Van Hee 2006

Strategy for Selection of Methods for
Separation of Bioparticles From Particle

Mixtures
P. van Hee, M.A. Hoeben, R.G.J.M. van der Lans, L.A.M. van der Wielen
Departmentof Biotechnology, Delft University of Technology Julianalaan 67, 2628 BC
Delft, The Netherlands; telephone: 0031-15-2792361; fax: 0031-15-2782355;
e-mail: L.A.M.vdWielen@tnw.tudelft.nl
Received 2 March 2005; accepted 10 January 2006
DOI: 10.1002/bit.20885
Abstract: The desired product of bioprocesses is often
produced in particulate form, either as an inclusion
body (IB) or as a crystal. Particle harvesting is then a
crucial and attractive form of product recovery. Because
the liquid phase often contains other bioparticles, such as
cell debris, whole cells, particulate biocatalysts or particulate by-products, the recovery of product particles is
a complex process. In most cases, the particulate product
is purified using selective solubilization or extraction.
However, if selective particle recovery is possible, the
already high purity of the particles makes this downstream process more favorable. This work gives an
overview of typical bioparticle mixtures that are encountered in industrial biotechnology and the various driving
forces that may be used for particleparticle separation,
such as the centrifugal force, the magnetic force, the
electric force, and forces related to interfaces. By coupling
these driving forces to the resisting forces, the limitations
of using these driving forces with respect to particle size
are calculated. It shows that centrifugation is not a general
solution for particleparticle separation in biotechnology
because the particle sizes of product and contaminating
particles are often very small, thus, causing their settling
velocities to be too low for efficient separation by
centrifugation. Examples of such separation problems
are the recovery of IBs or virus-like particles (VLPs) from
(microbial) cell debris. In these cases, separation processes that use electrical forces or fluidfluid interfaces
show to have a large potential for particleparticle
separation. These methods are not yet commonly applied
for large-scale particleparticle separation in biotechnology and more research is required on the separation
techniques and on particle characterization to facilitate
successful application of these methods in industry.
2006 Wiley Periodicals, Inc.
Keywords: particle-particle separation; bioparticle; inclusion body; cell debris; recovery
INTRODUCTION
There is a large and growing market for compounds that
are produced with biotechnological processes. Some of
these compounds obtain a particulate form during their
production. Examples of bulk compounds are b-carotene
with a production of &500 metric tons per year (Chemical
Correspondence to: L.A.M. van der Wielen
2006 Wiley Periodicals, Inc.
Week, April 19, 1995) and a turnover of &$320 million per

year (www.chemicalmarketreporter.com), vitamin E with a
production of &65,000 metric tons per year and a turnover of
approximately $2,000 million per year (Chemical Week,
March 21, 2001 and May 21, 1997), and L-tryptophan with a
production of &750 metric tons per year and a turnover of
approximately $38 million per year (Chemical Week,
November 5, 1997). Alternative processes for the production
of these compounds may avoid the formation of a particulate
product, but are not necessarily more favorable. Dealing with
particulate products thus is an important issue in biotechnology.
Bioparticles can be formed by aggregation of molecules or
by active accumulation of these molecules in specific
compartments inside cells. Active accumulation involves
active transport of components to specific locations inside the
cells where they may aggregate or where they are included in
polymers. The latter process does not necessarily yield solid
particles, whereas aggregation does. Aggregation requires
supersaturation of the liquid phase. A particulate product will
therefore only be formed by aggregation when the saturation
concentration is exceeded.
The saturation concentration of a compound is related to
its interactions with all of the surrounding molecules. If these
interactions are energetically favorable, the compound has a
high saturation concentration and vice versa. Figure 1 depicts
the octanolwater partitioning coefficient (Kow), which is a
measure for polarity, against the molar mass of various
organic compounds. The shaded areas indicate the solubility
of these compounds in water. The graph shows that a decrease
in solubility corresponds to an increase in molar mass and
Kow. Thus, small polar molecules like many amino acids
remain dissolved in water up to concentrations of 100 g/L or
higher, but with somewhat larger and less polar molecules,
the solubility is already exceeded at lower concentrations.
Most products in biotechnological processes consist of large
complex molecules with high molar mass. These products are
likely to be produced above their saturation concentration
and will thus form particles during the production process.
Moreover, it has been shown in many enzymatic conversion
processes that precipitation or crystallization of the product(s) has a favorable effect on the conversion rate (Ulijn
Log Kow
0-5 g/l
17
13
10
18
22
16
11
12
19
5-50 g/l
21
20
-1
-2
15
1
9
3
-3
14
50-250 g/l
5
7
-4
0
100
200
300
400
Mm (g/mol)
Figure 1. The relation between solubility, polarity, and molar mass for
various organic compounds. The shaded areas mark the solubility range of
the compounds. Kow is the octanolwater partitioning coefficient, which is a
measure for the polarity of the compounds. Compounds (Weast et al., 1922;
http://www.mdbio.com.tw/AM/am-trimethoprim.htm; http://chemfinder.cambridgesoft.com/result. asp; Sangster, 1997): (1) ampicillin, (2) trimethoprim, (3) alanine, (4) glycine, (5) methionine, (6) phenylalanine, (7) serine,
(8) tryptophan, (9) DL-valine, (10) codein, (11) phenylbutazone, (12)
quinidine, (13) phenytoin, (14) cimetidine, (15) chlorothiazide, (16)
theophylline, (17) ethacrynic acid, (18) furosemide, (19) phenobarbitol,
(20) caffein, (21) diazepam, (22) meprobamate. [Color figure can be seen in
the online version of this article, available at www.interscience.wiley.com.]
et al., 2003), thus leading to the design of processes yielding

particulate products.
A particulate product can be easily purified with particle
liquid separation techniques, such as centrifugation or filtration, if it is the only particle-phase present in the reaction
liquor. Generally, however, other particles are present
that make solidliquid separation useless. In literature,
many biotechnological processes are described that produce
a particulate product in the presence of other particles.
Examples of such processes are: (1) the production of intracellular particulate microbial products, such as inclusion
bodies (IBs), crystals, cell organelles, viruses, and virus-like
particles (VLPs) by microorganisms, and (2) enzymatic
conversions and biotransformations using (particulate)
biocatalysts that yield a particulate product and/or particulate
by-product. In these processes, the particulate bioproducts
need to be recovered from reaction liquors that contain other
particles. Recovery can be realized using selective solubilization or extraction, but since the particles contain the
product generally at a high purity, selective particle recovery
by particleparticle separation may be more favorable.
In particleparticle separation, particles are physically
separated from one another without affecting their (internal)
chemical composition or their morphology. A method that is
commonly used for particleparticle separation in biotechnology is centrifugation. This technique makes use of settling
velocity differences that result from size, shape, and/or
density differences of the particles. It has certain limitations
and cannot be used for all particleparticle separation
problems that are encountered in the biotech industry. When
centrifugation fails, other particleparticle separation methods using other driving forces, such as magnetic separation,
electrophoresis, and air flotation, should be considered.
This study gives an overview of bioparticle mixtures
that are encountered in biotechnological processes (Sections
Overview of Particle Mixtures in Biotechnological Processes and Properties of Typical Bioparticles and Their
Suspensions). Forces are evaluated that have potential for
physical separation of these mixtures. On the basis of this
evaluation, a strategy for particleparticle separation in
biotechnology is presented using the proposed driving forces.
Application of these forces may be limited because of
specific requirements for the process design. These possible
limitations are taken into consideration in Section Selection
of a ParticleParticle Separation Technique, but first the
potential of the forces for particleparticle separation is
determined by direct comparison (Section Forces in
ParticleParticle Separation). The work is restricted to
the application of single driving forces for separation.
OVERVIEW OF PARTICLE MIXTURES IN

BIOTECHNOLOGICAL PROCESSES
In this section, an overview is given of reported processes in
which bioparticles are formed in the presence of other particles.
These bioparticles may be intracellular particulate microbial
products, extracellular particulate microbial products, VLPs, or
solid products in biocatalysis. In Tables I and II, references are
Table I. References on biotechnological processes that yield particle mixtures.

Category
References
Intracellular particulate products produced

with microorganisms
Extracellular particulate products produced
with microorganisms
VLPs produced produced with microorganisms

Solid products produced with immobilized
catalysts
Production of solid products and solid by-products
with enzymes
Table II
(Beards et al., 1993; Buque-Taboada et al., 2004; Kometani et al.,
1997; Kumagai, 1999; Leuenberger, 1985; Matsumae et al., 1995;
Michielsen et al., 2000a,b; Miller, 1985; Nakayama, 1985; Spassov
et al., 1996; Takahashi, 2003)
(Andrews et al., 1995; Cruz et al., 2000; Kitano et al., 1987; Kuiper et al.,
2002; Meijer et al., 1996; Moran, 1999; Tsoka et al., 2000)
(Takahashi, 2003; Davison et al., 1997; Kasche and Galunsky, 1995;
Kuhl et al., 1990; Lee et al., 1999a; Zmijewski et al., 1991)
(Blacker and Holt, 1997; Youshko et al., 2002)
Biotechnology and Bioengineering, Vol. xx, No. x, month xx, xxxx

DOI 10.1002/bit
Van Hee et al.: Separation of Bioparticles from Particle Mixtures
Biotechnology and Bioengineering. DOI 10.1002/bit
0.0481
Polyphosphate granules
0.040.1
0.090.5
115
Magnetosomes
Carboxysomes
Other crystals
References
Hypothesis: sulfur storage
(Lins and Farina, 1999; Shively, 2003)
(Bowden et al., 1991; Fischer et al., 1992;

Fischer et al., 1995; Gram et al., 1994;
Hellebust et al., 1989; Hoess et al.,
1988; Honda et al., 2000; Koller et al., 1995;
LaVallie et al., 1993; Mitraki and
King, 1989; Rinas et al., 1992; Valax
and Georgiou, 1993; WangsaWirawan et al., 2001b; Wetzel et al., 1991;
Wong et al., 1997a)
Phosphate storage and regulation, (Bode et al., 1993; Lins and Farina, 1999;
energy storage, accumulation
Shively, 2003)
of unwanted (toxic) metals or
metals used in the metabolism
Energy storage
(Jane et al., 1994)
Nitrogen storage
(Oppermann-Sanio and Steinbuchel,
2002; Shively, 2003)
Hypothesis: energy/carbon storage (Shively, 2003)
Energy/carbon reserve
(Shively, 2003)
Aggregation, incorrect folding,

lacking the ability to excrete
Function
Helps in search for nutrients due to (Dunin-Borkowski et al., 1998; Lins and Farina,
magnetism
1999; Proksch et al., 1995)
(Shively, 2003)
Bluegreen algae, many nitrifying Hypothesis: storage of
bacteria and thiobacilli
ribulose-1,5-diphosphate
carboxylase
Various m.o.s
Due to increased production
(CN1294191; Eonseon et al., 2003;
Jeaong et al., 1999)
Thiorhodaceae and other

apochlorotic sulfur bacteria
Magnetotactic bacteria
*, indicates that the inclusion body is surrounded by a membrane and indicates that there is no membrane.
, variable shape
, contains Fe3O4 or iron sulphides
0.11
Plant cells
Cyanobacteria, recombinant
E. coli
Many prokaryotes
Bacteria, algae, etc.
, variable shape
, variable shape
/ variable shape
, spherical
Many m.o.s
, amorphous, spherical
0.020.3
0.10.8
Many (mainly recombinant)

microorganisms
Organism
, variable shape
Characteristics*
Glycogen granules
Polyhydroxyalkanoate
granules
Sulfur globules
1100
?>0.5
0.052.25
Proteins
Starch granules
Cyanophycin
Size (mm)
Literature overview of intracellular particulate microbial products.
Type of inclusion body
Table II.
listed of the various categories of particle mixtures that are

described in this section.
Intracellular Particulate Microbial Products
Non-excreted microbial products accumulate inside the
microorganism and often cause the formation of crystals or
IBs that have size in the range of 0.051.2 mm. Crystals are
formed when the product is produced above its saturation
concentration, such as in the case of b-carotene production by
Blakeslea trispora (Jeaong et al., 1999) and the production of
xanthophylls by microorganisms or microalgae (Eonseon
et al., 2003). IBs are formed when the product is accumulated
in a specific compartment inside the cells, such as with
polyhydroxyalkanoates (Lee et al., 1999b), or when it
aggregates inside the microorganism, which often happens
with peptides and proteins that are folded incorrectly due to
chaperones that are lacking in the organism (Buchner et al.,
1992; Honda et al., 2000; Wong et al., 1997a). Besides
protein IBs, crystals, and PHA granules, many other forms of
IBs can be found in nature, such as phosphate granules, starch
granules, sulfur globules, carboxysomes, magnetosomes,
glycogen inclusions, and cyanophycin inclusions. In Table II,
an overview is given of the characteristics of these IBs. It is
important to note that not all of these IBs are (yet) of
economical interest, but the overview gives a good
impression of what microorganisms are capable of.
The structure and characteristics of IBs may depend on the
cell compartment where the compound is accumulated. For
instance, b-lactamase IBs in E. coli were found to be
amorphous when produced in the periplasmic space and
highly regular-shaped when produced in the cytoplasm
(Bowden et al., 1991). It is difficult to predict the IB
properties and its location inside the cell just by looking at its
chemical composition. In addition, the surface properties of
the IBs may be influenced by intracellular dissolved
compounds that adsorb onto the IBs. This phenomenon
makes prediction of the IB properties even more difficult.
The separation of particulate intracellular microbial
products from biomass is generally performed by product
release through homogenization in combination with centrifugation. The IB enriched fraction is subsequently
dissolved and extracted with chemicals and, if required, the
product is refolded in a proper refolding buffer (Fischer et al.,
1992; Gram et al., 1994; Hellebust et al., 1989; Honda et al.,
2000). Since most of the IBs contain the product at high
purity, dissolution and extraction does not seem to be the
most efficient separation operation and direct recovery by
particleparticle separation may increase the separation
efficiency and reduce the number of process steps.
Extracellular Particulate Microbial Products
A reasonable number of cases have been reported where an
excreted microbial product forms crystals (5100 mm) due to
supersaturation of the liquid phase (Beards et al., 1993;
Buque-Taboada et al., 2004; Michielsen et al., 2000a,b;
Spassov et al., 1996). For instance, both the substrate and

