Mass transfer effects in fermentations
using immobilized whole cells
J O H N M. R A D O V I C H
School o f Chemical Engineering and Materials Science,
University o f Oklahoma, Norman, Oklahoma 73019, USA
Summary. The immobilization o f whole cells for
fermentation processes has many potential advantages
over fermentation with free cells, including higher cell
concentrations, higher productivites and a higher level
o f operational stability. Most o f the research reported in
the literature has been directed towards demonstrating
the feasibility o f using these systems for various fermenta-
tions. The ultimate goal o f research in this area is to bring
the understanding o f immobilized whole cells to the level
o f heterogeneous catalysis. Immobilized whole cell systems
are examined from a mass transfer perspective. Evidence
f o r external and internal mass transfer limitations is pre-
sented. Procedures for quantifying these effects in terms
o f effectiveness factors and determining the reaction
kinetics in their presence are reviewed. Development o f
the reactor design equations and the reactor performance
results for fermentations with immobilized cells are also
discussed.
Keywords: Fermentation; mass transfer effects; immobilized cells
Introduction
Immobilization of cells in the broadest sense can be
defined as physical confinement or localization of the
microorganisms that permits their economical reuse. 1
Immobilized cells can include dead cells which contain an
active enzyme that is used for a single reaction, as well as
viable, or growing, cells which are used for biochemical
conversions requiring multi-step enzyme reactions coupled
to cofactor regeneration and ATP-type energy sources. The
use of viable, immobilized whole cells as biocatalysts offers
an alternative for fermentations conventionally carried out
with free cells. The design of biochemical reactors to
maximize the advantages of immobilized cells requires an
understanding of inter- and intraphase mass transfer. This
review focuses on the effects of mass transfer limitations
on the fermentation reactions catalysed by these immobi-
lized cells. The nature o f the cells considered is also rather
limited. Systems using microbes belonging to the kingdom
of protists, e.g. bacteria or yeasts, are included. Fermenta-
tion reactions for the production of metabolites or the
transformation of substrates rather than the production
o f biomass (cell mass) are considered.
The focus of this review limits the usefulness of much
of the research literature on immobilized cells. Most of
this research on immobilized living cells has been directed
towards demonstrating the feasibility of the various
immobilization ~methods rather than towards analysis of
the mass transfer effects. An arbitrary classification of
2 Enzyme Microb. Technol., 1985, vol. 7, January
immobilization methods is as follows:
(1) immobilization without supports, which includes
the formation of cell aggregates by natural or c a t i o n i c -
polyelectrolyte-induced flocculation;
(2) covalent coupling, including crosslinking to an
activated support;
(3) adsorption on an inert, solid support;
(4) entrapment in inert semipermeable materials such as
hydrogels, fibres or membranes.
The last three methods, immobilization by 'synthetic'
means, or with supports will receive the greatest attention
in this review. Many of these techniques have been developed
in the last decade or so. In fact, a review of immobilization
methods alone could fill more than one volume. The reader
is referred to recent works of Mattiasson. z Other general
reviews on methods for immobilization and applications of
immobilized cells have been published quite regularly.3-1°
External mass transfer
External mass transfer can encompass all the transport steps
which bring substrates to the surface of the immobilized
cell matrix (ICM). ICM refers to the physical entity that
includes the cells in their immobilized state, but it is not
necessarily the cell itself. Moo-Young and Blanch, 11 in their
review of mass transfer design criteria for biochemical
reactors, list seven external mass transfer pathways which
can occur in an aerobic fermentation reactor. Resistance
to mass transfer exists in the gas film, at the gas liquid
interface, in the liquid film adjacent to that interface, in
the bulk liquid, in the liquid film surrounding the ICM
and at the liquid-ICM interface. The dominant resistance
is most often in the liquid film or at the liquid ICM
interface. This mass transfer resistance will be examined
more closely. The topics of aeration, or oxygen transfer,
and mixing in fermentation fluids are" beyond the scope
of this paper. They have been treated extensively in the
literature, which includes some recent reviews of mixing, 12
oxygen transfer 13 and aeration 14 as well as gas-liquid mass
transfer. Is
Definition ofan effectiveness factor
At steady state, the mass transfer rate of substrate from
the bulk liquid to a non-porous surface at which the sub-
strate is consumed by a chemical reaction will equal the
global rate of reaction, R : 16-18
kLam(C B - Cs) = R
where k L is the mass transfer coefficient, a m is the surface-
to-volume ratio for the ICM, and C B and C s are tile sub-
0141 --0229/85/010002--09 $03.00
© 1985 Butterworth & Co. (Publishers) Ltd
 Mass transfer effects in fermentations using immobilized cells: J. 114.Radovich
strate concentrations in the bulk liquid and at the ICM
surface. Since R is a function of C s , it will be a maximum
when kLa m becomes very large, i.e. (CB - Cs) goes to zero
since C s ~ C B.
