Showcasing work from the Laboratory of Inorganic Materials

Chemistry (CMI), University of Namur, Belgium and Laboratory

of Living Materials at the State Key Laboratory of Advanced
Technology for Materials Synthesis and Processing, Wuhan
University of Technology, China.

As featured in:

Title: Green and sustainable production of high value

compounds via a microalgae encapsulation technology that
relies on CO2 as a principle reactant
This research article highlights the extraordinarily long-term
viability and high activity of microalgae entrapped within an
alginatesilica hybrid matrix, holding much promise for the design
of new generation xed biomass photobioreactors that are based
on microencapsulation technology. This bionanotechnology opens
up exciting avenues for the design of photosynthetic solar cells and
articial trees for a green and sustainable production of high value
compounds and electricity on the basis of photosynthesis process.

See Joanna C. Rooke,

Bao-Lian Su et al.,
J. Mater. Chem. A, 2014, 2, 20560.

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PAPER

Cite this: J. Mater. Chem. A, 2014, 2,

20560

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Green and sustainable production of high value

compounds via a microalgae encapsulation
technology that relies on CO2 as a principle
reactant
Jonathan Desmet,a Christophe F. Meunier,a Emeric P. Danloy,a Marie-Eve Duprez,b
Anne-Lise Hantson,b Diane Thomas,b Pierre Cambier,c Joanna C. Rooke*a
and Bao-Lian Su*ad
A very promising and facile one-pot synthesis pathway is presented for the microencapsulation of live cells
in a very porous coreshell system based upon a robust matrix. (AlginateSiO2polycation)
shell@(alginateSiO2) core hybrid beads, on the millimeter scale, containing live cells are obtained
through cross-linking chemistry and the polycondensation of silicic acid in conjunction with the use of a
polycation to negate the surface charge on silica. Very interestingly it is revealed that the polycation used
(PDADMAC) plays a very important role in the formation of highly robust coreshell beads. PDADMAC
acts as a catalyst in the polycondensation of silicic acid, leading to the formation of a resistant double
layer shell comprising of an interior layer of alginateSiO2 with a very homogeneous distribution of
porous SiO2 and an external layer of porous PDADMAC that connes SiO2 within the bead. The
photosynthetic chlorophyta Dunaliella tertiolecta, which produces high value metabolites (such as antioxidants, pharmacologically active compounds, neutraceuticals etc.) via photosynthesis, has been
encapsulated within this coreshell system. Oximetry and uorescence measurements highlight how
this algal culture can remain photosynthetically active over an extraordinarily long period of 13 months
for high value compound production, whilst entrapped within a highly porous, mechanically and
chemically stable, optically transparent matrix, with no observable leaching of the cells from the core of
the beads. HPLC has been employed to highlight the presence of excreted metabolites, based on neutral
sugar building blocks such as arabinose, galactose and xylose, in the surrounding media. These results

Received 8th September 2014

Accepted 25th September 2014

reveal how this kind of high performance, low-cost, and easily scaleable coreshell living material could
be employed in large scale photobioreactors (PBRs), to potentially facilitate metabolite harvesting whilst

DOI: 10.1039/c4ta04659e
www.rsc.org/MaterialsA

protecting the culture from external contamination and for green energy production and environmental
(CO2) remediation.

Introduction
In recent years, the photosynthates of green algae have aroused
the interest of a range of industries owing to the elevated market
price of these compounds, their sustainable production from
cheap and readily available reagents and ultimately their
widespread applications1 such as in neutraceuticals,2 alimentary pigments,3 anti-inammatories4 and cosmetic additives.5

Laboratory of Inorganic Materials Chemistry (CMI), University of Namur, Rue de

Bruxelles 61, B-5000 Namur, Belgium. E-mail: bao-lian.su@unamur.be; joanna.
rooke@unamur.be; Fax: +32 (0)81725414; Tel: +32 (0)81724531

University of Mons, Rue de l'Epargne 56, B-7000 Mons, Belgium

Research Unit in Cell Biology (URBV), University of Namur, Rue de Bruxelles 61,
B-5000 Namur, Belgium
d

State Key Laboratory of Advanced Technology for Materials Synthesis and Processing,
Wuhan University of Technology, Hongshan Luoshi Road 122, 430070 Wuhan, China