product are crystals in the conversion of Ca-maleate to Ca-Dmalate by permeabilized Pseudomonas pseudoalcaligenes
(Michielsen et al., 2000a,b). These processes yield mixtures
of microbial cells and particulate product(s), again making particleparticle separation a key step in downstream
processing.
Currently, many of these products are separated from other
particles by dissolution in a second (organic) liquid phase
followed by crystallization (Buque-Taboada et al., 2004).
Particleparticle separation is only considered in those cases
where the density and/or size differences between the
product particles and the other particles are large enough
for separation by centrifugation.
Virus-Like Particles (VLPs)
Viruses and VLPs are particles with sizes between 20 and
200 nm that are produced intracellularly. The particles often
consist of an inactivated virus or a surface antigen of a virus
that is produced by a genetically modified microorganism.
For recovery of these intracellular VLPs, the cells have to be
disrupted, thus creating a mixture of cell debris and VLPs.
When the virus is still active, however, the cells may be lysed
spontaneously. Again it is clear that the production of these
bioparticles gives rise to a particleparticle separation
problem. Purification of VLPs is currently performed with
a wide variety of separation techniques, such as centrifugation (Cruz et al., 2000; Tsoka et al., 2000), filtration (Cruz
et al., 2000; Kuiper et al., 2002; Tsoka et al., 2000), extraction
without dissolution (Andrews et al., 1995; Kitano et al.,
1987), and chromatography (Cruz et al., 2000; Kuiper et al.,
2002; Tsoka et al., 2000) that are used in a large train of
separation operations. Direct VLP isolation by particle
particle separation would reduce the number of process steps
and might thus lower the downstream processing costs.
Particles in Enzyme Catalysis and
Biotransformation
Solid-to-solid conversion in enzymatic synthesis is thermodynamically possible if the apparent equilibrium constant is
larger than the solubility ratio of the product(s) and
substrate(s) (Diender et al., 1998). Besides the advantages
of conventional enzymatic synthesis, such as high regio- and
stereoselectivity, absence of racemization and reduced need
for side-chain protection, solid-to-solid conversion has
additional advantages. The reaction yields may increase
by product precipitation in water (Ulijn et al., 2003), which
eliminates the use of organic solvents to shift the thermodynamic equilibrium toward synthesis. Using water instead
of solvents is of course favorable for environmental reasons,
but more importantly, it may also avoid inactivation of the
biocatalyst by the solvent (Eichhorn et al., 1997).
Enzymatic reactions yielding solid products that have a
typical size of 5100 mm require particleparticle separation
in subsequent process steps when there are other particles

DOI 10.1002/bit
present. These other particles may be particulate biocatalysts, particulate by-products or crystals of unconverted
enantiomer in case of conversion of a mixture of racemic
crystals into chiral crystals (Straathof et al., 1998). When an
immobilized catalyst is the only other particle present in the
mixture, separation may be very easy by careful selection of
the catalyst carrier material with respect to size, density, and
structure, for example catalytic membranes (Kasche and
Galunsky, 1995) and catalytic expanded bed media (Van der
Wielen et al., 1990, 1996, 1997). With other particle mixtures
in enzymatic conversion processes particleparticle separation may be a key step in the purification process. Currently,
product purification is performed in most cases with
(selective) extraction and (selective) crystallization of the
product (Kometani et al., 1997; Kuhl et al., 1990; Matsumae
et al., 1995; Miller, 1985; Yan et al., 1999).
PROPERTIES OF TYPICAL BIOPARTICLES AND
THEIR SUSPENSIONS
In order to determine which separation process is suitable for
the recovery of a particulate bioproduct from a particle
mixture, the particle properties as well as the liquid phase
properties should be known. In this section, properties of
bioparticles and their suspensions are reviewed.
Particle Properties
Morphology, Shape, and Composition
Bioparticles have a large variation in shape, composition, and
morphology. Most extracellular particulate microbial products
and bioparticles that are formed in biocatalysis are produced as
highly pure crystals with varying shape. IBs, on the other hand,
may be crystalline or amorphous even when they are composed
of the same material but produced in different compartments of
a microbial cell (Bowden et al., 1991). In addition, IBs
composed of the same material may have a surrounding
membrane in one organism, while in another organism it lacks
this membrane (Shively, 2003). It is, therefore, impossible to
give a general impression of the morphology, shape, and
composition of intracellular particulate microbial products.
Particle Size Distribution
The particle size distribution is a key parameter for almost all
particleparticle separation processes and it will be treated in
more detail in Sections Forces in ParticleParticle Separation and Optimization and Control of Driving Forces.
The overview of bioparticle properties presented in Table III
shows that the particle size ranges from 0.05 to 100 mm and
that there is a large probability for the particle size
distributions of two types of bioparticles to overlap.
Particle Charge
Bioparticles have a surface charge density that is dependent
on their surface chemistry and the composition of the fluid
phase. In many cases, the surfaces of bioparticles contain

carbonyl, amino, and hydroxide groups. All of these groups
can exchange OH or H with the surrounding medium
causing a change in surface charge. When a charged particle
is in solution, it attracts ions with opposite charge and repels
ions with like charge. This phenomenon, in combination with
the thermal motion of the ions surrounding the particle,
causes the formation of an electrical double layer around
the particle. The thickness of this electrical double layer
is dependent on the ionic concentration of the liquid,
which is typically in the range of 0.01 mol/L to 1 mol/L in
biological systems, the valency of the ions and their thermal
motion.
A measure for the electrokinetic behavior of a particle is
given by its electrokinetic potential or zeta-potential, which
is the potential at the surface of shear between the charged
surface and the electrolyte solution. The relation between pH,
ionic strength, and the zeta-potential of bioparticles, such as
microbial cells and IBs, has been studied to a reasonable
extent (Egorova, 1994; Van der Wal et al., 1997; WangsaWirawan et al., 2001a,b; Yan et al., 1992). The typical zetapotential for bioparticles lies in the range of 100 mV to
30 mV (Van der Wal et al., 1997; Yan et al., 1992).
Suspension Characteristics
Density of suspension. The density of a suspension is
dependent on the density and concentration of the particles
and the density of the liquid. Table III shows that the
bioparticle density is in the range of 900 to 1,540 kg/m3.
Since most biotechnological processes are carried out
in aqueous systems with densities ranging from 1,000 to
1,050 kg/m3, the density of the suspension will be in between
900 and 1,540 kg/m3. In this work, the density of water will be
used for all particle suspensions because most products are
produced in water.
Viscosity of suspension. In most biotechnological processes, water is the main liquid phase. The viscosity of the
liquid phase may, however, be much higher than the viscosity
of water due to dissolved compounds. For example, in
fermentative processes that produce an intracellular product,
cells have to be disrupted in order to release the product. As
cells are ruptured, DNA and other molecules are released
causing a viscosity increase. This viscosity increase can be
limited by making use of cell disintegration methods that
hydrolyze these molecules. Nevertheless, the liquid phase in
fermentative processes is very likely to show non-Newtonian
behavior with viscosities exceeding the viscosity of water.
Besides dissolved molecules, the particle volume fraction
and the particle shape influence on the viscosity. Einstein
(Investigations on the theory of Brownian movement, 1926)
obtained the following theoretical relation for the viscosity of
identical non-interacting rigid spherical particle suspensions
at low particle volume fractions:
h h0 1 2:5c

DOI 10.1002/bit
0.55.0 (Agerkvist and Enfors, 1990; Bowden,

1985; Harrison, 1991; Hayashi et al., 2001b;
Kula et al., 1990)
2.010 (Bowden, 1985; Harrison, 1991; Kula et al.,
1990; Siddiqi et al., 1996)
4070 (Harrison, 1991)
540 (Bowden, 1985; Lipschutz et al., 2000; Nikolai
and Hu, 1992)
50100 (Bowden, 1985)
0.053.0 (Agerkvist and Enfors, 1990; Bowden, 1985;
Kula et al., 1990; Wong et al., 1997a;
Wong et al., 1997b)
0.058.0 (Kula et al., 1990; Siddiqi et al., 1996)
0.05-1.2 (Taylor et al., 1986; Walker and Lyddiatt,
1999; Wangsa-Wirawan et al., 2001b;
Wong et al., 1997b)
1.0100 (Bell et al., 1982, 1983; Jauregi et al., 2001;
Spassov et al., 1996; Wolff et al., 1997)
0.020.20 (Andrews et al., 1995; Cruz et al., 2000;
Kitano et al., 1987; Tsoka et al., 2000)
Diameter (mm)
Density (kg/m3)
1,000-1,540 (Absolom et al., 1986; Bell et al., 1983; Jauregi et al.,

2001; Leung et al., 1999; Matthews, 1974)
1,140-1,190 (Cruz et al., 2000)
900-1,260 (Preusting et al., 1993; Taylor et al., 1986; Wong

et al., 1997a,b)
1,0611,090 (Wong et al., 1997b)
1,040 (Lipschutz et al., 2000; Nikolai and Hu, 1992)
1,0801,120 (Erbeldinger et al., 1998; Wong et al., 1997b)
*These iso-electric points are available in literature but for other bioparticles different values may be obtained.
**If the iso-electric point of crystal is equal to the molecules of which it is composed.
VLPs
Crystals
Yeast
Inclusion bodies
Plant
Cell debris bacterial
Fungi and algae

Mammalian
Yeast
Whole cells bacterial
Particle
Table III. An overview of bioparticle characteristics.
311 (Weast et al., 1964; Chang, 1981)**
5.0 (Wangsa-Wirawan et al., 2001b)*
2.8 (Wangsa-Wirawan et al., 2001b)*
3.05.0 (Hayashi et al., 2001a,b)*
Iso-electric point
where c is the particle volume fraction, h the apparent

viscosity, and h0 the viscosity of the suspending medium.
This equation is valid for c 0.1. In the concentration
range, where this equation is applicable, the influence of
particle concentration is small. At higher particle concentrations, the influence can be much larger (Bird et al., 2002).
In general, it can be said that the viscosity of bioparticle
suspensions may vary from the viscosity of water to very
high values (Agerkvist and Enfors, 1990; Mosqueira et al.,
1981; Wong et al., 1997a). In this work, the viscosity of
water will be used, as it can be regarded as the common
lower limit in bioprocesses, which can be approached by
dilution and chemical or enzymatic treatment.
FORCES IN PARTICLEPARTICLE SEPARATION

The forces acting on a particle moving in a fluid are dependent on the presence of force fields, the particle properties,
the properties of the fluid phase(s), the properties of other
particles in the suspension and the mutual movement of each.
The resultant of these forces may be the driving force for
particleparticle separation. In this section, various forces
are estimated using the particle (and liquid phase) properties that were discussed in Properties of Typical Bioparticles and Their Suspensions. The particle shape is assumed
to be spherical to avoid complex calculations, even though
shape differences may be the basis for particleparticle
separation.
External Field Forces

Centrifugal Force
The centrifugal force, which is often applied in biotechnology, can be used for particleparticle separation if particles
in a mixture show different settling velocities under influence
of this external field. In the equation below, the net resultant
of the centrifugal force on a particle in a fluid phase is
shown.
FC a Dr V
2
2
where a is the centrifugal acceleration (m/s ), Dr the density

difference between the particle and the suspension (kg/m3),
and V the particle volume (m3). The difference in FC for two
types of particles depends on their differences in density
and volume. An increase in centrifugal acceleration may
cause an increase in the absolute force difference between
the particles. The typical upper limit of the centrifugal
acceleration that can be achieved with industrial scale
centrifuges is 20,000g. Therefore, the maximum FC that can
be achieved in bioparticle mixtures is approximately
5.5 105 N when the particles have a maximum size and
density of 100 mm and 1,540 kg/m3 respectively. The force
decreases rapidly with a decreasing particle diameter due to
its dependence on the particle volume and thus on the
cubical particle diameter.
Electric Force
The electric force is proportional to the electric field strength
and the particle charge. The effective charge of a particle can
be described as a function of its zeta-potential and the Debye
decay length (Van de Ven, 1989) that is a measure for the
thickness of its electrical double layer around the particle.
The reciprocal of the Debye decay length, k1, can be calculated
with the following equation (Schulze, 1984):
r
e2 Nav X

