Journal of Biotechnology 80 (2000) 203 – 215

www.elsevier.com/locate/jbiotec

Analysis and description of the evolution of alginate

immobilised cells systems
A. Laca, L.A. Garcı́a, M. Dı́az *
Department of Chemical Engineering and En6ironmental Technology (I.U.B.A.), Uni6ersity of O6iedo, c/Julián Cla6erı́a s/n,
33071 O6iedo, Spain
Received 18 January 1999; received in revised form 13 July 1999; accepted 14 March 2000

Abstract

Different immobilised cells models, including very simple ones, can be useful in the fitting of experimental results.
However, goodness or the ability to extrapolate results needs to be in accordance with basic observations and these
will also suggest models to be proposed. In this paper, observations of calcium alginate/bacteria systems are used to
show the ability of basic models to fit classic observations, as well as how new observations, in this case from
electronic microscopy, oblige us to think about more complex mechanisms and mathematical treatments. Nevertheless
it is not only important to discuss the model type, but also the type of kinetics assumed in the interior of the beads,
as well as the internal structure, the boundary conditions related to bead shredding and cell escape and finally,
geometrical effects. © 2000 Elsevier Science B.V. All rights reserved.

Keywords: Cell immobilisation; Diffusion; Escape; Model

1. Introduction transport phenomena models are looked for, par-

In recent years, at the same time as interest in
systems working with immobilised cells has arisen,
especially gel entrapment, a great number of au-
thors have been developing models supported by
experimental results. Some simple models only
aim at simulating data related to the global effi-
cacy of retention or production, for instance by
means of logarithmic equations, and in this way
good fits can be achieved. However, any extrapo-
lation of the results requires the physical basis of
the model to be correct, and for this reason
* Corresponding author.
ticularly ones that can be endorsed by different
items other than simple efficacies, such as mi-
croscopy observations for instance. In Table 1,
modelling studies combining diffusion and reac-
tion phenomena are presented. These models aim
at predicting the biomass growth, substrate con-
sumption and product formation that take place
in these systems.
Nakasaki et al. (1989) developed a model that
describes an unsteady state of a semi-batch reac-
tor containing cells immobilised in gel beads. This
model, once the initial conditions are given, pre-
dicts substrate consumption, product formation
and the variation of the cell concentration

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204 A. Laca et al. / Journal of Biotechnology 80 (2000) 203–215

throughout the beads, assuming that substrate
concentration is the same in the liquid and in the
surface of the support and that there is no cell
escape. The model was applied to a system of
immobilised yeasts for producing ethanol in a
packed-bed reactor, and the results obtained were
tested. However, no biomass profiles were avail-
able and the concave profile predicted by the
model could not be corroborated.
Monbouquette et al. (1990) carried out studies
employing a scanning microfluorometry technique
that demonstrated preferential growth in the sur-
face of the beads when cells of Zymomonas mo-
bilis were immobilised in Ca – alginate. These
authors developed a dynamic model in which the
influence of the concentration of the immobilised
biomass on the effective diffusion of nutrients and
products is taken into account. The model as-
sumes the existence of a critical concentration of
biomass that the support is able to bear inside;
once this maximum limit is exceeded, cell escape
begins. The simulated results obtained are very
close to the experimental ones, however an exper-
imental decrease in the surface cell concentration,
not predicted by the model, is observed as a
consequence of the breakage and abrasion of the
support.
Wijffels et al. (1991) also put forward a dy-
namic model that predicts the biomass and sub-
strate profiles throughout the support and the
substrate consumption by Nitrobacter agilis im-
mobilised in an airlift reactor; the limiting sub-
strate being the oxygen. By means of image
analysis, they found that after 42 days of fermen-
tation, 90% of the immobilised cells were situated
in a 140-mm thick external film of the support.
They also found a phenomenon not considered by
the model: cell escape and the consequent biomass
concentration decrease, which takes place in the
surface of the support.
Wolffberg and Sheintuch (1993) developed a
model that simulates cell growth and distribution
in a hollow-fiber or packed-with-gel-beads reac-
tor, solving the problem for different kinetics of
substrate consumption. However, they did not
achieve any experimental data that allows com-
parison of the model with the theoretical results.

