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Bioresource Technology 99 (2008) 3949–3964

Review

Microalgae immobilization: Current techniques and uses

Ignacio Moreno-Garrido *

Institute of Marine Sciences of Andalucı´a (CSIC), Campus Rı´o San Pedro, s/n 11510, Puerto Real, Ca´ diz, Spain

Received 19 July 2006; received in revised form 23 May 2007; accepted 23 May 2007
Available online 9 July 2007

Abstract

Information about advances in immobilization techniques and biotechnology use of freshwater and marine microalgae is scattered.
This work aims to bring together the main recent research about the topic. Passive and active immobilization techniques used on mic-
roalgae are listed and described in the text. Effect of immobilization on growth and metabolism of the cells is also reviewed. Current
uses of immobilized microalgae include metabolite production, culture collection handling, obtaining of energy and removing of
undesired or valuable substances from media (nutrients, metals and different pollutant agents). Applications of immobilized microalgae
in environ- mental aquatic research have been recently increased: novel immobilization techniques as well as the use of living
microalgae as biosen- sors in electronic devices designed to measure toxicity of substances and effluents demonstrated to be a very
promising topic in biotechnology research. Recent research pointed out the advantages of mixed bacterial–algal co-immobilized systems in
water treatment plants. Application of immobilized systems to the production of non-contaminant energy (as H 2 obtained from algal
cultures) is also an important topic to be explored in the next years.
© 2007 Elsevier Ltd. All rights reserved.

Keywords: Microalgae; Phytoplankton; Immobilization; Biotechnology

1. Introduction those problems. The use of immobilized algal cells in water

Microalgae (sensu lato, it means including prokaryotic
photosynthetic microorganisms such as cyanobacteria)
are organisms that play a key role in aquatic ecosystems.
It is estimated that around 40% of global photosynthesis
is performed by microalgae (Falkowsky, 1980). Microalgae
form the basis of most of trophic aquatic chain. In some
coastal environments, biomass of microphytobenthos can
match or even exceed the biomass of bacteria (La Rosa
et al., 2001). The use of microalgae in biotechnology has
been increased in recent years, these organisms being impli-
cated in food, cosmetic, aquaculture and pharmaceutical
industries (Borowitzka and Borowitzka, 1988), but small
size of single cells implies a problem in the application of
biotechnology processes to those organisms. Cell immobi-
lization techniques have been developed in order to solve
*
Tel.: +34 956832612; fax: +34 956834701.
E-mail address: ignacio.moreno@icman.csic.es
purification processes has been reported from long ago
(Robinson et al., 1988), as microalgae form part of the
organisms fixed in percolating filters of wastewater treat-
ment plants. But at the end of the sixties of the past cen-
tury, novel techniques for immobilizing biocatalysts in
general (from enzymes to whole cells) began to spread in
the literature (Papageorgiou, 1987), and the use of immobi-
lization techniques diversifies. Immobilized algae have been
used for biomass obtain and macronutrient removal. The
extremely high accumulation capacity of some of these
organisms for potentially dangerous substances (Maeda
and Sakaguchi, 1990) has been also exploited for bioreme-
diation techniques applied on polluted waters (specially
involving metals: Greene and Bedell, 1990). This capacity
has also been exploited in order to pre-concentrate these
substances and thus facilitate the measurement of traces
in environment (Carrilho et al., 2003; Singh and Prasad,
2000). Lower growth rates of immobilized algal cells, prob-
ably due to restrictions in the diffusivity of nutrients to the
immobilized cells, as well as protection of cells entrapped in

0960-8524/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2007.05.040
3950 I. Moreno-Garrido / Bioresource Technology 99 (2008) 3949–3964
immobilizing matrixes can also be exploited in order to
facilitate handling of culture collections (Lukavsky,
1988). Environmental uses of immobilized algal cells are
not restricted to pollutant removal. These techniques have
been recently used in field toxicity measurement experi-
ments (Admiraal et al., 1999; Moreira dos Santos et al.,
2002, 2004; Moreira et al., 2006). An important part of
the current work in the field of the toxicity measurement
is, however, focused in the design of microalgal-based bio-
sensors (Chouteau et al., 2004; Podola et al., 2004; Shit-
anda et al., 2005). Recent uses of microalgal technology
involving production of hydrogen (Dante, 2005; Kapdan
and Kargi, 2006) or electricity (Kadam, 2002) could be
improved by the use of immobilization techniques. Very
well known reviews on immobilization techniques and
uses for algae date from the end of the eighties (Robinson
et al., 1986; Codd, 1987; Papageorgiou, 1987). Cassidy et
al. (1996) made a revision of the environmental
application of immobilized cells (not only algae). Jen et al.
(1996) sum- marized in a review the new techniques till
that year on hydrogels for cell immobilization in general.
One of the most recent reviews on the topic is the work of
Mallick (2002), centred in the use of immobilized algae for
waste- water, nitrogen, phosphorus and metal removal.
But a big amount of information about all these and other
uses for immobilized cells still remains scattered. The
purpose of this review is, thus, to bring together all recent
informa- tion to date about microalgal immobilization
techniques and the current uses for this biological
material, in order to facilitate researchers the task of
finding references related to their work in this field.
2. Immobilization techniques for microalgal cells
Most of the immobilization techniques devised for
microorganisms in general can be applied to microalgae,
with the limitation of light transmission if living cells are
intended to be immobilized. Immobilization techniques
can be primarily divided into two groups: ‘‘passive’’ and
‘‘active’’ immobilization.
2.1. Passive immobilization
Many microorganisms (including some groups of micro-
algae) have a natural tendency to attach to surfaces and
grow on them (Robinson et al., 1986). This characteristic
can be exploited in order to immobilize cells on carriers
of different types (Codd, 1987). Normally, those processes
are easily reversible and contamination of effluents with
unstuck cells is unavoidable. Adsorbent materials (carriers)
for passive immobilization can be natural or synthetic.
With respect to natural carriers, recently efforts have been
made involving loofa biomass. Loofa sponges are the
fibrous support of the fruit of different species from the
genus Luffa (L. cylindrica – possible synonym: L.
aegypti- aca, L. operculata, L. acutangula). The sponge is
obtained from dry fruits after removing the pericarp
tissue. This car-
rier is non-toxic and (it is said to be) non-reactive, cheap,
mechanically strong and stable in long-term cultures (Liu
et al., 1998). Akhtar et al. (2004) used loofa sponge bio-
mass in order to immobilize cells of Chlorella sorokiniana,
to remove nickel(II) from aqueous solutions. This immobi-
lized system demonstrated to accumulate 25% more nickel
than free cells after 20 min exposition. This immobilizing
technique using loofa sponges has also been used for fun-
gus such as Phanaerochaete chrysosporium (Iqbal and
Edyvean, 2004, 2005; Ahmadi et al., 2006) in wastewater
treatments; bacteria as Zymomonas nobilis (Vignoli et al.,
2006) for sorbitol production; and microbial bio-systems
(Nagase et al., 2006) for degradation of carbendazim and
2,4-dichlorophenoxyacetic acid. For cells that have no nat-
ural trend to attach this type of support, Ogbonn et al.
(1996) reported the possibility of the co-use of chitosan
in order to increase the flocculating process of free cells
over the loofa surfaces. Liu et al. (1998) compared the cel-
lular adsorption capacity of loofa sponge cubes and poly-
urethane cubes, and found no differences for plant
(Coffea arabica) free cells. A problem on designing
research involving loofa sponge biomass is repeatability.
Structure of the skeleton of fruits varies from a plant to
another in function of culturing conditions: each loofa
sponge has dif- ferent structure (Liu et al., 1998). In any
case, for industry or commercial purposes, passive
immobilization of algal cells on loofa sponges seems to be
a very promising field.
Synthetic materials are also widely used in experiments
involving passive immobilization. Urrutia et al. (1995)
immobilized Scenedesmus obliquus cells in polyvinyl and
polyurethane in order to remove nitrate from water. Sur-
vival of adsorbed cells was compared with entrapped cells
(it means, cells immobilized by ‘‘active’’ immobilization),
by mixing concentrated cells with one of the pre-polymers.
Cellular growth is higher for adsorbed cells than that mea-
sured for entrapped cells, possibly due to the toxicity of
the pre-polymers (although these authors reported that
no toxic effects were found due to the residual presence
of pre-polymer reagents). Yamaguchi et al. (1999)
achieved a noticeable degradation of hydrocarbons by the
colorless hydrophobic microalgae Prototheca zopfii,
adsorbed to 8 mm-cubes of polyurethane foam in a
bubble reactor. Archambault et al. (1990) described a
reactor where plant cells attach by natural adhesion to
short-fiber polyester material named 7607.