The traditional chemical engineering approach has been
to compare the global rate o f reaction to that when
C s = C B, i.e. RB, chemical reaction is controlling.
Carberry, 18 among others, defines an external effectiveness
factor, ~, as: ~ = R / R B and shows that it is a function o f
the Damk6hler number, Da. Da is the ratio o f the maximum
chemical reaction rate to the maximum mass transport
rate. Carberry presents charts of ~ vs. Da for power-law
kinetics which require the reaction rate constant k and
Cs, parameters which are usually not observable. This
problem is avoided by defining ~/ as a function of ~Da,
which is a dimensionless parameter containing only obser-
vable parameters:
R
~Da - - -
kLam CB
Charts of ~/vs. ~Da are also given by Carberry. 18 When the
mass transfer is relatively rapid compared to the reaction
rate D a ' ~ 1 (or ~ D a ' ~ 1), the system behaves as if it were
homogeneous, i.e. R = R(CB). Under these conditions, the
reaction rate will show an Arrhenius temperature depen-
dence and vary linearly with the reaction phase volume.
When the surface reaction rate is much faster than mass
transfer, Da >> 1 (or ~Da >> 1), the reaction rate is given by
R = kLamC B since C s goes to zero. Under these conditions,
the reaction rate varies linearly with the area of the reacting
surface, and shows a much lower dependence on tempera-
ture since the activation energy for mass transfer is very
small. 18
A n a l y s i s f o r i m m o b i l i z e d cells
Very few articles in the literature analyse the effects
of external mass transfer for immobilized living cells. Too
many research papers usually assume that it is negligible,
but do not verify this assumption for the experimental
conditions. Venkatasubramanian e t al. 17 recommended
the use of a first order kinetics-type expression to account
for the mass transfer resistance. Shiotani and Yamani 19
studied the variation of ethanol productivity as a function
of space velocity in packed beds for yeast cells immobilized
in calcium alginate and polyacrylamide gels. Their horizontal
and vertical packed bed reactors showed increased produc-
tivity for higher space velocities. They did not analyse the
results in terms o f mass transfer effects. However, the cells
immobilized in polyacrylamide cubes had about 76% of
the productivity of cells immobilized in alginate spheres.
Assuming that ethanol production was mass transfer
limited, the ratio of specific surface areas should equal the
productivity ratio. Based on the dimensions of the gel
particles, this ratio is 75%! Williams and Munnecke 60
reported an increase in ethanol productivity ( g l - l h -~)
when the flow rate was increased in a packed bed o f yeast
cells immobilized in calcium alginate beads. Similar studies
were done by Murata et aI. 6°a and Amin and Verachert. 6°b
These are examples of the influence of external mass
transfer, which were not analysed as such.
Lamptey et al. 2° constructed Lineweaver-Burk plots
for the specific rate of ethanol formation for free cell
fermentation in a CSTR and for yeast cells immobilized
by adsorption on beechwood chips. The data from both
fermenters could be analysed by a Michaelis-Menten type
equation for non-competitive inhibition by ethanol. The
value o f the apparent saturation constant in this equation
was much higher in the immobilized cell reactor than in
the CSTR, which indicated the presence of an external
mass transfer resistance. Because o f this susbtrate concen-
tration gradient, a higher saturation constant would be
expected. No analysis to quantify this mass transfer effect
was attempted.
Analyses of external mass transfer for immobilized
n°n'viable cells which are used for their enzymic activity
are much more common in the literature. Although strictly
outside the scope of this paper, some examples of these
analyses are presented.
Linko et aL 21'22 concluded that external film diffusion
was not a factor in the isomerization of glucose to fructose
by Actinoplanes missouriensis whole-cell glucose isomerase
immobilized in cellulose beads. The degree of isomerization
was unaffected by flow rate 21 and conversion was linearly
related to the inverse flow rate in a plug flow, packed
column. If film diffusion were controlling, conversion
would drop with increased linear velocity, but to a power
less than o n e . 22 However, the presence or absence of
external diffusion limits can not be demonstrated by
changing mass velocity ( m a s s / t i m e ' a r e a ) alone, because
this also changes the contact time. Both the packed bed
depth and velocity must be changed in order to keep the
contact time constant. 23 This point is often ignored when
testing for external mass transfer effects. Saini and Vieth 24
and Boersma et al. 25 also looked at the external mass
transfer effects for immobilized whole cell glucose iso-
merase. Saini and Vieth saw no effect on the reaction
rate when the mass velocity was varied from 15 to 760 g
h -1 c m -2 through a packed bed o f Streptomyces venezuelae
ceils entrapped in collagen membrane chips. Boersma et aI.
measured the reaction rate and estimated the mass transfer
coefficient for glucose from empirical correlations for flow
in a packed bed of immobilized Arthrobacter cells. The
calculated concentration difference between the bulk
solution and biocatalyst surface was only 15% of the
maximum difference, which indicated that external mass
transfer was negligible. Wheatley and Phillips26 examined
the effect of stirrer speed on the apparent maximum
reaction velocity (Vmax) and apparent Michaelis-Menten
constant (Km) in a batch reactor for the /3-glucosidase
activity of Alcaligenes faecalis cells immobilized in poly-
acrylamide gels. E a d i e - S c a t c h a r d plots for initial rates
of/3-glucosidase showed a decrease in Vmax and an increase
!
in K m as the stirrer speed decreased. The effect was also
dependent on particle size. The initial rate of this reaction
in a packed column was also dependent upon flow rate.
