PAPER www.rsc.org/materials | Journal of Materials Chemistry

Immobilization of yeast and bacteria cells in alginate microbeads coated withsilica membranes: procedures, physico-chemical features and bioactivity†

Emanuela Callone,a Renzo Campostrini,a Giovanni Carturan,a Agostino Cavazzab and Raffaele Guzzon*b

Received 30th April 2008, Accepted 16th July 2008

First published as an Advance Article on the web 8th September 2008

DOI: 10.1039/b807301e

Salt alginate beads are used to entrap yeast or bacteria cells and exploited as protective environment for

deposition of silica gel membranes. This inorganic layer is obtained through different methods: by

dipping the beads in a silica sol (method A), by further consolidating them with tetraethoxysilane in an

apolar solvent (method B), and by coating the beads with methyltriethoxysilane in the gas phase

(method C). Physical–chemical and biological features are investigated. The pyrolysis study elucidates

the features of the polymeric organic layer. Elemental analysis and 29Si solid state NMR prove the

presence and the condensation degrees of the silica membranes. The bioactivity is studied by evaluating

both glucose (Saccharomyces cerevisiae) and L-malic acid (Oenococcus oeni) fermentations. The

fermentation performance is discussed considering possible limitations of mass transport across the

silica gel layer. Method A produces an inorganic layer made by of 57.3% Si(OSi^)4 and 42.7%

HO–Si(OSi^)3 units). In this case a linear relation is found among different contact times between

silica sol and beads and both silica amount and average thickness. Method B appears to be successful in

building up the layer, but it is also detrimental to cell viability. Method C is very efficient in terms of

mass deposit, cell viability maintenance and leakage reduction. In this case the membrane is constituted

by H3C–Si(OSi^)2OH and H3C–Si(OSi^)3 units in an almost 1 : 1 ratio.

Introduction

Immobilization of such biological agents as, for instance,

macromolecules, cells and microorganisms, is commonly

proposed as a successful technical answer to the increasing

demand of long-term bio-efficiency. It may also be claimed

that this approach provides controlled operation conditions

with maintenance of functions and selectivity proper to a free

biological load. For a wide field of possible applications, the

immobilization technique requires adjustments that concern

mainly the material used for heterogeneous-phase biomass

confinement. It should preferably possess the following features:

i) mechanical stiffness vs. stirring shear stress; ii) inertness vs.

substrate bioconversion; iii) stability vs. long-term/continuous

process leakage; iv) biocompatibility and non-toxicity vs. active

biological agents. Moreover, the material should show tailored

porosity for free diffusion of substrates and products.

An attractive field of exploitation is cell immobilization in

fermentation processes.1,2 Tested immobilization materials

are alginates,3–6 ceramics or glass beads,7 cellulosic materials,8

g-alumina,9 sol–gel silica films,10 polyacrylamide gels,11 and

combinations of different materials, according to the reviewed

classification of immobilization procedures.12

aDepartment of Materials Engineering and Industrial Technologies,University of Trento, via Mesiano 77, 38050 Trento (TN), Italy; Tel:+39 0461 882453bIASMA Research Center, via Mach 1, 38010 S. Michele all’Adige (TN),Italy. E-mail: raffaele.guzzon@iasma.it; Tel: +39 0461 615262

† Electronic supplementary information (ESI) available: Pyrolysis studyof the silica/alginate immobilization matrix. See DOI: 10.1039/b807301e

This journal is ª The Royal Society of Chemistry 2008

The entrapment of cells by such ionotropic hydrogels as

alginates is widely used13,14 for the ease of fabrication and for the

maintenance of functions and viability of immobilized cells.15

However, these materials present the disadvantage of both low

mechanical strength and poor chemical durability, which may

lead to cell leakage with active proliferation in the medium.16

Recently, Mørch et al. reported a complete study about the

complex relationship among several parameters: nature of the

bonding cation (Ca2+, Sr2+, Ba2+), type of alginate [rich M

(mannuronic acid) vs. G (glucuronic acid) regions], permeability,

gel strength, and alginate distribution.17

In recent years, an approach of wide applicability is based

on the construction of a covalent polymeric network around

the bio-molecules or cells, which remain definitely trapped.18–20

The advantages of this method derive from the enormous

advancements made in materials science and nanotechnology.

They could be applied also to solve biotechnology problems,

such as cell encapsulation.21–25 Most of the studied materials are

siliceous products obtained by sol–gel processes, i.e. the assembly

of ^Si–O– polymeric structures under mild conditions, starting

from suitable solution of molecular precursors.26–30

The attention for siliceous materials in cell immobilization is

determined by some outstanding chemical and physical features

of silica. The strength of the Si–O bond (452 kJ mol�1) ensures

both inertness and chemical stability; the choice of both

precursors and gelling conditions provides materials with

porosity suitable for free mass transport and stable cell trapping;

the mechanical features may be tuned from bulk silica to silicone

rubber behaviour, by increasing the percentage of alkoxide

precursors holding appropriate functional groups.

