Bioresource Technology 135 (2013) 142149

Contents lists available at SciVerse ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Biorenery of microalgae for food and fuel

Marieke Vanthoor-Koopmans a,b, Rene H. Wijffels a,, Maria J. Barbosa c, Michel H.M. Eppink a
a

Wageningen University, Bioprocess Engineering, P.O. Box 8129, 6700 EV Wageningen, The Netherlands
Universidad Autnoma de Quertaro, Biosistemas, Facultad de Ingeniera, Cerro de las Campanas, Quertaro, Quertaro, Mexico
c
Wageningen University and Research Centre, Food and Biobased Research, P.O. Box 17, 6700 AA Wageningen, The Netherlands
b

a r t i c l e

i n f o

Article history:
Available online 5 November 2012
Keywords:
Microalgae
Biorenery
Protein
Lipids
Cell disruption

a b s t r a c t
Microalgae are a promising source for proteins, lipids and carbohydrates for the food/feed and biofuel
industry. In comparison with soya and palm oil, microalgae can be grown in a more efcient and sustainable way. To make microalgae production economically feasible it is necessary to optimally use all produced compounds. To accomplish this focus needs to be put on biorenery techniques which are mild
and effective. Of the techniques described, Pulsed Electric Field (PEF) seems to be the most developed
technique compared to other cell disruption applications. For separation technology ionic liquids seems
most promising as they are able to both separate hydrophobic and hydrophilic compounds. But additional studies need to be evolved in the coming years to investigate their relevance as novel cell disruption and separation methods. We propose a complete downstream processing ow diagram that is
promising in terms of low energy use and state of the art knowledge.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
Microalgae have been of major interest for producing biofuels in
de last decade. Recently, development and focus was changed towards the use of microalgae in the food, feed, chemical and pharmaceutical sector as well. Microalgae contain high amounts of
lipids, proteins and carbohydrates, which all can be used for different markets. For biofuels only microalgae production appears to be
too costly (Wijffels and Barbosa, 2010), one of the main bottlenecks
in algae production is the amount of energy used in the whole process and the investment costs (Norsker et al., 2011). Microalgae are
grown in large water volumes requiring mixing and for harvesting
concentrating is needed, these are very energy-intensive processes.
Moreover downstream processing is one of the most inuential
parameters for high the production costs (Delrue et al., 2012). Most
microalgae species have a tough cell wall, which needs to be
cracked before being able to extract the product of interest, again
an energy-intensive process. When exploiting the whole potential
of microalgae ingredients many different products can be obtained
simultaneously and the market value will be higher than the production costs (Wijffels et al., 2010). Therefore focus should be put
on maximal exploitation of the microalgal biomass while minimizing energy use.
Lipids and proteins are the largest fractions of the microalgae
and globally the need for lipids and proteins is rising, carbohydrates are normally a minor part except for a few algae species
Corresponding author.
E-mail address: rene.wijffels@wur.nl (R.H. Wijffels).
0960-8524/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.10.135

(Carioca et al., 2009). Lipids can be used as a source for biofuels,

as building blocks in the chemical industry and edible oils for the
food and health market. The puried proteins can be used in the
food, feed, health and bulk chemical market and carbohydrates
for producing ethanol and chemicals (Radakovits et al., 2010;
http://www.oilgae.com/algae/pro/eth/eth.html). Several studies
have focussed on the use of microalgal product for different applications successfully (Chen et al., 2011; Ginzberg et al., 2000; Lu
et al., 2004; Mandal and Mallick, 2009). However all these studies
focussed on obtaining one specic product from the biomass and
therefore extraction methods were only developed for one specic
product. This mostly means that the other available and valuable
components in the microalgae were lost.
To be able to exploit the complete biomass of produced microalgae it is necessary to use mild extraction techniques. Several
commonly used lipid extraction techniques will harm and denature the protein fraction. Conventional extraction and separation
techniques, e.g. bead milling, homogenizers, high pressure, heating, osmotic shock and chemicals, are focused on obtaining one
product while damaging the other fractions. Biorenery is a facility
that integrates biomass conversion and separation, in which the
objective is to obtain several fractions by using mild separation.
Biorenery will be necessary to obtain different types of products
from one source. The conventional techniques, like e.g. the use of
chemicals or very high pressure, are proven technologies in the
downstream processing industry but nowhere near mild and
mainly focused on one product, the biorenery techniques appropriate for mild extraction are relatively new and should therefore
be studied thoroughly before commercial use is possible. This