The external mass transfer effects were greater in the
packed column than in the batch reactor for the same
normalized residence time. Kan and Shuler 27 took a
different approach. They measured the temperature depen-
dence, and calculated the activation energy for urocanic
acid production by Pseudomonas fluorescens cells immobi-
lized on the shell side o f a hollow fibre dialyser. The acti-
vation energy was 10-+2 kcal g-1 mol-1; less than 5 kcal
g-1 reel-1 would be expected if diffusion was important. 26
The diffusive flux of substrate across the hollow fibre
membrane and in the stagnant shell side fluid was, respec-
tively, about 3000 and 1000 times the reaction rate at
40'7/0 conversion. Toda z8 studied the effects of interparticle
mass transfer using immobilized, cell-bound enzymes. He
developed a Sherwood number correlation for the liquid
mass transfer coefficient in a packed bed of agar-gel spheres.
Enzyme Microb. Technol., 1985, vol. 7, January 3
Review
t
The Vmax and K m using cell-bound invertase o f Saccharo-
myces pastorianus were compared for the packed bed and
a well-mixed tank reactor. Toda also found that increasing
the liquid velocity through the bed decreased the observed
Vmax and K m for lactose hydrolysis using immobilized
whole-cell lactose of Escherichia coiL He summarized the
earlier work on the effects of external mass transfer on
the kinetic parameters o f immobilized enzymes.
Analysis f o r immobilized enzymes
The task of quantifying the external mass transfer
effects in terms o f an effectiveness factor and observable
Damk~Shler number (~Da) for immobilized living cells may
be simplified if one assumes that the kinetic behaviour is
mathematically similar to that of free cells20'29 or similar
to that o f immobilized enzyme systems. 6'3°'3z The latter
assumption appears to be valid in many o f the aforemen-
tioned applications of immobilized, dead cells containing
an active enzyme. The volume o f literature on external
mass transfer in immobilized enzyme systems is great;
some o f the more useful references follow.
Goldstein 33 has provided an excellent review of research
on external mass transfer in immobilized enzyme systems.
His mathematical analysis is largely based on the earlier
work of Horvath and Engasser. 34 They presented equations
and graphs of the external effectiveness factor versus a
modified Damk6hler number, Da' for various dimensionless
concentrations, C/Km. For Michaelis-Menten kinetics,
Da' was defined as:
Vmax
Da' - - -
kLamKm
where Vmax is the maximum reaction velocity and Km is
the Michaelis-Menten constant. Others 92 use the tradi-
ti0nal definition of Da:
Vmax
D a - m
kLam CB
When k L >> Vmax/Kmam, the reaction is kinetically con-
trolled; when k L < Vmax/gmam, the reaction is diffusion
controlled. The influence o f mass transfer on the Michaelis-
Menten kinetics is shown by curvature of the data on an
r
E a d i e - H o f s t e e plot o f experimental data. The Km is
higher than the free-enzyme Km when diffusional limita-
tions are present. Lee et al.3S analysed the external film
effects in a packed bed reactor for the reversible, one-
substrate, two-intermediate reaction kinetics of glucose
isomerase. The results were analysed in terms o f an effec-
tiveness factor which was a function of Da and CB/Km.
Patwardhan and Karanth 36 showed how the intrinsic
kinetic parameters in a plug-flow, packed-bed, immobilized
enzyme reactor can be obtained from the plots o f the
observed inlet substrate concentration times conversion
versus the log of one minus the conversion. They also
presented methods to estimate the film transfer resistance
from the kinetic data rather than from a mass transfer
correlation. Mazid and Laidler 37 analysed the flow kinetics
o f enzymes immobilized on the interior surface o f a tubular
reactor (see also review ref. 38). The observed kinetic
parameters were related to the diffusion-free parameters
and the mass transfer coefficient via a utilization factor
which is the effectiveness factor. This analysis was based
on earlier work for immobilization by attachment to
membranes 39 and inside tubes. 4° Numerical and approxi-
4 Enzyme Microb. Technol., 1985, vol. 7, January
mate solutions for the effectiveness factor were given for
Michaelis-Menten kinetics, substrate inhibition, and
competitive, non-competitive and anti-competitive kinetics.
Mukherjia et al. al examined the dependence of activation
energies on stirrer speed for hydrolysis of N-(a)-benzoyl-
L-arginine ethyl ester and casein by immobilized trypsin.