20560 | J. Mater. Chem. A, 2014, 2, 2056020569

Currently photosynthates are commonly obtained from

algae grown in photobioreactors (PBRs) via extraction processes
with organic solvents68 or supercritical uids such as CO2.9,10
The concentration of metabolites from microalgae cultivated in
suspension in PBRs is low, requiring voluminous systems.
Furthermore, the separation and purication steps required are
considerable and are far from optimal, reducing the yield and
signicantly increasing the cost and the production time.11
The demand for valuable metabolites necessitates sustainable and highly promising technologies that exploit the
photosynthetic processes performed by microalgae in a facile,
cost ecient and sustainable manner. The use of immobilised,
living biomass within a PBR is one potential solution to improve
yields as cellular density can be increased whilst being isolated
from predatory species, reducing contamination.12 The use of a
solid phase hybrid system (live cellinert matrix) allows the

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preservation of a quasi-constant, active cell population over

long periods of time that enables optimum yields and easy
handling at low cost. It can also protect the cells against
mechanical stresses typically encountered in PBRs, i.e. sheer
forces.13 Such living materials are part of an ongoing development of green chemistry as they use CO2 as a principle reactant
and light radiation as the energy source.14 Factors such as the
shape, porosity and mechanical and chemical stabilities of the
inert matrix are key points to be considered when engineering
such hybrid systems.
Over recent years microencapsulation has been favoured in
which a small number of cells are immobilised in small
discrete compartments, i.e. capsules. The widespread use of
biopolymers such as agarose, alginate, carrageenan, chitosan,
collagen and gelatine amongst others has been reported as
they impart a exible environment to allow for growth whilst
being biocompatible with the live cells.1521 Commonly
calcium alginate is employed as the synthesis procedure is
simple, based on the cross-linking of a sodium alginate
precursor with divalent cations, namely calcium. However
when used alone calcium alginate suers from major drawbacks such as poor thermal stability as well as fractures and
cavities (inhomogeneous porous networks) and poor
mechanical resistance since the gel strength decreases from
the surface to the core.22,23 Moreover, capsules are sensitive to
their external environment: i.e. modication of the osmotic
pressure that can lead to swelling and rupturing of the material, causing the leakage of immobilised entities.24 Coating the
surface of the droplet with polycationic polyelectrolytes25 (i.e.
chitosan,26 poly-L-lysine,27,28 polyallylamine29 etc.) can overcome these problems whilst forming a semi-permeable
membrane, as seen in the design of a bioarticial pancreas by
Lim and Sun where calcium alginate capsules containing
Langerhans' islets were incubated in a poly-L-lysine solution.27
Other groups have addressed these limitations with the use of
more resistant inorganic encapsulation matrices, focusing
mainly on silica;3037 a material that has improved thermostability, does not swell and can be synthesised via biocompatible methods.
In spite of this silica alone is also not ideal for cell encapsulation; its rigidity does not allow for cell growth, defects in
monolithic silica can result in cracking and cellular interactions
can result in the breakdown of the framework and internalisation of silica nanoparticles within the cell. A composite
material exploiting the benets of several dierent materials is
an excellent strategy. A combination of alginate (for its
outstanding viscoelasticity) with silica (for its mechanical
strength, chemical inertness, and that, unlike alginate, does not
swell) in a spherical morphology through a coacervation process
was reported in the literature by Fukushima et al. for the
immobilisation of enzymes or microorganisms.38 Other examples of biopolymersilica hybrid capsules have also been
reported in the literature.23,3942
However it has been found that silica is only deposited on
the surface of the forming calcium alginate and not throughout
the core of the bead, resulting in an insucient porosity for the
diusion of nutrients and metabolites. In addition, cracking

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Journal of Materials Chemistry A

