-
Methods of downstream processing for the production of biodiesel
frommicroalgae
Jungmin Kim, Gursong Yoo, Hansol Lee, Juntaek Lim, Kyochan
Kim,Chul Woong Kim, Min S. Park, Ji-Won Yang
PII: S0734-9750(13)00077-3DOI: doi:
10.1016/j.biotechadv.2013.04.006Reference: JBA 6676
To appear in: Biotechnology Advances
Received date: 7 November 2012Revised date: 13 April
2013Accepted date: 18 April 2013
Please cite this article as: Kim Jungmin, Yoo Gursong, Lee
Hansol, Lim Juntaek, KimKyochan, Kim Chul Woong, Park Min S., Yang
Ji-Won, Methods of downstream pro-cessing for the production of
biodiesel from microalgae, Biotechnology Advances (2013),doi:
10.1016/j.biotechadv.2013.04.006
This is a PDF le of an unedited manuscript that has been
accepted for publication.As a service to our customers we are
providing this early version of the manuscript.The manuscript will
undergo copyediting, typesetting, and review of the resulting
proofbefore it is published in its nal form. Please note that
during the production processerrors may be discovered which could
aect the content, and all legal disclaimers thatapply to the
journal pertain.
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
Methods of downstream processing for the production of biodiesel
from microalgae
Jungmin Kim1#, Gursong Yoo1#, Hansol Lee1#, Juntaek Lim1,
Kyochan Kim1, Chul Woong
Kim1, Min S. Park1,2,3*, and Ji-Won Yang1,2*
1Department of Chemical and Biomolecular Engineering, KAIST, 291
Daehak-ro, Yuseong-
gu, Daejeon 305-701, Republic of Korea
2Advanced Biomass R&D Center, KAIST, 291 Daehak-ro,
Yuseong-gu, Daejeon 305-701,
Republic of Korea
3Bioscience Division, Los Alamos National Laboratory, Los
Alamos, New Mexico, 87545,
USA
*Corresponding authors.
Tel.: +82 42 350 3924; fax: +82 42 350 8858.
E-mail address: [email protected] (J.-W. Yang)
Tel.: +82 42 350 5964; fax: +82 42 350 3910.
E-mail address: [email protected] (M. S. Park)
Author Contributions
#Jungmin Kim, Gursong Yoo and Hansol Lee contributed equally to
this work.
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
Abstract
Despite receiving increasing attention during the last few
decades, the production of
microalgal biofuels is not yet sufficiently 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 final production costs of microalgal biomass-based
biofuels.
Keywords
Biodiesel, downstream process, extraction, harvest, microalgae,
transesterification
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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
(Figure 1), microalgae also contribute to the quality of the
environment. These organisms can
fix CO2 from the atmosphere and thus contribute to greenhouse
gas (GHG) reduction. Despite
the various benefits 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
(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
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
nature of the thick microalgal cell wall (Lam and Lee, 2012b).
To date, increasing the oil
extraction efficiency 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 transesterification reaction
in methanol with an acidic or
an alkaline catalyst. This process is also a challenging task
due to the difficulty 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 significance 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
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
2.1. Introduction
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 efficiency of the
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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 purification
industry. Although there are
technical similarities between microalgal harvest and water
purification, 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
efficient harvesting strategy.
(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 efficient water and nutrient recycling after
separation to ensure that
the harvest process contributes only a small cost to the total
downstream process.
(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.
2.2. Harvest methods
2.2.1. Centrifugation
Most microalgae can be recovered from dilute suspension through
centrifugal force. Both
an improved harvest efficiency and an increased concentration of
microalgal biomass are
achieved within a short time by centrifugation. Particularly for
high-value products, such as
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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 flocculating microalgae (Ankistrodesmus falcatus,
Ettlia texensis, Neochloris
oleoabundans, and Tetraselmis suecica) to harvest
non-flocculating microalgae (Chlorella
vulgaris and Scenedesmus obliquus) before employing the
centrifuge. This bio-
flocculation/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
microfiltration as a pre-harvest technique and centrifugation as
a post-concentration method.
By combining submerged filtration and centrifugation, the total
harvest energy of C. vulgaris
and P. tricornutum decreased from 8 to 0.84 and 0.91 kWh/m3,
respectively. These low
energy consumptions could be achieved because only a small
volume of medium (6.7%)
remained after the pre-concentration step was repeated 15 times
(93.3%).