Park et al. 42 studied the external mass transfer resistance
in the hydrolysis of benzylpenicillin by cylindrical pellets
of immobilized penicillin amidase in a recirculation batch
reactor. The mass transfer coefficient was determined
experimentally as a function of flow rate and substrate
concentrations. A C h i l t o n - C o l b u r n correlation fitted the
experimental data quite well. Buchholz determined mass
transfer coefficients and the dependence of the initial
reaction rates o f immobilized enzymes on stirrer speed
in a vessel and flow rate in a packed bed. 42a
Determination o f kr
The mass transfer coefficient which is used in the
Damk6hler r ~ m b e r to estimate the relative importance
o f external mass transfer can be estimated from corre-
lations available in the literature.43 Satterfield's text 23a
described methods for determining kL in various liquid
solid reactors. With respect to mass transfer in fermenta-
tion systems, the early review of Calderbank,44 and the
more recent works of Moo-Young and Blanch 11'45 and
Atkinson and Mavituna 46 presented experimental results
and correlations to predict kL in bioreactors. Those works
placed a heavy emphasis on gas-liquid mass transfer for
aerobic fermentations. Buchholz 16,42a provided correla-
tions which were applicable to his packed bed and agitated
vessel bioreactors. Additional empirical correlations for
traditional chemical engineering systems have been sum-
marized recently for l i q u i d - s o l i d mass transfer in agitated
vessels,47-49 packed beds,5 ° - s 3 and fluidized beds. s 3 - s 4
These results should be used with caution because the
physical properties of the biological systems (e.g. viscosity
non-Newtonian behaviour, partical size, density differences)
may be outside the database used in the correlation. Infor-
mation on the physical properties o f fermentation fluids
can be found in Moo-Young and Blanch's review 11 as well
as in the 'Biochemical Engineering and Biotechnology
Handbook', s5 Taguchi's earlier reveiw s6 and Schiigerl's
review,r3
I n t e r n a l m a s s transfer
The mass transfer resistance of the immobilization matrix
is important in determining the operational activity of the
immobilized cells. At steady state, a concentration profile
of substrate (and product) is established within the ICM,
i.e. the interior sees a different concentration from that at
the surface. If the reaction occurs simultaneously with
diffusion in the ICM, the overall process cannot be modelled
in terms of a mass transfer resistance in series with a
chemical reaction, as was the case for external mass transfer.
This type of intraparticle mass transfer has been analysed
in the chemical engineering literature for porous, hetero-
geneous catalysts.23a
Definition o f the effectiveness factor
Internal mass transfer effects can be quantified in terms
of an effectiveness factor, r/, which is defined as the ratio
of the actual reaction rate to the reaction rate when all
the interior surface of the catalyst particle is exposed to
 Mass transfer effects in fermentations using immobilized cells: J. 114.Radovich
the same substrate concentration as that of the exterior
surface. The reaction kinetics of interest for immobilized
cells fall into three categories: 6
(a) intrinsic kinetics, the kinetic behaviour of the enzyme
activities of the free cells;
(b) inherent kinetics, the kinetic behaviour of the immo-
bilized cell in the absence of diffusional limitations;
(c) effective kinetics, the kinetic behaviour of the immo-
bilized cells when diffusional limitations are significant.
r / f o r immobilized cells should be the ratio of the effective
to the inherent kinetics; however, r/found in the biochemical
literature is not always consistent with this definition.
Satterfield's text TM is a comprehensive summary of
traditional chemical engineering analysis of the effectiveness
factor. The important parameters - effective diffusivity,
particle size and kinetic constants - that determine the
extent of intraparticle diffusion effects are given by the
Thiele diffusion modulus, 4). q~ is defined as q~= L3`, where
L is a length parameter equal to the volume of the catalyst
particle divided by the surface area for reactant diffusion
(L = r/3 for a sphere, r/2 for a cylinder, and thickness/2 for
a flat plate), and 3` is a reaction-diffusion rate parameter.
For Michaelis-Menten kinetics:
Vmax
3,-
KmDe
where D e is the effective substrate diffusivity within the
catalyst. 32 Satterfield presented graphs of ~ vs. ~ for
different reaction orders, 2aa while Cheetham a2 and Wang
et al. 31 presented ~ vs. ~ plots with K m / C s as a parameter
for the Michaelis-Menten kinetics. The usefulness of
these plots is limited by the need to know the intrinsic
kinetic parameters which are usually not available or
measurable. Following the analysis of Aris s7 and Bischoff,s8
Weisz s9 developed plots of r/versus an observable modulus,
cb, which is expressed only in terms of measurable quantities:
R r2
qg-
Cs De
where r is the radius of the spherical particle. Weisz s9 also
showed that all the curves of 7? vs. qb for Langmuir-
Hinshelwood kinetics (which have the same form as the
Michaelis-Menten kinetics) fall between the curves for zero
and first order kinetics. Satterfield 23a presented more
detailed plots for these types of kinetics.
