Biotechnology Advances 30 (2012) 709–732

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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 a b s t r a c t

Article history: The rapid increase of CO2 concentration in the atmosphere combined with depleted supplies of fossil fuels
Received 8 March 2011 has led to an increased commercial interest in renewable fuels. Due to their high biomass productivity,
Received in revised form 14 November 2011 rapid lipid accumulation, and ability to survive in saline water, microalgae have been identified as promising
Accepted 4 January 2012
feedstocks for industrial-scale production of carbon-neutral biodiesel. This study examines the principles
Available online 11 January 2012
involved in lipid extraction from microalgal cells, a crucial downstream processing step in the production
Keywords:
of microalgal biodiesel. We analyze the different technological options currently available for laboratory-
Microalgae scale microalgal lipid extraction, with a primary focus on the prospect of organic solvent and supercritical
Biodiesel fluid extraction. The study also provides an assessment of recent breakthroughs in this rapidly developing
Oil extraction field and reports on the suitability of microalgal lipid compositions for biodiesel conversion.
Lipid extraction © 2012 Elsevier Inc. All rights reserved.
Organic solvent
Supercritical carbon dioxide
Downstream process
Pre-treatment
Direct transesterification

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 710
2. Microalgal lipid composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 710
3. Overview of downstream processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712
4. Lipid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 715
4.1. Organic solvent extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 716
4.1.1. Basic principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 716
4.1.2. Solubility parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 718
4.1.3. Selection of organic solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 718
4.1.4. Operating variables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 720
4.1.5. Modifications to organic solvent extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 721
4.2. Supercritical fluid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 722
4.2.1. Basic principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 722
4.2.2. Operating variables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 723
4.3. Comparison between organic solvent extraction and SCCO2 extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 724
5. Effect of cellular pre-treatment on lipid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 726
6. Simultaneous extraction and transesterification of microalgal lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 729
7. Microalgal biorefinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 730
8. OriginOil Single-Step Extraction of microalgal lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 730
9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 731
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 731
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 731

⁎ 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
710 R. Halim et al. / Biotechnology Advances 30 (2012) 709–732
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 transesterifica-
tion 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 addition-
al strains on food production (Widjaja et al., 2009). Their high photo-
synthetic 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 oleagi-
nous 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
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.
Modified from Nelson and Cox (2000).
hand, the development of an effective and energetically efficient
lipid extraction process from the microalgal cells is critical for the suc-
cessful 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 field. We report on the suitability of microalgal lipid
compositions for biodiesel conversion and review the different con-
ventional downstream bioprocessing steps required for microalgal
biodiesel production. We then examine the technologies currently
available for laboratory-scale microalgal lipid extraction, paying spe-
cial attention to the use of organic solvent extraction and supercritical
fluid 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 field, such as
the development of a simultaneous microalgal lipid extraction-
methylation 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
 R. Halim et al. / Biotechnology Advances 30 (2012) 709–732 711

acids consist of at least one double bond (Nelson and Cox, 2000). since acylglycerols generally have a lower degree of unsaturation
When the carboxylate end of the fatty acid molecule is bonded to than other lipid fractions (i.e. polar lipids), they produce FAME with
an uncharged head group (e.g. glycerol), a neutral lipid molecule is higher oxidation stability. As shown by the lipid profiles of three
formed (e.g. triacylglycerol). On the other hand, the association of a microalgal species (Nannochloropsis oculata, Pavlova lutheri, Isochrysis
fatty acid molecule to a charged head group (e.g. glycerol and phos- sp.) in Fig. 2, microalgal lipids usually comprise high levels of polar
phate complex) forms a polar lipid molecule (e.g. phospholipid). lipids and non-acylglycerol neutral lipids (HC, ST, K, FFA). As such,
Lipids can be defined as any biological molecule which is soluble they often need to be purified before they can be transesterified.
in an organic solvent. As mentioned above, most lipids contain fatty When comparing lipids obtained from logarithmic and stationary
acids (Fig. 1) and can generally be classified into two categories growth phase, stationary-phased lipids, despite having an abundance
based on the polarity of the molecular head group (Kates, 1986a): of polar lipids at 51–57 wt.%, contain higher levels of TG (20–41 wt.%
(1) neutral lipids which comprise acylglycerols and free fatty acids of total lipid) and appear more attractive for biodiesel processing
(FFA) and (2) polar lipids which can be further sub-categorized into than logarithmic-phased lipids (Dunstan et al., 1993).
phospholipids (PL) and glycolipids (GL). Neutral lipids are used Microalgal fatty acids range from 12 to 22 carbons in length and
primarily in the microalgal cells as energy storage, while polar lipids can be either saturated or unsaturated. The number of double bonds
pack in parallel to form bilayer cell membranes. Acylglycerol consists in the fatty acid chains, however, never exceeds 6 and almost all of
of fatty acids ester-bonded to a glycerol backbone and is categorized the unsaturated fatty acids are cis isomers (Medina et al., 1998).
according to its number of fatty acids: triacylglycerols (TG), diacylgly- The fatty acid profile of lipid extracted from Tetraselmis suecica during
cerols (DG), monoacylglycerols (MG). FFA is a fatty acid bonded to a early stationary phase is shown in Fig. 3 (Volkman et al., 1989). T. sue-
hydrogen atom. cica is a common green microalga and its fatty acid profile is used
It is noted that there are also some types of neutral lipids that do here as an example to illustrate the suitability of microalgal lipids
not contain fatty acids, such as hydrocarbons (HC), sterols (ST), for biodiesel synthesis. Having C16:0, C18:1, and C18:2 as its the
ketones (K), pigments (carotenes and chlorophylls). Even though principal fatty acids, Tetraselmis lipid appears to have the required
these lipid fractions are soluble in organic solvents (hence fitting fatty acid profile for conversion to high-quality biodiesel. Saturated
the definition 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
neutral lipids breakdown
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 nu-
tritional 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
neutral lipids breakdown
species, Lv et al. (2010) demonstrated that some microalgal species
are richer in neutral lipids than other species.
The composition and fatty acid profile 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 station-
ary 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
neutral lipids breakdown
(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 sacrificing growth and through
the cessation of cell division. Due to the aforementioned inter- and
intraspecific variations, the suitability of microalgal lipids for biodie-
sel production is difficult to assess and often needs to be examined
on a case-by-case basis.
Acylglycerols are desirable for commercial-scale biodiesel produc-
tion for two main reasons. Firstly, industrial-scale alkaline-catalyzed Fig. 2. Compositions of crude lipids extracted from three microalgal species during
logarithmic phase and stationary phase. Top: Nannochloropsis oculata, middle: Pavlova
transesterification is designed to process acylglycerols (TG, DG, and lutheri, bottom: Isochrysis sp. For neutral lipids, TG: triacylglycerols, HC: hydrocarbons,
MG) and has limited efficacies on other lipid fractions, such as polar ST: sterols, K: ketones, FFA: free fatty acids.
lipids and free fatty acids (Christie, 2007; Lang et al., 2001). Secondly, Modified from Dunstan et al. (1993).
712 R. Halim et al. / Biotechnology Advances 30 (2012) 709–732

a the transesterification step. Each of the downstream processes is

wt% of total fatty acids

30 explained in more detail below.

