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JBA-06676; No of Pages 15

Biotechnology Advances xxx (2013) xxxxxx

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Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Methods of downstream processing for the production of biodiesel


from microalgae
Jungmin Kim a, 1, Gursong Yoo a, 1, Hansol Lee a, 1, Juntaek Lim a, b, Kyochan Kim a, Chul Woong Kim a,
Min S. Park a, b, c,, Ji-Won Yang a, b,
a
b
c

Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701, Republic of Korea
Advanced Biomass R&D Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701, Republic of Korea
Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, 87545, USA

a r t i c l e
Available online xxxx
Keywords:
Biodiesel
Downstream process
Extraction
Harvest
Microalgae
Transesterication

i n f o

a b s t r a c t
Despite receiving increasing attention during the last few decades, the production of microalgal biofuels is
not yet sufciently cost-effective to compete with that of petroleum-based conventional fuels. Among the
steps required for the production of microalgal biofuels, the harvest of the microalgal biomass and the extraction of lipids from microalgae are two of the most expensive. In this review article, we surveyed a substantial
amount of previous work in microalgal harvesting and lipid extraction to highlight recent progress in these
areas. We also discuss new developments in the biodiesel conversion technology due to the importance of
the connectivity of this step with the lipid extraction process. Furthermore, we propose possible future directions for technological or process improvements that will directly affect the nal production costs of
microalgal biomass-based biofuels.
2013 Elsevier Inc. All rights reserved.

Contents
1.
2.

3.

Introduction . . . . . . . . . . . . . .
Harvest . . . . . . . . . . . . . . . . .
2.1.
Introduction . . . . . . . . . . .
2.2.
Harvest methods . . . . . . . . .
2.2.1.
Centrifugation . . . . . .
2.2.2.
Flocculation . . . . . . .
2.2.3.
Filtration . . . . . . . .
2.2.4.
Flotation . . . . . . . . .
2.2.5.
Magnetic separation . . .
2.2.6.
Electrolysis . . . . . . .
2.2.7.
Ultrasound . . . . . . .
2.2.8.
Immobilization . . . . . .
Extraction . . . . . . . . . . . . . . .
3.1.
Introduction . . . . . . . . . . .
3.2.
Cell disruption methods . . . . . .
3.2.1.
Mechanical methods . . .
3.2.2.
Chemical methods . . . .
3.2.3.
Biological methods . . . .
3.3.
Soxhlet extraction and its derivatives
3.4.
Direct transesterication . . . . .

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Correspondence to: M.S. Park, Advanced Biomass R&D Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701, Republic of Korea. Tel.: +82 42 350 5964; fax: +82 42 350
3910.
Correspondence to: J.-W. Yang, Advanced Biomass R&D Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701, Republic of Korea. Tel.: +82 42 350 3924; fax: +82 42
350 8858.
E-mail addresses: minsungpark0@kaist.ac.kr (M.S. Park), jwyang@kaist.ac.kr (J.-W. Yang).
1
Jungmin Kim, Gursong Yoo and Hansol Lee contributed equally to this work.
0734-9750/$ see front matter 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.biotechadv.2013.04.006

Please cite this article as: Kim J, et al, Methods of downstream processing for the production of biodiesel from microalgae, Biotechnology
Advances (2013), http://dx.doi.org/10.1016/j.biotechadv.2013.04.006

J. Kim et al. / Biotechnology Advances xxx (2013) xxxxxx

3.5.
Milking . . . . . . . . . . . . . . . . . . . . . . . .
3.6.
Concluding remarks . . . . . . . . . . . . . . . . . .
4.
Conversion: transesterication of microalgal lipid . . . . . . .
4.1.
Introduction . . . . . . . . . . . . . . . . . . . . .
4.2.
Catalytic transestercation . . . . . . . . . . . . . . .
4.2.1.
Homogeneous base, acid, and enzyme catalysts .
4.2.2.
Heterogeneous catalysts . . . . . . . . . . . .
4.3.
Non-catalytic transesterication . . . . . . . . . . . .
4.4.
In-situ transesterication . . . . . . . . . . . . . . .
4.4.1.
Mechanically catalyzed in-situ transesterication
4.4.2.
Chemically catalyzed in-situ transesterication .
4.5.
Concluding remarks . . . . . . . . . . . . . . . . . .
5.
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction
Recently, microalgae have received much attention as an attractive
biomass for the commercial production of advanced biofuels, including
biodiesel and aviation fuels. In addition to their potential as an alternative biomass for advanced biofuels and bioproducts (Fig. 1), microalgae
also contribute to the quality of the environment. These organisms can
x CO2 from the atmosphere and thus contribute to greenhouse gas
(GHG) reduction. Despite the various benets associated with the
production of biofuels using microalgae, an economic feasibility of the
microalgae-based biofuels industry comparable to that of either the petroleum or the bioethanol industry has not yet been achieved. One of the
main reasons for the high production cost of algal biofuels is the lack of a
highly economic process that integrates the multiple steps associated
with the harvest, extraction, and conversion of biomass to biodiesel.
Biodiesel production from microalgal biomass is a sequential process
that consists of the cultivation, harvest, oil extraction, and conversion of
algal lipids into advanced biofuels. With the exception of cultivation, the
downstream process contributes to 60% of the total biodiesel production
cost. Therefore, it is essential to reduce the total combined cost of
harvest, extraction, and conversion through a number of technical
breakthroughs. The cost for microalgal harvest is as high as 20% of the
total production cost of biodiesel, although it varies based on the type
of harvest technology used and the density of the microalgal culture

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(Mata et al., 2010). The oil extraction from dried biomass can be accomplished using various cell rupturing techniques, including autoclave,
ultrasound, homogenization, and bead milling. Treatments with organic
solvents, acids, alkalis, or enzymes can be used for the chemical or biological breakdown of the cell wall. Physical methods, such as freezing
and osmotic shock, have also been used for the oil extraction process.
The mechanical methods that have been developed for the extraction
of oil from microalgal biomass are not recommended due to the nature
of the thick microalgal cell wall (Lam and Lee, 2012b). To date, increasing the oil extraction efciency from algal biomass has been a
challenging task in the development of an economically viable biodiesel production process from microalgae. After the oil is extracted,
the biodiesel is produced through a transesterication reaction in methanol with an acidic or an alkaline catalyst. This process is also a challenging task due to the difculty in the recovery of the product and the
production of toxic chemicals.
There is a tremendous potential to improve the economics of
microalgal biofuels. Although the signicance of cultivation is acknowledged as the single component that contributes the most to
the total production cost of microalgae-based biofuels, this review
limits its discussions to the recent technical developments in the
harvest of microalgae, the extraction of algal lipids, and the conversion of lipids to biodiesel. Therefore, this article reviews the current
status and recent advances in the relevant technologies for the

Fig. 1. Downstream ow for various microalgal products.

Please cite this article as: Kim J, et al, Methods of downstream processing for the production of biodiesel from microalgae, Biotechnology
Advances (2013), http://dx.doi.org/10.1016/j.biotechadv.2013.04.006

J. Kim et al. / Biotechnology Advances xxx (2013) xxxxxx

downstream processes of biodiesel production from microalgal


biomass. In addition, this review provides perspectives on new directions for technological improvements that will enable the commercialization of microalgae-based biofuels and chemicals.
2. Harvest

(4) The effect of the chosen harvest technique on the subsequent


processes, such as lipid extraction and biodiesel conversion,
needs be minimized.
(5) More studies of marine microalgae harvesting techniques are
strongly encouraged to facilitate the development of harvest
technology in the future.

2.1. Introduction

2.2. Harvest methods

In addition to the economics aspect, the typically tiny size of a


microalgal cell (less than 10 m in diameter) in a diluted culture
medium (less than 2 g/L) and a density similar to that of water
make microalgal harvesting one of the key bottlenecks for the production of biodiesel from microalgae. Additionally, the negatively
charged surfaces of the microalgae prevent these organisms from easily settling by gravity. Unfortunately, the best way to harvest various
microalgal species has not yet been determined (Uduman et al.,
2010). Thus, a proper harvesting method that can improve both the
economics and the efciency of the process according to the desired
products and/or the biology of the microalgal species needs to be
developed.
Most microalgal harvest and recovery techniques have been
developed based on technologies that have been used in the water purication industry. Although there are technical similarities between
microalgal harvest and water purication, it is necessary to develop
approaches that can address the technical needs that are unique to
microalgal harvest. The following points should be considered when
designing an efcient harvesting strategy.

2.2.1. Centrifugation
Most microalgae can be recovered from dilute suspension through
centrifugal force. Both an improved harvest efciency and an increased
concentration of microalgal biomass are achieved within a short time
by centrifugation. Particularly for high-value products, such as food or
aquaculture applications, centrifugation is often recommended to recover high-quality algae without chemical and bacterial contamination
of the raw product (Mata et al., 2010). However, the intensive energy
input of centrifugation has a negative effect on the net energy and CO2
balances in microalgal biodiesel production (Beach et al., 2012; Sander
and Murthy, 2010). Recently, several newly designed centrifuges have
been used in microalgae harvesting for biodiesel production. Nevertheless, these centrifuges still require a high capital investment and high
operating costs compared to other approaches. As a result, recent research has suggested that the centrifugal energy consumption can be
saved by applying other pre-concentration methods prior to the centrifugation. Salim et al. (2012) used four different occulating microalgae
(Ankistrodesmus falcatus, Ettlia texensis, Neochloris oleoabundans, and
Tetraselmis suecica) to harvest non-occulating microalgae (Chlorella
vulgaris and Scenedesmus obliquus) before employing the centrifuge.
This bio-occulation/pre-concentration step greatly reduced the operational energy of centrifugation from 13.8 to 1.83 MJkgDW 1. Additionally, Bilad et al. (2012) used submerged microltration as a pre-harvest
technique and centrifugation as a post-concentration method. By
combining submerged ltration and centrifugation, the total harvest
energy of C. vulgaris and P. tricornutum decreased from 8 to 0.84 and
0.91 kWh/m 3, respectively. These low energy consumptions could be
achieved because only a small volume of medium (6.7%) remained
after the rst 15 times pre-concentration (93.3%).

