Chemical Engineer& Science, Vol. 40, No. 8, pp. 1321-1354. 1985. ooo9-2x9/85 53.00+0.

00
Printed in Great Britain. Pergamon Press Ltd.

REVIEW ARTICLE NUMBER 17

THE IMMOBILIZATION OF WHOLE CELLS: ENGINEERING

PRINCIPLES

STEVEN F. KAREL, SHARI B. LIBICKI and CHANNING R. ROBERTSON

Department of Chemical Engineering, Stanford University, CA 94305, U.S.A.

(Received 18 June 1984)

Abstract-The immobilization of whole cells involves the retention of catalytically active cells within a
restricted region of a bioreactor. Techniques which have been used to immobilize whole cells include
adsorption, aggregation, confinement and entrapment. These techniques can be applied to essentially all of
the viable or non-viable whole cell systems of potential interest: microorganisms, animal and plant cells. The
fact that immobilized cells may be living leads to unique effects in this form ofheterogeneous catalysis. These
include the impact of immobilization on cell physiology and cell mobility, physical interactions of
immobilized cells with the support, and the creation of a microenvironment. The theory of mass-transfer and
reaction in these types of systems is well understood, but schemes capable of predicting substrate and product
diffusivities and the intrinsic kinetics in the aggregate must still be developed. New experimental approaches
are being used to elucidate the basic mechanisms that determine these physical and metabolic properties of
immobilized cells. Several reactor configurations have been successfully used with immobilized cells, and
many more have been proposed. The choice of a particular design for a given process depends on the
requirements for mass-transfer, the growth behaviour of the cells, and the structural properties of the
aggregate.

CONTENTS
1. INTRODUCTION 1322
1.1. Scope of the review 1322
1.1.1. Previous reviews
1.2. Objectives of whole-cell immobilization 1322
1.3. Definition of whole-cell immobilization 1323
2. CLASSIFICATION OF IMMOBILIZED CELL SYSTEMS 1324
2.1. Cells attached to a surface 1324
2.2. Cells entrapped in a porous matrix 1326
2.2.1. Preformed porous matrices
2.2.2. Porous matrices formed in sirtl
2.3. Cells contained behind a barrier 1327
2.4. Self-aggregating cells 1327
2.5. Ethanol production using immobilized cells: an example 1327
3. PHYSICAL AND CHEMICAL PROPERTIES OF IMMOBILIZED CELL
AGGREGATES 1328
3.1. Effective diffusivity in cell aggregates 1328
3.1.1. Effective diffusivity measurements where the tracer is not consumed
3.1.2. Effective diffusivity measurements where the tracer is consumed
3.1.3. Effective diffusivity measurements in animal tissue
3.1.4. Sources of difFusive resistance in cell aggregates
3.2. Interactions among components in the aggregate 1330
3.2.1. Cell mobility within the aggregate
3.2.2. Chemical interactions between the support and the solution
3.2.3. Direct interactions between the cells and the support
3.3. Physical interactions of the aggregate with the environment 1332
4. BIOLOGICAL PROPERTIES OF IMMOBILIZED CELLS 1333
4.1. Cell morphology 1333
4.2. Cell physiology 1333
4.2.1. Effect of surfaces on the growth of cells
4.2.2. Influence of immobilization method on cell viability
4.2.3. Permeabilization of cells
4.2.4. Catalytic stability of immobilized cells
4.2.5. Immobilized resting cells
4.2.6. Techniques for characterizing the metabolic and physical state of immobilized
cells
5. INTtiRACTIONS BETWEEN REACTION AND DIFFUSION IN
IMMOBILIZED CELL SYSTEMS 1338
5.1. Intrinsic kinetics of immobilized cells 1339
5.2. Mass transfer and the observed reaction rate: examples 1339

CES 40:x-* 1321

 1322 STEVEN F. KAREL et al.
5.3. Mass transfer and the observed reaction rate: theory
5.3.1. Problem formulation and simplification
53.2. Asymptotic solutions and generalized moduli
5.4. Experimental detection of mass-transfer limitations
6. REACTOR CONFIGURATIONS FOR IMMOBILIZED
6.1. Stationary particle or surface reactors
6.2. Moving surface reactors
6.3. Mixed particle reactors
7. SUMMARY
NOTATION
REFERENCES
1. INTRODUCTION
Although the intentional use of immobilized cells by
man is a recent development, the self-immobilization
of cells is a widespread process in nature. The attach-
ment of microorganisms to surfaces plays an import-
ant role in microbial ecology and pathogenicity [ 143.
“Biofilms” resulting from the growth of cells on surfaces
are often present in conventional industrial processes
using microorganisms [5]. Interest in enzyme im-
mobilization during the last two decades motivated the
development of new immobilization techniques, many
of which are applicable to cells, and provided a new
perspective for examining the conventional processes
which involve surface-attached cells. New techniques
to genetically manipulate industrially important
microorganisms have provided additional incentive
for the development of novel techniques for the
microbial production of biochemicals, and immobi-
lized cells may play a significant role in this regard.
1.1. Scope of the review
Although a wide variety of natural as well as
artificial systems are characterized by the retention of
cells at a surface or within a particle, we will focus our
attention on systems which may be of technological
interest, and exclude from consideration the ecological
and pathogenic roles of microbial attachment [Z, 63.
The state-of-the-art in immobilized cell technology
has developed to the point that methods for im-
mobilizing any given cell type are considered routine.
Microbial processes in which immobilized cells have
the potential to be applied far outnumber those where.
they are not applicable. Methods and applications
have already been reviewed in some detail (see below).
On the other hand, two important issues which have
not been adequately reviewed are:
(1) to evaluate whether immobilized cell systems can
be successfully adapted to an industrial scale and
(2) to elucidate the physical and biochemical prin-
ciples underlying whole-cell immobilization.
The former topic addresses the question of process
design and will not be considered here. As for the latter,
we will give an overview of previous fundamental
studies on those aspects of whole-cell immobilization
which are important in understanding the behaviour of
immobilized cell catalysts, and particularly on studies
focusing on events occurring on a microscopic scale,
that is, at the level of a cell aggregate.
1340
1346
CELLS 1346
1347
1347
1347
1347
1348
1349
1.1.1. Previous reviews. The review by Birnbaum et
al. [7] is an excellent overall introduction to the field of
immobilized cells. The collection of reviews edited by
Chibata and Wingard [S] offers a thorough treatment
of methods and applications. The two volumes edited
by Mattiasson [9] consider a number of sub-topics
associated with immobilized cells in considerable
depth. Overviews of immobilized cell technology, its
history and its future directions from several different
perspectives have been given in several reviews
[l&21].
The engineering aspects of immobilized cells have
not been reviewed in as much detail. A number of
recent publications have addressed specific engineering
problems associated with immobilized enzyme systems
[22-241, and Vieth and Venkatasubramanian give a
framework for discussion of some of the reactor
engineering concerns which apply to immobilized cells
[25]. Other reviews are concerned more specifically
with the interaction between kinetics and mass transfer
in biological systems [26-281. This subject will be
discussed in more detail in Section 5.3.
A number of topics that might be considered within
the realm of immobilized cell technology have also
been the subject of recent reviews:
-use of surface-attached films in wastewater treat-
ment [S, 29-321;
-formation of pellets or floes in suspension culture
c33, 341;
-use of immobilized cells in biosensors [35, 361;
-immobilization of plant cells in culture [37-391;
-growth of surface-attached animal cells in culture
[4042 ]_
1.2. Objectives of whole-cell immobilization
In most technological applications of immobilized
cells, the objective is to increase the extent of reaction.
Specifically, one might desire to:
-increase the volumetric productivity;
-increase the product concentration in the outlet
stream;
-decrease the substrate concentration in the outlet
stream.
Immobilizing cells may simplify process design in
two ways. Confinement of the cells to large particles or
to surfaces facilitates the separation of cells from
products in solution, and may permit the use of
 Immobilization of whole cells
continuous reactors while avoiding washout. It may
also allow reactors to contain more cells and therefore
presumably to have higher volumetric reaction rates.
In certain immobilized cell applications, however,
the extent of reaction is not of primary interest in
design. For example, immobilized cells have been
considered for use as a hybrid artificial pancreas
[4346]. Here, the objective is to mimic the dynamics
of insulin secretion by the pancreatic islet cells in a
healthy individual. In another application, immobi-
lized cells have been coupled to analytical devices for
use as biosensors [35,36]. In this instance a linear and
rapid response of the output signal to changes in the
concentration of the detected species is generally the
design objective.
Immobilized cell systems may be viewed as an
alternative either to the use of immobilized enzymes or
to free cells. Two primary considerations in the choice
of a biological catalyst are the difficulty of producing
the catalyst, and the ability to maintain the desired
activity and specificity of the catalyst under reaction
conditions. In the former case, the more difficult it is to
produce the catalyst, the longer is the lifetime required
for a feasible process. In general, immobilized cells
must have a considerably longer working lifetime than
free cells in order to be a reasonable alternative. In the
latter case, immobilized enzymes may have better
specificity and higher activity than immobilized cells or
free cells. Reduction of the observed reaction rate due
to mass transport limitations in substrate supply and
product removal must be considered in all of these
cases. In considering the use of immobilized cells, we
will therefore be concerned with activity, specificity,
and longevity.
1.3. De$nition of whole-cell immobilization
Whole-cell immobilization may be defined as the
physical confinement or localization of intact cells to a
certain defined region of space with the preservation of
some desired catalytic activity. This definition is
similar to those given in other reviews [47,48], which in
turn are extensions of a widely accepted definition
applied to immobilized enzymes [49]. Three issues are
implicit in this definition: the extent of the region of
confinement, the catalytic activity and the longevity of
the catalyst.
The first issue concerns the size of the region of space
in which the cells or enzymes are immobilized. For
example, certain reviewers have considered cells sus-
pended in bulk fluid in a cell recycle reactor to be
immobilized [l 1, 501. On the other hand, there is the
question of how large a cell aggregate must be for the
cells contained in it to be considered immobilized. For
the purposes of this paper, this question is resolved by
considering only those systems where cells are local-
ized within a region of space within which diffusion is
the dominant mass transfer mechanism, that is, where
no appreciable convective mixing takes place.
In the remainder of this paper we will refer to the
region in which the cells are localized as the im-
mobilized ceil “aggregate”, regardless of the method by
I323
which the cells are immobilized. Within the aggregate
three components can be distinguished, as shown in
Fig. 1. These are the cells; the support, which may be a
solid or a gel; and the solution that fills the remainder
of the space in the aggregate. The chemical properties
of the solution in the aggregate may be quite different
from those in the bulk solution. This phenomenon has
been referred to in the literature as the establishment of
a “microenvironment”. In spite of this complex struc-
ture, for engineering purposes the aggregate is usually
thought of as a homogeneous phase in a simple
geometry (e.g. a sphere or a thin film).
The second issue pertains to the extent of the
retention of catalytic activity. A wide variety of
catalytic activities are associated with a living cell. If
loss of viability accompanies immobilization, the cells
ought at least to retain the enzymatic activity as-
sociated with the desired application. The degree of
retention of each particular activity normally present
in free cells will depend on the immobilization tech-
nique and on the reaction conditions. If the desired
activity consists only of a single enzymatic step, the
elimination of competing activities may be advan-
tageous. On the other hand, if the continued growth of
cells is desired, the bulk of the enzymatic complement
should be retained. In Table 1 a number of processes to
which immobilized cells have been applied are ar-
ranged by the complexity of the reaction pathway
involved. The use of immobilized cells as ligands in
afinity chromatography [Sl] is a special case in which
no retention of enzymatic activity in the cells is
required.
The third issue implicit in the definition is that of
catalyst stability. To be useful, an immobilized cell
preparation should have an operating lifetime ranging
(b)
Fig. 1. (a) The three phases that comprise many immobi-
lized cell systems: cells, cell support, interstitial fluid. (b) An
engineering representation of an immobilized cell system.
I324 STEVEN F. KAREL~~~~.
Table 1. Typical biochemical processes performed by immobilized cells (arranged in order of
increasing enzymatic complexity)

Isomerization of glucose to fructose [55] Single-step catalysis

Production of aspartic acid from fumaric acid [56]
Transformation of steroids [57]
Oxidation of glucose to gluconic acid [SS]
Regeneration of ATP [59] I
Fermentation of glucose to ethanol [52] Multiple-step catalysis
Oxidation of caffeine [60]
Synthesis of glutamic acid [61]
Synthesis of proinsulin [62]
Growth on dilute organics (wastewater treatment) I
C631 Growth of celts

from weeks to months. Prospects for long operating immobilized cell systems is presented in Section 5. The
lifetimes are extremely good because viable cells will review concludes in Section 6 with a brief overview of
replicate under conditions permitting growth, so that immobilized cell reactor design.
the catalysts can be “regenerated”. This regeneration
by growth may be continuous [52] or periodic [53]. It 2. CLASSIFICATION OF IMMOBLLlZED CELL SYSTEMS
is therefore important in considering literature reports A multitude of whole cell immobilization methods
of catalytic lifetime to consider whether cell growth exist. These can be divided into four major categories
could have been taking place during the experiment. A based on the physical mechanism causing im-
comparison of catalytic lifetime between viable and mobilization:
non-viable immobilized cells is valuable only in the
sense of a practical assessment of processes, as the (1) attachment to a surface;
underlying causes of deactivation in the two cases are (2) entrapment within a porous matrix;
distinct. With immobilized non-viable cells, the factors (3) containment behind a barrier and
affecting catalytic lifetime will be similar to those that (4) self aggregation.
apply to immobilized enzymes, and which have been A schematic showing the different methods of
examined in some depth [54]. With viable cells, the immobilization, and their interrelationship is shown in
presence of environmental constraints that may limit Fig. 2. Examples of the various types of immobiliz-
the ability of the cells to continue to grow will ation are sketched in Fig. 3.
determine the useful lifetime of the system.
In Section 2 of this review, the diverse forms of cell 2.1. Cells attached to a surface
immobilization are classified and discussed with exam- Any immobilization method in which cells are
ples. Sections 3 and 4 treat the physical and bio- bound to a surface, regardless of the type of binding,
chemical properties of immobilized cell preparations. can be classified as a surface-attached process. The
The application of the mathematical theory of reaction thickness of the cell layer may range from less than a
and diffusion in permeable media as this applies to monolayer of cells to a film one millimeter or more in