25
Cultivation of microalgae is performed in either an indoor or an
outdoor system (Chisti, 2007). Indoor cultivation systems normally
20
use photobioreactors, while outdoor systems employ either raceway
15
ponds or photobioreactors. In an outdoor system, the microalgae are
10 grown in the open environment where cultivation parameters
5 (temperature and light intensity) are dependant on day-to-day
0 weather conditions. The microalgae grown in such a system often
suffer from inconsistent growth rates and are more susceptible to
:0

:0

:0

C 1

:1

:1

:2

:2

:3

:3

:4

C 4

:5
C t

:4
:
:1

:
14

16

18

16

18

20

16

18

16

18

16

18

20
20
16

local species invasion. On the other hand, the microalgae grown in

C

C
fatty acid chain
b No. of double bonds in the fatty acid chain
wt% of total fatty acids
≥4
0
20.8%
27.6%
3 cost of downstream processing (Danquah et al., 2009; Molina Grima
14.2%
1
2 1-trans
16.2% 20.3%
0.8%
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-configuration.
Modified 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 flow 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 consequential-
ly 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 avail-
able 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 pre-
pare 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 finally converted to biodiesel 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). Through-
out 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 cul-
tivation method) and needs to be concentrated in order to reduce the
et al., 2003). Solid–liquid separation methods, such as centrifugation,
filtration, and flocculation, 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-efficient dewatering technology is currently an active field of
research.
Among the myriads of dewatering technologies (Table 1),
flocculation appears to be the most advantageous due to its low ener-
gy requirement (Uduman et al., 2010; Wijffels and Barbosa, 2010).
During flocculation, 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 flocculate microalgal cells. More details on microalgal dewa-
tering and flocculation can be found elsewhere (Uduman et al., 2010).
Post-dewatering, the concentrate undergoes pre-treatment pro-
cess aimed at enhancing the efficiency 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 alter-
ations. 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 concen-
trate. In a typical 2-step laboratory-scale pre-treatment pathway, the
concentrate is completely dried and then milled into fine powders.
This pre-treatment eliminates residual water known to prohibit effec-
tive 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 matri-
ces. 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 ex-
traction are discussed in detail in the following section (Section 4).
 R. Halim et al. / Biotechnology Advances 30 (2012) 709–732 713

Fig. 4. Process flow diagram showing the downstream processing steps needed to produce biodiesel from microalgal biomass.

Microalgal lipid extraction generally uses either organic solvent or
supercritical fluid (such as supercritical carbon dioxide) as an
extraction solvent.
After lipid extraction, the resulting mixture, consisting of extrac-
tion solvent, residual water (only when extraction is performed on
concentrate or disrupted concentrate), lipids, and cell debris, is
submitted to a solid–liquid separation method, such as filtration, to
remove the cell debris. For organic solvent extraction, a liquid–liquid
separation method, such as distillation, vacuum evaporation, or solid-
phase solvent adsorption, is then employed to remove the extraction
714 R. Halim et al. / Biotechnology Advances 30 (2012) 709–732

