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

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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: jwyang@kaist.ac.kr (J.-W. Yang)

Tel.: +82 42 350 5964; fax: +82 42 350 3910.

E-mail address: minsungpark0@kaist.ac.kr (M. S. Park)

Author Contributions

#Jungmin Kim, Gursong Yoo and Hansol Lee contributed equally to this work.

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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

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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

(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

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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

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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

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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.

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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.

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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

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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).

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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,

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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 (

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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

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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

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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).

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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.

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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

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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

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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,

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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

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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

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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

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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

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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

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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

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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

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microalgal oil with a low FFA concentration (

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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-

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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

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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,

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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

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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

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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