Table 1
Summary of some models proposed for immobilised cells systems

Reference Support Micro-organism Considerations in the model

Substrate/products diffusion affected Cell escape

by biomass

Nakasaki et al. Gel Yeast No No

(1989)
Monbouquette Alginate Z. mobilis Yes Yes
et al. (1990)
Wijffels et al. Carrageenan N. agilis No No
(1991)
Wolffberg and Hollow-fiber/gel – Yes No
Sheintuch
(1993)
Hunic et al. Carrageenan N. europaea and N. agilis No No
(1994) co-immobilised
Wang et al. Alginate Lactobacillus delbrueckii Yes No
(1995)
Cachon et al. Alginate Lactoccocus diacetylactis Yes Yes
(1995)
Wijffels et al. Carrageenan N. europaea Yes Yes
(1995)
dos Santos et Alginate and N. europaea and Pseudomonas sp. No Yes
al. (1996) carrageenan co-immobilised
 A. Laca et al. / Journal of Biotechnology 80 (2000) 203–215
Hunic et al. (1994) presented a model that
characterises ammonium nitrification with Nitro-
somonas europaea and N. agilis co-immobilised in
k-carrageenan beads and they validated the model
by comparing it with experimental data. In order
to solve the model, they assumed constant effec-
tive diffusivity and the same kinetics that are
presented by the free cells. Concave profiles were
achieved experimentally for both micro-organ-
isms, fitting the results predicted by the model
very well. Nevertheless, once more an experimen-
tal decrease in biomass concentration near the
surface of the support is observed that is not
predicted by the model.
Wang et al. (1995) proposed a mathematical
model to analyse the mass transfer behaviour in
lactic acid fermentation using immobilised cells.
Simulations indicated a cell concentration gradi-
ent in the gel beads, a gradient that the authors
claimed to be due to inhibition products instead
of substrate limitation. Experimental and theoreti-
cal data are compared successfully, but real
biomass profiles inside the support were not avail-
able. Cachon et al. (1995) also established a model
for studying lactic acid fermentation, bioconver-
sion of citric acid and cell escape in a reactor
containing immobilised cells. Again, simulation
lead the authors to a concave profile, but once
more no experimental data of biomass profiles
inside the support are available in order to vali-
date the model.
Wijffels et al. (1995) went further by developing
a colony expansion model, which took into ac-
count the fact that cell growth inside the support
is produced by the formation of cell colonies from
the initial individual cells. They also considered
two biomass release mechanisms: either growth
resulting in continuous leakage of single bacteria
or eruption of entire colonies at once. Moreover,
diffusional limitations on these micro-colonies are
introduced in this model.
dos Santos et al. (1996) developed a model that
describes nitrification and denitrification by N.
europaea and Pseudomonas sp. co-immobilised in
separate layers of gel beads. Comparing experi-
mental and theoretical results, they found that the
trend is quite well predicted, although some dis-
crepancies, probably due to diffusional limitations
205
caused by the presence of micro-colonies, were
found. On the other hand, concentration profiles
inside the support were not compared with exper-
imental data.
As can be seen, there are a great number of
authors that have been involved in developing this
type of model. In spite of the assumptions not
always being the same, a good correlation be-
tween the experimental data and the theoretical
model is achieved in nearly every case studied. To
obtain a model which experimental data fits prop-
erly is not a very difficult task and different
assumptions for the same phenomenon are valid.
However, it seems obvious that in order to obtain
a really correct modelisation, it is necessary to
know exactly what is going on inside the immobil-
isation support during the fermentation process.
With this aim in mind, the use of different existing
microscopic techniques proves to be a very power-
ful working method. The final aim of the present
study was to call attention to the need for basic
studies, and to contribute to the understanding,
by means of these microscopic techniques, of the
phenomena that take place during the fermenta-
tion process in order to establish the basis of a
more correct modelisation of the system.
2. Materials and methods
Serratia marcescens (ATCC 25419) was chosen
for this work due to its proven ability for protease
production and also for its adequate behaviour
when immobilized (Vuillemard et al., 1988; Quirós
et al., 1994). Sweet dehydrated whey enriched
with proteins and supplied by a dairy (I.L.A.S.,
Anleo, Asturias, Spain) was employed. The com-