Attachment of periphyton to different surfaces can also
be used in ecology studies. Admiraal et al. (1999) used
microbenthic algae and bacteria colonizing glass discs to
measure the response of these organisms to different levels
of metal pollution. Danilov and Ekelund (2001) compare
settlement patterns of periphyton on glass, wood and
plas- tic in lakes of different trophic status, this order
(glass > wood > plastic) being the preference of the
periphytic spe- cies to attach on. Studies on glass attached
periphyton have also been used to measure the influence
of water current in streams on the structure of colonizing
periphytic algae (Ghosh and Gaur, 1998). Periphyton
attachment to glass
 I. Moreno-Garrido / Bioresource Technology 99 (2008) 3949–3964
slides (mainly diatoms) and population growth on this type
of surfaces has been studied by Brandini et al. (2001) in a
subtropical estuary during a whole year, permitting
researchers to compare differences caused by depth, light,
temperature and grazing pressure. Nayar et al. (2005) also
studied the settlement of periphytic algae in glasses in a
tropical estuary (Singapore), characterizing production in
terms of 14C uptake. Following those authors, planktonic
diatom species such as Skeletonema costatum and
Thalass- iosira rotula, together with cyanophytes such as
Synecho- coccus sp., dominated the assemblages. Glass-
immobilized filaments of Anabaena sp., have also been
used in hydrogen production experiments (Robinson et al.,
1986). These sup- port materials can be only used with
cells from species showing natural trend to attach o
aggregate (mainly dia- toms and cyanophytes), if the
material is not previously
treated or a co-flocculant is not used. Other carriers such
as Biolite® (a ceramic material) have been used for immo-
bilizing bacteria (Prieto et al., 2002), but no references
related to photosynthetic microorganisms have been found.
2.2. Active immobilization
Regarding active immobilization techniques, the use of
flocculant agents, chemical attachment and gel
entrapment should be distinguished.
2.2.1. Flocculant agents
Flocculant agents were primarily used in order to avoid
tedious and expensive centrifugation when algae are
intended to be removed from a liquid medium. Among
the commonly used flocculants, chitosan has been the
most widely employed. Chitosan is a linear amino
polysaccha- ride of b-D-glucosamine (2-amino-2-deoxy-b-
! by (1
D-glucan) units joined 4)-linkages (Oungbho
and Mu¨ ller, 1997). It is obtained through a chitin
(obtained from exo- skeletons of crustaceans) alkaline
deacetylation. This poly- saccharide presents positive-
charged amino groups, providing very interesting
properties for adsorbing nega- tive-charged particles and it
is demonstrated to be useful for a large number of
microalgal species (Lubia´n, 1989). This substance is
biodegradable, and thus can be used in harvesting of algae
for nutritional purposes. Large amounts of Euglena
gracilis have been removed from
media by Gualtieri et al. (1988), reaching a 96–98% reduc-
tion of suspended cells with 200 mg L—1 of chitosan at pH
7.5. The inconvenience of chitosan in immobilizing tech-
niques is its weak stability. Kaya and Picard (1996) tried
to solve this problem using high viscosity chitosan and
konjac flour (glucomannan, obtained from tuberous root
of the konjac tree – Amorphophalus konjac) in order to
enhance the stability of floccules immobilizing viable cells
of Scenedesmus bicelularis to use them in tertiary treatment
of wastewaters, but concluded that konjac flour did not
sig- nificantly modify the rheological properties of mixed
chito- san solutions. High viscosity chitosan gels (2% p/v)
showed a higher chemical stability in the experiments
described by
3951
these authors. It seems that the content of phosphate
anions in the media helps to maintain the stability of chito-
san gels. Chitosan can interfere in the growth of immobi-
lized algae: Moreira et al. (2006) found low growth rates
(increasing factor not higher than 4 after 3 days) of Phae-
odactylum tricornutum immobilized in alginate treated with
chitosan as additional hardener, while cells immobilized in
alginate beads hardened only with CaCl2 or SrCl2 showed
increasing factors of 31 and 76 times, respectively.
2.2.2. Chemical attachment
Chemical attachment presents some great disadvantages
when living cells are intended to be immobilized, because
the chemical interaction (mainly due to covalent bonding,
cross-linking – involving glutaraldehyde, for instance – or
photocrosslinkable resins) causes damages in cellular sur-
face and drastically reduces viability of cells. Ion attraction
is not so harmful to living organisms, but effectiveness of
this technique depends on pH and ionic strength of the
sur- rounding media (Codd, 1987). It is necessary to
remind that pH in the immediate medium of a living
microalgae can reach very high values in light, due to
photosynthetic metabolism. Nevertheless, some
experiments do not require active metabolism of cells, and
non-living organisms are used in chemical attachment
immobilization techniques. Thus, Seki and Suzuki (2002)
compared the adsorption capacity of two floc-type
biosorbents in order to remove an ‘‘inactivated’’ marine
microalga which is able to accu- mulate Cd and Pb from
aqueous media. The species used, Heterosigma akashiwo,
forms red tides in coasts of Japan, thus being an
inexhaustible and attractive material for the biosorbent,
following those authors. The two materials compared
were milk casein floccules (used as an immobiliz- ing
flocculant protein and as a biosorbent at the same time)
and glutaraldehyde. The technique developed comprised
as much as 67% of microorganisms present in the
cultures.
2.2.3. Gel entrapment
This method is the most widely used technique for algal
immobilization. Following Codd (1987), gel entrapment
can be performed by the use of synthetic polymers (acryl-
amide, photocrosslinkable resins, polyurethanes), proteins
(gelatine, collagen or egg white) or natural polysaccharides
(agars, carrageenans or alginates).
2.2.3.1. Synthetic polymers for gel entrapment. Blanco et
al. (1999) described the use of polysulphone (a
thermoplastic material) and epoxy resin entrapping cells
of the cyanobac- teria Phormidium laminosum, in order to
check the capacity of biosorption (and desorption, by the
use of 0.1 M HCl) of Cu(II), Fe(II), Ni(II) and Zn(II). Epoxy
resins consist of two components that react with each
other forming a hard, inert material. One of them is a
bisphenol and the other is epichlorohydrin. Toxicity of the
components makes epoxy resins not suitable for
entrapping living cells. In the exper-
iments described by Blanco et al. (1999), cells were previ-
ously dried (and thus killed) at 55 °C. Authors concluded
3952 I. Moreno-Garrido / Bioresource Technology 99 (2008) 3949–3964
that metal accumulation in the beads directly depends on
the entrapped cyanobacterial biomass. The same cyano-
bacterial species has been used by Garbisu et al. (1991) in
experiments designed in order to remove nitrate from water
by cells immobilized in polyvinyl and polyurethane foams.
In the described case, polyvinyl was hydrophilic (PV-50)
and the polyurethane was prepared from Hypol FHP
2002, a special grade of polyether polyisocyanate pre-
poly- mer that is mixed with a hydroxyl group. Entrapment
rap- idly leads to the death of cells, and only slow passive
colonization of foams was used for experiments involving
nitrate removal. These authors also found that recently
made foams were toxic to cells and inhibited even passive
colonization, possibly due to the leftover pre-polymers.
Toxicity disappears when foams were pre-autoclaved. Por-
phyridium cruentum cells have been immobilized in polyure-
thane foam by Thepenier et al. (1985) in order to produce
polysaccharides. After immobilization, these authors did
not detect any oxygen evolution, due to the high degree
of cellular destruction during the immobilization reaction.
At least four days had to pass before the immobilized col-
onies began to grow (after intensive rinses). The genus
Por- phyridium produces high amounts of exo-
polysaccharides, which could protect inner cells in
colonies or cellular groups from toxicity of pre-polymers.
The same could occur with mucilaginous cyanobacteria
(Streble and Kra- uter, 1987), protected by sheaths. These
sheaths can stick one to another forming colonies of
filaments. Thus, it is possible that inner filaments in a
colony could survive to toxicity of the pre-polymers of
polyurethane foams and after rinsing of pre-polymer
leftovers, and then begin to grow and colonize the foams.