(1) The choice of harvesting technique depends on the characteristics of the microalgae species and the type(s) of the desired
product(s).
(2) The combination of different harvest techniques can compensate
for the weaknesses of the individual techniques and often results
in a synergistic effect on the harvesting process (Table 1).
(3) It is necessary to develop a process that achieves complete cell
separation in a dilute suspension and efcient water and nutrient recycling after separation to ensure that the harvest process
contributes only a small cost to the total downstream process.

Table 1
The combination of different separation techniques.
Combination

Performance

Reference

Bio-occulation and
centrifugation

90% of centrifugal energy input reduced for


N. oleoabundans harvesting, using S. obliquus
of bio-occulation before centrifugation.
Pre-concentration by submerged ltration
saved both a large volume of broth (>90%)
and an energy consumption of centrifuge.
No retarded algal growth of Scenedesmus sp.
was observed in reused medium from both
bio-occulant and metallic salt.
Pre-concentration by polyaluminum chloride
(PAC) at 10 ppm effectively reduced the
moisture contents of ltered biomass,
resulting in 34% w/w of solid concentration.
Metallic coagulant enhanced the
performance of microotation generated
by uidic oscillator, resulting in 99.2% of
harvest efciency.
Neutralization caused by metallic coagulants
enhanced otation efciency of both cells and
algogenic organic matter (AOM).
When the PAC dose was 0.5 mg/l, 5 s of ultrasonic irradiation increased algae removal
efciency of M. aeruginosa from 35% to 67%.

Salim et al.
(2012)

Filtration and
centrifugation
Bio-occulation and
inorganic
occulation
Inorganic occulation
and ltration

Inorganic occulation
and otation

Inorganic occulation
and otation
Inorganic occulation
and ultrasound

Bilad et al.
(2012)
Kim et al.
(2011)
Lee et al.
(2012)

Hanotu et al.
(2012)

Henderson
et al. (2010)
Zhang et al.
(2009)

2.2.2. Flocculation
Various approaches have been used to occulate individual
microalgal cells to build algal ocs that are more suitable for separation.
Flocculation techniques can be used alone or can be applied as a
pre-concentration step prior to other harvesting methods.
Microalgal cells have a negatively charged surface that makes
the cells stable in a dilute solution. The negatively charged cells can
be neutralized and destabilized with positively charged coagulants,
such as polyvalent cations and cationic polymers. Several studies
have employed aluminum- and iron-based metal salts as occulants.
In metallic salt-induced occulation, however, a high dosage of costly
occulant and an acidic pH are required to achieve a satisfactory
result (Zhang and Hu, 2012). Additionally, cell lysis was induced
by the addition of aluminum salts (Papazi et al., 2010). Residual
metal salts after harvesting may negatively affect both the medium
recycling and the quality of the desired products (Estevez et al.,
2001; Mojaat et al., 2008; Perreault et al., 2010). In contrast, organic
polymer occulants, such as chitosan and grafted starch, exhibited a
more acceptable recovery of microalgae with both a lower dosage
and a reduced impact on the environment compared with metallic
salts (Banerjee et al., 2012; Beach et al., 2012). No growth inhibition
was observed when inorganic polyelectrolytes, which are commonly
used in wastewater treatment, were applied to harvest freshwater
microalgal species. Recently, as an alternative to conventional occulants, cationic metal-bound aminoclays were synthesized and successfully applied in microalgae harvesting (Farooq et al., 2013; Lee et al.,
2013). The authors of these previous studies used Mg- and Al-bound

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aminoclays for bulk harvesting and an Fe-bound aminoclay for an coating material on a membrane lter. Despite the satisfactory harvesting
performance and reusability of these materials, the raw material cost
should be further reduced before this approach becomes a viable option
for the harvest of microalgae. Regardless of the occulant types, the
effectiveness of the harvest signicantly decreases when these techniques are applied to marine microalgae due to the high ionic strength
of seawater (Bilanovic and Shelef, 1988; Sukenik et al., 1988). Therefore,
further modications and improvement are necessary for the use of this
alternative technique in the harvesting of marine microalgae.
Extracellular polymeric substances (EPSs) have emerged as an
environmentally friendly occulant in microalgal harvesting. An EPS
is dened as a bioocculant, such as polysaccharides, functional
proteins, and glycoproteins, synthesized by organisms, such as bacteria,
algae, fungi, and actinomyces (Abd-El-Haleem et al., 2008). According
to Zheng et al. (2012), poly (-glutamic acid) (-PGA) from Bacillus
subtilis was effective in harvesting both freshwater and marine
microalgae. Moreover, maintenance of the cell integrity and the
low material price of this occulant (approximately US$5/kg) are merits
of -PGA. A bioocculant from Paenibacillus polymyxa AM49 was
successfully combined with cationic chemicals for the harvest of
95% of Scenedesmus sp. (Kim et al., 2011). Additionally, the use of a
bioocculant enhanced the growth rate of microalgae in a recycled
medium, whereas the growth activity was inhibited when a cationic
salt was applied alone. A mixture of microbes, including Pseudomonas
stutzeri and Bacillus cereus, induced effective harvesting of the marine
microalgae Pleurochrysis carterae (CCMP647) (Lee et al., 2009). In this
study, inexpensive organic substrates, such as glycerol and acetate,
were used instead of glucose to grow the EPS-producing microbes.
Even in the absence of EPS, whole microbes can be used as occulating
agents to induce bioocculation (Salim et al., 2012). In a recent study,
occulating microalgae was used to harvest the oleaginous microalgae
C. vulgaris and S. obliquus to ultimately reduce the energy of centrifugation. By optimizing the concentration ratio of the occulating and oleaginous microalgae, the sedimentation rate and the recovery efciency
was increased.
Auto-occulation is the phenomenon of chemical occulation of
microalgal cells in the presence of calcium and magnesium ions at a
high pH (Vandamme et al., 2012). Lee et al. (1998b) compared the occulating activity of Botryococcus braunii with the activities of three different occulation methods at various cultivation times: auto-, inorganicand polymer-occulation. Of these methods, auto-occulation showed
the highest harvest efciency for a cultivation of up to three weeks.
Vandamme et al. (2012) investigated different methods to induce the
auto-occulation of the microalga Chlorella vulgaris. The use of calcium
hydroxide achieved a 50-fold increase in the concentration with both a
low cost ($18/ton of biomass) and a low environmental risk (>85% of
viability). Nevertheless, careful consideration is necessary in the selection of auto-occulation as an acceptable harvest method. For the efcient aggregation of microalgae, the presence of calcium, magnesium,
and phosphorus ions in the culture broth should be sufcient, i.e., ion
rich-seawater and wastewater might be the optimal medium conditions for auto-occulation. It is also necessary to consider the consumption of iron by the replacement of magnesium hydroxide during
auto-occulation because iron can enhance the biomass productivity
in cultivation that are conducted using a recycled medium (Kim et al.,
2011). The effects of both the base used for occulation and the acid
used for pH neutralization on the economic feasibility and the environmental impact of the process should be considered (Wu et al., 2012).
2.2.3. Filtration
Membrane ltration has been widely utilized in biotechnological
applications due to its high separation efciency, simple and continuous operation, and no requirement of chemicals in the process. For
microalgae-based biofuel production, membrane ltration can also
facilitate recycling of the culture medium used for the cultivation of

microalgae to retain the residual nutrients in the culture medium and


to remove the protozoans and viruses (Ahmad et al., 2012). In addition,
membrane ltration can simplify subsequent processes, e.g., extraction,
conversion, rening, and the use of the residual biomass, without the
use of coagulants (Zhang et al., 2010).
Nevertheless, the signicant reduction in the permeate ux
caused by membrane fouling is the critical constraint of membrane
ltration. During microalgae harvesting, membrane fouling is caused
by the attachment of algogenic organic matter (AOM) and the accumulation of the algal cake layer on the membrane surface (Ahmad
et al., 2012; Zhang et al., 2010).
Cross-ow ltration is widely used to decrease fouling with the
tangential ow and performs more efciently than does dead-end
ltration. In cross-ow ltration, backwashing and ventilation of the
algae medium can help control the fouling and recover ux (Chen
et al., 2012). Zhang et al. (2010) found that ultraltration concentrated
an algal culture by 150-fold (from 1 to 154.85 g/L) under conditions of
pulsated air scouring combined with backwashing. In addition, the
modication of the membrane module in cross-ow ltration is an
option to improve the harvest efciency and reduce the energy
consumption. An integrated system composed of a ceramic tubular
membrane and a hollow ber membrane accomplished 99% media recovery and concentrated the biomass from 1.5 to 150 g/L using a low
energy input (Bhave et al., 2012). Submerged microltration can be applied to microalgal harvest as the rst stage of the up-concentration
step because the shear induced by coarse air bubbles is expected to alleviate membrane fouling (Bilad et al., 2012).
Furthermore, dynamic ltration is an improvement for the microalgal
harvest method because this method uses the turbulence over the membrane lter to generate higher shear stress on the membrane surface
compared with cross-ow ltration. Dynamic microltration achieved
an approximately 3-fold higher plateau ux with a lower energy usage
compared with a ltration system with no rotation because the rotational
system increased the turbulence and shear stress (Rios et al., 2011). At
the same shear rate, rotation-based dynamic ltration obtained an almost
twofold higher ux compared with cross-ow ltration because the low
ow rate of dynamic ltration decreased the fouling induced by broken
algal cells and their AOM (Frappart et al., 2011). Additionally, despite
its high electricity requirement, dynamic ltration can reduce both the
expense associated with the equipment and membrane replacement
and the total cost compared with cross-ow ltration (Ros et al., 2012).
In addition to the progress that has been made to control membrane fouling and to produce a high ux, it is also necessary to develop
membranes with properties that can address the unique characteristics of various microalgal species to ultimately make membrane ltration an effective technology for microalgal harvest. Furthermore, the
development of a continuous harvest system that integrates cultivation and extraction processes will signicantly improves the effective
use of ltration-based harvest technology because the integrated
system should be able to harvest a large-scale microalgal culture and
the water can be recycled for further cultivation.
2.2.4. Flotation
Microalgal cells are captured by upward gas bubbles in otation, and
the microalgal biomass is then collected in the vacuole layer on top
of the suspension. Microalgal cells with a diameter from 1030 m to
500 m are preferred for effective otation. Due to the reduced surface
charges on microalgal cells, the pre-aggregation of various microalgal
species was shown to be effective to attain the mass required for effective otation (Hanotu et al., 2012; Henderson et al., 2010).
In general, the otation efciency is dependent on the size of the
created bubble: nanobubbles (b1 m), microbubbles (1999 m), and
ne bubbles (12 mm). A smaller bubble size will exhibit a longer
longevity and a larger carrying capacity due to the increased surface
area-to-volume ratio (Zimmerman et al., 2011). Hanotu et al. (2012)
emphasized that small bubbles, which rise slowly, can more easily