position of this whey powder is: fatty matter
(4.0% w/w), protein (34.98% w/w), water (3.97%
w/w), ashes (7.07% w/w), lactic acid (1.3% w/w),
lactose (37.72% w/w), others (10.96% w/w). All
experiments were carried out in a liquid medium,
thus the whey powder was dissolved in distilled
water until reaching a concentration of 9.5% (w/v)
since this was found to be the optimum concen-
tration for protease productivity. Sterilisation of
the whey was carried out by tangential mi-
crofiltration (0.3 mm). The final concentration of
proteins in the culture broth was 7.85 mg ml − 1.
206 A. Laca et al. / Journal of Biotechnology 80 (2000) 203–215
2.1. Obtaining the pre-inoculum
Erlenmeyer flasks (250 ml) containing 50 ml of
NBG, nutrient broth at 2% (w/v), supplied with
glucose 1% (w/v), were inoculated with S. marces-
cens and incubated in an orbital shaker (NBS,
Mod. G25) for 10 h at 37°C and 250 rpm (Quirós
et al., 1995). Subsequently, cells were separated
from the broth by centrifugation and re-sus-
pended in NaCl 0.7% (p/v) in an aseptic way.
2.2. Immobilisation and fermentation procedure
The technique of cell immobilisation employed
was that of entrapment in calcium alginate beads
(Vuillemard et al., 1988) and all the process was
carried out under sterile conditions. The cells
obtained as pre-inoculum were mixed with
sodium alginate (Janssen Chimica) (2.1% w/v)
according to the following relationship: 10 ml of
alginate/8 ×1010 colony forming units (cfu); the
mixture being agitated until homogeneous. The
obtained mix was then allowed to drop at a
constant rate onto a solution of CaCl2 (3% w/v)
that was being continuously agitated. The beads
obtained in this way had an average diameter of
0.27 cm. The beads remained in the CaCl2 solu-
tion for half an hour, completing the alginate
gelification process and were subsequently re-
moved from the liquid medium and distributed
among the flasks containing the whey. Every flask
was inoculated with beads containing the total
amount of 8x1010 cfu centrifuged. Fermentation
of the whey by S. marcescens immobilised in the
way described was carried out in 250 ml Erlen-
meyer flasks containing 50 ml of medium at 37°C
and 250 rpm (Quirós et al., 1995).
2.3. Microscopic techniques
Samples of beads containing immobilised S.
marcescens were taken at different fermentation
times up until 25 h. These samples were kept in
saline solution, NaCl (0.7% w/v), at 4°C for one
day and afterwards the fixing process began
(Braña et al., 1981). Firstly, the alginate beads
were treated with a solution of: osmium tetroxide
(1% w/v), sodium barbital (0.6% w/v), sodium
acetate (0.4% w/v), sodium chloride (0.57% w/v),
hydrogen chloride (0.029 N) and calcium chloride
(0.01 M) during 16 h. Subsequently, the beads
were washed with veronal buffer: sodium barbital
(0.58% w/v), sodium acetate (0.38% w/v), sodium
chloride (0.55% w/v), hydrogen chloride (0.028 N)
and calcium chloride (0.01 M), until the liquid
appeared clear. Next, a post-fixation treatment,
submerging the beads in uranile acetate (2% w/v)
in veronal buffer for 2 h in the dark. The beads
were washed once again in veronal buffer. The
beads were then progressively dehydrated in ace-
tone solutions of increasing concentrations (from
25 to 100%). These beads, once dehydrated, were
soaked up by Epon812 resin in a progressive way,
starting with a mixture of one part of Epon and
three of acetone and ending with pure Epon, and
were kept for 16 h at room temperature in said
resin. Finally, the beads with pure resin were
placed in an inclusion mould of silicone and the
polymerization took place for 2 days at 60°C.
2.4. Optical microscopy
One micrometer thick sections at different radii
were made to the beads included in the resin by
means of an LKBV ultramicrotome equipped
with glass blades. Sections were stained during a
number of seconds with toluidine blue and subse-
quently washed with distilled water, with the aim
of forming a contrast between the gel and the
colonies of S. marcescens. These samples were
analysed by means of the ‘IMCO 1’ system, which
consists of an IMCO 10 (MIP software) image
processor, an Olympus BHT microscope equipped
for transmitted light observations and a JVC
TK870E TV camera. Pictures were taken at
100× and 1000× magnifications with the afore-
mentioned equipment and a Mitsubishi CP100E
video printer.
2.5. Transmission electron microscopy
Once more, cuts of sections quite close to the
surface and of  700 A, of thickness were made to
the beads included in the resin, by means of an
LKBV ultramicrotome equipped with diamond
blades. Sections were stained during the first 10
 A. Laca et al. / Journal of Biotechnology 80 (2000) 203–215 207

