcum sp. (Halim et al., 2011), the inclusion of isopropanol
as a co-solvent was shown to improve the total lipid yield of pure
hexane system by more than 300% (nal total lipid yield of pure
concentrate
microalgal culture
dried
hexane/IPA (3/2 v/v)
powder
cell
debris
hexane/
IPA
methanol
KOH
crude
lipids
biodiesel,
hexane/IPA
glycerol,
un-transesterified
hot plate stirrer
lipids
Fig. 7. Schematic diagram showing the experimental steps typically undertaken for laboratory-scale production of microalgal biodiesel using an organic solvent mixture as a lipid
extraction technology. Hexane/isopropanol (IPA) (3/2 v/v) mixture is used for lipid extraction. Cell disruption, lipid fractionation, and post-transesterication purication are not
performed. (a) Cultivation with photobioreactors. (b) Dewatering with a centrifuge. (c) Drying (pre-treatment) with an oven. (d) Particulate size reduction (pre-treatment) with a
pestle and a mortar. (e) Lipid extraction with hexane/isopropanol mixture. (f) Removal of cell debris with a lter. (g) Removal of extraction solvent with a distillation unit.
(h) Transesterication with an alkali catalyst.
718
the organic phase (a mixture of non-polar organic solvent and polar

organic solvent), while the aqueous phase (a mixture of water and
polar organic solvent) will contain primarily non-lipid contaminants
(proteins and carbohydrates) (Kates, 1986b; Medina et al., 1998).
As such, biphasic separation removes not only residual water but
also non-lipid contaminants from the mixture of organic solvents
and lipids. The organic phase is decanted and evaporated to yield
dry crude lipids, which are then fractionated and transesteried.
Fig. 7 shows the experimental steps typically undertaken for
laboratory-scale production of microalgal biodiesel using an organic
solvent mixture as a lipid extraction technology. Table 3 summarizes
the methods and the ndings of recent studies which investigated
organic solvent extraction of microalgal lipids.
4.1.2. Solubility parameters
Among the myriad of thermodynamic parameters (polarity index,
Kauri-butanol value, and Hildebrand solubility parameter) attempting to predict the solubility of a solute in a solvent, Hansen solubility
parameters (HSP) appears to be one of the more widely accepted and
promising systems (Gupta et al., 1997; Hansen, 2008; Snyder, 1974).
HSP characterization predicts that a solute will dissolve in a solvent if
the molecules of either substance have similar force of interaction.
With HSP system, total energy of cohesion (E) can be quantitatively divided into three components (Hansen, 2008). These components
account for the atomic dispersion (or Van der Waals) interactions
(ED), the molecular dipolar interactions (Ep), and the molecular
hydrogen bonding (electron exchange) interactions (EH).
E ED Ep EH
E is experimentally determined by measuring the energy required

to evaporate the liquid (solute or solvent), thus breaking all of its
cohesive bonds.
Dividing Eq. (1) by the molar volume, V, yields the three components of Hansen solubility parameters.
E=V ED =V Ep=V EH =V
where E/V = 2, ED/V = (D) 2, Ep/V = (P) 2, and EH/V = (H) 2

2
D P H
where is the Hildebrand total solubility parameter, D is the Hansen

dispersion parameter, P is the Hansen dipolar parameter, and H is
the Hansen hydrogen bond parameter. The SI unit for all of the
parameters is MPa 1/2. HSP characterization can be conveniently visualized with a spherical representation. Hansen solubility parameters
of the solute are at the center of the solubility sphere and the radius
of the solubility sphere (Ro) indicates the extent of interaction for
solubilization to occur. Good solvents lie within the solubility sphere
and poor ones lie outside. Ra is the distance of the solvent from the
center of the solubility sphere.
2
Ra 4DS DP PS PP HS HP
Subscript s is for the solvent and subscript p is for the solute.

If Ra b Ro, the solvent lies within the solubility sphere and the
solute is soluble in the solvent. Relative Energy Difference (RED) is
numerical representation of this graphical visualization. The smaller
its RED value, the better the solvent is at dissolving the solute.
RED Ra=Ro
For a solvent mixture, composite Hansen solubility parameter,

i, mix, is calculated with:
i;mix 1 i;1 2 i;2
Subscript i signies one of the three components of Hansen solubility parameters, is volume fraction of each solvent in the mixture.
We have computed the HSP characterizations for organic solvents
and organic solvent mixtures commonly employed to extract lipids
from microalgal biomass in Table 2. All of the Hansen solubility
parameters of pure organic solvents were obtained from Hansen,
2007. Eq. (6) was used to calculate composite Hansen solubility
parameters for organic solvent mixtures. Triacetin was used as a
representative for all types of triacylglycerols. Ra was calculated
with triacetin as a solute (Eq. (4)). Ro value of 12 MPa 1/2 indicates
marginal solubility of a solute in a solvent and was assigned for RED
estimation (Eq. (5)).
As can be seen from Table 2, triacetin appears to be highly soluble
in hexane/isopropanol mixture (3:2 v/v) (RED = 0.3) and chloroform
(RED = 0.4). On the other hand, chloroform/methanol/water mixture
(1:2:0.8 v/v/v) appears to be poor solvents for triacetin (RED = 1.2).
Such a nding is contradictory to previous lipid extraction works
which recommend the use of chloroform/methanol/water mixture
(Molina Grima et al., 1994). We note that the current triacetinbased HSP characterizations must be treated with caution. Triacylglycerols with higher carbon number will have different Hansen solubility parameters to triacetin. Additionally, limited availability of Hansen
solubility parameters for other desirable neutral-lipid derivatives,
such as diacylglycerols, monoacylglycerols, and neutral lipids/polar
lipids complexes, prevents the complete characterization of microalgal lipids. More research is needed before the mechanisms underlying
organic solvent extraction of microalgal lipids (as outlined in
Section 4.1.1.) can be fully explained with HSP characterization.
4.1.3. Selection of organic solvents
In addition to satisfying the previously mentioned criteria for an
ideal lipid extraction technology, the selected organic solvents should
Table 2
HSP characterizations for organic solvents and organic solvent mixtures commonly employed to extract lipids from microalgal biomass. Ra was calculated with triacetin as a solute.
Hansen solubility parameters for triacetin: D = 16.5 MPa1/2, P = 4.5 MPa1/2, H = 9.1 MPa1/2.
Organic solvent or
organic solvent
mixture
Hansen dispersion
parameter or D
(MPa1/2)
Hansen dipolar
parameter or P
(MPa1/2)
Hansen hydrogen
bond parameter
or H (MPa1/2)
Afnity of solvent
with triacetin
or Ra (MPa1/2)
Relative energy
difference or
RED
Solubility of
triacetin in
the solvent
Chloroform
Methanol
Water
Chloroform/methanol (1:2 v/v)
Chloroform/methanol/water (1:2:0.8 v/v/v)
Hexane
Isopropanol
Hexane/isopropanol (3:2 v/v)
Ethanol
17.8
15.1
15.6
19.0
15.9
14.9
15.8
15.3
15.8
3.1
12.3
16.0
9.8
10.7
0.0
6.1
2.4
8.8
5.7
22.3
42.3
17.7
22.1
0.0
16.4
6.6
19.4
4.5
15.6
35.2
11.2
14.5
10.6
7.6
4.1
11.2
0.4
1.3
2.9
0.9
1.2
0.9
0.6
0.3
0.9
Very good
Poor
Very poor
Good
Poor
Good
Good
Very good
Good
Table 3
Methods and results summary of recent studies investigating organic solvent extraction of microalgal lipids.
Kates, 1986b
(method for
microalgae)
Guckert et al., 1988
Nagle and Lemke,