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attach to microalgal cells and more stably transport them to the top
surface of the suspension compared with large bubbles. In addition to
the bubble size, the surface characteristics, such as the hydrophobicity
and the charge of the microalgae, are also crucial factors that determine
the interaction between the cells and the bubbles. In an aqueous
solution, the opposing surface characteristics of the microalgal cells
(negatively charged hydrophilic) and the air bubbles (negatively
charged hydrophobic) can be modied to ensure a better contact. Compared with conventional aeration, the interaction between freshwater
microalgae, such as C. vulgaris and S. obliquus FSP-3, and bubbles was
enhanced by ozone otation even though the negative surface charge
of the algal cells became stronger by ozonation (Cheng et al., 2010;
Cheng et al., 2011). Ozonation effectively produced protein-like substances through cell lysis during otation. The released proteins were
suggested to be biopolymers that make the bubble surface more hydrophilic to ultimately obtain effective contact between the microalgal
cells and the bubbles; this nding is consistent with the result reported
by Henderson et al. (2010). Due to the ozone scavenging activity of
humic-like substances, the selection of microalgae species and a cultivation strategy that produces a lower quantity of humic acids are strongly
preferred to ensure ozone-induced otation performance. Garg et al.
(2012) demonstrated that the otation performance of the marine
microalgae Tetraselmis sp. M8 was improved with increased algal hydrophobicity, which was achieved by addition of the cationic surfactant
C14TAB. These researchers emphasized that the algal hydrophobicity
played a more crucial role in the otation of marine microalgae than
did ionic strength, which is generally regarded as a primary inhibition
factor in marine microalgae otation.
2.2.5. Magnetic separation
Magnetic microalgal harvest involves the use of both functionalized
magnetic particles and an external magnetic eld. Because both the
microalgal cells and the magnetic particles have negatively charged
surfaces in an aqueous medium, cationic polyelectrolytes are needed
as bridges between the magnetic particles on the algal cells (Toh et al.,
2012). Cationic binder-modied magnetic particles and microalgal
cells are incorporated through direct linking or electrostatic interactions. After the microalgal cells are linked with the magnetic particles,
the cells can be harvested with an external magnetic eld from the
aqueous solution.
There are two strategies that use cationic binders to encourage
the binding of magnetic particles on algal cells: attached-to and
immobilized-on (Lim et al., 2012). For the attached-to approach, the
surface of the microalgal cells are rst modied with cationic binders,
and then the magnetic particles are added. In contrast, the magnetic particles are functionalized with cationic binders in the immobilized-on
strategy. Lim et al. (2012) applied iron oxide nanoparticles (NPs) and
the cationic polyelectrolyte poly (diallyldimethylammonium chloride)
(PDDA) to the harvesting of the freshwater microalgae Chlorella sp. and
demonstrated that a higher removal efciency with a lower dosage of
NPs was achieved through the immobilized-on approach mainly due to
the better colloidal stability of the NPs. The separation performance
signicantly varied with the shape of the NPs (e.g., 87.1% with nanorod
and 9.9% with nanosphere at a concentration of NPs of 50 mg/L). The
attractive features of magnetic separation include the completion of the
algal harvest within a few minutes (one order of magnitude lower) and
the regeneration of magnetic particles without a decrease in the efciency (Xu et al., 2011a). However, the study noted that the procedures used
for the regeneration of the magnetic particles can vary for different
microalgae species. For instance, the magnetic nanoparticles incorporated with Chlorella ellipsoidea were simply dissolved by HCl, whereas HCl
was not suitable for the regeneration of the magnetic particles aggregated
with B. braunii due to cell leakage. The nding by Cerff et al. (2012) indicated that the magnetic separation of microalgae depends on the applied
pH and the composition of the culture medium. The elevation of the
pH and the presence of di- and trivalent ions, such as Ca2+, PO43-, and

Mg2+, enhanced the occulation between the microalgae and the


magnetic particles, which resulted in increased separation efciency.
2.2.6. Electrolysis
Electrolysis-based technologies have been widely adopted in the
water industry for the removal of various contaminants, including
microalgae. Particularly in water treatment, the advanced oxidation
process (AOP) that generates reactive oxygen species (ROS) is a crucial
process for the inactivation of microorganisms and the mineralization
of organic molecules, whereas AOP is not suitable for the recovery of
cells and the efcient reuse of culture medium in electrolysis-based
microalgal harvest. Therefore, the strategy used for the development
of electrochemical technologies for microalgae harvest should be different from that used in water industries.
When employing an electrolytic technology, polyvalent cations,
such as Al3+ and Fe2+/Fe3+, are dissolved from the sacricial anode
throughout the harvest period. These metal ions react with water
molecules to form metal hydroxides. Consequently, the positively
charged metal hydroxides bind to the negative surface of the microalgal
cells and destabilize the microalgal suspension through charge neutralization (electro-coagulation). Simultaneously, bubbles, such as O2 and
H2, from the anode and cathode, respectively, are continuously generated by water electrolysis. These bubbles can separate the algal ocs by
attaching to their surfaces and by enhancing the attachment between
the metal hydroxides and the algal cells (electro-otation). Compared
with conventional cationic metal salts, metal ions released from a sacricial anode offer several advantages, including high efciencies with a
low dose, a wide working pH range, and the absence of coupled anions
in the electrolytic harvest technology (Gao et al., 2010).
Particularly in marine species harvest, the energy requirement
associated with the electrolysis approach is advantageous compared
with other harvest techniques (Kim et al., 2012a; Poelman et al.,
1997). The electrical energy input of the electrolytic harvest approach
is 10-fold lower with marine microalgae than with freshwater species
(Vandamme et al., 2011). The high ionic strength of seawater can be
a major problem that blocks the effectiveness of other harvesting
methods. In contrast, the high conductivity induced by the high concentration of ions in seawater was proven to substantially reduce the
amount of electricity required to release metal ions and bubbles from
the electrodes. Nevertheless, careful approaches may be sensible in
the application of electrolytic recovery for marine species due to the
high concentration of chloride ions (approximately 19 g/L) in the
culture medium. Because the redox potentials for chlorine dioxide
(1.57 V) and chlorine (1.36 V) are not signicantly different from
that of O2 (1.23 V), it is likely that chlorine species with potent germicidal activity against microalgae are produced. Kim et al. (2012b) continuously harvested the marine microalgae Nannochloris oculata using
the electrolytic method and noted that the color of the harvested
biomass turned from green to white after 20 min of operation, likely
due to the bleaching activity of chlorine species. Additionally, the
residual chlorine species after the harvest can diminish the reusability
of the medium and the viability of the cells. It might be possible to
solve this problem through the neutralization of the chlorine species
by the addition of reducing agents. Jorquera et al. (2002) demonstrated that the inclusion of a reducing agent (sodium thiosulfate) allowed
better growth of the microalgae Isochrysis galbana in electrolytically
treated seawater. However, further study of the neutralization of
reactive chlorine species produced during electrolysis is needed to
make the use of the electrolytic technique more attractive in the
marine microalgae harvest sector.
2.2.7. Ultrasound
In the down-stream process of microalgal biodiesel production,
ultrasound can be used for both algal harvest and lipid extraction.
Ultrasound with a high frequency (on the order of MHz) and a low
amplitude enables cells to aggregate, whereas ultrasound with a low

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frequency (on the order of KHz) and a high amplitude induces cell
rupture (Bosma et al., 2003).
In an acoustic eld, the algal cells are moved and aggregated into
knots in which the cell experiences no shear stress. An acoustic wave
(resonance frequency of 2.1 MHz) was applied to the continuous separation of the freshwater microalgae Monodus subterraneus UTEX 151
(Bosma et al., 2003). Despite some merits, such as no cell damage
and a small footprint in a small-scale operation, ultrasound-induced
harvesting may be unsuccessful on an industrial scale due to the high
energy input and the low separation efciency. However, if used as an
assisting method, ultrasound might be applied for microalgal harvesting.
Zhang et al. (2009) combined ultrasound and polyaluminum chloride
(PAC) to harvest the freshwater microalgae Microcystis aeruginosa. The
destruction of the gas vacuoles inside cells by sonication resulted in a
loss of buoyancy and an increased ability to settle. A short application
of sonication (15 s) was sufcient to improve the occulation performance of algal cells. Additionally, the effect of ultrasound was greater
when the PAC dosage was lower.

2.2.8. Immobilization
There is no separate harvest step (usually subsequent to the cultivation step) in the immobilization approach. An entrapment matrix
in which algal cells are embedded and grow is employed at the beginning of the cultivation. Consequently, the beads where microalgae
grow into maturity are easily separated through simple sieving without a large energy input.
Lam and Lee (2012a) used alginate to immobilize the freshwater
microalgae C. vulgaris and indicated that alginate beads may be suitable
for simplifying the overall separation process. The co-immobilization of
microalgae and nutrients might be a solution for the low growth rate of
immobilized microalgae compared to the culture of free cells. Additionally, the co-immobilization of microalgae with plant-growth-promoting
bacteria may be another solution to enhance the microalgal growth
achieved through immobilization technology (Gonzalez and Bashan,
2000). As a possible entrapment matrix using lamentous fungi, pelletization was used to immobilize and grow the freshwater microalgae
C. vulgaris (Zhang and Hu, 2012). As a result, 63% and 24% of C. vulgaris
was harvested by the pelletization of Aspergillus niger under photoautotrophic and heterotrophic growth conditions, respectively. Importantly,
the study proposed that pelletization by oleaginous lamentous fungi

may contribute to the enhancement of the total oil yield and the fatty
acid quality in microalgal-based biodiesel production.