Table 1 solvent and the residual water from the lipids. When non-polar/polar
Different laboratory-scale technologies available for each downstream processing
organic solvent mixture is used (refer to Section 4.1.1), residual water
step required to produce biodiesel from microalgae. The scale-up potential of each
technology is examined. . * * *: highly scalable. *: lack scalability. is removed from the extraction solvent and the lipids via biphasic
separation and decantation. The liquid–liquid separation then
Process step Technologies Scale-up potential removes the extraction solvent from the lipids. On the other hand,
raceway ponds
for supercritical fluid extraction, pressure decompression returns
***
Cultivation the extraction solvent as well as the residual water to their gaseous
photobioreactors **
states and results in forced precipitation of the lipids (refer to
agglomeration *** Section 4.2.1). As such, no extra step is needed for the removal of
centrifugation ** extraction solvent and residual water.
Dewatering filtration ** The isolated lipids, referred to as crude lipids or total lipids, can
flocculation ***
now be gravimetrically quantified. The term ‘total lipids’ is primarily
used for analytical purposes. As previously mentioned in Section 2,
pressure dewatering *
in addition to acylglycerols, crude lipids obtained from microalgal
ultrasonication ** biomass frequently contain polar lipids and non-acylglycerol neutral
lipids (such as free fatty acids, hydrocarbons, sterols, ketones,
high-pressure homogenization ***
carotenes, and chlorophylls). From the perspective of biodiesel
french pressing ** production, any non-acylglycerol biochemical fraction is a contami-
Pre-Treatment: cell
disruption
bead miling ** * nant and will have to be removed from the crude lipids. As such,
microwave ** crude lipids arising from microalgal biomass are often subjected to a
chemical lysis (acids & fractionation step before they are transesterified. Different purifica-
** tion methods, such as liquid chromatography, acid precipitation,
enzymes)
osmotic shock **
and urea crystallizations, are used for lipid fractionation (Medina
et al., 1998).
oven drying * During transesterification, the fatty-acid-containing lipid fractions
Pre-Treatment: drying
freeze drying
in the crude lipids are reacted with alcohol (methanol, ethanol,
*
isopropanol, butanol) and converted to fatty acid alkyl esters. When
spray drying *
methanol is used, the reaction produces fatty acid methyl ester
Pre-Treatment: particulate milling with specific sieve ** (FAME) or biodiesel. Either an acid (such as H2SO4) or an alkali
size reduction (such as NaOH or KOH) can be used as a catalyst for transesterifica-
crushing with pestle and mortar * tion (Christie, 2007; Volkman et al., 1989). Since alkali catalysts
organic solvent extraction ** have faster reaction rates (estimated at 4000× faster) and higher con-
supercritical fluid extraction ** versions than acid catalysts for the transesterification of acylglycerols,
organic solvent extraction with they are commercially used in the chemical industry for conversion of
* plant and animal oils to biodiesel (Huang et al., 2010). As previously
Soxhlet apparatus
noted in Section 2, alkaline-catalyzed transesterification has limited
Lipid extraction ultrasound-assisted organic
solvent extraction
** efficacies when applied to non-acylglycerol fatty-acid-containing
lipid fractions, such as polar lipids and free fatty acids. During alkaline
transesterification of acylglycerols, the catalyst cleaves the ester
microwave-assisted organic
solvent extraction
** 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
accelerated organic solvent
** amounts of crude microalgal lipids are available, a large amount of
extraction
methanol (substantially in excess of stoichiometric requirement) is
sub-critical organic solvent often added to ensure quantitative transesterification.
**
extraction Once transesterification is completed, the reaction mixture,
Removal of cell debris
containing biodiesel, glycerol, reformed alkali catalyst, excess
filtration **
from extraction solvent- methanol, and un-transesterified lipids, then undergoes post-
residual water-lipids centrifugation (not applicable transesterification purification to remove by-product contaminants
mixture **
to supercritical fluid extraction) (glycerol, alkali catalyst, and excess methanol). A laboratory-scale
distillation ** post-transesterification purification method typically consists of 2
Removal of extraction
vacuum evaporation *
steps. The reaction mixture is left to settle under gravity to induce bi-
solvent and residual water
from lipids. This step is phasic partitioning (top biodiesel/un-transesterified lipids phase and
not applicable to bottom glycerol phase). Once the biodiesel/un-transesterified lipids
supercritical fluid solid-phase solvent adsorption ** phase is decanted off, it is washed repeatedly with water to eliminate
extraction.
any alkali catalyst and excess methanol (Chisti, 2007; Demirbas,
liquid chromatography
2008; Demirbas and Karslioglu, 2007). More details on alkaline trans-
*
esterification of acylglycerols and post-transesterification purification
silicic acid column
chromatography
* can be found elsewhere (Lang et al., 2001; Wahlen et al., 2011).
Lipid fractionation
Analyses of the FAME composition of the purified biodiesel/
acid precipitation **
untransesterified lipids phase are carried out using a gas chromatog-
urea crystallization ** raphy (GC) system.
acid catalyst *** Variables that affect FAME conversion during alkaline transesteri-
Transesterification
alkali catalyst ***
fication 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
 R. Halim et al. / Biotechnology Advances 30 (2012) 709–732 715

Triacylglycerol Methanol FAME Glycerol

(biodiesel)

Diacylglycerol Methanol FAME Glycerol

(biodiesel)

FAME
Monoacylglycerol Methanol (biodiesel) Glycerol

Free Fatty Acid Potassium Soap Water

Hydroxide

Monoacylglycerol Water Free Fatty Acid Glycerol

Fig. 5. Various reactions involving the alkali catalyst, KOH or potassium hydroxide. [1], [2], and [3] illustrate the alkaline transesterification 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 (saponification).
[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.
Modified 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 (saponification). 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 saponification loss (Huang
et al., 2010). Past works on alkaline transesterification 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 transesterification 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 pow-
der. 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 specificity
716 R. Halim et al. / Biotechnology Advances 30 (2012) 709–732

towards lipids in order to minimize the co-extraction of non-lipid
contaminants, such as protein and carbohydrates. To reduce down-
stream fractionation/purification, 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 efficient (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 bio-
mass beyond a paste consistency (200 g dried microalgal biomass/L
culture) is energy intensive, it will be economically beneficial 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 fluid extraction, an emerging green technology that is
currently gaining considerable research attention, for microalgal
lipid quantification.
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 attrac-
tions 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
solvent–lipids complex, driven by a concentration gradient, diffuses
across the cell membrane (step 4) and the static organic solvent
film 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
film is formed due to the interaction between organic solvent and
cell wall. This film surrounds the microalgal cell and remains
undisturbed by any solvent flow 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 lipid–protein associations. On the other hand,
polar organic solvent (such as methanol or isopropanol) is able to
disrupt the lipid–protein 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 pro-
posed in lower half of Fig. 6 and can be divided into 5 steps. The or-
ganic 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 associ-
ations 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 lipid–protein associations binding the lipid
complex to the cell membrane. An organic solvent–lipids complex is
formed and dissociates away from the cell membrane (step 3). The

static organic 5 bulk organic

solvent film solvent

cell membrane
4 and cell wall

1
3
2
cytoplasm
1
2
nucleus

4 3

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 solvent–lipids com-
plex. Step 4: diffusion of organic solvent–lipids complex across the cell membrane. Step 5: diffusion of organic solvent–lipids complex across the static organic solvent film into the
bulk organic solvent.
 R. Halim et al. / Biotechnology Advances 30 (2012) 709–732 717

organic solvent–lipids complex then diffuses across the cell mem-
brane (step 4) and the static organic solvent film 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 ex-
traction 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 free-
standing globules and in the form of membrane-associated com-
plexes. 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% (final total lipid yield of pure
hexane system = 0.015 g lipid/g dried microalgal biomass, final 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 solid–liquid separation method (such as filtration),
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 chloro-
form for chloroform/methanol mixture) and water. Upon complete
biphasic separation, neutral and polar lipids will mainly partition in

a b c

concentrate

microalgal culture

e
dried hexane/IPA (3/2 v/v)
powder
f
d
cell
debris

hexane/
IPA

h
methanol KOH g

crude
lipids

biodiesel, hexane/IPA
glycerol,
hot plate stirrer
un-transesterified
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-transesterification purification 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 filter. (g) Removal of extraction solvent with a distillation unit.
(h) Transesterification with an alkali catalyst.
718 R. Halim et al. / Biotechnology Advances 30 (2012) 709–732