1990
Molina Grima et al., 1994
Lee et al., 1998
Fajardo et al., 2007 Halim et al., 2011
Microalgal species
N/A
Any
Chlorella sp.
Chaetoceros muelleri
and Monoraphidium
minutum
Isochrysis galbana
Botryococcus braunii
Phaeodactylum
tricornutum
Chlorococcum sp.
State of microalgal
biomass at the
start of extraction
Concentrate
Concentrate
(residual water
content = 90 wt.%)
Dried powder by lyophilization
Concentrate (residual
Dried powder by lyophilization
water content = 85 wt.%)
Concentrate
Dried powder
by lyophilization
Concentrate
(residual water
content = 70 wt.%)
or dried powder by
thermal drying
Mass of dried
microalgal
biomass (g)
5 to 6
0.1
Not
specied.
Mass of wet
paste = 1.5 g.
60
0.12
10
Organic solvents or
organic solvent
mixtures
Water/methanol/
chloroform
(0.8:2:1 v/v/v)
Water/
isopropanol
(1:5 v/v)
For Soxhlet: methylene chloride/

methanol (2:1 v/v). For batch
extraction 1: chloroform/
methanol/50 mM phosphate
buffer (35:70:28 v/v/v/). For
batch extraction 2, n-hexane/IPA/
distilled water (70:47.7:3 v/v/v).
1-butanol, ethanol,
hexane/2-propanol
(2/3 v/v), water/
methanol/chloroform
as a control system
Chloroform/methanol/water
(1:2:0.8 v/v/v), hexane/ethanol
(1:2.5 v/v), hexane/ethanol
(1:0.9 v/v), butanol, ethanol,
EtOH/water (1:1 v/v), hexane/
isopropanol (1:1.5 v/v)
Chloroform/methanol (2:1 v/v), Ethanol

hexane/isopropanol (3:2 v/v),
dichloroethane/methanol
(1:1 v/v), dichloroethane/
ethanol (1:1 v/v), acetone/
dichloromethane (1:1 v/v)
Amounts of organic
solvent or organic
solvent mixture
added
76 ml organic
solvent mixture/ g
dried microalgal
biomass
16 ml
organic
solvent
mixture/ g
concentrate
For Soxhlet: 1000 ml organic

solvent mixture/ g dried
microalgal biomass.
For batch extraction 1: 1330 ml
organic solvent mixture/g dried
microalgal biomass. For batch
extraction 2: 1207 ml organic
solvent mixture/ g dried
microalgal biomass.
20 g organic solvent
mixture/ g dried
microalgal biomass
76 ml organic solvent mixture/ g 250 ml organic solvent

dried microalgal biomass
mixture/ g dried
microalgal biomass
5 ml organic
solvent mixture/g
dried microalgal
biomass
75 ml organic solvent
mixture/ g dried
microalgal biomass
Duration (min)
60
90
For Soxhlet: 180. For batch
extraction 1: 2160.
For batch extraction 2: overnight.
60
50
1440
450
Degree of agitation
(rpm)
Not specied
Not specied
For Soxhlet: N/A. For batch

extraction 1: not specied. For
batch extraction 2: not specied.
High (not specied)
Not specied, but stated to be

constant
High (not specied)
500
800
Extraction
temperature
(C)
25
5060
For Soxhlet: not specied. For

batch extraction 1: not specied.
For batch extraction 2: room.
Near boiling point

of each solvent
(not specied)
25
Not specied
25
25
N/A
11.9; Soxhlet with methylene

chloride/methanol (2:1 v/v)
Not specied as results

are expressed as
efciencies to a control
system (assigned at
100%). 2nd Highest
efciency with 1-butanol
at 94%
8.9; chloroform/methanol/water
(1:2:0.8 v/v/v).
2nd Highest efciency with
ethanol at 8.0 wt.% of dried
microalgal biomass
28.6; chloroform/
methanol (2:1 v/v)
6.3; ethanol
6.8; hexane/
isopropanol (3:2 v/v)
N/A
Maximum nal
total lipid yield
(wt.% of dried
microalgal
biomass); the
organic solvent or
the organic
solvent mixture
to achieve it
Hexane, hexane/
isopropanol (3:2 v/v)
Kates, 1986b
(method for
microorganisms)
719
preferably be volatile for low-energy distillation from the crude lipids

(Kates, 1986b; Medina et al., 1998).
Chloroform/methanol (1/2 v/v) is the most frequently used organic solvent mixture for lipid extraction from any living tissue. Using
this organic solvent system, residual endogenous water in the microalgal cells acts as a ternary component that enables the complete
extraction of both neutral and polar lipids. It is noted that this method
does not require the complete drying of microalgal biomass. Once the
cell debris is removed, more chloroform and water are added to
induce biphasic partitioning. The lower organic phase (chloroform
with some methanol) contains most of the lipids (both neutral and
polar) while the upper aqueous phase (water with some methanol)
constitutes most of the non-lipids (proteins and carbohydrates)
(Medina et al., 1998).
Extraction using chloroform/methanol (1/2 v/v) is fast and quantitative. Chloroform, however, is highly toxic and its usage is undesirable. The method was originally developed by Folch et al. (1951) for
the isolation of total lipids from brain tissues. For this reason, its
efcacy in extracting lipids from microalgal biomass still needs
further assessment. In a study by Lee et al. (1998), the performance
of ve different organic solvent mixtures in extracting lipids from
bead-beaten Botryococcus braunii cells was compared. As can be
seen in Table 3, chloroform/methanol obtained the highest nal
total lipid yield at ~ 0.29 g/g dried microalgal biomass. On the other
hand,
dichloroethane-based
organic
solvent
mixtures
(dichloroethane/methanol and dichloroethane/ethanol), previously
recommended for lipid extraction from the green algae Cladofora,
were found to have limited efcacies when applied to B. braunii.
Hexane/isopropanol (3/2 v/v) mixture has been suggested as a
low-toxicity substitute to chloroform/methanol system (Halim et al.,
2011). The mixture works in a similar fashion with chloroform/
methanol system. Upon biphasic separation, the upper organic
phase (hexane with some isopropanol) contains most of the lipids
(both neutral and polar) while the lower aqueous phase (water
with some isopropanol) contains most of the non-lipids (proteins
and carbohydrates). When evaluated for microalgal lipid extraction,
hexane/isopropanol mixture was found to be more selective towards
neutral lipids compared to chloroform/methanol system (Guckert et
al., 1988; Lee et al., 1998; Nagle and Lemke, 1990). As previously
mentioned, segregation of neutral lipid class at the lipid extraction
step is highly desirable as it would allow microalgal biodiesel production to occur with minimal downstream purication. Guckert et al.
(1988) attributed the neutral lipid selectivity of hexane/isopropanol
mixture to its inability to extract the polar lipid constituents of microalgal membranes (chloroplast membranes contain glycolipids and
cell membranes contain phospholipids). The hexane/isopropanol
system, however, yielded a surprisingly low total lipid recovery
when applied to B. braunii (Lee et al., 1998).
Pure alcohol (such as butanol, isopropanol, and ethanol) is cheap,
volatile, and has a strong afnity to membrane-associated lipid
complex due to its ability to form hydrogen bonds. However, its
polar nature is also a disadvantage as it limits interaction with freestanding neutral lipid globules. For this reason, when used as a
microalgal lipid extraction solvent, alcohol is almost always combined with a non-polar organic solvent, such as hexane or chloroform,
to ensure the total extraction of both forms of neutral lipids (freestanding globules and as membrane-associated complexes) (Halim
et al., 2011).
In their study, Nagle and Lemke (1990) evaluated the efciencies
of three organic solvents (butanol, hexane/2-propanol mixture,
ethanol) in extracting crude lipids from Chaetoceros muelleri and
compared them to a control water/methanol/chloroform mixture
(Table 3). Even though the control polar/non-polar mixture was
veried to be the most effective organic solvent system (assigned
an arbitrary extraction efciency of 100%), butanol (with an average
extraction efciency of 94%) was found to be highly promising with
a nal total lipid yield consistently higher than hexane/2-propanol

mixture or ethanol in all triplicates. The authors argued that, even
though all of the organic solvents used were safe to handle, the
consistency of butanol yields from one replicate to another (standard
deviation of 3%) indicated a lower sensitivity to changes in the extraction procedure, a benecial attribute if the process were to be scaled
up. Due to its propensity to inactivate many phosphatidases and
lipases, Kates (1986b) recommended the use of isopropanolcontaining organic solvent mixture to extract lipids from unicellular
algal species that produces lipid degradative enzymes.
4.1.4. Operating variables
The evolution of lipids during the organic solvent extraction on
microalgal biomass is observed to follow a rst-order kinetics
equation (Halim et al., 2011; Harrison et al., 2003b):

kt
me ms;o 1e
where me is the amount of lipid extracted in the organic solvent at

time t (g lipid/g dried microalgal biomass), ms,o is the amount of
lipid originally present in the cells (g lipid/g dried microalgal
biomass), k is the lipid mass transfer coefcient from the microalgal
cells into the organic solvent (min 1), and t is the extraction time
(min).
The parameter k itself is a function of several operating variables
and can be described generally as:
k f ag; s=b; T
where ag is the degree of agitation (revolution per minute), s/b is the

ratio of organic solvent to dried microalgal biomass (ml organic solvent/g dried microalgal biomass), and T is the extraction temperature
(C). Table 3 provides a summary of the levels of operating variables
used in various studies investigating organic solvent extraction of
microalgal lipids.
Fig. 8 (Fajardo et al., 2007) shows typical extraction curves
obtained when using an organic solvent to extract lipids from microalgal biomass. These curves conform to the model of Eq. (1), where
the rate of lipid recovery is observed to decrease with extraction
time. During the 24-hour extraction, a majority of the lipids is recovered within the rst 8 h (6070% of all extractable lipids) and extending the extraction time beyond 12 h does not seem to make any
signicant contribution to the nal total lipid yield. This kind of
asymptotic behavior is attributed to the diffusion-driven nature of
lipid extraction where the rate of lipid evolution is controlled by the
100
Total lipid yield