M. Vanthoor-Koopmans et al. / Bioresource Technology 135 (2013) 142149

paper will focus on biorenery of microalgae, the possible bottlenecks it implies and the markets it can replace or add onto. The
main focus is put on the isolation of proteins and lipids as these
are the largest fractions that microalgae contain, however other
fractions like carbohydrates and pigments will add signicant value to the total production process when separated as well. In
the future these products are also necessary to study.

2. Current European lipid and protein market

As the biggest fraction of microalgae consist of lipids and proteins the market possibilities of these two products are explored
and described in this paper. However, microalgae contain more
valuable products such as carbohydrates and pigments. Carbohydrates such as starch or glycogen (cytoplasm) and cellulose (inner
cell wall), which are ideal components for producing ethanol and
chemicals (Radakovits et al., 2010). Pigments as chlorophyll can
be used as anti inammatory and wound healing additive to pharmaceuticals, carotenoids reduce the risk of cancer, and astaxanthin
is a powerful antioxidant (Christaki et al., 2011). These higher valuable products make microalgae even more attractive as food and
feed additives.
Microalgae oils and proteins have the potential to replace and
add to the current oil and protein markets. Globally the demand
is rising due to the rising population. Therefore it is necessary to
nd sustainable sources of lipids and proteins. The energy and food
market demands high quality products at large quantities. The major imported sources for lipids and proteins in Europe are palm oil
and soybeans and cake of soybeans (www.faostat.fao.org). Both
these sources lead to deforestation in their region of origins (Lima
et al., 2011; Wicke et al., 2011), thus further increasing the production of these crops is highly unwanted. Alternative sources for biofuels, vegetable oils or proteins derived from terrestrial crops, e.g.
maize, sugarcane, rapeseed, contribute to water scarcity, forest devastation and they impose pressure on the food market (Timilsina
et al., 2012). Algae have much higher areal oil productivities and
therefore less area will be necessary to produce similar quantities
(Mata et al., 2010). Moreover microalgae are not bound to arable
land and can be grown in sterile places or even on the sea. Therefore, microalgal production is not competitive with other food production sources and a sustainable source for lipids and proteins.
Ninety percents of global palm oil production and trade comes
from Asia, more specically Malaysia and Indonesia (Thoenes,
2009). A total amount of 6.8 million tonnes of palm oil is imported
in Europe, this corresponds to 17 million tonnes of microalgal biomass containing 40% lipids. In 2010 soy beans were mainly imported
from Brazil (45%), USA (22%) and Paraguay (19%). The soybeans are
mostly imported into The Netherlands (20%), Germany (19%) and
Spain (18%) where they are processed and mostly further exported
as soy oil or soybean meal towards other countries in and out of Europe (http://comtrade.un.org/db/dqBasicQuery.aspx). Soy oil is the
main competitor with palm oil but its total market is much smaller.
Soybean meal is also imported into Europe, again The Netherlands
and Germany are the main receivers, 24% and 20% respectively. A total amount of 6.5 thousand tonnes of soy bean meal is imported into
Europe, which is neglectable, in respect to protein, to the total
amount of soybean imported, less than 0.2%. The total amount of
soy beans imported in Europe is 7.5 million tonnes, containing
40% protein this means 3 million tonnes of protein. This corresponds
to 10 million tonnes of algal dry weight having a 30% protein content. Producing 17 million tonnes of microalgae as a lipid source
generates sufcient biomass for the necessary protein production
if both products can be separated efciently. 17 million tonnes of algae can be grown, considering a production of 50 tonnes per Ha per
year, on 340.000 Ha land, which is as big as 0.7% of the total area of