bed, o *=
L 0.88; radius of the particles, R*=0.135
cm; yield factor, Y *s/x =0.79; effective diffusivity
of the biomass and the substrate in the support,
Dx = 3× 10 − 10 cm2 s − 1, Ds = 10 − 9 cm2 s − 1; ini-
tial concentrations of biomass and substrate in the
support and in the bulk; C *(0)=x 0.954 mg ml − 1,
C *(0)=
s 0, C *bx(0)= 0, C *bs(0)= 7.8 mg ml − 1; mass
transfer coefficient in the external film (and/or
bead interface) of the biomass and the substrate,
Kx = 111 cm − 1; Ks =  cm − 1 (* experimental
data). A FORTRAN subroutine called PDECOL was
used to solve the differential equations system.
Fig. 1. Experimental results of S. marcescens profiles in the
support. Cell concentration is given in mg of cell dry weight
per ml of support.
3. Results and discussion

min with uranile acetate and afterwards with lead
citrate, and were then washed with distilled water.
Pictures were taken with a ZEISSEM-109 micro-
scope at 80 kV and with a resolution of 7 A, . The
screens employed were of copper. The pictures
were taken at 4400 × , 7000 × and 12 000 ×
magnifications.
2.6. Model
The homogeneous model developed in a previ-
ous study (Laca et al., 1998), was employed. The
equations and boundary conditions are indicated
next (being i =x, s)
3.1. Modelisation
A first glance at what was happening inside the
beads was achieved by optical microscopy tech-
niques; the distribution of the biomass concentra-
tion through the immobilisation support being
determined (Laca et al., 1998) (Fig. 1) in this way.
This analysis clearly indicated a preferential cell
growth in the border of support, whereas in the
inner part of the beads no cell growth was de-
tected up until zones quite close to the border,
where a slight cell concentration gradient was
observed, even though the cell concentration val-
ues obtained just inside the border were much
higher than those in the interior.

 
Mass balance inside the support: The origin of these profiles seems to be the
(Ci 1 ( 2 (Ci difficulty that the substrate presents for reaching
=r%i + 2 r Di the interior of the support, either due to its in-
(t r (r (r
abilty to diffuse through the alginate or because it