In the experiment of Thepe- nier et al. (1985), after 10
days, oxygen production in batch cultures began to
decrease dramatically and stabilized at low values on day
30, but polysaccharide production began to increase from
day 40. This foam also permitted the release of cells to the
media. Kawabata et al. (1990) suc- cessfully immobilized
bacterial cells (Escherichia coli) on the surface of cross-
linked poly(N-benzyl-4-vinylpyridi- nium bromide)
containing styrene (BVPS). The cells remained alive and
were able to produce L-aspartic acid from ammonium
fumarate. Although this technique has not been applied
to microalgae, it seems to be suitable for immobilizing
living algal cells. Willke et al. (1994) also successfully
immobilized the bacteria Paracoccus denitrifi- cans in
polycarbamoylsulphonate (PCS), a very low toxic hydrogel
matrix. Survival of cells to immobilization proce- dure was
greater than 99%. Rarely, references concerning
immobilization with this technique can be found, but it
could be a very suitable technique in order to avoid the
sta- bility problems of alginate gels (over all in marine
environ- ments, as will be further discussed). Other
techniques, as those described in Jeon et al. (2002), imply
polyvinyl alco- hols and glutaraldehyde. This technique
does not seem to be recommendable if living cells are
intended to be immo- bilized. Silica gels can be used for
immobilizing microalgal cells. Rangasayatorn et al. (2004)
described a bioassay
involving Spirulina platensis in order to check cadmium
adsorption of immobilized cells in alginate and silica gel.
The latter technique is also called sol–gel technique, and
it is described in Weller (2000) for immobilizing antibodies.
Gelation of the sol occurs when pH of alkali silicate is car-
ried under values of 10. But in the case described by
Rangasayatorn et al. (2004), particles of silica gel obtained
after crushing were dried at 80 °C, and thus survival of
cells
is not a target point of the experiment. Nevertheless, cad-
mium adsorption of silica gel entrapping cells is as high
as in the alginate entrapped cells (near 100% of Cd adsorp-
tion after 1 h exposition, 10 mg L—1 being the initial Cd
concentration). Overnight drying (80 °C) of polysilicate
matrix seems to be a common procedure, as it is also used
by Stark and Rayson (2000) in a work comparing metal–
ion binding capacity of different immobilized materials
(Sphagnum peat, top soil, peat, compost peat, organic
peat, peat replacer, dead Chlorella vulgaris cells and cells
from Datura innoxia – a solanacean plant). In this case,
biomass from Chlorella was obtained by a commercial
algae-based biosorbent (Algasorb®), and this material
shown to be very
efficient in metal removal (but surpassed by peat and
organic compost peat) (Singh and Prasad, 2000).
2.2.3.2. Proteins for gel entrapment. Proteins are not
widely used in microalgal immobilization. In the reviews of
Rob- inson et al. (1986) and Papageorgiou (1987),
immobiliza- tion by proteins does not appear among the
techniques used. Only Codd (1987) mentioned egg white,
collagen and gelatine as examples of possible
immobilization tech- niques involving proteins. Other
organisms apart from microalgae have been immobilized
by these techniques (yeasts in hen egg white, for instance:
Kubal and D’Souza, 2004). The use of glutaraldehyde as a
cross-linker for egg white does not predict good results
with living microalgae, but could be used for dead cells.
2.2.3.3. Natural polysaccharides for gel entrapment. Gel
entrapment in natural polysaccharide matrixes is the most
widely used immobilization technique for microorganisms
in general (and microalgae in particular). Among them,
carrageenan, agar and alginate are the most employed.
Carrageenan is a collective term for polysaccharides
pre- pared by water alkaline extraction from some
Rhodophy- ceae (red algae), over all from the families of
Gigartinacieae and Solieriaceae. Carrageenan consists of
alternating 3-linked-b-D-galactopyranose and 4-linked-a- D-
galactopiranose units. It precipitates as a gel in the pres-
ence of cationic compounds: metal ions, amines, amino
acid derivates and water-miscible organic solvents (Tosa
et al., 1979). Different isomeric forms of carrageenan can
be found, in function of the algal specific origin. For
instance, i, j and k-carrageenan are primarily produced
by Aghardhiella subulata and the gametophyte and
tetrasp- orophyte of Chondrus crispus, respectively (Burdin
and Bird, 1994). Hardening processes in order to increase
the mechanical stability of carrageenan-based matrixes
have
 I. Moreno-Garrido / Bioresource Technology 99 (2008) 3949–3964
also been designed: Chamy et al. (1990) described a tech-
nique of hardening for j-carrageenan immobilizing Sac-
charomyces cerevisiae by treatment with Al(NO3)3. This
treatment revealed to increase cellular retention, a better
evacuation of gas from the reactor where immobilized
cells were cultured and an increase of 20% in ethanol
productiv- ity by immobilized yeasts. Bacteria have also
been success- fully immobilized (and stored during years)
in carrageenan matrixes (Cassidy et al., 1997). Travieso et
al. (1996) com- pared the nutrient removal capacity of
three microalgal species (C. vulgaris, Chlorella kessleri and
Scenedesmus quadricauda) immobilized in different
matrixes (including j-carrageenan hardened with 0.3 M
KCl). But the stability of j-carrageenan beads was quite
lower than that of Ca- alginate beads in the experimental
conditions. The j-carra- geenan beads were partially
destroyed after one week, the cells were liberated from
the beads and a loss of bead struc- ture was reported after
this time. Thus, the authors selected Ca-alginate instead of
j-carrageenan for the described nutrient removal
experiment.
Agar is a sulphated galactan obtained from some
species of red algae (mainly from the genus Gelidium,
Pterocladia or Gracilaria) (Burdin and Bird, 1994). Agar is a
thermo- reversible gel. The major gel forming component
of agar (agarose) consists of a linear chain of sequences of
(1–3)- linked-b-D-galactopyranosyl units and (1–4)-linkages
to 3,6-anhydro-a-D-galactopyranosyl units. Robinson et al.
(1986), Codd (1987) and Papageorgiou (1987) cite this
polymer as suitable for immobilizing microalgal cells. Agar
matrixes for immobilizing living cells present a capital lim-
itation: as it has been said, agar is a thermo-reversible gel.
Dissolved at concentrations that will provide a good
mechanical structure, agar melts around 85 °C and solidi-
fies between 35 and 40 °C. Species able to resist a short
thermal shock of this level should be selected for agar
immobilization. Attending to our experience,
temperatures over 30 °C could damage a wide variety of
(at least) marine microalgae (except cyanophytes and
some eukaryotic algae well adapted to marshes and salt
marshes environments, like some species from the genus
Dunaliella, Nannochlorop- sis or Tetraselmis). Agarose has
been used as an immobili- zation matrix type fixed-bed for
C. vulgaris in experiments of Cu(II) biosorption (Aksu et
al., 1998). In the agarose
particle formation described by the authors, the agarose
solution containing C. vulgaris cells was dropped into edi-
ble oil at 40 °C, although the temperature decreased after
dropping to 15 °C. The particles were then removed from
oily to aqueous phase by the addition of phosphate buffer
solution. These authors reported that metal adsorption
capacity of agarose and agarose–microorganisms system
was lower than that reported for Ca-alginate in the same
conditions. Nevertheless, the presence of immobilized cells
increased metal uptake capacity of both matrixes. No
efforts were made during this experiment in order to check
if immobilized cells were alive or not.
The most widely used polysaccharide gel for entrapping
living cells is alginate. Alginates constitute a family of
3953
unbranched binary copolymers of 1–4-linked-b-D-mannu-
ronic acid and a-L-guluronic acid in different proportions
and sequences (Smidsrød and Skja˚k-Braek, 1990), in
func- tion of the organism and tissue they are isolated
from. Commercial alginates are extracted from brown
algae, mainly different species from the genus
Laminaria (L. hyperborea, L. digitata, L. japonica) the
species Macrocystis pyrifera, Ascophyllum nodosum, Lesonia
negrescens or spe- cies of the genus Sargassum, although
all brown algae con- tain alginate in different proportions
reaching up to 40% of dry weight (Ertesva˚g and Valla,
1998). This substance has a structural function in those
organisms. Some bacteria can also produce alginates, as
Azotobacter vinelandii (Smidsrød and Skja˚k-Braek, 1990)
or Azotobacter chroococcum (Ert- esva˚g and Valla,
1998). Kidambi et al. (1995) describe the production of
alginate by several phytopathogenic species of the genus
Pseudomonas as a response to the addition of copper
salts. The capacity of producing this substance seems to
be an evolutive strategy against the human use of
copper-based phytosanitary products (high affinity of
alginates for copper will be further discussed in the text:
Jang et al., 1995c), and additionally enhances bacterial
ability of adhesion to solid surfaces (Boyd and Chakra-
barty, 1995).
A major advantage of alginate gel entrapment is that
immobilized cells do not suffer extreme physical–chemical
condition changes during the immobilization process. Per-
meability, null toxicity and transparency of formed matrix
imply a very gentle environment for immobilized cells
(Smidsrød and Skja˚k-Braek, 1990; Arau´ jo and
Andrade Santana, 1996). Alginates are used in industry as
viscosifi- ers, stabilizers and gel-formers, film-formers or
water-bind- ing agents (Ertesva˚g and Valla, 1998).