3. Extraction
3.1. Introduction
Lipid extraction, along with the dewatering of the biomass, is an
energy-intensive process in microalgal biodiesel production. The high
energy consumption is caused by a combination of various factors,
including the temperature and pressure conditions of the extraction
process, the distillation cost that is associated with separating lipids
from organic solvents or supercritical uids, and the cost of biomass
drying.
The major reason for high energy consumption is that microalgae
possess a cell wall, which is a thick and rigid layer composed of complex carbohydrates and glycoproteins with high mechanical strength
and chemical resistance.
Because of the energy consumption associated with the cell wall,
which is a cost-related problem, biodiesel production is not yet considered an economically feasible process. Therefore, it is necessary to develop an extraction process that does not require drying the microalgae.
As a result, there are many challenges that need to be overcome. One
of the largest challenges is the low extraction yield from wet biomass
due to the immiscibility of water in wet biomass with non-polar organic
solvents, which dissolve neutral lipids. Traditional lipid extraction
methods, such as those developed by Folch (Folch et al., 1957) and
Bligh & Dyer (Bligh and Dyer, 1959), use a co-solvent system, which is
a mixture of a non-polar solvent (chloroform) and a polar solvent
(methanol), to extract the lipids from dry biological material. When
these techniques are directly applied to a wet microalgal sample, the
microalgal cells tend to remain in the water phase due to their surface
charges, which prevents them from making direct contact with the
organic phase. This phenomenon, which is the main cause of the low
extraction yields obtained, also occurs when supercritical uids are
employed for lipid extraction (Halim et al., 2012). Fig. 2 summarizes
the cons and pros of dry and wet extractions of microalgal biomass.
We summarize various lipid extraction methods that combine cell
disruption techniques and suggest future research directions for the development of improved methods for lipid extraction from microalgae.

Fig. 2. Comparison of dry route and wet route of lipid extraction from microalgae.

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3.2. Cell disruption methods


The cell disruption methods can be categorized into three types:
mechanical, chemical, and biological methods. There are diverse
mechanical methods, including the use of microwave, ultrasonication,
bead beating, high pressure homogenization (HPH), and electroporation. Chemical methods comprise the use of chemical treatments and
osmotic shock. Biological methods mostly involve the use of enzymes
to degrade polysaccharides and/or proteins.
3.2.1. Mechanical methods
Mechanical methods directly break cells through physical force,
and their largest advantage is that these methods can be universally
applied to biomass regardless of its species. In addition, there is less
risk of degradation or degeneration of the target products during
cell disruption. Harrison (1991) provided various options for mechanical cell disruption, such as HPH, bead beating, and grinding
using mortar and pestle, but there are few methods that are applicable to wet biomass. For example, grinding or simple pressing cannot
be efciently utilized with algal paste or a dilute algal suspension
with a water content higher than 60%. In contrast, some methods,
such as ultrasonication, can be more effectively applied to wet biomass. This section of the review discusses mechanical cell disruption
methods based on various mechanisms.
3.2.1.1. Microwave. Microwave is an electromagnetic wave with a
frequency between 300 MHz and 300 GHz, which is lower than that
of infrared and higher than that of radio waves. However, only
small-range microwaves of approximately 2450 MHz are used in
microwave ovens, and this frequency is also used for cell disruption
because it can rotate the dipole of \OH bonds in water or alcohols.
Through this mechanism, the microwave can rapidly heat the biomass to cause damage to the cell envelopes and directly break the
weak hydrogen bonds in the cell envelopes. Microwave radiation is
advantageous due to its quick penetration into biomass, which results
in rapid cell disruption. For example, the efciency of supercritical
carbon dioxide extraction from lyophilized C. vulgaris was improved
2.6-fold after 6 min of microwave radiation (800 W) (Dejoye et al.,
2011). The effects of heating by microwave radiation and water
bath on a S. obliquus slurry were compared at two temperatures (80
and 95 C) (Balasubramanian et al., 2011). The results showed that
microwave radiation was signicantly preferable over the use of a
water bath due to the rapid heating, and pretreatment at 95 C
showed a two-fold improvement in lipid extraction compared to
pretreatment at 80 C when the slurry was extracted with hexane via
liquid-liquid extraction. In another study (Cheng et al., 2010), a dilute
biomass (5 g/L) of Botryococcus sp., C. vulgaris, and Scenedesmus sp.
was treated with microwave, autoclaving, bead beating, ultrasonication,
and osmotic shock and then subjected to 5 min of liquidliquid extraction using a mixture with an equivalent volume of chloroform and
methanol (1:1, v/v). The results demonstrated that microwave was
the most efcient method because it resulted in a 2- to 4-fold higher
extraction yield compared with the control (without cell disruption
treatment). A similar study conducted by Prabakaran and Ravindran
(2011) evaluated various cell disruption methods for Chlorella sp.,
Nostoc sp., Tolypothrix sp. and found that microwave and ultrasonication
exhibited the best performance. Notably, microwave yielded similar results with all of the species, whereas the other methods were inefcient
for certain species. Despite the strong advantages of microwave radiation, it also has disadvantages. It requires a vast cooling system due to
the high temperature and pressure used, and thermally labile products
can be degraded during the process. In addition, the large-scale use of
microwave radiation will consume a tremendous amount of electricity.
For example, a commercial large-scale microwave oven (Votsch
Hephaistos VHM 180/300) has a usable volume of 7000 L and consumes

electricity at a rate of 68 kW. Therefore, the energy consumption required for microwave cell disruption should be evaluated thoroughly.
3.2.1.2. Ultrasonication. Ultrasonication, which utilizes the cavitation
effect caused by ultrasound in a liquid, is also a well-known method
for the cell disruption of microorganisms. When ultrasound is radiated
to liquid media, small vacant regions, which are called microbubbles,
are momentarily formed as the liquid molecules are moved by the
acoustic waves. If ultrasound with a sufcient intensity is used, the
microbubbles are compressed to their minimum radii and implode,
thereby producing heat, light (sonoluminescence), free radicals, and
shockwaves, which can damage the cell envelopes of microorganisms
(Miller et al., 1996; Miller et al., 2002). Ultrasonic cavitation is affected
by the viscosity and the temperature of the liquid media, and a low temperature is favorable for effective sonolysis (Jiang et al., 2006). Therefore,
one should continuously cool the liquid media because the temperature
increases rapidly due to heat dissipation. In addition, ultrasonic cavitation is signicantly more intense at low frequency (1840 kHz) than
at high frequency (400800 kHz) (Cravotto et al., 2008). Some examples of the use of ultrasonication for the disruption of microalgal biomass
will now be discussed.
To enhance the fermentation yield, Yoo et al. (2012) applied ultrasound (40 kHz) to S. obliquus YSW15 biomass for up to 60 min. The
yield dramatically increased after 15 min of the pretreatment, and
denite destruction of the cell envelope was observed after 60 min
through energy-ltering transmission electron microscopy (EF-TEM)
and atomic force microscopy (AFM). Ultrasonication can also increase
the biogas fermentation yield of a dilute biomass (4 g/L) of Scenedesmus
sp. (Gonzlez-Fernndez et al., 2012). In this article, the authors suggested a good criterion for the energy consumption of ultrasonication:
ES supplied energy

Pt
V  TS0

In this equation, Es is the amount of ultrasonic energy consumed


per unit mass of biomass (dry weight), P is the ultrasonic power, t is
the treatment time, V is the volume, and TS0 is the concentration
of biomass. As a result, an approximately 2-fold increase in the
fermentation yield was observed at an Es of 100129 MJ/kg. Another
study (Adam et al., 2012) investigated solvent-free extraction
using ultrasonication. N. oculata with a water content of 7095%
was subjected to ultrasound (20 kHz) for up to 25 min, and the lipids
were separated in the absence of a solvent. Although the extraction
yield was only 0.21%, the process itself has some advantages because
it omits the evaporation of the extraction solvents used to separate
the lipids from the solvents. Researchers at Los Alamos National Laboratory (LANL) developed a separation method for the extraction of lipids,
cell residues, and culture media using ultrasounds with two different
frequencies and are optimizing the process variables, such as shapes
of ultrasonic probes (Marrone et al., 2011). Compared with other
methods, ultrasonication appears to be the best cell disruption method
for some algal species (Prabakaran and Ravindran, 2011). Ranjan et al.
(2010) conducted an in-depth study of ultrasonication utilizing simulations of microbubble sizes and microturbulence velocity depending on
different extraction solvents and found that ultrasonication could greatly increase the extraction yield from dry Scenedesmus sp. cells compared
with the methods developed by Soxhlet and Bligh and Dyer. Table 2
summarizes the results of some of the studies that investigated the
use of ultrasonication for lipid extraction. We attempted to compare
the results by calculating the Es, but the data from many of the articles
were insufcient. Therefore, we suggest that it would be benecial to
calculate the value of Es in further studies for the integration of various
research results. The main advantage of ultrasonication is the achievement of strong cell disruption based on the cavitation effect. However,
there are also disadvantages. The energy consumption is high due to
the high ultrasonic power and extensive cooling, and it is difcult to

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Table 2
Summary of various studies regarding ultrasonic cell disruption to wet biomass.
Reference

Prabakaran and
Ravindran (2011)

Lee et al. (2010)

Zheng et al. (2011)

Choi et al. (2011)

Miranda et al. (2012)

Gonzlez-Fernndez
et al. (2012)

Purpose
Species
Biomass concentration (g/L)
Working volume (mL)
Frequency (kHz)
Power (W)
Optimal treatment time
Performance a
Supplied energy (Es) (MJ/kg)

Lipid extraction
Chlorella sp.
5
100
50

15 min
**

Lipid extraction
C. vulgaris
5
100
10

5 min
*

Lipid extraction
C. vulgaris
2.5
50

17.1 min
***

Ethanol fermentation
S. obliquus YSW15

40

15 min
**

Ethanol fermentation
S. obliquus
100
5

200
150 s
**
60

Methanol fermentation
Scenedesmus sp.
4
250

1530 min
**
100129

a
The number of asterisks means the performance of ultrasonic cell disruption in corresponding reference. More asterisks mean higher performance. One asterisk means that
there was little improvement of the process yield of the authors purpose (e.g. lipid extraction) by ultrasonication, two asterisks mean the yield was improved by 23 times,
and three asterisks mean the yield was increased by approximately 5 times.