)
Mass balance in the stirred tank reactor:
is consumed by the external cells more quickly
dCbi 3 (1−oL ) (Ci than it is able to diffuse, which seems to be the
=− Di +r %%i
dt R oL (r r = R most probable reason. This phenomenon has been
Boundary conditions: described previously by other authors (Monbou-
quette et al., 1990; Arnaud and Lacroix, 1991;
(Ci (Ci
r = 0, =0; r = R, =Ki (Cbi −Ci (R)) Hunic et al., 1994; Wijffels et al., 1995). Another
(r (r possible explanation might be the very low con-
General assumptions for the simulation were: centration of oxygen that exists in the inner zones
Monod’s equation for kinetics (r %,i internal and r %%,
i of the support (Kwak and Rhee, 1992; Kurata
external) (maximum external and internal specific and Furusaki, 1993; Vives et al., 1993), and al-
growth rate mmaxint =9 × 10 − 5 s − 1, mmaxext =4 × though S. marcescens is a facultatively anaerobic
10 − 5 s − 1; external and internal Monod’s con- micro-organism, the anaerobic metabolism usu-
stant, ks ext,int = 0.001 mg ml − 1); porosity of the ally implies a much lower Yx/s. Other studies have
208 A. Laca et al. / Journal of Biotechnology 80 (2000) 203–215
pointed out the possibility that the concave
profile obtained is caused by inhibitor com-
pounds that may be accumulated inside the sup-
port (Arnaud and Lacroix, 1991; Wang et al.,
1995), although this is not what happens in our
case because it was observed that if the ex-
hausted whey was supplemented with fresh whey,
cells grew again once the culture had reached the
stationary phase. On the other hand, the cells
themselves seem to present a very low ‘diffusiv-
ity’ through the gel, since a high cell ‘diffusivity’
(Laca et al., 1998) would imply homogeneous
cell distribution through the whole support,
which does not occur.
A great number of authors have indicated that
the gel entrapment technique allows a maximum
cell concentration in the immobilisation support
to be reached (Monbouquette et al., 1990; Wolff-
berg and Sheintuch, 1993; Wang et al., 1995; dos
Santos et al., 1996). Some of these authors
(Monbouquette et al., 1990; Cachon et al., 1995)
have even pointed out that is from this defined
concentration onwards, and not before, when cell
escape begins. In the present case, it is worth
noting that this was not observed, because cell
growth in the liquid medium caused by cell es-
cape of the beads took place from the very first
moment of the fermentation process, when the
cells located in the surface were still growing and
so rising their concentration. Besides, the cell
growth inside the support was observed for 25 h
(Laca et al., 1998), and until this moment, a
maximum in the cell concentration was not
achieved. Nevertheless, the cell growth rate was
lower at the end of the process, which might
imply the proximity of the maximum concentra-
tion referred to by other authors. However, we
think it is more likely that this decrease is due
more to the proximity of the stationary phase of
growth, because the same rate decrease was also
observed among the cells that were outside the
beads, than to the incapacity of the gel to bear a
greater amount of cells.
Another question to be taken into account is
the fact revealed in most studies with alginate or
carrageenan supports (Wijffels et al., 1991, 1995;
Hunic et al., 1994) that a cell concentration de-
crease was observed in zones quite close to the
beads surface as a direct consequence of cell
escape. This phenomenon is not observed in Fig.
1, probably because the thickness of the zone
where this fall should exist is too narrow to
appreciate any concentration difference with the
employed method. Another possible explanation
might be that our fermentation lasted for only 25
h instead of 40 days, as occurs in the related
works, and this period may be too short to
achieve said phenomenon.
Figs. 2–5, corresponding to pictures from the
optical microscopy technique in which all the
previous comments are confirmed, are shown
next. Said pictures show support sections, where
the cells can be observed in an obscure tone, a
consequence of the staining process, and the algi-
nate appears in a clearer tone. At the very begin-
ning of fermentation (Fig. 2a–c) an
homogeneous distribution of the micro-organ-
isms is observed since no differences between the
inside and the border of the beads were detected.
As the fermentation time increased, no differ-
ences in the centre of the support were observed
and no growth was achieved (Fig. 5a,b). On the
other hand, in zones close to the border of the
support, the appearance and later enlargement of
cell colonies were observed. Although the general
trend was for significant cell growth to be ob-
served only in the border of the beads, 3a and 4a
show the presence of colonies in inner parts of
the beads, probably due to a poor initial distri-
bution. However, it must be kept in mind that
although said pictures seem to show the inner
parts of the support, really these cuts were made
in zones quite close to the border (r/R= 0.89
and 0.97, respectively), so they are still surface
zones. Fig. 4b (arrow) clearly shows breakages in
the surface of the alginate beads, these holes
appearing where colonies would have existed pre-
viously and which are now empty, since cells
have escaped to the liquid medium. On the other
hand, it is necessary to point out that the algi-
nate support behaves like a continuous and ho-
mogeneous medium in which cells are entrapped,
which can be observed in the figures.
With prior observations, a well-defined model
such as the homogeneous one can be used and is
well established in the literature (Laca et al.,
 A. Laca et al. / Journal of Biotechnology 80 (2000) 203–215 209

1998). This model of general application was con-
sidered because it assumes that the support is a
continuous homogeneous medium in which cells
are entrapped and optical microscopy has shown
that this is the behaviour of the alginate.
Boundary conditions for the modelisation have
been previously given (Laca et al., 1998). A high
external resistance for the biomass was consid-
ered, since the maximum cell growth reached in
the liquid medium was much lower than that
reached inside the support, although it is known
that a certain degree of cell escape that provokes
cell growth in the liquid medium exists. With
respect to the substrate, it was assumed that no
external resistance exists, since agitation of the
system was very high. Monod’s equation was the
one considered for kinetics. Fig. 6 shows the
results obtained when applying this model; as can
be observed a concave profile that fits the experi-
mental results quite well (see Fig. 1) appears. The

Fig. 2. Picture of immobilised S. marcescens. Fermentation time = 0 h. (Optic microscopy): (a) Central section. (b) Section in
r/R = 0.98. (c) Edge of a section in r/R= 0.98.
210 A. Laca et al. / Journal of Biotechnology 80 (2000) 203–215

Fig. 3. Picture of immobilised S. marcescens. Edge of a section in r/R =0.89. Fermentation time = 7 h. (Optic microscopy).