Ci z2i
k
ekT
where 1/k is the Debye decay length (m), e the elementary

charge (C), Nav the Avogadro number, E the dielectric
permittivity of the fluid (F/m), k the Boltzmann constant (J/
K), T the temperature (K), Ci the ionic concentration of the
solution (mol/m3), and zi the ion valency (). In the equation
below, the influence of the Debye decay length on the electric
force exerted onto a particle is shown (Schulze, 1984).
1
Fe q E 2 p dp 1 k dp e zp E
2
where q is the particle charge (C), E the electrical field

strength (V/m), dp the particle diameter (m), and zp the zetapotential of the particle (V). Equation 4 shows that the
difference in Fe for two types of particles is governed by their
radius and zeta-potential differences. The absolute force
difference increases with an increase of the electrical field
strength.
Bioparticles have sizes up to 100 mm and zeta-potentials as
low as 100 mV. They are generally suspended in water
(E 7.12 1010 F/m) with ionic concentrations ranging
from 0.01 mol/L to 1 mol/L. This causes the maximum Fe that
can be reached in biological systems with a typical maximum
electrical field of 6 106 V/m (Perry et al., 1998), to be in the
order of 3.8 107 N. To our knowledge, the electric force
has not yet been applied for large-scale particleparticle
separation in biotechnology, though it is used in the mineral
industry for the recovery of minerals. In biotechnology, the
electric force has been mainly used for particle surface
charge determination as well as the recovery/purification of
bioparticles and biomolecules on analytical scale by electrophoresis using membranes, gels, and capillaries (Henskens
and Dieijen-Visser, 2000; Jones et al., 1983; Micale et al.,
1980; Netz, 2003; Preece and Luckman, 1981; Young, 1976),
and by dielectrophoresis (WO200196857; Washizu et al.,
2000). Besides these small-scale applications, there are
indications that the force has potential for large-scale
applications as well (Douglas et al., 1995; Ivory, 1993;
Morgan et al., 1997).
Magnetic Force
Some bioparticles contain compounds that are (para)magnetic. Examples of these bioparticles are magnetotactic

bacteria and red blood cells. Magnetotactic bacteria use

magnetosomes, which are intracellular magnetic particles,
to find the optimal oxygen concentration and/or redox
potential in water and at sedimentwater interfaces
(Dunin- Borkowski et al., 1998). Magnetosomes consist
of iron oxide magnetite crystals or iron sulphate crystals,
which are enveloped by a membrane (Lins and Farina, 1999).
Red blood cells contain hemoglobin, which gives them
paramagnetic properties (Melville et al., 1975). Besides
bioparticles with natural magnetic properties, non-magnetic
bioparticles can be magnetized. Microorganisms and microbial products can be highly efficient bioaccumulators of
soluble and particulate forms of metals (Lins and Farina,
1999). Because the metals are (para)magnetic, the bioparticles will obtain magnetic properties as well. The extent to
which the bioparticles become magnetic depends on the
absorption time and metal concentration (Bahaj et al., 1989).
Adsorption of magnetically labeled antibodies onto cells or
other bioparticles can have a similar effect (McCloskey et al.,
2003). This process is very selective because of the specific
interactions between the antibodies and their binding sites.
Magnetized bioparticles can move in a magnetic field,
where the magnetic force exerted onto the particle can be
described with the following equation:
dB
FM w H V
dx
where w is the magnetic susceptibility of the particle (), H

the magnetic field intensity (A/m), dB/dx the magnetic field
gradient (T/m). Equation 5 shows that the magnetic force
difference is proportional to the differences in particle
volume and particle magnetization, which is the product of
susceptibility and magnetic field intensity.
The magnetic susceptibility is the intensity of magnetization of a particle when it is placed in a uniform magnetic field.
For microorganisms with absorbed metal ions, this susceptibility is in the range of 028.7 105 (SI units) (Bahaj et al.,
1989), but other conditions may yield higher susceptibilities.
The magnetization of magnetosomes in magnetotactic
bacteria has been reported to be close to that of bulk
magnetite (Dunin-Borkowski et al., 1998; Proksch et al.,
1995), which has a saturation magnetization of 4.8 105 A/
m with a susceptibility of about 3.1 (SI units) (Heider et al.,
1996). These high values will, however, only hold for
particles containing a very large fraction of magnetite.
Therefore, the data for magnetized microorganisms, as
presented in Bahaj et al. (1989), will be used for the
estimation of the maximal magnetic force in this work.
The magnetic field gradient, which has a typical maximum
value of 2.5 105 T/m in an industrial-scale superconducting
electromagnet (Perry et al., 1998), influences the absolute force
difference between the particles. The maximum magnetic field
intensity that can be obtained in such a device is 3.98 106 A/
m. Using this value for the magnetic field intensity may give an
overestimation of the magnetic force since the saturation
magnetization of the bioparticles is probably lower than this
magnetic field intensity. Nevertheless, if the saturation

magnetization is neglected and the maximum magnetic field
gradient can be applied, the maximal FM that can be reached in
biological systems is in the order of 1.5 104 N for 100 mm
particles. Even though this force can possibly exceed the
centrifugal force, it has been applied in biotechnology mostly
on a small scale for the concentration and separation of cells
(Comella et al., 2001; Cullison and Jaykus, 2002; McCloskey
et al., 2003; Melville et al., 1975; Owen, 1978; Radbruch et al.,
1994; Zborowski et al., 2003) and the purification of molecules
by their adsorption onto magnetic adsorbents followed by
recovery of the magnetic adsorbents (Heeboll-Nielsen et al.,
2003; Hubbuch and Thomas, 2003).
Interaction Forces
Besides the external field forces, there are forces acting between
particles and objects, such as other particles, part of the
separation device or fluidfluid interfaces. The magnitude of
these forces depends on the particle properties and the properties
of the object/interface that the particle is interacting with. Here,
only spherical and planar geometries are considered.
Van der Waals Interaction
The Van der Waals force results from interactions between
atoms that are dipoles and/or induced dipoles. In most cases,
this force is attractive since the free energy of a system is
lower when the dipoles and/or induced dipoles are aligned
and thus attract one another. In Equation 6, an approximation
of the interaction force between two particles is given
(Schulze, 1984).

dp d
dp d
A
FvdW

if
>> h
12 h2 dp d
dp d
6
where A is the Hamaker constant (J), h the distances
between the surfaces of the two particles, and d the diameter
of the other sphere (m).
For the interaction between a flat surface and a spherical
particle at close distances, the diameter of the other sphere, d,
can be chosen much larger than the particle diameter dp,
which results in the following equation (Schulze, 1984):
A dp
for
h << dp << d 7
FvdW
12 h2
The parameters determining the selectivity of particle
particle separation with the Van der Waals force are the
particle diameter and the Hamaker constant. The latter
typically lies between 1021 and 1019 J (Fielden, 1996;
Han, 2002; Hayashi et al., 2001b; Koliadima, 1999; Okada
et al., 1990b; Shaw, 1966).
Electrostatic Interaction
For two spherical particles with zeta-potentials between
60 mV and 60 mV (Hogg et al., 1965) the electrostatic

DOI 10.1002/bit
interaction force can be approximated with the following

equation (Hogg et al., 1965; Okada et al., 1990a):
dp d
k zp z
FE 2 p e
dp d
"
#
zp z2 e2kh
ekh

1 ekh 2 zp z 1 e2kh

dp d
for
>> h and k dp >> 1
dp d
where z is the zeta-potential of the surface interacting

with the particle (V). In order to calculate the interaction
force between a surface and a spherical particle at close
distances the diameter of one of the two spheres can again
be chosen much larger than the other. This results in the
following approximation (Hogg et al., 1965; Okada et al.,
1990a):
kh
e
FE 2 p e d p k z p z
1 ekh
2
for h << dp << d; k dp >> 1 and
zp z
<< 1
zp z
9
The selectivity of the electrostatic interaction force for
particleparticle separation is determined by the differences
in radius and zeta-potential of the particles that need to be
separated. The absolute force difference can be increased by
an increase of the zeta-potential of the surface the particles
are interacting with.
The Van der Waals force and the electrostatic interaction
force are both dependent on the distance between the particle
and the interacting surface. The resultant of these two forces
is called the DLVO force. In Figure 2, a typical force
distance curve is presented for the DLVO force, the Van der
Waals force, and a repulsive electrostatic interaction force.
Forces larger than zero represent repulsive forces and
negative forces are attractive. When two types of particles
interact with the same object or fluidfluid interface, their
DLVO forcedistance curves will be different. Particles with
a repulsive DLVO force will move away from the surface and
particles with an attractive DLVO force will move towards
the surface. This phenomenon has been applied for separation in potential barrier field-flow fractionation (Hansen and
Giddings, 1989; Koliadima and Karaiskakis, 1990), a method
in which the particle suspension flows parallel to a (charged)
surface. Another example of the use of the DLVO force for
bioparticle separation was given by Hayashi et al. (2001a) in
a study that showed that various microorganisms having
different zeta-potentials can be separated on the basis of these
differences by adsorption onto a charged surface.
The magnitude of the maximum DLVO force difference
for two particles can be approximated by calculating the
force difference at the Debye decay length for a particle with
the largest Hamaker constant and maximal electrostatic
attraction and another particle with the lowest Hamaker
constant and maximal electrostatic repulsion. As mentioned
Figure 2. A typical force distance curve for the DLVO force between two
particles with charges of equal sign. [Color figure can be seen in the online
version of this article, available at www.interscience.wiley.com.]
before, the Hamaker constant for bioparticles lies in the range

of 1021 1019 J and their zeta-potentials are in the range of
100 mV to 30 mV. Thus, the maximal driving force for
particleparticle separation of 100 mm particles using the
DLVO force is approximately 3.8 107 N. In this estimation, the zeta-potential limits for Equations 8 and 9 are
ignored. The magnitude of the DLVO is thus reasonable
compared to the centrifugal force but it has not yet been
applied for large-scale particleparticle separation in biotechnology.
There is another interaction force that depends on the
distance between the interacting objects, that is, the hydrophobic interaction force. This force will not be discussed
because there is a lack of literature data for bioparticles,
which makes estimation of the magnitude of this force
impossible. It is worth mentioning, however, that an
expression similar to that for the Van der Waals force has
been proposed to quantify the hydrophobic force with a
constant that is different from the Hamaker constant
(Rabinovich and Yoon, 1994).
Interfacial Tension Force

The interfacial tension between two fluid phases can be
lowered by adsorption of particles at the fluidfluid interface.
The energy reduction that is related to this process can be
calculated using (Binks, 2002):
EI p=4 dp2 g 1 cosy2
10
where g is the interfacial tension (N/m) and y is the

equilibrium contact angle between the particlefluid interface and the fluidfluid interface measured through the fluid

where the particle is pulled into. The increase in energy of

the system that is caused by removal of the particle from the
interface gives rise to a force that pulls the particle back into
the interface. This force is related to the static and dynamic
fluidfluid interfacial tension, the particle diameter, and the
velocity of the particle during its removal from the interface.
Exact calculation of the interfacial tension force would
require a detailed description of the particle removal process
(Nguyen, 2003), but it is approximated with the derivative of
Equation 10 (Clarke and Wilson, 1983) that is shown in
Equation 11. This equation only takes into account the
energy difference between the initial state when the particle
is at its equilibrium position in the interface and the final
state when the particle is fully immerged into one of the
fluid phases and neglects the phenomena at intermediate
particle positions.
@p=4 dp2 g 1 cosy2