J. Mater. Chem., 2008, 18, 4839–4848 | 4839

The methods of achieving a cell-safe encapsulation by silica gel

are characterized by the need to avoid the toxicity proper to

classic sol–gel processing. Such toxicity could be related to both

the use of silane precursors, the release of alcohol and the pH

values of the medium.

The principal expedients overcoming sol–gel toxicity are:

a. use of gas-phase Si alkoxides,31

b. use of purge inert gas flow to remove quickly the alcoholic

by-products by venting,31

c. rapid evaporation of alcohol from the liquid phase before

cell encapsulation,32

d. use of non-alkoxide Si precursors, such as silicates and

colloids.33–35

However, the sol–gel immobilization of cells remains difficult

and with tricky handling steps requiring the expertise of scientists

who otherwise may not be familiar with cell handling. This fact

may constitute a severe limitation to its exploitation in the food

industry, which requires large biomass and easy immobilization

procedures. Moreover, the formation of the Si–O cage around

the cells or cell aggregates may be not an innocent procedure, in

the sense that mutual interactions between sol–gel silica and

biological agents are possible; this fact constitutes a negative

aspect in the case of the properly suspicious food industry.

With these premises, it is here presented an alternative

approach for immobilization of yeast and bacteria, based on

a double layer preparation. Thus, the cells remain encapsulated

in ordinary M alginate (M ¼ Ca2+, Ba2+) beads coated by a silica

gel layer. As shown in Fig. 1, ionotropic hydrogel is used here

for encapsulation of cells in beads with predictable geometry.

It represents a protective microenvironment that allows the

deposition of silica under safe conditions, because the cells

remain in a heterogeneous phase with respect to silica coatings.

The silica gel layer is obtained by appropriate choice of the

functional groups bonded to the Si atom. The method, depicted

in Fig. 1, was tested for immobilization of different cells. It is here

applied to Saccharomyces cerevisiae and Oenococcus oeni cells,

both used in ordinary wine fermentation, to provide an alter-

native to the traditional use of free microorganism culture. The

work focuses on the features of different silica coatings and on

the activity of immobilized cells in comparison with free ones.

Materials and methods

Materials

Na alginate was purchased from Sigma Aldrich (Alginic acid

sodium salt, low viscosity) as 2 wt% solution. Other chemicals

Fig. 1 General scheme of M2+ alginate + sol–gel silica immobilization of

cells.

4840 | J. Mater. Chem., 2008, 18, 4839–4848

were reagent-grade products (Fluka) used without further puri-

fication. Tetraethoxysilane (TEOS) was purchased from Fluka

and methyltriethoxysilane (MTES) from Sigma Aldrich.

Saccharomyces cerevisiae cells were cultured in modified yeast

malt extract broth: tryptone (Oxoid) 5 g dm�3; yeast extract and

malt extract (Oxoid) 3 g dm�3; Tween 80 (Acros Organics)

0.1 cm3 dm�3; glucose 70 g dm�3 (Carlo Erba); Oenococcus oeni

cells were cultured in Tomato Juice broth (Difco).

Microorganisms

The Saccharomyces cerevisiae cells, belonging to IASMA

Research Centre collection, were cultured in yeast medium broth

for 48 h at 25 �C up to 109 cfu cm�3. The culture medium was

centrifuged at 4000 rpm for 10 min; the broth was eliminated and

cells were washed with physiological solution and stored at 5 �C

before immobilization treatment. The strain Oenococcus oeni

IASMA Pn4, also belonging to IASMA Research Centre collec-

tion, was produced and freeze-dried by Lallemand Inc. with

NBRª technology. The cells were stored in a freezer at �20 �C,

then directly immobilized. No reactivation was necessary.

Immobilization procedures

Yeast or bacteria cells were dispersed in sterile Na alginate

solution, to obtain a cell density of 109 cfu cm�3 of Na alginate

suspension for both Saccharomyces cerevisiae and Oenococcus

oeni. The suspension was extruded through a series of needles

(diameter ¼ 0.2 mm; rate ¼ 0.5 cm3 min�1) with an injector in the

presence of a constant airflow (2 dm3 min�1) in a sterile 0.1 M

CaCl2 (or BaCl2) solution under continuous agitation. Details of

the procedure were already reported.27 The beads were main-

tained in solution for 10 min, racked and washed with sterile

distilled water.