The polymer is soluble in cold water and forms
thermostable gels. Gelation of monovalent salts of this
polysaccharide (normally Na- alginate) dissolved in water
occurs when droplets of a mix- ture of cells (or enzymes)
and alginate monovalent salts are mixed with a solution
containing gel-forming ions. Gel for- mation is a very quick
process. The most common cation used to form alginate
gels is Ca2+. When the gel is intended to be re-dissolved,
media with sodium citrate (Hertzberg and Jensen, 1989) or
phosphate (hexametaphosphate could be also used) can be
used: calcium cations can be seques- tered by soluble
anions or can be substituted in the matrix by monovalent
cations in order to destabilize the structure. Na-alginate
does not dissolve appropriately in sea or salt water.
Hertzberg and Jensen (1989) described a protocol for
immobilizing marine microalgae in alginate beads: Sodium
chloride in adequate proportions for reaching sea- water
salinity values and Na-alginate must be previously
(separately) dissolved in distilled water and mixed after
dis- solving. Marine microalgal cells can be then added to
the salty Na-alginate and dropped to the Ca 2+ seawater
solu- tion. These authors (Hertzberg and Jensen, 1989)
immobi- lized seven marine microalgal species and
checked the growth of entrapped cells. Pane et al. (1998)
developed a study of the viability of Tetraselmis suecica
immobilized
3954 I. Moreno-Garrido / Bioresource Technology 99 (2008) 3949–3964
in Ca-alginate beads and compared the cellular growth of
immobilized and free cells, finding that the latter was
higher, although chlorophyll content per cell in
immobilized cells was higher, possibly as a response to
shading condi- tions provided by immobilization. Moreno-
Garrido et al. (2005) checked the growth of immobilized
cells and Ca-algi- nate bead stability in a 17-day
experiment involving 11 mar- ine microalgal species, finding
that the stability of beads can also depend on the
immobilized species. Attending to the resistance of the
species to toxics, growth rates when immo- bilized and
maintenance of the bead structure, those authors
selected T. suecica as a good target organism for toxic
accumulation experiments and P. tricornutum or
Chaetoceros gracilis as good target organisms for toxicity
bioassays involving Ca-alginate immobilized microalgal
cells. Loss of stability is a limitation for the use of Ca-algi-
nate matrixes in sea, estuarine and brackish waters. The dis-
solved monovalent cations (mainly sodium) in the media
can substitute divalent cations and results in the loss of
structure of matrixes. When shaking is applied, Ca-alginate
beads of 3–5 mm can dissolve in seawater in 24 h (unpub-
lished data). In order to avoid these problems, some
recent strategies have been developed. Moreira et al.
(2006) per- formed a study of alginate bead stability
checking different proportions of two types of alginates
(isolated from M. pyrifera and L. hyperborea) hardened
with Ca2+ or Sr2+, immobilizing cells of P. tricornutum.
Strontium divalent cations, as well as Ba2+ (Santos-Rosa et
al., 1989), have been proposed as hardeners for alginates
providing more stable gels in sodium and phosphate-
enriched media. In a study performed by Widerøe and
Danielsen (2001), viability of entrapped cells in Ba, Ca and
Sr-alginates was checked on a human leukemic T cell line.
Ba-alginate provided lower cellular growth rates, while
cells exposed to calcium and strontium grew in a similar
way. In any case, inhibition of growth of barium exposed
cells was less than 20% in com- parison to the others. In
support of the work described in Moreira et al. (2006),
those authors confirmed that the beads prepared using
4.9% of alginate from L. hyperborea and hardened with a
4% strontium solution were found to be the most stable
and the most suitable for microalgal growth when they
were exposed to natural field conditions. Small beads of
Ca-alginate encapsulating bioactive sub- stances can be
produced by emulsification and internal- ionotropic
gelation (Poncelet et al., 1999). This method pro- poses
dispersion of alginate and a calcium salt mixture (cit- rate)
in canola oil by stirring. Then, certain volume of canola oil
containing glacial acetic acid is added to the emulsion.
After few minutes, this oil-bead suspension is added to a
calcium chloride solution in order to stabilize beads and
the oil is removed by washing with a surfactant. This
technique has also been used for Ca-alginate encapsu-
lating DNA (Quong et al., 1998) and it was improved by
Chan et al. (2002) in order to avoid possible clumping of
microspheres and excessive waste of calcium salts. Microen-
capsulation has also been developed by co-use of alginate
and polylysine (Jen et al., 1996). Other microencapsulation
strategy for microalgae is described by Joo et al. (2001): a
mixture of 2% sodium carboxymethyl cellulose, 2% CaCl2
and microalgae is stirred in 0.8% sodium alginate till the
capsules are formed. After washing, the capsules are sub-
merged in 2% CaCl2 for 20 min for the purpose of harden-
ing. Those authors affirm that capsules had a better
resistance when compared with beads manufactured by
the ‘‘traditional’’ way (sodium salt dissolved and dropped
in calcium solution), but do not report numerical data. Algi-
nate is relatively cheap (overall when compared with other
immobilizing matrixes), non-toxic, transparent enough, rel-
atively stable and easy to use. As stability problems in
sodium or phosphate rich media have been avoided, this
immobilization technique seems to be a very promising tool
in microalgal biotechnology.
3. Effect of immobilization on microalgal cells
Toxicity of some immobilizing techniques on cells has
been discussed above (Blanco et al., 1999; Garbisu et al.,
1991; Thepenier et al., 1985; Rangasayatorn et al., 2004;
Urrutia et al., 1995). Overall, pre-polymers of synthetic
foams and resins use to be highly toxic for microalgae.
When not so toxic immobilization techniques are applied,
immobilized microalgal cells show, as minimum, a longer
lag period when these are compared with free cells (Mal-
lick, 2002; V´ılchez et al., 1997). Some authors consider
this similar to that occurring in free cultures (Lukavsky et
al., 1986). After this period the specific growth rate (k)
can be very similar in free and immobilized cells (Lau et al.,
1998; Mallick, 2002) although some reports confirmed
that it is lower than in the latter case: Pane et al. (1998)
reported a lag-phase of 5–6 days for immobilized T.
suecica in Ca- alginate beads. It is common to find higher
chlorophyll production in immobilized cells (Lau et al.,
1998; Pane et al., 1998; Robinson et al., 1986). This is a
phenomenon that should be taken into account when
estimations of bio- mass by chlorophyll are intended to be
done. Hertzberg and Jensen (1989) demonstrated that the
maximum amount of cells in culture could be higher for
immobilized algae (P. tricornutum, in this case) than for
free cells. The type of alginate and, overall, the initial
cellular concentra- tion seem to be very important in
maximum cellular den- sity that can be reached in
immobilized cultures: the higher initial cellular density,
the higher final cellular den- sity to be reached in cultures.
The latter phenomenon can be explained because growing
cells inside the immobilizing matrix tend to form colonies.
Maximum number of cells per colony must be limited by
some factors as nutrient dif- fusion or light (colonies are
smaller in the inner part of the beads than in the surface).
Thus, the higher number of col- onies, the higher number
of cells in the culture. In the same work, the authors found
that S. costatum did not thrive so well when immobilized:
very high initial cell densities were needed to obtain stable
cultures. Chaetoceros ceratosporum and Thalassiosira
pseudonana also grew well in those exper- iments, while
Emiliania huxleyi, Amphydinium carterae and
 I. Moreno-Garrido / Bioresource Technology 99 (2008) 3949–3964
Scripsiella trochoidea did not grow when immobilized,
although some of them appear to be metabolically active
because during long periods of time these exhibited red
autoflouorescence. Moreno-Garrido et al. (2005) per-
formed a 17-day experiment on Ca-alginate beads
immobi- lizing 11 different species belonging to eight
different microalgal taxonomic classes: Nannochloropsis
gaditana (Eustigmatophyceae); Heterocapsa sp.
(Dinophyceae); Rhodomonas salina (Cryptophyceae);
Isochrysis aff. galbana (Prymnesiophyceae); T. pseudonana,
C. gracilis, P. tricor- nutum and S. costatum
(Bacillariophyceae); Tetraselmis chui (Prasinophyceae); P.
cruentum (Rhodophyceae) and Dunaliella salina
(Chlorophyceae). Growth of cells and sta- bility of beads
were checked during the performance of the experiment.
As Hertzberg and Jensen (1989) studied in the work cited
before, Moreno-Garrido et al. (2005) found that
Skeletonema costaum did not grow when immobilized.