Fig. 2. Immobilizaticn methods and their classification in terms of the physical mechanism of localization.
 Immobilization
(a) cells attached to a surface
biofilm support
semi-permeable
membrane
Fig. 3. The four classes of immobilization of whole cells.
depth. Adsorbed cells are ubiquitous in nature (e.g.
dental plaque) as well as in biotechnological processes.
The adsorption of microorganisms is a major factor in
the fouling of heat transfer equipment and other
surfaces [64]. One of the earliest immobilized cell
processes used cells adsorbed to woodchips in a packed
bed reactor to produce acetic acid (vinegar) [65].
Schematics of some surface-attached systems are
shown in Fig. 3(a).
Systems in which cells are immobilized by adsorp-
tion are popular due to the ease of this type of
immobilization. The strength with which the cells are
bonded to the support varies with cell type and support
type [66,67], thus the system is more useful for some
cell strains than for others. As there is no barrier
between the cells and the solution in this system, this
method cannot be used where a cell-free efRuent
is desired. The depth of the biofilm often varies,
especially with feed flow rate and is not readily
determined. Thus, it is often difficult to accurately
control biofilm processes, such as those used in sewage
treatment [S]. Even with these drawbacks this type of
immobilization has been widely employed in industry.
The industrial use of surface-attached cell systems
(biofilms) has been extensively examined in the field of
wastewater treatment [32]. These biofilms are often
comprised of an uncharacterized mixed culture of cells
growing in a film on sand or rocks. There is a wealth of
engineering data and models for these kind of im-
of whole cells I325
(b) cells entrapped I,, a porous n,atr,x
mobilized systems. Much of this data can be gener-
alized and applied to many of the other immobilized
cell systems currently under investigation, although
they by no means solve or address all the relevant
engineering questions in this field.
Another type of adsorbed cell system is mammalian
cell culture. As a general rule, the only mammalian cells
that do not require a solid support for growth are so-
called transformed cells which are often tumorigenic
[68]. In addition, mammalian cells consume oxygen.
and produce carbon dioxide and the concentrations of
these two gases must be controlled over a narrow range
[42]. The requirements for the the accurate control of
oxygen and carbon dioxide concentrations and the
need for surface attachment make large scale pro-
duction with mammalian cells difficult using standard
submerged culture technologies.
The majority of industrial mammalian cell culture
takes place in roller bottles [68]. These are semi-batch
cultures in which cells adhere to the inside of bottles
rotating around their axes of symmetry. The bottles
are approximately one-third full of medium. This
allows the cells access to both liquid nutrients and
oxygen from the air. Because the medium must be
changed periodically to introduce fresh nutrients and
discard waste products, this is a very labour intensive
approach to large scale cell culture. Techniques which
offer both surface attachment and high gas contacting
rates have been reviewed [69]. One such method is the
i 326 STEVEN F. KAREL et al.
use of microcarriers as a support for the adsorption
of cells. These are small porous beads (lOC-200 p in
diameter) to which the cells adhere [70, 711. After
initial seeding, the cell laden microcarriers are placed in
a packed bed reactor or continuous stirred tank
reactor (CSTR).
Cells can be chemically bound to a surface by a
variety of methods which include crosslinking by
glutaraldehyde, silanization to a silica support and
chelation to metal oxides [72,73). From an engineer-
ing viewpoint, covalently bonded cells are similar to
cells that are immobilized by adsorption. Because of
the relative size of the cell (major axis ca. 1 p) to the
covalent bonds, the strength of the covalent bond to
the surface is similar to the strength of the hydrogen
bonds or electrostatic attractions characteristic of
simple adsorption. They will be considered as one
category for the purposes of this review.
2.2. Cells entrapped in a porous matrix
The second major category of cell immobiij~tion is
entrapment within a porous matrix. Two methods of
entrapment are possible. In the first, cells are allowed
to diffuse into a preformed porous matrix. After the
cells begin to grow, their mobility is hindered by the
presence of other cells and the matrix and they are thus
effectively entrapped. In the second method, the
porous matrix is synthesized in situ around the cells to
be immobilized. Both these types of methods are fertile
ground for research and will be discussed in more
detail below. A schematic of this type of immobiliz-
ation is shown as Fig. 3(b).
2.2.1. Preformed porous matrices. Whole cell en-
trapment within a porous matrix is rapidly coming into
widespread use as an immobilization method [7]. The
cells are entrapped in a matrix which protects them
from the shear field outside the particles. The distri-
bution of microorganisms is easily measured by
examining particles removed from the reactor. As with
the adsorption method, cells are not completely sep-
arated from the efAuent in these systems. A high degree
of cell viability is retained in most entrapment
methods. These systems are therefore useful for a
variety of growth-related processes.
Many types of porous matrices have been used to
entrap cells. These include cordierite, bricks, kieseiguhr,
volcanic rock and various types of ceramics [74-761.
Messing [63] used controlled pore glass to immobilize
cells for the production of methane from waste
products. The preformed supports have two main
advantages over the in situ formation of matrices
around cells. First, the preformed support particles are
more resistant to the compression and disintegration
that can occur in packed beds and stirred vessels.
Second, adsorption of cells to the preformed supports
can take place under conditions which are not harmful
to the cells. A disadvantage of entrapment within
preformed porous matrices is that the high cell volu-
metric Racking densities associated with other im-
mobilization methods are difficult to achieve.
The use of porous matrices with very large pores
(100 p and greater) represents a form of immobiliz-
ation which lies between self-flocculation and entrap-
ment. These supports encourage the growth and
aggregation of cells in the quiescent zones formed by
the large pores. Surface-attached cells play a minor role
in this system as a result of the small surface area for
attachment. Cell growth is controlled by the continu-
ous flow of fluid past the particles, since cells beyond
the boundary defined by the flow field around the
particle are swept into the eflluent [77,78].
2.2.2. Porous matrices formed in situ. Most of the
current research in the general area of immobilized
cells involves porous supports that are formed around
ceeils. A variety of compounds can be gelled into
hydrophilic porous matrices under conditions mild
enough to allow cell entrapment with a minimal loss of
viability [79]. The polymer-cell mixture is either gelled
immediately into the final desired shape and size, or
gelled in sheets and then cut into particles of the correct
dimensions. The most common forms are small beads,
about l-5 mm in diameter, although gels have been
cast into membrane form. The gel beads can be used as
catalyst particles in a packed bed or fluidized bed
reactor. Methods for gel entrapment have been
thoroughly discussed elsewhere [7,48,80].
The first demonstration of whole cell immobiliz-
ation in porous gels was the use of polyacrylamide gel
to immobilize the lichen Unbilicaria pustuiata [Sl].
Since then, a variety of polymer matrices have been
used to immobilize whole cells, including K-
carrageenan, agar and alginate gels [7]. There are
many examples of the use of gel entrapment in the
literature, including industrial processes for the pro-
duction of amino acids [ 133. Some of the immobilizing
matrices are highly hydrated, allowing almost un-
hindered diffusion for low molecular weight ( < 10,000
Daltons) products and substrates [SZ]. The retention
of cellular viability permits the catalyst to be used for
multi-step reactions and reactions which require cofac-
tors and allows growth in the gel after immobilization.
It has been proposed that inoculation of gels with a
small number of cells and subsequent growth improves
the quality of the catalyst [52]. Gel entrapment is a
relatively straightforward immobilization technique
and can be used with a variety of systems.
Catalytic ultrafiltration membranes consisting of gel
entrapped microorganisms have also been investigated
[83---851. Membranes of different polymeric com-
positions were used to entrap the thermophilic micro-
organism Caldarieikz acidophiln. The use of this heat
resistant culture was necessitated by the high tempera-
tures encountered in the membrane casting procedure.
Reactant was ultrafiltered through the 50 p thick
membranes and high activities were reported in all
cases. A rapid decay in activity was noted for cells
immobilized in a polysulfone membrane formed by a
phase inversion process. The same cells demonstrated
long-range stability (> 140 h) when immobilized in a
glutaraldehyde cross-linked albumin membrane.
 Immobilization of whole cells
2.3. Cells contained behind a barrier
The third major category of cell immobilization is
containment behind a barrier. Again, this barrier can
be preformed, or formed around the cells to be
immobilized. Schematics of this type of immobiliz-
ation are shown in Fig. 3(c).
This kind of cell immobilization is ideal for several
specialized systems. When cell separation from the
ef8uent is required, or when some high molecular
weight product needs to be separated from the effluent,
these systems are highly useful [86]. These advantages
are countered by the fact that it is often difficult to
sample cells immobilized behind a barrier and the
uniform supply of sparingly soluble nutrients to the
cell mass is often problematic [87].
The barrier which immobilizes cells can be as simple
as the liquid/liquid phase interface between two
immiscible fluids. Mohan and Li [SS] immobilized the
denitrifying bacteria, Micrococcus denitrificans, by
emulsifying the cell suspension with a surfactant into
an hydrocarbon solvent. The surfactant-enclosed
aqueous droplets were then resuspended in the
aqueous phase containing the substrate. The authors
report that cells did not leak from the aqueous phase
through the organic phase to the cell-free aqueous
phase surrounding the droplets. The liquid boundary
could also be in the geometry of a planar liquid
membrane [89].
Microencapsulation has been recently applied as a
method for whole cell immobilization. This technique
was first used in biotechnology for the immobilization
of enzymes [90]. Recently, mammalian cells have been
successfully grown in polylysine enclosed droplets
suspended in culture broth [86]. The semipermeable
membrane allows nutrients to diffuse to the cells, but
retains the cells and some of the higher molecular
weight products produced by the cells. It has been
reported that bacterial cells have been similarly im-
mobilized [91]_
The growth of cells behind a preformed semi-
permeable membrane is the third major area within the
classification of whole cell immobilization behind a
barrier. This technique was also pioneered with the
immobilization of enzymes [92,93]. Since the cells are
retained behind a semi-permeable wall or membrane,
this type of system is very similar to microencapsu-
lation in an engineering sense. Nutrients are supplied
to, and products are removed from, the cell mass by
diffusion. In addition, the geometry used in these
reactors could conceivably be manipulated to allow
nutrient supply by convection across the semi-
permeable membrane. Immobilized cell systems have
been demonstrated using microbes in both the non-
growing [94] and the growing state [95-973 for a
variety of microbial species and membrane types. Plant
cells [98] and mammalian cells [99] have been grown
in this configuration. This type of system has also been
demonstrated successfully for the large scale growth of
mammalian cells [100] and for use as an artificial
pancreas [4345]. Both ultrafiltration and microfil-
tration membranes have been used for this purpose.
1327
2.4. Self-aggregating cells
Cells that naturally aggregate or flocculate can also
be considered immobilized within the scope of the
definition of immobilization presented earlier. The
large size of the aggregates makes their use possible in
reactors designed for immobilized cells, such as packed
bed reactors, fluidized beds and CSTR’s. Cell types
that belong in this category include molds which
naturally form pellets and plant cells in culture.
Artificial flocculating agents or cross-linkers may be
added to enhance the process of aggregation for cells
that do not naturally flocculate. It has been shown that
similar mechanisms govern both artificially and nat-
urally induced flocculation [lOI]_ Aspects of microbial
flocculation have been reviewed elsewhere [33]. This
approach to cell immobilization is shown sche-
matically in Fig. 3(d).
2.5. Ethanol production using immobilized cells:
an example
As a possible application of immobilized cells we
consider the production of ethanol from simple sugars.
This process, carried out by yeast or certain bacteria,
has been extensively studied from a technological
point of view. Novel continuous processes proposed
for this fermentation include tower fermentors, ex-
tractive or vacuum fermentation and cell recycle
reactors [102, 1031. More recently effort has focused
on the development of continuous processes using
immobilized living cells. This particular application is a
good example of the interchangeability of immo-
bilization techniques, since, as detailed in Table 2,
essentially all of the techniques mentioned in
Sections 2.1-2.4 have been used with some success.
This table lists a number of particular examples of the
use of immobilized cells for ethanol production, an
exhaustive compilation of which can be found in other
reviews [104, 1051.
Future efforts need to be focused on the develop-
ments of principles to guide the selection of an
immobilization technique for a given application and
its particular requirements. Goals in the design of a
commercial ethanol fermentation often include a long
operating lifetime for the reactor, a high ethanol
concentration in the outlet stream and a high volu-
metric productivity. The ability to use inexpensive but
ill-defined substrates (e.g. whey) is also important. In
certain ethanol processes, subjective criteria such as the
taste of the final product are important.
The prediction of the rate of ethanol production by
immobilized cells is a difficult problem. Under the low
growth conditions prevalent in immobilized cell pro-
cesses, the regulation of glycolysis with respect to the
production and destruction of biomass is not well
understood. In addition, substrate inhibition and
especially product inhibition may have a significant
effect on both the rates of growth and ethanol
production. Mass transfer within the reactor and
within the immobilized cell aggregate is also likely to
play an important role in all of the proposed processes.
We therefore suggest that a fundamental description
 I328 STEVEN F. KAREL et al.
Table 2. Immobilized cells for ethanol production

Immobilization method Medium Oreanism Ref.