was still observed that resulted in cells leaching into the

surrounding culture medium in which they can proliferate.
Moreover, the synthesis of alginatesilica capsules oen
involves a complex multi-step process (layer-by-layer).43 Dissolution of silica is also a well-known phenomenon that occurs
with time and must be controlled.44
In this present work, novel hybrid alginatesilica beads have
been engineered by successfully combining several dierent
technologies in order to entrap the microalgae Dunaliella
tertiolecta via a very ecient one-step biocompatible coacervation process. The structure of the composite material has been
optimised to yield a solid core comprising a mixture of exible
alginate and mineralised porous silica, homogeneously
distributed throughout the entire core, ensuring not only
excellent diusion within the bead but also contributing to
better mechanical and chemical stabilities. Furthermore, the
process results in the formation of a semi-permeable shell, rich
in a biocompatible polyamine, namely polydiallyldimethylammonium chloride (PDADMAC). Polyamines can catalyse the
hydrolysis and condensation reactions of a silica sol and act as
occulating agents.4550 Moreover, adding a polycation outer
layer such as poly-L-lysine to alginate capsules or to hybrid
alginatesilica beads can overcome the swelling phenomenon
and also lead to an additional mechanical resistance. This shell
allows the diusion of metabolites and nutrients to and from
the external medium whilst reinforcing the mechanical
strength of the bead and reducing or preventing the dissolution
of sodium alginate and silica species or the leakage of entrapped living cells into the biological medium.
This approach enables a rapid and simple synthesis of
hybrid capsules in high volumes but at low cost whilst
combining the benets of several technologies in one step
without the need for successive manufacturing processes. The
suitability of these hybrid beads for use in a xed biomass
photobioreactor for the production of high value metabolites
is evaluated in terms of cell preservation, silica dissolution,
material robustness, etc. It is revealed that the photosynthetic
chlorophyta Dunaliella tertiolecta, which is known to produce
commercially interesting metabolites via photosynthesis, can
be encapsulated using this coreshell strategy and the cells
remain photosynthetically active for an extraordinarily long
period of at least 13 months, whilst the hybrid beads themselves remain intact with no degradation. This bionanotechnology opens up exciting avenues for a green and
sustainable production of high value compounds using CO2
as a principle reagent whilst it could overcome current PBR
limitations such as contamination, water evaporation and
biofouling.

Experimental
Microalgae cultivation
Dunaliella tertiolecta (ATCC 30929) suspension cultures were
maintained in asks at ambient temperature under uorescent
strip lighting and transferred into fresh Johnsons culture
medium at regular intervals.

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Entrapment of microalgae in hybrid alginatesilica beads

Hybrid silicaalginate beads were prepared using a silica sol
obtained from sodium silicate (1.5 M, assay 25.528.5%, Merck)
passed over an acid ion exchange resin (Amberlite IR 120, Hform, Acros). 5 mL preformed silicic acid sol (1.5 M),
[SiOx(OH)4
2x]n, was adjusted to pH 5.1 (0.1) with NaOH 0.1 M
and mixed with 5 g 3 wt% sodium alginate solution (alginic acid
sodium salt, from brown algae, Aldrich) and 0.5 mL cell
suspension of Dunaliella tertiolecta (ATCC 30929). In order to
establish this cell suspension, a predetermined volume of cell
culture in the stationary phase was centrifuged and the supernatant discarded. The pellet of cells was resuspended in fresh
culture media, thus providing essential nutrients within the
beads as they form to allow for any delay in external media
diusing into the beads. This mixture was then dropped into an
aqueous solution of polydiallyldimethylammonium chloride
(PDADMAC) (0.4 wt%) (20 wt% in H2O, Aldrich) containing
CaCl2 (20 mM) to catalyse the polycondensation of the silica
precursor and the cross-linking and gelication of the alginate,
respectively. Aer incubation within this mixture, the hybrid
alginatesilica beads that formed were washed three times with
fresh culture medium prior to being transferred into sterile
asks in the presence of Johnsons culture medium.
Characterisation techniques
Photosynthetic activity. The photosynthetic activity of hybrid
beads containing microalgae was monitored using a Clark
electrode (Pt/Ag), purchased from HansaTech, to determine the
oxygen concentration in a closed system. A suspension of three
hybrid beads in 1.0 mL of Johnsons culture medium, mixed
with NaHCO3 (10 mL, 0.6 M), was placed in the chamber and the
system was ushed with argon to remove any atmospheric
oxygen. The chamber was then closed and subsequently
obscured from natural light. An external light source, l 650
nm, 1200 mmol m
2 s
1, was used to stimulate photosynthesis.
HPAEC-PAD analysis. Photosynthates based on neutral sugar
building blocks that were excreted by the encapsulated microalgae were identied by High-Performance Anion Exchange
Chromatography with Pulsed Amperometric Detection (HPAECPAD). An aliquot of media was taken from the external phase of
the hybrid alginatesilica bead suspension aer 32 days postsynthesis to which a pre-determined amount of fucose was
added as an internal standard. Before carrying out the analysis,
the aliquot was treated in triuoroacetic acid (1.5 mL, 3 M) at
120  C for 3 h to hydrolyse the polysaccharides. The sample was
then evaporated to dryness. The resulting white powder was
resuspended in 1 mL MilliQ water and again evaporated to
dryness, and the process repeated. Concurrently sugar standards (arabinose, galactose, glucose, glucosamine, mannose,
rhamnose and xylose) of known concentrations were also
prepared for peak identication, as well as two controls (MilliQ
H2O and fresh culture media), which were hydrolysed and dried
in an identical fashion, each with a fucose internal standard.
Aer centrifugation (13 000 rpm, 10 min), the supernatants
were injected into the HPAEC system. By using an anionexchange column CarboPac PA-20 (0.4  150 mm) from Dionex,