scale-up this process because cavitation only occurs in small regions


near ultrasonic probes.
3.2.1.3. Bead beating. Bead beating, also known as bead mill or ball mill,
is a very simple cell disruption technique that breaks cells by shaking a
closed container lled with the target cells and beads made of quartz or
metal. The cells are disrupted by collision or friction with the beads. This
is a common method used for extracting DNA from biological samples
(Robe et al., 2003). Bead beating can disrupt a cell within minutes,
and it can be applied to biomass in situ without any preparation. Compared with ultrasonication, HPH, and homogenization, bead beating
showed the highest extraction yield from wet pellets of B. braunii
UTEX 572. In fact, 28.6% (dry weight basis) of the lipids was extracted
using a mixture of chloroform and methanol (2:1, v/v), and this yield
was 1.96-fold higher than that obtained with the control (without any
cell disruption treatment) (Lee et al., 1998b). However, other studies
that compared bead beating to other cell disruption methods showed
that bead beating was not as efcient as the other approaches (Cheng
et al., 2010; Prabakaran and Ravindran, 2011; Sheng et al., 2012;
Zheng et al., 2011). There are various factors that affect the efciency
of bead beating, such as the container shape, the shaking rate, the
bead size, the amount of beads used, and the types of beads. In addition,
these factors will inuence not only the cell disruption efciency but
also the energy consumption. However, we did not nd a detailed discussion of these factors in the literature. The advantages of bead beating
are the simplicity of the equipment and the rapidness of the treatment,
but this method is disadvantageous because it is hard to scale-up and it
requires an extensive cooling system to prevent the thermal degradation of the target products.
3.2.1.4. High pressure homogenization. HPH, which is also known as
French press, was invented by Charles Stacy French. This cell disruption process utilizes hydraulic shear force generated when the slurry
under high pressure is sprayed through a narrow tube. This approach
has commonly been used for the extraction of the internal substances
of microorganisms and for sterilization. It has many advantages, such
as low heat formation, low risk of thermal degradation, low cooling
cost, no dead volume in the reactor, and easy scale-up. Various investigations found that HPH exhibited the highest cell disruption efciency among the various cell disruption methods. Sheng et al.
(2012) reported that HPH (2600 psi, mid-speed) was the best cell
disruption method for Synechocystis PCC 6803 biomass (20.6 g/L).
These researchers evaluated the cell disruption efciency by measuring the increase in the soluble chemical oxygen demand (SCOD)
during the cell disruption. Halim et al. (2012) compared the efciency
of HPH, ultrasonication, bead beating, and sulfuric acid treatment for
the disruption of the wet biomass of Chlorococcum sp. through cell
counting and measuring the colony diameters. The results showed
that HPH can destroy 70% of the total cells. When operated at
500800 bar and 13 mL/min, the number of ruptured cells increased

and saturated until the biomass was passed through the apparatus
four times. A higher efciency was observed with higher pressure
and cell concentration. In another investigation conducted by Zheng
et al. (2011), C. vulgaris culture was directly passed through HPH
(10,00020,000 psi, 400 mL/min) for lutein extraction. After the
treatment, the particle size of the solution decreased by 85%, whereas
the concentration of eluted lutein increased by only 1316%. However, the amount of lutein, which was accumulated by human intestinal Caco-2 cells, increased threefold, which means that the digestion
availability of lutein was signicantly enhanced. The stability of lutein
was also conrmed. This nding implies that HPH can rupture cells
while preserving thermally labile substances. Despite its many advantages, HPH requires a relatively long treatment time and consumes a
considerable amount of energy. Therefore, the improvement of the
HPH apparatus is required to shorten the treatment time and reduce
the energy consumption.
3.2.1.5. Electroporation. Electroporation is the disarrangement of molecules on the cell envelope with dipole moments by applying an electromagnetic eld (EF) to the biomass. This method has been used to
insert DNA into cells and to extract DNA from cells. The application
of an EF of suitable intensity to the target cells leads to the formation
of pores on the cell envelopes of the cells, and the pores are closed by
a healing process when the EF is removed. However, a much stronger
EF damages the cell envelopes beyond their healing ability and can
thus induce permanent cell disruption. Sheng et al. (2011) applied a
pulsed electric eld (PEF) to a Synechocystis PCC 6803 suspension
(0.3 g/L) and compared the cell disruption efciency obtained when
the same biomass was treated with heating. Almost every cell treated
with PEF was ruptured and stained with SYTOX green, whereas a
small number of cells were stained when the culture was treated with
heating. The authors of this article suggested a variable to represent
the intensity of the PEF, which can be calculated by the following
equation:
TI K

V 2 DfHRT
L2

In this equation, TI is the intensity of the PEF, V is the applied voltage,


D is the pulse width, f is the pulse frequency, is the sample conductivity, L is the distance between the electrodes, HRT is the residence time of
the liquid media, and K is a constant for unit conversion. The authors'
subsequent investigation compared PEF (TI = 36 kWh/m 3) to other
cell disruption methods (Sheng et al., 2012) and found that PEF
exhibited a similar performance to bead beating and microwave with
temperature control. Because temperature control (cooling) has a negative effect on cell disruption, it appears that EF and heating exhibit a
synergistic effect. Electroporation is receiving attention from industry.
OriginOil developed tabular and tubular equipment that can lyse cells
using EF (Eckelberry et al., 2011), and a patent led by NLP includes

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the electrolysis of microalgae for biodiesel production (Zheng et al.,


2011). Electroporation is promising because of its simplicity and high
energy efciency, which is due to the direct usage of electricity. However, more objective comparisons with other cell disruption methods
need to be performed.
3.2.2. Chemical methods
Microalgal cells can be disrupted by chemical means, such as treatments with acids, alkalis, and surfactants, which can degrade chemical
linkages on the cell envelope, or osmotic pressure, which induces the
pop-out of the cells. Its main advantage is its lower energy consumption compared with the mechanical methods because it does not
require a large amount of heat or electricity.
3.2.2.1. Chemical treatment. The permeability of cells can be increased
by diverse chemicals, such as polymyxin, lysine polymers, protamine,
polycationic peptides, and cationic detergents (Vaara, 1992). If
the permeability exceeds a certain limit, the cells will rupture. Acids
and alkalis induce hydrolysis of the cell envelope. The cell envelope
can also be weakened by heating, which can result in hydrolysis,
and proteins on the cell envelope can be denatured. The treatment
of dry S. obliquus with 2 N sulfuric acid increased the ethanol fermentation yield to 95.6% of the yield of obtained from control cells subjected
to harsh quantitative acid hydrolysis with 76% sulfuric acid (Miranda
et al., 2012). The performance of this method for the disruption of
wet biomass with a water content of 80% was 60% of that obtained in
the dry biomass experiment. Therefore, it appears that a relatively
benign acidity can adequately treat microalgal biomass. Sathish and
Sims (2012) utilized a step-wise extraction using acids and alkalis for
the disruption of wet biomass composed mainly of Chlorella sp. and
Scenedesmus sp. The cell envelopes were hydrolyzed by 1 M sulfuric
acid and 5 M sodium hydroxide at 90 C for 30 min each, and 0.5 M
sulfuric acid was then added to dissolve chlorophyll and precipitate
the free fatty acids. The precipitate was extracted by hexane, which
led to a recovery of 60% of the total lipids. Although it used a large number of steps that required centrifugation, this was an interesting investigation because it attempted to separate lipids and chlorophyll, which
is a by-product of conventional lipid extraction. Chemical treatment
was also applied to extract astaxanthin, which is an antioxidant supplement with a high economic value, from H. pluvialis (Sarada et al., 2006).
Among various chemicals, including acetone, methanol, dimethyl sulfoxide (DMSO), hydrochloric acid (HCl), and organic acids, 4 N HCl
was the best cell disruption chemical reagent, which led to the recovery
of 94% of the total astaxanthin from the cell body. The performance of
4 N HCl was much higher than that of DMSO (67%) and methanol
(19%). Despite the high cell-disruption performance of the chemical
treatments, chemical methods have some disadvantages. The chemicals
must be continuously consumed, and acids and alkalis can corrode the
surface of reactors. The neutralization of the acids and alkalis doubles
the cost, and the cost increases if the biomass is dilute. In addition, the
chemicals can react with the target products. Therefore, various synergistic approaches with mechanical methods should be investigated to
reduce the chemical usage.
3.2.2.2. Osmotic shock. Osmotic shock disrupts cells through a sudden increase or decrease in the salt concentration of the liquid media, which
disturbs the balance of osmotic pressure between the interior and the
exterior of the cells. There are two osmotic stresses that can damage
cells: hyper-osmotic stress and hypo-osmotic stress. When the salt concentration is higher in the exterior, the cells suffer hyper-osmotic stress.
As a result, the cells shrink as uids inside the cells diffuse outwards, and
damage is caused to the cell envelopes. In contrast, hypo-osmotic stress
occurs when the salt concentration is lower in the exterior. The water
ows into the cells to balance the osmotic pressure, and the cells swell
or burst if the stress is too high. Hypo-osmotic shock is a general procedure that is used for the extraction of substances from microorganisms.