Wet coating (method A). Hydrolysis of Si(OEt)4 (TEOS) in an

ethanol–water solution (ethanol 508.57 cm3 dm�3, TEOS

371.46 cm3 dm�3, HCl 0.01 M 119.97 cm3 dm�3, nominal SiO2

concentration ¼ 100 g dm�3) was carried out under acid catalysis.

24 h after mixing, the solution was diluted with the same water

volume and evaporated at 30 �C under reduced pressure to about

half of the original volume until a nominal SiO2 concentration of

100 g dm�3. Alginate beads containing the cells were immersed in

this suspension and collected after 30 s, 5, 15 and 30 min.

Samples were recovered by filtration, washed with sterile distilled

water and stored in a low nutrient broth (yeast medium broth

diluted 1 : 1 with water for S. cerevisiae, tomato juice broth

diluted 1 : 1 with water for O. oeni).

Two-phase coating (method B). A suspension of beads prepared

according to method A (5 cm3 of beads in 20 cm3 of H2O) was

mixed with 20 cm3 of a CH2Cl2–hexane (50 : 50 wt/wt) solution

under vigorous stirring producing an emulsion of the two immis-

cible liquids. The mixture was poured into a glass tube closed by

a filter frit mounted on a 100 cm3 flask, with a lateral gas inlet,

containing 10 cm3 of TEOS and stored at 80 �C in an oil bath. A

5 dm3 min�1 N2 flux was passed into the flask, providing the TEOS

stripping and bubbling into the two-phase suspension across the

filter frit. The operation was prolonged until complete TEOS

This journal is ª The Royal Society of Chemistry 2008

evaporation. Beads were recovered by sedimentation, washed

with distilled water and stored at 5 �C in low nutrient broth.

Gas coating (method C). 5 cm3 of wet beads prepared as

described in method A were dried in a filter frit by crossing an air

flow until evidence of free movement of single beads. 10 cm3 of

CH3Si(OEt)3 (MTES) were stripped by an air flow across the

microspheres as described in method B, until complete MTES

evaporation. Beads were poured into distilled water, filtered and

stored at 5 �C in low nutrient broth.

Physical and chemical characterization

Dimensions of beads before and after silica gel coating in the

presence of immobilized cells, and produced according to

reported procedures, were determined by optical microscopy

(100 beads for each trial). Data are reported in Table 1. Electron

microscopy, SEM Cambridge Stereoscan 200 (Oxford Instru-

ments Analytical) and TMP ESEM (Philips), were used to define

the surface morphology of specimens.

Solid state NMR experiments were carried out on a Bruker

Avance 400 WB spectrometer, operating at 79.493 MHz for 29Si.

Samples were dried at 120 �C for 24 h and packed in 4 mm diameter

zirconia rotors. Experimental conditions: 29Si ramp CP-MAS:

2.7ms 90� pulse, 5 s for recycle delay, 5 ms of contact time and 5 kHz

of rotating speed. Q8M8 [octakis(trimethyl siloxy)silsesquioxane]

was used as primary shift scale references for 29Si.

Thermogravimetric (TG) and differential thermal analyses

(DTA) were performed on a LabSys Setaram thermobalance

operating in the 20–1000 �C range, with a heating rate of 10 �C

min�1. Thermal analyses were recorded working under 120 cm3

min�1 He (99.999%) flux. Powdered samples (ca. 10–20 mg) were

analyzed using alumina crucibles and a-Al2O3 as reference.

A direct and continuous sampling of the gas phase evolved from

the solid during the thermal analysis was realized with TG-MS

coupling by means of a lab-assembled transfer line, which inter-

faced the thermobalance’s furnace with the mass spectrometer.

The mass spectral analyses (MS) were carried out using a TRIO

1 VG quadrupole mass spectrometer. Electron mass spectra

(70 eV) were continuously recorded with frequency 1 scan s�1 in

the 2–500 amu range.

The instrumental interface was made of 2 m of a deactivated

silica capillary column (i.d. 0.19 mm) thermostatted at 250 �C,

which allows the detection of any gaseous released species

Table 1 Labelling of samples and main analytical data

SampleEncapsulationmethod

Mean beaddiameter/mm

DepositSiO2

a/g

1 Ca alginate 428 � 93 —2 Ba alginate 420 � 85 —3a Ca alginate + method A (30 s) 470 � 92 —3b Ca alginate + method A (5 min) 478 � 93 5.23c Ca alginate + method A (15 min) 489 � 94 5.73d Ca alginate + method A (30 min) 511 � 91 6.24 Ca alginate + method B 529 � 88 —5 Ca alginate + method C 403 � 67 14.0

a Determined on specimen dried at 120 �C for 4 h. Analysis performed by C

This journal is ª The Royal Society of Chemistry 2008

throughout the pyrolysis process. MS data were recorded as

a continuous sequence of mass spectra from which it was possible

to extrapolate both the total ion current (TIC) plot and

the contribution of any m/z ion current (IC) graphs vs. time

(i.e. temperature of the pyrolysis process).