143

Spain. These numbers show that Europe does not need to be dependent on other continents for its production of lipids and proteins.
Moreover, extension of production for biofuels will also be possible
when the demand will rise.
3. Lipid and protein quality
To replace the current oil and protein market not only the quantity but also product quality is important. Edible oils and proteins
have a nutritional and functional property in foods, such as taste,
structure and stability. These functionalities should be considered
when replacing the current sources. For most applications, oil present as triacylglycerides (TAGs) is preferred. These TAGs are widely
present in different species of microalgae. TAGs are used to store
fatty acids under stress conditions (Hu et al., 2008). When stress
conditions, e.g. nutrient depletion, pH changes, high salinity, are
applied during microalgal cultivation, accumulation of fatty acids
were found in TAGs up to 50% of total biomass (Hu et al., 2008).
Moreover, algae contain the two essential fatty acids, EPA
(C20:5) and DHA (C22:6) and other omega 3 fatty acids that most
other crops do not contain. These essential fatty acids are necessary in human and animal metabolism even though they are not
able to synthesize these fatty acids themselves. Therefore it is very
important to obtain EPA and DHA from food sources. Fig. 1 shows a
comparison of fatty acid composition of different algal species and
palm oil. Here can be seen that algae oil contain the same fatty
acids as palm oil and have a more diverse range scale of fatty acids,
moreover they contain more healthy unsaturated fatty acids.
In terms of proteins, the amino acid content is the major quality
factor. 20 amino acids exist of which 8 are essential. Human and
animals require these essential fatty acids sufciently in their
nutrition. In animal feed soya and maize are mostly used as a feed
source. These crops do contain the complete range of amino acids,
however not always in the right ratio. Addition of specic amino
acids, such as methionine and lysine, in the chicken branch increases growth rate (Lima et al., 2008), however this is costly. Different types of algae contain different amounts of the essential
amino acids and therefore selecting the right species for specic
applications can lower the costs in animal production. Fig. 2 shows
the comparison between soya and three different species of algae
in their essential amino acid content. It is shown that a balanced
diet can be made using algae instead of or in combination with
soya.
To achieve feasible algae production rst focus should be put on
improving the production efciency and with that lowering
production costs. Moreover the produced algae should be used to
obtain both the oil and the protein fraction from the same biomass,
therefore biorenery is necessary. Biorenery used for microalgae
is not yet developed, in this review the potential of applying
different biorenery techniques in the eld of microalgae is
described. Advances made so far for microalgae and other elds
are discussed.
4. Biorenery
Biorenery techniques are necessary to exploit all products produced by microalgae after cultivation. The main bottleneck is to
separate the different fractions without damaging one or more of
the product fractions. Technologies to overcome these bottlenecks
need to be developed, and they should be applicable for a variety of
end products of sufcient quality at large quantities. For that, the
developed techniques should be mild, inexpensive and low in energy consumption. First focus in obtaining the products should
be on cell disruption to release the products or to make them available for extraction. When the products are released from the cells,

144

M. Vanthoor-Koopmans et al. / Bioresource Technology 135 (2013) 142149

Percentage of total fatty acids (%)

60

50

40

30

20

10

0
14:0

16:0

16:1

16:2

Palm oil
Neochloris oleoabundans

16:3

16:4

18:0

18:1

18:2

Chlorella vulgaris
Pheaodactylum tricornutum

18:3

18:4

20:5

Scenedesmus obliquus

Fig. 1. Fatty acid composition of palm oil and different species of algae (Breuer et al., 2012; Dauqan et al., 2011).

Percentage of total amino acids (%)

12
10
8
6
4
2

Chlorella vulgaris

Scenedesmus obliquus

*
ni
ne
Th
re
o

n*
Tr
yp
to
ph
a

ni
ne
hi
o
et
M

ny
la
l
Ph
e

Soymeal

e*
an
in

ne
*
si
Ly

in
e*
Va
l

e*
Le
uc
in

Is
ol
eu

ci
n

e*

Spirulina platensis

Fig. 2. Amino acid composition of soymeal and three different algal species (Becker, 2007).

different extraction and separation techniques need to be used for

purication. It is important to keep in mind that the different technologies should be tailored to the different species of interest and
should therefore be exible. Some algal species do not have a cell
wall, which makes cell disruption less energy intensive. These differences need to be taken into account when selecting the cell disruption and separation approach for specic algae species.
The algal cell consists of different organelles, such as, lysosomes, mitochondria, and endoplasmatic reticulum. Each type of
organelle contains specic components which can be used in our
advantage to fractionate the different components. It was proposed
to rst disrupt the cells to release the lipids, proteins and carbohydrates from the cytoplasm after which the different larger cell
compartments (organelles) can be fractioned to later obtain their
specic compounds by disruption (Eppink et al., 2012).
Proposed procedure is:
(1) Specic cell disruption using mild technology, rst whole
cell wall disruption followed by organelle disruption.