adsorption to wood chips glucose + nutrients Sacchoromyces CJW

glucose + buffer cereuisiae
adsorption to ion exchange resins glucose + nutrients S. cerevisiae Cl071
adsorption to glass fiber filters glucose + nutrients Zymomonas Cl@31
mobilis
adsorption to gelatin-coated sup- glucose + nutrients S. cereuisine CJW
port
entrapment in polyurethane foam glucose + nutrients S. cerevisiae C78f
or in stainless steel mesh S. uvarum
entrapment in rc-carrageenan glucose + nutrients S. cerevisiae
coentrapment in Ca alginate with glucose buffer + S. cereuisiae [::]
magnetite periodic nutrient
SUPPlY
coentrapment in Ca alginate with cellobiose + nutrients Z. mobilis ClJOl
fi-D-glucosidase
entrapment in polyacrylamide glucose + salts S. formosensis ClJll
glucose + nutrients
entrapment in Na alginate inulin sugars Kluveromyces IJ 121
+ nutrients marxianus
entrapmentin polyacrylamideor in whey KI. fragi CJ 131
K-carrageenan
entrapment in pectin glucose + nutrients S. cerevisiae
containment by hollow fiber mem- glucose + salts S. cerevisiae
branes giucose + nutrients
containment of cells with cellulase cellulose S. cerevisiae CJJSI
and cellobiase by means of a
liquid-liquid phase boundary
flocculation of yeast glucose + nutrients Schizosaccharomyces CJJ61
pombe
flocculation of yeast with albu- glucose + nutrients S. cerevisiae CJJ71
min/phosphatidyl choline com-
plex

of an immobilized living cell system is not possible with
the information available at the present time. Many of
the physical and biochemical parameters that are
required to describe the system are, at best, only known
to within an order of magnitude. A description of, and
means for estimating, these parameters is presented in
Sections 3 and 4.
3. PHYSICAL AND CHEMICAL PROPERTIES OF
IMMOBILIZED CELL AGGREGATES
To understand the effects of immobilization on cells,
one must first understand the physical and chemical
properties of the aggregate composed of the cells and
the support. The diffusive transport and distribution
of substrates and products are of the utmost import-
ance. In addition, one requires an understanding of the
mechanism by which the cells are retained within the
aggregate. The durability of the aggregate will depend
on its hydrodynamic interactions with the en-
vironment.
3.1. Eflective dljiisivity in cell aggregates
The physical most relevant to the study of
property
immobilized cells is the effective diffusivity of solutes in
the aggregate. Knowledge of the diffusivity of both
substrates and products of cellular reactions is re-
quired for the accurate prediction of overall reaction
rates. Cells comprise some of the diffusive resistance in
almost all immobilized cell systems. Figure 3 shows the
configuration of three types of systems discussed
earlier. Substrates must diffuse through the dense cell
mass and often through a catalyst support to reach
individual cells. Information concerning the effective
diffusivity of some chemical species in various sup-
ports is available [82, 118, 1191. In general, the ac-
quisition of this type of data is straightforward.
On the other hand, little is known about the
diffusivity of solutes, specifically nutrients and fermen-
tation products, in dense cell masses. The volume
fraction of cells in the aggregate (cell packing) may
affect the diffusivity of solutes in the cell mass. Very
little work has been done to correlate the diffusive
properties of a microbial mass with cell packing or
other cell properties. Measurements of diffusivities of
various substances in different types of cell masses are
summarized in Table 3 and their significance is dis-
cussed below.
In general, effective diffusivity measurements are
made by imposing a known concentration of a tracer
on one boundary of a cell aggregate, and measuring
either the steady-state flux through the aggregate or
the transient rate of effusion into or out of the
aggregate. In either case a model that incorporates
certain assumptions about the diffusion process is
required. The diffusion process is generally assumed to
be Fickian in nature, and dependent only on the
concentration gradient and effective diffusivity of the
substrate in the aggregate. Convective transport within
 Immobilization of whole cells I329

Table 3. Values of diffusivities of- substances in cell masses

Relative
Type of diffusivity+ Temperature Measurement Is tracer
cell mass Tracer ( 70,) (“Cl technique consumed? Ref.

Nitrosomona.s oxygen 85
biofilm nitrite 85 20 transient flux no c1201
nitrare 93-100
waste treatment oxygen Z&80 20 transient flux no c1211
biofilm glucose 3C50
waste treatment oxygen 110 20 steady-state flux no c1221
biofilm glucose 110
dental plaque sucrose 2&100 37 concentration unclear cl231
inulin i I%90 profile
tritiated water 25-35
dental plaque sucrose 2&2.5 35 transient flux no cl241
glucose 25-30
helium 62
hydrogen 49
rat muscle ethylene 52 37 transient flux no Cl331
nitrous oxide 59
mammalian tissue dextran 140 37 transient flux no cl321
tumor cells dextran 1O-90
tumor cells glucose 25-50 37 transient flux no Cl311
Zoogloea
ramigera floe oxygen 1+1.5 30 transient flux yes Cl281
mycelial pellet oxygen 25-104 20 transient flux yes cl251
fungal pellet oxygen US100 20 transient flux yes Cl381
microbial biofilm oxygen 20 27 transient flux yes cl291
’ Relative diffusivity = effective diffusivity of tracer in cell aggregate/diffusivityof tracerin water.