FI
p dp g 1 cosy
d
@ 2p 1 cosy
11
where dp (1 cosy) is the immersion depth of the
particle into the fluid where it will be pulled out of, which is
equal to the distance the particle has to be transported for its
removal from the interface. Equation 11 shows that the
selectivity in particleparticle separation using the interfacial tension force is dependent on differences in contact
angle, particle diameter, and the fluidfluid interfacial
tension.
Contact angles of bioparticles depend on the properties of
the two fluid phases at the interface. Theoretically, this
contact angle can be between 08 and 1808 but in practice, less
extreme values are encountered, such as the airwater
contact angle for bacterial cells that ranges from 58 to 1078
(Absolom et al., 1986; Busscher et al., 2000; Chattopadhyay
et al., 1995). The large variation in these contact angles is
caused by variation of the surface properties of the bacterial
cells, as well as the variation in composition of the aqueous
phase. For polyhydroxybutyrate, airwater contact angles
have been measured with a capillary rise technique that vary
from 218 to 908 (Marchessault et al., 2001). The large
variation in these values is due to the use of various film
preparation techniques. When these contact angles are used,
the maximum interfacial tension force that can be encountered for bioparticles of 100 mm diameter is in the order of
5.8 105 N when adsorbed to an airwater interface
(g 0.072 N/m). The force thus has a large potential for
particleparticle separation in biotechnology.
Particleparticle separation using the interfacial tension
force has only been studied to a small extent (Boucher, 1989;
Jauregi et al., 2001; Winitzer, 1973a,b). In biotechnology,
interfaces have been used for the recovery and/or separation
of bioparticles and solutes from mixtures. In these applications, aqueous two-phase systems (Andrews et al., 1995;
Asenjo et al., 1991; Guereca et al., 1994; HeywoodWaddington et al., 1986; Kula, 1986, 1993; Walker and
10
Lyddiatt, 1999) and other liquidliquid systems (Borbas

et al., 2001; Dennison and Lovrien, 1997; Hoeben et al.,
2004; Jauregi et al., 2001, 2002; Kiss et al., 1998; Pike and
Denisson, 1989; Tan and Lovrien, 1972) were used to capture
the desired products in the interface between the two liquids.
In addition, air flotation which makes use of the adsorption of
particles and/or molecules to air bubbles that rise in the liquid
phase due to buoyancy, has been applied for the recovery of
whole cells (Bahr and Schugerl, 1992; De Dousa et al., 2003;
Gahr and Schugerl, 1992; Gaudin, 1975; Grieves and Wang,
1966, 1967; Kalyuzhnyi et al., 1965; Palmieri et al., 1996;
Sadowski and Golab, 1991; Tybussek et al., 1994; Vlaski
et al., 1996; Wang et al., 1994). In all of these examples,
interfaces are used to capture the product, but this does not
necessarily mean that separation is accomplished solely with
the interfacial tension force.
The use of the interfacial tension force for complete
particleparticle separation in a single stage requires one particle to adsorb into the interface while the other particle
remains in one of the fluid phases. When this is accomplished, the adsorbed particle can be selectively removed
from the system by separating the interface from the fluid
phase that contains the other particle. There are two methods
that can be applied for obtaining a fluidfluid interface that is
enriched with one of the two particles that are present in the
system. One method is to create conditions that allow both
particles to adsorb but forces one of the two particles to
detach from the interface. In this case, detachment can be
accomplished by using another force such as the drag force
and the centrifugal force, or by competition between the two
particles for adsorption to the interface as was reported by
Jauregi et al. (2001, 2002). Another method is to create
conditions that prevent one of the types of particles from
adsorbing into the interface. This can be accomplished by
using fluid phases that make it energetically unfavorable for
one of the particles to adsorb at the interface or by creating an
energy barrier for one of the particles that prevents the
particle from approaching the interface close enough for
adsorption to take place. This energy barrier could be related
to the DLVO force or the hydrodynamic conditions in the
system. It is clear that both methods require careful selection
of the fluid phases and the hydrodynamic conditions in the
separation device. There are many possible fluid phase
combinations that have a wide variety of interfacial tensions.
For instance, the interfacial tension for aqueous two-phase
systems is in the order of 0.3 103 N/m, while for an air
water interface, it is in the order of 0.072 N/m. This indicates
that the range of possibilities for particleparticle separation
using the interfacial tension force is comprehensive.
Dynamic Interactions Between Particle and Fluid
Forces resulting from the dynamic interactions between
particles and fluids depend on particle movement and the
change in particle movement relative to the fluid phase. This
movement is governed by all forces that act on the particle.
The forces discussed in this section are therefore in many

DOI 10.1002/bit
cases dependent on other forces and can only be calculated

when these other forces are known. Moreover, the dynamic
interactions between particles and fluids are often only
applicable in combination with other forces. For this reason,
they end up in the force balance on the opposite side of the
other forces, which from now on will be regarded as driving
forces, as is shown in Equation 12.
X
i
Fi
Fj
where CD () is the drag coefficient, A\ is the crosssectional area of the particle (m2) and v (m/s) is the particle
velocity relative to the fluid phase. The drag coefficient is
dependent on the shape of the particle, on its orientation in
the fluid and on its relative velocity. The drag coefficient for
spherical particles at Reynolds numbers below 0.1 is given
by Stokes law:
CD
12
where Fi are the driving forces and Fj are the dynamic

particlefluid interaction forces. Furthermore the dynamic
particlefluid interaction forces will be regarded as inevitable forces that influence separation using the driving
forces even though they can be useful for separation. Their
applicability for particle separation will not be discussed in
this work.
There are three dynamic particlefluid interaction forces
that are frequently mentioned in literature. These are the drag
force, the inertia force and the force resulting from the virtual
mass of a particle. Besides these forces, many other forces
exist such as the Magnus force that results from a spinning
motion of a moving particle and the Kutta-Joukowski lift
force that results from the fluid-flow profiles around a particle.
These forces are not often applied for particleparticle
separation on a large scale but they have been shown to be
applicable on a small-scale through for instance field-flow
fractionation (Giddings et al., 1976). In field-flow fractionation (FFF), a force field acts perpendicular to a flowing liquid
that has a certain flow profile. This force field can be the
gravitational force or one of the other field forces that were
discussed in Section External Field Forces. The applied
force field causes the particles to move through the flow
profile in the flowing liquid. This causes a differentiation of
the particle velocities in the separation device that is the basis
for their separation. This process is influenced by the dynamic
particlefluid interaction forces and Brownian motion (see
Section Other Phenomena: Brownian Motion). In flow
FFF, a cross-flow liquid stream is applied perpendicular to a
flowing suspension (Giddings et al., 1976; Wahlund and
Litzen, 1989). In this process, the particles are distributed on
the basis of the dynamic particlefluid interaction forces, the
gravitational force and Brownian motion. Separation on the
basis of the lift force is feasible in FFF when it is large enough
in comparison to the applied force field (Giddings, 2000).
Below the drag force, the inertia force and virtual mass are
discussed.
Drag Force
The drag force results from friction between the fluid and the
particle (friction drag) and the displacement of the fluid phase
by the particle (form drag). In the equation below, the general
form of the drag force is given.
Ff CD A? 1=2 rl v2
13
24
Re
for Re < 0:1:
This relation can also be used for Reynolds numbers

between 0.1 and 1 but then the drag force is underestimated
with about 10%. For Reynolds numbers between 0.5 and
6 103, the drag coefficient can be calculated with the
following expression (Bird et al., 2002):
CD
!2
r
24
0:5407
Re
for 0:5 < Re < 6 103 :
At Reynolds numbers between 5 102 and 105 the drag

coefficient obtains a constant value of about 0.44 (Bird et al.,
2002).
Inertia Force
An expression for the inertial force is given below:
FIE V rp
dv
dt
14
where rp is the particle density (kg/m3). If the inertia force is

included in the force balance, particle acceleration becomes
a function of particle density, particle size, and the other
forces. This causes a differentiation in acceleration of particles with different driving forces, sizes, and/or densities,
which could be the basis for particleparticle separation.
Application of this principle on an industrial scale will be
very difficult. In addition, including the inertia force in
the force balance requires time iterations for calculation of
the relative particle velocities and their derivative to time,
making simple estimation of the particleparticle separation efficiency impossible. Therefore, the inertia force will
not be considered in any force calculations in this work.
For small particles, the inertia force generally is very
small because these particles tend to follow the liquid
streamlines (Caulet et al., 1996), which makes this a proper
assumption. With larger particles, the inertia force may
reduce or even prevent particle acceleration. This can
prevent the particles from reaching their terminal velocity
that was calculated on the basis of the drag force and the
driving forces.
Virtual Mass
The virtual mass of a particle accounts for the resistance of
the fluid against changes in particle velocity relative to that of
the fluid. For spherical particles in a constant fluid flow

11
profile, the virtual mass is equal to half of the particle volume

(Zhang et al., 2000), so that
FVM
1
dv
V rl
2
dt
15
where rl is the liquid density (kg/m3) and dv/dt is the

acceleration (m/s2). The same argument holds for this
force as for the inertia force with respect to its influence
on the relative particle velocity and its applicability for
particleparticle separation. Therefore this force will be
excluded from the force balance as well.
Other Phenomena: Brownian Motion

The time-average kinetic energy of a particle, regardless of
its size, is equal to the average thermal energy of the
surrounding molecules, which is 3/2 kT (1/2 kT in one
dimension). The kinetic energy of a particle may fluctuate in
time and may thus differ from the time-average value. This
makes estimation of the microscopic effects of the Brownian
motion very difficult. The time-average value will, therefore,
be used for estimation of the macroscopic effect of Brownian
motion. Equation 16 shows the relation between the onedimensional concentration gradient and the Brownian force
(Van de Ven, 1989).
FB
k T dn

n dx
16
where dn/dx is the particle concentration gradient (mol/m4).

Particles can thus move from a high particle concentration
to a low concentration as a result of the Brownian force.
In addition, particle motion occurs in homogeneous
suspensions where dn/dx is zero, but will be random. This
random motion does not cause a net particle transport and
is therefore not useful for particleparticle separation by
itself. However, it can act as a dominant transport
mechanism and facilitate other processes such as particle
adsorption at interfaces. Brownian motion has been used in
biotechnology mainly for the separation of molecules by
making use of the differences in their transport through
membranes and in matrices. Transport in these systems
depends on the thermal motion of the compounds, their
partitioning in the media and the hindrance they experience
within the matrix or membrane. In addition, Brownian
motion can play an important role in particle separation
using field-flow fractionation (Giddings, 2000).
For estimation of the maximum Brownian force in
bioparticle mixtures, the maximum obtainable particle
concentration gradient is required. If it is assumed that
the highest particle concentration is reached at 60 %v/v
particles (dense packing of spheres) and decreases to 0 %v/v
over a distance of 103 m, then 1/n dn/dx is approximately
2 103/m at an average value of n resulting in a Brownian
force of 8.1 1018 N at a temperature of 298 K. Although
this estimation may be off by a couple of orders of magnitude,
we can readily see that the Brownian force acting on 100 mm
12
particles is very small compared to the other forces that were

discussed in this section. For very small particles the
Brownian force becomes more important because it is
independent of particle size while the other forces decrease
with particle size.
Overview of Driving Force Differences

Between Particles
The upper limit of the driving force difference for two
bioparticles with similar sizes and different properties is
estimated for bioparticles and liquid phases with properties
that are presented in the Section Properties of Typical
Bioparticles and Their Suspensions and Section Forces in
ParticleParticle Separation. The result of this estimation is
depicted in Figure 3 for the whole range of particle sizes that
may be encountered in biotechnology. The graph shows that
for particle diameters between 20 nm and 100 mm, the interfacial tension force has the largest potential for separation of
particles with the same size. For particle diameters below
0.5 mm, the electrical force also has large potential. The
centrifugal force has a much lower potential than these two
forces and the graph confirms that it may even be inapplicable
for the separation of small particles. Whether or not the
interfacial tension force and the electric force are the largest
driving forces for particleparticle separation in practice
depends on the liquid phase properties and the particle
properties for each specific separation problem. These
properties can be manipulated to some extent to increase
the selectivity for many of the driving forces (Section
Optimization and Control of Driving Forces).
Direct comparison of the driving forces (Fig. 3) can help to
determine the forces and processes that should be investigated in more detail. After this initial screening, the
limitation to the applicability of the driving forces has to be
force (N)
10-2
electrical force
10-4
interfacial tension force
10-6
10-8
centrifugal force
10-10
10-12
magnetic force
-14
DLVO force
10
Brownian force
-16
10
10-18
10-20
100
101
102
103
104
105
particle diameter (nm)

Figure 3. An overview of the maximal force difference that can be
obtained with the driving forces when they act on two bioparticles with
similar sizes and different properties.