2.2.2. Flocculation
Various approaches have been used to flocculate individual
microalgal cells to build
algal flocs 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.
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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 flocculants. In
metallic salt-induced
flocculation, however, a high dosage of costly flocculant 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
flocculants, 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 flocculants, 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
aminoclays for bulk harvesting and
an Fe-bound aminoclay for an coating material on a membrane
filter. 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 flocculant types, the effectiveness of the
harvest significantly 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 modifications
and improvement are necessary for the use of this alternative
technique in the harvesting of
marine microalgae.
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
Extracellular polymeric substances (EPSs) have emerged as an
environmentally friendly
flocculant in microalgal harvesting. An EPS is defined as a
bioflocculant, 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 flocculant (approximately US$5/kg)
are merits of -PGA. A
bioflocculant from Paenibacillus polymyxa AM49 was successfully
combined with cationic
chemicals for the harvest of 95% of Scenedesmus sp. (Kim et al.,
2011). Additionally, the
use of a bioflocculant enhanced the growth rate of microalgae in
a recycled medium, whereas
the growth activity was inhibited when a cationic salt was
applied alone. A mixture of
microbes, including Pseudomonas stutzeri and Bacillus cereus,
induced effective harvesting
of the marine microalgae Pleurochrysis carterae (CCMP647) (Lee
et al., 2009). In this study,
inexpensive organic substrates, such as glycerol and acetate,
were used instead of glucose to
grow the EPS-producing microbes. Even in the absence of EPS,
whole microbes can be used
as flocculating agents to induce bioflocculation (Salim et al.,
2012). In a recent study,
flocculating microalgae was used to harvest the oleaginous
microalgae Chlorella vulgaris and
Scenedesmus obliquus to ultimately reduce the energy of
centrifugation. By optimizing the
concentration ratio of the flocculating and oleaginous
microalgae, the sedimentation rate and
the recovery efficiency was increased.
Auto-flocculation is the phenomenon of chemical flocculation of
microalgal cells in the
presence of calcium and magnesium ions at a high pH (Vandamme et
al., 2012). Lee et al.
(1998b) compared the flocculating activity of Botryococcus
braunii with the activities of
three different flocculation methods at various cultivation
times: auto-, inorganic- and
polymer-flocculation. Of these methods, auto-flocculation showed
the highest harvest
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
efficiency for a cultivation of up to three weeks. Vandamme et
al. (2012) investigated
different methods to induce the auto-flocculation of the
microalga Chlorella vulgaris. The
use of calcium hydroxide achieved a 50-fold increase in the
concentration with both a low
cost (18$/ton of biomass) and a low environmental risk (>85%
of viability). Nevertheless,
careful consideration is necessary in the selection of
auto-flocculation as an acceptable
harvest method. For the efficient aggregation of microalgae, the
presence of calcium,
magnesium, and phosphorus ions in the culture broth should be
sufficient, i.e., ion rich-
seawater and wastewater might be the optimal medium conditions
for auto-flocculation. It is
also necessary to consider the consumption of iron by the
replacement of magnesium
hydroxide during auto-flocculation 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 flocculation 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 filtration has been widely utilized in biotechnological
applications due to its
high separation efficiency, simple and continuous operation, and
need of chemicals required
in the process. For microalgae-based biofuel production,
membrane filtration 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 filtration can simplify
subsequent processes, e.g.,
extraction, conversion, refining, and the use of the residual
biomass, without the use of
coagulants (Zhang et al., 2010).
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
Nevertheless, the significant reduction in the permeate flux
caused by membrane fouling
is the critical constraint of membrane filtration. 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-flow filtration is widely used to decrease fouling with
the tangential flow and
performs more efficiently than does dead-end filtration. In
cross-flow filtration, backwashing
and ventilation of the algae medium can help control the fouling
and recover flux (Chen et
al., 2012). Zhang et al. (2010) found that ultrafiltration
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 modification of the membrane
module in cross-flow filtration is
an option to improve the harvest efficiency and reduce the
energy consumption. An
integrated system composed of a ceramic tubular membrane and a
hollow fiber 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 microfiltration
can be applied to
microalgal harvest as the first 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 filtration is an improvement for the
microalgal harvest method
because this method uses the turbulence over the membrane filter
to generate higher shear
stress on the membrane surface compared with cross-flow
filtration. Dynamic microfiltration
achieved an approximately 3-fold higher plateau flux with a
lower energy usage compared
with a filtration 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
filtration obtained an almost twofold higher flux compared with
cross-flow filtration because
the low flow rate of dynamic filtration decreased the fouling
induced by broken algal cells
and their AOM (Frappart et al., 2011). Additionally, despite its
high electricity requirement,
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
dynamic filtration can reduce both the expense associated with
the equipment and membrane
replacement and the total cost compared with cross-flow
filtration (Ros et al., 2012).