the aggregate is assumed to be negligible. Two exper-
imental approaches are generally used. In one, care is
taken to ensure that consumption of the tracer does
not affect the results. In the other, both diffusive
transport and consumption of the substance are
considered. Some assumptions must be made about
the form of the kinetic expression governing the tracer
consumption. These are incorporated into a model
that calculates the effective diffusivity from the exper-
imental data. The method that considers only diffusion
is more desirable, as fewer assumptions are required
calculate the transport properties.
3.1.1. Efective d@utsivity measurements where the
tracer is not consumed. The effective diffusivities of a
variety of substances in microbial biofilms isolated
from waste treatment facilities have been studied by
several groups. Williamson and McCarty [120]
measured the diffusivity of oxygen and of various ions
through a layer of Nirrosomonas cells and debris filtered
onto a membrane. To insure that no consumption of
substrates occurred during the measurements, the
diffusivity of each substrate was determined in the
absence of other substrates needed by the microbes for
reaction. The values of the diffusivites for each of the
substances measured had a large standard deviation,
probably due in part to the unevenness of the bacterial
film and the varying cell densities used in the
experiments.
Matson and Characklis [121] measured the diffus-
ivity of glucose and oxygen through a layer of waste
treatment biofilm which was scraped onto a supported
filter mesh. The microbes were deactivated by mercuric
chloride and ultraviolet light to prevent consumption
of the tracers in the system. Transient concentration
measurements were made to determine the diffusivity
through the cell mass. A similar method was used to
measure the effective diffusivity of glucose, oxygen and
ions through a layer of cells by Onuma and Omura
to [122].
Dental plaque, a naturally occurring biofilm, is a
mixture of oral microbes and extracellular products.
Tatevossian [123] measured the diffusivities of sugars
in this type of microbial mass using a plaque-filled
tube, one end of which was placed in a radiolabeled
solution for a specified period. The tube was sectioned
and each piece was assayed for radioactivity. The
diffusivity through the plaque was calculated using a
one-dimensional infinite slab diffusion model. Non-
reactivity appears to have been assumed. The large
standard deviation in the measurements is probably
due to the uncharacterized nature of dental plaque.
Another measure of diffusivity through dental plaques
yielded values in the same range 11241. The plaque was
heat-killed before being placed in a two-cell diffusion
apparatus. Radiolabeled tracers were used to measure
the diffusivity through the plaques. There was no
measure of cell density reported in this study and no
attempt was made to characterize the plaques.
1330 STEVEN
3.1.2. Efective d@iusivity measurements where
tracer is consumed. A number of studies have been
performed to estimate the effective diffusivity under
conditions where reactions and diffusion occur simul-
taneously. The assumptions made about the kinetic
expression describing the reaction rate in each case are
discussed along with the experimental results in this
section. The application of theory to combined
reaction-diffusion will bc discussed in Section 5.
Oxygen transfer is an important problem in the
growth of mold in the form of pellets. In an early study,
consumption and diffusion parameters were estimated
by assuming zero-order oxygen uptake and homo-
geneous cell distribution in a spherical mold pellet
[125]. The zero-order uptake assumption was later
questioned and the data reinterpreted according to
Monod-type kinetics [126]. In an extension to this
model, the age and, therefore, reaction rate of the mold
mycelia was permitted to vary with location. The
experimental data showed good agreement with the
three parameter representation that was used [ 1271.
Mueller et al. [ 1281 employed an anoxic centre model
to describe diffusion in a Zoogloea ramigera floe. A two
parameter model was used with the data to calculate
the values of effective diffusivity and the kinetic
constant. A similar procedure has been used to es-
timate the effective difiusivity through a bacterial film
modeled as an infinite slab [129, 1303.
3.1.3. Ejkctive dl@usivity measurements in animal
tissue. Measurements of diffusivity of gases in mam-
malian muscle have also been made. Aside from the
lack of cell wall, mammalian tissue is structurally
similar to a dense bacterial mass. In a study to
determine the effective diffusivity of several gases in rat
muscle it was determined that the effective diffusivity is
similar to that in dense bacterial cells [ 1311. Other
investigations have been performed to measure the
rate of dextran, fluorescein, oxygen and glucose diffu-
sion in normal and tumor tissue [131--1331, and the
rate of oxygen diffusion in salamander muscle [134].
3.1.4. Sources of d@iisive resistance in cell ag-
gregates. Although many studies have been conducted
to determine the diffusivity of various substances
through cell masses, very little is understood about the
source of the diffusive resistances. It has been ac-
knowledged [121] that the type of cell and the
structure of the ccl1 mass might have an impact on the
diffusivity, but a fundamental understanding of this
problem has not yet been achieved. Another parameter
affecting the diffusive resistance in a cell mass is the
production and excretion of extracellular. substances.
Many cells produce extracellular polysaccharides,
some of which can act to promote the cohesion of a
biofihn. These substances might be important de-
terminants of the diffusive resistance in these films.
The density of the cell mass was measured in some of
the studies mentioned above [120, 121, 1291, but the
F. KAREL~~ al.
the only correlation of density with diffusive resistance in a
cell mass is presented by Yano et al. [125]. This shows
a weak increase of diffusive resistance with increasing
cell density. The data of Williamson and McCarty
[120] revealed no correlation between dry cell weight
and diffusivity. Although Matson and Characklis
[121] measured the dry weight of the cells in each
experiment, no correlation between this and diffusivity
was reported. In one of the dental plaque studies,
plaques of varying cell densities were used. Higher
diffusive resistances were measured in the denser
plaques. Unfortunately, the density data were pre-
sented in terms of centrifugation speed used to sep-
arate the plaque from the plaque fluid (the packing
density of the cells in the sample increases with
increasing ccntrifugation speed) and not density or dry
cell weight [123].
Using a different approach, Matsunaga et al. [135]
measured the diffusive resistance in a membrane
consisting of gel-entrapped Clostridium butyricum at
various microbial volumetric packing densities. They
found that the diffusivity of glucose through the
composite membrane decreased with increasing cell
packing. The diffusivity of hydrogen through a densely
packed cell/gel membrane was about two-thirds of its
value through the empty gel.
Klein and Schara [136] have measured the diffus-
ivity of phenol in polyacrylamide discs containing
entrapped cells over a range of polymer volume
fractions and cell loadings. They suggested an em-
pirical correlation of the form:
~ = (1 -vv,)Zexp(-qv,)
a’*
where 2 is the effective diffusivity of the tracer, B*
is the diffusivity of the tracer in free solution, vX is the
volume fraction of cells, vp is the volume fraction of the
polymer and q is a parameter characterizing
the molecular size of the tracer. For small molecules,
the parameter q was assumed to be equal to 4. Klein
and Manecke [118] later proposed an alternate ex-
pression of the form:
- = exp ( - q(v, + up)).
9*
A theoretical model describing oxygen transport in
tissues as transport though a heterogeneous material
has been proposed by Tai and Chang [137].
Information to correlate diffusive resistances with
cell packing and microorganism type is necessary to
allow prediction of the diffusivity of substances
(specifically pro&cts and substrates) through the
dense cell masses found in immobilized whole-cell
reactors. The effect of extracellular products on the
effective diffusivity in a cell mass also needs to be
investigated.
3.2. Interactions among components in the aggregate
Among the other physical or chemical influ-
ences acting on immobilized cells, we are especially
interested in those responsible for the immobilization.
 Immobilization
In the case of entrapped or contained cells, the impetus
for immobilization is a restriction of the mobility of
the cells within the porous matrix or across the
confining barrier. In cases where cells are attached to a
surface or to each other, the immobilizing force is a
physical or chemical interaction among cells or be-
tween the cells and the support.
3.2.1. Cell mobility within the aggregate. The degree
to which the restriction of cell mobility in the aggregate
is desirable depends on the particular process. With
non-viable cells, the major problem may be the leakage
of desired enzymes from the immobilizing material; it
is therefore useful to make the confinement barrier
impermeable to high molecular weight species. On the
other hand, if living immobilized cells are used, a
greater degree of mobility may be desired to allow
waste products and dead cells to be removed from the
aggregate as the cells grow. A steady-state may thus be
achieved in which the rate of synthesis of new cellular
material is balanced by the rate of loss of cell material
in the effluent of a continuous reactor. In this sense, the
role of immobilization is to increase the cell hold-up in
a continuous reactor as compared to a case where free
cells are used [25].
A number, of different mechanisms mediate cell
transport within the aggregate. Cells may diffuse
passively as a result of Brownian motion. In addition,
certain strains exhibit motility and chemotaxis [139].
The volume expansion caused by the synthesis of new
cell material is itself a form of transport. The large size
and consequent low diffusion coefficient of the cells
make the presence of even a small degree of convection
within the aggregate a significant factor in facilitating
the transport of cells.
Very little has been done to directly measure the
mobility of cells under any of the conditions typical of
immobilized cells. In the case of biofilms, it is often
assumed that a steady-state is achieved with a spatially
uniform population of cells [14O, 1411. In this steady-
state, the growth rate of the cells is matched by the
combined rates of cell death and the physical removal
of cells. The relative importance of these two rates may
vary from system to system [142, 1431. The transport
of cells also occurs as a result of sloughing, the
occasional detachment of large sections of the biofilm.
Sloughing is thought in some instances to be a result of
the formation of an anaerobic or otherwise nutrient-
limited zone within the film owing to mass transfer
limitations. The formation of bubbles due to anaerobic
metabolism may also lead to sloughing [S]. The fact
that there is little resistance to expansion and contrac-
tion of the film also helps to provide an even distri-
bution of cells within the film.
One indirect method of assessing the degree of
immobility of immobilized cells is to examine the
pattern of growth under diffusional limitations. If the
cells appear to form a uniform layer, then the rate at
which cells move about within the aggregate is rela-
tively rapid; on the other hand, if the cells are less
mobile, a dense layer of cells may form near the
of whole cells 1331
nutrient source. One example of this effect observed in
the absence of any physical support is the formation of
mycelial pellets with hollow cores surrounded by a
dense layer of growing cells under conditions of
diffusion-limited growth [34, 1441. The growth of cells
in large numbers in a thin layer near the surface of the
gel while the interior contains very few cells also attests
to the relative immobility of cells in gel particles [145].
In gels containing living microbes, the most import-
ant aspect of mobility is the rate of cell effusion from
the gel. A number of investigators have reported the
rate of appearance of cells in the medium in exper-
iments using gel-entrapped cells [52, 113, 1461. The
rate of leakage generally is associated with the growth
of the organisms. It can be reduced significantly either
by slowing the growth rate of the organism [108, 1471
or by altering the gelation procedure to engender a
tighter matrix [146, 1481. The appearance of “micro-
interstices” on the surface of a gel particle may indicate
that cell division expands and fractures the gel [149].
Expansion of the cell-containing pockets in the gels has
also been observed [ 150, 1513. Since cell leakage is
associated with growth, it probably is not a major
cause of deactivation of the catalyst. The growth of
cells exterior to gels may, however, lead to obstruction
of flow within the reactor [96, 108, 1523.
In preformed porous matrices, mobility is important
in allowing the support to be inoculated as well as
preventing leakage after inoculation. Messing and co-
workers [153, 1541 have examined the effect of pore
size on cell immobilization using controlled pore glass.
The optimal pore size is determined by a balance
between two effects: the pores must be large enough to
allow penetration and growth of cells within the
matrix, yet they must be small enough to hinder
leakage of the growing cells. The optimal pore size for
bacteria or yeast was determined to be between one to
four times the major dimension of the cells. When
mycelial organisms were grown after entrapment of
spores, the optimal size range was somewhat larger.
This does not preclude the use of porous materials with
a larger pore size, which has been shown to be useful in
certain situations [78, 1551.
Systems using preformed membranes for cell en-
trapment offer the possibility of obtaining a sterile
product stream from an immobilized cell reactor by
ensuring that absolutely no cells cross the confining
membrane. Growing cells have, however, shown an
amazing ability to penetrate membranes with pore
sizes smaller than the cells. The ultrathin retentive wall
characteristic of certain asymmetric hollow fiber ultra-
filtration membranes apparently does not have the
mechanical stability to resist the pressure of growing
cells [96]. The stress caused by cell growth may induce
fissures in the membrane, allowing cells to proliferate
throughout the reactor. Other investigators have also
noticed that growing cells can penetrate and eventually
pass through membranes with pore structures signifi-
cantly smaller than the ceils themselves [ 1X%158]. The
mechanism for this passage is unclear. In our own
experience, some types of hollow microporous iso-
1332 STEVEN F.
tropic fibers can prevent cell breakthrough, although
the membrane may collapse under the concerted
pressure of cell growth around the fiber.
3.2.2. Chemical interactions between the support ana’
the solution. The chemical properties of the phase in
which the cells are immobilized may exert a direct
effect on the reactivity by changing the solubilities of
substrates and products in the local environment.
Indirect effects arise from changes in the enzymatic
properties of the cell as a result of the changed
environment. It has been argued that the high polymer
concentrations found in gels can alter the activities of
metabolic pathways by depressing the water activity
[163]. In this regard one may note that biofilms can
have high concentrations of extracellular polymers,
especially under conditions of carbon excess [29].
The effects of gel composition have been quantified
in terms of a partition coefficient relating the concen-
trations in the aggregate and in the bulk of a given
chemical species. Many substrates are relatively in-
soluble in water, so that organic solvents have been
used to increase the substrate concentration in im-
mobilized cell processes [159,160]. The work of Fukui
and his associates [160, 1611 on the bioconversion of
steroids by cells immobilized in hydrophobic gels
demonstrates the importance of understanding par-
titioning effects. Polymer mixtures were used to form
gels having a range of chemical properties. The analysis
of such systems is complicated by the possible toxicity
of the organic solvents, and by the fact that the cells
themselves may affect the partitioning behaviour of
organic substrates [162].
3.2.3. Direct interactions between the cells and the
support. An examination of the wide variety of sur-
faces onto which cells adhere suggests that no single
mechanism can be responsible for all the effects
reported. Attachment is observed on surfaces having
high surface free energies such as glasses or clays as
well as on low energy surfaces such as polyethylene or
wood. The accumulation of cells at liquid-liquid or at
gas-liquid interfaces has been exploited as the basis for
separation processes [164-l 663.
The colonization of surfaces by cells may be as-
sociated with an ecological advantage Cl673 due to
adsorption of nutrients or to the microbial degra-
dation of the support to provide nutrients for growth
[168-1701. Even artificial supports such as PVC may
be degraded [171].
Either hydrophobic or ionic interactions can cause
cell adsorption. Attempts have been made to build a
theoretical framework in both cases. Hydrophobic
interactions have been quantified by measurements of
contact angle [172] and of the liquid-liquid partition-
ing properties of the cells [173]. Theories have been
proposed to treat cells as macroscopic ions to explain
binding of cells to charged surfaces [67, 174, 1751. The
adsorption of proteins and other organic material on
surfaces prior to cell attachment and the possibility
that cells produce extracellular or membrane polymers
KAREL~I al.
which enhance adhension cast doubt on the universal
applicability of these theoretical approaches. Many
experiments suggest that cell adhesion is a two-step
process consisting of a reversible weak binding fol-
lowed by a time-dependent irreversible attachment
which may be due to the synthesis of extracellular
polymers [29, 1761.
The growth of animal cells in culture represents an
extreme case of the sensitivity of cells to the chemical
nature of the support. Levine and co-workers Cl773
found that both adhesion and growth of animal cells
on DEAE-dextran microcarriers were strongly de-
pendent on the surface charge of the support. The
ability to alter the strength of adhesion by manipulat-
ing the calcium concentration in the medium has been
used to facilitate bead-to-bead transfer of cells [178]. A
number of investigators are continuing to examine the
effects of different surface chemistries on the growth of
animal cells, including collagen [ 1791, glass [ 1801 and
derivatized polyacrylamides [ 181, 1821. The role of
specific cell surface glycoproteins such as fibronectins
in the adhesion of animal cells has been considered in
some depth [183].
3.3. Physical interactions of the aggregate with the
environment
In reactors using immobilized cells, a certain
amount of fluid motion is required to enhance mass
and heat transfer within the reactor. Hydrodynamic
interactions between the particles and the environment
fall into three categories: (1) structural changes in the
aggregate caused by hydrodynamic shear stresses and
by inter-particle collisions; (2) changes in the flow
pattern and mixing of fluid and particles within a
reactor and (3) enhancement of mass transfer of
solutes and/or cells to and from the aggregate by
increasing the relative fluid velocity at the surface. The
structural effects include abrasion and compression of
support particles, which may have detrimental effects
on the stability of the reactor. Detachment ofadsorbed
cells by shear stress or by interparticle collisions may
be detrimental or advantageous, depending on the
particular process.
The shape and size of immobilized cell particles or
films is of central importance in modeling hydro-
dynamic interactions. Nevertheless, even these simple
parameters are not always easily characterized. Both
cell growth and physical interactions can change the
size of the particles. Many gels and ion-exchange resins
swell or shrink with varying solute concentrations.
Physical processes leading to the breakdown of the
immobilized cell aggregate have been examined pri-
marily in particulate systems and then mostly with gel
particles. Specifically, attention has been focused on
the compression of particles in packed columns, and
on the abrasion of particles in agitated systems [22,23,
1841. It has been observed that the mechanical prop-
erties of the aggregate deteriorate at very high ag-
gregate cell concentrations [22, 1481. In a number of
cases the treatment of gel particles with cross-linking
agents such as dialdehydes or diamines results in an
 Immobilization of whole cells
aggregate with improved mechanical properties ES,
185, 1863.
In certain cases the confinement of cells to large
particles may enhance the overall rate of mass transfer.
The formation of mycelial pellets rather than the more
open hyphal structure usually associated with mycelial
growth dramatically reduces the solution viscosity,
thus facilitating agitation and gas-liquid mass transfer
11871. For mycelia immobilized within porous beads,
Gbewonyo and Wang [188] found that the limitation
to oxygen transfer imposed by internal mass transfer
resistance within the beads was less severe than the
limitation to oxygen transfer created by the increased
viscosity of the free cells in solution.
Hydrodynamic effects on the cells at the surface
depend on the velocity field exterior to the aggregate.
For suspended particles, the relative velocity depends
critically on the density difference between the ag-
gregate and the suspending solution. Thus, the predic-
tion of external mass transfer coefficients for im-
mobilized ceil particles is difficult [ 1891. Other prop-
erties depending on buoyancy such as fluidization
criteria are also affected [190].
The velocity field near a surface is also thought to
have an effect on the transport of cells to and from the
surface. A number of experimental studies have been
performed to investigate the effects of surface shear
stress on the overall rate of adsorption and desorption
with clean surfaces, but no clear trend has emerged
[143, 191-1933.
A complete knowledge of the physical and chemical
properties of immobilized cell aggregates, even if it
were available, would still be insufficient for an
understanding of their behaviour, since cell physiology
must certainly be considered. The biochemical prop-
erties of the cells are determined not only by the
intrinsic attributes of the cells themselves, but also by
the environment in the aggregate, which in turn
depends on physical processes in the aggregate. In the
following section, we will consider the current state of
research concerning the biological properties ofcells in
the immobihzed state.
4. BIOLOGICAL PROPERTIES OF IMMOBILIZED
CELLS
There are many examples in the literature showing
that cellular physiology and morphology sometimes
change upon immobilization. There are a number of
possible reasons for this. The immobilization pro-
cedure can alter the metabolic activity or viability of
the cells. As mentioned earlier, the microenvironment
is often different than that of cells in a suspension
culture. In addition, the physical stresses that closely
packed growing cells exert on one another and on the
support are not present in traditional fermentations.
These properties of immobilization often affect the
cells in a variety of ways which will be discussed below.
4.1. Cell morphology
The most obvious difference between an immobil-
ized cell culture and a suspension culture is the fraction
I333
of total volume occupied by ceils. The volumetric
fraction of cells in conventional suspension cultures
varies between 1 and 30 % [194]. Methods to increase
cell density in suspension cultures such as vacuum
fermentation to selectively remove growth inhibitors
and cell recycle systems have been successfully used to
increase this fraction to as much as 607;. The cell
packing density in some immobilized cell reactors,
however, approaches 100 “/, of the available volume
C961.
There are several examples in the literature where
electron microscopy has been used to examine the
effects of the close cell packing characteristic of some
immobilization procedures. Scanning electron micro-
graphs of cells isolated from raw sewage adsorbed to
alumina and cordierite shown in Fig. 4 illustrate the
high cell packing that can be achieved with this type of
immobilization [63]. The high volumetric fraction of
Psetufomunascells immobilized on a Millipore filter as
well as some of the retained extracellular products
surrounding the cells [29] is shown in Fig. 5. Scanning
electron microscopy has been used to distinguish the
different biofilm morphologies caused by distinct
organisms in mixed cultures [ 195, 1961.
Transmission electron micrographs of E. coli and
S. cerevisiae grown around asymmetric hollow fiber
membranes are shown in Figs 6 and 7 187, 961. Fission
and budding, each indicative of cellular viability, can
be seen in both micrographs. The microbes are dis-
torted by the pressure of the surrounding organisms,
especially in the case of the individual yeast cells.
Similar deformation of cells was also reported [197]
for growing yeast cells entrapped in a polycrylamide
gel. Laretta-Garde [198] reported that growing
Rhodopseudomonas capsulata cells exerted sufficient
pressure to induce deformation of the surrounding
calcium alginate gel.
4.2. Cell physiology
The fundamental assumption usually made in mod-
eling the behaviour of immobilized cells is that the
cellular metabolism depends only on the microen-
vironment. This assumption cannot be correct for the
case of mammalian cells, many of which absolutely
require surface attachment for growth; therefore,
physical interactions at the cell surface in some way
regulate the metabolism of the cells. A number of
reports have also appeared suggesting that certain
unicellular microorganisms may also exhibit an ac-
celeration of their metabolic activities as a result of
surface attachment. An alarming trend has developed
in whichanomalous results obtained with immobilized
cells can be justified by an “enhanced activity” for cells
at a surface. It is therefore of some interest to review
the available evidence for this effect and to consider if
and how it should be incorporated in models that
predict the behaviour of immobilized cells.
4.2.1. Efect of surfaces on the growrh of cells. The
evidence for surface enhancement of growth rate falls
into three categories. In the first category, the growth
 STEVEN F. KAREL er al.