( wt% of dried microalgal
biomass)
720
90
80
70
60
50
40
30
20
10
0
0
12
16
20
24
Time (h)
Fig. 8. Typical rst-order extraction curves obtained for organic solvent extraction of
microalgal lipids. Microalgae: Phaeodactylum tricornutum, organic solvent: ethanol.
Ratio of organic solvent to dried microalgal biomass (s/b) was optimized. X 5 ml
ethanol/g dried microalgal biomass, 10 ml ethanol/g dried microalgal biomass,
15 ml ethanol/g dried microalgal biomass.
Modied from Fajardo et al. (2007).
lipid concentration gradient between the microalgal cells and the

organic solvent. Extraction is most rapid in the beginning when
concentration gradient is at its steepest. As lipids are removed from
the microalgal cells into the organic solvent, the concentration
gradient diminishes and lipid extraction slows down.
As can be seen in Table 3, every study indicates a different ratio of
organic solvent to dried microalgal biomass (s/b). The appropriate s/b
value for each microalgal strain varies depending on its lipid content
and its intrinsic solvent-cellular interaction. During their investigation of lipid extraction from Phaeodactylum tricornutum, Fajardo
et al. (2007) found extraction efciency to increase with decreasing
s/b ratio (Fig. 8). The nal total lipid yields obtained with 10 and
15 ml ethanol per gram of dried microalgal biomass were around
80 wt.%. With 5 ml ethanol per gram of dried microalgal biomass,
the nal total lipid yield was roughly 90 wt.%. Even though this
claim was rather counter-intuitive, the authors ascribed the higher
total lipid yield at lower organic solvent ratio to its higher agitation
intensity per volume unit. It is important to nd the optimum s/b
value for a specic microalgal strain. An s/b value that is too high results in excessive consumption of organic solvent, while a value that
is too low leads to handling difculties and incomplete extraction.
Variation in the extraction temperature has been reported to
inuence lipid yield. Increasing temperature from 30 C to 60 C was
observed to enhance lipid extraction rate from animal tissues
(Fajardo et al., 2007). However, a rise beyond 70 C led to an oxidative degradation of thermo-labile components which resulted in a
lower lipid yield. In a study by Balasubramanian et al. (2010), increasing temperature during lipid extraction from Scenedesmus obliquus
resulted in a signicant increase in the nal total lipid yield. The
authors attributed this increase to enhanced mass transfer kinetics
at higher temperature.
The kinetics and the mechanism underlying organic solvent extraction of microalgal lipids are not yet well understood and require
further research. It is noted that organic solvent extraction has several
disadvantages. The method generally uses large amounts of toxic
organic solvents, is slow, and requires energy-intensive evaporation
for solvent removal. The extent of lipid extraction by a volume of
water out
condenser
water in
Soxhlet extractor
with a thimble to
hold the
microalgal
biomass
heating mantle
round-bottom flask
containing the
organic solvent
Fig. 9. The Soxhlet apparatus.
Modied from De Castro and Ayuso (2000).
721
organic solvent is also thermodynamically restricted by the lipid

mass transfer equilibrium (Wang and Weller, 2006). Once lipid concentration between the bulk organic solvent and the cellular matrices
has reached its partition equilibrium level, no further transfer of lipids
from the cells to the organic solvent will take place.
4.1.5. Modications to organic solvent extraction

A majority of the laboratory-scale organic solvent extractions
reported in the literature have been performed as a batch process.
Even though batch extraction is limited by lipid mass transfer equilibrium, a continuous organic solvent extraction able to overcome this limitation requires a large amount of organic solvent and becomes too
expensive. Through its ingenious cycles of solvent evaporation and condensation, the Soxhlet apparatus continuously replenishes cells with
fresh organic solvent (hence evading equilibrium limitation) while simultaneously minimizing solvent consumption (Luque de Castro and
Garcia-Ayuso, 1998; Wang and Weller, 2006). The apparatus is shown
in Fig. 9 and has 3 compartments: a continuously heated roundbottom ask to store the extracting organic solvent, the Soxhlet extractor to hold the microalgal biomass (existing as concentrate or disrupted
concentrate or dried powder), and the continuously cooled condenser.
Organic solvent from the heated round-bottom ask enters the condenser and is immediately channeled into the Soxhlet extractor. The organic solvent comes in contact with the microalgal biomass and
performs lipid extraction. The thimble in the extractor prevents the
microalgal biomass from being carried away by the organic solvent
ow and, as such, serves as a lter to remove cell debris. Once the organic solvent in the extractor reaches the overow level, a siphon unloads the organic solvent-lipids mixture from the extractor back into
the round-bottom ask. The organic solvent is heated and evaporates
again while the extracted crude lipids remain in the round-bottom
ask. This cycle is repeated until no more crude lipids are extracted in
the Soxhlet extractor. Despite its advantageous design in avoiding equilibrium limitation, the Soxhlet apparatus suffers from high energy requirement for continuous distillation (Luque de Castro and GarciaAyuso, 1998; Wang and Weller, 2006).
Independent studies by Guckert et al. (1988) and Halim et al. (2011)
veried the superior efcacy of Soxhlet extraction when compared to a
batch extraction. Among the three systems experimented by Guckert
et al. (1988) to extract lipids from Chlorella sp., Soxhlet extraction
using methylene chloride:methanol (2:1 v/v) mixture obtained the
highest nal total lipid yield (Table 3). In terms of the dry microalgal
weight, the nal total lipid recovered was approximately 11.9% by
Soxhlet extraction using methylene chloride: methanol, 11.1% by
batch extraction using chloroform/methanol/50 mM phosphate buffer,
and 5.8% by batch extraction using n-hexane/isopropanol/distilled
water. Halim et al. (2011) found Soxhlet operation of hexane extraction
to be signicantly more efcient than its batch counterpart when used
to extract lipids from Chlorococcum sp. (nal total lipid yield of batch
extraction = 0.015 g lipid/g dried microalgal biomass, nal total lipid
yield of soxhlet extraction = 0.057 g lipid/g dried microalgal biomass).
Despite its improved total lipid recovery, Soxhlet extraction potentially
suffered from lipid degradation resulting from the use of elevated temperature throughout the process. Guckert et al. (1988) noted that the
crude lipids recovered using a Soxhlet system contained less PUFAs
then those obtained by batch extractions and ascribed this observation
to potential thermal degradation due to the harshness of the Soxhlet
method.
A couple of modications to organic solvent extraction have also
been introduced: microwave-assisted organic solvent extraction and
accelerated or subcritical organic solvent extraction. Each modication utilizes an auxiliary process that enhances the kinetics of lipid
extraction by the organic solvent through speedy disruption of the
cellular structures (Luque de Castro and Garcia-Ayuso, 1998; Wang
and Weller, 2006). Modied organic solvent extraction synergistically
722
combines cell disruption pre-treatment (to be reviewed in Section 5)