(2) Extraction and fractionation of the different fractions (proteins, lipids, carbohydrates) using mild conditions, all available components need to be functional after this separation
step.
(3) Further separation of the different fractions to obtain specic products (e.g. omega 3 fatty acids, saturated and
mono-unsaturated fatty acids).
In this review the rst two steps will be described in detail. Focus is put on making the different components available and separating the different fractions. Further purication towards specic
products is described briey in combination with some of the
extraction and fractionation techniques.
4.1. Cell disruption
To keep all components intact, mild breakage is necessary to
disrupt the cell wall. First breakage of the outer cell wall is necessary to make the cellular components available. Currently available

M. Vanthoor-Koopmans et al. / Bioresource Technology 135 (2013) 142149

cell disruption techniques cause complete disruption of the cells

and are focused on obtaining one specic product while damaging
the other available components. Techniques used are homogenizers, high pressure, bead milling, heating, microwaves, osmotic
shock and chemicals. Alternative continuous ow techniques are
proposed to be applicable to disrupt the cells mildly, for example:
Pulsed Electric Field, Supersonic Flow Fluid Processing and ultrasound. Moreover, the new generation of enzymes available nowadays can also be suitable as a mild cell disruption technique.
The continuous ow techniques are receiving more and more
attention in research the last decade and all are focussed on perforating the outer cell wall. Possible applications are; (1) Analyzing
single cellular content, (2) transfection of cells, (3) inactivating
cells in the food industry, and (4) analyzing cellular properties
e.g. blood samples. The major advantage of these techniques is that
they do not harm the intracellular content, the low sample volumes required for analyses due to effective separation of the intracellular content and the effective cell inactivation. Cell inactivation
occurs due to the perforation of the cell wall or cell membrane, this
causes irreversible damage and the cell dies (Fox et al., 2006).
Moreover due to the perforation the intracellular components will
be easily extractable. The challenge for biorenery of microalgae is
to apply these principles at large scale and low cost. An advantage
of these types of techniques is that they can handle low concentrated streams, therefore no or less concentration is necessary
and this will lower energy use signicantly. However, it will require larger volume processing in the continuous ow technique.
The Pulsed Electric Field, Supersonic Fluid Flow Processing, and
ultrasound are techniques that perforate cell walls or membranes
(electroporation), the actual perforation is done using different
mechanisms. Depending on the energy requirement and the efciency of the different perforation techniques the best technology
can be chosen. Enzymes work very specically on cell membrane
compounds and can cause cell wall disruption effectively. These
technologies all cause electroporation, however, the effectiveness
of each technique should be studied before clear conclusions can
be drawn and breakthroughs are achieved.

4.1.1. Pulsed Electric Field

Pulsed Electric Field (PEF) is a technique in which high electric
eld strengths are used to perforate a cell wall or cell membrane.
Depending on the strength and length of the electric eld this
can cause reversible or irreversible electroporation. This ability
can be used in different applications like transfecting cells by introducing foreign DNA when causing reversible perforation or analyzing cellular content and inactivating or pasteurizing cells by
irreversible perforation (Fox et al., 2006). Several studies have
shown very effective separation of cellular content in microuidic
devices using PEF (Gao et al., 2004; McClain et al., 2003). Both studies were focussed on single cell analysis, by immobilizing the cells
in a micro device and in that way separating the soluble compounds from the cells. To analyze intracellular content this technique is very effective, however, to extract the content of cells on
an industrial scale immobilization will not be efcient.
El Belghiti and Vorobiev (2004) have used PEF to extract sugar
from sugar beets. They have shown that PEF can be efciently used
in large scale processes and that it enhanced the extraction of sugar
from the sugar beets. Increasing electric eld, number of pulses,
extraction time and stirring speed during extraction all improved
the amount of sugar extraction until an optimum was found. First
sugar beet slices were exposed to pulses of a certain electrical
intensity after which the sugar beet slices were put in an aqueous
solution to start the extraction. The improved extraction results
obtained are promising for applying this technique for algae
extraction as well.