Fig. 4. (a) Scanning electron micrograph of cells immobilized on alumina. (b) Scanning electron micro-
graph of cells immobilized on cordierite (with permission [63]).

of cells at a surface leads to the periodic release of cells
into the medium. This effect has been ascribed to
synchronous growth of the cells with desorption of one
of the daughter cells following cell division, and the
period has therefore been identified with the gener-
ation time of the cells [199-2041, although this may
not apply in all cases. In two of these experimental
studies, it has been observed that the overall metabolic
activity of the cells is lower than that of free cells for
similar systems while the growth rate as determined
from the “generation time” seems to have increased
[200, 2051. This discrepancy has yet to be adequately
explained.
In the second category, adsorption is observed to
lead to a short-term increase in the respiration rate of
the cells at the surface [72,206,207]. It is unclear if the
 Immobilization of whole cells
Fig. 5. Scanning electronmicrograph of Pseudomonas urruyinosa grown on a Millipore filter. Extracellular
polysaccharide (EPS) filaments bind the microbes together (with permission (291).
increase in substrate utilization leads to an increase in
the growth rate of the cells. The third category of
results reflects the importance of surfaces as a selective
factor in microbial ecology. A number of investigators
have examined the relative contributions to the overall
metabolic activity in undefined mixed cultures of
surface attached cells and free cells [208]. This topic
has been considered in some depth by Fletcher and
Marshall [6], who point out that both increases and
decreases in activity can be observed.
A number of theories have been advanced to explain
these observations. There may be a selection of a sub-
population of cells by the adsorption process [5,209].
Alternatively, it has been proposed that attachment
somehow alters the permeability of the cell membrane
[6,206]. There may also be chemical interactions of the
surface with compounds in the solution leading to a
more favourable environment for the cells at the
surface. It has been argued that surfaces are environ-
ments of high nutrient concentration because of the
adsorption of proteins or other organic materials
[5,6]. This argument seems reasonable only when
nutrient solutions are dilute and undefined as in
natural rather than in laboratory conditions, and when
the number of cells attached to the surface is small.
Given the variability of the results reported, the only
conclusion to draw is that under certain conditions the
metabolic activity of cells at surfaces will be different
1335
from that of free cells. There is no consistent method
for predidting how the behaviour will differ. In a wide
variety of cases the reaction behaviour of immobilized
cells is essentially identical to the activity of free cells
[54, 142, 210-2141. Since the simplest assumption is
that the activity does not change on immobilization,
the use of activity data obtained in solution to model
the behaviour of immobilized cells is advocated.
4.2.2. Influence of immobilization method on cell
t;iability.The method of entrapment or immobiliz-
ation can reduce the culture viability. Some studies
show that entrapment in a polyacrylamide gel destroys
viability in 5&90°/0 of the cells [149, 1971. The toxic
side effects to cells of polyacrylamide entrapment have
been studied in depth [215]. One investigation, how-
ever, indicates the respiration rate of a variety of cells
was unchanged after entrapment in a variety of
polymers [SO]. In a study to determine the sensitivity
of cells to immobilization during different growth
phases, it was found that cells entrapped in a polymer
gel while in a lag phase retained a greater fraction of
viability compared with those immobilized during the
exponential growth phase [149]. As long as some of
the entrapped cells remain viable, the cells in the gel can
grow to a desired density after entrapment. The
retention of viability is dependent both on the
microorganism and the gelation conditions [197].
1336 STEVEN F. KAREL et al.

c
L
Fig. 6. Transmissionelectronmicrographof cross-sectionof E. coli immobilizedin the macropores of an
asymmetric hollow fiber membrane. The lumen fL) and the outer wall (S) are shown [227].

Comparable data on the retention of viability after
entrapment behind a semipermeable membrane or
adsorption onto a porous matrix shows that the per
cent viability retained is very high. This is expected
because the conditions of immobilization for both of
these methods are very mild.
4.2.3. Permeabilization of cells. The permeabiiity of
the cell membrane can be changed upon immobiliz-
ation, either as a side effect of the immobilization
method or by design. Several investigators have at-
tempted, with some success, to increase the per-
meability of plant cell membranes with dimethylsui-
foxide (DMSO) to allow enhanced release of products
[216-2181. The subject of cell permeabilization has
been recently reviewed by Felix [2 193. Drioli et al. [83]
reported that the activity of Calda~~e~~a Qriduphiia
increased when immobilized within cellulose acetate
and polysulfone membranes and hypothesize that this
is a result of the permeabilization of the ceil membrane
during the immobilization process. Intentional per-
meabilization of the cell membrane to increase pro-
duction can succeed, but may also resuit in lower
stability of the enzymes in the cell, perhaps due to the
toss of enzyme from the cells through the membrane
[220-J
 Immobilization of whole ceils 1337

Fig. 7. Transmission electron micrograph of cross-section of yeast cells immobilized in the macropores ofan
asymmetric holiow fiber membrane. The lumen (L) of the fiber is designated [227].

Cell lysis is an extreme limit of cell membrane
permeabilization. If cell viability is not important to
the desired catalytic activity, a crude enzyme mixture
can be obtained by cell growth to synthesize a desired
enzyme followed by cell lysis to expose the enzyme to
the solution. Several investigators [221,224] have used
this procedure to obtain reactors with high aspartase
activity. In both cases, increased activity following lysis
indicated that intact cell membranes provided a signifi-
cant diffusive resistance.
42.4. Catalytic stability of immobilized ceils.
lmmobili~tion affords whole cells some protection
from physical and chemical challenges. Takata et al.
[222,223] showed that the enzymatic activity of whole
cells immobilized in K-carrageenan gel beads is more
stable to environmental challenges than either free
whole cells or native enzymes. These challenges include
heat, pH changes, organic solvents or protein denatur-
ing agents. Similar results have been found in other gel-
entrapped immobilized systems [220]. The authors
CBS40:8-B
hypothesize that the reason for the increased stability
to the protein denaturing agents is the physical
structure of the matrix which may hinder the action of
the denaturing agents. The partitioning effect of the gel
may explain the increased resistance of the immobi-
lized cells to the organic solvents. Another reason for
increased enzyme stability in immobilized cell systems
is suggested by Klibanov (I543. He notes that in the
event of cell lysis, the catalyst support may stabilize the
enzymatic activity of the lysate. This stems from the
buffering capacity attributed to some supports and the
physical protection that would help prevent the dis-
sociation of oligomeric enzymes.
One of the main concerns in the use of immobilized
cell reactors is that of catalytic longevity. Cell protein is
constantly degraded and regenerated in whole cells
[224]. Only preliminary work has been performed to
determine whether protein degradation in immobi-
lized cells occurs at the same rate as in free cells in
suspension culture [225]. If the cells do not reproduce
themselves, and only degrade protein, a constant
1338 STEVEN F. KAREL
decrease in catalytic activity will take place. In
nutrient-limited sections of the reactor, continued cell
activity can take the form of cryptic growth, that is,
proliferation of cells on the nutrients released by
nearby lysed cells. Cryptic growth has been implicated
in nutrient-limited areas of immobilized cell aggre-
gates [ 197, 2261.
4.2.5. Immobilized resting ceils. Although some cell
growth is desired in many microbial reactor systems, a
high cell growth rate in an immobilized system can be
deleterious in two ways. The cell growth diverts
substrates and nutrients from product formation to
biomass production, and, as noted previously, often
leads to operational difficulties [87, 1081.
The disadvantages of continued microbial growth in
immobilized cell systems have led investigators to
search for methods of maintaining cell productivity
while slowing or stopping cell growth. One method
which has been used is nutrient deprivation, usually of
nitrogen [146, 226, 227-J. Alternatively, antibiotics
which dissociate growth from metabolism or other
metabolic inhibitors can be added to the medium [60,
62, 149, 228, 2291. Temperature sensitive organisms
which cease to grow but maintain their viability above
a certain temperature may also be useful.
Nutrient deprivation has been the most common
method used to slow or stop cell growth. Inloes [226]
grew ethanol-producing S. cerevisiae around hollow
fibers and then attempted to stop growth while
sustaining ethanol production by substituting a ni-
trogen deficient medium for the growth medium.
Although the cell growth rate sharply decreased, the
production of ethanol dropped to less than loo/, of the
rate in the growth medium. Repeated day-long in-
fusions of growth medium at intervals of about 5 days
revived the cells, but increased production was only
apparent while the growth medium perfused the
system. Similar results were seen in other studies
involving ethanol producing yeast [52,230] and with
other microorganisms [147,227,231]. Furusaki ef al.
[ill J successfully placed a different type of yeast,
S. formosensis, into an ethanol-producing resting state
using nitrogen deprivation. The success of this study
may have been related to the fact that unlike the
L. delbruckii and S. cerevisiae described above, this
yeast is not a fastidious organism and can grow on a
defined medium.
The productivity of a nutrient deprived reactor can
be superior to that of a reactor operating under full
growth conditions. Forberg et al. [146] attempted to
reduce growth and retard the production of unwanted
byproducts in a gel entrapped butanol producing
Clostridium acetoburylicum fermentation by perfusing
the reactor with nitrogen free medium and pulsing the
reactor with growth medium for 15 min every 2,4 or 8.
The butanol production increased to 170% of its
growth phase value during the pulse-feed phase, while
the cell production dropped to 5 o/0of its value during
the growth phase.
Substrate inhibition was used to control cell growth
et al.
in a rc-carrageenan immobilized yeast system [232].
Cell growth was reduced 50% and the volumetric
productivity of ethanol was increased 120 o/0by intro-
ducing successively higher glucose concentrations in
the feed. The initial glucose concentration of 5 y0 (w/v)
was increased in steps of 5% (w/v) until the final
concentration of 25 o/0(w/v) was reached.
4.2.6. Techniques for characterizing the metabolic
and physical state of immobilized cells. There are a
variety of methods for investigating the metabolic state
of immobilized whole cells. These vary from micro-
scopic investigation of the catalyst particle to on-line
measurements of respiration and cellular activity
[233]. Calorimetric protein assays have been adapted
for use with immobilized cells [234]. Cells that are
reversibly immobilized can be removed from their
support and then tested for their metabolic rate and
viability. This method presumes that the actual ad-
herence or entrapment of the cell does not affect the
cellular metabolism.
A number of non-invasive spectroscopic techniques
may be used to investigate the behaviour of im-
mobilized cells. Methods for using nuclear magnetic
resonance spectroscopy (NMR) to allow in uivo obser-
vation of immobilized cell metabolism are being
developed in our laboratory and elsewhere [235]. The
fate of an isotope (e.g. 31P) can be determined by
sequential scans which indicate the relative amounts of
phosphorylated intermediates and products. In ad-
dition, 31P can yield some information about the pH
distribution in the cell mass. Work on the investigation
of free cell metabolism using NMR has been reviewed
[236]. Electron paramagnetic resonance spectroscopy
(EPR) is a similar technique which has been used in the
investigation of the structure of immobilized enzymes
[237, 2383. Total reflection infrared spectroscopy
[ 1721 and total reflection fluorescence [239] have been
used to investigate the chemistry of cell-surface inter-
actions. Flow cytometry, a method recently developed
for the investigation of the metabolism of free cells, has
been applied to immobilized systems [240].
Another non-invasive technique which can be used
to probe cell metabolism in the immobilized state is
radioisotope labeling. This technique has been used to
study protein degradation [225] and the metabolic
activity of immobilized cells [225, 2411.
It is clear that immobilization affects cellular metab-
olism in at least some instances. The elucidation of the
effects of immobilization on the physiological and
morphological state of the cell is an important area of
research. The special stresses and environment to
which an immobilized cell is subject need to be better
understood so that the variables which affect the cell
can be manipulated advantageously.
5. INTERACHONS BETWEEN REACTION AND
DIFFUSION IN IMMOBILIZED CELL SYSTEMS
The most important question which arises in the use
of immobilized cells is that of mass transfer resistance
either external to or within the immobilized cell
 Immobilization
aggregate. The reduced surface area of ceil aggregates
and the presence of the support comprise additional
barriers to mass transfer relative to free cells in a well-
mixed solution. This tends to lower the overall rate of
reaction, as well as creating an environment within the
aggregate different from the bulk solution. Living cells
are quite sensitive to such changes in their environ-
ment. If thecells are growing within theaggregate, then
the existence of internal mass transfer limitations may
createa spatial distribution of the growth rate and thus
redistribute the catalytic activity. The behaviour of
living cells is therefore a complex process, which only
in certain simple cases is amenable to the type of
theoretical treatment which has been successful in
describing immobilized enzyme systems.
5.1. Intrinsic kinetics of immobilized cetls
To understand the kinetics of immobilized cells, it
seems appropriate to start with the kinetics of free cells
exposed to similar solution conditions. The rate of a
cellular reaction typically is first-order at vanishing@
small substrate concentrations and zero-order at
higher concentrations. The empirical Monod equation
which is often used to describe cell growth is ident-
ical in form to both the first-order Langmuir-
Hinshelwood and Michaelis-Menten equations.
Many other rate expressions nevertheless have the
same asymptotic behaviour and give equally good
results when applied to cell systems [242, 2431.
Because these rate laws are bounded by the zero- and
first-order cases, the solutions of the problems combin-
ing diffusion with these two simple rate laws are
valuable in that they can be applied as lower or upper
bounds to the general problem without requiring
detailed knowledge of the rate expression.
In some cases, more complex rate laws must be used.
Very high substrate concentrations can inhibit reac-
tions. Product inhibition is even more likely to be
observed in a typical immobilized cell process [244].
The wide variety of reactions catalyzed by a single cell
means that side products cannot generally be ignored.
The product selectivity in immobilized cell processes
has been investigated in a number of studies [SS, 146,
226, 229, 2451.
If the immobilized cells are not growing, and
especially if the cell membrane is no longer intact, the
reaction rate behaviour may be simple and formally
similar to that of immobilized enzymes. If the ceils are
intact and living, then the reaction rates are often
determined by regulatory processes or membrane
transport and therefore depend on the nutritional
status of the cells. In that case, rate expressions may
only be applicable over a narrow range of environ-
mental conditions.
Two important categories of processes using im-
mobilized cells involve the use ofvery low but non-zero
net growth rates. With primary metabolites, which are
direct products of metabolism, rates of production are
highest at high cell growth rates. High growth rates
cannot, however, be sustained in immobilized cel1
systems. In contrast, antibiotics are generally produced
of whole cells I339
in batch culture during the so-called stationary phase
when little if any growth occurs. Secondary metabohte
production with immobilized cells has attracted atten-
tion in hope that the stationary phase may be pro-
longed [231,2465248]. Most experience shows, how-
ever, that non-growing cells are unstable (see Sections
4.2.4-4.2.5.).
In each of these cases, the emerging consensus is that
in most cases immobilized living cell processes should
be operated so that growth is very slow or intermittent.
Under such conditions, any reasonable model must
consider both rates of synthesis and degradation of cel1
material, as well as the regulation of primary and
secondary metabolism. This means that maintenance
energy and cell lysis must certainly be taken into
account. Unfortunately, even in suspension cufture the
available experimental results have not been ad-
equately modelled [249]. Considerably more work is
required to elucidate the regulation of the various
cellular pathways at low growth rates or during
starvation [250, 2.51). Cell recycle systems may be
useful in determining intrinsic properties of cells under
physiological conditions similar to those prevailing in
immobilized cell systems.
5.2. Mass transfer and the observed reaction rate:
examples
Mass transfer limitations are most striking in im-
mobilized cell systems when the supply of oxygen to
cells and the removal of carbon dioxide is required.
The transfer of oxygen from the gas phase to the liquid
phase has long been recognized as the major rate-
limiting step in the aerobic growth of cells in suspen-
sion [252). The high volumetric reaction rates
achieved with immobiiized cells shift the relative
importance of the resistance terms so that mass
transfer within the aggregate may be rate-limiting. To
the extent that oxygen consumption can be modelled
as a zero-order reaction, experimental and theoretical
results can be expressed in terms of the depth of a cell
layer in which the oxygen is consumed. In biofilms, this
“critical distance” has been estimated in a number of
situations as being between 50-100 p [IS]. Using gel-
entrapped Bacillus umy~oiiq~efaciens, Shinmyo and co-
workers [253] found that cells grew only within 50 g of
the outer surface. in work with mold pellets, the range
of values reported by various observers for the “critical
radius” is considerably wider (80-2000 p) [34].
A variety of methods have been suggested for
increasing the rate of oxygen transfer to immobilized
cells. Increasing the partial pressure of oxygen will
increase the rate of mass transfer, but at the risk of
substrate inhibition [254]. Soluble oxygen carriers
such as hemoglobin and perfluorocarbons [256] have
been used. Hydrogen peroxide can be used to supply
oxygen either through the natural catalase activity of
the cells or by an enzyme coimmobilized with the cells
[257,258]. The local generation of oxygen from water
by photosynthetic or electrolytic means can also be
achieved [259, 2603. Among these methods only
1340 STEVEN F.
increasing the partial pressure of oxygen is suitable for
large-scale use at the present time.
As we mentions above, high concentrations of
waste products also limit the reaction rate. Mass
transfer of products away from the aggregate is
therefore important. A common product is carbon
dioxide, which can inhibit growth either directly [262]
or through its effect on the pH in the microenviron-
ment. At sufficiently high concentrations, nucleation
and bubble formation can occur [263, 2641. Bubbles
formed in this manner can have a detrimental effect on
the physical state of the catalyst [113, 1141. Gas
removal is often an important aspect of reactor design
in the use of immobilized cells [265, 2661.
There may be compounds that are released and/or
taken up by cells that are apparently unrelated to the
reaction of practical interest yet play an important role
in determining the metabolic behaviour of the ceils.
Mass transfer of such compounds in the aggregate may
be important. An example is the role of metabolic
intermediates and hormones in plant or animal cell
culture. Plant cells in culture differentiate preferen-
tially in the center of large aggregates [267]. The
growth of surface-dependent mammalian cells is de-
pendent on the number of ceils per unit surface area,
declining both at low and high surface coverages f268].
Both of these effects probably reflect the intervention
of some hormone or metabolic intermediate. A second
example involves the scavenging of toxic materials.
Strictly anaerobic organisms may be protected from
trace quantities of oxygen by immobilization, as a
result of the lowering ofthe diffusion rate of oxygen to
the cells while the scavenging activity of the cells is
maintained [266].
5.3. Mass trawfer and the observed reaction rate:
theory
Given the difficulty of conducting detailed and well-
controlled experiments with immobilized cells or en-
zymes, considerable effort has been exerted in applying
the mathematical theory of reaction and diffusion in
porous media to these systems. A vast number of
solutions of the differential equations involved using
various particular geometries and reaction rate ex-
pressions have been reviewed in the literature in the
context of biochemical reactions [23,26-28,189,269,
270-J. The results of these calculations provide an
estimate of the actual reaction rate in an immobilized
system, based on estimates of the intrinsic reactivity
and the diffusivity of substrates, intermediates and
products in the aggregate.
We will present in some detail the solutions most
generally applicable to immobilized cells with an
emphasis on the asymptotic results. The various
particular solutions are cataloged in Table 4, but a
minimum of discussion is devoted to them. We will rely
extensively on the excellent two-volume treatise of Aris
[271] in this section and the interested reader is
referred to his book for a more detailed mathematical
treatment.
KAREL er ai.
5.3.1. Problem formulation and simplz@cation. The
complex metabolism of a living cell encompasses many
reactions with many substrates and products. In
assembling a framework for the mathematical rep-
resentation of the mass transfer problem we allow an
arbitrary number of reacting species S with an ar-
bitrary number of independent reactions N. Since the
distribution of cells and of cell activity within the
immobilized region may be extremely non-uniform,
we allow both the intrinsic reactivity R* (k = 1 . . . N)
and the effective diffusivity aj(j = 1 . . . S) to vary
with position r as welI as with the concentrations
C(r, t). ft is especially important to consider the effects
of non-uniform effective diffusivity in that a number of
observers have reported a decline in total reaction rate
with an increase in the number of cells, and have
attributed this effect to a decrease in effective diffus-
ivity with increasing cell number in the aggregate
[118, 135).
To solve the equations governing the transport
processes and reactions in the aggregate, a number of
simplifying assumptions are required. These will be
given below as {A I)-(AlO) in order of decreasing
generality, along with justification for each assumption
and an indication of where and how previous workers
have diverged from these assumptions.
(Al) The aggregate is isothermal. Unlike the situ-
ation with heterogeneous catalysis in the gas phase on
supported metal catalysts, temperature gradients in an
immobilized cell aggregate will generally be negligible.
The Prater temperature AT is the maximum possible
temperature difference between the interior and ex-
terior of a catalyst particle (or cell aggregate), hased on
a balance of the rates of substrate diffusion and heat
conduction [272]:
where AN is the heat of reaction, C? the bulk substrate
concentration, Z@is the effective substrate diffusivity in
the aggregate, K is the partition coefficient of the
substrate in the aggregate and k is the thermal
conductivity in the aggregate. Microbial processes that
are aerobic have large heats of reaction, but the
solubility of oxygen in water (Cb) is quite low, which
leads to very small values of AT in essentially all
situations. For anaerobic processes such as the fermen-
tation of glucose to ethanol, the AT’ values may be
higher. For the fermentation of glucose to ethanol and
carbon dioxide by yeast, AH is approximately
35 kcal/mol [273]. A calculation using the figures for
diffusivity and thermal conductivity from free solution
[274] shows that the maximum AT for the complete
conversion of a 1 M glucose solution is 0.13”G. Such a
small temperature rise will have little effect on the
reaction rate. A heat balance is therefore not required
for modelling the reaction behaviour at the level of the
aggregate. On the other hand, heat transfer from the
particles in the reactor to the environment is an
 Immobilization of whole cells 1341