Fig. 4. Picture of immobilised S. marcescens. Edge of a section in r/R= 0.96. Fermentation time = 25 h. (Optic microscopy).

exception arises in what happens between values
of r/R: 0.9 and 1, where the model predicts a
greater degree of cell growth than that found
experimentally.
3.2. Diffusional and kinetic effects
The experimental data fitting does not imply
the existence of a correct modelization, since ki-
netics and substrate diffusivity also have to be
taken into account. Variations in these consider-
ations will thus lead to completely different re-
sults, as shown in Figs. 7 and 8.
In Fig. 7, a kinetic equation of the Ricatti type
was employed for the support, as well as for the
liquid medium (rx = aCx (1−tCx ) with a=9×
10 − 5 s − 1, t= 1.3×10 − 4 ml mg − 1). This kinetic
equation does not consider substrate concentra-
 A. Laca et al. / Journal of Biotechnology 80 (2000) 203–215 211

Fig. 5. Picture of immobilised S. marcescens. Central section. (Optic microscopy): (a) Fermentation time = 7 h. (b) Fermentation
time= 25 h.

tion, so, although it might be valid for the free cell the optical one. All the pictures were taken from
growth (Quirós et al., 1995), it does not prove 25-h fermentation samples and corresponded to
adequate for what is happening inside the sup- sections close to the surface, focusing on the
port, because the main cause for cells not growing border of the beads, in order to observe those
there is precisely the absence of substrate. The zones where cell density was the highest (Fig.
resulting profiles are convex, completely different 9a–c).
to those obtained previously. These pictures corroborate the practically null
In Fig. 8, Monod kinetics were maintained, ability of S. marcescens for moving through the
with the single variation of an increase in the alginate support. This had been reported by dos
substrate diffusivity values through the support Santos et al. (1996), when nitrifying and denitrify-
(from 10 − 9 to 10 − 7 cm2 s − 1). What is being ing bacteria were immobilised in alginate and
assumed here is that the substrate presents a carrageenan supports. Observing the pictures, it
higher rate for penetrating into the alginate and is
therefore able to reach inner zones where cell
growth is produced. The profiles obtained here
are rather different, and can be considered of a
concave–convex type. Because of all the previ-
ously mentioned facts, a more in-depth study of
the mechanism of immobilisation was required in
order to understand what is really happening
inside the support.

3.3. Complexity of the model

Transmission electron microscopy was used to

obtain a more detailed vision of the biomass Fig. 6. Theoretical results of biomass profiles when the homo-
growth process inside the alginate support, since geneous model is applied and Monod-type kinetics is sup-
this technique allows a greater magnification than posed.
212 A. Laca et al. / Journal of Biotechnology 80 (2000) 203–215
Fig. 7. Theoretical results of biomass profiles when the homo-
geneous model is applied and Ricatti-type kinetics is supposed.
Fig. 8. Theoretical results of biomass profiles when the homo-
geneous model is applied and Monod-type kinetics and a
substrate diffusivity higher than in Fig. 6 are supposed.
seems that bacteria were entrapped inside the
alginate without any possibility of movement.
Thus, those cells that were close to the surface
and therefore had easy access to oxygen and the
rest of the nutrients began to reproduce them-
selves, finally forming colonies, which are the
spherical shapes that appear in the pictures. In
order to form these colonies, the growing bacteria
had to press against the walls of the alginate
surrounding them. This leads to the appearance of
what can be observed in the pictures as a mem-
brane that surrounds each single colony, which
keeps them independent from the rest, even
though colonies are situated quite close to one
another, as can be clearly observed in Fig. 9c.
The system is therefore far from what can be
considered an homogeneous model, since alginate
support and colonies differ clearly, making up
two phases. Thus, in the final model, the consider-
ations previously taken into account by Wijffels et
al. (1995) when describing his colony expansion
model, could be considered:
“ Nutrient diffusivity through the colonies is not
the same as through the alginate support. A
large list of authors have highlighted the de-
crease in the effective diffusivity of the nutri-
ents through the immobilisation support that
takes place as cell density increases (Monbou-
quette et al., 1990; Arnaud et al., 1992; Korgel
et al., 1992; Wolffberg and Sheintuch, 1993;
Wang et al., 1995).
“ Cells that are located in the central zone of the
colonies will present an additional diffusional
restriction, since nutrients have to get through
all the surrounding cells to reach them.
The resulting granule model will be similar to
that used in catalysis when a pellet is formed by
compact grains (Levenspiel, 1979). In this case,
there is one pore size in the grains and another in
the rest of the particle, and therefore two different
difusivities.
3.4. Boundary conditions
When relating cell concentration inside and
outside the support, the boundary conditions of
the model should take into account which process
is the one that provokes cell escape. Transmission
electronic microscopy has allowed us to under-
stand how this escape process takes place. Grow-
ing colonies situated very close to the surface
press against the alginate wrapper until it breaks
and releases the cells into the liquid medium. It
seems quite logical to think that some abrasion in
the external surface, caused by the external agita-
tion, might contribute to facilitating this phe-
nomenon (Monbouquette et al., 1990; Kuhn et
al., 1991; Wijffels et al., 1995). In this sense, it is
also possible that the nature of the culture
medium affects the stability of the support, pro-
voking greater damage. On the right-hand side of
the picture in Fig. 9a, it can be observed how a
colony situated on the surface of the bead has
broken the alginate wall and how most of the cells
have left, leaving the former colony practically
 A. Laca et al. / Journal of Biotechnology 80 (2000) 203–215 213