Het- erocapsa sp. also did not grow (as far as I know, there
are no good results of immobilizing photosynthetic
dinoflagel- lates in the literature), and N. gaditana
resulted in unstable beads in experimental conditions. The
rest of the assayed species showed growth inside the
beads, reaching sooner or later a stable number of cells
(stationary equilibrium phase), except C. gracilis and I.
galbana, which on day 17 still showed continuous growth.
Joo et al. (2001) immobi- lized four microalgal species
(Chlorella minutissima, Pav- lova lutheri, Haematococcus
pluvialis and Dunaliella bardawil) by two methods, and
compared the growth of immobilized and free cells. The
authors studied that cells immobilized by encapsulation by
the use of 2% sodium carboxymethyl cellulose, as
described before, grew better for the four species in
bubble column bioreactors. Inhibi- tion, enhancement or no
growth differences between free and immobilized
microalgal cells have been reported in dif- ferent works,
such as Mallick (2002). Lukavsky et al. (1986) studied the
morphology of immobilized cells in agar and Ca-alginate.
Following those authors, which designed an experiment
involving S. quadricauda and C. kessleri, cells suffered an
initial lag phase (considered as comparable to that
reported for free cells), followed by one to three divisions.
After that, cell division stops but giant cells in
C. kessleri (which, following these authors, are common
in suspension exposed to sublethal conditions) are not
usu- ally produced: cell shape and size do not show visible
changes, organelles are recognizable and grains of starch
appear inside the cells. In S. quadricauda, however, mon-
strous cells use to appear. In any case, cells immobilized
in agar were less affected than those immobilized in algi-
nate. For other immobilized microorganisms, many
changes in size and shape have been reported (Cassidy
et al., 1996). Nevertheless, Hatanaka et al. (1999) did not
find differences in shape or chlorophyll content between
immobilized and free cells of Dunaliella parva. In general,
alterations in shape of colonies are more frequent than
alterations in cell shapes (Mallick, 2002) when microalgal
cells are immobilized. V´ılchez et al. (1997) studied the via-
bility of agar-immobilized Chlamydomonas reindhartii
cells,
3955
confirming that maximum growth was noticed near the
sur- face of the beads. Diffusion of nutrients must also be
important, apart from light, because no-photosynthetic
organisms such as the bacteria Nitrosomonas europea show
the same pattern of growth in carrageenan beads (Wijffels
and Tramper, 1989). Attending to V´ılchez et al. (1997),
these authors confirmed the presence of a thin skin of
poly- mer surrounding colonies of C. reindhartii and
attribute the constant number of cells to the equilibrium
between cellu- lar growth and release from the beads. The
use of co-immo- bilized systems (Mun˜ oz and Guieysse,
2006) can improve the growth of immobilized organisms,
avoiding gas diffu- sive problems inside the immobilizing
matrixes, as will be discussed in Section 4.8.
Zeglinska (2005) (personal communication) reported
active escape of filamentous cyanobacteria (Nodularia
spumigena) from alginate beads. Normally, motility of cells
is not strong enough to provide escape from beads, but in
the case of slithering filamentous cyanobacteria this
move- ment seems to permit algae to escape from
immobilizing matrixes.
4. Current use of immobilized microalgal cells
Most frequent current uses of immobilized algal cells
are the culturing for metabolite production, improvement
of culture collections handling, obtaining of energy (via
H2 or electricity power), nutrients, metal or organic
pollutant removal from aquatic media, measurement of
toxicity and co-immobilization system production for
different purposes.
4.1. Culturing for metabolite production
Microalgae has been widely used as biocatalysts in bio-
transformation and de novo biosynthesis (Borowitzka and
Borowitzka, 1988). Different types of bioreactors are able
to be used in microalgal biotechnology for different pur-
poses (Mallick, 2002), immobilization techniques being a
response to the problem of biomass losses in outflows
(Nakasaki et al., 1989). Hatanaka et al. (1999) described
a reduction process mediated by Ca-alginate immobilized
D. parva, from hydroxyacetone to (R) 1,2-propanediol,
which is a highly priced glycol. Production of propanediol
by immobilized cells was found to be similar to that
con- firmed for free cells. Tripathi et al. (2002) described
bio- transformation of phenylpropanoid compounds to
HPLC-detectable vanilla flavour metabolites by free and
Ca-alginate immobilized cells of H. pluvialis. Vanillin accu-
mulation in immobilized cells is higher than in free cells
(no explanation of this phenomenon is given in the text,
but references from other works with different
microorganisms reviewed by those authors confirmed that
it is not a rare case). Transformation of nitrite into
ammonium in a med- ium containing L-methionine-D,L-
sulphoximine (MSX), and the inhibitor of glutamine
synthetase, has been described by Santos-Rosa et al.
(1989) by the use of a Ba-alginate immobilized mutant
strain of C. reindhartii.
3956 I. Moreno-Garrido / Bioresource Technology 99 (2008) 3949–3964
Maximum volumetric activity was 2700 lM h—1 in a packed-
bed reactor. Ba-alginate was used in this case because a
previous study demonstrated that cell leakage was minor
than that found in the same conditions for
Ca-alginate. Thepenier et al. (1985) studied production of
polysaccharides by the unicellular red algae P. cruentum
immobilized in polyurethane foams. As it has been com-
mented in Section 2, jelly colonies of microalgae could sur-
vive to the toxicity of foam pre-polymers. At the beginning
of the tests, no oxygen evolution was observed in immobi-
lized cultures, possibly due to high degree of cellular
destruction, although after many flushing rinses, survival
colonies began to grow again. When culture reaches sta-
tionary phase, production of polysaccharides was notice-
able (measured as an increase of medium viscosity).
4.2. Culture collection handling
Handling of a large microalgal culture collections is a
tedious, time-consuming task. Long-term storage is a very
interesting topic related to microalgae culture handling,
saving human and economic resources. Chen (2001)
reported the maintenance of physiological activities of Ca-
alginate entrapped S. quadricauda cells after three years
of storage in darkness at 4 °C without culture medium,
with the sole starving symptom of pyrenoid disappearance.
After
replacing long-term stored beads in fresh media, the num-
ber of immobilized coenobia increased by near by 40 times
in four weeks (and pyrenoid was re-constructed). The same
author (Chen, 2003) repeated the experience with I. galbana,
achieving good results after one year of storage. Hertzberg
and Jensen (1989) stored Ca-alginate cultures of P. tricornu-
tum during one year in diminished light at 4 °C and con-
firmed immediate production of oxygen in a Clark-type
electrode when beads were transferred to light. Lukavsky
(1988) immobilized 31 strains of cyanophytes and eukary-
otic algae in 2% agar (inside the warm agar, not seeded on
the surface), and kept cultures at reduced temperature
(10 °C). After 32 months most of the strains were still met-
abolically active. Joo et al. (2001) immobilized four micro-
algal species (D. bardawil, C. minutissima, P. lutheri and H.
pluvialis) in Ca-alginate capsules and beads in order to get
highly dense cultures. Those authors achieved higher con-
centration of encapsulated cells than in the case of
cultures
with free cells or bead-type immobilized cells. Response of
D. bardawil and H. pluvialis in bubble column reactors
was specially high, reaching values five times higher than
that for free cells. Romo and Pe´rez-Mart´ınez (1997)
also stored Pseudanabaena galeata in Ca-alginate beads for
14– 18 months. Recuperation of cells in fresh cultures was
not different to that checked for standard cultures.
Hertzberg and Jensen (1989) also pointed out that
immobilization is an interesting way to produce colonies
from disperse cul- tures. Extending their affirmation, the
technique could also be used as a way of producing clonal
colonies from natural microalgal assemblages in order to
isolate scarce cells of a determinate species via posterior
micromanipulation. Novel
techniques have been developed in order to facilitate
culti- vation of algae. Nowak et al. (2005) designed a
system based in a 96-well plate where a membrane filter,
which immobilizes algal strains, constitutes the bottom of
each well. This system allows culturing of several
microalgal strains with less effort, time and money.
4.3. Obtaining energy (electricity or hydrogen)
It is said that hydrogen is the fuel of the future due to
its high conversion efficiency, recyclability and non-
polluting nature (Das and Vezirog˘lu, 2001).
Bioproduction of hydro- gen has demonstrated to be
environmental friendly and less energy consumer when
compared with thermochemical and electrochemical
processes (Kapdan and Kargi, 2006). Some algae are able
to produce hydrogen in stress conditions (Melis, 2002)
such as the deprivation of sulphur-nutrients in green
algae, which causes a reversible inhibition of the oxygenic
photosynthetic processes. Lack of sulphur pre- vents the
protein synthesis and algae cannot perform the turnover
of specific photosystem-II reaction centre protein. Then,
photochemical activity of PSII declines in the absence of
sulphur and oxygen is released to the media at lower rates
than O2 consumption, achieving anaerobic algal cultures
in the dark if they are sealed and sulphur- deprived. The
same occurs when O 2 is directly removed from the media.