Table 4. Theoretical analyses of reaction-diffusion with immobilized cells and enzymes: literature summary

Authors Year Kinetics+ CommentsS Ref.

Harremoes 1976 0 A, B, F, Z
Shieh et al. 1982 0 A, B, W, Z
Rodrigues et al. 1983 0 B,KZ 323
Goldman and Katchalski
Rovito and Kittrell
1971
1973
1
1
E.R.T,Y
E.T,X
E3
[;g;
Kim and Cooney I976 1 &WV
Gondo 1977 1 J%R,X
Rony 1971 0, 1 E,H,W [;:;I
Matson and Characklis 1975 0. 1 p, w Cl211
Webster and Shuler 1978-1981 0, 1 CE,H,T,X [286, 326, 3271
Atkinson er al. 1968 3 4WP.W [33, 269, 3281
Atkinson et al. 1968 3 .%B,F,V
Engasser and Horvath 1973-1976 3 C,E,X E:? 2821
Fink, Na and Schultz 1973 3 C.&X I3291
Atkinson ef al. 19741976 3 A,B,E,M,W [303, 330,331]
Hamilton er al. 1974 3 A,E,X [:;y, 3321
Maxham and Hickman 1974 3 A,CP,X
Waterland et al. 1974, 1975 3 E,H,F,V [93, 2831
Georgakis ef al. 1975 3 E,G.R,W E:::j 3341
Dahodwala et al. 1976 3 C,E,F,MX
Jennings er al. 1976 3 RXZ C3361
Williamson and McCarty 1976 3 B,W,A 1120, 2971
Rittmann and McCarty 1978-1982 3 AB,S,F,X.Z [140, 142, 210, 300, 3373
Buchholz et al. 1979-1982 3 C,E,F.X.B 5;;g;4. 3 161
Pedersen and Horvath 1981 3 E,Y,Z
Yamane er al. 1981 3 A,C,E,G.X E:~:j 3391
Andrews 1982 3 P,S,W,Z.A
Do er al. 1982 3 A,C,E,N,W [;;T, 34&342]
Ruckenstein and Kalthod 1982 3 E.M,W.C
Benelield and Molz 1983 3 P,M,Z,W
Rittmann and Dovantzis 1983 3 B,M,X [Zj
Moo-Young and Kobayashi 1972, 1973 3,4 C,E,G,P,F.W [ 127,296, 3443
Shuler et al. 1972, 1973 3,4 C,E,Y,C 1278, 2791
Lin 1977-1979 3,4 E,X,Z [345-347)
Atkinson and Rahman 1979, 1982 3.4 A,P.X,Z,D
Engasser and Horvath 1973-1976 4 C,E,Y E::? 3491
Bailey and Chow 1974 4 E, M,W,C c3@51
Ramachandran et al. 1975, 1976 4 E,M,R,X 5:::j35”l
Jin and Wang 1982 5 M,P,R,W
Leypoldt and Gough 1981, 1982 6 A,E,M.G,X
Verhoff and Goldstein 1982 7 D,E,M,W

+O,Zero-order kinetics, R = k, ; 1, first-order kinetics, R = k,C; 3, hyperbolic kinetics (e.g. Michaelis Menten),
R = k,C/(k, +C); 4, substrate and/or product inhibition, R = k,C/(k, +k,C +C’); 5, substrate and product inhibition,
R = k, exp (k2 ~ li,C); 6, “ping-pang” kinetics, R = k,ClC2/(k2CI + k3C2 +CIC2); 7, reversible enzymatic reaction, R
= k, (C, - k,C,)/(k, + kdC2 + C,). Where: R = intrinsic reaction rate; Ci = concentration; ki = rate constant.
*A, Algebraic approximation or asymptotic results; B, biofilms; C, effectiveness factor charts; D, decay of enzymatic activity;
E, immobilized enzymes; F, comparison to experimental data; G, generalized Thiele modulus; H, hollow fiber reactor;
M, multiple substrates or products; N, non-uniform cell distribution; P, mycelial pellets or floes; R, reaction network;
S, calculation of steady-state cell number; T, transient analysis; V, internal mass transfer plus laminar flow in outer region;
W, internal mass transfer only; X, external and internal mass transfer resistance; Y, external mass transfer only; Z, includes
overall reactor performance model; A, “ deep” film; B, “operational” effectiveness factor; C, includes electrostatic potential;
D. particle size distribution.