However, the literature only includes studies that utilize hyper-osmotic


shock (Cheng et al., 2010; Prabakaran and Ravindran, 2011) because
hypo-osmotic shock requires a large amount of water for the dilution
of the liquid media, which makes the process unrealistic at the industrial
scale. We have shown that hyper-osmotic shock using sorbitol and sodium chloride (NaCl) increased the liquid-liquid extraction yield of lipids
from wildtype and cell wall-less mutant strains of Chlamydomonas
reinhardtii (Yoo et al., 2012). We optimized the growth phase of the
microalgae and the extraction solvents and increased the extraction
yield by twofold. Osmotic shock uses inexpensive materials and a simple
process. However, according to the literature, the performance of this
process is not as efcient as that obtained with other approaches, and it
results in a tremendous amount of wastewater with high salinity.
3.2.3. Biological methods
Biological methods refer to methods that degrade the cell envelope
using enzymes. Although there are other biological methods that use
phages or autolysis (Geciova et al., 2002; Harrison, 1991), most investigations of biological cell disruption utilize enzymes because enzymes
are the most commercially available and the most easily controlled biological materials. The advantages of enzymatic methods are the mild
reaction conditions and the high selectivity. An enzyme can selectively
degrade a specic chemical linkage, whereas mechanical methods
destroy almost every particle existing in the solution, and chemical
methods sometimes induce side-reactions of the target products. The
cell envelope of microalgae, such as Chlorella, has very resistant sporopollenin layers, but these can be degraded by a mixture of enzymes
(Braun and Aach, 1975). In this study, Braun and Aach incubated
Chlorella sp. with a mixture of cellulase, hemicellulase, and pectinase
for 90 h and found that 80% of the cells were converted into cells in
an osmotic labile state without rigid cell walls. Young et al. (2011)
tested six types of enzymes (papain, pectinase, snailase, neutrase,
alcalase, and cellulase) in the degradation of the cell wall of Mortierella
alpina for arachidonic acid extraction. As a single enzyme, neutrase
exhibited the best performance by increasing extraction yield by threefold. The analysis of various combinations of different enzymes showed
that the mixture of pectinase and papain was the best recipe because
these enzymes degrade carbohydrates and proteins, respectively. The
authors noted that the combination of different enzymes does not
always give better results because reaction inhibition can occur if
these if competitive absorption on substrates. Compared with mechanical methods, the enzymatic methods exhibited very competitive
results (Zheng et al., 2011). If the enzymes are chosen carefully, enzymatic cell disruption is effective. However, the critical downfall of this
method is the high cost of the enzymes. There are two ways to reduce
the cost of an enzymatic process: immobilization of the enzymes and
the combination of this process with other methods. Immobilized
enzymes can efciently degrade the cell envelopes of C. pyrenoidosa,
and they increased the lipid extraction yield by 75%. However, the enzyme activity was signicantly reduced when the enzyme was recycled.
After the enzyme was recycled 5 times, the relative hydrolysis yield
decreased to 40%, which indicates that the recyclability of immobilized
enzymes is a serious problem (Zhang et al., 2010). The combination
of the enzymatic method and the microwave approach appeared promising. Jin et al. (2012) applied a dialyzed plMAN5C solution (enzyme
mixture) and microwave to the wet biomass (water content of 92%)
of Rhodosporidium toruloides Y4 to enhance the lipid extraction. The
lipid yield was dramatically increased by the combination of enzyme
and microwave compared with the enzyme or microwave approach
alone. However, further research is required to reduce the cost and
the treatment time of this process.
3.3. Soxhlet extraction and its derivatives
Soxhlet extraction, which was invented by Franz Von Soxhlet in 1879,
is an improvement of the simple solidliquid extraction technique. In a

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10

J. Kim et al. / Biotechnology Advances xxx (2013) xxxxxx

Soxhlet extractor, the extraction solvent is evaporated, re-condensed, and


dropped into the sample container. Because the sample is in continuous
contact with fresh solvent with a limited amount of solvent through
recycling, this process can extract lipids with high efciency. These advantages make Soxhlet extraction a popular method for quantication
of lipids in biological samples, but its long extraction time and high energy consumption for evaporation are problematic. Furthermore, it is difcult to scale-up this process because of the complexity of the apparatus,
and it is nearly impossible to make this a continuous process (Halim
et al., 2012). Therefore, recent investigations have attempted to increase
the extraction rate by omitting the recycling of the solvent and increasing
the temperature and the pressure to a supercritical state to achieve better
mixing and a higher diffusion rate. Pressurized liquid extraction (PLE),
which is also known as accelerated solvent extraction (ASE), is a novel extraction method. In the apparatus, the solvent and the highly pressured
nitrogen gas are transported to the extraction cell, which is heated from
the outside to maintain a supercritical state. PLE shows superior performance over conventional methods. Cescut et al. (2011) compared PLE
to Soxhlet extraction and a modied Bligh and Dyer method and found
that PLE (using a mixture of chloroform and methanol) exhibited a
2-fold higher yield, a 5-fold higher extraction rate, and a 20-fold lower
solvent consumption compared with the other methods without sacricing the lipid quality. PLE can also be applied to wet biomass. Blue-green
microalgal biomass with a water content of 91% was extracted using a
method similar to that developed by Cescut et al. (2011), and 99.7% of
the lipids were extracted with 100 g of solvent (Kanda and Li, 2011).
Although this type of extraction exhibits a high extraction rate and a
high yield, its performance can seriously deteriorate when a dilute
biomass is used due to the inadequate contact between the cells and
the solvents; in this case, a dispersant, such as glass beads, is required,
which adds to the cost.
3.4. Direct transesterication
Direct transesterication (in-situ transesterication) is a combination of lipid extraction and biodiesel conversion. When an oleaginous
biomass, alcohol, and catalyst are mixed together and heated to a high
temperature, lipid extraction by alcohol and transesterication occur simultaneously, and biodiesel is produced directly. This can signicantly
reduce the energy consumption because this process does not require
the separation of lipids from the extraction solvents, such as organic
solvents and supercritical carbon dioxide. There are many studies that
have evaluated and optimized direct transesterication. According to
Patil et al. (2011), the microwave-assisted direct transesterication of
Nannochloropsis sp. dry biomass can yield FAME (biodiesel) at a yield
of up to 77% under optimum conditions. In their subsequent investigation (Ranjan et al., 2010), these researchers conducted an in-depth
optimization of the process variables, including the methanol dosage, the catalyst (potassium hydroxide, KOH) dosage, the reaction
time, and the microwave power dissipation, using response surface
methodology (RSM). The optimal conditions were the following:
biomass:methanol = 1:13 (w/w), KOH dosage = 2.5 wt.%, reaction
time = 810 min, and microwave power dissipation = 1,400 W.
Another study applied direct transesterication to wet biomass with a
water content of 90% and found that wet biomass can also be converted
to biodiesel if harsher conditions are applied (Patil et al., 2011). Wahlen
et al. (2011) tested the application of direct transesterication to dry
and wet biomass of various microalgal species (Chaetoceros gracilis,
Phaeodactylum tricornutum, Tetraselmis suecica, Neochloris oleoabundans,
Chlorella sorokiniana, Synechocystis sp., Synechococcus elongatus, and a
mixed culture from a municipal wastewater lagoon). The results revealed
that the FAME yield can range from 40% (S. elongatus) to 82% (C. gracilis)
depending on the species, which implies that an optimization should be
conducted for each species. In addition, these researchers applied direct
transesterication to wet biomass with various water contents and
found that only a small amount of water (50%) could severely reduce

the FAME yield. These results imply that a cell disruption pretreatment
should be utilized before the process. Moreover, the conversion of the
whole biomass at high temperature and pressure will cause an enormous
number of side reactions between the cell materials and the alcohol; thus,
the cost for the separation of the nal product (biodiesel) should be
considered if direct transesterication is utilized.
3.5. Milking
Milking is slightly different from other extraction methods. Normally, extraction from a microorganism is conducted by rst
harvesting the biomass, followed by cell disruption and substance recovery. Milking refers to the extraction of the target materials directly
from live cells without harvesting or killing the cells (Hejazi and
Wijffels, 2004). The simplest milking process involves a two-phase
reactor. In this system, the microorganism is cultivated in an aqueous
medium under an organic phase, which extracts the target product
excreted from the microorganism. The milking of carotenoids from
Dunaliella bardawil and Dunaliella salina was performed using
dodecane, and the highest recovery (5.3%) was observed when D. salina
was mixed by aeration (Kleinegris et al., 2010). The toxicity of the organic solvent is an important issue in two-phase cultivation, and it is
governed by a partition coefcient (logP). The log Poctanol value should
be higher than 5.5 to ensure cell growth because solvents with a low
log Poctanol are hydrophilic and dissolve into the aqueous phase, which
kills the microorganisms (Zhang et al., 2011). Therefore, a long-chain
organic solvent, such as dodecane, must be chosen for milking, which
is problematic due to the high cost of these solvents. Although milking
can completely omit the costs associated with harvesting and cell disruption, its extraction efciency is too low. Kleinegris et al. (2011)
claimed that the milking efciency can be improved by permeabilizing
or rupturing some of the cells and re-cultivating the others. Furthermore, as suggested by Wijffels and Barbosa (2010), efcient milking
might be possible if an ideal microalgal strain that excretes lipids,
such as B. braunii, and has other advantageous traits for mass cultivation
is developed.
3.6. Concluding remarks
This chapter covered various technologies for the separation of
lipids from microalgal biomass. However, an ideal method has not
yet been identied. There are three major problems associated with
lipid extraction:
1) No efcient cell disruption method has been developed for wet biomass. Researchers have tested diverse techniques and performed
optimizations, but their results are not economically feasible for
large-scale process. To disrupt the rigid cell envelope of microalgae,
a synergistic approach that combines different techniques might be
preferable to using a single method.
2) There is no reasonable way to compare the different methods that
have been developed. The energy consumption or material cost
should be considered when comparing the different methods,
but each investigation was performed under very different conditions, which makes it difcult to compare them to each other. For
example, the water content of wet biomass critically affects the
extraction or cell disruption efciency. Thus, the process variables,
such as the water content, should be standardized for biodiesel
production, which would help integrate the research results
obtained from various investigations.
3) The post-extraction processes were not considered. Various lipid
extraction techniques affect the conversion (transesterication)
or purication processes differently. Thus, the economic feasibility
of a lipid extraction method should be assessed as a whole, including the subsequent post-extraction processes. This approach is essential for the establishment of a biorenery based on microalgae.