The inherent kinetics of the immobilized cells can be
greatly affected by internal diffusion when low substrate
concentrations, very active preparations or large particles
are used. Weisz indicated that if qb/> 0.3 to 3, significant
diffusional modifications of the kinetics can be expected, s9
The existence of internal mass transfer limitations can be
experimentally detected in a number of ways:
( I ) as a lowering of the apparent energy of activation
for the reaction;
(2) by an increase in the productivity/activity when
the particle size is decreased, thus decreasing the diffusion
length;
(3) by a decrease in productivity/activity when the
active cell content is increased.
In general, the effectiveness factor will be close to one
if high substrate concentrations, small, very porous bio-
catalyst particles, immobilized cells of low activity or
immobilization just at the surface of a support are used.
There will usually be a trade-off between a highly active,
immobilized cell preparation with low 7? and a less active
preparation with a higher rl. The goal is to achieve the
highest reaction rate per unit volume of biocatalyst.
D e t e r m i n a t i o n o f 71
The most reliable data for r/ are obtained from experi-
mental measurement. In the absence of experimental data,
r/can be estimated from tabulated or graphical correlations.
A number of workers have studied the internal mass
transfer effects by varying the size of the ICM,16'61-65
and varying the viable cell loading in the immobilization
matrix. 16'60-63'65'66 The particle size at which the overall
reaction rate per unit mass of biocatalyst no longer increases
is free of internal diffusional effects, i.e. rt = 1. This diffusion-
free reaction rate can be compared with the observed
reaction rates for the larger particles to calculate 7?. This
procedure was used by Klein and coworkers for degradation
of phenol by Candida tropicalis immobilized in polyacryl-
amide gels; 16,61 for gluconic acid production from glucose
by Acetobacter simplex cells 12 and glucose oxidation by
Gluconobacter oxydans immobilized in calcium alginate. 66
Nilsson et al.65 also determined 77 in this manner for the
denitrification of water using alginate-immobilized Pseudo-
monas denitrificans ceils. Navarro and coworkers 63 reported
decreases in ethanol productivity with increasing particle
sizes for yeast immobilized in pectin, and Scherer e t aL 64
showed that the methanol conversion rate was unaffected
by changes in the size of alginate beads containingMethano-
sarcina bakeri. Neither of these groups analysed their
results in terms of r/.
The observed reaction rate per unit of biocatalyst should
change in direct proportion to the changes in cell loading
in the ICM for a given particle size. If it does not, internal
diffusion is important. Klein and coworkers presented the
results of these types of studies, 16'61'62 but they also
changed the cell activity by immobilizing different strains
of E. coli in epoxy beads. 16,62 They also fitted the data
with expressions for r/ as a function of ~b. Williams and
Munnecke 6° and Navarro et aL 63 reported increases in
ethanol production rates ( g h -1) when the cell loading
increased, but the ethanol production rate per gram of
cells decreased. 6° Tramper et al.66 varied the cell loading
and looked at the kinetics of glucose oxidation and the
variation in effectiveness factor.
Sliniger et aL 67 determined the effectiveness factor by
calculating the ratio of the observed, specific ethanol
production rate by immobilized Pachysolen tannophilus
cells to the observed, specific rate for free cells. Hiemstra
et al. 68 did the same thing for the rate of oxygen consump-
tion during methanol oxidation by Hansenula polymorpha.
None of these results gave an effectiveness factor according
to the basic definition. The ratio they used might more
accurately be described as an efficiency factor. The effici-
ency factor would equal the effectiveness factor if the
diffusion-free specific reaction rate of the immobilized
cells was equal to the specific reaction rate of the free
cells. This equivalence was not verified.
The effectiveness factor was measured experimentally
for a number of immobilized, non-viable cell systems having
single enzyme activity. The usual experimental technique
was to measure activity or reaction rate as a function of
particle size. For example, calculation of 7? in this manner
was done for immobilized cells containing glucose iso-
merase 2z'24-26 and for sucrose inversion by whole cell
invertase of Saccharomyces pastorianis. 69 Boersma et al. ,2s
Enzyme Microb. Technol., 1985, vol. 7, January 5
Review
Table 1 Summary of r/derivations for biocatalysts

Kinetic expression type Biocatalyst Geometry Reference

M--M; r/ for diffusion a, and reaction rate controlling b IWC invertase Sphere 69
Zero order IWC glucose isomerase Flat plate 24
Modified M--M for reversible, one-substrate reaction IWC glucose isomerase Sphere 25
M--M for substrate and product inhibition Galactosidase in mould pellets Sphere 71
Zero and first order limits of M--M IWC in hollow fibres Cylinder 72
Monod equation for cell growth Biological film Sphere 74
M--M, Hill equation for respiration Mycetial pellets Flat plate 75
Sphere 76
M--M, substrate and product inhibition IE or IWX Sphere 77
Monod for cell growth Biological films Flat plate 78
Bacterial flocs Sphere 79
Monod Bacterial flocs Flat plate 80
Sphere
Cylinder
Monod for cell growth Yeast flocs Sphere 81
M--M, non-competitive substrate and product inhibition, competitive IE Flat plate 82
product inhibition
M--M IE Flat plate 34
Ping-pong mechanism for two-substrate system IE Flat Plate 83

aRef. 70;b ref. 58; M--M, Michaelis--Menten; IWC, immobilized whole cell; IE, immobilized enzyme

Saini and Vieth 24 and Hiemstra e t al. 68 compared their
experimental r7 to one derived from solution of the mathe-
matical model in terms of a modified Thiele modulus.