A semiquantitative evaluation of pyrolysis data was performed

by processing the ion currents of appropriate m/z signals, the

mass spectra of the evolved species and the integrated values of

suitable ion currents; procedure details are reported elsewhere.36,37

Microorganism viability evaluation during immobilization

treatments

1 g of microspheres loaded with microbial cells was mechanically

crushed and homogenized with 9 cm3 of distilled water in

a Stomacher 400 Blender (Birkmann Istrument) to obtain

complete and homogeneous dispersion of cells. The Saccharo-

myces cerevisiae cell density and viability were monitored by

methylene blue staining and cell count in a Burker Chamber,

with an optical microscope. The cell density of Oenococcus oeni

was evaluated by plate count on tomato juice agar (Difco). The

viability was determined for both free and entrapped cells, in

alginate beads with or without silica gel coatings (different

procedures and different contact times). The results were

expressed as living cell %, after treatments, of the biological load

mixed in the alginate suspension.

Cell leakage

5 g of microspheres were stored in 100 cm3 of low nutrient broth at

10 �C. The cell release was evaluated by measuring the cell density

in the medium after 24 h of immersion. The measure was performed

with a plate count in WL nutrient agar (Oxoid) for yeast, and with

a plate count in tomato juice agar (Difco) for lactic acid bacteria.

Fermentation activity

The activity was determined by the kinetic study of alcoholic

(immobilized yeast) and of malolactic (immobilized bacteria)

fermentations at different substrate concentrations, using an

appropriate amount of beads. Yeast alcoholic fermentation was

performed at 25 �C, in modified yeast malt extract broth, without

carbon sources, at five different glucose concentrations (0.4, 0.6,

0.7, 0.8, 1.1 M). The density of free and immobilized yeast was

regulated to 107 cfu cm�3 of medium, for each trial. The

edm�2

SiO2

thickness/mm

Yeast celldensity/108 cfu g�1

Bacteria celldensity/108 cfu g�1

Cell leakageafter 24 h

— 8.2 8.4 57%— 6.9 6.3 35%— 7.9 7.8 28%5.2 7.4 7.4 22%5.8 9.2 7.6 23%6.2 9.3 7.9 24%— — 0.4 —9.2 9.2 7.8 <1%

entro Sperimentale Del Vetro (Murano, Venice, Italy).

J. Mater. Chem., 2008, 18, 4839–4848 | 4841

advancement of the alcoholic fermentation was evaluated by the

mass loss related to CO2 production.38 The malolactic fermen-

tation was performed in tomato juice broth without sugars; the

density of free and immobilized bacteria was adjusted to 107 cfu

cm�3 of medium, for each trial, using an appropriate amount of

beads. The concentration of L-malic acid was adjusted to 1, 3, 4,

6, 7 � 10�2 M. The reaction advancement was followed by

measuring the volume of CO2 released vs. time, with a gas burette

(detection limit: 0.1 cm3), jacketed by circulating water at

a constant temperature of 22 �C. All fermentation curves were

fitted using the Gompertz function39 and the program Table

Curve 2D� (Systat Software Inc., Richmond, CA) Results were

treated according to the Lineweaver–Burk method.

Results and discussion

Chemical and physical characterization

According to Fig. 1, microorganisms were dispersed in a 2 wt%

Na alginate solution, and were further processed to obtain Ca

(or Ba) alginate beads. Suspension viscosity, inlet gas flow and

aging time in CaCl2 (or BaCl2) solution were studied previously

to achieve reliable control of microsphere diameter.40 The

method used here involves the coaxial air-flow dropping of the

Na alginate suspension to produce microcapsules in the 400–

800 mm diameter range.40 The general conditions are reported in

Fig. 2. Prepared microspheres displayed an average diameter of

450 mm with value distributions reported in Fig. 3. Fig. 4 shows

typical optical micrographs of the specimens: entrapped

Saccharomyces cerevisiae (Fig. 4A) and Oenococcus oeni

(Fig. 4B) cells were detectable. The evidence of both the budding

of yeast and the preferential chain-line structure of bacteria

suggested that the immobilized biological load maintains its

viability. The whole beads, i.e. Ca alginate microspheres holding

immobilized yeast cells, showed a spherical geometry that dete-

riorated over a period of 30–35 days, breaking up into almost

hemispherical parts. In contrast, the beads containing bacteria,

the Ba alginate capsules, and the Ca alginate beads coated by

Fig. 2 Apparatus and experimental conditions for production of M2+

alginate beads.