20562 | J. Mater. Chem. A, 2014, 2, 2056020569

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the chromatographic separation of neutral sugars was made

possible. A gradient elution (5 mM NaOH) was used as the
mobile phase under constant helium pressure for the initial
separation. Further analysis with an isocratic ow (10 mM
NaOH) was required to identify the arabinose and rhamnose
peaks. A post-column derivatisation was performed by the
addition of 0.75 M NaOH. The sample injection volume was
10 mL and the column oven temperature was xed at 35  C. Aer
measurement, the column was reconditioned by washing with
200 mM NaOH for 10 min and then re-equilibrated with the
starting buer.
Material characterisation. Scanning electron microscopy
images were collected on a JEOL JSM-7500F microscope to
establish the morphology of the hybrid beads. Samples were
dehydrated using ethanol baths with progressively increasing
concentrations prior to supercritical drying with carbon dioxide
in order to remove the water within the porous network without
destroying it. The beads were nally subjected to a sputter
coating with gold to overcome charging eects under the electron beam.
Optical and uorescence microscopy techniques were performed using a Nikon microscope with a multizoom AZ100 to
observe the shape of the hybrid beads and to determine their
size. The autouorescence of photosynthetic pigments found
within microalgae was exploited to identify the viability of cells
by uorescence microscopy. In parallel, the silica distribution
throughout the entire bead was determined with the aid of a
uorescent probe, 2-(4-pyridyl)-5-((4-(2-dimethylaminocarbamoyl)methoxy)-phenyl)oxazole (PDMPO, 20 mL, 10 mM), which
interacts with silica species to exhibit an intense blue uorescence. Aer incubation of the beads with PDMPO, micrographs
were collected at 447/60 nm with a colour camera (DSRi1).
Textural properties of hybrid alginatesilica beads were
evaluated through nitrogen adsorptiondesorption measurements at
196  C on a Micromeritics TRISTAR 3000 porosimeter. Prior to performing the analysis, the beads were
dehydrated with anhydrous ethanol, dried with supercritical
CO2 and degassed at room temperature under vacuum
(70 mTorr) overnight. The specic surface area was calculated
using the BET (BrunauerEmmettTeller) equations and the
average pore size was established by the BJH (BarrettJoyner
Halenda) method.
The Young's moduli of beads of various chemical compositions were estimated by placing beads of pre-determined
diameter (determined by microscopy) on an Instron 4505
compressiontraction bench. A stress was subsequently applied
to the bead and the compression of the bead was measured as a
function of the load applied. From these measurements the
modulus was elucidated, E (kPa).

Results and discussion

Engineering highly robust and biocompatible (alginateSiO2
polycation) shell(alginateSiO2) core composite systems
The formation mechanism of the hybrid coreshell alginate
silica beads relied on a simple eco-friendly and very ecient
one-step coacervation process that was initiated by a decrease in

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solubility of sodium alginate caused by the addition of the

polycation PDADMAC. A typical procedure required the preparation of a sodium alginatesilica precursor mixture which
contained the microalgae to be immobilised. This was added
dropwise, via a pipette or a syringe with a needle, into a coacervation solution containing the polycation and a divalent salt
such as CaCl2, resulting in the gelation of the droplets owing to
cross-linking of the alginate via complexation of sodium alginate with divalent cations (Ca2+) and the polymerisation of the
silica precursor induced by the pH variation of the silica sol
(silicic acid), catalysed by the presence of a basic polycation in
the gelation medium. Through this very simple and gentle
approach, novel hybrid spherical beads composed of a solid
porous core, homogeneous in composition, with a semipermeable double-layer shell could be obtained via a one-pot
synthesis strategy. These beads can be formed on an adjustable
millimeter-scale size, dependent on the diameter of the needle,
and possessed an excellent mechanical strength with no localised regions rich in either alginate or silica, negating the
deposition of silica on the alginate surface. The core part of the
bead was based on a sodium alginatesilica composite, in
which the microalgae were entrapped, and the shell was
composed of an intermediate layer of calcium alginatesilica
and an outer layer of the polycation PDADMAC (Fig. 1).
As the cross-linking of an alginate droplet within a divalent
cation bath becomes progressively less towards its core, the
resultant mechanical strength of an alginate capsule decreases
from the shell to the core. Via the homogeneous distribution of
a silica network throughout an alginate droplet, a microcapsule
can be signicantly reinforced. To ensure a homogeneous
composition, the silica network needs to form in situ and at the
same time as the alginate cross-linkage. This can be achieved by
catalysing the polycondensation of a siliceous sol which in turn
is achieved by adjusting the pH, hence the use of a polyamine
(base) with a silicic acid sol (pH 2). The polyamine had a double
role in this process. Its presence in the coacervation solution
not only catalysed the formation of the 3D silica network, but
also allowed the formation of an external polyamine layer
around the bead. The PDADMAC layer was intended to reduce
or even prevent any swelling of the bead or leakage of silica
species, sodium alginate or microalgae outside of the bead. The
interest to design this kind of hybrid bead stemmed from the
combination of the viscoelastic properties of alginate with the