Some experiments related to this topic have recently been
developed on C. reindhartii and other species (Dante,
2005; Laurinavichene et al., 2006; Markov et al., 2006;
Polle et al., 2002). C. reindhartii seems to be a promising
organism for further studies in this field. Co-fir- ing of
microalgal biomass with coal is another power-gen-
erating procedure. CO2 produced in power-plant fuel gas
can be used to improve microalgal growth. Microalgal bio-
mass obtained in this way is burned again in the same
power plant (Kadam, 2002). This could be a suitable way
of reducing the high amounts of carbon dioxide (around
26 Gt per year, following the same author) produced by
humans and helps the high CO 2 producing countries (The
United States generates 5.7 Gt, it means about 22% of
worldwide anthropogenic emissions) to reach CO2 levels
in agreement with the Kyoto Protocol (van Vuuren et al.,
2006). In any case, molecular hydrogen production seems
to be the target in microalgal-based energy production, as
medium and long-term endurance of thermal electric plants
based in firing materials should be severely studied by
envi- ronmentalists and governments, due to the
recognized pol- lutant capacity of those types of plants.
Some efforts had also been made during the eighties in
order to generate electricity by photovoltaic cells
containing photosynthetic biocatalysts, as reviewed by
Papageorgiou (1987), but no recent references have been
found with respect to this topic.
4.4. Nutrient removal
Cultivation of algae in wastewater containing nutrients
offers the combined advantages of treating water and pro-
 I. Moreno-Garrido / Bioresource Technology 99 (2008) 3949–3964
duction of algal biomass, which can be industrially
exploited (Mallick, 2002). A limitation in these processes
is the high price and time-consumption of centrifugation
or filtration. Immobilization techniques have tried to solve
these problems (Proulx and de la Noue, 1988; Tam and
Wong, 2000; Travieso et al., 1996; V´ılchez and Vega,
1994, 1995; V´ılchez et al., 2001). Immobilized P. laminosum
in polyurethane and polyvinyl foam has been used in con-
tinuous flow reactors designed in order to remove nitrate
from water (Garbisu et al., 1991). Comparison was done
between adsorbed and entrapped cells. As it has been
said, pre-polymers of polyurethane foams are toxic to
cells. Those authors concluded that entrapment was not a
suit- able method for immobilizing living cells in those
matrixes, and experiments were only performed on
adsorbed cells. In the work described, only nitrate was
removed from the medium by adsorbed cells in light,
and nitrite or ammo-
nium was not released (less than 1 mg L—1 of both
nutrients in all the samples). Using chlorophyll as an
indicator of
microalgal biomass (this can lead to errors if a correlation
between chlorophyll and number of cells is done based on
free cells, as it has been commented), immobilized P. lami-
nosum removed nitrate at 1.1 lM mg—1 chl h—1, while free
cells removed 2.6 lM mg—1 chl h—1 in batch cultures. In
reactors (no free cells can be used here), removal of nitrate
had an efficiency of 90% during three months. The authors
comment that a packed bed is a more adequate system than
a fluidized reactor, because in the latter tumbling and col-
lisions between particles result in desorption of cells. At
low concentrations, consumption of nutrients by immobi-
lized microalgal cells is limited, possibly due to limitation
in nutrient diffusion through immobilizing matrixes. Thus,
Garbayo et al. (2000) found that Ca-alginate immobilized
C. reindhartii cells did not consume nitrate when concen-
tration of this nutrient was below 0.14 mM, while free cells
almost fully consumed it. The authors postulated that
nitrate consumption inhibition could occur due to the pres-
ence of nitrite. Mallick (2002) reviewed some works stating
that exponentially growing microalgal cells removed more
nutrients (nitrate and phosphate) than aged cultures.
Strains of Scenedesmus intermedius and Nannochloris sp.
were isolated from different sources of pig manure in order
to design a depuration system for the removal of macronu-
trients from farm wastewaters (Jime´nez-Pe´rez et al., 2004).
Nitrogen and phosphorus uptake from free and Ca-algi-
nate immobilized cells were compared in this work, finding
to be slightly lower in immobilized systems (but in the same
order of magnitude). Nevertheless, the uptake of nutrients
by those autochthonous species was markedly higher than
that measured for common commercial species, probably
due to a better adaptation to high nutrient concentrations.
In seawater selection of resistant taxons for depuration
assays is also an important topic. Thakur and Kumar
(1999) selected D. salina in order to perform nutrient
(ammonium, nitrate and phosphate) uptake experiments.
This alga is halotolerant and could be used in waters of a
wide range of salinities. In this concrete experiment, immo-
3957
bilized cells always removed more nutrients than free
cells. After 36 h, the levels of removed nitrate, ammonium
and phosphate were 62%, 42% and 65% of initial
concentra- tions, respectively. Dunaliella species, thus,
seems to be a good target organism for accumulation
assays in waters of variable salinity (Do¨ nmez and Aksu,
2002). Immobiliza- tion techniques improve cost-effective
phycoremediation processes (Olgu´ın, 2003). The removal
of nutrients from animal wastewaters is a very interesting
topic as many underground water bodies in several
regions of the world are threatened of suffering
eutrophication (Travieso Co´ r- doba et al., 1995a,b).
Recently, co-immobilized systems have been developed in
order to enhance nutrient removal from wastewaters (see
Section 4.8).
4.5. Metal removal
Vegetal biomass (Spinti et al., 1995) and especially
macro- (Valdman et al., 2001; Al-Rub et al., 2004) and mic-
roalgae are known to accumulate high amounts of metal
from their environment (Stark and Rayson, 2000; Travieso
et al., 2002). This capacity has been exploited for different
purposes (Aksu and Ac¸ikel, 1999; Burdin and Bird,
1994; Greene and Bedell, 1990; Hashim et al., 2000). Silica
immo- bilized-algal biomass (Pilayella littoralis) has been
pro- posed to be used as a biosorbent for metal pre-
concentration before measuring in analytical devices
(inductively coupled plasma optical emission
spectrometry) (Carrilho et al., 2003). But the main purpose
of the use of immobilizing algae binding metals has been
detoxification and metal recovery (Greene and Bedell,
1990). The latter is possible because a great part of the
metal bound to cel- lular surfaces and immobilizing
systems is able to be des- orbed via acid treatment.
Other resins, such as
Amberlite®, have been used in order to immobilize micro-
organisms set aside for metal removal (Baytak and Tu¨
rker, 2005). Sorption of metals on algae seems to be not
only a simple adsorption process, but also an exchange of
ions, where Ca2+ is often replaced (Crist et al., 1994).
Binding of metals to algal surface occurs in living and
non-living algae (Greene and Bedell, 1990), cell surface
area being a major parameter in the uptake of metals by
microalgae (Khoshmanesh et al., 1997). Dead cells can be
very efficient in accumulating metals (Do¨ nmez et al.,
1999). Blanco et al. (1999) used biomass of the
cyanobacterium P. laminosum immobilized in
polysulphone and epoxy resin beads for Cu(II), Fe(II),
Ni(II) and Zn(II) sequestering. They found that the
amount of biosorbed metal increased with biomass and the
amount of metal available. Biosorption–desorption (acid-
mediated) cycles with this immobilized system main- tain
the efficiency after at least 10 cycles. Copper is selec-
tively adsorbed by alginates (Alhakawati and Banks,
2004; Jang et al., 1995b,c; Nestle and Kimmich, 1996). Jang
et al. (1995a) considered the possibility of adding copper-
sequestering agents to alginate gels in order to enhance
copper recovery from the media. Addition of sodium poly-
styrenesulphonate (NaPSS) to the sodium alginate
resulted
3958 I. Moreno-Garrido / Bioresource Technology 99 (2008) 3949–3964
in an increase of copper adsorption, but the addition of
cells of the cyanophyte Microcystis sp. resulted in an even
better result. Accumulation of metals by immobilizing sys-
tems containing microalgae seems to present two phases
(Malik, 2004). Garnham et al. (1992a) developed an exper-
iment involving immobilized Chlorella salina in Ca-alginate
beads, accumulating radioisotopes of metals ( 60Co, 54Mn
and 65Zn). The authors found a rapid biosorption non-
dependent on light, temperature or metabolic inhibitor
(carbonylcyanide n-chlorohydrazone: CCCP), followed by
a slower accumulation phase depending on cellular
metab- olism. Similar results have been found by Moreno-
Garrido et al. (1998, 2002): Part of the metal accumulated
cannot be washed by solutions containing high
concentrations of che- lating EDTA. It is supposed that this
metal is absorbed into the cells by active or passive
methods (Greene and Bedell, 1990) and not adsorbed to
the cell surface or the immobilizing matrix. Packed-bed
columns containing immobilized cells seem to be very
efficient in the removal of metals from aquatic media
(more than fluidized-bed or air-lift reactors, Moreno-
Garrido et al., 2002). Aksu et al. (1998) studied biosorption
of copper(II) in Ca-alginate and agarose immobilized C.
vulgaris. Those authors did not find a significant increase
in metal adsorption due to the presence of immobilized
algae. But Moreno-Garrido et al. (2002) found great
differences in Ca-alginate beads system immobilizing cells
of N. gaditana when compared with systems without
immobilized cells: Beads containing cells accumulate
practically all Cu in media (as free cells did) and 80% of Zn,
while beads without cells accumulated near by 80% of Cu
but did not accumulate measurable amounts of Zn. Similar
results can be found when dead cells were employed: no
Zn was removed when dead cells are immobilized in Ca-
alginate beads.