4842 | J. Mater. Chem., 2008, 18, 4839–4848

silica gel (methods A, B, and C) were stable in time. A conceivable

interpretation can be advanced: yeast cells continue to grow even

at the coalescence density, entailing a valuable volume increase.

The resulting stress could promote the observed bead breaking.

In contrast, bacteria have slower growth rates, and Ba alginate

capsules show higher strength than Ca alginate ones (Young’s

modulus E was reported in Mørch et al.,41 and Smidsrød42), so

that the original geometry was indefinitely preserved.

The mechanical aspects of the antagonism between cell growth

and material stiffness were already studied and discussed,

considering a biaxial model of the stress field.10 If applied to the

present case of fracture into two hemispheres, it agrees with

a preferential cell volume increase at the centre of the bead, so

that fracture starts from the centre and propagates along the

maximum diameter. Moreover, it was shown that M alginate

linking, according to the egg-box model,43,44 decreases from the

surface to the centre of beads;45 consequently, internal cells

growth was favoured owing to minor M alginate constraint.

For a clear description of the encapsulating material both 29Si

solid state NMR and thermal analysis were done on the samples.

Because of its good bio-performances summarized in Table 1,

sample 5 was extensively studied. The details are reported in the

ESI.† It is interesting to notice that the thermogravimetric curve

showed three main events with a total weight loss of 61.1%. They

were centred at 273, 480 and 738 �C with relative intensities of 40,

7, 9 and 10 wt%, respectively. The released species were CO2 and

H2O at first, followed by various hydrocarbons, and only CO2

during the third event.

These data indicated that during alginate polymerization the

complete substitution of Na+, originally coordinated to the

alginate units, with a semistoichiometric amount of Ca2+

occurred. In fact, the thermal formation of CaCO3, which

decomposes at around 800 �C releasing CO2, and the presence of

CaO in the pyrolyzed residue accounted for this. The release of

water in the first event accounted for the complete condensation

of both the ^Si–OH groups of the silica gel and the ^C–OH

groups of the alginate structure. The residual carbons of the

alginate in part were released as complex hydrocarbon species

(Diels–Alder reaction) and in part pyrolyzed to graphitic carbon.

Moreover, the mass balance combined with the peak analysis

of NMR data was used to propose a nominal composition of the

inorganic layer of (OH)0.427SiO1.786, in agreement with the

elemental analysis and the residual SiO2 in the pyrolyzed solid.

Comparison of bead coating

Wet coating (method A). The silica sol was prepared by TEOS

hydrolysis under acid catalysis in hydro-alcoholic medium,

which was evaporated to eliminate ethanol, and re-diluted with

water to restore the original 100 g dm�3 SiO2 nominal concen-

tration. The alginate beads were immersed in the silica sol with

different contact times (Table 1). The SiO2 coating affects the

bead diameter, which linearly increases with the contact time,

according to the Dd ¼ kt law, where d is the bead diameter, t is

the contact time between beads and silica sol and k ¼ 1.2 mm s�1.

Elemental analysis of SiO2 indicated that deposited silica

increases from 5.23 to 6.20 g (m2 of microspheres surface)�1.

Considering the silica density ¼ 1 g cm�3, the SiO2 layer thickness

was calculated (Table 1).

This journal is ª The Royal Society of Chemistry 2008

Fig. 3 Diameter distribution of beads. A: sample 1, B: sample 3d, C: sample 4, D: sample 5.

Fig. 4 Optical microscopy observations of Ca alginate microspheres

(sample 1). A: budding of yeast cells inside the bead; B: immobilized cells

of Oenococcus oeni organized in typical chains.

Some comments about the mechanism of SiO2 sol deposition

on the M alginate surface are necessary. Firstly, the process of

deposition clearly combined two different mechanisms, an initial

This journal is ª The Royal Society of Chemistry 2008

very fast step, accounting for the immediate Dd ¼ 30 mm,

followed by a second one, corresponding to a linear Dd (or SiO2

thickness) increase vs. contact time. Secondly, the dimensional

stability of the silica sol vs. coalescence is determined by the

electrostatic repulsion among negatively charged micrometric

particles.46 Both hydrophilic character, due to surface ^C–OH

exposed M and G units, and positive surface charge, due to

incomplete coordination of M2+, may be envisaged for alginate

beads. Accordingly, at the moment of mixing, immediate SiO2

particle flocculation occurred on the alginate surface. Both the

favourable electrostatic interaction and the possible condensa-

tion of ^C–OH of saccharide species vs. ^Si–OH47 justify the

stability of deposited silica gel. The diameter increase of 50 mm is

not in contrast with a homogeneous deposition of silica gel

particles that have an average diameter of 20–30 mm. Thus,

alginate beads immediately achieved the same nature as silica sol

particles, which coalesce according to the second-order kinetic

law proposed by Smoluchowski.47 At the beginning, the process

follows a pseudo-first order kinetic, because the SiO2 sol particle

concentration largely exceeds the alginate bead concentration;

then it becomes a pseudo-zero order process, because the alginate

bead concentration is constant. This interpretation agrees with

the experimental evidence of linear dimensional and thickness

increases vs. contact time (Fig. 5).