DOI 10.1002/bit
taken into consideration. This is done in Section Selection

of a ParticleParticle Separation Technique.
Before discussing the optimization of driving forces, a
small comment has to be made with respect to combining
driving forces in particleparticle separation. In many cases,
we are forced to deal with combinations of forces because
some forces cannot be excluded from the process. Examples
of these forces are the Brownian force, the gravitational force
and many of the forces that are related to the dynamic
interactions between particles and fluids. It is therefore
essential to take into account the influences of these forces
when designing a process. Furthermore, combinations of
driving forces may give additional options in particle
particle separation. Force estimations like those presented in
Figure 3 can aid the selection process. Evaluation of these
combinations is only valuable when a specific separation
problem is regarded because of the larger number of possible
combinations and the difficulty of combining some of the
forces. Therefore this topic will not be dealt with in this work.
In literature, this strategy is discussed to some extent. For
instance, the thesis of Tils (Tils and Tels, 1992) deals with
particle separation by flotation in a centrifugal force field, and
in field-flow fractionation dynamic particle-fluid interaction
forces are often combined with field forces and Brownian
motion (see section Dynamic Interactions Between Particle
and Fluid).
OPTIMIZATION AND CONTROL OF DRIVING
FORCES
Particle properties can be manipulated by changing their
surface chemistry, which influences the electrical force, the
magnetic force, the DLVO force, the interfacial tension force,
and possibly the aggregate size.
The surface properties of particles can be controlled by
adsorption of surfactants. This technique is commonly
applied in flotation and may change the zeta-potential of
particles and their contact angle with the fluidfluid interface
(Fraunholcz and Dalmijn, 1998; Hanumantha Rao and
Forssberg, 1997). Polymer or polyelectrolyte adsorption
may have a similar effect as the surfactants that are used in
flotation.
The particle size distribution can be manipulated using
aggregation. This process only has a positive effect on
particleparticle separation if it is selective for one kind of
particle or if the particles aggregate specifically with their
own kind resulting in aggregate sizes that yield a larger force
difference compared to the force difference for the primary
particles. It is therefore very important that heteroaggregation and entrapment of particles in aggregates of other
particles is prevented. The aggregation rate of particles in a
suspension may be increased by reducing the electrostatic
repulsion between the particles, by adding polymers or
polyelectrolytes, with shear, by changing the temperature,
with high pressure treatment or by enzymatic action
(renneting) (Dickinson, 2003). The first two methods will
be briefly discussed.
The electrostatic repulsion between particles can be

reduced by changing the nature and concentration of the
ions in solution (Eq. 3), by reducing their zeta-potential
through pH change (Eq. 8) or by specific adsorption of
surfactants or ions on their surface (Chang and Hshieh,
1991). The latter strategy has been shown to work
successfully for airwater interfaces with aluminium (Li
and Somasundaran, 1992) and magnesium ions (Li and
Somasundaran, 1991) as well as for polystyrene particles
with surfactants (Okada et al., 1990b). Addition of polymers
or polyelectrolytes can also lower the electrostatic repulsion,
but more importantly these compounds may form bridges
between the particles by adsorbing onto the particle surface.
Bridging causes an increase in particle aggregation rate due
to more effective particle collisions and a decrease in
aggregate break-up rate. Examples of chemicals that can be
used for particle size manipulation are borax, which is an
effective flocculation agent for cell debris, as reported by
Tsoka et al. (2000) and calcium ions that can be used to
flocculate microorganisms by forming metal-polymer complexes with the polymers on the surface of the microorganism, as reported by Sanin and Vesilind (1996).
SELECTION OF A PARTICLEPARTICLE
SEPARATION TECHNIQUE
In the following sections, a method for creating a window
of operation for the driving forces is presented and the
selection procedure for suitable separation methods is
discussed for typical particle mixtures that are encountered
in biotechnology.
Window of Operation for the Driving Forces in
ParticleParticle Separation
The window of operation for the field forces depends on the
particle properties and the minimum velocity difference that
is required for their separation. The velocity of a particle can
be calculated by solving the force balance between the
driving forces and the dynamic particlefluid interaction
forces (Eq. 12). From the dynamic particlefluid interaction
forces only the drag force will be taken into account for
reasons that were discussed in Section Overview of Driving
Force Differences Between Particles. The driving forces for
separation of bioparticles will often be small near their
separation limits causing particle velocities to be small as
well. Stokes law can therefore be applied for the drag force
in most cases. As an example, in Equation 17 Stokes drag is
included in the force balance of Equation 12 in case the
centrifugal force is the only driving force.
p
FC Ff ! a Dr dp3
6
3 p dp v ! dp;lim
s
18 vmin
a Dr

17
13
The limiting particle diameter that can be separated with the

driving force is a function of the particle properties, the fluid
properties, the centrifugal acceleration, and the minimum
required terminal particle velocity. By subtracting the minimum required terminal particle velocities of all particles in a
mixture, the minimum particle diameters that are required
for the separation of the mixture can be calculated. The
relation between the minimum particle diameters and
the minimum required velocity difference in a binary particle mixture is shown below for the field forces in case of
Stokes drag. A similar approach can be taken when other
hydrodynamic conditions apply by including the appropriate expression for the drag coefficient (see section Drag
Force).
a
Centrifugal Force : Dvmin jv1 v2 j
18

2
Dr1 dp lim;1 Dr2 dp2 lim;2
18
Electric Force : Dvmin
2eE
j1 0:5 k dp lim;1
3
z1 1 0:5 k dp lim;2 z2 j
19
H dB
Magnetic Force : Dvmin

18 dx

20

2
2
w1 dp lim;1 w2 dp lim;2
These equations show that the field forces can be applied for
all particle sizes that cause the particle velocity difference to
exceed the minimum required difference. For the DLVO
force, a similar expression can be obtained (Eq. 21), which
shows that the window of operation of the DLVO force is
independent on the particle diameter. It can thus be applied to
all particle sizes as long as the actual velocity difference
exceeds the minimum required difference. Since this velocity
difference is a function of the distance between the particles
and the interacting surface, it will be estimated by using the
Debye decay length.

2 e k z
ekh
DLVO Force : Dvmin

z1 z2
3
1 ekh

A2 A 1
:
36 p h2
21
The window of operation for the interfacial tension force
cannot be constructed in a similar manner as for the field
forces and the DLVO force because it completely depends
on the design of the separation device, as was discussed
in Section Dynamic Interactions Between Particle and
Fluid. In Section Selection of a Driving Force for ParticleParticle Separation, this design will be treated in
14
more detail to facilitate construction of the operation

window for the interfacial tension force. The operation
window of the Brownian force will not be discussed because
estimation of the particle concentration gradient is arbitrary
and will therefore not give an accurate selection criterion.
Nevertheless, the Brownian force may be applicable if the
differences in particle transport rates through membranes, in
matrices, and in field-flow fractionation are large enough.
Estimation of the minimum required velocity difference
requires knowledge of the feed flux, particle concentration in
the feed, and the area available for separation. All of these
parameters depend on the design of the separation device. To
make comparison of the driving forces possible, we will limit
ourselves to using the minimum particle velocity that is
required for centrifugal separation. This velocity can be
calculated with the Sigma theory (Ambler, 1961), which
states that separation of a particle from the liquid phase with
an efficiency of 100% requires a minimum particle velocity
perpendicular to the liquid flow equal to the ratio of the
volumetric liquid flux and the available settling area
perpendicular to the liquid flow. In order to make this theory
applicable for particleparticle separation instead of particleliquid separation, we have to allow for different
apparatus designs dependent on the direction and magnitude
of the particle velocities. In Figure 4, two possible designs are
presented (Giddings, 1991). These designs show that for
complete separation in devices with similar dimensions that
are used for processing equal volume fluxes, the minimum
required velocity difference between the particles should be
in the same order as the terminal velocity of a single particle
that is required for particleliquid separation. With a feed
v1
particle 1
v2
particle 2
feed suspension
perpendicular
feed suspension
liquid flow
particle 2
particle 1
v1
v2
vf
parallel
Figure 4. Examples of the design of separation devices for cases where
particle movement is perpendicular and parallel to the liquid flow. In case of
particle movement parallel to the liquid flow, the velocity difference between
the two types of particles should be such that the net velocity of Particle 2 is in
the direction of the liquid flow (v2 vf < 0), while the net velocity of
Particle 1 is in opposite direction of the liquid ow (v1 vf > 0). Feed
distribution will become an issue in this design. The vectors indicate the
direction of particle movement relative to the liquid phase.

DOI 10.1002/bit
flux of 1 m3/h that is reasonable for large-scale processes and

an equivalent settling area of 2 104 m2 (Perry et al., 1998),
which is approximately the upper limit for industrial centrifugation at high gravitational acceleration (11,500g), the
minimum required particle velocity difference is approximately (1 11,500)/(3,600 2 104) 1.6 104 m/s.
For the magnetic and electric force, this minimum required
velocity difference is in the same order when industrial-scale
electric and magnetic separators having a separation section
with an approximate length, width, and height of 3, 1, and
0.05 m (dimensions are estimated from handbook data (Perry
et al., 1998)) are considered for treating the same feed.
Therefore, the same velocity difference will be applied in all
calculations
In the following section, a window of operation is created
for each of the driving forces when applied to typical particle
mixtures in biotechnology.
Selection of a Driving Force for
ParticleParticle Separation
The overview of particle mixtures that was presented in
section Overview of Particle Mixtures in Biotechnological
Processes shows that there are different classes of separation problems that may require different purification strategies. In most of these separation problems product
purification requires removal of whole cells or cell debris.
Construction of the operation window for the centrifugal
force requires information on particle size and density, which
is available in literature for most particle mixtures. Estimation of the electric, and magnetic force, on the other hand,
requires the magnetic susceptibility and particle charge,
which are scarcely reported in literature. These particle
properties are therefore estimated on the basis of the data that
were presented in Sections Electric Force and Magnetic
Force. In Figure 5, the relation between the limiting particle
diameters for the centrifugal, electric and magnetic force are
shown for extreme cases of IB purification (Fig. 5A and B),
VLP purification (Fig. 5C), and extracellular particulate
microbial product purification (Fig. 5D and E) that may be
encountered in industry. In the calculation of these limits,
the liquid phase density is assumed to be 1,000 kg/m3, since
most biotechnological processes are carried out in water.
Higher liquid densities may give larger velocity differences
in centrifugation and may thus lower separation limits,
but possibilities for density manipulation depend on each
specific separation problem and will not be considered here.
Furthermore, the density of whole cells and cell debris is
assumed to be 1,085 kg/m3 (Wong et al., 1997b).
In Figure 5, the area marked with horizontal dotted lines
represents all particle diameter combinations that cannot be
separated with the magnetic force and the area marked with
vertical dotted lines represents the combinations that cannot
be separated with the centrifugal force. The electric force
only fails to give separation for particle diameter combinations exactly on the boundary line that is depicted in Figure 5.
If larger velocity differences are required for the electric
force the two particle diameter limits will move apart in a

similar manner as with the magnetic force. Furthermore, the
DLVO force may be applied to all particle diameter
combinations since the minimum particle velocity difference
is exceeded in all cases. However, applicability of this force
will depend very much on process design. The limits of
the interfacial tension force are not depicted for reasons that
were discussed in Section Window of Operation for the
Driving Forces in ParticleParticle Separation.
In each of the graphs depicted in Figure 5, the area that is
marked with dashed lines confines the range of particle sizes
that are encountered in biotechnology. These areas show that
separation of extracellular particulate microbial products
from whole cells can be performed with centrifugation in
most cases regardless of the density of the product. Purification of IBs and VLPs by centrifugation, on the other hand,
will be difficult or even impossible in many cases because
many particle size combinations lie outside of the working
area for centrifugation. In addition, cell debris may have a
broad particle size distribution causing part of the cell debris
particle size to be below the separation limit for centrifugal
separation in many cases. This inevitably leads to incomplete
separation. When the centrifugal force fails, one of the other
driving forces must be selected.
The magnetic force, the interfacial tension force (in
combination with the DLVO force), and the electric force are
used in the paper and/or mineral industry, which may make
application of these forces in biotechnology possible within a
reasonable time span. The electric force and the interfacial
tension force are most favorable because in theory there are
very few separation problems that cannot be resolved with
these forces. Even though the actual zeta-potentials may be
different from those that were assumed for constructing
Figure 5, a small difference in zeta-potential is sufficient
for particle separation with the electric force (Dvmin still
exceeded). Since the zeta-potential can be easily manipulated
in biological systems by varying the pH, nearly all particles
with a different relation between pH and zeta-potential can be
separated with the electric force. It is much more difficult to
indicate what particle mixtures can be separated with the
interfacial tension force (in combination with the DLVO
force) because the mechanisms of flotation and interfacial
partitioning are not fully understood yet. In addition, the
mixtures that need to be treated in biotechnology are often
very complex due to the large number of components that are
present, which makes prediction of the particle behavior very
difficult. Nevertheless, an attempt will be made to construct a
window of operation for the interfacial tension force. As
indicated in Section Dynamic Interactions Between Particle
and Fluid, there is a number of strategies for the use of the
interfacial tension force for particleparticle separation.
These strategies have in common that one type of particles
needs to remain adsorbed at the interface while the other
particles remain in the continuous phase, for instance, due to
an energy barrier that prevents adsorption of these particles at
the interface or due to competition between the particles
for adsorption at the interface. In most biotechnological

15
diameter particle 2 (m): IBs
diameter particle 2 (m): IBs
10-4
10-4
10-5
10-5
electric force
electric force
10-6
10-6
centrifugal force
centrifugal force
10-7
10-7
magnetic force
magnetic force
10-8
10-8
10-8
10-7
10-6
10-5
10-4
10-8
10-7
10-6
10-5
10-4
diameter particle 1 (m): cell debris
diameter particle 2 (m): VLPs
diameter particle 2 (m): product
10-4
10-4
10-5
10-5
electric force
electric force
10-6
10-6
centrifugal force
10-7
10-7
centrifugal force
magnetic force
10-8
10-8
magnetic force
10-7
10-6
10-5
10-4
10-8
10-8
10-7
10-6
10-5
10-4
diameter particle 1 (m): whole cells
Figure 5. An overview of the particleparticle separation limits for the centrifugal force, the electric force, and the magnetic force for extreme particle
particle separation problems that are encountered in biotechnology. Particle 1 represents cell material with a magnetic susceptibility of 2.5 104 and a zetapotential of 0.030 V. Particle 2 represents the particulate product with a magnetic susceptibility of 1.0 104 and a zeta-potential of 0.010 V. The vertical
lines indicate the conditions where centrifugation is not applicable for separation and the horizontal lines indicate the area where the magnetic force is not
applicable for separation. The electric force can be applied for all particle size combinations that are not on the line that indicates the separation limit. The area
confined by the dashed lines indicates all particle size combinations that are encountered in biotechnology for each class of separation problems. A: Separation
of IBs with a high density (1,260 kg/m3) from microbial cell debris. B: Separation of IBs with a low density (900 kg/m3) from microbial cell debris. C: Separation
of VLPs with a density of 1,190 kg/m3 from microbial cell debris. D: Separation of extracellular particulate microbial products with a high density (1,540 kg/m3)
from whole cells. E: Separation of extracellular particulate microbial products with a low density (1,000 kg/m3) from whole cells.
16