and lipid extraction as a single step.
Microwave-assisted organic solvent extraction uses the aid of
electromagnetic radiation within a specic frequency range to
deliver large amount of thermal energy to the microalgal cells
(Balasubramanian et al., 2010). When the cells receive this energy,
local internal superheating occurs leading to instantaneous temperature rise within the matrices and rapid pressure effects on the cell
wall/membrane structure. Cell structures are immediately ruptured
forcing cell constituents to spill out. This effective expulsion of cell materials facilitates a more rapid diffusion of microalgal lipids into the
extracting organic solvent. Microwave-assisted heating is substantially
more rapid than conventional heating as heat is delivered via radiation
rather than convection and conduction. Balasubramanian et al. (2010)
examined the use of microwave-assisted hexane extraction to recover
lipids from S. obliquus. Microwave-assisted hexane extractions were
found to result in higher oil yields compared to conventionally waterheated hexane extraction control methods at all extraction temperatures and times.
During accelerated or subcritical organic solvent extraction, lipid extraction is performed at an elevated pressure and temperature in order
to accelerate extraction kinetics and to disintegrate cellular structures.
Subcritical organic solvent extraction has some of the benets of supercritical uid extraction (described in Section 4.2), but is still performed
below critical conditions in order to minimize operating cost (Herrero
et al., 2005). Chen et al. (2011) examined the use of subcritical ethanol
to extract lipids from wet microalgal paste of Nannochloropsis sp. and
found the extraction process to be highly efcient (maximum nal
lipid recovery= 90.21% of total lipids). Neither of the modications
described above (microwave-assisted or subcritical organic solvent
extraction) has been applied to an industrial scale due to their high energy requirements. It is also noted that there is currently limited understanding on the key variables affecting the performances of these
modied extraction processes (Luque de Castro and Garcia-Ayuso,
1998; Wang and Weller, 2006). The scale-up potentials of organic
solvent extraction and its modications are examined in Table 1.
4.2. Supercritical uid extraction
Supercritical uid extraction (SFE) is an emerging green technology that has the potential to replace traditional organic solvent
extraction.
4.2.1. Basic principles
When the temperature and the pressure of a uid are raised over their
critical values (Tc and Pc), the uid enters the supercritical region (Fig. 10)
(Pourmortazavi and Hajimirsadeghi, 2007; Reverchon and De Marco,
2006; Taylor, 1996). Supercritical uid appears suitable to be used as an
extraction solvent for lipid recovery from microalgal biomass due to the
following reasons (Mendes et al., 1995, 2003, 2006; Taylor, 1996):
Tunable solvent power
Fig. 10. PT phase diagram for carbon dioxide, showing the supercritical region.
Table 4
Physical properties of a typical uid in different states. Modied from Taylor (1996).
Gas
Supercritical uid
Liquid
Density
(kg/m3)
Viscosity
(Pas)
Diffusion coefcient
(mm/s)
1
1001000
1000
10
50100
5001000
110
0.010.1
0.001
The solvent power of a supercritical uid is a function of its density

which can be continuously adjusted by changing the extraction
pressure and the extraction temperature. As such, solvent power of
the uid can be tuned such that it interacts primarily with neutral
lipids (i.e. acylglycerols).
Favorable mass transfer
As shown in Table 4, supercritical uid displays physical properties
intermediate to a liquid and a gas (Taylor, 1996). These transitional
properties allow for rapid penetration of the uid through cellular
matrices, thus resulting in a higher total lipid yield and a shorter
extraction time.
Production of solvent-free crude lipids
Crude lipids obtained from supercritical uid extraction are free from
extraction solvent. Therefore, no energy is expended for extraction
solvent removal.
Supercritical carbon dioxide (SCCO2) is the primary solvent used
in the majority of supercritical uid extractions. Its moderate critical
pressure (72.9 atm) allows for a modest compression cost, while its
low critical temperature (31.1 C) enables successful extraction of
thermally sensitive lipid fractions without degradation. SCCO2 facilitates a safe extraction due to its low toxicity, low ammability, and
lack of reactivity (Macias-Sanchez et al., 2007; Taylor, 1996). If the
microalgal cells are to be cultivated at a coal-red power station,
the CO2 required for supercritical conversion can be conveniently
obtained from the scrubbed ue gas of the station. This paper will
focus on the use of SCCO2 for microalgal lipid extraction.
Fig. 11 shows a lab-scale supercritical carbon dioxide (SCCO2)
extraction unit used for the recovery of microalgal lipids (Applied
separations, 2007). A mixture of microalgal biomass (existing as
concentrate or disrupted concentrate or dried powder) and packing
materials (normally diatomaceous earth or diatoms) in a specic
ratio is placed inside the extraction vessel equipped with a heating
element. A feed pump delivers CO2 from its source to the extraction
vessel at a pressure greater than Pc. As soon as the vessel is heated
(T > Tc), the compressed CO2 is converted to its supercritical state
and performs lipid extraction on the microalgal biomass (Fig. 12).
During the lipid extraction process, the microalgal biomass and
diatoms are packed tightly inside the cylindrical extraction vessel. The
supercritical carbon dioxide travels on the surface of the packed
mixture and lipids are desorbed from the microalgal biomass.
Immediately upon dissolution, the SCCO2 encloses the lipids to form a
SCCO2lipids complex. The complex, driven by concentration gradient,
diffuses across the static SCCO2 lm and enters the bulk SCCO2 ow.
The frits placed at both ends of the extraction vessel prevent the
mixture of microalgal biomass and diatoms from being carried away
by the SCCO2 ow. As such, the frits serve as a lter to remove cell
debris. The SCCO2lipids mixture, as well as some residual water (if
lipid extraction was performed on concentrate or disrupted concentrate), then leaves the extraction vessel to enter the collection vessel,
where a micrometering valve is used to rapidly depressurize the incoming uid (Fig. 11). Upon complete depressurization, the SCCO2,
together with any residual water, returns to gaseous state and the
extracted crude lipids precipitate in the collection vessel. As such,
723
Fig. 11. Schematic diagram of a laboratory-scale SCCO2 extraction system. The unit is used to extract lipids from microalgal biomass.
Modied from Applied separations (2007).
SFE-derived crude lipids are free from any extraction solvent and do
not need to undergo an extraction solvent removal step. Even though
SCCO2 extraction can be operated as either a batch (static) or a continuous (dynamic) process, it is generally exercised as a continuous
extraction as this often results in an improved yield (Taylor, 1996).
The process described above is based on a dynamic extraction. The
feasibility of applying SCCO2 process to extract microalgal lipids has
been demonstrated (Andrich et al., 2005, 2006; Canela et al., 2002;
Cheung, 1999; Halim et al., 2011; Herrero et al., 2006; Mendes et al.,
1995, 2003, 2006; Mendiola et al., 2007; Sajilata et al., 2008; Thana
et al., 2008).
4.2.2. Operating variables

Operating variables which inuence the performance of SCCO2
extraction of microalgal lipids include pressure, temperature, modier addition, and uid ow rate (Pourmortazavi and Hajimirsadeghi,
2007). Table 5 summarizes the levels of operating variables and the
ndings of recent studies investigating SCCO2 extraction of microalgal
lipids.
Fig. 12. Schematic diagram of the proposed supercritical carbon dioxide extraction
mechanism. Microalgal biomass and diatoms are packed tightly as a mixture inside
the cylindrical extraction vessel. Supercritical carbon dioxide ows on the surface of
the packed mixture. lipids, SCCO2. Static SCCO2 lm enclosing the packed mixture
is formed due to the interaction between SCCO2 and microalgal biomass. The
mechanism can be described in 3 steps. Step 1: desorption of lipids from the microalgal
biomass into the static SCCO2 lm. Step 2: solubilization of the released lipids by
SCCO2. Step 3: formation of a SCCO2-lipids complex. Step 4: diffusion of the SCCO2lipid complex across the static SCCO2 lm into the bulk SCCO2 ow.
The evolution of lipids during SCCO2 extraction on microalgal biomass can be described by the following rst-order kinetics equation
(Goto et al., 1993; Halim et al., 2011; Ozkal et al., 2005):