The chemical nature of deposited silica gel was defined by

solid state 29Si NMR experiments (Fig. 6A, sample 3d and

Table 2). The experiments indicated that Si was involved in

a network constituted by 57.3% Si(OSi^)4 (Q4) and by 42.7%

HO–Si(OSi^) 3 (Q3) units. The chemical shift values for Q3

(�101.2 ppm) and Q4 (�109.0 ppm) agree with literature

data.48,49 These results showed that deposited silica sol particles

did not present residual ^Si–OEt moieties and condensation

among Si–OH groups had extensively occurred, as evidenced by

J. Mater. Chem., 2008, 18, 4839–4848 | 4843

Fig. 5 Wet SiO2 deposition on Ca-alginate microspheres (method A).

Diameter increase (6d) and layer thickness vs. contact time.

Fig. 6 29Si NMR spectra: A, sample 3d (method A); B, sample 5

(method C).

Table 2 29Si NMR data. T2 ¼ RSi(OSi)2OH, T3 ¼ RSi(OSi)3; Q3 ¼HOSi(OSi)3 Q4 ¼ Si(OSi)4

T2 T3 Q3 Q4

Chemical shift, ppm �54.6 �63.3 �101.2 �109.0Sample 3d — — 42.7% 57.3%Sample 5 33.0% 34.9% 12.5% 19.6%

Fig. 7 SEM micrographs of Ca alginate beads. A: uncoated Ca alginate

microbeads (sample 1); B: microbeads coated by silica sol (method A,

sample 3d).

the absence of Q1 and Q2 and by the important percentage of Q4.

Considering the small dimension of sol particles, it may

reasonably be assumed that most of the ^Si–OH (Q3) units were

preferably exposed at the surface. The morphology change after

silica sol coating was evidenced by ESEM observations reported

in Fig. 7A and B. The silica gel layer (Fig. 7B) clearly reduced the

surface roughness (Fig. 7B) and it was homogenously distributed

on the alginate surface.

Two-phase coating (method B). This method was proposed to

favour the reaction between alginate and hydrolyzed silane,

stiffening the silica gel layer–alginate surface interaction. As

a matter of fact, TEOS is quite soluble in apolar solvents, such as

4844 | J. Mater. Chem., 2008, 18, 4839–4848

hexane–CH2Cl2, but the water suspension of beads, prepared

according to method A, presents a clear hydrophilic character;

thus the two phases are not miscible. As the two phases were put

in contact, hydrolysis of TEOS leads to exclusion of Si(OH)4

from the organic phase, with favourable aggregation of this

product to alginate beads. Moreover, the use of gas bubbling

allows both operating with a high dispersion of the two phases

and possible removal of ethanol, produced during TEOS

hydrolysis. The method is quite fast and suitable for handling

high loads of microbeads. These features have been considered

for immobilization of bacteria which do not tolerate prolonged

exposure to toxic and non-nutritive environments. Recovered

alginate beads did not aggregate and preserved the original

shape. Unfortunately, a preliminary bacteria viability test

(sample 4, Table 1) indicated a high mortality of the biological

load. Consequently, the method was dropped.

Gas-coating (method C). The method provides a siliceous layer

deposition, around the microbeads, using silanes in the gas

phase. It was performed by direct reaction between MTES in the

gas phase and M alginate beads, already coated by silica sol

particles (method A). The high reactivity of MTES vs. surface

exposed Si–OH was exploited in order to stabilize the sol-particle

layer. The aim was the build-up of a siliceous membrane with

narrow porosity and plastic-like behaviour. These expectations

were based on previous experience with organic modified sili-

ceous materials.50 Moreover, the use of alkoxides in the gas phase

was known to be effective in producing stable coatings on M

alginate beads, without detrimental effects on cell viability.31,51

Obtained specimens showed a very smooth surface (Fig. 8B) and

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Fig. 8 ESEM micrographs of Ca alginate beads. A: Ca alginate beads

after 30 min wet coating (method A, sample 3d). B: Microbeads after

30 min wet coating + MTES gas coating (method C, sample 5).