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 transesterified.
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 findings 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) attempt-
ing 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 quantitative-
ly 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 ¼ ED þ Ep þ 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 compo-
nents of Hansen solubility parameters.
E=V ¼ ðED =VÞ þ ðEp=VÞ þ ðEH =VÞ
where E/V = δ 2, ED/V = (δD) 2, Ep/V = (δP) 2, and EH/V = (δH) 2
2 2 2 2
δ ¼ ðδD Þ þ ðδP Þ þ ðδH Þ
where δ is the Hildebrand total solubility parameter, δD is the Hansen
dispersion parameter, δP is the Hansen dipolar parameter, and δH is
the Hansen hydrogen bond parameter. The SI unit for all of the
parameters is MPa 1/2. HSP characterization can be conveniently visu-
alized 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 2 2 2
Ra ¼ 4ðδDS −δDP Þ þ ðδPS –δPP Þ þ ðδHS –δHP Þ ð4Þ
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 ¼ Ra=Ro ð5Þ
For a solvent mixture, composite Hansen solubility parameter,
δi, mix, is calculated with:
δi;mix ¼ φ1 δi;1 þ φ2 δi;2 þ … ð6Þ
Subscript i signifies one of the three components of Hansen solu-
bility parameters, φ 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
ð1Þ 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 finding is contradictory to previous lipid extraction works
which recommend the use of chloroform/methanol/water mixture
ð2Þ (Molina Grima et al., 1994). We note that the current triacetin-
based HSP characterizations must be treated with caution. Triacylgly-
cerols with higher carbon number will have different Hansen solubil-
ity parameters to triacetin. Additionally, limited availability of Hansen
ð3Þ solubility parameters for other desirable neutral-lipid derivatives,
such as diacylglycerols, monoacylglycerols, and neutral lipids/polar
lipids complexes, prevents the complete characterization of microal-
gal 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: δD = 16.5 MPa1/2, δP = 4.5 MPa1/2, δH = 9.1 MPa1/2.

Organic solvent or Hansen dispersion Hansen dipolar Hansen hydrogen Affinity of solvent Relative energy Solubility of
organic solvent parameter or δD parameter or δP bond parameter with triacetin difference or triacetin in
mixture (MPa1/2) (MPa1/2) or δH (MPa1/2) or Ra (MPa1/2) RED the solvent

Chloroform 17.8 3.1 5.7 4.5 0.4 Very good

Methanol 15.1 12.3 22.3 15.6 1.3 Poor
Water 15.6 16.0 42.3 35.2 2.9 Very poor
Chloroform/methanol (1:2 v/v) 19.0 9.8 17.7 11.2 0.9 Good
Chloroform/methanol/water (1:2:0.8 v/v/v) 15.9 10.7 22.1 14.5 1.2 Poor
Hexane 14.9 0.0 0.0 10.6 0.9 Good
Isopropanol 15.8 6.1 16.4 7.6 0.6 Good
Hexane/isopropanol (3:2 v/v) 15.3 2.4 6.6 4.1 0.3 Very good
Ethanol 15.8 8.8 19.4 11.2 0.9 Good
Table 3
Methods and results summary of recent studies investigating organic solvent extraction of microalgal lipids.

Kates, 1986b Kates, 1986b Guckert et al., 1988 Nagle and Lemke, Molina Grima et al., 1994 Lee et al., 1998 Fajardo et al., 2007 Halim et al., 2011
(method for (method for 1990
microorganisms) microalgae)

Microalgal species N/A Any Chlorella sp. Chaetoceros muelleri Isochrysis galbana Botryococcus braunii Phaeodactylum Chlorococcum sp.
and Monoraphidium tricornutum
minutum

State of microalgal Concentrate Concentrate Dried powder by lyophilization Concentrate (residual Dried powder by lyophilization Concentrate Dried powder Concentrate
biomass at the (residual water water content = 85 wt.%) by lyophilization (residual water
start of extraction content = 90 wt.%) content = 70 wt.%)
or dried powder by
thermal drying

Mass of dried 5 to 6 Not 0.1 60 5 0.12 10 4

microalgal specified.
biomass (g) Mass of wet
paste = 1.5 g.

R. Halim et al. / Biotechnology Advances 30 (2012) 709–732

Organic solvents or Water/methanol/ Water/ For Soxhlet: methylene chloride/ 1-butanol, ethanol, Chloroform/methanol/water Chloroform/methanol (2:1 v/v), Ethanol Hexane, hexane/
organic solvent chloroform isopropanol methanol (2:1 v/v). For batch hexane/2-propanol (1:2:0.8 v/v/v), hexane/ethanol hexane/isopropanol (3:2 v/v), isopropanol (3:2 v/v)
mixtures (0.8:2:1 v/v/v) (1:5 v/v) extraction 1: chloroform/ (2/3 v/v), water/ (1:2.5 v/v), hexane/ethanol dichloroethane/methanol
methanol/50 mM phosphate methanol/chloroform (1:0.9 v/v), butanol, ethanol, (1:1 v/v), dichloroethane/
buffer (35:70:28 v/v/v/). For as a control system EtOH/water (1:1 v/v), hexane/ ethanol (1:1 v/v), acetone/
batch extraction 2, n-hexane/IPA/ isopropanol (1:1.5 v/v) dichloromethane (1:1 v/v)
distilled water (70:47.7:3 v/v/v).

Amounts of organic 76 ml organic 16 ml For Soxhlet: 1000 ml organic 20 g organic solvent 76 ml organic solvent mixture/ g 250 ml organic solvent 5 ml organic 75 ml organic solvent
solvent or organic solvent mixture/ g organic solvent mixture/ g dried mixture/ g dried dried microalgal biomass mixture/ g dried solvent mixture/g mixture/ g dried
solvent mixture dried microalgal solvent microalgal biomass. microalgal biomass microalgal biomass dried microalgal microalgal biomass
added biomass mixture/ g For batch extraction 1: 1330 ml biomass
concentrate organic solvent mixture/g dried
microalgal biomass. For batch
extraction 2: 1207 ml organic
solvent mixture/ g dried
microalgal biomass.

Duration (min) 60 3 For Soxhlet: 180. For batch 90 60 50 1440 450

extraction 1: 2160.
For batch extraction 2: overnight.

Degree of agitation Not specified Not specified For Soxhlet: N/A. For batch High (not specified) Not specified, but stated to be High (not specified) 500 800
(rpm) extraction 1: not specified. For constant
batch extraction 2: not specified.