important consideration in reactors that are poorly
mixed (e.g. packed beds) [275].
(A2) Fick’s law governs the process of diffision in
the aggregate. In general, the driving force for dif-
fusion of a single component depends on the concen-
tration gradients of all the components in the mixture.
This is, however, only important when large absolute
gradients in concentration occur, which is rarely the
case with immobilized cells. We will assume that the
simplest form of Fick’s law governs the diffusive
transport (each component flux depends only on its
own concentration gradient). Other effects such as
convective transport within the aggregate [276, 2771
and the transport of ions in the presence of an
electrostatic potential [278-2811 are also neglected in
this treatment.
(A3) The aggregate can be represented as a homo-
geneous phase. The immobilized cell aggregate is
composed of a number of different materials which
may or may not be homogeneously dispersed.
Nevertheless, it is generally assumed that the aggregate
may be represented as a locally homogeneous phase
1342 STEVEN
characterized at any point by a single concentration of
each chemical species and that these concentrations
can be used in the kinetic expressions for the cell
activity. We will assume that transport across cell
membranes is incorporated in the intrinsic cellular
reaction rate expressions.
The assumption of a homogeneous aggregate is
required because of the difficulty of describing both the
local structure of, and the interactions of the com-
ponents within, the aggregate. These interactions may
modify some of the parameters in the rate law.
Unfortunately, the unambiguous determination of
such parameters from experiments with immobilized
cells is rarely possible because of the very mass transfer
limitations being modelled. The use of rate constants
determined for free cells in solution in the absence of
any mass transfer limitations is therefore advocated.
Using (Alk(A3), a set of non-linear partial differen-
tial equations can be derived from local mass balances
to describe the spatial and temporal variations of the
concentrations Cj(r, t) of the S species based on the N
independent reaction rates R,(C, r). Each rate is
expressed in terms of consumption of a given substrate
k:
ac.
I=V*G@jVC,- 2 q5*Rk, j=l,2 ,..., S.
at L=,
Here Sj(C, r) is the effective diffusivity of species j and
& is a stoichiometric coefficient defined as the number
of moles of species j consumed per mole of species k
consumed. According to this definition, qj = 1.
(A4) A partition coeficient and a mass transfer
coeficient define external transport to the particle. The
boundary conditions are given by flux matching and
the assumption of equilibrium between internal (Cf’)
and external (Cf’) concentrations at the surface is
represented by a partition coefficient (Kj):
C+ = K .C”’
I I J.
We assume that the external flux ofeach component to
the surface ( Jj) depends only on a single mass transfer
coefficient (k,)j which characterizes the hydrody-
namics in the bulk, and on the bulk concentration Cg.
Thus the flux per unit area evaluated at the surface in
the direction normal to the surface (n) is:
Jj = (k&(Cjb -Cf’) = - Sjo-VCj.
In some reactor geometries it is possible to avoid the
use of a mass transfer coefficient by solving both the
differential equations for convection-diffusion out-
side the aggregate and the reaction-diffusion problem
inside the aggregate and coupling the two solutions
directly. This works best in well-defined laminar flow
situations such as those found in certain fixed-film
reactors [282] and hollow-fiber reactors [283].
(A5) The aggregate is at steady-state. If the time-
dependent form of eq. (2) is to be solved, an initial
condition specifying the concentrations of all the
F. KAREL~~ al.
species everywhere in the aggregate is required. Full
solutions of eq. (2) are rarely needed in the context of
immobilized cell processes. The time dependence of
eq. (2) is, however, of primary importance in traveling
wave solutions of the diffusion equation. One appli-
cation of these solutions has been as models for
morphogenesis and differentiation in biological sys-
tems. These models are outside the scope of this review
but have been considered in depth elsewhere [271,284,
2851.
To neglect the time dependence, the characteristic
time for the establishment of a steady-state concen-
tration profile (L.‘/g) must be short compared to the
time scale over which conditions in the reactor change.
If so, one can set the left-hand side of eq. (2) equal to
zero. This assumption is valid in most practical cases,
so that it is easier to consider the exceptions case by
case.
One such exception is the experimental determi-
nation of effective diffusivity in immobilized cell
aggregates from the uptake or release of a tracer [ 118,
286). A second example of practical importance is the
discontinuous use of immobilized cell sensors [3S].
Equations similar to those that would apply for an
immobilized cell sensor have been examined in the
context of dissolved oxygen probes [287].
(2) A second exception arises when there are changes in
the intrinsic activity of the catalyst. The deactivation of
enzymes under conditions of mass transfer limitation
has been considered by a number of authors
[288-2911. The most important result of these investi-
gations is the suggestion that diffusional artifacts may
be responsible for the apparent longevity of im-
mobilized enzyme or cell preparations. With im-
mobilized cells, the possibility of growth under mass
transfer limitations leads to additional complications,
since the evolution in time of the catalyst distribution
itself is thus determined at least in part by the ability of
substrates to diffuse in the aggregate.
Oscillatory behaviour might also preclude the
(2a) steady-state assumption. As we mentioned previously,
sustained periodic behaviour has been observed for the
growth of cells on surfaces [198-2031. The intentional
periodic supply of nutrients has also been used with
immobilized cells [147, 2921. It is as yet unclear
whether mass transfer is important in these situations.
(A6) The efects of non-unijorm cell distribution
(2b) can be separated from species concentration eficts. A
cell is typically described as being a self-contained unit
whose reactive properties depend only on the local
environment. The spatial distribution of various en-
zymatic activities within the aggregate is then given
only by the distribution of active cells. The intrinsic
reaction rate fk (C) can therefore be separated from the
cell distribution X(r):
& (C r) = X (r)X (C). (3)
An exception to this assumption would be the case
of coimmobilized mixed cultures in which the spatial
distributions of the species are different. This excep-
 Immobilization of whole cells
tion is commonly observed in biofilms in waste
treatment processes [29, 1951. Differing spatial distri-
butions have been analysed theoretically oniy for the
case of simple competition of two motile species for a
single diffusing substrate [293 J_ Interactions among
populations are complicated by the heterogeneous
environment caused by immobilization, which for
example allows the coexistence of competitors with
differing surface affinities [294].
Similarly, one might assume that the diffusivity is
independent of substrate and product concentrations,
but not necessarily independent of the concentration
of cells. The influence of cell number X(r) is given in
this case by a spatial distribution of the effective
diffusivity of a reference component g1 (r). One might
also assume that the diffusivity of all the species varies
with cell number in the same manner, so that for each
species:
6, = gj(C, r)/s,(r) = af/g:. (4)
One possible instance in which this assumption might
fail arises from the fact that the diffusivity of a gas may
vary with cell concentration differently than the diffus-
ivity of an ion because of the hydrophobicity of the cell
membrane. The assumption may also fail if high
molecular weight compounds are involved (see Section
3.1).
(A7) The problem can be reduced to depend on a
single spatial variable.If only the overall reaction rate
of the system is of primary interest, it has been shown
that the solutions for essentially all geometries depend
primarily on a single length scale (L) which is given by
the ratio of aggregate volume (V,) to aggregate surface
area (A,) [271]. We can therefore express eq. (2) in
terms of a single spatial component of r normal to the
surface which we will refer to as z.
At this point we prefer to rewrite the simplified
version of eq. (2) incorporating assumptions (Al k(A7)
in terms of non-dimensional parameters. A parameter
p is used to allow a generalized coordinate system
(p = 0 Cartesian; p = 1 cylindrical; p = 2 spherical).
Physical properties are non-dimensionalized with re-
spect to those of the reference compound j = 1.
0 = djx-p& xPh(x)z -,$, ajk+th(l)
> term and integrating:
x a(x)g,(w(x)), j = 1, . . . , S
dwj -0
dx
(5a)
(5b)
x= 1: pj(l-Wj(1)) =
[
2
1_X=* .
Parameter definitions are given in the Nomenclature.
The two important dimensionless parameters which
govern the behaviour of the problem are the
Thiele modulus &, which is a ratio of rates of reaction
and internal diffusion, and the modified Biot number,
1343
/Ij, which is a ratio of rates of external and internal
mass transfer.
The full solution to the problem posed above is
given by the set of concentration profiles wj (x). For the
most part, one is not interested in these detailed
profiles. The most useful way to present results is in
terms of an effectiveness factor given by:
4k =
U v.
RI,(C(r), r) d v
I/[s “,
R, (C*,
1(64
r) d V
or, in our case:
1
‘Ik = b+ l) +‘aCQk (w(x)) dx
s
(6b)
As defined here, the effectiveness factor is the ratio of
the actual overall rate of a given reaction to the rate
which would prevail in the absence of mass transfer
limitations. With this definition, the effectiveness fac-
tors ‘I* approach unity when the Thiele moduli &k
become vanishingly small. Other authors have pre-
ferred to define effectiveness factors with respect to the
rate evaluated at the bulk concentrations, in which case
a partition coefficient appears in the asymptotic value
of qk for small & [278, 279, 29.51.
(A8) The problem can be reduced to depend only on
a single concentration. Many problems involving sev-
eral chemical species can be simplified. If the number of
diffusing species (S) is greater than the assumed
number of independent reactions (N), then the number
of differential equations to be solved can be reduced by
using appropriate combinations of the equations.
As an example, the applicable analysis for the
important case of two co-diffusing substrates or the
case of a single substrate and a single product of a
single reaction is shown below. For each species
j= 1,2:
0 = 6ixPP&[xPh(x) z)-or,d’a(x)h(l)g(w(x)).
(7)
Combining the two equations to eliminate the rate
(5)
x x-~$ xPh(x)$$
(8)
>
wt(l)-wl(x) = (S,a,/S,a2)(wI(1)-wWI(x)).
Equation (9) gives an algebraic relationship between
(9)
the two concentrations which is valid at all points
within the aggregate independent of the form of the
reaction rate expression. If the external mass transfer
resistance is negligible, w1(1) = w2( 1) = 1. This ex-
pression can be substituted back into the rate term
1344 STEVEN F.
g(w,(x), w2(x)) to obtain a new expression g’(w(x)).
This substitution drastically reduces the parameter
space which must be examined when using a numerical
solution technique, as has been shown most effectively
by Moo-Young and Kobayashi in their calculations
using Michaelis-Menten kinetics with product in-
hibition [296].
When more than one substrate is involved in the
reaction, (e.g. living cells), it is essential to determine
which substrate is exhausted first. This influences the
cell physiology and indirectly the physical properties
of the aggregate. This problem has been examined for a
number of immobilized enzyme and cell processes
[297-3021. The combination of equations given above
can be used to derive substrate exhaustion criteria in a
direct manner [303, 3043.
The use of theoretical results to predict the effects of
product inhibition is of special interest because it is
particularly difficult to diagnose product inhibition
effects directly from experiments [305]. A formidable
problem arises from product inhibition caused by the
generation of hydrogen ions. Here the buffering
capacity of the cells and of the medium must be
considered, as well as the highly non-linear dependence
of reaction rate on the pH. Theoretical treatments have
been advanced for certain simplifications of the prob-
lem [ 130,306,307].
(A9) There is only a single reaction taking
place. Although cells have a number of independent
enzymatic activities which operate simultaneously, we
will assume that only one reaction of importance needs
to be considered. The ability to model reaction net-
works in immobilized cells is likely to be important,
especially with the development of-systems with mul-
tiple populations which are coimmobilized, as for
example in methanogenesis [308]. There is very little
work which relates to this problem specifically with
immobilized cells, although various reaction networks
have been considered in the context of immobilized
enzymes [30!-3 161. This work demonstrates the feasi-
bility of solving problems numerically, but also points
out the difficulty of generalizing the results to other
systems. The effect of diffusion on the selectivity (ratio
of reaction rates of two reactions in a network) has
been considered extensively in the chemical engineer-
ing literature [271,317]. Most of the focus in the
biochemical engineering literature has been on the case
where the end-product of a series of consecutive
enzymatic reactions is desired [3 121.
(AlO) There is no spatial dependence of the diffus-
iviry or the intrinsic activity. As noted previously, the
growth or death of cells can lead to a spatial distri-
bution of the catalytic activity a(x). Using a step-
function rate law for growth, Lauffenburger and co-
workers [3 1S] have predicted steady-state distri-
butions of motile cell populations growing in the
presence of nutrient mass transfer limitations. Do and
Bailey [319] have considered the effects of non-
uniform activity distributions on Michaelis-Menten
KAREL~I al.
kinetics under various conditions, as well as deriving
the asymptotic expressions for q at large and small 4
for arbitrary kinetics. They found that the effects were
relatively small. The assumption of a uniform distri-
bution has been used to derive a lower bound for the
effectiveness factor for the non-uniform case [320].
The inability to measure an activity distribution which
prevails in most experimental conditions precludes the
use of refined models with an activity distribution. In
the most common case of a non-uniform activity
distribution, that of a thin active layer near the surface
of the aggregate, results are easily obtained by a
redefinition of the length scale (L) as the ratio of the
volume of the active layer to the external surface area.
This also applies to the common case of a biofilm on an
inert support particle [321].
Using all of the preceding assumptions (Alk(Al0)
the form of the equation obtained is that which is most
often considered: a single reaction at steady-state, with
uniform intrinsic activity and constant effective
diffusivity.
d
X -P_ = d2g'(w(x)) (10)
dX
BC 1: [dw/dx],,, = 0 (104
BC 2: [dw/dx], = 1 = /?(l -w:(l)) (lob)
BC 2’: for p $ 4, w(3) .= 1 (lk)
note also that ri = [dw/dx], = i /&2 (1Od)
42 = X,,f (c*)L2/c*Y (1Oe)
# = k,L/WK. (IOf)
If solutions of the reaction-diffusion eq. (10) are to
be used to make quantitative predictions of reaction
rate, especially in the vicinity of r#~= 1, then numerical
techniques are required for the solution of the equa-
tion with a particular kinetic expression g’(w). In
Table 4, a list of solutions which have been generated
to equations of the general form of eqs (5) and (10) in
the context of immobilized cells and enzymes is given,
classified by the kinetic expression used. Where general
solutions are given, these may take the form of a plot of
Thiele modulus vs effectiveness factor, or of algebraic
approximations to the exact solution, as noted in the
table.
The definition of the Thiele modulus 4 given in eq.
(lOe), when applied to MichaelissMenten kinetics,
gives:
C#P = V_L2/(Km +c*)Y. (11)
In addition to this definition [339], previous investi-
gators have used a number of different definitions for
the Thiele modulus when modeling Michaelis-Menten
kinetics, including the moduli which apply in the zero-
order limit [126,346] and the first-order limit [Zl, 241.
While technically acceptable, the utility is diminished
as the Thiele modulus no longer reflects the ratio of the
bulk reaction rate to the maximum rate of diffusion,

and 4 > 1 can no longer unequivocally serve as a test
of diffusional intrusions in the kinetics.
Situations where the rate of reaction increases with
extent of conversion (e.g. substrate inhibition) can
result in the existence of more than one steady-state
solution for a given value of the external concen-
tration. In such cases the effectiveness factor for the
substrate can exceed unity. Qualitative results dictat-
ing the existence or absence of multiple solutions can
be obtained by considering an algebraic analogue of
the differential equations describing reaction-diffusion
[352].
5.3.2. Asymptotic solutions and generalized moduli.
The Dirichlet problem applies when external mass
transfer resistance is negligible or fl% 4. In this case
asymptotic solutions for the effectiveness factor q are
easily obtained for arbitrary kinetics for either 4 % 1
or + G 1. For I#JB 1, the concentration profile is
homogeneous throughout the aggregate, so that q = 1
by defintion. For C#+ 1, the reaction occurs in a thin
layer near the outer surface so that one is justified in
using the equation in Cartesian coordinates [353]:
d2w/dxZ = d’g’(w).
Changing variables to u = dw/dx, and integrating:
W(X)
I/2
u2(x) = 242 2g’(w’) dw’
I V(X= 0)
1
and q=+-’ ’ 2g’(w’)dw’
[S w(I=o)
For very large 4, the substrate concentration dimin-
ishes rapidly within the aggregate so that w(x = 0) - 0.
Thus the asymptotic value of 9 is given by:
112
1
qG4-1 1 2g’(w’) dw’ . (124
U 0
Traditionally, a generalized Thiele modulus 4’
-I,2
[I1
defined so that q = l/4* when #* + 1, so that
+*=##
0
2g’(w’) dw’
1 .
Fig. 8. Effectiveness factor as a function of the generalized Thiele modulus c#Pfor Michaelis-Menten
(redrawn from [271]).
Immobilization of whole cells 1345
The results of using the generalized modulus defined
in (12b) are shown in Fig. 8 for Michaelis-Menten
kinetics. The use of this form of the Thiele modulus
accounts for almost all of the variation in the effective-
ness factor except in the vicinity of c$* = 1.
The major objection to this general approach is that
this modulus may be difEcuIt to calculate. This objec-
tion, however, applies equally well to any approach
requiring a knowledge of the intrinsic rate parameters.
One way to circumvent this problem may be the use of
an observable modulus as suggested by Weisz [354]:
Q, = ?$2 = ROb,L2/C*9. (13)
The parameter Q, contains only the observed reaction
rate without any assumption about the form of the rate
law. For 4 d 1, r~+ 1 and 0 4 1. For r$ $=-1, r,~-D l/#~
and @ + 1. This observable parameter can thus serve as
a test for diffusional limitations. The calculated ef-
fectiveness factor can be plotted against this modulus,
as shown in Fig. 9 [354]. On the other hand, for the
case of immobilized cells, the evaluation of this
modulus should not be regarded as an infallible test.
For example, reductions in reaction rate as a result of
product inhibition coupled with diffusion cannot be
directly assessed in this manner.
The solution to the Dirichlet problem suggests that
the overall rate increases proportionally to r# as
4 + a\. For finite values of the Biot number p, the
boundary condition given in (lob) indicates that this
. cannot be the case, and that for 1 4 /3 4 4 the appro-
priate expression is:
rl = b/92 = kLC*/X,,, / (C*) LK = l/Da (14a)
where Da is a Damkohler number. As before, an
external observable modulus can be defined:
R = qDa = R,b,L/kLCb. (14b)
is
No explicit assumptions about the rate law are required
to evaluate the parameter Ll, which indicates external
UW
diffusion control as R -+ 1 and kinetic control for
R6 1.
kinetics
I346 STEVEN F. KAREL et al.
1.0 _ , 1 , , , ,,,I

ZERO ORDER -

FIRST ORDER
0.1 _=

Fig. 9. Calculated effectiveness factor as a function of the observable modulus Q, (redrawn from [3543).