kt
me ms;o 1e
where me is the amount of lipid extracted by the SCCO2 at time t

(g lipid/g dried microalgal biomass), ms,o is the amount of lipid
originally present in the microalgal cells (g lipid/g dried microalgal
biomass), k is the lipid mass transfer coefcient from the microalgal
cells into the eluting SCCO2 (min 1), and t is the extraction time
(min).
The parameter k itself is a function of several operating variables
and can be described generally as:
k f P; T; M; Q
where P is the extraction pressure (bar), T is the extraction temperature (C), [M] is the concentration of polar modier (mol% of CO2), Q
is the SCCO2 ow rate (l/min).
Fig. 13 (Andrich et al., 2005) shows typical extraction curves
obtained when using SCCO2 to extract lipids from microalgal biomass.
These curves conform to the model proposed in Eq. (3), where the
rate of lipid recovery is observed to decrease with extraction time.
During their studies investigating lipid extraction from Nannochloropsis sp., Andrich et al. (2005) found majority of the total lipids (>80%)
to be extracted within 5000 s. Continuing the extraction run beyond
10,000 s did not seem to dramatically increase the total lipid yield.
This kind of asymptotic behavior is ascribed to the diffusion-driven
nature of lipid extraction where the rate of lipid evolution is controlled by the lipid concentration gradient between the microalgal
biomass and the SCCO2. In our previous study evaluating the feasibility of SCCO2 process to extract lipids from Chlorococcum sp. (Halim
et al., 2011), we found the rst-kinetic model described by Eq. (3)
to accurately describe experimental data (minimum r 2 value for all
extraction curves = 0.92).
Solvent power of SCCO2 during lipid extraction is a direct function
of the extraction pressure and the extraction temperature (Taylor,
1996). Higher extraction pressure leads to a higher uid density
and, thus, to an increase in solvent power. However, increasing
extraction pressure also increases operating cost, lowers selectivity,
and often results in the co-extraction of unwanted cellular components. Temperature increase leads to two competing phenomena.
The decrease in uid density lowers SCCO2 solvent power while a
simultaneous increase in lipid volatility enhances the lipid mass
transfer into the bulk SCCO2 ow (Cheung, 1999; Soares et al.,
724
Table 5
Methods and results summary of recent studies investigating supercritical carbon dioxide extraction of microalgal lipids. Special attention is given to the effect of pressure change
and of temperature change on the total lipid yield. Optimum condition is dened as the experimental condition that produces the highest nal total lipid yield.
Study
Microalgal
species
SCCO2 ow rate; Polar modier; Results and optimum condition

Extraction Extraction
temperature extraction
quantity of
pressure
polar modier
or P (bar) or T (C)
duration (min)
Sajilata
et al.,
2008
Spirulina
platensis
316, 350,
400, 450,
484
Andrich
et al.,
2005
Mendes
et al.,
2003
Final total lipid yield

at the optimum
condition (wt.% of dried
microalgal biomass)
40
0.7 l/min; 26.4,

40, 60, 80, 94
Ethanol; 9.64,
11, 13, 15,
16.36 ml
Total lipid yield increased with P. Optimum condition

was found at 400 bar, 60 min, and 13.7 ml ethanol.
8.6
Nannochloropsis 400, 550,

sp.
700
40, 55
0.17 kg/min;
360
None
At constant T, lipid extraction rate increased with P.

At constant P, lipid extraction rate slightly increased
with T.
Final total lipid yield was the same at any T and P.
25.0
Spirulina
maxima
100, 250,
350
50, 60
Not specied;
not specied
ethanol;
At constant T, total lipid yield increased with P.
3.1
10 mol% of CO2 At constant P, total lipid yield decreased with T.
At constant T and P, polar modier addition signicantly
increased total lipid yield.
Optimum condition was found at 350 bar, 60 C with
ethanol addition (10 mol%).
Cheung, Hypnea
1999 charoides
241, 310,
379
40, 50
1 l/min; 120
none

At low P (241 bar), total lipid yield decreased with T.
At medium to high P (310 and 379 bar), total lipid
yield increased with T.
Optimum condition was found at 379 bar and 50 C.
6.7
200, 350
40, 55
0.4 l/min; 500
none

At low P (200 bar), total lipid yield decreased with T.
At high P (350 bar), total lipid yield increased with T.
Optimum condition was found at 350 bar and 55 C.
13.0
Mendes
et al.,
1995
Chlorella
vulgaris
2007; Taylor, 1996). Table 5 compiles ndings from previous studies

on the effect of pressure change and of temperature change on
SCCO2 lipid extraction from microalgal biomass.
Because of its non-polar nature, SCCO2 is unable to interact with
either polar lipids or neutral lipids that form complexes with polar
lipids. The addition of a polar modier, often referred to as
co-solvent or entrainer, enhances the uid afnity towards polar
lipids as well as lipid complexes that contain both neutral lipids and
polar lipids. It further facilitates complete lipid extraction by
diminishing the uid viscosity and allowing the SCCO2 to rapidly
permeate through the cellular matrices (Pourmortazavi and
Hajimirsadeghi, 2007; Taylor, 1996). Common polar modiers
include methanol, ethanol, toluene and methanolwater mixture.
Mendes et al. (2006) demonstrated that the addition of methanol to
SCCO2 signicantly increased the extraction of -linolenic acid
(from 0.05 wt.% of dried microalgal biomass to 0.44 wt.%) from
Spirulina maxima.
The ow rate of SCCO2 through the extraction vessel affects lipid
extraction kinetics. Even though increasing SCCO2 ow rate enables
Fig. 13. Typical rst-order extraction curves obtained for SCCO2 extraction of microalgal lipids. Microalgae: Nannochloropsis sp., all extractions were performed at a constant
temperature (40 C). Pressure was optimized. 70 MPa, 55 MPa, 40 MPa.
Modied from Andrich et al. (2005).
more effective contact between the extraction uid and the lipids, it
often results in uneven uid penetration and dead volumes within
the extraction vessel (Pourmortazavi and Hajimirsadeghi, 2007).
The density in which microalgal biomass is packed to form a xed
bed within the extraction vessel plays an important role in inuencing extraction efciency (Pourmortazavi and Hajimirsadeghi, 2007).
In the case of extraction from dried microalgal powder, packing density is directly related to microalgal powder particulate size and the
volumetric ratio of packing materials (normally diatomaceous earth
or diatoms) to microalgal powder. Even though higher packing
density increases the amount of lipids in the vessel, it reduces the
vessel's porosity and can adversely affect the extraction kinetics via
uid channeling effects (Pourmortazavi and Hajimirsadeghi, 2007).
4.3. Comparison between organic solvent extraction and SCCO2
extraction
Table 6 provides a comparison between organic solvent extraction
and SCCO2 extraction when they are used to extract lipids from
microalgae. Despite having low reactivity with lipids and being
effective when directly applied to a wet feedstock (concentrate or
disrupted concentrate), organic solvent extraction is slow and uses
large amounts of expensive/toxic solvents. It has a limited selectivity
towards biodiesel-desirable lipid fractions (acylglycerols containing
mainly cis-unsaturated fatty acids with less than 4 double bonds)
and requires energy-intensive liquidliquid separation method
(such as distillation) to remove the organic solvent from the lipids.
On the other hand, SCCO2 extraction is rapid and non-toxic. It has
high selectivity towards biodiesel-desirable lipid fractions due to
SCCO2 tuneable density and produces solvent-free crude lipids. It is
also non-reactive with the lipids. It remains effective when applied
to a wet feedstock (concentrate or disrupted concentrate) though
high residual water content in the microalgal biomass tends to lead
to ow impedance and restrictor plugging. High installation costs of
the extraction pressure vessel as well as unfavorable energy requirements for the uid compression and heating remain the primary
obstacles for scaling-up SCCO2 extraction (Crespo and Yusty, 2005;
Table 6
Comparison between organic solvent extraction and SCCO2 extraction for microalgal lipid extraction. * * *: good. *: poor.
Organic solvent extraction
SCCO2 extraction
**
Selectivity is not easily tuned. When non-polar organic solvent is used, only limited
amount of neutral lipids can be extracted. When non-polar/polar organic solvent
mixture is used, both neutral lipids and polar lipids are extracted.
***
SCCO2 tunable selectivity when combined with exible polar modier arrangement should enable
specic extraction of acylglycerols and minimize co-extraction of contaminants (polar lipids and
non-acylglycerol neutral lipids).
Total lipid yield
**
***
Due to its intermediate liquidgas properties, SCCO2 can penetrate through cellular matrices rapidly
and produces a higher total lipid yield.
Extraction time
**
Lipid extraction rate is slow and lipid extraction requires a long time for completion.
***
Due to its intermediate liquid-gaseous properties, SCCO2 can penetrate through cellular matrices
rapidly. As such, lipid extraction rate is fast and lipid extraction can be completed within a short period.
Energy requirement
**
It consumes little energy as lipid extraction is conducted near ambient conditions.
However, organic solvent needs to be removed from the lipids via energy-intensive
liquidliquid separation method (such as distillation).
*
It is highly energy-intensive as uid compression and heating are needed to convert CO2 to supercritical
state. However, crude lipids are free from extraction solvent and do not need to undergo an extraction
solvent removal.
Installation and operating