Derivations of the relationship between r/ and a Thiele-
type modulus have appeared quite frequently for bio-
catalysts. Table 1 contains a summary o f the kinetic
expressions of interest which have been used in those
derivations. Analyses of immobilized enzymes, biological
films, mycelial pellets, microbial flocs and immobilized
whole cells have been included. Engasser and Horvath 84
and Goldstein 33 provided a review of the pertinent litera-
ture for immobilized enzymes. Depending on the system,
the fermentation kinetics of immobilized living cells may
be more closely represented by microbial flocs rather than
immobilized enzymes. 6 Evaluation of r/ from these expres-
sions requires a knowledge of the substrate diffusivity
in the ICM.
The effective diffusivity, De, o f the substrate in the
immobilization matrix can be related to the diffusivity
in solution, D, by the equation:
De 0 unambiguously clear that such mass transport effects have
D r
where 0 is the porosity, and r is the tortuosity o f the
material. 2s'32 In general, De will be proportional to the
water content of the immobilized gel and inversely propor-
tional to the molecular weight of the substrate. 32 Usually,
the porosity, tortuosity and most of the mechanical proper-
ties of the materials chosen for immobilization are not
known (see Krouwel et al. 8s for a review of mechanical
properties of some common immobilization materials).
Cheetham e t aL 86 estimated that 0 for calcium alginate
beads was 0.85. The scanning electron micrographs of
Sherer et al.64 showed the macroporous structure of these
gels. Klein et al. 87 used inverse steric exclusion chromato-
graphy to estimate the maximum pore sizes of alginate
gels from dextran standards.
An unsteady state method is usually used to measure
De .62'69'88'89 The change in substrate concentration in
a well-stirred solution containing the ICM particles is
measured as a function of time. The measured concentra-
as a parameter. Other methods have also been used.as ,68,86,91
De should be measured for particles containing inactivated
immobilized cells because the cell activity or growth within
the immobilization matrix can affect the ICM structure. 88
Determination of kinetic parameters
If the Thiele modulus, rather than the observable
modulus, is used to estimate ~, the kinetic parameters
must be known. The effective and inherent kinetics of the
immobilized cells may be quite different from the intrinsic
kinetics of the free cells. Too often a Michaelis-Menten
type kinetic expression has been used to analyse the kinetic
behaviour of immobilized enzymes without knowing the
intrinsic kinetic parameters. 92 The intrinsic kinetics, the
kinetic parameters for the enzyme activities of the free
cells, are of less importance for estimating r/ than the
inherent kinetics, the kinetic parameters of the immo-
bilized cells in the absence of internal mass transfer limita-
tions (and without external mass transfer effects, also!).
Most kinetic investigations of immobilized cells are not
been eliminated. 16 However, Boersma et al. 2s and Scherer
et al.64 measured inherent kinetic parameters after experi-
mentally verifying that mass transport phenomena were
negligible. Kobayashi et al. 7s took the same precautions
prior to measuring the respiration rates for mould
pellets. These researchers determined the initial reaction
rates as a function of substrate concentration and used
Lineweaver-Burk plots to calculate Vmax and K m . Other
w o r k e r s 26,62,6s,66,68 did not eliminate diffusional effects.
They attempted to determine Vmax and K m from Line-
w e a v e r - B u r k or E a d i e - S c a t c h a r d plots despite the pre-
ponderance of papers concerning the fallacies of such
an analysis for immobilized enzyme systems affected by
mass transport (see Goldstein 33 for a discussion and review
o f the experimental literature as well as refs 93 95). Similar
discussions for flocculating microorganisms have also been
reported. 96-1°1 When diffusional effects exist, the Line-
weaver Burk and Eadie Hofstee plots are non-linear.
The apparent Vmax and K m vary with changes in particle
size and cell loading.2 6 ' 6 6 The apparent K m will be higher
•

tions are fitted by the unsteady state diffusion equations when diffusional limitations are present. Values of Vmax
given by Crank 9° for the particle geometry with diffusivity and K m obtained from extrapolation of experimental

6 Enzyme Microb. Technol., 1985, vol. 7, January

 Mass transfer effects in fermentations using immobilized cells: J. M. Radovich
data presented in Lineweaver-Burk or Eadie-Hofstee
plots are not valid because of the error in such analysis.