Fig. 1 Schematic of a hybrid alginatesilica bead. Shading from black

to white indicates calcium-alginatesodium-alginate concentration
gradient within the bead.

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Journal of Materials Chemistry A

robustness and optical transparency of silica in order to obtain

a resistant durable material, with signicant porosity, suitable
for cell encapsulation. The properties of this material as well as
its suitability for microencapsulation are outlined below
through the study of the cellular activity and metabolite
production of immobilised Dunaliella tertiolecta.
Evaluation of the (alginateSiO2polycation) shell(alginate
SiO2) core composite system
Polycation choice. A polycation, PDADMAC, was employed in
the formation of the hybrid alginatesilica beads for several
reasons, namely it acted as a catalyst to accelerate the hydrolysis
and polycondensation of the silicic acid sol into a 3D SiO2 gel
network within the core and also in the external shell of the
bead, thus creating a porous crust. It also formed a quasi
selectively permeable outer membrane that allowed nutrients,
gases and metabolites to diuse through the bead from the
external culture medium and vice versa whilst stopping dissolved silica, sodium alginate or microalgae from leaching out
of the bead. The polycondensation mechanism of silica with
polyamine is not yet established, however preliminary studies
have shown that the amino groups of the polyamine can stabilise the transition state of the condensation reaction by
promoting the proton transfer.51
Herein the processes of bead formation are discussed independently of the concomitant cross-linking of the sodium
alginate, a process that is occurring in parallel. Under aqueous
conditions the polycation can form a microemulsion where the
polymer forms an aggregate with a positively charged surface.
Thus when brought into contact, the silicic acid and the polycation can interact through ionic interactions with SiO32
.
Moreover, as it catalyses the polycondensation of the silicic
acid, the forming silica will also be negatively charged at slightly
acidic pH. Owing to these microscopic interactions PDADMAC
can play the role of a occulating agent (a chemical that
promotes the aggregation of colloids and ne particles suspended in liquids). In other words, PDADMAC and silica act as
coacervates, an aggregate of colloidal droplets held together by
electrostatic attractive forces. On addition of the reagent
mixture to the cross-linking bath (CaCl2(aq)PDADMAC),
adsorption and/or the dissolution of the silica precursor is
possible within the polyamine droplets that form in the
aqueous bath. As these aggregates surround the silica sol they
form a barrier, which becomes the shell of the hybrid bead, with
silica mineralisation occurring in the interior (complex coacervation). Complex coacervation involves the phase separation
that occurs when two hydrophilic colloids are mixed, in this
case the polycation and the silicic acid sol. Three immiscible
phases can be formed, the liquid vehicle phase (water), the core
phase (silica precursor) and the coating phase (polycation),
when the silica precursor is dispersed into the polymer/water
microemulsion, enabling the precipitation of the complex
coacervate (polycationsilicic acid) via electrostatic interactions.
The polymer coating adsorbs at the interface between the core
and the liquid vehicle phase which results in the polycation
forming an external layer around the silicic acid drop.

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Concurrently the polycation is also catalysing the formation of