Isolating microalgal strains from polluted waters is a
suitable tool for selecting metal-tolerant and highly accu-
mulating cells. Khattar et al. (1999) isolated a strain of
Anacystis nidulans from polluted sites. This strain was able
to grow in a medium up to 100 lM of chromium, being
able to accumulate 43 nM of Cr per mg of microalgal pro-
tein (as estimative of biomass) immobilized in agar (free
cells were able to accumulate 35 nM of Cr) in the experi-
mental conditions. Akhtar et al. (2004) reported recovery
of nickel from electroplating industrial effluents by the
use of C. sorokiniana immobilized in loofa sponges by nat-
ural adsorption. These authors achieved a maximum C.
sorokiniana biomass of 261 ± 22 mg g—1 of dry sponge
after 24 days of incubation. Adsorption was always higher
in immobilized systems than that measured for free cells
(loofa sponges without immobilized cells adsorbed a small
amount of this metal). After adsorption, addition of acids
such as HCl and H2SO4 resulted in liberation of more
than 99% of adsorbed metal. Moreno-Garrido et al. (2005)
used
Tetraselmis chuii, a very stress-resistant taxon, as an immo-
bilized organism to remove Cu and Cd from marine waters.
After 24 h exposition, all Cu and 20% Cd were removed
from the media by Ca-alginate immobilized populations
of T. chuii. Rangasayatorn et al. (2004) found a maximum
Cd accumulation capacity of 70.9 mg g—1 biomass for
immobilized cells in Ca-alginate matrixes. Cells were suc-
cessfully used during five adsorption–desorption consecu-
tive processes. During these cycles, system adsorption
capacity was reduced from around 94% to near by 66%
of total cadmium. Microalgal living cells used to accumu-
late higher levels of metals than dead microalgal biomass
(Moreno-Garrido et al., 1998). First, divalent cations
became less soluble at high pH values. Active (photosyn-
thetic) cells provide a high-pH environment in the immedi-
ate surface cellular layer, increasing thus adsorption to the
cellular surface. Second, there are processes of absorption
(dependent on cellular metabolism) of metals by microal-
gae (Garnham et al., 1992a,b; Moreno-Garrido et al.,
2002), slower than the adsorption process that increases
the capacity of living algae to accumulate those pollutants.
Noble metals can also be accumulated by algae and
selectively eluted from them (Guo et al., 2000). Gee and
Dudeney (1987) described the adsorption of gold from a
dissolved metal mixture by C. vulgaris and S. platensis
immobilized in Ca-alginate. In this case, selective desorp-
tion of Fe and Au was performed by acidic pH adjustment
and acidic thiourea addition, respectively. Guo et al. (2000)
also described the uptake of neodymium, a rare lantha-
noid, by living and fossil (570 million years old) algae.
4.6. Organic pollutants removal
Aksu (2005) made a review about the biosorption of
organic pollutants and found only few data on microalgae.
In the same year, Aksu and Tezer (2005) studied the
bio- sorption of three reactive dyes onto dead biomass of
C. vul- garis, finding that dye sorption was highly
dependent on pH: the optimum pH value for adsorption
was 2.0. Temper- ature also affected the process in a
proportional inverse way. Oh et al. (2000) immobilized
yeast cells in polyure- thane foams in order to absorb and
degrade oil on water surface. Few works describing algal
capacity for degrading oil have been published. A recent
paper from Chaillan et al. (2006) described the appearance
of cyanobacterial mats (Phormidium animale) in petroleum-
polluted site. They con- cluded that there is no evidence of
biodegradation of crude oil by cyanophytes, but from
other organisms present in the mat formed by
Phormidium involved in degradation pro- cesses, Biofilm-
forming bacteria covering macroalgae can also be able to
degrade oil (Radwan et al., 2002).
C. sorokiniana in aggregation with bacteria was able to
successfully remove salicylate from a photobioreactor in
an experiment described by Mun˜ oz et al. (2006). The
role of each organism in the biosystem is not clear
enough, but degradation seems to be performed by the
bacterial part of the symbiosis system. Normally, organic
pollutants are more easily degraded by bacteria than by
algae (Prieto et al., 2002). Hydrocarbons, nevertheless,
have been reported to be degraded by Ca-alginate
immobilized color- less P. zopfii (Suzuki et al., 1998;
Yamaguchi et al., 1999).
 I. Moreno-Garrido / Bioresource Technology 99 (2008) 3949–3964
In free and immobilized systems, 100% of a mixture of
tetradecane, pentadecane and hexadecane (0.1% (v/v) each
one) was degraded. Special care in the experimental
design was taken in order to ensure that evaporation was
not the cause of the elimination of hydrocarbons from the
media. Every 14 days, cells were washed and re-used,
resulting in identical response from immobilized cells.
Other cases of trophic use of organic pollutants seem to
be the Chryso- phyte Ocrhomonas danica (Semple and
Cain, 1996; Semple, 1998), which is able to
heterotrophically grow on phenol and phenolic mixtures.
No reports about immobilization of this species have
been found.
4.7. Measuring toxicity
Bulk chemical characterization of waters and sediments
do not provide information about toxicity (Munawar and
Munawar, 1987), except in cases of extreme pollution.
Thus, bioassays have been designed in order to detect tox-
icity of effluents, sediments or substances on organisms.
Microalgae have been found to be sensitive organisms to
different pollutants (Leo´ n et al., 2001; Radix et al.,
2000) in toxicity bioassays (Bitton and Dutka, 1986),
possibly due to their high surface/volume ratio. Their key
role in freshwater and marine aquatic trophic nets implies
the necessity of developing suitable toxicity tests in order
to count with efficient tools to be used by researchers and
authorities when it is required. In situ experiments have
been designed in order to increase environmental relevance
of toxicity tests (Moreira dos Santos et al., 2002, 2004;
Moreira et al., 2006; Munawar and Munawar, 1987), as
manipulation of samples when carried to the laboratory
is then avoided and natural conditions of light, tempera-
ture or pH fluctuations are maintained. Bozeman et al.
(1989), in a pioneering work, compared the toxicity of
seven pollutants of different origin (Cd, Cu, Glyphosate,
Hydrothol, Paraquat, pentachlorophenol and sodium
dodecyl sulphate) to free and immobilized cells of the
green microalga Selenastrum capricornutum (currently
Pseud- okirchneriella subcapitata), suggesting the
possibility of the use of immobilized systems in in situ
toxicity experi- ments. Following those authors,
differences in toxicity for free and immobilized algae
varied from no significant dif- ferences for copper and
pentachlorophenol to nearby four times more sensitive for
free cells in the case of Glyphosate or Paraquat. Admiraal
et al. (1999) performed an experi- ment on sand and
natural glass-attached microbentic assemblages of algae
and bacteria in a metal polluted stream in the river
Dommel (Belgium). The authors com- pared the sensitivity
of those assemblages to zinc, finding different sensitivities
in function of the origin of the assem- blages (the most
polluted origin, the lower sensitivity). Pro- tection against
toxicity in immobilized cells is reported in different works
(Cassidy et al., 1996). Awasthi and Rai (2005)
demonstrated lower inhibition of nitrate uptake in
S. quadricauda immobilized (with respect to free cells) when
exposed to Ni, Zn or Cd. In this work, no metal measure-
3959
ments were performed in the media, and the easiest
expla- nation is the removal of part of the metals by the
entrapping matrix, being thus less available for cells. But
removal of toxicants by immobilizing matrixes would not
explain all cases of less toxicity of immobilized cells. Sur-
factants are not so selectively adsorbed by Ca-alginates.