In addition to the progress that has been made to control
membrane fouling and to
produce a high flux, it is also necessary to develop membranes
with properties that can
address the unique characteristics of various microalgal species
to ultimately make membrane
filtration an effective technology for microalgal harvest.
Furthermore, the development of a
continuous harvest system that integrates cultivation and
extraction processes will
significantly improves the effective use of filtration-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
flotation, and the microalgal
biomass is then collected in the vacuole layer on top of the
suspension. Microalgal cells with
a diameter from 10-30 m to 500 m are preferred for effective
flotation. 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
flotation (Hanotu et al., 2012,
Henderson et al., 2010).
In general, the flotation efficiency is dependent on the size of
the created bubble:
nanobubbles (
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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 modified to ensure a better contact. Compared with
conventional aeration, the
interaction between freshwater microalgae, such as Chlorella
vulgaris and Scenedesmus
obliquus FSP-3, and bubbles was enhanced by ozone flotation 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
flotation. 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 finding 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 flotation performance. Garg et
al. (2012) demonstrated
that the flotation 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 flotation of marine microalgae than did
ionic strength, which is
generally regarded as a primary inhibition factor in marine
microalgae flotation.
2.2.5. Magnetic separation
Magnetic microalgal harvest involves the use of both
functionalized magnetic particles
and an external magnetic field. 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
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
binder-modified 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
field 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 first modified
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 efficiency 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 significantly 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 efficiency (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 Botryococcus braunii due to cell leakage. The
finding 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
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
trivalent ions, such as Ca2+, PO43-, and Mg2+, enhanced the
flocculation between the
microalgae and the magnetic particles, which resulted in
increased separation efficiency.
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
efficient 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 sacrificial 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 flocs by
attaching to their surfaces and by enhancing the attachment
between the metal hydroxides
and the algal cells (electro-flotation). Compared with
conventional cationic metal salts, metal
ions released from a sacrificial anode offer several advantages,
including high efficiencies
with a low dose, a wide working pH range, and the absence of
coupled anions in the
electrolytic harvest technology (Gao et al., 2010).
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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 significantly 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
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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
frequency (on the order of KHz) and a high amplitude induces
cell rupture (Bosma et al.,
2003).
In an acoustic field, 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
efficiency. 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 (1-5 s) was sufficient to improve the flocculation
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 chlorella
vulgaris and indicated that alginate beads may be suitable for
simplifying the overall
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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 filamentous
fungi, pelletization was used to immobilize and grow the
freshwater microalgae Chlorella
vulgaris (Zhang and Hu, 2012). As a result, 63% and 24% of
Chlorella vulgaris was
harvested by the pelletization of Aspergillus niger under
photoautotrophic and heterotrophic
growth conditions, respectively. Importantly, the study proposed
that pelletization by
oleaginous filamentous 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 fluids, 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
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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 fluids are
employed for lipid extraction (Halim et al., 2012). Figure 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.
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
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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 efficiently 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
efficiency of supercritical carbon
dioxide extraction from lyophilized Chlorella 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 Scenedesmus obliquus slurry were
compared at two
temperatures (80 and 95 C) (Balasubramanian et al., 2011). The
results showed that
microwave radiation was significantly preferable over the use of
a water bath due to the rapid
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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 liquid-liquid
extraction using a mixture with an equivalent volume of
chloroform and methanol (1:1, v/v).
The results demonstrated that microwave was the most efficient
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 inefficient 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 7,000 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
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
with a sufficient 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., 2002,
Miller et al., 1996). 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 significantly
more intense at low frequency (18-40 kHz) than at high frequency
(400-800 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
Scenedesmus obliquus YSW15 biomass for up to 60 min. The yield
dramatically increased
after 15 min of the pretreatment, and definite destruction of
the cell envelope was observed
after 60 min through energy-filtering 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:
0
)energysupplied(TSV
tPE S
=
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 100-129 MJ/kg.