silica within that drop.
Through the use of a polycation, very stable, porous, transparent and tough hybrid beads that entrapped microalgae could
be prepared. Bhatia et al. described the use of polyelectrolytes
for cell encapsulation in which a large library of potential
polyamines were revealed.25 Inspired by this, the polycation
PDADMAC was chosen in this work due to its biocompatibility
with living cells.
Mechanical resistance. The mechanical resistance of the
beads was determined by establishing the Young's modulus; a
measure of stiness of an elastic material within the limits of
Hooke's law. This value was then compared to beads of various
compositions based on the components of the hybrid material
(Table 1).
As expected, the mechanical strength of a bead depends
upon its composition. The values obtained highlight not only
the advantage of combining alginate with silica (almost four
fold increase in resistance) but also the fact that the polycation
can impart strength to both alginate and alginatesilica beads.
This added strength reveals the utility of using a polycation to
form a tougher shell around the hybrid alginatesilica beads.
Silica distribution. The distribution of silica within the core
part of the bead was successfully determined using the uorescent probe, 2-(4-pyridyl)-5-[(4-(2-dimethylaminoethylaminocarbamoyl)methoxy)-phenyl]oxazole (PDMPO).52 This probe
molecule produces intense blue uorescence in the presence of
silica with which it interacts. The quantum yield of this uorescence is dependent upon the silica concentration. By
combining the eect of this probe molecule with optical
microscopy it is possible to establish whether the silica is
distributed homogeneously throughout the hybrid structure or
whether it is concentrated in various zones. This analysis
technique highlighted that in the presence of PDADMAC a zone
rich in silica can be identied by the bright blue spots in the
shell of the bead yet the remaining silica is still evenly distributed throughout the core of the bead (Fig. 2a), whereas silica
species are uniformly distributed throughout the entire bead in
the absence of this polycation (Fig. 2b) with no localised regions
of silica. This means that PDADMAC facilitates the enrichment
of the shell of the bead with silica.
Furthermore, the chemical stability of the material was also
improved by using PDADMAC. As outlined in Fig. 3a a core
shell bead was obtained only when in the presence of

Fig. 2 Fluorescent microscopy images of a cross-section of a hybrid

alginatesilica bead in the presence of PDMPO (silica tracer) (a) with
and (b) without PDADMAC. (c) Graph of the uorescence intensity of
hybrid beads with and without PDADMAC.

PDADMAC. The shell, which was mainly composed of the polycation, was clearly distinguished from the core part of the
hybrid alginatesilica bead. Some cracking of the bead surface
and even fragmentation of the beads and leaching of silica
species into the culture medium could be observed aer a few
days when PDADMAC was omitted from the protocol.
Characterisation of hybrid alginatesilica beads. The
textural properties of the beads were characterised by adsorptiondesorption of nitrogen. Prior to analysis the beads were
dried with supercritical CO2 to carefully remove the liquid
phase from the capillaries whilst leaving the porous network
intact. For comparisons sake pure alginate beads were also
prepared and subjected to the same treatment to evaluate the
porosity of alginate alone compared with the hybrid alginate
silica material proposed herein. The analysis of both alginate

Table 1 Mechanical resistance comparison of beads of various

compositions. Each type of bead was incubated for 3 hours in an
appropriate bath (either CaCl2 alone or PDADMAC and CaCl2). Their
mechanical resistance was expressed in terms of their Young's
modulus

Bead composition

Young's modulus E (kPa)

Alginate
AlginatePDADMAC
AlginateSiO2
AlginateSiO2PDADMAC

78
117
296
325

20564 | J. Mater. Chem. A, 2014, 2, 2056020569

Scanning electron microscopy images of hybrid alginatesilica

beads (a) with and (b) without PDADMAC.

Fig. 3

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and hybrid alginatesilica beads yielded a type II isotherm

according to the IUPAC classication, which is attributed to a
macroporous material (Fig. 4). Hybrid alginatesilica beads (C,
D, G and H) possessed a much higher specic surface area and a
much higher porous volume in comparison to pure alginate
beads in all cases (Table 2). This increase in porous volume is a
key factor that can improve diusion within the hybrid beads.
Diusion is a vital process, required not only in the supply of
nutrients and gases essential to the survival of the microalgae
but also to harvest the metabolites produced by the entrapped
live microorganisms. This would strongly suggest that hybrid
alginatesilica beads are superior to alginate beads in terms of
cell encapsulation technology.
To conrm these results, images were taken of the hybrid
material by scanning electron microscopy (SEM) (Fig. 5). Crosssection images reveal a 3 dimensional porosity throughout the

Fig. 4 Nitrogen adsorptiondesorption isotherms of (a) algae immobilised in alginate capsules (-, C) and alginatesilica beads (:, *)
incubated in a CaCl2 bath (-, :) or a CaCl2PDADMAC bath (C, *)
and (b) analogous blank samples fabricated under the same conditions
in the absence of cells. Isotherms B, C, D, F, G and H have been oset
by 900, 1000, 1500, 1100, 1100 and 1600 cm3 g
1 respectively for
clarity.