Moreno-Garrido et al. (2007) found less toxicity for immo-
bilized cells of P. tricornutum exposed to sediments spiked
with surfactant lineal alkylbenzene sulphonate (LAS) than
for free cells. Lower diffusivity of toxicants in beads (Jang,
1993) should also explain, at least in part, lower toxicity to
entrapped cells. This species (P. tricornutum) seems to be
a good microalgal species to be used in toxicity tests
involv- ing immobilized algae. It is one of the species
suggested by ISO (1995) to test water quality by the use of
growth inhi- bition tests: it is cosmopolitan, with low
nutrient require- ments, fast growth and good sensitivity
to toxicants. Additionally, it has been used by several
authors in free and immobilized toxicity tests (Cid et al.,
1995; Kos- akowska et al., 2004; Mayasich et al., 1986;
Moreira et al., 2006; Moreira dos Santos et al., 2002;
Morelli and Pratesi, 1997; Morelli and Scarano, 2001;
Moreno-Garrido et al., 2007; Overnell, 1975; Pavlic´ et al.,
2005; Wiegman et al., 2002). Immobilization by gel
entrapment is also a very interesting topic for in situ
microalgal experiment design because it provides
protection to microalgae in front of grazers (Cassidy et al.,
1996; Faafeng et al., 1994). Mic- roalgal grazers cannot be
easily eliminated from waters or sediments due to the
small size that those organisms can achieve: nematodes
and, over all, amoebas or ciliates can predate on
microalgal cells slightly smaller than them. Twist et al.
(1997) developed a method of in situ biomoni- toring using
Ca-alginate immobilized Scenedesmus subspic- atus for the
assessment of eutrophication in surface waters. A clear
advantage of this technique is that local flora can be
isolated and incorporated to the biomonitor. Of course,
when Ca-alginate is used in natural (or micro and meso-
cosms simulating natural) environment, limitation of the
technique is degradation of beads. In freshwater streams
this limitation seems to be a couple of weeks. In marine
environments (Faafeng et al., 1994), duration of beads is
quite shorter (a few days) (Moreira dos Santos et al.,
2002). Improvement in bead fabrication is trying to avoid
those problems (Moreira et al., 2006). Periphytic algae
nat- urally established on glass slides have been used in in
situ toxicity tests conducted to test the impact of heavy
metals resuspended by dredging works on microalgae
(Nayar et al., 2003). High concentrations of Zn
(up to
17,240 mg L—1), Cu (up to 11 mg L—1) and Cd (up to
1.8 mg L—1) were found, as a result of dredging, in the
aqueous phase, reducing periphyton biomass between
95% and 100% for polluted waters.
Biosensors of different types involving microalgal cells
have been designed in order to detect pollutants in the envi-
ronment. Lukavsky and Marsˇa´lek (1997) immobilized
S. capricornutum in 2% agar in order to use it as a
biosensor for Cr6+ toxicity. Sensitivity of this biosensor was
the same
3960 I. Moreno-Garrido / Bioresource Technology 99 (2008) 3949–3964
found for other bioassays, including growth inhibition test.
Frense et al. (1998) used S. subspicatus immobilized on a
filter paper and covered with alginate in an optical biosen-
sor based in the chlorophyll a fluorescence as biomarker
for pollutants in water and soil extracts, as a pre-selection
protocol before sending samples to high-cost standard
analysis. C. vulgaris has also been used in optic biosensors
in order to determine the toxicity of herbicides (Naessens
et al., 2000) such as atrazine, simazine and diuron, com-
monly used in cereal cultures. In this case, algal immobili-
zation was performed on GF/C Whatman filters. Filter
paper disks immobilizing algae have also been used by
Sanders et al. (2001) in biosensors designed in order to
detect chemical warfare agents. Not optic, but ampero-
metic algal-based sensors have also been designed (Shit-
anda et al., 2005), taking advantage of variations in the
photosynthetically produced oxygen. Limitations of toxic-
ity testing using free or immobilizing microalgae are
restricted to those toxicants that affect structures present
in the algal cells. It means that pollutants affecting bone
development or nervous system will not be easily
detected with microalgal-based bioassays. In contrast,
toxicants affecting photosynthesis (as copper ions or
herbicides) will be more appropriately detected with
vegetal cells (such as microalgae) bioassays. Adequate
aquatic toxicity studies should cover organisms from
different levels, but microal- gae should never be
forgotten due its basal position in the trophic chain.
4.8. Co-immobilized systems
Recent efforts have been made in the field of co-
immobi- lization (Nagase et al., 2006). De-Bashan et al.
(2002a,b, 2004) co-immobilized Chlorella with a
microalgae growth-promoting bacterium (Azospirillum
brasiliense) in Ca-alginate beads. This bacterium is not
able to remove nutrients from wastewaters, but enhances
growth of immo- bilized algae. Co-immobilized biological
system removed higher percentages of nutrients from
wastewaters (100% of ammonium, 15% nitrate and 36% of
phosphorus) when compared with immobilized algal cells
without bacteria (75% ammonium, 6% nitrate and 19%
phosphorus). Mun˜ oz and Guieysse (2006) reviewed
the interactions between algae and bacteria in processes
designed for the treatment of hazardous contaminants.
Production of oxy- gen by algae improves degradation of
substances that must be degraded aerobically. Both
bacteria and algae could produce defending substances
against the other co-immobi- lized organism. Increase of pH
values due to photosynthe- sis and increase of oxygen in
the media could also slow down bacterial growth when co-
immobilized with algae. On the other hand, consumption
of CO2 and extracellular matter production (such as
exopolysaccharydes) by algae can enhance bacterial
growth rate, as well as CO2 and growth promoter
substances production by bacteria can enhance microalgal
growth. The same authors recognized that most widely
used immobilization techniques involve
weak and expensive matrices (when used in large scale),
and propose other approaches such as photobioreactors
with the bacterial–algal microcosm attached on the
reactor walls.
5. Future perspectives
Immobilization techniques can be used in molecular
biology, as plasmid stability in immobilized cells has been
reported (Cassidy et al., 1996). Risk of unwanted muta-
tions is reduced when cells are immobilized (Codd, 1987).
Genetic manipulation of immobilized cyanobacteria
(hydrogenase negative gene) could also improve hydrogen
generation processes (Das and Vezirog˘lu, 2001).
Immobi- lized microalgae have been recently used as a
tool for water quality control in fish culture. Chen (2001)
studied that ammonium concentrations decreased in
tilapia cultures containing immobilized cells of S.
quadricauda (freshwater cultures) and clam cultures
(Chen, 2003) containing I. gal- bana (seawater cultures). In
the latter case, slow liberation of cells from immobilizing
beads provided a continuous input of food for filtering
clams. This could reduce costs of clam culturing when
compared with traditional methods.
The use of microalgae in the design of biosensors is a
very recent and interesting topic in biotechnology. Chou-
teau et al. (2004) designed a conductometric biosensor with
C. vulgaris cells (the authors pointed out that the use of
whole cells is quite cheaper than that of isolated enzymes)
in order to measure the inhibition of alkaline phosphatase
activity as a bio-indicator of toxic stress. Volatile com-
pounds (such as formaldehyde) have also been detected
by the use of multiple-strain algal sensor chips designed
by Podola et al. (2004). Genetically modified organisms
can also be used in sensors, such as the strain of Synecho-
coccus employed by Schreiter et al. (2001): this organism
harbours a gene encoding for the luciferase from Vibrio
harveyi under the control of an inducible alkaline phospha-
tase promoter, which can be induced by phosphorus
limita- tion. The resultant ‘‘CyanoSensor’’ is able to
detect 0.3–
8 lM of PO 3—
and respond to other organic phosphorus
4
sources. This sensor is also storable for three weeks at 4 °C.
Combinations of solar energy and algal immobilization
technologies can be successfully used in industrial processes
(Mallick, 2002). In the same way, studies about production
of energy via photosynthetically generated H 2 are a recent,
promising field of research that has been reduced to date
to green freshwater algae. Other taxonomic groups should
be tested in order to optimize molecular hydrogen
production.
Acknowledgements
This work has been granted by the Spanish National
Plan of Science and Technology Research, under the pro-
jects ‘‘Use of immobilized systems for marine microalgae
in the incorporation and evaluation of toxic substances in
marine ecosystems’’ (REN2001-2095/MAR) and ‘‘A new
 I. Moreno-Garrido / Bioresource Technology 99 (2008) 3949–3964 3961

ecologically relevant tool in environmental toxicology: Carrilho, E.N.V.M., No´ brega, J.A., Gilbert, T.R., 2003. The use of
microphytobenthos for the environmental quality assess- silica- immobilized brown alga (Pilayella littoralis) for metal
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