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The downstream process for microalgal-based biodiesel production, including the extraction step, is receiving increasing attention.
It is necessary to reduce the downstream costs to ensure the economic feasibility of microalgal-based biodiesel production. By obtaining
additional biological knowledge of the target species and through
the integration of the harvesting, lipid extraction, and conversion processes, we will be able to reduce the cost and increase the efciency of
the entire process.
4. Conversion: transesterication of microalgal lipid
4.1. Introduction
After the extraction of lipids from microalgae, a conversion process
is necessary to produce biodiesel because the extracted microalgal
oil's viscosity is too high for the oil to be used directly as a fuel (Fuls
et al., 1984). If oils with high viscosity were used in engines, engines
would fail quickly due to the rapid accumulation of oil sludge. Therefore, to produce a sustainable fuel that offers smooth engine operation,
the viscosity of microalgal oil must be reduced. A common method
that reduces the viscosity of microalgal oil is the implementation of
the transesterication reaction, a chemical reaction that converts
microalgal oils (TAG) into their corresponding FAME, which is also
known as biodiesel (Bala, 2005). In the presence of catalysts and an alcohol in excess, the reaction is accelerated and pushes the equilibrium
toward the formation of the products FAME and glycerol. Acids, bases,
and enzymes are well-known catalysts that mediate transesterication.
One of the major drawbacks of the transesterication reaction is the
difculty of recovering products from toxic liquid catalysts, which can
adversely affect human health and the environment. Furthermore, the
transesterication process is a species-dependent reaction, specically,
an in-situ reaction using a co-solvent system, where extraction and
transesterication occur at the same time. Moreover, moisture and
free fatty acid content are critical factors that affect the production of
high-quality biodiesel.
Future transesterication strategies should be independent of
these elements to achieve the ultimate goal: economically feasible
biodiesel production. The following sections review the traditional
use of acids, bases, and enzymes for transesterication and discuss
their drawbacks; moreover, the potential of recent advances for the
production of microalgal biodiesel with improved efciency and
cost-effectiveness is evaluated.
4.2. Catalytic transestercation
4.2.1. Homogeneous base, acid, and enzyme catalysts
Base catalysts are commonly used for the transesterication reaction
because they are low-cost and allow for moderate reaction temperatures
and pressures to be used, which provides an economic advantage for the
commercial production of biodiesel with low capital and operational
costs. In addition, the fast reaction kinetics of transesterication allows
for the production of biodiesel in high yield and a relatively short time
compared with those achieved using other catalysts (Schuchardt et al.,
1998).
However, a high concentration of free fatty acids (FFAs) in feedstocks is one of the key factors that prevents the recovery of biodiesel
in high yields due to the apparent saponication that results from a direct reaction between the hydroxide groups in alkali catalysts and TAG
in microalgae (Vicente et al., 2004). It is reported that vegetable oil with
3% FFAs by weight can cause saponication, which reduces yield
(Ramadhas et al., 2005). Thus, it is recommended that base-catalyzed
transesterication be performed only with pure microalgal oil with a
low FFA concentration (b0.5 wt.%) (Freedman et al., 1984). Furthermore, it is difcult to develop a combined process for the separation
and purication of FAME from glycerol because it is challenging to
completely eliminate saponication, which increases the solubility of

11

glycerol and dissolves FAME back into the solvent phase. This additional
process makes homogenous basic catalyst-based transesterication
less attractive for large-scale industrialization (Lam and Lee, 2012b).
Additionally, the removal of the chemical waste that results from
the neutralization of base catalysts is another disadvantage of the
transesterication of microalgal oil using base catalysts.
The most commonly used acid catalyst is sulfuric acid, which is widely
used because it is inexpensive and highly catalytic. However, acid catalysts are not as commonly used as base catalysts for transesterication.
The slow reaction kinetics of acid catalysts is one of the most signicant
reasons for this disparity; acid catalysts have a reaction rate that is
4,000 times slower than that of base catalysts (Fukuda et al., 2001). However, acid catalysts can be used to process oils with high FFA concentrations because saponication does not occur when acid catalysts are
used for transesterication. Moreover, acid catalysts can simultaneously
esterify and transesterify TAG. Despite the advantages mentioned
above, acid-catalyzed transesterication is not popular in the commercial
biodiesel industry because it requires additional processes and costs. Unlike base-catalyzed reactions, acid-based reactions must occur at high
temperature and pressure and require a long reaction time due to their
low catalytic activity (Kawashima et al., 2009). Thus, reactions are usually
performed over 12 h at a higher temperature than that used in
base-catalyzed reactions, which results in greater energy consumption
and lower yields (Vyas et al., 2010). In addition, the longer reaction
time required at high temperature causes the rapid corrosion of
equipment, which thus must be replaced frequently. Acid-catalyzed
transesterication also suffers from the problem of chemical waste
generation associated with neutralization, which requires additional
equipment, chemicals, and time (Leung et al., 2010). Because of these
apparent disadvantages, acid-catalyzed transesterication is not popular in the biodiesel industry.
Because homogeneous acid and base catalysts exhibit undesirable
properties, such as saponication, chemical waste generation, and
high reaction temperature or pressure and the complex processes
and costs associated with them, researchers in the eld have been
devising new methods to support transesterication, such as enzymatic
catalysis. The enzyme-based transesterication platform is an attractive
alternative to the homogeneous acid- or base-based approaches described above. For example, lipase-based transesterication has been
used effectively because of its tolerance to FFA concentrations and
water, as well as its mild reaction conditions and moderate temperature
and pressure requirements. With no saponication occurring during
the process, there is no need for additional separation and purication
steps for products and waste. After the reaction, biodiesel is easily separated from glycerol. In addition, the ability to reuse the enzyme makes
the process efcient for the production of biodiesel in high yield per unit
production cost (Jegannathan et al., 2008).
Unfortunately, these attractive enzyme-based systems also face
several challenges that prevent them from being routinely used as
transesterication platforms. These difculties arise from the fact that
enzyme activity is inuenced by several factors that are associated
with the transesterication process itself, such as the pH of the reaction,
concentrations of substrates and enzymes, and interaction distances
between substrates and enzymes (Suali and Sarbatly, 2012). These conditions must be carefully studied and optimized to produce maximum
yields. Enzymes can be denatured and destabilized by excess methanol
and glycerol produced during transesterication. Moreover, enzymes
are notably expensive; therefore, it is difcult to use them on a commercial scale.
To avoid these problems, enzymes can be immobilized on a suitable
surface. There are several ways to immobilize lipase catalysts: adsorption,
entrapment, encapsulation, and cross-linking. Adsorption, also known as
the carrier-binding method, binds lipase to a carrier by weak forces, such
as dispersion forces (Jegannathan et al., 2008). This approach is the oldest
method, but it still widely used because it costs less than other methods
and can be performed under moderate conditions with easy recovery of

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J. Kim et al. / Biotechnology Advances xxx (2013) xxxxxx

the carrier. Other immobilization methods, such as entrapment, encapsulation, and cross-linking, have been used but are not highly effective due
to their intensive immobilization conditions (Tan et al., 2010).
4.2.2. Heterogeneous catalysts
To exploit the advantages of both acid and base catalysts, the further
development of heterogeneous catalysts seems inevitable for biodiesel
production. These catalysts have several advantages that are very attractive for industrialization. With the advantages of both acids and
bases, having the ability to simultaneously esterify and transesterify
lipids while being non-corrosive, heterogeneous catalysts are also environmentally friendly because they are recyclable and last longer than
homogeneous catalysts. In addition, the easy separation of catalysts
from products via simple ltration also provides an economic advantage (Lam and Lee, 2012b; Leung et al., 2010).
Researchers (Umdu et al., 2009) recently reported the use of
heterogeneous catalysts, CaO and MgO, supported on Al2O3 to enhance the density of basic sites and the basic strength of the catalysts,
which produced a conversion yield of 97.5% at 50 C. This nding provides evidence for the potential use of heterogeneous catalyst-based
transesterication platforms for biodiesel production at a reasonable
cost. Table 3 summarizes all of the catalytic transesterication methods
mentioned above.
4.3. Non-catalytic transesterication
In non-catalytic transesterication, methanol is employed at a critical temperature to extract and transesterify algal lipids simultaneously
in a single reaction. Combining the two processes saves time and
money, which makes supercritical methanol (SCM) attractive for industrial biodiesel production. When the SCM process is performed, wet biomass is typically used based on the hypothesis that watermethanol
mixtures exhibit both hydrophobic and hydrophilic characteristics at
high temperature, which helps to reduce the reaction time and product
separation (Akiya and Savage, 2002). The water in the wet biomass
also plays an important role as a solvent and reactant, which is the
same role played by methanol (Kusdiana and Saka, 2004). Although
transesterication with SCM has not been widely studied to date, a
recent report has shown that under optimum conditions, using SCM
with N. oculata (CCMP 1776) produces an 84.2% conversion yield at
250 C in 25 min with an algae-to-methanol ratio of 1:8 (wt./vol)
(Patil et al., 2012). Even with the attractive characteristics of obtaining
a reasonable yield in a relatively short reaction time, it still is a difcult
task to study or produce biodiesel using any supercritical uid (SCF)
method because the associated energy input requirements, the capital
cost of building a high-temperature, high-pressure chamber, and the
cost of monitoring the system are excessively high. Thus, industries do

not appear to be investing signicantly in research related to the SCF


method. Conversely, several studies have demonstrated the appeal of
the SCF method by comparing capital costs and production prots,
showing that the latter can outweigh the former (Patel et al., 2006).
4.4. In-situ transesterication
In-situ transesterication, often called direct transesterication, is a
process that is similar to the SCM, where the extraction and
transesterication processes occur simultaneously. This simpler process
provides an advantage over the conventional biodiesel production process, where extraction and conversion are separate. Thus, similarly to
SCM, in-situ transesterication has the advantages of the minimal use
of solvents, easy separation of products, and reduced reaction time.
However, the most important factor in in situ transesterication is the
state of the feedstock (whether it is wet or dry), which signicantly
affects the yield of biodiesel. The use of dry biomass produces a
better yield than that of wet biomass due to the higher percolation of
chemicals, which is inhibited by water. Table 4 summarizes the following in-situ transesterication methods.
4.4.1. Mechanically catalyzed in-situ transesterication
Mechanically catalyzed transesterication involves the use of mechanical processes rather than chemical reactions. Microwave radiation,
ultrasound radiation (sonication), and autoclaving are examples of mechanical catalysis, which improve reaction parameters such as reaction
time and temperature. These mechanical processes can also be used
in the extraction process, as mentioned earlier. However, the yields
achieved by these processes are not as high as those achieved by solvent
extraction. Conversely, mechanical forces can increase the yield obtained
during transesterication. Though mechanical catalysts do not directly
facilitate the transesterication reaction, they improve the nal yield by
increasing the penetration of solvents into cells for improved lipid extraction. With mechanical agitation, such chemicals as sodium hydroxide
or sulfuric acid can transesterify more lipids, leading to high yields.
Patil et al. (2012) recently reported data regarding microwave-assisted
in-situ transesterication with dried Nannochloropsis. The reported
conversion yield was 80.1% with an algae-to-methanol ratio of 1:12
(wt./vol), a KOH concentration of 2% by weight, and a reaction time of
45 min at 6064 C. The energy consumption was lower than that for
SCM, although the energy consumed during the drying process was not
taken into account. If the energy used in the drying process had been
calculated, the amount of energy consumed during microwave-assisted
in situ transesterication may have been higher than that consumed
by SCM. Another recently reported mechanically catalyzed in-situ
transesterication approach involved the use of ultrasound radiation.
Ehimen et al. (2012) improved the in-situ transesterication of Chlorella