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Journal of Materials Chemistry A

entire hybrid alginatesilica bead, with the porosity extending

from the distal layer to the core. The pore sizes were in the range
of 50100 nm, which is much smaller than the size of the algae
to be immobilised. Furthermore, no fractures were observed on
the surface of the hybrid bead (Fig. 5a) meaning that cell
leaching into the surrounding media would be prevented.
Microencapsulation of Dunaliella tertiolecta within the core
shell alginatesilica hybrid system
Cell viability and photosynthetic activity. In order to prove
the viability of live hybrid alginatesilica beads for use in a
PBR, the entrapped cells were examined in terms of dispersion,
intactness and activity. Firstly, encapsulated algae were characterised by SEM. Fig. 5b and d show how the cells were
entrapped and well dispersed throughout the core of the bead
and that the integrity of the cell was preserved with minimal
adhesion of the hybrid matrix upon the cell surface. The
nucleation of nanoparticulate silica upon the surface of an algal
cell wall has already been shown to interfere with the metabolic
pathways of the cell.53,54 Thus this encapsulating hybrid-matrix
is more adaptable to the preservation of photosynthetic activity
than SiO2 alone.
Fig. 6a and b shows how there is a minimal electrostatic
interaction between the beads, highlighting their potential use
in a uidised bed or air-li PBR where the media and gases can
circulate between the beads with no agregation of the beads.
The entrapment of algae within hybrid beads did not aect
the properties of the beads, in particular their morphology and
robustness. Fluorescence microscopy images of a cross-section
of a hybrid bead with microalgae showed that the photosynthetic pigments, namely chlorophylls a and b, were well
preserved aer encapsulation and dispersed throughout the
bead (Fig. 6c). The formation of aggregates of cells, seen in this
image as regions where there is a greater density of uorescence, may have occurred during centrifugation. As observed
under a UV-excitation light in Fig. 6d with a bead marked with
PDMPO, the silica species are homogeneously dispersed
throughout the bead. Even though alginate and silica carry a
negative surface charge at slightly acidic pH, it is still possible to
obtain a good dispersion of these two components within the
composite material owing to hydrogen bond interactions
between alginate and silica.23 Image (e) is a superposition of the
two uorescent images, in red are the living microorganisms
and in blue the silica proving the dierent origins of the
uorescence.
The autouorescence of chlorophyll shows that the photosynthetic apparatus was intact, however, it gives no evidence
that the light harvesting mechanism is fully functional postencapsulation. Thus the oxygen production of the hybrid beads,
within an illuminated closed system enriched with NaHCO3
(CO2 source), was determined via electrochemical methods,
commonly known as oximetry. Fig. 7 shows how entrapped
Dunaliella tertiolecta can produce oxygen for over 13 months
within this encapsulation technology. Time zero corresponds to
the time when the microalgae were encapsulated within the
hybrid beads. The graph depicts how cellular activity increases

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Table 2

Paper

Textural properties of various alginate capsules and alginatesilica beads synthesised with and without PDADMAC

Sample

Alginate

A
B
C
D
E
F
G
H

7
7
7
7
7
7
7
7

Silica

7
7
7
7

CaCl2
7
7
7
7
7
7
7
7

PDADMAC

Algal cells

SBET m2 g
1

VBJH cm3 g
1

7
7
7
7

351
194
714
641
286
126
673
631

1.5
0.89
2.0
2.2
1.9
0.84
2.3
2.2

7
7
7
7

(a) Bright eld and (b) dark eld optical microscopy images of a
cluster of hybrid alginatesilica beads with a PDADMAC outer
membrane containing entrapped microalgae; (cf) a cross-section of
one of these beads where (ce) are uorescent microscopy images
revealing (c) the chlorophylls contained within live algal cells, (d) the
distribution of silica via the aid of a PDMPO tracer and (e) the superposition of (c) and (d); (f) is the corresponding bright eld optical
microscopy image for comparison.

Fig. 6

Fig. 5 Scanning electron microscopy images of (a, c, e, and g) the

distal layer and (b, d, f, and h) the core part of a hybrid alginatesilica
bead with the PDADMAC outer membrane containing entrapped
microalgae.

over the rst 3 to 4 months as the cells adapt to their new

environment before reaching a steady state of between 3 and
4 mmol O2 per gram of hybrid material per hour.
Fig. 8 shows how a hybrid alginatesilica material preserves
cellular activity, in this case the production of oxygen, better

20566 | J. Mater. Chem. A, 2014, 2, 2056020569

than with alginate alone. For the same amount of encapsulated

matter, the activity of cells inside a hybrid matrix is consistently
higher than for those cells encapsulated within simple alginate
beads. This may come from a loss of cells through leaching as
the alginate beads swell over time. Globally this experiment
shows how an encapsulating matrix based on silicaalginate is
superior to alginate alone in order to maintain signicant cell
activity.
Carbohydrate production. In conjunction with oximetry
methods it is possible to determine the photosynthetic activity

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Paper

Fig. 7 Photochemical production of oxygen by entrapped microalgae

within hybrid alginatesilica beads with the PDADMAC outer
membrane.