(non-energy related) cost
**
Expensive organic solvent is needed. Not all of the organic solvents can be recycled.
The pressure vessel needed for SCCO2 extraction can be extremely expensive to install.
***
Lipid extraction can be applied to microalgal concentrate without additional

pre-treatment step and with minimal loss of efciency.
High residual water content within the microalgal biomass results in ow impedance and restrictor
plugging.
Hazard and toxicity
*
Toxic organic solvents are used.
***
Reactivity with lipids
**
Organic solvent is non-reactive with lipids. However, distillation carried out to
remove the organic solvent from the lipids exposes the lipids to high
temperature and possible artifact formation.
***
SCCO2 is non-reactive with lipids.
Applicability to microalgal
concentrate or wet feedstock
Criteria
Neutral lipids selectivity
725
726
cloud point and a high pour point), while biodiesel made from PUFA
tends to be volatile and has a low oxidation stability.
Schematic diagrams for an industrial-scale organic solvent
extraction system and an industrial-scale SCCO2 extraction system
are proposed in Fig. 15. For SCCO2 extraction, compressor is used to
pressurize the uid to a supercritical state. The scale-up potential of
each lipid extraction technology is examined in Table 1.
5. Effect of cellular pre-treatment on lipid extraction
Fig. 14. Comparison between dynamic SCCO2 extraction and dynamic hexane
extraction (using a Soxhlet apparatus). (a) total lipid yield. (b) FAME composition of
the crude lipids. Microalgae: Chlorococcum sp. In (a), SCCO2 extraction, Hexane
extraction (using a Soxhlet apparatus). In (b), the letter t after the fatty acid name
(C16:1t) denotes trans-isomerism. When no letter t appears, fatty acid is of
cis-isomerism. For SCCO2 extraction, mass of microalgal dried powder = 20 g, dried
powder : diatomaceous earth = 2/1 w/w, T = 60 C, P = 30 MPa. For hexane extraction
(using a Soxhlet apparatus), mass of microalgal dried powder = 4 g, total number of
cycles or equilibrium establishments = 55.
Modied from Halim et al. (2011).
Halim et al., 2011). For a more extensive evaluation than Table 6, a

thorough understanding of mass transfer mechanisms and of kinetic
parameters involved in lipid extraction is required.
In our previous study extracting lipids from dried Chlorococcum
sp. (Halim et al., 2011), we compared the performance of dynamic
SCCO2 extraction with that of dynamic hexane extraction (using a
Soxhlet apparatus). As shown in Fig. 14, SCCO2 extraction was found
to be more efcient than hexane extraction. Eighty minutes of
SCCO2 extraction (total lipid yield = 0.058 g lipid/g dried microalgal
biomass) achieved a higher total lipid yield than 5.5 h of Soxhlet extraction (total lipid yield = 0.032 g lipid/g dried microalgal biomass).
This outcome was expected since supercritical uid has more
favorable physicochemical properties and facilitates more rapid
cellular permeation (Cheung, 1999; Mendes et al., 2003). Andrich
et al. (2005) reported similar results. Despite demonstrating that
both extractions eventually obtained equivalent nal total lipid yields
from Nannochloropsis sp., they measured a lower lipid mass transfer
coefcient for extraction with hexane than with SCCO2. In terms of
fatty acid composition (Fig. 14), Chlorococcum crude lipids extracted
by SCCO2 comprised a substantially higher quantity of C18:1 than
the corresponding crude lipids extracted by hexane. Such selectivity
was benecial since C18:1, as a cis-unsaturated fatty acid with less
than 4 double bonds, is highly desirable for biodiesel production. As
previously mentioned (Section 2), biodiesel derived from saturated
fatty acids often has disadvantageous cold ow properties (a high
The effects of cellular pre-treatment on microalgal lipid extraction

have not been investigated extensively. As previously described, the
pre-treatment process can take alternative pathways depending on
the desired biomass alterations (Fig. 4). The process can be
performed in a single step or multiple steps. It is noted that most of
the pre-treatment steps (such as thermal drying for complete water
removal or high-pressure homogenization for cell disruption) are
energy intensive and should only be carried out if they substantially
enhance the efciency of microalgal lipid extraction. Based on the
combination of technologies available in Table 1, the pre-treatment
process can alter the following conditions of the microalgal biomass:
degree of cell disruption, residual water content, and, in the case of
dried microalgal powder, particulate size.
The efciency of microalgal lipid extraction is known to increase
with the degree of cell disruption. When intact cells are disintegrated
during cell disruption, intracellular lipids are liberated from the cellular structures and released into the surrounding medium (Chisti and
Moo-Young, 1986; Gouveia et al., 2007; Lee et al., 2010;
Mendes-Pinto et al., 2001). During subsequent lipid extraction, the
eluting extraction solvent can directly interact with these free lipids
without penetrating into the cellular structures. The lipid extraction
process is thus no longer restricted by the transportation of extraction
solvent and lipids across the cell membrane. It is completed more
rapidly and results in a higher lipid recovery. It is noted that most
cell disruption methods (such as bead milling, ultrasonication, and
high-pressure homogenization) require certain degree of water in
the microalgal biomass for their successful operation. For this reason,
cell disruption step in a pre-treatment pathway is always performed
before the drying step (as illustrated in Fig. 4). Additionally, most
cell disruption methods will not be able to process microalgal
concentrate with exceedingly low water contents (i.e. microalgal
paste or pellet).
Laboratory-scale cell disruption methods (Fig. 16) are classied
based on the manner in which they achieve microalgal cellular disintegration: mechanical or non-mechanical (Chisti and Moo-Young,
1986; Harrison et al., 2003a). Mechanical methods include bead
mill, press, high-pressure homogenization, ultrasonication, autoclave,
lyophilization, and microwave, while non-mechanical methods often
involve lysing the microalgal cells with acids, alkalis, enzymes, or
osmotic shocks (Chisti and Moo-Young, 1986).
Bead mill, high-pressure homogenization, and ultrasonication are
three of the most widely used methods for laboratory-scale microalgal cell disruption (Chisti and Moo-Young, 1986; Harrison et al.,
2003a). Bead mill achieves cellular disruption by physically grinding
the microalgal cells against the solid surfaces of glass beads in a violent agitation. Among the myriad of cell disruption methods, bead
mill appears most suitable for large-scale application due to its low
operating cost (Chisti and Moo-Young, 1986). High-pressure homogenization pumps microalgal concentrate through narrow orice of a
valve under high pressure. It then releases the concentrate into a
low-pressure chamber. Cellular disintegration is thus achieved
through high-pressure impingement of accelerated cellular jet on
the stationary valve surface as well as through pressure-drop induced
shear stress that the microalgal concentrate experiences as it passes
from the valve to the chamber (Chisti and Moo-Young, 1986).
Ultrasonication disrupts microalgal cells via transmission of sonic
727
Fig. 15. Proposed schematic diagrams of (a) an industrial-scale organic solvent extraction system and (b) an industrial-scale SCCO2 extraction system. The systems are intended for
lipid extraction from microalgal biomass.
waves. These waves create a series of microbubble cavitations on the

cell surface and eventually disintegrate the cell membrane/wall
(Chisti and Moo-Young, 1986). Detailed working mechanisms of
bead mill, high-pressure homogenization and ultrasonication can be
found elsewhere (Chisti and Moo-Young, 1986; Harrison et al.,
2003a).
Lee et al. (1998) assessed the effect of prior mechanical cell

disruption on lipid extraction from the species B. braunii. They used
chloroform/methanol mixture (2/1 v/v) as an extraction solvent and
found completely disrupted microalgal cells to yield almost twice
the amount of crude lipids of intact microalgal cells. Also, among
the different mechanical cell disruption methods investigated
Fig. 16. The classication of laboratory-scale cell disruption methods.
728
Botryococcus sp.
30
20
10

(wt% of dried microalgal
biomass)
Chlorella vulgaris
30
20
10
Scenedesmus sp.
30
20
10
0
s
ve
wa
o
r
ic
tin
lav
toc
Au
n-
No
ing
on
pti
u
isr
ea
b
d-
Be
k
oc
on
ati
ic
on
c
oti
sh
Os
Fig. 17. Effect of prior cell disruption on total lipid yield of organic solvent extraction.
Three microalgal species (Botryococcus sp., Chlorella vulgaris, and Scenedesmus sp.)
were investigated. Organic solvents: chloroformmethanol (1/1 v/v) mixture.
Modied from Lee et al. (2010).