145

At commercial scale PEF is used to pasteurize foods at low

heats. The shelf life of orange juice and tomato juice is comparable
using conventional heat or PEF treatment (Min et al., 2003a; Min
et al., 2003b), taste was not affected after PEF treatment in different fruit juices (Mosqueda-Melgar et al., 2012) and fatty acid composition was unaffected in orange juice (Zulueta et al., 2007). The
rst studies on commercial scale have been performed with no loss
of quality of the product, the production rates range from 400 L/h
to 2000 L/h (Min et al., 2003a; Min et al., 2003b) showing the potential to scale up.
Using PEF in microalgae research is starting to get attention
nowadays. PEF has been successfully applied to recover lipids from
microalgae at low energy intensities, PEF caused that the extraction solvent could more easily reach the lipids. It was suggested
as a suitable technique to be used in large scale processes (Sheng
et al., 2012). In another study electroporation was caused using
PEF in Auxenochlorella protothecoides, the spontaneous release of
intracellular products was studied after electroporation (Gottel
et al., 2011). It was found that 1015% of the initial biomass could
be found in the supernatant after centrifugation, however, high
molecular compounds were not released from the cells. The increase in dissolved organic compounds in the supernatant after
electroporation was mostly caused by small molecules like carbohydrates and amino acids. Releasing the larger compounds could
be improved by larger perforation or by the use of solvents for
extraction. Moreover it was found that longer storage time at
ambient conditions before centrifugation increased the release of
organic carbon signicantly.
Overall, PEF treatment shows to be a very promising technique
useful for cell perforation at large scale. The energy consumption of
PEF is much lower than for the conventional techniques (Table 1)
(de Boer et al., 2012). This technique has already been implemented in the single step extraction technique by OriginOil. They
use electromagnetic waves to cause electroporation and at the
same time it causes the cells to come out of solution, which makes
cell concentrating easier and less energy intensive (www.originoil.com). Still more research is necessary on the efciency of perforating all cells in different cell density cultures; the equipment
design, the optimal peak voltage and pulse time need to be found.
We are dealing with single cells and all cells need to be exposed
long enough to the electrical pulses applied to make sure that all
cells will be sufciently perforated.

4.1.2. Supersonic Flow Fluid Processing

Supersonic Flow Fluid Processing is a highly sophisticated technology. Supersonic steam is introduced into a special annular conditioning chamber that is wrapped around the core of the unit and
injected through nanopore channels into the fermentation uid
(biomass) whereby a thermo uid interaction with the cells is obtained for an optimal mass transfer. With increased steam ow, the
steam exit velocity becomes supersonic and starts to form a controllable shock wave. This shockwave continues to grow and forms
a low density, low temperature, low pressure, supersonic velocity
zone across the bore diameter increasing energy transfer and cell
disruption. Using this technique a homogeneous disruption is obtained. Although steam is used, the temperature does not exceed
35 C. Therefore this technique does not cause protein or other
valuable component to be denaturated.
The company PDX has patented this technique as atomisation
and claims that it can be used effectively as a disinfection technique in the food industry. Not much research can be found on
using this novel technique specically to disrupt cells for improvement of extraction. However, as PEF, it causes electroporation and
can therefore be a promising technique for the biorenery of
microalgae.

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M. Vanthoor-Koopmans et al. / Bioresource Technology 135 (2013) 142149

Table 1
Energy requirements of different cell disruption techniques (de Boer et al., 2012).

Incoming concentration (dw)

Energy required to process (kW h/m3)
Effectiveness (cells destroyed)
Specic energy consumption (kW h/kg)
Energy consumption per energy content in produced algae (%)a
Specic energy consumption (MJ/m3)
a

Homogeniser

Bead milling

Ultrasound

Pulsed Electric Field

15%
38
96%
0.25
4.2
136.8

15%
1500
90%
10
164.4
5400

15%
10
Not given
0.07
1.1
36

25%
15
Not given
0.06
1.0
54

Energy content of algae is taken as 21 kJ/g biomass (Weyer et al., 2010).