empty. In the same way, on the upper left-hand
side of the same picture in Fig. 9a and more
clearly in Fig. 9b, a colony pressing strongly
against the alginate surface can be observed. It
seems that if the colony grew a little more or were
simply subjected to very smooth friction, the thin
layer that is surrounding it could be broken, thus
releasing its content into the liquid medium.
3.5. System geometry
With regard to geometry effects, Kurata and
Furusaki (1993) found that the spherical diameter
of the bead increases during the fermentation
process. This occurs when the cells grow and form
aggregates on and in the vicinity of the gel sur-
face. This swelling of the beads should be taken
into account in the model. Equations involving
swelling/shrinking of the beads coupled with fer-
mentation are much more complicated to solve,
though how to do this has still not been
demonstrated.
Moreover, on looking at the immobilised cells
located in the beads, it needs to be said that cells
forming the colonies appear smaller and some-
what deformed compared to free cells; which
seems logical since in order to grow they had to
overcome the resistance of the surrounding algi-

Fig. 9. Picture of immobilised S. marcescens. Edge of a superficial section. Fermentation time = 25 h. (Transmission electronic
microscopy): (a) Final magnification × 3732. (b) Final magnification ×5938. (c) Final magnification ×10 179.
214 A. Laca et al. / Journal of Biotechnology 80 (2000) 203–215
Fig. 10. Schematic summary of the aspects commented on in
this paper concerning immobilised cells systems.
nate wall. This cell deformation has previously
been reported by Wolffberg and Sheintuch (1993).
Finally, the superficial colonies may create un-
even, non-spherical beads. Strictly, this would
give rise to the need for 3D models, which in turn
give rise to much more complicated mathematical
equations than the 1D models usually considered.
All the subject matters introduced in this paper,
from geometry through differential equations to
kinetics, are displayed in Fig. 10, which presents
the different aspects that give rise to more and
more sophisticated models. The models at work in
each case can be simplified up to a certain degree,
depending on the relative importance of each
subject matter, the available data and the aim
sought after with the model.
4. Conclusions
Optical microscopy allows us to obtain quanti-
tative results regarding cell distribution inside cal-
cium alginate beads. The experimental results
obtained here fit an homogeneous model based on
two ‘species’, substrate and cells, quite well. Nev-
ertheless, electronic microscopy observations
show cell behaviour inside the bead forming
colonies, so that a model of the granule type
should be considered. This model has more
parameters to be considered, such as the size of
the granules or the diffusivity inside them. The
problem of including a large number of parame-
ters gives rise to the key question of selecting a
given acceptable level of complexity.
More difficulties or decisions have to be dis-
cussed when applying models. One of these is the
kinetics inside the beads as said kinetics may be
equal to or different from the outside kinetics and
also the parameters may present different values.
This decision affects even the shape of the concen-
tration profiles inside the beads.
A very important decision with regards to the
model is the type of boundary conditions, or the
equation for the interface with different types of
assumptions for cell escape. This consideration is
clearly different for the distinct confinement types.
In the alginate beads, the swelling and shrinkage
observed may need to be introduced, thus making
it more difficult to solve the mathematical models.
Finally, the modifications observed in the geome-
try during the growth evolution process may
modify the first basic assumption of sphericity.
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