the deposited layer covers the residual surface unevenness of

beads previously coated by method A. It is noteworthy that the

average diameter was reduced from 511 (sample 3c, Table 1) to

403 mm (sample 5, Table 1), resulting in a 21% decrease. This fact

has been previously observed and interpreted in terms of

mechanical stress exerted by the siliceous membrane on the soft

M alginate inner part.40 Indeed, the amount of deposited silica

gel layers increased from 5.23 to 14.02 g m�2, evidencing the

effectiveness of the mass deposition.29Si NMR spectra (Fig. 6B and data of Table 2) gave infor-

mation about the chemical nature of the silica gel deposit:

CH3Si(OSi^)2OH (T2) units, at 54.6 ppm, and CH3Si(OSi^)3

(T3)units, at 63.6 ppm were detected in 48% and 52% propor-

tion respectively. The chemical shift values were in accordance

with literature data on resonances of CH3Si(OSi^)3�n(OH)n

species.49 Thus, reaction of MTES with M alginate beads coated

by silica sol (sample 5, Table 1) involved the formation of

a well-connected silica gel network, as supported also by the

absence of T1 units and the important presence of T3 units. The

signals corresponding to Q3 and Q4 units referred to the

primary sol layer and were identical, in terms of chemical

shift and relative abundance, to the signals of sample 3d

(Fig. 6A).

Cell leakage and viability

The yeast cell density was almost constant for samples from 1 to

3d (7.7 � 1 � 108 cfu cm�3), but it increased for sample 5 (1.3 � 0.2

� 109 cfu cm�3), i.e. for Ca alginate beads coated using method C.

The same trend was observed in the case of immobilized bacteria

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(sample 1–3d ¼ 1.9 � 0.7 � 108 cfu cm�3, sample 5 ¼ 3.7 � 0.6 �108 cfu cm�3). As sample 5 undergoes a considerable shrinkage,

corresponding to a volume contraction of 21% (with respect to

sample 3d), the increase of cell concentration for sample 5 was

clearly justified.

Cell leakage is an important parameter to define the effec-

tiveness of the immobilization material. Data are reported in

Table 1. The high cell loss observed after 24 h for Ca alginate

beads was explained by the low stiffness of this immobilization

matrix. The phenomenon was reduced for Ba alginate (sample 2)

and Ca alginate beads coated by the silica gel layers (samples

3a–3d). The absence of leakage observed for sample 5 must be

emphasized; in this case the siliceous MTES membrane behaves

as a totally immobilizing material. This fact reflects the physical

and chemical nature of the MTES-derived silica gel, which

usually presents very low and narrow porosity.50 The positive

feature of sample 5 finds a favourable counterpart in cell viability

data, which are the highest of all the samples. These results

prompt the development of method C as an efficient and

promising technology for safe microorganism confinement in

heterogeneous phase.

Bioactivity of immobilized microorganisms

Fermentation tests were performed to verify the efficiency of

immobilized cells, in comparison with free ones. It is well

recognized that in food fermentations cell multiplication and

substrate conversion are not directly related.52 Within the

immobilization matrix, the cell growth may be excluded, due to

cell coalescence density at the moment of immobilization. Under

these conditions, the cellular energy drives the fermentative

metabolism. Several authors, reviewed by Groboillot et al.,53

observed that immobilized microorganisms show valuable

increases of fermentation efficiency.

Saccharomyces cerevisiae kinetic tests. Glucose degradation by

a pure culture of Saccharomyces cerevisiae was used to study the

fermentation activity of both free and immobilized cells by

method A and method C. The glucose concentrations were

chosen within the range of those found in wines. Evolution of

CO2 vs. time was followed measuring the weight loss during the

process;54 curves were fitted using a modified Gompertz equa-

tion39 and lag time. Maximum fermentation rate and the

asymptote were calculated for each trial (Fig. 9). The lag time of

each curve was 17 h � 7 min, consistent with Saccharomyces

cerevisiae metabolism; the asymptotes agreed with the stoichio-

metric balance of CO2 produced from glucose fermentation. The

maximum rates were linearly linked to the initial concentrations.

Fig. 10 reports the Lineweaver–Burk plots for experiments

carried out with free and immobilized yeast cells. Calculated

maximum rates (Vmax) and Michaelis–Menten constants (Km)

are reported in Table 3. An important result concerns the

increase of Vmax of cells immobilized with method A (sample 3d,

Table 3), in agreement with the above general statements. On the

other hand, the immobilization of cells in microbeads coated

with gas-deposed silica gel (method C, sample 5, Table 3) reduced

the maximum rate to an extent comparable with free cells. This

behaviour was found even in bacteria and will be discussed in the

next section.

J. Mater. Chem., 2008, 18, 4839–4848 | 4845

Fig. 9 Typical glucose fermentation fitted with the Gompertz equation.

a: Asymptote (ml CO2), b: Vmax (ml CO2 day�1), c: lag time (day). Sample

5, [glucose] ¼ 10% w/w, t ¼ 25 �C, immobilized cell load ¼ 109 cfu g�1,

bead weight ¼ 10 g.