4.1.3. Ultrasound
Ultrasound utilizes the process of cavitation to disrupt the cell
wall. Collapsing bubbles driven by bulk pressure variation due to
ultrasound waves can cause cell and also molecule breakage
(Ranjan et al., 2010). Many years ago ultrasound was already
effectively used to kill bacteria and viruses (Alliger, 1975). Other
applications involve cell disruption for the release or extraction
of intracellular components, disinfection, wastewater treatment,
synthesis of biodiesel, emulsication, freezing, crystallization and
gene transfer into cells (Gogate and Kabadi, 2009). For microalgae,
ultrasound has been used to improve oil extraction from the cells,
the complete process for microalgae has been patented (Echevarria
Parres, 2011).
The operational parameters should be chosen carefully as two
different types of cavitation can be caused, either transient or stable. When transient cavitation is reached, the generated bubble
will eventually collapse completely at which point the local temperature and pressure can increase tremendously causing damage.
Stable cavitation is milder as the small bubbles are forced to oscillate and not even always completely collapse. Using this technique
it should be investigated how efcient the cell wall rupture can be
done without causing intramolecular damage. Ultrasound is a very
low energy intensive technique compared to the conventional cell
disruption techniques (Table 1).
4.1.4. Enzymes
Enzymatic degradation is another mild cell wall disruption
method. Enzymes are not widely used in industry because of the
high costs of cell lysing enzymes. However, enzymes are very
selective in their degradation process dependent on the composition of the cell wall, therefore using enzymes is a mild and effective
technique and might therefore become interesting (Chisti and
Moo-Young, 1986). Successful cell lysing of Pseudomonas putida
has already been done in 1983 using lysozyme for the isolation
of alkane hydroxylase (Fish et al., 1983). For the purpose of algae
enzymes might become interesting as microalgal cell walls contain
(hemi)cellulose with glycoproteins or polysaccharides, which
makes the cell wall thick and stable. To degrade such strong cell
walls mechanically a lot of energy is required. Specic enzymes degrade specic compounds and might weaken the cell wall so that
mechanical degradation can be carried out at low energy costs.
The exact composition of the cell wall is important to be able to
choose the right (mixture) of enzymes necessary.
In macroalgae several studies were performed using enzymatic
degradation of the cell wall to increase the extraction yield (Amano
and Noda, 1992; Denis et al., 2009; Fleurence et al., 1995). For different types of macroalgae, green, red and brown, different types of
enzymes or enzyme mixtures were used. For green algae a mixture
of cellulase and Macerozyme; for red algae different digestive enzymes from the gut of abalone and Macerozyme; and for brown
seaweeds a cellulase and Macerozyme mixture in combination
with an extract of gut from abalone was used. For all treated seaweeds a signicant difference was found in extracted protein composition (Amano and Noda, 1992). For the red seaweed Grateloupia

turuturu different polysaccharidases or combinations were used to

digest the cell wall Grateloupia turuturu, they found that cellulase
alone and combination of cellulase with k-carrageenase resulted
in the best digestion conditions (Denis et al., 2009). These studies
conrm that different types of genera will have differences in their
cell wall composition and therefore different types of enzymes are
necessary.
For microalgae not many studies have been performed with enzymes to degrade cell walls. In a preliminary study two different
enzymes were used for degradation. Cellulase and lipase were used
to degrade cellulose and phospholipids, respectively. Formation of
glucose and glycerol as degradation products were observed,
showing that degradation occurred (Sander and Murthy, 2009).
However death cells were used here, which may not be representative for living cells. Further research is necessary to study the
effectiveness of cell wall degradation in living cells.
4.2. Extraction and separation techniques
There has been an increased interest in separation technologies
as a result of the rapid development of the biotechnology market.
Cost efcient separation of e.g. therapeutic proteins has led to major efforts towards different extraction and separation techniques
that are scalable and economical. There are several promising
extraction and separation methods under research at the moment,
which we will further describe towards the use for microalgae.
These promising extraction methods are the use of ionic liquids
and surfactants, the main objective here is to separate all fractions
without losing any product.
4.2.1. Ionic liquids
In recent years, ionic liquids have been widely considered as
alternatives to classical organic solvents and applied in organic
synthesis, electrochemistry, liquid phase extraction, catalysis for
clean technology, and separations (Berthod et al., 2008; Vidal
et al., 2012). Ionic liquids are made of cations and anions as they
are simple molten salts. The advantage and novelty of ionic liquids
are their low melting temperature, generally below 100 C. Ionic
liquids can be an alternative to classical organic solvents by their
ability to extract hydrophilic from hydrophobic compounds. They
are easily recyclable and therefore produce minimum pollution
compared to organic solvents. The range of different ionic liquids
is enormous and therefore the specic applications as well.
First attempts have been made to extract lipids from algal biomass using ionic liquids. A combination of ionic liquids and methanol increased the yield of lipids obtained from a culture of
Chlorella sp. from 11.1% to 19% compared to the original lipid
extraction method of Bligh and Dyer method (Kim et al., 2012). It
was proposed that the organic solvent was necessary to break
the cell-wall and that the ionic liquid functioned as facilitator in
transferring the lipids towards the surface (Young et al., 2010).
Moreover, they mentioned that simultaneous protein extraction
can occur when using the right ionic liquids. Therefore, the method
we propose will be very promising, as we propose to rst rupture