DOI 10.1002/bit
maximum particle diameter (m)
10-1
diameter particle 2 (m): product
organic phase/
aqueous phase
ATPS
10-4
gas/liquid
10-2
= 107 o
10-5
10-3
electric force
10-6
10-4
10-7
10-5
10-4
centrifugal force
=5o
10-3
10-2
10-1
interfacial tension (N/m)

magnetic force
10-8
10-8
10-7
10-6
10-5
10-4
diameter particle 1 (m): whole cells
Figure 6. Window of operation for the interfacial tension force showing

the relation between contact angle, interfacial tension and the maximum
particle size that remains adsorbed at the interface under gravity. The particle
density and continuous fluid phase density are assumed to be 1,540 and
1,000 kg/m3, respectively. In the graph approximate ranges of the interfacial
tension of different types of fluidfluid systems are given.
Figure 5. (Continued )
processes, the density of the continuous (often aqueous)

phase will be higher than the density of the dispersed phase.
This density difference can be utilized in the process by
making the dispersed phase rise through the continuous phase
under influence of the gravitational force or centrifugal force.
The interfacial tension force acting on the particle that is
adsorbed at the interface should therefore exceed the
gravitational (and shear) force(s) that acts on the particle to
prevent detachment from the interface. In Figure 6, the relation
is given between contact angle, interfacial tension, and the
maximum particle diameter that still allows particles to remain
at the interface under gravity. For construction of this graph,
the particle density and continuous phase density are assumed
to be 1,540 and 1,000 kg/m3, respectively and the contact
angles are assumed to be in the range that was reported in
section Forces in ParticleParticle Separation. The operation window shows that the interfacial tension force can be
applied for separation of particle sizes ranging from molecular
scale up to approximately 0.01 m regardless of the differences
in particle densities as long as particle adsorption at the
interface is selective for one of the particles in the mixture. The
process of creating selectivity can be very complex, however. It
depends on the hydrodynamics near the interface, the
interaction forces (Section Interaction Forces) and competition effects. Thorough discussions on the application of
interfaces for particle separation lie outside of the scope of this
work and can be found elsewhere (Clarke and Wilson, 1983;
Finch and Dobby, 1990; Gaudin, 1975; Schulze, 1984).
CONCLUSION
Selection of a particleparticle separation technique requires
knowledge of the particle properties, such as density,
magnetic susceptibility, zeta-potential, and hydrophobicity.

When these properties are known, the optimal particle
particle separation technique can be chosen by solving the
force balance between the drag force and the driving forces.
Force estimations reported in this work show that the
interfacial tension force (in combination with the DLVO
force) and the electrical force, in whatever way applicable,
have large potential for particleparticle separation in
biotechnology. The electrical force requires very small
differences in zeta-potential to make complete separation
possible and the window of operation of the interfacial
tension force indicates its applicability to a very broad range
of particle sizes (up to 0.01 m). The use of the electrical force
and the interfacial tension force becomes desirable when
centrifugation cannot be applied for particleparticle
separation. This is generally the case when particle sizes
are small (1 mm) and/or when the density differences
between the particles are small. Examples of such separation
problems are the separation of IBs and VLPs from cell debris.
Currently, only limited use is made of these alternative
particleparticle separation techniques due to a lack of
knowledge. Therefore R&D efforts should increase in this
area to extend the downstream processing toolbox that is
currently available to the biotech industry.
NOMENCLATURE
a
A
A\
B
c
CD
Ci
centrifugal acceleration (m/s2)

Hamaker constant (J)
cross-sectional area of particle (m2)
magnetic field (T)
particle volume fraction (-)
drag coefficient (-)
ionic concentration (mol/m3)

17
d
dp
dp,lim
e
E
EI
FB
Fe
FE
FF
FC
Fi
FI
FIE
Fj
FM
FvdW
FVM
h
H
k
n
Nav
t
T
q
Re
v
vmin
V
x
zi
w
E
g
h
h0
k1
y
rl
rp
z
zp
diameter of sphere (m)

particle diameter (m)
limiting particle diameter (m)
elementary charge (C)
electric field (V/m)
energy required to pull a particle out of a fluid-fluid interface (J)
Brownian force (N)
electric force (N)
electrostatic interaction force (N)
drag force (N)
centrifugal force (N)
general expression for the driving forces (N)
interfacial tension force (N)
inertia force (N)
general expression for the dynamic particlefluid interaction
forces (N)
magnetic force (N)
Van der Waals force (N)
force caused by virtual mass of particle (N)
distance between the surfaces of a particle and an object (m)
magnetic field intensity (A/m)
Boltzmann constant (J/K)
particle concentration (mol/m3)
Avogadro number (#/mol)
time (s)
temperature (K)
particle charge (C)
Reynolds number ()
relative velocity between particle and fluid (m/s)
minimum required particle velocity (m/s)
particle volume (m3)
distance (m)
ion valency ()
magnetic susceptibility ()
dielectric permittivity of the fluid (F/m)
interfacial tension (N/m)
apparent viscosity (Pa s)
liquid viscosity (Pa s)
Debye decay length (m)
contact angle between particle and fluid-fluid interface (8)
liquid phase density (kg/m3)
particle density (kg/m3)
zeta-potential of a surface (V)
particle zeta-potential (V)
References
Absolom DR, Zingg WA, Neumann AW. 1986. Measurement of contact
angles on biological and other highly hydrated surfaces. J Colloid
Interface Sci 112:599601.
Agerkvist I, Enfors S-O. 1990. Characterization of E. coli cell disintegrates
from bead mill and high pressure homogenisers. Biotechnol Bioeng
36:10831089.
Ambler CM. 1961. Theory-The fundamentals of separation, including
Sharples Sigma value for predicting equipment performance. Ind Eng
Chem 53:430433.
Andrews BA, Huang RB, Asenjo JA. 1995. Purification of virus like particles
from yeast cells using aqueous two-phase systems. Bioseparation 5:
105112.
Asenjo JA, Franco T, Andrews AT, Andrews BA. 1991. Affinity separation of
proteins in aqueous two-phase systems. In: White MD, Reuveny S,
Shafferman A, editors. Biologicals from recombinant microorganisms and
animal cells: Production and recovery. Mannheim: VCH Publishers. 439 p.
Bahaj AS, Watson JHP, Ellwood DC. 1989. Determination of magnetic
susceptibility of loaded micro-organisms in bio-magnetic separation.
IEEE Trans Magn 25:38093811.
18
Bahr KH, Schugerl K. 1992. Recovery of yeast from cultivation medium by

continuous flotation and its dependence on cultivation conditions. Chem
Eng Sci 47:1120.
Beards T, Cohen MA, Parratt JS, Turner NJ. 1993a. Stereoselective
hydrolysis fo nitriles and amides under mild conditions using a whole
cell catalyst. Tetrahedron: Assymetry 4:10851104.
Bell DJ, Heywood-Waddington D, Hoare M, Dunnill P. 1982. The density of
protein precipitates and its effect on centrifugal separation. Biotechnol
Bioeng 24:127141.
Bell DJ, Hoare M, Dunnill P. 1983. The formation of protein precipitates and
their centrifugal recovery. In: Scheper T, editor. Advances in
biochemical engineering/biotechnology 26. New York: Springer. 1 p.
Binks BP. 2002. Particles as surfactants-similarities and differences. Curr
Opin Colloid Interface Sci 7:2141.
Bird RB, Stewart WE, Lightfoot EN. 2002. Transport phenomena. New
York: John Wiley & Sons, Inc. 186 p.
Blacker AJ, Holt RA. 1997. Development of a multi-stage chemical and
biological process for an optically active intermediate for an antiglaucoma drug. In: Collins AN, Sheldrake GN, Crosby J, editors.
Chirality in industry II. New York: John Wiley & Sons Ltd. 245 p.
Bode G, Mauch F, Ditschuneit H, Malfertheiner P. 1993. Identification of
structures containing polyhosphate in Helicobacter pylori. J Gen
Microbiol 139:30293033.
Borbas R, Turza S, Szamos J, Kiss E. 2001. Analysis of protein gels formed
by interfacial partitioning. Colloid Polymer Sci 279:705713.
Boucher EA. 1989. Separation of small-particle dispersions by the
preferential accumulation in one of two liquid phases, or by static
flotation at their interface. J Chem Soc Faraday Trans 85:29632972.
Bowden C. 1985. Recovery of micro-organisms from fermented broth. Chem
Eng 415:5054.
Bowden GA, Paredes AM, Georgiou G. 1991. Structure and morphology of
protein inclusion bodies in Escherichia coli. Biotechnology 9:725730.
Buchner J, Brinkmann U, Pastan I. 1992. Renaturation of a single-chain
immunotoxin facilitated by chaperones and protein disulsude isomerase.
Biotechnology 10:68685.
Buque-Taboada EM, Straathof AJJ, Heijnen JJ, Van der Wielen LAM. 2004.
In-situ removal using a crystallization loop in the asymmetric reduction
of 4-oxoisophorone by S.cerevisiae. Biotechnol Bioeng 86:795800.
Busscher HJ, Bos R, Van der Mei HC. 2000. Physicochemistry of microbial
adhesion from an overall approach to the limits. In: Baszkin A, Norde W,
editors. Physical chemistry of biological interfaces. New York: Dekker.
431 p.
Caulet P, Van der Lans RGJM, Luyben KCAM. 1996. Hydrodynamical
interactions between particles and liquid flows in biochemical applications. Chem Eng J 62:193206.
Chang R. 1981. Physical chemistry with applications to biological systems.
New York: MacMillian publishing co.,inc.
Chang YI, Hshieh CY. 1991. The effect of cationic electrolytes on the
eletrophoretic properties of cells. Colloids and Surf 53:2131.
Chattopadhyay D, Rathman JF, Chalmers JJ. 1995. Thermodynamic
approach to explain cell adhesion to air-medium interaces. Biotechnol
Bioeng 48:649658.
Clarke AN, Wilson DJ. 1983. Foam flotation, theory and application. New
York: Marcel Dekker.
Comella K, Nakamura M, Melnik K, Chosy J, Zborowski M, Cooper MA,
Fehniger TA, Caligiuri MA, Chalmers JJ. 2001. Effects of antibody
concentration on the separation of human natural killer cells in a
commercial immunomagnetic separation system. Cytometry 45:285293.
Cruz PE, Peixoto CC, Devos K, Moreira JL, Saman E, Carrondo MJT. 2000.
Characterization and downstream processing of HIV-1 core and viruslike-particles produced in serum free medium. Enzyme Microb Technol
26:6170.
Cullison MA, Jaykus L-A. 2002. Magnetized carbonyl iron and insoluble
zirconium hydroxide mixture facilitates bacterial concentration and
separation from nonfat dry milk. J Food Prot 65:18061810.
Davison BH, Barton JW, Petersen GR. 1997. Nomenclature and methodology for classification of nontraditional biocatalysis. Biotechnol Prog
13:512518.