Table 3
Different methods for catalytic transesterication.
Catalytic transesterication

Specication

Remark

Reference

Homogeneous base

High reaction rate


Low cost
Moderate reaction condition
Not FFA dependent
Simultaneous esterication and
transesterication

FFA and water dependent (saponication)


Waste management

Schuchardt et al. (1998),


Ramadhas et al. (2005)

Low reaction rate


Corrosion
High reaction temperature
Waste management

Vicente et al. (2004),


Fukuda et al. (2001),
Kawashima et al. (2009),
Vyas et al. (2010),
Leung et al. (2010)
Lam and Lee (2012b),
Leung et al. (2010),
Umdu et al. (2009)
Jegannathan et al. (2008),
Suali and Sarbatly (2012)

Homogeneous acid

Heterogeneous catalyst

Homogeneous enzymes

Simultaneous esterication and


transesterication (acid)
Less waste management
Not FFA dependent
Less waste management
(Easy product separation)

FFA and water dependent, high reaction


temperature (base)
High cost
pH control
Denaturation (alcohol dependent)
Immobilization
High cost

Please cite this article as: Kim J, et al, Methods of downstream processing for the production of biodiesel from microalgae, Biotechnology
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J. Kim et al. / Biotechnology Advances xxx (2013) xxxxxx

13

Table 4
Summary of in-situ transesterication.
In-situ transesterication

Specication

Remark

Reference

Mechanical assistance

High conversion yield


Moderate reaction condition

Patil et al. (2012),


Ehimen et al. (2012)

Co-solvent system

Can be environmental friendly

Ionic liquid assistance

Not FFA dependent


Easy optimization for specic reaction
Negligible vapor pressure
Recyclable

Water dependent
High energy consumption due to
prerequisite drying process
Water dependent
Species dependent
Low conversion yield
High cost
Aquatic toxicity
Water dependent

sp. by sonication (24 kHz). The conversion yield was in the range
of 9196% with a reaction time of 20 min to 2 h. The molar ratio of
algae to methanol in ultrasound-assisted reactions is much higher
(1:1051:315) but still lower than that used in microwave-assisted
transesterication when converted to a wt./vol ratio (1:1.31:4). However, the reaction time of ultrasound-agitated transesterication was
reported to be longer (02 h), thought it delivered a higher yield at a
similar temperature (60 C).
4.4.2. Chemically catalyzed in-situ transesterication
Chemically catalyzed in situ transesterication involves the use of
chemicals only. No mechanical process is used during the reaction. Drying the biomass feedstock is preferred in chemically catalyzed in situ
transesterication reactions. It is reported that feedstock containing
more than 31.7% water may likely inhibit in situ transesterication
(Ehimen et al., 2010). Two explanations for this behavior were recently
proposed (Lam and Lee, 2012b): inhibition occurs due to hydrolysis
during transesterication or water could react with TAG to form diglyceride and FFA, leading to the esterication instead of transesterication
of FFA and producing no FAME.
4.4.2.1. Chemically catalyzed in-situ transesterication via a co-solvent
system. In chemically catalyzed in-situ transesterication, a co-solvent
system is used to maximize the yield of FAME by improving the
efciency of lipid extraction. The co-solvent system uses a mixture of
two different organic solvents, one of which is typically ethanol. The
solvent must be miscible with methanol, insoluble in water, and environmentally friendly while being chemically inert such that there few
side reactions (Xu et al., 2011b). Because the co-solvent serves as a
lipid extractor, transesterication at extraction sites must be performed
by a concentrated base or acid in the absence of water. However,
though the co-solvent system offers the various advantages described
above, it must be further developed and optimized for individual
microalgal strains. For examples, (Xu et al., 2011b) used a toluene/
toluene system for Spirulina, but the biodiesel yields obtained differed
between Cladofora and B. braunii when the species were subjected to
the same co-solvent system (dichloromethane/methanol) (Lee et al.,
1998a).

Xu and Mi (2011),
Lee et al. (1998a)
Kim et al. (2012c),
Young et al. (2010)

acidic catalysts also provides the advantages of easy separation and the
ability to recycle catalysts without using additional chemicals. By
exploiting these advantages, researchers are now trying to produce
biodiesel with ILs via in-situ transesterication. (Young et al., 2011)
performed in-situ transesterication with 1-ethyl-3-methylimidazolium
derivatives, methanol, and acetyl chloride. The conversion yield was notably high (85100%), but the molar ratio of alcohol to TAG (1000:1
3000:1) also was high. The only drawback of using ILs is high cost; they
are usually as expensive as lipase enzymes, although the prices vary
among different ILs. However, it is still debatable whether the use of
ILs would produce any prot in the future because no comprehensive
life cycle assessment (LCA) of microalgal biodiesel production has
been made using the costs associated with the IL-based in-situ
transesterication of wet microalgal biomass.
4.5. Concluding remarks
It is still too early to conclude what conversion method will best
achieve the ultimate goal of economic feasibility. Every method possesses advantages and disadvantages. Homogeneous catalysts are inexpensive chemicals, but they produce abundant toxic waste at the end of
reactions due to the neutralization of the catalyst and separation of the
products. Heterogeneous catalysts do not produce notable amounts of
waste, but their costs are too high to use in a large-scale operation. Similar problems also arise with the use of enzymes, which have the additional problems of sensitive storage and reaction conditions. Because
of these cost issues, in-situ transesterication was developed to minimize production costs by combining extraction and conversion into
a single-step process. These in situ reactions can be mechanically or
chemically assisted to maximize biodiesel yield but only with dried
feedstock. Because these reactions require dried feedstock, the cost advantage of combining the extraction and conversion processes is lost to
the cost of supplying the energy required for the drying process. Several
researchers assert that the use of ILs could reduce production costs.
However, it is uncertain whether or not ILs are the answer for future
biodiesel production. Further research in this area must be performed
to determine the best conversion process for future generations.
5. Conclusions

4.4.2.2. Chemically catalyzed in-situ transesterication via ionic liquids.


In recent years, ionic liquids (ILs), salts in liquid form, have been
found to be useful in biodiesel production by degrading the lignin
from lignocellulosic biomass. ILs have many advantages with respect
to in-situ transesterication for biodiesel production. ILs have high
solubility, negligible vapor pressure, and inherent basicity or acidity.
Moreover, ILs are non-volatile, thermally stable, and recyclable after
transesterication reactions and can immobilize non-ionic liquid
catalysts. Their negligible vapor pressure and non-volatility suggest
that ILs can be used in reactions at ambient pressure, which helps to
reduce capital costs. The recyclability of ILs reduces the amount of
waste generated after reactions take place, which helps to minimize
environmental issues. The ability of ILs to immobilize both basic and

Though biofuels produced from microalgae biomass has been


touted as one of the best alternatives to petroleum-based transportation fuels, there have been limited technological breakthroughs to
make the commercialization of microalgal biodiesel a reality. In addition to cultivation, the costs associated with algal harvest and lipid
extraction are high and require further technical innovations. Therefore,
it is essential that new strategies be developed to effectively incorporate
the positive elements of the existing approaches reviewed in this paper
and to develop new approaches that can signicantly reduce costs
while achieving positive energy gains and net negative CO2 production.
These new approaches must consider the following aspects, although
not necessarily at the same time: 1) the approaches must be algal-

Please cite this article as: Kim J, et al, Methods of downstream processing for the production of biodiesel from microalgae, Biotechnology
Advances (2013), http://dx.doi.org/10.1016/j.biotechadv.2013.04.006

14

J. Kim et al. / Biotechnology Advances xxx (2013) xxxxxx

species agnostic, meaning that new approaches can be applicable to


any microalgal species without signicant differences in effectiveness;
2) the technologies must have a strong potential to be integrated into
existing upstream and downstream steps such that a continuous
process can be developed for biocrude or biodiesel production; and
3) the technologies must be scalable to meet the challenge of commercial biocrude production. These goals will be met when researchers
begin to apply knowledge from other elds, including cellulosic biofuels
for pretreatment, nanomaterials or scaffolds for the development of
new catalysts for deoxygenation, and synthetic biology for improved
autoocculation, easy cell wall dissolution, and designer lipid composition. The direct conversion of wet microalgal biomass to biocrudes,
which can be transformed into a variety of transportation fuels, may
be one of the best solutions to be achieved in the near future.
Regardless of what set of technological approaches are used for
harvest, extraction, and conversion, these solutions must be environmentally sustainable while being sufciently inexpensive to make
microalgal biofuels competitive with petroleum-based transportation
fuels.
Acknowledgments
This work was supported by the Advanced Biomass R&D Center
(ABC) of Global Frontier Project funded by the Ministry of Education,
Science and Technology (ABC-2011-K000908). This work was also supported by the National Energy Technology Innovation of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) grant
funded by the Korea government Ministry of Knowledge Economy
(No. 2012 T100201665). MSP was supported by the Brain Pool Program
funded by the Korean Federation of Science and Technology Societies
Grant by Korea Government (MEST, Basic Research Promotion Fund).
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Please cite this article as: Kim J, et al, Methods of downstream processing for the production of biodiesel from microalgae, Biotechnology
Advances (2013), http://dx.doi.org/10.1016/j.biotechadv.2013.04.006