M. Vanthoor-Koopmans et al. / Bioresource Technology 135 (2013) 142149

the cell wall in a mild way so the lipids and proteins will be available for extraction. Ionic liquids can then be applied as an additive
to the aqueous phase before separating the biomass from the proteins and lipids. Adding the ionic liquid in this stage makes that
both the hydrophobic and hydrophilic compounds are soluble.
After this, separation of the remaining biomass can take place by
micro- or ultra ltration or centrifugation.
Other separation techniques in combination with ionic liquids
can be a very strong tool to further separate the different fractions
into separate compounds or groups of compounds. For gas chromatography (GC), liquid chromatography (LC), and capillary electrophoresis (CE) ionic liquids have been successfully used (Berthod
et al., 2008; Han and Row, 2010). It showed an unusual selectivity
to both polar and non-polar compounds. For GC, ionic liquids have
been used as a stable stationary phase to enhance separation selectivity (Anderson and Armstrong, 2003; Armstrong et al., 1999). For
LC ionic liquids have been used as a mobile phase additive for the
separation of e.g. amines (Xiaohua et al., 2004). However, the high
viscosity and UV absorbance of ionic liquids limit the applicability
as a mobile phase additives in LC techniques. In CE, ionic liquids
have been used as electrolyte additives to separate proteins (Jiang
et al., 2003), phenols and aromatic acids (Vaher et al., 2002).
For microalgae it would be useful to use LC techniques having
the ionic liquid as a stationary phase. The aqueous phase containing the different products of interest of the algae will pass this stationary phase while effectively separating the different fractions
without using organic solvents. Not much research has been reported in the use of ionic liquids as a stationary phase in LC. However, lately more attention was given to this promising technique
as higher selectivity can be obtained using ionic liquids as the stationary phase in LC, moreover, the mobile phase used can be aqueous without containing solvents. First attempts have been made
using silica particles as the basis for the stationary phase. Two
types of imidazolium room temperature ionic liquids were bonded
to silica particles and acted successfully as a stationary phase separating organic acids in high performance liquid chromatography
(Wang et al., 2006). Further research and development will be necessary to make ionic liquids an applicable tool in separating fractions selectively using liquid chromatography.
4.2.2. Surfactants
Surfactants have been used to separate specic proteins. Proteins can be solubilized in organic solvents with surfactants, maintaining their functional properties (Pires et al., 1996). The to be
separated proteins can be transferred between an aqueous solution
and a reversed micellar organic phase. Conventional techniques
use a centrifugation or ltration step to separate cellular material
from the soluble components in the liquid medium. In this way
the non secreted proteins are lost. By using liquidliquid extraction
technology, after cell disruption, the product of interest can be extracted from the cellular material before further separation. For
this liquidliquid extraction, water in oil microemulsions (with
surfactants) can be used efciently (Cklen and Hatton, 1985).
The protein transfer between the aqueous phase and the micellar organic phase is largely dependent on the pH, ionic strength
and type of salt used in the bulk aqueous phase. However, the type
and concentration of surfactant and type of solvent also inuences
extractability. It was shown that electrostatic interactions between
the surfactant head groups and the protein can promote the transfer of the protein into the organic phase (Cklen and Hatton, 1985).
This electrostatic interaction can be inuenced by determining the
net charge of the proteins by changing the pH of the aqueous solution. After having separated the protein of interest into the micellar
organic phase, the proteins and also the surfactants need to be
recovered. Here again changing pH can cause the required effect
of back-extraction.