Fig. 8 Photochemical production of oxygen by entrapped microalgae

within beads of dierent compositions.

of the hybrid beads by analysing the culture media in which

they are suspended to detect the presence of excreted metabolites. It is well known that in photosynthesis, carbon dioxide is
reduced to glucose and eventually converted into more complex
polysaccharides. Thus the culture media were monitored using
High-Performance Anion Exchange Chromatography with
Pulsed Amperometric Detection (HPAEC-PAD) to identify any
metabolite derivatives (neutral sugars) present. Before analysis,
the supernatant was treated in triuoric acid (TFA) to hydrolyse
any potential oligo- and polysaccharides into monosaccharides.
The carbohydrates detected were composed of neutral sugars
such as glucose, mannose and xylose units. Fig. 9 shows the
chromatogram obtained from media that had been in contact
with the hybrid system for 32 days. By comparing the retention
times in the chromatogram to those of a stoichiometric mix of
the hydrolysed standard sugars (20, 50, and 100 ppm) it was

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Journal of Materials Chemistry A

Chromatogram of culture media in contact with alginatesilica

beads containing D. tertiolecta for 32 days. The inset reveals the
separation of peaks corresponding to rhamnose and arabinose via an
isocratic elution.

Fig. 9

possible to assign the majority of the peaks thus revealing the

presence of arabinose, galactose, glucose, glucosamine,
mannose, rhamnose and xylose in the hydrolysed media from
the alginatesilica hybrid system. The retention times for
arabinose and rhamnose were quasi identical, resulting in a
superposition of the peaks which could only be resolved aer
reanalysis under an isocratic elution. Table 3 reveals the
proportions of each metabolite obtained where it can be seen
that the major building block detected was glucose, which could
indicate the presence of glucans in the growth media. (1 / 4)-aD-Glucan, a polysaccharide comprised of D-glucose monomers
linked by glycosidic bonds, is a known extracellular polysaccharide produced by D. tertiolecta.55
These preliminary results imply that the entrapped microalgae within the spherical hybrid beads are able to produce
primary products over the course of one month and excrete
these photosynthates through the porous network of the beads
and into the surrounding media.

Percentages of neutral sugar building blocks detected within

a culture media exposed to hybrid photosynthetic beads for 32 days

Table 3

Sugar
Arabinose
Rhamnose
Glucosamine
Galactose
Glucose
Xylose
Mannose

Gradient elution%
3.8a
2.3
5.2
75.4
6.4
6.9

Isocratic elution%
1.3
1.1
2.1
5.8
77.1
12.5a

Arabinose and rhamnose could not be separated via a negative

gradient elution, whilst xylose and mannose could not be separated
by isocratic elution.

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Conclusions
The extraordinarily long-term viability and high activity of
microalgae entrapped within an alginatesilica hybrid matrix
hold much promise for the design of new generation xed
biomass photobioreactors that are based on the microencapsulation technology described within. These PBRs could be
employed in the sustainable production of high value metabolites based on a judicious choice of the cell line. This work has
shown how it is possible to engineer more robust beads than
previously seen by combining two dierent technological
concepts; the use of a hybrid alginatesilica material in a core
shell system. This is made possible by the use of a polycation
such as PDADMAC, which not only catalysed the formation of a
silica network on the atomic scale but also acted as a occulating agent on the nano(micro)metric scale, thereby aggregating the silica colloid and forming a silica rich outer
membrane around the core of the alginatesilica bead. These
novel hybrid coreshell spherical beads having an adjustable
millimeter scale size and exhibiting good mechanical and
chemical stabilities were prepared through a simple ecofriendly and ecient one-pot coacervation method, overcoming
the need for multi-step manufacturing processes making
this technology inexpensive and ideal for industrial
implementation.

Acknowledgements
This work was realised in the framework of FOTOBIOMAT
funded by the Walloon Region and Redugaz, an Interreg IV
(France-Wallonia) project funded by the European Union and
the Walloon Region. B.-L. Su acknowledges the Chinese Central
Government for an Expert of the State position in the programme of Thousand talents and the Chinese Ministry of
Education for a Changjiang Scholar position at the Wuhan
University of Technology.

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