Another study (Adam et al.,
2012) investigated solvent-free extraction using
ultrasonication. Nannochloropsis oculata
with a water content of 70-95% was subjected to ultrasound (20
kHz) for up to 25 min, and
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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 insufficient. Therefore, we suggest that it
would be beneficial 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
difficult to 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 filled
with the target cells and beads
made of quartz or metal. The cells are disrupted by collision or
friction with the beads. This is
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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
Botryococcus 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 efficient 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
efficiency 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 influence not
only the cell disruption efficiency but also the energy
consumption. However, we did not find
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
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
exhibited the highest cell disruption efficiency among the
various cell disruption methods.
Sheng et al. (2012) reported that HPH (2,600 psi, mid-speed) was
the best cell disruption
method for Synechocystis PCC 6803 biomass (20.6 g/L). These
researchers evaluated the cell
disruption efficiency by measuring the increase in the soluble
chemical oxygen demand
(SCOD) during the cell disruption. Halim et al. (2012) compared
the efficiency 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 500-800 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 efficiency 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 (10,000-20,000
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 13-16%.
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 significantly
enhanced. The stability of lutein was
also confirmed. This finding 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 field (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
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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 field (PEF) to
a Synechocystis PCC 6803 suspension (0.3 g/L) and compared the
cell disruption efficiency
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:
2
2
LHRTDfVKTI =
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/m3) 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 filed by NLP includes the electrolysis of
microalgae for biodiesel
production (Zheng et al., 2011). Electroporation is promising
because of its simplicity and
high energy efficiency, which is due to the direct usage of
electricity. However, more
objective comparisons with other cell disruption methods need to
be performed.
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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 fluids 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 flows 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
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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 efficient 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
specific 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 hours
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,
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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
efficiently degrade the cell
envelopes of Chlorella pyrenoidosa, and they increased the lipid
extraction yield by 75%.
However, the enzyme activity was significantly 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
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
Soxhlet extraction, which was invented by Franz Von Soxhlet in
1879, is an
improvement of the simple solid-liquid extraction technique. In
a 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 efficiency. These
advantages make Soxhlet extraction a popular method for
quantification of lipids in
biological samples, but its long extraction time and high energy
consumption for evaporation
are problematic. Furthermore, it is difficult 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 modified 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 sacrificing 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
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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 transesterification
Direct transesterification (in-situ transesterification) 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 transesterification
occur simultaneously, and biodiesel is produced directly. This
can significantly 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
transesterification. According to Patil et al.
(2011), the microwave-assisted direct transesterification 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 = 8-10 min, and
microwave power dissipation = 1,400 W. Another study applied
direct transesterification 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 transesterification 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
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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 transesterification 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 final product (biodiesel) should
be considered if direct
transesterification is utilized.
3.5. Milking
Milking is slightly different from other extraction methods.
Normally, extraction from a
microorganism is conducted by first 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 coefficient
(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
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
solvents. Although milking can completely omit the costs
associated with harvesting and cell
disruption, its extraction efficiency is too low. Kleinegris et
al. (2011) claimed that the
milking efficiency can be improved by permeabilizing or
rupturing some of the cells and re-
cultivating the others. Furthermore, as suggested by Wijffels
and Barbosa (2010), efficient
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 identified.
There are three major
problems associated with lipid extraction:
1) No efficient 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 difficult to compare them to each other. For
example, the water content of wet
biomass critically affects the extraction or cell disruption
efficiency. 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 (transesterification) or purification
processes differently. Thus, the
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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
biorefinery based on microalgae.
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 efficiency of the entire process.
4. Conversion: Transesterification 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 oils
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 transesterification 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
transesterification.
One of the major drawbacks of the transesterification reaction
is the difficulty of
recovering products from toxic liquid catalysts, which can
adversely affect human health and
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
the environment. Furthermore, the transesterification process is
a species-dependent reaction,
specifically, an in-situ reaction using a co-solvent system,
where extraction and
transesterification occur at the same time. Moreover, moisture
and free fatty acid content are
critical factors that affect the production of high-quality
biodiesel.
Future transesterification 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
transesterification and discuss their
drawbacks; moreover, the potential of recent advances for the
production of microalgal
biodiesel with improved efficiency and cost-effectiveness is
evaluated.
4.2. Catalytic transesterfication
4.2.1. Homogeneous base, acid, and enzyme catalysts
Base catalysts are commonly used for the transesterification
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 transesterification allow 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
saponification 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 saponification, which reduces yield
(Ramadhas et al., 2005).