Extraction 25 50–60 For Soxhlet: not specified. For Near boiling point 25 Not specified 25 25
temperature batch extraction 1: not specified. of each solvent
(°C) For batch extraction 2: room. (not specified)

Maximum final N/A N/A 11.9; Soxhlet with methylene Not specified as results 8.9; chloroform/methanol/water 28.6; chloroform/ 6.3; ethanol 6.8; hexane/
total lipid yield chloride/methanol (2:1 v/v) are expressed as (1:2:0.8 v/v/v). methanol (2:1 v/v) isopropanol (3:2 v/v)
(wt.% of dried efficiencies to a control 2nd Highest efficiency with
microalgal system (assigned at ethanol at 8.0 wt.% of dried
biomass); the 100%). 2nd Highest microalgal biomass
organic solvent or efficiency with 1-butanol
the organic at 94%
solvent mixture
to achieve it

719
720 R. Halim et al. / Biotechnology Advances 30 (2012) 709–732
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 organ-
ic solvent mixture for lipid extraction from any living tissue. Using
this organic solvent system, residual endogenous water in the micro-
algal 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 quanti-
tative. Chloroform, however, is highly toxic and its usage is undesir-
able. The method was originally developed by Folch et al. (1951) for
the isolation of total lipids from brain tissues. For this reason, its
efficacy in extracting lipids from microalgal biomass still needs
further assessment. In a study by Lee et al. (1998), the performance
of five 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 final
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 efficacies 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 produc-
tion to occur with minimal downstream purification. Guckert et al.
(1988) attributed the neutral lipid selectivity of hexane/isopropanol
mixture to its inability to extract the polar lipid constituents of micro-
algal 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 affinity 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 free-
standing neutral lipid globules. For this reason, when used as a
microalgal lipid extraction solvent, alcohol is almost always com-
bined with a non-polar organic solvent, such as hexane or chloroform,
to ensure the total extraction of both forms of neutral lipids (free-
standing globules and as membrane-associated complexes) (Halim
et al., 2011).
In their study, Nagle and Lemke (1990) evaluated the efficiencies
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
verified to be the most effective organic solvent system (assigned
an arbitrary extraction efficiency of 100%), butanol (with an average
extraction efficiency of 94%) was found to be highly promising with
a final 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 extrac-
tion procedure, a beneficial 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 isopropanol-
containing 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 first-order kinetics
equation (Halim et al., 2011; Harrison et al., 2003b):
 
−kt
me ¼ ms;o 1−e ð1Þ
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 coefficient 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 ¼ f ðag; s=b; TÞ ð2Þ
where ag is the degree of agitation (revolution per minute), s/b is the
ratio of organic solvent to dried microalgal biomass (ml organic sol-
vent/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 micro-
algal 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 recov-
ered within the first 8 h (60–70% of all extractable lipids) and extend-
ing the extraction time beyond 12 h does not seem to make any
significant contribution to the final 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
90
( wt% of dried microalgal
80
Total lipid yield
70
biomass)
60
50
40
30
20
10
0
0 4 8 12 16 20 24
Time (h)
Fig. 8. Typical first-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.
Modified from Fajardo et al. (2007).
 R. Halim et al. / Biotechnology Advances 30 (2012) 709–732
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 investiga-
tion of lipid extraction from Phaeodactylum tricornutum, Fajardo
et al. (2007) found extraction efficiency to increase with decreasing
s/b ratio (Fig. 8). The final 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 final 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 find the optimum s/b
value for a specific microalgal strain. An s/b value that is too high re-
sults in excessive consumption of organic solvent, while a value that
is too low leads to handling difficulties and incomplete extraction.
Variation in the extraction temperature has been reported to
influence 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 oxida-
tive degradation of thermo-labile components which resulted in a
lower lipid yield. In a study by Balasubramanian et al. (2010), increas-
ing temperature during lipid extraction from Scenedesmus obliquus
resulted in a significant increase in the final total lipid yield. The
authors attributed this increase to enhanced mass transfer kinetics
at higher temperature.
The kinetics and the mechanism underlying organic solvent ex-
traction 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.
Modified 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 con-
centration 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. Modifications 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 equilibri-
um, a continuous organic solvent extraction able to overcome this lim-
itation requires a large amount of organic solvent and becomes too
expensive. Through its ingenious cycles of solvent evaporation and con-
densation, the Soxhlet apparatus continuously replenishes cells with
fresh organic solvent (hence evading equilibrium limitation) while si-
multaneously 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 round-
bottom flask to store the extracting organic solvent, the Soxhlet extrac-
tor 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 flask enters the con-
denser and is immediately channeled into the Soxhlet extractor. The or-
ganic 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
flow and, as such, serves as a filter to remove cell debris. Once the or-
ganic solvent in the extractor reaches the overflow level, a siphon un-
loads the organic solvent-lipids mixture from the extractor back into
the round-bottom flask. The organic solvent is heated and evaporates
again while the extracted crude lipids remain in the round-bottom
flask. This cycle is repeated until no more crude lipids are extracted in
the Soxhlet extractor. Despite its advantageous design in avoiding equi-
librium limitation, the Soxhlet apparatus suffers from high energy re-
quirement for continuous distillation (Luque de Castro and Garcia-
Ayuso, 1998; Wang and Weller, 2006).
Independent studies by Guckert et al. (1988) and Halim et al. (2011)
verified the superior efficacy 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 final total lipid yield (Table 3). In terms of the dry microalgal
weight, the final 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 significantly more efficient than its batch counterpart when used
to extract lipids from Chlorococcum sp. (final total lipid yield of batch
extraction = 0.015 g lipid/g dried microalgal biomass, final 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 tem-
perature 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 modifications to organic solvent extraction have also
been introduced: microwave-assisted organic solvent extraction and
accelerated or subcritical organic solvent extraction. Each modifica-
tion 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). Modified organic solvent extraction synergistically
722 R. Halim et al. / Biotechnology Advances 30 (2012) 709–732
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 specific 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 tempera-
ture 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 ma-
terials 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 water-
heated hexane extraction control methods at all extraction tempera-
tures and times.
During accelerated or subcritical organic solvent extraction, lipid ex-
traction 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 benefits of super-
critical fluid 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 efficient (maximum final
lipid recovery= 90.21% of total lipids). Neither of the modifications
described above (microwave-assisted or subcritical organic solvent
extraction) has been applied to an industrial scale due to their high en-
ergy requirements. It is also noted that there is currently limited under-
standing on the key variables affecting the performances of these
modified extraction processes (Luque de Castro and Garcia-Ayuso,
1998; Wang and Weller, 2006). The scale-up potentials of organic
solvent extraction and its modifications are examined in Table 1.
4.2. Supercritical fluid extraction
Supercritical fluid extraction (SFE) is an emerging green technolo-
gy that has the potential to replace traditional organic solvent
extraction.
4.2.1. Basic principles
When the temperature and the pressure of a fluid are raised over their
critical values (Tc and Pc), the fluid enters the supercritical region (Fig. 10)
(Pourmortazavi and Hajimirsadeghi, 2007; Reverchon and De Marco,
2006; Taylor, 1996). Supercritical fluid 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. P–T phase diagram for carbon dioxide, showing the supercritical region.
Table 4
Physical properties of a typical fluid in different states. Modified from Taylor (1996).
Density Viscosity Diffusion coefficient
(kg/m3) (μPa∙s) (mm²/s)
Gas 1 10 1–10
Supercritical fluid 100–1000 50–100 0.01–0.1
Liquid 1000 500–1000 0.001
The solvent power of a supercritical fluid 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 fluid can be tuned such that it interacts primarily with neutral
lipids (i.e. acylglycerols).
• Favorable mass transfer
As shown in Table 4, supercritical fluid displays physical properties
intermediate to a liquid and a gas (Taylor, 1996). These transitional
properties allow for rapid penetration of the fluid 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 fluid 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 fluid 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 facili-
tates a safe extraction due to its low toxicity, low flammability, and
lack of reactivity (Macias-Sanchez et al., 2007; Taylor, 1996). If the
microalgal cells are to be cultivated at a coal-fired power station,
the CO2 required for supercritical conversion can be conveniently
obtained from the scrubbed flue 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 specific
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
SCCO2–lipids complex. The complex, driven by concentration gradient,
diffuses across the static SCCO2 film and enters the bulk SCCO2 flow.
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 flow. As such, the frits serve as a filter to remove cell
debris. The SCCO2–lipids mixture, as well as some residual water (if
lipid extraction was performed on concentrate or disrupted concen-
trate), then leaves the extraction vessel to enter the collection vessel,
where a micrometering valve is used to rapidly depressurize the in-
coming fluid (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,
 R. Halim et al. / Biotechnology Advances 30 (2012) 709–732
Fig. 11. Schematic diagram of a laboratory-scale SCCO2 extraction system. The unit is used to extract lipids from microalgal biomass.
Modified 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 con-
tinuous (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 influence the performance of SCCO2
extraction of microalgal lipids include pressure, temperature, modifi-
er addition, and fluid flow rate (Pourmortazavi and Hajimirsadeghi,
2007). Table 5 summarizes the levels of operating variables and the
findings 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 flows on the surface of
the packed mixture. lipids, ○ SCCO2. Static SCCO2 film 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 film. Step 2: solubilization of the released lipids by
SCCO2. Step 3: formation of a SCCO2-lipids complex. Step 4: diffusion of the SCCO2-
lipid complex across the static SCCO2 film into the bulk SCCO2 flow.
723
The evolution of lipids during SCCO2 extraction on microalgal bio-
mass can be described by the following first-order kinetics equation
(Goto et al., 1993; Halim et al., 2011; Ozkal et al., 2005):
 