147

Recently, new developments in using surfactants for protein

extraction arose. Anionic surfactants have been used to bind specic protein (lysozyme) in order to make them insoluble in aqueous solutions, and therefore the proteinsurfactant bond
precipitated without losing protein activity (Cheng and Stuckey,
2012; Shin et al., 2004). Surfactant precipitation reduces the
amount of surfactant required in comparison with reverse micellar
extraction. Moreover, less energy is required to recover the precipitated proteins in comparison with solubilized proteins.

5. Sustainability and future prospect

Microalgae production is very promising to replace the current
vegetable oil and protein commodities in a sustainable way.
Advantages as higher oil areal production rates, non competitiveness with food crops due to the ability to culture algae on non arable land and lower greenhouse gas emission make algae this
promising. However, algae production as a commodity source is
not yet feasible. The main bottleneck lies in the scale of production
and the total production and processing costs. These costs are largely caused by the energy consumption of the different process
steps in the total production and extraction chain. Algae research
is relatively young and many improvements can still be made to
improve production and extraction efciency. Especially when
more products can be obtained from one microalgae production
process, the value of algae will increase signicantly and will make
algae production economically feasible (Wijffels et al., 2010).
In this review we have described different biorenery techniques, which can be used to obtain the different available products from microalgae. Main reason for the success of biorenery
is that the techniques used are mild and energy efcient. First step
is to disrupt the cell wall after which efcient extraction needs to
take place. We have described four cell disruption techniques,
Pulsed Electric Field, Supersonic Fluid Flow, Ultrasound and enzymes. Ultrasound and Pulsed Electric Field both are very energy
efcient (Table 1), for Supersonic Fluid Flow this is not well known,
but to be expected in the same range. Enzymes can be promising,
however to obtain the necessary enzymes is a very expensive
and long process. Pulsed Electric Field is the most developed technique of the discussed techniques and is therefore be promising to
further develop for microalgal biomass application. However, all
techniques should be studied further to be able to evaluate thoroughly which technique is most applicable with regard to efciency, energy consumption and scalability.
To extract the different components, separation efciency and
possible reuse of the used extractor is important. Ionic liquids
are very promising as they can be used to both extract lipids and
protein, moreover reusing the ionic liquids using chromatography
separation techniques is highly efcient. Surfactants have the
advantage that the separated proteins are precipitated and therefore easier to recover. A combination of these extractors can also
be used.
At this moment, taking into account how far the development of
the different described cell disruption and extraction techniques
are in combination with the energy consumption, we suggest to focus on the use of Pulsed Electric Field as a cell disruption technique
and using ionic liquids for extraction. Fig. 3 shows the proposed
downstream processing scheme that we believe can be promising
to obtain all available and useful products produced by algae. In
the ow diagram can be seen that no concentration step is taken
into account before cell disruption, this might be possible using a
continuous ow cell disruption technique and will decrease energy
consumption signicantly although larger process ows need to be
processed. It must be said that in most parts of the total scheme research still is necessary to nd optimal process conditions for

148

M. Vanthoor-Koopmans et al. / Bioresource Technology 135 (2013) 142149

Fig. 3. Proposed biorenery approach using Pulsed Electric Field as a cell disruption technique and ionic liquids as extractor in different phases of the process.

efcient cell disruption and separation. Moreover, as said before

the other described techniques do need to be further developed
as well to really nd the best techniques and process ow necessary to make biorenery for microalgae successful.

6. Conclusion
For economical production of microalgae it is necessary to use
most biomass compounds. For that biorenery is necessary in
which cell disruption techniques are mild and effective. Pulsed
Electric Field (PEF) is the best industrially developed technique.
Large scale PEF for microalgae needs further development. Supersonic uid ow and ultrasound are also promising for continuous
cell disruption. For separation technology the ionic liquids are
most promising to separate both hydrophobic and hydrophilic
compounds (e.g. proteins, lipids). We propose a complete downstream processing ow diagram that is promising in terms of low
energy use and state of the art knowledge.

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