Some researchers 1°2 have recommended that experimental
results be presented as plots of conversion versus residence
time for various substrate concentrations. If Michaelis-
Menten type kinetics can be assumed for the immobilized
cell reactions, the procedures recommended by Hamilton
et al.,9a Engasser and Horvath, 94 Kobayashi and Laidler 9s
or Lee et al.l°3 should be used to extract the inherent
kinetic parameters. (Engasser and Horvath's procedure 94
can also be used to determine D e . )
Miscellaneous
The effects of internal mass transfer limitations of
nutrients are responsible in many cases for the uneven
distribution of living cells within the immobilization
matrix after exposure to a growth medium. Examples of
this effect have been reported by Inloes et al. 104 for yeasts
entrapped in hollow fibres, by Wada et al. los-loT and Wang
and Hettwer 1°8 for yeasts immobilized in carrageenan, by
Siess and Divies 1°9 for yeast entrapped in polyacrylamide
gel, and by Polack et al. 88 for yeast immobilized in agar.
Techniques for quantifying the non-uniform growth have
been discussed recently for yeasts in polyacrylamide
gel lie and bacteria in carrageenanJ °9'111
The overall effectiveness factors for situations in which
both internal and external mass transfer limitations occur
can be predicted from the equations and plots presented
by Carberry 18 for power-law kinetics, by Webster et aL va
for the first order and zero order limits of Michaelis-
Menten kinetics, by Kuu and Polack 77 for Michaelis
Menten kinetics with and without substrate or product
inhibition, by Toro and Gaudioso 112 for a first order
approximation of Micbaelis-Menten kinetics, and by
Ilias et aL lla for substrate, non-competitive, inhibited
kinetics. In these cases, the overall effectiveness factor
is a function of a Thiele modulus, a Sherwood number or
mass Blot number and dimensionless concentration ratio.
Kasche lISa defined overall effectiveness factors with respect
to the reaction with free biocatalyst and presented experi-
mental calculations for immobilized enzymes and cells.
I m m o b i l i z e d cell r e a c t o r p e r f o r m a n c e a n d d e s i g n
Reactor performance is expressed in terms of the activity,
stability and selectivity of the biocatalyst, and the product
yield and conversion of substrate to product at given sub-
strate concentrations. Reactor design equations (RDE)
relate substrate concentration and conversion (or product
concentrations) to the reactor hydrodynamics (fluid flow
rate and extent of mixing) and reaction kinetics. The size
of a reactor or the amount of biocatalyst needed to achieve
a desired production rate can be calculated from these
equations. The development of reactor design equations
for immobilized cell reactors will proceed differently for
non-viable immobilized cells which catalyse a single enzyme
reaction compared to viable or growing immobilized cells
which catalyse a multi-step conversionJ 7 This review
focuses more on the latter systems but each is considered.
Single-enzyme immobilized cells
Venkatasubramamian et al. 17 indicated that reactors
using cells immobilized for single enzyme activity can be
analysed according to the equations developed for immobi-
lized enzyme reactors. They gave an excellent summary of
the reactor design equations for ideal, continuous stirred
tank reactors (CSTR) and plug flow reactors (PFR). Their
presentation was based largely on an earlier review of
Vieth et al. for immobilized enzyme reactorsJ 14 Reactor
design equations are presented for the following types of
enzyme kinetics: Michaelis-Menten, substrate inhibition,
competitive and non-competitive product inhibition. The
important reactor design parameters are reactor space time
(reactor volume/volumetric throughput), fractional conver-
sion of substrate, and volumetric reactor productivity
(initial substrate concentration times conversion/reactor
space time). When external mass transfer is important, the
mass transfer coefficient is incorporated into the apparent
or effective kinetic parameters. The effective kinetic
parameters are used in the kinetic equations. If internal
mass transfer limitations occur, the actual rate expression
is multiplied by an experimentally determined or predicted
effectiveness factor and included in the RDE. Vieth et al.
presented details of this analysis for different types of
reactors and immobilized-enzyme systems, as well as
combined internal and external mass transfer limitations. 114
There have been many articles published concerning
immobilized enzyme reactor performance and design since
Vieth's review. Although their discussion is outside the
scope of this paper, recent articles on external mass transfer
effects in a packed bed for a reversible reaction system a3
and in a trickle bed containing co-immobilized glucose
oxidase and catalase 1is are of particular interest, as also
are the articles on combined external and internal mass
transfer for invertase and glucose oxidase co-immobilized
within a polyacrylamide gel 116'117 and invertase immobi-
lized on anion-exchange resin. 118 Kasche 118a presented
reactor design equations and experimental results in terms
of an overall, operational effectiveness factor, 770. r/o was
defined as the ratio of the times needed for a specified
substrate conversion using the same amount of free and
immobilized biocatalyst when the apparent K m equals
K m . Saini and Vieth 24 and Webster and Shuler 72 presented
reactor design equations for single-enzyme immobilized
cells for a packed bed and hollow fibre reactor with recycle,
respectively. Both groups assumed that internal and external
mass transfer were negligible. Linko et al. ,22 Wheatley and
Phillips 26 and Yamamotu et al. 119 presented packed bed
reactor performance data but did not attempt to analyse
it in terms of the reactor design equations.