−kt
me ¼ ms;o 1−e ð3Þ
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 coefficient 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 ¼ f ðP; T; ½M; Q Þ ð4Þ
where P is the extraction pressure (bar), T is the extraction tempera-
ture (°C), [M] is the concentration of polar modifier (mol% of CO2), Q
is the SCCO2 flow 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 Nannochlorop-
sis 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 con-
trolled by the lipid concentration gradient between the microalgal
biomass and the SCCO2. In our previous study evaluating the feasibil-
ity of SCCO2 process to extract lipids from Chlorococcum sp. (Halim
et al., 2011), we found the first-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 fluid 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 compo-
nents. Temperature increase leads to two competing phenomena.
The decrease in fluid density lowers SCCO2 solvent power while a
simultaneous increase in lipid volatility enhances the lipid mass
transfer into the bulk SCCO2 flow (Cheung, 1999; Soares et al.,
724 R. Halim et al. / Biotechnology Advances 30 (2012) 709–732

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 defined as the experimental condition that produces the highest final total lipid yield.

Study Microalgal Extraction Extraction SCCO2 flow rate; Polar modifier; Results and optimum condition Final total lipid yield
species pressure temperature extraction quantity of at the optimum
or P (bar) or T (°C) duration (min) polar modifier condition (wt.% of dried
microalgal biomass)

Sajilata Spirulina 316, 350, 40 0.7 l/min; 26.4, Ethanol; 9.64, Total lipid yield increased with P. Optimum condition 8.6
et al., platensis 400, 450, 40, 60, 80, 94 11, 13, 15, was found at 400 bar, 60 min, and 13.7 ml ethanol.
2008 484 16.36 ml

Andrich Nannochloropsis 400, 550, 40, 55 0.17 kg/min; None At constant T, lipid extraction rate increased with P. 25.0
et al., sp. 700 360 At constant P, lipid extraction rate slightly increased
2005 with T.
Final total lipid yield was the same at any T and P.

Mendes Spirulina 100, 250, 50, 60 Not specified; ethanol; At constant T, total lipid yield increased with P. 3.1
et al., maxima 350 not specified 10 mol% of CO2 At constant P, total lipid yield decreased with T.
2003 At constant T and P, polar modifier addition significantly
increased total lipid yield.
Optimum condition was found at 350 bar, 60 °C with
ethanol addition (10 mol%).

Cheung, Hypnea 241, 310, 40, 50 1 l/min; 120 none At constant T, total lipid yield increased with P. 6.7
1999 charoides 379 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.

Mendes Chlorella 200, 350 40, 55 0.4 l/min; 500 none At constant T, total lipid yield increased with P. 13.0
et al., vulgaris At low P (200 bar), total lipid yield decreased with T.
1995 At high P (350 bar), total lipid yield increased with T.
Optimum condition was found at 350 bar and 55 °C.