The mass transfer coefficient kL depends on the depth [269], the cell concentration [ 136,305,355] and
particular reactor configuration. Expressions for the the enzymatic activity ofthe individual cells [290,356].
estimation of k, are available for suspended particles, Modifying the hydrodynamics by manipulating the
for particles in a packed bed, and for flow over surfaces rate of agitation in a vessel or the superficial velocity in
[189, 3241. It is difficult to estimate k, accurately in a fixed bed allows for a test of external mass transfer
many situations, most notably in the case of im- resistance [22,325]. In these direct methods, care must
mobilized cell particles that are almost neutrally be taken that the mechanical properties of the ag-
buoyant. For suspended particles kL is estimated using gregate are not altered.
a correlation for the Sherwood number Sh = k,d,/B * There are also indirect methods to infer the presence
where d, is the aggregate diameter and a* is the of mass transfer resistance. Variation of the reaction
diffusivity in the bulk solution. For a sphere suspended temperature allows the calculation of an apparent
in a stagnant fluid, Sh = 2, while for spheres moving in activation energy. Under mass transfer control, the
a fluid the values of Sh increase somewhat, depending apparent activation energy is depressed [23, 26, 2911.
on the buoyancy [189]. The so-called unstirred or The variation of substrate concentration to determine
“Nernst” layer thickness is a fictitious distance that an apparent rate law may also be an indicator.
reflects the value of (d,/Sh), and thus cannot be Observation of an increased apparent K, is often
measured directly. associated with mass transfer resistance. Although the
Since in general Sh > 1, the Biot number Lineweaver-Burk plot commonly used to determine
p = k,L./zZK is likely to be less than unity only when the parameters for Michaelis-Menten kinetics is non-
reaction is localized near a surface (d, g L). External linear when mass transfer resistances are present, the
mass transfer control in the absence of internal normal scatter in experimental data may obscure this
limitations is therefore expected to be observed pri- effect [26,336].
marily with surface attached cells. Nevertheless, the If information can be obtained about local substrate
presence of external diffusion resistance is an import- concentration or cell distribution within the aggregate,
ant limiting factor at very high reaction rates. these may also serve as a test of the presence of mass
transfer. Microscopic techniques are useful for this
5.4. Experimental detection of mass-transfer purpose, as discussed in Section 4.1. Microprobes have
limitations been used to measure local oxygen concentrations
The detailed experimental verification of a reaction- within immobilized cell aggregates [134, 3571.
diffusion model of immobilized cell metabolism is a As a result of the difficulty of characterizing im-
difficult proposition, owing to experimental uncer- mobilized cell aggregates, a detailed experimental
tainties in assessing the activity of the cells, the effective verification of the theory of combined reaction and
diffusivity and the distribution of cells in the aggregate. diffusion has not been possible to date. The available
Those studies in which some comparison between evidence does, however, suggest that understanding
theory and experiment was attempted are noted in the effects of mass transfer is of the utmost importance
Table 4. in the use of immobilized cells.
A simple and useful technique which can be used is
the variation of experimental conditions over a wide 6. REACTOR CONFIGURATIONS FOR IMMOBILIZED

range of at least one of the parameters making up the CELLS

Thiele modulus. This serves as a test for the presence of The choice of a reactor design for an immobilized
mass transfer limitations. Experimentally accessible cell process is dictated by the nature of the mass
parameters which have been used for this purpose transfer requirements. Such concerns are the supply
include the particle size [125, 127, 136, 3053 or film and removal of gases, the supply and removal of
 Immobilization of whole cells
solutes in the liquid phase and possibly the removal of
solids (cells) from the reactor. To meet these varied
requirements, a number of different reactor configur-
ations have been considered for use with immobilized
cells. These form three categories, depending on the
location of the cell aggregates:
(1) particles or large surfaces which are fixed in space;
(2) large moving surfaces and
(3) particles suspended and mixed in solution.
Reactors can be further subdivided into two classes
based on the gas transport requirements, and therefore
whether they contain two (liquid-solid) or three
(gas-liquid-solid) phases in the reactor. The design of
two-phase reactors is considerably simpler than the
design of three-phase reactors. Two-phase reactors are
therefore preferable if bubble formation can be
avoided. Even in fermentations in which all the
nutrients are liquids, gaseous end-products (CO1, N,,
CH,) must be removed if large amounts are formed,
thus necessitating the design of a three-phase reactor.
Three-phase reactors are generally required when
gaseous nutrients must be supplied, although in a
limited number of applications all the reactants and
products can be transferred directly between solid
(cell) and gas phases, so that two-phase gas-solid
reactors can be used [358].
6.1. Stationary particle or surface reactors
The most common reactor configuration used with
immobilized cells is that of a packed bed of particles.
Packed beds of immobilized cell aggregates have been
used at the industrial scale [I3 3. The advantages of
packed beds include simplicity of operation and
reasonably high mass transfer rates. Problems in the
operation of packed beds include obstruction by
uncontrolled cell growth [109], compression of the
particles leading to excessive pressure drops [22, 251
and inadvertent formation of gas bubbles [265, 2661.
For these reasons, simple packed beds are applicable
primarily when nonviable cells are used.
There are a number of similar systems in which the
cells are fixed at a surface while the liquid flows past.
These include hollow-fiber [96,97] or spiral-wound
[359] membrane cartridges, the “Biological Film
Reactor” [282] and gels coated onto a fixed wire
support [266]. Cell growth and product removal is
more easily controlled in such systems than in packed
beds.
In the three-phase versions of these reactors, the
flow of liquid and gas alternates over any point on the
support, so that gas transport occurs across a thin film
wetting the surface. This reactor type is typified by the
trickle bed [65, 3601, which is the three-phasecorollary
of the packed bed. Spiral wound membranes [85] and
porous ceramic monoliths [74] with immobilized cells
have also been operated in this fashion.
The use of membrane reactors with two types of
membrane, one liquid-permeable and one gas-
permeable, allows transport across two distinct sur-
faces, both in close proximity to the cells [99, 1581. The
1347
use of gas-permeable tubing as a support has also been
applied to animal cell culture, where the cells adhere to
the interior surface while liquid passes through the
inside [361].
6.2. Moving surface reactors
In this reactor category, mass transport is enhanced
by the movement of large surfaces within the reactor.
Rotating biological contactors (RBCs) contain a
number of rotating discs on a shaft in a vessel partially
or completely filled with liquid. Biofilms grow on the
surfaces of the discs. RBCs have been operated in a
completely submerged mode for the denitrilication of
wastewater [362,363].
In the three-phase case, the surface is alternately
exposed to gas and liquid, and as before gas transport
occurs across a thin liquid film. In addition to partially
submerged RBCs [30,364], roller bottles and other
more complex devices used for mammalian cell culture
[69] fall into this category. These systems are well-
defined and easily controlled, but it is difficult to
maintain a reasonable surface area to volume ratio in
scale-up.
6.3. Mixed particle reactors
Reactors in which liquid and solid phases are mixed
include fluidized beds and stirred tanks. Two-phase
reactors are generally limited to anaerobic operation.
In microcarrier culture of animal cells, however,
sufficient oxygen supply has been achieved by diffusion
through silicone tubing [365]. Nevertheless, oxygen
transfer usually requires the intimate mixing of all
three phases.
Three-phase mixed particle reactors are quite widely
used. A well-established example of such systems is the
use of tower fermentors with flocculant organisms
[349, 3661. A variety of fluidized, semi-fluidized or
expanded bed reactors in which the particles are
suspended and mixed by the upflow of gas and liquid
has been envisaged [25, 3663691. The flow pattern,
and consequently the liquid and gas hold-up which can
be achieved, depends on the particle density and flow
rates [78, 3691. Particle densities can change with
growth of the biofilm, thus complicating the analysis
[370,196]. The design of two-phase and three-phase
fiuidized bed bioreactors is an area of intense interest,
but as yet there has been little agreement on the correct
approach to the design of these systems [141,321,366,
369-3731.
7. SUMMARY
Immobilized cell systems can be prepared using a
variety of adsorption, entrapment, containment or
aggregation techniques. The methodology is well
developed and has been applied to microorganisms,
cultured animal and plant cells, and subcellular or-
ganelles for a number of applications [9]. Early studies
have focused primarily on the direct measurement of
the catalytic activity and stability of the immobilized
cell system. These measurements, combined with the
prudent application of the theory of reaction and
1348 STEVENF. KAREL et al.

diffusion in porous catalysts, may be adequate for the 4 aggregate diameter, L

design of systems using non-living cells as enzymatic Da Damkohler number, X,,,f(C*)L./k,C*
catalysts. This information is in general not sufficient T2,(C. r) effective diffusivity of species j in aggregate,
for analysis and design in the emerging technology L2T-’
using immobilized living cells. diffusivity of species j in free solution
In this review we call attention to certain aspects of diffusivity of reference species (j = 1) in free
the behaviour of immobilized cell systems which have solution
an indirect but important influence on the overall X(C) rate expression for reaction k, (mol k) T-l
reactivity. An understanding of the fundamental (cell mass) - ’
properties underlying the behaviour of immobilized c?*(w) =f*(C)/&(C*), dimensionless rate expres-
cells is critical to both the improvement of established sion for reaction k
techniques and the development of novel ones. These g’ (w) pseudo one substrate rate expression (dimen-
properties include: sionless)
h(x) = 9i (.z)/_%?:, dimensionless effective diffus-
dell physiology. The environment and growth rates
ivity distribution
characteristic of immobilized cell processes are quite
different from those in conventional processes, and JI flux of species j to the surface, (mol j)
L-sT-’
the behaviour of the cells under these conditions
k thermal conductivity, cal/“C cm s
must be elucidated.
(kL)i mass transfer coefficient for species j to ag-
-cell mobility. The degree to which living cells can
gregate surface, L T - ’
move within and escape from the support is an
K, partition coefficient for species j
important determining factor of the dynamic be-
K, Michaelis constant, mol L- 3
haviour of immobilized cell catalysts.
L characteristic length scale, L
-interactions among the cells, support and
surface normal vector
solution. Physical properties of an immobilized cell
fG number of independent reactions
aggregate depend not only on the support material
P = (0, 1,2), symmetry parameter
but also on the cell loading, and as such complicates
4 characteristic parameter in effective diffus-
any attempts to estimate a priori physical properties
ivity estimation
such as the effective diffusivity of solutes in the
position vector, L
aggregate.
k,CC, r) rate of reaction (consumption) of species k,
With an improved understanding of these physical (molk)L-3T-1 {k=l,...,N}
and biochemical properties, fundamental engineering R ohs overall observed rate, mol L- 3 T-i
techniques and formalisms developed in the chemical S number of chemical species
engineering literature for describing combined reac- Sh (k,),d,@T, Sherwood number
tion and mass transfer could be applied to analyse the t time, T
overall performance of immobilized living-cell sys- f4 dwldx
tems. The ability to predict the behaviour of these “X volume fraction of cells in aggregate
systems is required to facilitate the design and optimiz- “P
volume fraction of polymer in aggregate
ation of immobilized cell reactors and to make them a K aggregate volume, L3
viable alternative to conventional microbial processes. V-
rate constant in Michaelis-Menten equation,
mol L-’ T-i
wJ (x) = C, (z)/CT, dimensionless concentration of
NOTATION species j
Note: subscripts j and k, which refer to diffusing = z/L, dimensionless distance
and reacting components respectively, are omitted in %r, distribution of active cells, (cell mass) Lm3
cases where only one species or one reaction is X BYC catalytic activity per unit volume, (cell mass)
involved. L-3T-1

yield coefficient, (mol j) (mol k)-’

44 = x wx,,,9 dimensionless activity distri-
component of r normal to the surface of the
bution
aggregate surface area, L2 particle or film, L
A
Bi = (kL)i L/g:. characteristic Biot number
C, tr, t) concentration of species j, (mol j) L- 3 Greek letters
{j = 1,. . . ) S} = qkC$/Cy, stoichiometric coefficient for
C(r, t) concentration vector, {C,, . . . , C,} species j, reaction k
Cp concentration of species j in the bulk = Bi/SjKjh(l), modified Biot number for
C?’ concentration of species j in the fluid layer species j
J
adjacent to the aggregate surface = 9i*/9:
C+
J concentration of species j at the aggregate effectiveness factor [see eq. (6)]
surface = K,/C*
CT = KjCp, concentration of species j in the (X,,,X (C’) L.‘/h(l) 3: C:) (ek = Thiele
aggregate at equilibrium modulus for reaction k)
 Immobilization of whole cells 1349

+* = (X,,,f(C*)L*/S@c*) (4 = Thiele modu- E311 Switzenbaum M. S., Enzyme Micrab. Technof. I983 J
lus, one component problem) 242.

cb* generalized Thiele modulus [see eq. (12b) J c321 Henze M. and Harremoes P., Water Sci. Technol. 1983
15 1.
4 = vrsp’. observable modulus for internal
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1982 28 1.
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