Immobilized viable cell reactors
The performance of reactors containing immobilized
viable cells has been the subject of a number of recent
articles. Performance primarily in terms of substrate con-
centration/conversion is discussed rather than in terms of
stability. Stability studies are commonly done as part of
feasibility demonstrations for an immobilization method.
Such studies are beyond the scope of this review. The
analysis of immobilized viable cell reactors must include
equations for biomass maintenance and growth in the
formulation of the reactor design equations. 17
Many papers have been published which present experi-
mental performance data for C S T R s 6 7 , 6 8 a , 1 2 ° - 1 2 3 and
packed bed reactors (PBRs) 19'121'124-126 but none of
these research workers used reactor design equations to
analyse the results.
Dale et aL 127 presented experimental substrate and
product concentration profiles for a PBR. They developed
a mathematical model which included a cell growth term
Enzyme Microb. Technol., 1985, vol. 7, January 7
Review
for the cells immobilized by adsorption on packing, but
did not include any details. Sitton et al. 128 presented the
performance data but modelled the substrate consumption
rate in the PBR by a first order relationship. Internal
and external mass transfer effects were assumed to be negli-
gible. This assumption was also made by Gencer and
Mutharasan, 129 who examined the performance of a PBR by
varying the residence time of the feed. Their model included
specific cell growth rates and ethanol formation rates, as
did that of Tyagi and Ghose. 13° Venkatasubramanian
et al. 17 made the same assumption in their development
of reactor design equations for a CSTR to account for all
growth, growth associated products and secondary meta-
bolites. Yamani and Shimizu 131 presented design equations
in terms of substrate concentration rather than fractional
conversion for a CSTR and PFR. Krouwel et al. 132 neglected
external mass transfer, but included an effectiveness factor
for internal diffusion for cell growth and substrate con-
sumption in their model for a CSTR containing ClostrMium
beijerinckii cells immobilized in alginate. Heyman and
Nguyen 3° incorporated external and internal diffusional
effectiveness lactors into the maximum reaction rate
equation used in their computer simulation of a batch
reactor, a CSTR, and a PBR containing immobilized
yeast ceils for ethanol production. A similar comparison
including a fluidized-bed reactor was made by Dean and
Venkatasubramamian, but they presented no details of
the model. 133
Ghommidh and Navarro 134 and Wallis et al. 13s pre-
sented results of a dynamic analysis of fixed film bio-
reactors based on a biofilm growth reactor model for
a fixed-bed pulse reactor and fluidized-bed reactor, respec-
tively. The applicability of these biofilm models is often
overlooked in the analysis of immobilized viable cell
reactors. Atkinson 136 presented the detailed development
of the biological rate equations for design of ideal stirred-
tank and plug-flow fermenters containing microbial films
of flocs. His analysis, which accounts for mass transfer
effects, is applicable to simple growth associated systems
without substrate or product inhibition. Atkinson and
Mavituna 137 summarized recent applications of this model
for reactor performance and design equations of trickle
bed and fluidized beds. Details of the CSTR analysis
incorporating diffusional limitations for microbial flocs
are given by Atkinson and Rahman.8° Shieh 138 pregented
the derivation of the model for the fluidized bed biofilm
reactor, whose performance is primarily affected by the
biofihn thickness and particle size. Bungay et al. presented
a selected review of the performance of the microbial films
used in waste treatment. 139
Conclusion
The influence of interparticle and intraparticle mass transfer
in the performance of immobilized living cells has not been
thoroughly examined in the literature, although a useful
start has been made. The importance of these effects on
reactor design should not be underestimated. Researchers
in this area can draw upon previous developments in hetero-
geneous catalysis, immobilized enzyme systems and biofilm
reactors. As the technology develops, the applicability of
the effectiveness factor relationships needs to be experi-
mentally verified for immobilized cells. Such a task is
relatively straightforward for the external mass transfer
limitation, but additional characterization of the diffusional
properties of the immobilization materials is also needed
8 Enzyme Microb. Technol., 1985, vol. 7, January
for the internal mass transfer limitation. Development
and verification of kinetic equations for product forma-
tion by immobilized cells, especially viable cells, should
also be emphasized. Finally, much work is needed on the
development of reactor design equations which account
for the mass transfer effects, and the analysis of the experi-
mental performance data in terms of these equations. The
ultimate goal is to provide a database to bring the design
of immobilized cell reactors up to the level of sophisti-
cation found in the design of heterogeneous, catalytic
reactors.
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