2007; Taylor, 1996). Table 5 compiles findings 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 modifier, often referred to as
co-solvent or entrainer, enhances the fluid affinity 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 fluid viscosity and allowing the SCCO2 to rapidly
permeate through the cellular matrices (Pourmortazavi and
Hajimirsadeghi, 2007; Taylor, 1996). Common polar modifiers
include methanol, ethanol, toluene and methanol–water mixture.
Mendes et al. (2006) demonstrated that the addition of methanol to
SCCO2 significantly increased the extraction of γ-linolenic acid
(from 0.05 wt.% of dried microalgal biomass to 0.44 wt.%) from
Spirulina maxima.
The flow rate of SCCO2 through the extraction vessel affects lipid
extraction kinetics. Even though increasing SCCO2 flow rate enables
Fig. 13. Typical first-order extraction curves obtained for SCCO2 extraction of microal-
gal lipids. Microalgae: Nannochloropsis sp., all extractions were performed at a constant
temperature (40 °C). Pressure was optimized. ● 70 MPa, ■ 55 MPa, ♦ 40 MPa.
Modified from Andrich et al. (2005).
more effective contact between the extraction fluid and the lipids, it
often results in uneven fluid penetration and dead volumes within
the extraction vessel (Pourmortazavi and Hajimirsadeghi, 2007).
The density in which microalgal biomass is packed to form a fixed
bed within the extraction vessel plays an important role in influenc-
ing extraction efficiency (Pourmortazavi and Hajimirsadeghi, 2007).
In the case of extraction from dried microalgal powder, packing den-
sity 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
fluid 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 liquid–liquid 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 flow impedance and restrictor plugging. High installation costs of
the extraction pressure vessel as well as unfavorable energy require-
ments for the fluid 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.

Criteria Organic solvent extraction SCCO2 extraction

Neutral lipids selectivity ** ***

Selectivity is not easily tuned. When non-polar organic solvent is used, only limited SCCO2 tunable selectivity when combined with flexible polar modifier arrangement should enable
amount of neutral lipids can be extracted. When non-polar/polar organic solvent specific extraction of acylglycerols and minimize co-extraction of contaminants (polar lipids and

R. Halim et al. / Biotechnology Advances 30 (2012) 709–732

mixture is used, both neutral lipids and polar lipids are extracted. non-acylglycerol neutral lipids).

Total lipid yield ** ***

Due to its intermediate liquid–gas 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. It is highly energy-intensive as fluid compression and heating are needed to convert CO2 to supercritical
However, organic solvent needs to be removed from the lipids via energy-intensive state. However, crude lipids are free from extraction solvent and do not need to undergo an extraction
liquid–liquid separation method (such as distillation). 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.

Applicability to microalgal *** *

concentrate or wet feedstock
Lipid extraction can be applied to microalgal concentrate without additional High residual water content within the microalgal biomass results in flow impedance and restrictor
pre-treatment step and with minimal loss of efficiency. plugging.

Hazard and toxicity * ***

Toxic organic solvents are used.

Reactivity with lipids ** ***

Organic solvent is non-reactive with lipids. However, distillation carried out to SCCO2 is non-reactive with lipids.
remove the organic solvent from the lipids exposes the lipids to high
temperature and possible artifact formation.

725
726 R. Halim et al. / Biotechnology Advances 30 (2012) 709–732
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.
Modified 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 efficient 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 ex-
traction (total lipid yield = 0.032 g lipid/g dried microalgal biomass).
This outcome was expected since supercritical fluid 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 final total lipid yields
from Nannochloropsis sp., they measured a lower lipid mass transfer
coefficient 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 beneficial 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 flow properties (a high
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 fluid 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
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 efficiency 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 efficiency 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 cellu-
lar 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 classified
based on the manner in which they achieve microalgal cellular disin-
tegration: 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 microal-
gal 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 vio-
lent 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 homog-
enization pumps microalgal concentrate through narrow orifice 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
 R. Halim et al. / Biotechnology Advances 30 (2012) 709–732 727

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 classification of laboratory-scale cell disruption methods.

728 R. Halim et al. / Biotechnology Advances 30 (2012) 709–732

Botryococcus sp. extraction from three microalgal species (Botryococcus sp., Chlorella
30 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
20
beating, microwave, sonication, and osmotic shocks), microwave
obtained the highest final total lipid yield and appeared to be the
10 most efficient for all three microalgal strains. For Botryococcus sp.,
bead beating and microwave obtained the highest final total lipid
yields with respectively 0.281 and 0.286 g lipid/g dried microalgal
0 biomass, while sonication seemed to be the least efficient at 0.088 g
lipid/g dried microalgal biomass. For C. vulgaris, autoclave and micro-
(wt% of dried microalgal

Chlorella vulgaris
30 wave appeared to be the most efficient, whereas bead beating pro-
Final total lipid yield

duced a low final total lipid yield at 0.079 g lipid/g dried microalgal
biomass. With Scenedesmus sp., microwave was again found to
biomass)

20 show the highest extraction efficiency, while yields from the other
methods were similar. For all three microalgal species, prior disrup-
tion of the cells by any of the assessed method was found to improve
10
final total lipid yield during the lipid extraction step (refer to Fig. 17
and compare all of the methods with the control non-disruption).
0 The mechanism in which residual water in the microalgal biomass
affects lipid extraction is not well understood and warrants future in-
Scenedesmus sp. vestigation. One hypothesis speculates that the presence of residual
30
water in the microalgal biomass will adversely affect lipid extraction
efficiency. Water forms a barrier that prohibits effective lipid mass
20 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
10 postulates that the presence of residual water in the microalgal
biomass will improve lipid extraction efficiency. Water swells the
cells and facilitates better solvent access to the lipids. Drying of
0
g s k microalgal concentrate prior to lipid extraction is deemed unneces-
on ing tin ve on oc
upti lav ea wa icati sh sary and may hinder lipid mass transfer. Various microorganisms
isr toc d-
b
ic r o on oti
c
n-
d Au Be
a M S m (bacteria, yeasts, and viruses) have been successfully extracted in
No Os
their wet state (~ 90 wt.% water) using non-polar/polar organic
Fig. 17. Effect of prior cell disruption on total lipid yield of organic solvent extraction. solvent mixtures (Kates, 1986b; Medina et al., 1998).
Three microalgal species (Botryococcus sp., Chlorella vulgaris, and Scenedesmus sp.) With regards to SCCO2 extraction, abundance of residual water in
were investigated. Organic solvents: chloroform–methanol (1/1 v/v) mixture. the microalgal biomass results in a highly compacted particle bed
Modified from Lee et al. (2010).
within the extraction vessel. This often leads to flow impedance and
restrictor plugging (Pourmortazavi and Hajimirsadeghi, 2007;
(sonication, homogenization, high-pressure French press, bead beat- Schwartzberg, 1997).
ing or bead mill, and lyophilization), mechanical shearing with bead During their investigation of lipid extraction from Chlorococcum
mill obtained the highest final total lipid yield. In a different study sp., Halim et al. (2011) assessed the effect of residual water content
by Lee et al. (2010), the effect of prior cell disruption on lipid within the microalgal biomass on total lipid yield. As shown in
Fig. 18 (modified from Halim et al., 2011), the presence of residual
0.07 water in the microalgal biomass did not appear to substantially affect
(g lipid / g dried microalgal