Fig. 10 Lineweaver–Burk plots of glucose fermentation by Saccharo-

myces cerevisiae.

Table 3 Kinetic parameters of glucose fermentation by Saccharomycescerevisiae

Sample 108 Vmax/M s�1 g(cells)�1 Km/M�1

Free cell 4.8 � 0.5 0.3 � 0.13d 7.8 � 0.3 0.4 � 0.25 3.3 � 0.02 0.1 � 0.03

Fig. 11 L-Malic acid fermentation by Oenococcus oeni. Sample 5,

[L-malic acid] ¼ 0.1 M, t ¼ 22 �C, total immobilized cell load ¼ 108 cfu g�1,

bead weight ¼ 5 g.

Fig. 12 Lineweaver–Burk plot of L-malic fermentation by Oenococcus

oeni.

Table 4 Kinetic parameters of malolactic fermentation by Oenococcusoeni

Sample 106 Vmax/M s�1 g(cells)�1 102 Km/M�1

Free cell 4.2 � 0.4 4 � 0.13d 11 � 0.6 4 � 0.25 4.0 � 0.2 2 � 0.6

Oenococcus oeni kinetic tests. Malolactic bacteria are quite

sensitive to the environment: high cell death is often observed

after their inoculation in wine, and complete inactivity may result

from harmful compounds, even if they are present at concen-

tration levels bearable by yeast cells. Despite this, under the

working conditions described in the Experimental section (high

cellular concentration, continuous agitation, and pH control),

the malolactic fermentation advancement was linearly dependent

on time (Fig. 11), lag phase was not observed, and the slope of

fermentation curve was directly related to the initial concentra-

tion of L-malic acid. These results agreed with the ordinary

4846 | J. Mater. Chem., 2008, 18, 4839–4848

biochemical behaviour of L-malic degradation, which is a one-

step first-order reaction, catalyzed by the malolactic enzyme.52

Data in Fig. 12 and Table 4 summarize the bioactivity of free and

immobilized Oenococcus oeni cells. If compared to free cells,

immobilized bacteria of sample 3d showed a valuable increase of

the maximum fermentation rate, but displayed a comparable Km.

These results indicate that method A is compatible with the

activity of microorganism requirements, and that the silica gel

coating did not interfere with the transport of substrate and

products from the medium to cells and vice versa.

On the other hand, the siliceous coating deposited from MTES

treatment (method C, sample 5) changed the cell activity: Vmax

was near to that of free cells, but their Km was lower. Discussion

of these facts may involve the properties of the silica gel

membrane, deposited by reaction with gaseous MTES. It is

known that siliceous gel materials prepared from MTES display

This journal is ª The Royal Society of Chemistry 2008

low surface area, narrow porosity and mechanical behaviour

approaching that of silicon rubber.50 It seems acceptable to

attribute the observed Vmax and Km changes, compared to those

of sample 3d (Tables 3 and 4) to these features, involving limi-

tations of mass transport across the MTES derived membrane.

Indeed, the presence of CH3–Si groups modified the surface

wettability, the acid/basic character, and the hydrophilic

behaviour of the siliceous layer and may also affect substrate and

product transport.

Conclusions

This work defines some aspects concerning safe and general

immobilization of microorganisms of interest for fermentation

processes. In particular, the research confirms the effectiveness of

entrapment in a double-layer matrix. The method of Ca alginate

bead production by using coaxial air-flow dropping of a Na

alginate cell suspension in CaCl2 allows the production of beads

with a narrow diameter distribution centred at 450 mm and it is

suitable for continuous processing. The subsequent coating with

a silica gel layer improves the mechanical stability and reduces

the leakage. Optimization of the cellular entrapment is achieved

by deposition of a siliceous membrane from gaseous MTES on

Ca alginate beads already coated by silica sol particles: the

viability of immobilized cells is good, leakage is absent and

bioactivity is maintained. These positive performances suggest

the possibility to extend the immobilizing process to other useful

microorganisms. Before doing that the research requires:

a. testing of immobilized cells (yeast and bacteria) fermenta-

tion experiments in real beverages;

b. deeper investigation of the chemical interactions among the

Ca alginate surface, the deposited silica sol layer and the MTES-

deposed membrane.

Acknowledgements

Edmund Mach Foundation (IASMA) is acknowledged for

financial support, granting a PhD bursary to Dr R. Guzzon

and a research fellowship to Dr E. Callone; Dr S. Krieger and

Dr P. Vagnoli (Lallemand Inc.) are deeply acknowledged for

financial support, interest, suggestions, and stimulating discus-

sions during the advancement of this work.

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