(g lipid / g dried microalgal
biomass)
(sonication, homogenization, high-pressure French press, bead beating or bead mill, and lyophilization), mechanical shearing with bead
mill obtained the highest nal total lipid yield. In a different study
by Lee et al. (2010), the effect of prior cell disruption on lipid
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
A
C1
C2
D1
D2
Organic solvent
Fig. 18. Effect of residual water content within the microalgal biomass on total lipid
yield of organic solvent extraction. Microalgae: Chlorococcum sp. A: Hexane extraction
of microalgal dried powder. B: Hexane extraction of microalgal concentrate. C: Hexane/
isopropanol (3/2 v/v) extraction of microalgal dried powder (C1: organic phase, C2:
aqueous phase). D: Hexane/isopropanol (3/2 v/v) extraction of microalgal concentrate
(D1: organic phase, D2: aqueous phase). For A and C: mass of dried powder = 4 g. For B
and D: mass of concentrate = 13.3 g, residual water content = 70 wt.% of concentrate.
For A, B, C, D: mass of dried microalgal biomass = 4 g, volume of organic solvent
mixture = 300 ml, duration = 7.5 h.
Modied from Halim et al. (2011).
extraction from three microalgal species (Botryococcus sp., Chlorella

vulgaris, and Scenedesmus sp.) was evaluated (Fig. 17). Chloroform
methanol (1/1 v/v) mixture was used as an extraction solvent in all
cases. Among the cell disruption methods assessed (autoclave, bead
beating, microwave, sonication, and osmotic shocks), microwave
obtained the highest nal total lipid yield and appeared to be the
most efcient for all three microalgal strains. For Botryococcus sp.,
bead beating and microwave obtained the highest nal total lipid
yields with respectively 0.281 and 0.286 g lipid/g dried microalgal
biomass, while sonication seemed to be the least efcient at 0.088 g
lipid/g dried microalgal biomass. For C. vulgaris, autoclave and microwave appeared to be the most efcient, whereas bead beating produced a low nal total lipid yield at 0.079 g lipid/g dried microalgal
biomass. With Scenedesmus sp., microwave was again found to
show the highest extraction efciency, while yields from the other
methods were similar. For all three microalgal species, prior disruption of the cells by any of the assessed method was found to improve
nal total lipid yield during the lipid extraction step (refer to Fig. 17
and compare all of the methods with the control non-disruption).
The mechanism in which residual water in the microalgal biomass
affects lipid extraction is not well understood and warrants future investigation. One hypothesis speculates that the presence of residual
water in the microalgal biomass will adversely affect lipid extraction
efciency. Water forms a barrier that prohibits effective lipid mass
transfer from the cells to the extraction solvent. As such, drying of
microalgal concentrate is non-optional and has to be performed
prior to the lipid extraction. On the other hand, another hypothesis
postulates that the presence of residual water in the microalgal
biomass will improve lipid extraction efciency. Water swells the
cells and facilitates better solvent access to the lipids. Drying of
microalgal concentrate prior to lipid extraction is deemed unnecessary and may hinder lipid mass transfer. Various microorganisms
(bacteria, yeasts, and viruses) have been successfully extracted in
their wet state (~ 90 wt.% water) using non-polar/polar organic
solvent mixtures (Kates, 1986b; Medina et al., 1998).
With regards to SCCO2 extraction, abundance of residual water in
the microalgal biomass results in a highly compacted particle bed
within the extraction vessel. This often leads to ow impedance and
restrictor plugging (Pourmortazavi and Hajimirsadeghi, 2007;
Schwartzberg, 1997).
During their investigation of lipid extraction from Chlorococcum
sp., Halim et al. (2011) assessed the effect of residual water content
within the microalgal biomass on total lipid yield. As shown in
Fig. 18 (modied from Halim et al., 2011), the presence of residual
water in the microalgal biomass did not appear to substantially affect
total lipid yield. Hexane extraction of concentrate (nal total lipid
yield = 0.010 g lipid/g dried microalgal biomass) obtained a slightly
lower lipid recovery than its dry powder counterpart (nal total
lipid yield = 0.015 g lipid/g dried microalgal biomass), while
hexane/isopropanol extraction of concentrate (nal total lipid
yield = 0.123 g lipid/g dried microalgal biomass) surprisingly
obtained a higher total lipid yield than hexane/isopropanol extraction
of dried powder (nal total lipid yield = 0.068 g lipid/g dried microalgal biomass). These ndings were encouraging particularly since the
organic solvent extraction of wet biomass did not require any additional pre-treatment step.
Among the drying technologies that can be applied to microalgal
concentrate, freeze drying is preferred for its mild operating conditions. Thermal drying, though commonly used in laboratory practice,
is not recommended as it degrades thermolabile lipids, results in
evaporative loss of volatile lipids, and yields powder with nonuniform particulate size (Pourmortazavi and Hajimirsadeghi, 2007).
Once dried, microalgal biomass forms powder (or agglomeration)
that can be milled into different particulate size. Reducing the particulate size of microalgal powder prior to lipid extraction generally
enhances lipid recovery as it increases the interfacial surface area
729
Fig. 19. Alternative process ow diagram showing the downstream processing steps needed with a simultaneous extraction and transesterication step to produce biodiesel from
microalgae.
available for biomass-solvent contacts and shortens the diffusion

pathway of the extraction solvent. However, exceedingly small
particulate size of the microalgal powder may lead to a higher
tendency of lipid re-adsorption, uid channeling effects in the
extraction vessel (for SCCO2 extraction), and inhomogeneous lipid
extraction (Pourmortazavi and Hajimirsadeghi, 2007). Sabio et al.
(Pourmortazavi and Hajimirsadeghi, 2007) conducted a study on
SCCO2 extraction of oil from tomato skins and veried that smaller
tomato skins resulted in higher oil recoveries.
6. Simultaneous extraction and transesterication of
microalgal lipids
Recent studies investigating biodiesel production from microalgae
have focused their efforts on the development of an alternative
downstream processing step termed simultaneous extraction and

transesterication (Wahlen et al., 2011). This step, also known as direct transesterication or in-situ transesterication, combines lipid
extraction and transesterication in a single step, thereby simplifying
the downstream pathway required for biodiesel production from
microalgal biomass (Fig. 19). The method involves the simultaneous
addition of acid catalyst and pure methanol to microalgal biomass
(generally in the form of dried powder). The methanol extracts the
lipids from the microalgal biomass and, catalyzed by the acid,
concurrently transesteries the extracted lipids to produce fatty
acid methyl esters. Similar downstream processing steps as those
required in the traditional pathway are then followed, where the
reaction mixture (consisting of methanol, biodiesel, glycerol, reformed acid catalyst, un-transesteried lipids, and cell debris) undergoes cell-debris removal and post-transesterication purication
730
Fig. 20. Schematic diagram of a laboratory-scale OriginOil Single-Step Extraction system.

Extracted from OriginOil, 2010.
(Fig. 19). Filtration is used for cell-debris removal. A laboratory-scale

post-transesterication purication consists of multiple steps. The reaction mixture is rst distilled to remove methanol. It is then left to
settle under gravity to induce biphasic partitioning (top biodiesel/
un-transesteried lipids phase and bottom glycerol phase). The
biodiesel/un-transesteried lipids phase is decanted off and washed
repeatedly with water to eliminate any acid catalyst. It is noted that
studies investigating direct transesterication of microalgal biomass
have, so far, only used acid catalysts (acetyl chloride and H2SO4).
Direct transesterication was originally derived by Lepage and
Roy (1984) as a rapid method to analyze the fatty acid contents of adipose tissues and milk. The method is highly desirable as it reduces
the amount of downstream manipulation required for biodiesel production from any given biomass. Compared to the conventional
lipid-extraction-followed-by-transesterication approach, direct
transesterication has been shown to improve biodiesel yields of
various animal and plant tissues. However, the efcacy of direct
transesterication on microalgal biomass has not been sufciently
investigated. Parameters that affect the performance of direct
transesterication include the ratio of methanol to dried microalgal
biomass (ml methanol/g dried microalgal biomass), the reaction
temperature (C),
Wahlen et al. (2011) examined the effect of residual water content
within the microalgal biomass on the kinetics of direct transesterication. Chaetoceros gracilis biomass with residual water contents
ranging from 9 to 50% of microalgal paste weight was subjected to
direct transesterication. Increasing residual water content within
the microalgal biomass was found to progressively decrease FAME
yield. For biomass with residual water content equal to 50% of the
microalgal paste weight, FAME yield was half that obtained from
the direct transesterication of dried powder (biomass with residual
water content = 0%).
7. Microalgal biorenery
The cost of producing microalgal biodiesel can theoretically be offset by revenues generated from other co-products of the microalgal
biomass. Microalgae contain signicant quantities of proteins and