0.06 total lipid yield. Hexane extraction of concentrate (final total lipid
Final total lipid yield

yield = 0.010 g lipid/g dried microalgal biomass) obtained a slightly

0.05 lower lipid recovery than its dry powder counterpart (final total
biomass)

0.04 lipid yield = 0.015 g lipid/g dried microalgal biomass), while

0.03
0.02
0.01
0 organic solvent extraction of wet biomass did not require any addi-
A B C1
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.
Modified from Halim et al. (2011).
hexane/isopropanol extraction of concentrate (final 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 (final total lipid yield = 0.068 g lipid/g dried microal-
gal biomass). These findings were encouraging particularly since the
C2 D1 D2 tional pre-treatment step.
Among the drying technologies that can be applied to microalgal
concentrate, freeze drying is preferred for its mild operating condi-
tions. 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 non-
uniform particulate size (Pourmortazavi and Hajimirsadeghi, 2007).
Once dried, microalgal biomass forms powder (or agglomeration)
that can be milled into different particulate size. Reducing the partic-
ulate size of microalgal powder prior to lipid extraction generally
enhances lipid recovery as it increases the interfacial surface area
 R. Halim et al. / Biotechnology Advances 30 (2012) 709–732 729

Fig. 19. Alternative process flow diagram showing the downstream processing steps needed with a simultaneous extraction and transesterification 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, fluid 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 verified that smaller
tomato skins resulted in higher oil recoveries.
6. Simultaneous extraction and transesterification 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
transesterification (Wahlen et al., 2011). This step, also known as di-
rect transesterification or in-situ transesterification, combines lipid
extraction and transesterification 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 transesterifies 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, re-
formed acid catalyst, un-transesterified lipids, and cell debris) under-
goes cell-debris removal and post-transesterification purification
730 R. Halim et al. / Biotechnology Advances 30 (2012) 709–732
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-transesterification purification consists of multiple steps. The re-
action mixture is first distilled to remove methanol. It is then left to
settle under gravity to induce biphasic partitioning (top biodiesel/
un-transesterified lipids phase and bottom glycerol phase). The
biodiesel/un-transesterified lipids phase is decanted off and washed
repeatedly with water to eliminate any acid catalyst. It is noted that
studies investigating direct transesterification of microalgal biomass
have, so far, only used acid catalysts (acetyl chloride and H2SO4).
Direct transesterification was originally derived by Lepage and
Roy (1984) as a rapid method to analyze the fatty acid contents of ad-
ipose tissues and milk. The method is highly desirable as it reduces
the amount of downstream manipulation required for biodiesel pro-
duction from any given biomass. Compared to the conventional
lipid-extraction-followed-by-transesterification approach, direct
transesterification has been shown to improve biodiesel yields of
various animal and plant tissues. However, the efficacy of direct
transesterification on microalgal biomass has not been sufficiently
investigated. Parameters that affect the performance of direct
transesterification 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 transesterifi-
cation. Chaetoceros gracilis biomass with residual water contents
ranging from 9 to 50% of microalgal paste weight was subjected to
direct transesterification. 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 transesterification of dried powder (biomass with residual
water content = 0%).
7. Microalgal biorefinery
The cost of producing microalgal biodiesel can theoretically be off-
set by revenues generated from other co-products of the microalgal
biomass. Microalgae contain significant quantities of proteins and
carbohydrates as well as smaller amounts of high-value functional
ingredients (astaxanthin, canthaxanthin, carotenes, chlorophylls, Ω3
free fatty acids, and γ-linolenic acid). Each of these cell components
can be appropriately utilized to co-generate a useable product in a
biorefinery. Recent studies have concluded that industrial-scale
production of microalgal biodiesel can only be made economically
sustainable if a biorefinery based production strategy is pursued
(Wijffels et al., 2010). In a biorefinery, the crude lipids are to be frac-
tionated into high-value functional ingredients and lipids for biodie-
sel (acylglycerols). Functional ingredients have been linked with the
promotion of anti-oxidant, anti-inflammatory, as well as anti-
carcinogenic 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 bioetha-
nol. As such, microalgal biorefinery will simultaneously produce bio-
diesel, high-value products, livestock feeds, and bioethanol.
Unfortunately, the combination of technologies needed to implement
a microalgal biorefinery 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 fluid fracturing, combined with pulsed
 R. Halim et al. / Biotechnology Advances 30 (2012) 709–732
electromagnetic fields and pH modification (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
clarifier for skimming, lipid fractionation, and transesterification to
biodiesel, while the cell debris settles to the bottom of gravity clarifier
for further processing as fuel and other valuable by-products. The
water (or growth medium) in the middle of the gravity clarifier 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 elec-
tromagnetic fields delivery and to assist in cell disruption, 2) Quan-
tum Fracturing™ creates fluid effect to mechanically stress
microalgal cells, 3) Pulsed electromagnetic fields deliver the force
that disrupt the microalgal cells.
OriginOil Single-Step Extraction™ has several benefits (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 produc-
tion of microalgal biodiesel are still in the early stages of develop-
ment. Lipid extraction from microalgal biomass has not received
sufficient attention and represents one of the many bottlenecks hin-
dering economic industrial-scale production of microalgal biodiesel.
Future research on microalgal biodiesel should focus on developing
an effective and energetically efficient 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 specific in order to minimize
the co-extraction of non-lipid contaminants (such as protein and
carbohydrates) but also selective towards acylglycerols in order to re-
duce downstream fractionation/purification (Fajardo et al., 2007;
Medina et al., 1998). Additionally, the selected technology should be
efficient (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 di-
lute 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 microal-
gal biomass/L culture) is energetically prohibitive. For this reason, it
will be economically beneficial if the selected lipid extraction tech-
nology can be directly applied to relatively wet feedstock, i.e. concen-
trate 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 extrac-
tion 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 microal-
gal lipid extraction. It is rapid, non-toxic, has high selectivity towards
acylglycerols, and produces solvent-free lipids. Its main disadvan-
tages are associated with the high capital cost and the high energy
requirement for supercritical fluid compression. Several studies
have shown that the disruption of microalgal cells prior to the lipid
extraction step enhances the extraction efficiency.
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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