Thus, it is recommended that base-catalyzed transesterification
be performed only with pure
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
microalgal oil with a low FFA concentration (
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
additional equipment, chemicals, and time (Leung et al., 2010).
Because of these apparent
disadvantages, acid-catalyzed transesterification is not popular
in the biodiesel industry.
Because homogeneous acid and base catalysts exhibit undesirable
properties, such as
saponification, chemical waste generation, and high reaction
temperature or pressure and the
complex processes and costs associated with them, researchers in
the field have been devising
new methods to support transesterification, such as enzymatic
catalysis. The enzyme-based
transesterification platform is an attractive alternative to the
homogeneous acid- or base-
based approaches described above. For example, lipase-based
transesterification 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
saponification occurring during the process, there is no need
for additional separation and
purification 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 efficient 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 transesterification
platforms. These difficulties
arise from the fact that enzyme activity is influenced by
several factors that are associated
with the transesterification 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 transesterification. Moreover, enzymes
are notably expensive;
therefore, it is difficult 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-
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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 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 filtration 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 finding
provides evidence for the potential use of heterogeneous
catalyst-based transesterification
platforms for biodiesel production at a reasonable cost. Table 3
summarizes all of the
catalytic transesterification methods mentioned above.
4.3. Non-catalytic transesterification
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
In non-catalytic transesterification, 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 water-methanol
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 transesterification
with SCM has not been
widely studied to date, a recent report has shown that under
optimum conditions, using SCM
with Nannochloropsis oculata (CCMP 1776) produces an 84.2%
conversion yield at 250 C
in 25 minutes with an algae-to-methanol ratio of 1:8 (wt/v)
(Patil et al., 2012). Even with the
attractive characteristics of obtaining a reasonable yield in a
relatively short reaction time, it
still is a difficult task to study or produce biodiesel using
any supercritical fluid (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
significantly in research related to the
SCF method. Conversely, several studies have demonstrated the
appeal of the SCF method
by comparing capital costs and production profits, showing that
the latter can outweigh the
former (Patel et al., 2006).
4.4. In-situ transesterification
In-situ transesterification, often called direct
transesterification, is a process that is similar
to the SCM, where the extraction and transesterification
processes occur simultaneously. This
simpler process provides an advantage over the conventional
biodiesel production process,
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
where extraction and conversion are separate. Thus, similarly to
SCM, in-situ
transesterification 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
transesterification is the state of the feedstock (whether it is
wet or dry), which significantly
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 transesterification
methods.
4.4.1. Mechanically catalyzed in-situ transesterification
Mechanically catalyzed transesterification 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 transesterification. Though mechanical
catalysts do not directly
facilitate the transesterification reaction, they improve the
final 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
transesterification 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 4-5 min at 60-64 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
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
during microwave-assisted in situ transesterification may have
been higher than that
consumed by SCM. Another recently reported mechanically
catalyzed in-situ
transesterification approach involved the use of ultrasound
radiation. Ehimen et al. (2012)
improved the in-situ transesterification of Chlorella sp. by
sonication (24 kHz). The
conversion yield was in the range of 91-96% with a reaction time
of 20 min to 2 hours. The
molar ratio of algae to methanol in ultrasound-assisted
reactions is much higher
(1:105~1:315) but still lower than that used in
microwave-assisted transesterification when
converted to a wt./vol ratio (1:1.3~1:4). However, the reaction
time of ultrasound-agitated
transesterification was reported to be longer (0~2 hours),
thought it delivered a higher yield at
a similar temperature (60 C).
4.4.2. Chemically catalyzed in-situ transesterification
Chemically catalyzed in situ transesterification 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 transesterification reactions. It
is reported that feedstock
containing more than 31.7% water may likely inhibit in situ
transesterification (Ehimen et al.,
2010). Two explanations for this behavior were recently proposed
(Lam and Lee, 2012b):
inhibition occurs due to hydrolysis during transesterification
or water could react with TAG
to form diglyceride and FFA, leading to the esterification
instead of transesterification of
FFA and producing no FAME.
4.4.2.1. Chemically catalyzed in-situ transesterification via a
co-solvent system.
In chemically catalyzed in-situ transesterification, a
co-solvent system is used to maximize
the yield of FAME by improving the efficiency of lipid
extraction. The co-solvent system
uses a mixture of two different organic solvents, one of which
is typically ethanol. The
-
ACCE
PTED
MAN
USCR
IPT
ACCEPTED MANUSCRIPT
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, transesterification 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 microalga