carbohydrates as well as smaller amounts of high-value functional
ingredients (astaxanthin, canthaxanthin, carotenes, chlorophylls, 3
free fatty acids, and -linolenic acid). Each of these cell components
can be appropriately utilized to co-generate a useable product in a
biorenery. Recent studies have concluded that industrial-scale
production of microalgal biodiesel can only be made economically
sustainable if a biorenery based production strategy is pursued
(Wijffels et al., 2010). In a biorenery, the crude lipids are to be fractionated into high-value functional ingredients and lipids for biodiesel (acylglycerols). Functional ingredients have been linked with the
promotion of anti-oxidant, anti-inammatory, as well as anticarcinogenic activities in the human bodies and are typically used as
food supplements. If the microalgal species contains a high level of
proteins, the residual biomass from biodiesel production processes
can be used as livestock feeds. If the species has high carbohydrate
contents, the residual biomass can be fermented to produce bioethanol. As such, microalgal biorenery will simultaneously produce biodiesel, high-value products, livestock feeds, and bioethanol.
Unfortunately, the combination of technologies needed to implement
a microalgal biorenery is still in the early stages of development.
Milder cell disruption/lipid extraction process needs to be explored
to ensure that the functionalities of different cell components are
retained.
8. OriginOil Single-Step Extraction of microalgal lipids
OriginOil, Inc has established a novel method for microalgal lipid
extraction (OriginOil, 2010). Instead of following the traditional
sequence-based downstream processing pathway outlined in Fig. 4,
the method devised by OriginOil performs three simultaneous
functions (dewatering, cell disruption, and lipid extraction) in a
single downstream step (Fig. 20). This approach is referred to as
OriginOil Single-Step Extraction.
Within a single step (OriginOil, 2010), microalgal concentrate is
exposed to Quantum Fracturing, a patent-pending technology
based on the science of uid fracturing, combined with pulsed
electromagnetic elds and pH modication (Fig. 20). Microalgal cells

are instantly disrupted and intracellular lipids are released from the
microalgal biomass. The microalgal lipids rise to the top of the gravity
clarier for skimming, lipid fractionation, and transesterication to
biodiesel, while the cell debris settles to the bottom of gravity clarier
for further processing as fuel and other valuable by-products. The
water (or growth medium) in the middle of the gravity clarier can
be decanted and recycled for microalgal cultivation. There are three
components to OriginOil Single-Step Extraction (OriginOil, 2010):
1) CO2 injection lowers pH of the growth medium to optimize electromagnetic elds delivery and to assist in cell disruption, 2) Quantum Fracturing creates uid effect to mechanically stress
microalgal cells, 3) Pulsed electromagnetic elds deliver the force
that disrupt the microalgal cells.
OriginOil Single-Step Extraction has several benets (OriginOil,
2010). By combining three functions (dewatering, cell disruption,
and lipid extraction) in a single downstream step, it substantially
reduces the energy expenditure required to produce biodiesel from
microalgal biomass. The lipid extraction method does not use any
toxic extraction solvent and, thus, does not require a solvent recovery
step. Finally, the lipid extraction method is highly effective when
directly applied to wet feedstock (concentrate).
9. Conclusions
The downstream technologies needed for industrial-scale production of microalgal biodiesel are still in the early stages of development. Lipid extraction from microalgal biomass has not received
sufcient attention and represents one of the many bottlenecks hindering economic industrial-scale production of microalgal biodiesel.
Future research on microalgal biodiesel should focus on developing
an effective and energetically efcient lipid extraction process. A
fundamental understanding of lipid mass transfer mechanisms from
the microalgal biomass to the extraction solvent is needed to scale
up the lipid extraction process.
An ideal lipid extraction technology for microalgal biodiesel
production needs to be not only lipid specic in order to minimize
the co-extraction of non-lipid contaminants (such as protein and
carbohydrates) but also selective towards acylglycerols in order to reduce downstream fractionation/purication (Fajardo et al., 2007;
Medina et al., 1998). Additionally, the selected technology should be
efcient (both in terms of time and energy), non-reactive with the
lipids, relatively cheap (both in terms of capital cost and operating
cost), and safe (Kates, 1986b). Microalgal culture is harvested as a dilute aqueous suspension (from 0.1 to 2 g dried microalgal biomass/L
culture) and is substantially concentrated. However, dewatering the
microalgal biomass beyond a paste consistency (200 g dried microalgal biomass/L culture) is energetically prohibitive. For this reason, it
will be economically benecial if the selected lipid extraction technology can be directly applied to relatively wet feedstock, i.e. concentrate or disrupted concentrate with concentrations between 10 and
200 g dried microalgal biomass/L culture (Halim et al., 2011).
Studies in the past decade have demonstrated the use of organic
solvents and the use of supercritical carbon dioxide to extract lipids
from microalgal biomass. However, each technology has its merits
and limitations. Despite having low reactivity with lipids and being
directly applicable to relatively wet feedstock, organic solvent extraction is slow and uses a large amount of expensive/toxic solvents. On
the other hand, supercritical carbon dioxide extraction is a promising
green technology that can potentially be used for large-scale microalgal lipid extraction. It is rapid, non-toxic, has high selectivity towards
acylglycerols, and produces solvent-free lipids. Its main disadvantages are associated with the high capital cost and the high energy
requirement for supercritical uid compression. Several studies
have shown that the disruption of microalgal cells prior to the lipid
extraction step enhances the extraction efciency.
731
Acknowledgments
This work was supported by an Australian Research Council (ARC)
Linkage grant between the Department of Chemical Engineering in
Monash University (Victoria, Australia) and Bio-Fuel Pty Ltd (Victoria,
Australia).
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Biotechnology Advances 30 (2012) 709732

Contents lists available at SciVerse ScienceDirect

Research review paper

Extraction of oil from microalgae for biodiesel production: A review

Corresponding author. Tel.: + 61 3 990 53420.

R. Halim et al. / Biotechnology Advances 30 (2012) 709732

hand, the development of an effective and energetically efcient

R. Halim et al. / Biotechnology Advances 30 (2012) 709732

since acylglycerols generally have a lower degree of unsaturation

neutral lipids breakdown

neutral lipids breakdown

neutral lipids breakdown

R. Halim et al. / Biotechnology Advances 30 (2012) 709732

wt% of total fatty acids

fatty acid chain

No. of double bonds in the fatty acid chain

the transesterication step. Each of the downstream processes is

R. Halim et al. / Biotechnology Advances 30 (2012) 709732

Microalgal lipid extraction generally uses either organic solvent or

concentrate or disrupted concentrate), lipids, and cell debris, is

R. Halim et al. / Biotechnology Advances 30 (2012) 709732

Removal of cell debris

chemical lysis (acids &

milling with specific sieve

crushing with pestle and mortar

organic solvent extraction

supercritical fluid extraction

organic solvent extraction with

accelerated organic solvent

sub-critical organic solvent

centrifugation (not applicable

solid-phase solvent adsorption

silicic acid column

R. Halim et al. / Biotechnology Advances 30 (2012) 709732

Free Fatty Acid

Free Fatty Acid

R. Halim et al. / Biotechnology Advances 30 (2012) 709732

towards lipids in order to minimize the co-extraction of non-lipid

solvent, such as hexane or chloroform, the organic solvent penetrates

R. Halim et al. / Biotechnology Advances 30 (2012) 709732

hexane system = 0.015 g lipid/g dried microalgal biomass, nal total

hexane/IPA (3/2 v/v)

hot plate stirrer

R. Halim et al. / Biotechnology Advances 30 (2012) 709732

the organic phase (a mixture of non-polar organic solvent and polar

E is experimentally determined by measuring the energy required

where E/V = 2, ED/V = (D) 2, Ep/V = (P) 2, and EH/V = (H) 2

where is the Hildebrand total solubility parameter, D is the Hansen

Subscript s is for the solvent and subscript p is for the solute.

For a solvent mixture, composite Hansen solubility parameter,

Guckert et al., 1988

Nagle and Lemke,

Molina Grima et al., 1994

Lee et al., 1998

Fajardo et al., 2007 Halim et al., 2011

Dried powder by lyophilization

For Soxhlet: methylene chloride/

Chloroform/methanol (2:1 v/v), Ethanol

For Soxhlet: 1000 ml organic

76 ml organic solvent mixture/ g 250 ml organic solvent

For Soxhlet: N/A. For batch

High (not specied)

Not specied, but stated to be