DOI 10.1002/bit
De Dousa SR, Oliveira KF, Souza CS, Kilikian BV, Laluce C. 2003. Yeast
flotation viewed as the result of the interplay of supernatant composition
and cell-wall hydrophobicity. Colloids surf B 29:309319.
Dennison C, Lovrien R. 1997. Three phase partitioning: Concentration and
purification of proteins. Protein Expression Purif 11:149161.
Dickinson E. 2003. Colloidal aggregation: Mechanisms and implications. In:
Dickinson E, Van Vliet T, editors. Food colloids, biopolmers and
materials. Cambridge: The royal society of chemistry. 68 p.
Diender MB, Straathof AJJ, Van der Wielen LAM, Ras C, Heijnen JJ. 1998.
Feasibility of the thermodynamically controlled synthesis of amoxicillin. J Mol Catal B 5:249253.
Douglas NG, Humffray AA, Pratt HRC, Stevens GW. 1995. Electrophoretic
mobilities of proteins and protein mixtures. Chem Eng Sci 50:743754.
Dunin-Borkowski RE, McCartney MR, Frankel RB, Bazylinski DA, Posfai
M, Buseck PR. 1998. Magnetic microstructure of magnetotactic bacteria
by electron holography. Science 282:18681870.
Egorova EM. 1994. The validity of the Smoluchowski equation in electrophoretic studies of lipid membranes. Electrophoresis 15:11251131.
Eichhorn U, Bommarius AS, Drauz K, Jakubke HD. 1997. Syntesis of
dipeptides by suspension-to-suspension conversion via thermolysin
catalysis; from analytical to preparative scale. J Pept Sci 3:245
251.
Eonseon J, Polle JEW, Lee HK, Hyun SM, Chang M. 2003. Xanthophylls in
microalgae: From biosynthesis to biotechnological mass production and
application. J Microbiol Biotechnol 13:165174.
Erbeldinger M, Ni X, Halling PJ. 1998. Enzymatic synthesis with mainly
undissolved substrates at very high concentrations. Enzyme Microb
Technol 23:141148.
Fielden ML, et al. 1996. Surface and capillary forces affecting air bubbleparticle interactions in aqueous electrolyte. Langmuir 12:37213727.
Finch JA, Dobby GS. 1990. Column flotation. New York: Pergamon Press.
Fischer B, Sumner I, Goodenough P. 1992. Isolation and renaturation of bioactive proteins expressed in Escherichia coli as inclusion bodies. Drug
Res 42:15121516.
Fischer M, Ittah A, Gorecki M, Werber MM. 1995. Recombinant human
acetylcholinesterase expressed in Escherichia coli: Refolding, purification and characterization. Biotechnol Appl Biochem 21:295311.
Fraunholcz N, Dalmijn WL. 1998. Selective wetting of polymers by
surfactant adsorption during froth flotation. J Dispersion Sci Technol 19:
859873.
Gahr KH, Schugerl K. 1992. Recovery of yeast from cultivation medium by
continuous flotation and its dependence on cultivation conditions. Chem
Eng Sci 47:1120.
Gaudin. 1975. Flotation. New York: McGraw-Hill.
Giddings JC. 1991. Unified separation science. New York: Wiley. 145, 200
219 p.
Giddings JC. 2000. The field-flow fractionation family: Underlying
principles. In: Schimpf ME, Caldwell K, Giddings JC, editors. Fieldflow fractionation handbook. New York: Wiley. 330 p.
Giddings JC, Yang FJ, Myers MN. 1976. Flow field-flow fractionationVersatile new separation method. Science 193:12441245.
Gram H, Ramage P, Memmert K, Gamse R, Kocher HP. 1994. A novel
approach for high level production of a recombinant human parathyroid
hormone fragment in E.coli. Biotechnology 12:10171023.
Grieves RB, Wang SL. 1966. Foam separation of Escherichia coli with a
cationic surfactant. Biotechnol Bioeng 8:323336.
Grieves RB, Wang SL. 1967. Foam separation of bacteria with a cationic
surfactant. Biotechnol Bioeng 9:187194.
Guereca L, Bravo A, Quintero R. 1994. Design of an aqueous two-phase
system for the purification of ICP from Bacillus thuringiensis. Process
Biochem 29:181185.
Han MY. 2002. Modeling of DAF: The effect of particle and bubble
characteristics. J Water Supply-AQUA 50:2734.
Hansen ME, Giddings JC. 1989. Retention perturbations due to particle-wall
interactions in sedimentation field-flow fractionation. Anal. Chem.
61:811819.
Hanumantha Rao K, Forssberg KSE. 1997. Mixed collector systems in
flotation. Int J Min Process 51:6779.
Harrison STL. 1991. Bacteral cell disruption: A key unit operation in the
recovery of intracellular products. Biotech Adv 9:217240.
Hayashi H, Nihei T, Ono M, Tsuneda S, Hirata A, Sasaki H. 2001a. Rapid
recovery of bacterial cells from a stable dispersion by heterocoagulation
to a fibrous collector. J Colloid Interface Sci 243:109115.
Hayashi H, Tsuneda S, Hirata A, Sasaki H. 2001b. Soft particle analysis of
bacterial cells and its interpretation of cell adhesion behaviors in terms
of DLVO theory. Colloids surf B 22:149157.
Heeboll-Nielsen A, Choe W-S, Middelberg APJ, Thomas ORT. 2003.
Efficient inclusion body processing using chemical extraction and high
gradient magnetic fishing. Biotechnol Prog 19:887898.
Heider F, Zitzelsberger A, Fabian K. 1996. Magnetic susceptibility and
remanent coercive force in grown magnetite crystals from 0.1 mm to
6 mm. Phys Earth Planet Interiors 93:239256.
Hellebust H, Murby M, Abrahmsen L, Uhlen M, Enfors S-O. 1989. Different
approaches to stabilize a recombinant fusion protein. Bio/technology 7:
165168.
Henskens YMC, Dieijen-Visser MP. 2000. Capillary zone electrophoresis as
a tool to detect proteins in body fluids: Reproducibility, comparison with
conventional methods and a review of the literature. Ned Tijdschr Klin
Chem 25:219229.
Heywood-Waddington D, Peters TJ, Sutherland IA. 1986. The dynamics of
phase partition: A study of parameters affecting rat liver organelle
partitioning in aquous two-polymer phase systems. Biochem J 235:245
249.
Hoeben MA, Van der Lans RGJM, Van der Wielen LAM. 2004. Kinetic
model for separation of particle mixtures by interfacial partitioning.
AIChE J 50:11561168.
Hoess A, Arthur AK, Wanner G, Fanning E. 1988. Recovery of soluble,
biologically active recombinant proteins from total bacterial lysates.
Biotechnology 6:12141217.
Hogg R, Healy TW, Fuerstenau DW. 1965. Mutual coagulation of colloidal
dispersions. Trans Faraday Soc 62:16381651.
Honda J, Andou H, Mannen T, Sugimoto S. 2000. Direct refolding of
recombinant human frowth differentiation factor 5 for large-scale
production process. J Biosci and Bioeng 89:582589.
Hubbuch JJ, Thomas ORT. 2003. High-gradient magnetic affinity separation
of trypsin from porcine pancreatin. Biotechnol Bioeng 79:301313.
Ivory CF. 1993. Free-flow electrophoresis in bench-top and large-scale
bioprocessing. J Cell Biochem Suppl 17A:43.
Jane J-L, Kasemsuwan T, Leas S, Ames IA, Zobel H, Darien I, Robyt JF.
1994. Anthology of starch granule morphology by scanning electron
microscopy. Starke 46:121129.
Jauregi P, Hoeben MA, Van der Lans RGJM, Kwant G, Van der Wielen LAM.
2001. Selective Separation of physically near-identical microparicle
mixtures by interfacial partitioning. Ind Eng Chem Res 40:5815
5821.
Jauregi P, Hoeben MA, Van der Lans RGJM, Kwant G, Van der Wielen LAM.
2002. Recovery of small bioparticles by interfacial partitioning.
Biotechnol Bioeng 78:355364.
Jeaong J-C, Lee I-Y, Kin S-W, Park Y-H. 1999. Stimulation of b-carotene
synthesis by hydrogen peroxide in Blakeslea trispora. Biotechnol Lett
21:683686.
Jones R, Preece AW, Luckman NP, Forrester JA. 1983. The analysis of the red
cell unsaturated fatty acid test for multiple sclerosis using laser
cytopherometry. Phys Med Biol 28:11451151.
Kalyuzhnyi MY, Petrushko GM, Novikova GP. 1965. Flocculation of
candida utilis and candida tropicalis yeast cells and its relation to
flotation. Microbiol 34:918924.
Kasche V, Galunsky B. 1995. Enzyme catalyzed biotransformations in
aqueous two-phase systems with precipitated substrate and/or product.
Kiss E, Szamos J, Tamas B, Borbas R. 1998. Interfacial behaviour of proteins
in three-phase partitioning using salt-containing water/tert-butanol
systems. Colloids Surf A 142:295302.
Kitano K, Nakao M, Itoh Y, Fujisawa Y. 1987. Recombinant hepatitis B virus
surface antigen P31 accumulates as particles in Saccharomyces
cerevisiae. Biotechnology 5:281283.

19
Koliadima A. 1999. Optimization of experimental conditions for characterizing colloids and macromolecules by field-flow fractionation. J Liq
Chrom Rel Technol 22:27632777.
Koliadima A, Karaiskakis G. 1990. Potential-barrier field-flow fractionation,
a versatile new separation method. J Chromatogr 517:345359.
Koller G, Graumann K, Kramer W, Sara M, Jungbauer A. 1995. Laboratoryscale production and purification of recombinant HIV-1 reverse
transcriptase. J Chromatogr B 664:107118.
Kometani T, Sakai Y, Matsumae H, Shibatani T, Matsuno R. 1997.
Production of (s2,3s)-2,3-dihydro-3-hydroxy-2-(4-methoxyphenyl)1,5-benzothiazepin-4(5h)-one, a key intermediate for diltiazem synthesis, by bakers yeast-mediated reduction. J Fermentation Bioeng 84:
195199.
Kuhl P, Halling PJ, Jakubke HD. 1990. Chymotrypsin suspended in organic
solvents with salt hydrates is a good catalyst for peptide synthesis from
mainly undissolved reactants. Tetrahedron Lett 31:5216.
Kuiper M, Sanches RM, Walford JA, Slater NKH. 2002. Purification of a
functional gene therapy vector from Moloney Murine Leukaemia Virus
using membrane filtration and ceramic hydroxyapatite chromatography.
Kula MR. 1986. Aqueous phase separation. Bioact Microb Prod 3:103120.
Kula MR. 1993. Scaling-up protein extraction in aqueous two-phase
systems. J Cell Biochem Suppl 17A:40.
Kula MR, Schutte H, Vogels G, Frank A. 1990. Cell disintegration for the
purification of intracellular proteins. Food Biotechnol 4:169183.
Kumagai H. 1999. Dihydroxyphenylalanine. In: Flickinger MC, Drew SJ,
editors. Encyclopedia of bioprocess technology. New York: Marcel
Dekker. 821 p.
LaVallie ER, DiBlasio EA, Kovacic S, Grant KL, Schendel PF, McCoy JM.
1993. A thioredoxin gene fusion expression system that circumvents
inclusion body formation in E.coli cytoplasm. Biotechnology 11:187
193.
Lee DC, Park JH, Kim GJ, Kim HS. 1999a. Modeling, simulation, and kinetic
analysis of a heterogeneous reaction system for the enzymatic
conversion of poorly soluble substrate. Biotechnol Bioeng 64:272283.
Lee SY, Choi J-I, Wong HH. 1999b. Recent advances in polyhydroxyalkanoate production by bacterial fermentation: Mini-review. Int J Biol
Macromol 25:3136.
Leuenberger HGW. 1985. Microbiologically catalysed reaction steps in the
field of vitamin and carotenoid synthesis. In: Tramper J, Van der Plas
HC, Linko P, editors. Biocatalysts in organic syntheses. Amsterdam:
Elsevier. 99 p.
Leung AKW, Park MMV, Borhani DW. 1999. An improved method for
protein crystal density measurement. J Appl Crystallogr 32:1006
1009.
Li C, Somasundaran P. 1991. Reversal of Bubble Charge in multivalent
inorganic salt solutions- Effect of magnesium. J Colloid Interface Sci
146:215218.
Li C, Somasundaran P. 1992. Reversal of bubble charge in multivalent
inorganic salt solutionsEffect of aluminum. J Colloid Interface Sci
148:587591.
Lins U, Farina M. 1999. Phosphorus-rich granules in uncultured magnetotactic bacteria. FEMS Microbiol Lett 172:2328.
Lipschutz JH, Guo W, OBrien LE, Nguyen YH, Novick P, Mostov KE. 2000.
Exocyst is involved in cystogenesis and tubulogenesis and acts by
modulating synthesis and delivery of basolateral plasma membrane and
secretory proteins. Mol Biol Cell 11:42594275.
Marchessault RH, Monasterios CJ, Lepoutre P. 2001. Properties of poly-hydroxyalkanoate latex: Nascent morphology, film formation and
surface chemistry. In: Dawes EA, editor. Novel biodegradable microbial
polymers. Dordrecht: Kluwer. 97 p.
Matsumae H, Douno H, Yamada S, Nishida T, Ozaki Y, Shibatani T, Tosa T.
1995. Microbial asymmetric reduction of (RS)-2-(4-methoxyphenyl)1,5-benzothiazepin-3,4(2H,5H)-dione, the key intermediate in the
synthesis of diltiazem hydrochloride. J Fermentation Bioeng 79:
2832.
Matthews BW. 1974. Determination of molecular weight from protein
crystals. J Mol Biol 82:513526.
20
McCloskey KE, Chalmers JJ, Zborowski M. 2003. Magnetic cell separation:

Characterization of magnetophoretic mobility. Anal Chem 75:6868
6874.
Meijer A, Vallinga CE, Ossewaarde JM. 1996. A microcarrier culture
method for the production of large quantities of viable Chlamydia
pneumoniae. Appl Microbiol Biotechnol 46:132137.
Melville D, Paul F, Roath S. 1975. Direct magnetic separation of red-cells
from whole-blood. Nature 255:706.
Micale FJ, Krumrine PH, Vital TJ, Van der Hoff JW. 1980. Experimental
parameters involved in the separation of colloidal particles by
continuous electrophoresis. Colloids Surf 1:373386.
Michielsen MJF, Frielink C, Wijffels RH, Tramper J, Beeftink HH. 2000a.
Growth of Ca-D-malate crystals in a bioreactor. Biotechnology and
Bioengineering 69:548558.
Michielsen MJF, Frielink C, Wijffels RH, Tramper J, Beeftink HH. 2000b.
Modeling solid-to-solid biocatalysis: Integration of six consecutive
steps. Biotechnol Bioeng 69:597606.
Miller TL. 1985. Steroid fermentations. In: Moo-Young M, et al., editors.
The principles, applications and regulations of biotechnology in
industry, agriculture and medicine. Oxford: Pergamon Press. 297 p.
Mitraki A, King J. 1989. Protein folding intermediates and inclusion body
formation. Bio/technology 7:690697.
Moran E. 1999. A microcarrier-based cell culture process for the production
of a bovine respiratory syncytial virus vaccine. Cytotechnol 29:135
148.
Morgan H, Green NG, Hughes MP, Monaghan W, Tan TC. 1997. Large-area
travelling-wave dielectrophoresis particle separator. J Micromech
Microeng 7:6570.
Mosqueira FG, Higgins JJ, Dunnill P, Lilly MD. 1981. Characteristics of
mechanically disrupted bakers yeast in relation to its separation in
industrial centrifuges. Biotechnol Bioeng 23:335343.
Nakayama K. 1985. Tryptophan. In: Moo-Young M, editor. Comprehensive
biotechnology. Oxford: Pergamon Press. 621 p.
Netz RR. 2003. Polyelectrolytes in electric fields. J PhysChem B 107:8208
8217.
Nguyen AV. 2003. New method and equations for determining attachment
tenacity and particle size limit in flotation. Int J Miner Process 68:
167182.
Nikolai TJ, Hu W-S. 1992. Cultivation of mammalian cells on macroporous
microcarriers. Enzyme Microb Technol 14:203208.
Okada K, Akagi Y, Kogure M, Yoshioka N. 1990a. Analysis of particle
trajectories of small particles in flotation when the particles and bubbles
are both charged. Can J Chem Eng 68:614621.
Okada K, Akagi Y, Kogure M, Yoshioka N. 1990b. Effect on surface charges
of bubbles and fine particles on air flotation process. Can J Chem Eng
68:393399.
Oppermann-Sanio FB, Steinbuchel A. 2002. Occurence, functions and
biosynthesis of polyamides in microorganisms and biotechnological
production. Naturwissenschaften 89:1122.
Owen CS. 1978. High-gradient magnetic separation of erythrocytes.
Biophys J 22:171178.
Palmieri MC, Greenhalf W, Laluce C. 1996. Efficient flotation of Yeast cells
grown in batch culture. Biotechnol and Bioeng 50:248256.
Perry RH, Green DW, Maloney JO. 1998. Perrys chemical engineers
handbook. New York: McGraw-Hill. p 1948, 1953.
Pike RN, Denisson C. 1989. Protein fractionation by three phase partitioning
(TTP) in aqueous/t-butanol mixtures. Biotechnol Bioeng 33:221228.
Preece AW, Luckman NP. 1981. A laser doppler cytopherometer for
measurement of electrophoretic mobility of bioparticles. Phys Med Biol
26:1118.
Preusting H, Hazenberg W, Witholt B. 1993. Continuous production of
poly(3-hydroxyalkanoates) of Pseudomonas oleovorans in a high-celldensity, two-liquid-phase chemostat. Enzyme Microb Technol 15:311
316.
Proksch RB, Schaffer TE, Moskowitz BM, Dahlberg ED, Bazylinski DA,
Frankel RB. 1995. Magnetic force microscopy of the submicron
magnetic assembly in a magnetotactic bacterium. Appl Phys Lett
66:25822584.

DOI 10.1002/bit
Rabinovich YI, Yoon R-H. 1994. Use of atomic force microscope for the
measurement of hydrophobic forces between silanated silica plate and
glass sphere. Langmuir 10:19031909.
Radbruch A, Mechtold B, Thiel S, Miltenyi S, Pfluger E. 1994. High-gradient
magnetic cell sorting. Methods Cell Biol 42:387403.
Rinas U, Tsai LB, Lyons D, Fox GM, Stearns G, Fieschko J, Fenton D, Bailey
JE. 1992. Cysteine to serine substitutions in basic fibroblast growth
factor: Effect on inclusion body formation and proteolytic susceptibility
during in vitro refolding. Biotechnology 10:435440.
Sadowski Z, Golab Z. 1991. Flotation of Streptomyces pilosus after lead
accumulation. Biotechnol Bioeng 37:955959.
Sangster J. 1997. Octanol-Water partition coefficients: Fundamentals and
physical chemistry. New York: Wiley. 134 p.
Sanin FD, Vesilind PA. 1996. Synthetic sludge: A physical/chemical model
in understanding bioflocculation. Water Environ Res 68:927933.
Schulze HJ. 1984. Physico-chemical elementary processes in flotation.
Developments in mineral processing. Amsterdam: Elsevier.
Shaw DJ. 1966. Introduction to colloid and surface chemistry. London:
Butterworths.
Shively JM. 2003. Inclusion bodies of prokaryotes. Annu Rev Microbiol
28:167187.
Siddiqi SF, Titchener-Hooker NJ, Ayazi Shamlou P. 1996. Simulation of
particle size distribution changes occuring during high-pressure
disruption of bakers yeast. Biotechnol Bioeng 50:145150.
Spassov G, Pramatarova V, Wandrey C. 1996. Conversion of corticosteroid
hormones by immobilized cells of Flavobacterium dehydrogenans,
Curvularia Iunata and Arthrobacter simplex on ceramic carriers. Appl
Microbiol Biotechnol 46:481488.
Straathof AJJ, Wolff A, Heijnen JJ. 1998. Solid-to-solid kinetic resolution
Determination of the enantiomeric ratio. J Mol Catal 5:5561.
Takahashi S. 2003. Microbial production of D-p-hydroxyphenylglycine. In:
Aida K, editor. Biotechnology of amino acid production. Tokyo:
Kodansha LTD. p. 269279.
Tan KH, Lovrien R. 1972. Enzymology in aqueous-organis cosolvent binary
mixtures. J Biol Chem 247:32783285.
Taylor G, Hoare M, Gray DR, Marston FAO. 1986. Size and density of
protein inclusion bodies. Biotechnology 4:553557.
Tils HMGC, Tels M. 1992. A study into fine particle flotation separation
characteristics with application to centrifugal force field flotation. Int J
Min Process 36:201217.
Tsoka S, Ciniawskyj OC, Thomas ORT. 2000. Selective Flocculation and
precipitation for the improvement of virus-like particle recovery from
yeast homogenate. Biotechnol Progr 16:661667.
Tybussek R, Linz F, Schugerl K, Moses N, Leonard AJ, Rouxhet PG. 1994.
Comparison of the continuous flotation performances of Saccharomyces
cervisiae LBG H620. Appl Microbiol Biotechnol 41:1322.
Ulijn RV, De Martin L, Gardossi L, Halling PJ. 2003. Biocatalysis in reaction
mixtures with undissolved solid substrates and products. Curr Org Chem
7:13331346.
Valax P, Georgiou G. 1993. Molecular characterization of b-lactamse
inclusion bodies produced in Escherichia coli. 1. Composition. Biotechnol Progr 9:539547.
Van de Ven TGM. 1989. Colloidal hydrodynamics. London: Academic Press
Inc. p 69, 277.
Van der Wal A, Minor M, Norde W, Zehnder AJB, Lyklema J. 1997.
Electrokinetic potential of bacterial cells. Langmuir 13:165171.
Van der Wielen LAM, Potters JJM, Straathof AJJ, Luyben KCAM. 1990.
Integration of bioconversion and continuous product separation
by means of countercurrent adsorption. Chem Eng Sci 45:23972404.
Van der Wielen LAM, Diepen PJ, Houwers J, Luyben KCAM. 1996. A
countercurrent adsorptive reactor for acidifying bioconversions. Chem
Eng Sci 51:23152325.
Van der Wielen LAM, Van Dam MHH, Luyben KCAM. 1997. Trickle flow of
dense particles through a fluidised bed of others. Chem Eng Sci 52:553
565.
Vlaski A, Van Breemen AN, Alaerts GJ. 1996. Optimisation of coagulation

conditions for the removal of cyanobacteria by dissolved air flotation or
sedimentation. J Water SRT 45:253261.
Wahlund KG, Litzen A. 1989. Application of an asymetrical flow field-flow
fractionation channel to the separation and characterisation of proteins,
plasmids, plasmid fragments, polysaccharides and unicellular algae.
J Chomatogr 461:7387.
Walker SG, Lyddiatt A. 1999. Processing of nanoparticulate bioproducts:
Application and optimisation of aqueous two-phase systems. J Chem
Technol Biotechnol 74:250255.
Wang S, Kretzemer G, Schugerl K. 1994. Comparison of the morphologies of
a flotable and non-flotable strain of Saccharomyces cerevisiae. Appl
Microbiol Biotechnol 41:537543.
Wangsa-Wirawan ND, Ikai A, ONeill BK, Middelberg APJ. 2001a.
Measuring the interaction forces between protein inclusion bodies and
an air bubble using an atomic force microscope. Biotechnol Progr
17:963969.
Wangsa-Wirawan ND, ONeill BK, Middelberg APJ. 2001b. Physicochemical characteristics of LR3-IGF1 protein inclusion bodies: Electrophoretic mobility studies. Biotechnol Progr 17:786790.
Washizu M, Suzuki S, Kurosawa O, Kawabata T. 2000. Dielectrophoretic
handling of bio-molecules and its application to bio-separation. Trans
Institute Electron Inf Comm Eng C (J83-C):18.
Weast RC, Astle MJ, Beyer WH. 1922. CRC Handbook of chemistry and
physics. Boca Raton: CRC Press. C722 p.
Weast RC, Selby SM, Hodgman CD. 1964. Handbook of chemistry and
physics. Cleveland: The chemical rubber co.
Wetzel R, Perry LJ, Veilleux C. 1991. Mutations in human interferon gamma
affecting inclusion body formation identified by a general immunochemical screen. Biotechnology 9:731737.
Winitzer S. 1973a. Separation of solids at liquid-liquid interface. Sep Sci
8:4556.
Winitzer S. 1973b. Separation of solids at liquid-liquid interface: Theory.
Sep Sci 8:647659.
Wolff A, Zhu L, Kielland V, Straathof AJJ, Jongejan JA, Heijnen JJ. 1997.
Simple dissolution-reaction model for enzymatic conversion of
suspension of solid substrate. Biotechnol Bioeng 56:440.
Wong HH, ONeill BK, Middelberg APJ. 1997a. Centrifugal processing of
cell debris and inclusion bodies from recombinant Escherichia coli.
Bioseparation 6:361372.
Wong HH, ONeill BK, Middelberg APJ. 1997b. Cumulative sedimentation
analyses of Escherichia coli debris size. Biotechnol Bioeng 55:556
564.
Yan N, Zhang M, Ni P. 1992. Size distribution and zeta-potential of
polyamide microcapsules. J Membr Sci 72:163169.
Yan Y, Bronscheuer UT, Cao L, Schmid RD. 1999. Lipase-catalyzed
solid-phase synthesis of sugar fatty acid esters. Removal of byproduct
by azeotropic distillation. Enzyme Microb Technol 25:725728.
Young RAL. 1976. The applications and techniques of cell electrophoresis
and the Zeiss rectangular chamber cytopherometer. Biomed Eng 11:
373377.
Youshko MI, Van Langen LM, De Vroom E, Van Rantwijk F, Sheldon RA,
Svedas VK. 2002. Penicillin acylase-catalysed ampicillin synthesis
using a pH gradient: A new approach to optimization. Biotechnol
Bioeng 78:589593.
Zborowski M, Ostera GR, Moor LR, Milliron S, Chalmers JJ, Schechter
AN. 2003. Red blood cell magnetophoresis. Biophys J 84:263
2645.
Zhang J, Li Y, Fan L-S. 2000. Discrete phase simulation of gas-liquid-solid
fluidization systems: Single bubble rising behaviour. Powder Technol
113:310326.
Zmijewski MJ, Briggs BS, Thompson AR, Wright IG. 1991. Enantioselective acylation of a beta-lactam intermediate in the synthesis of
loracarbef using penicillin G amidase. Tetrahedron Lett 32:1621
1622.

21

Van Hee 2006

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Van Hee 2006

Uploaded by

Copyright:

Available Formats

Strategy for Selection of Methods for

Separation of Bioparticles From Particle

Keywords: particle-particle separation; bioparticle; inclusion body; cell debris; recovery

2006 Wiley Periodicals, Inc.

Week, April 19, 1995) and a turnover of &$320 million per

et al., 2003), thus leading to the design of processes yielding

OVERVIEW OF PARTICLE MIXTURES IN

Table I. References on biotechnological processes that yield particle mixtures.

Intracellular particulate products produced

VLPs produced produced with microorganisms

Biotechnology and Bioengineering, Vol. xx, No. x, month xx, xxxx

Van Hee et al.: Separation of Bioparticles from Particle Mixtures

Biotechnology and Bioengineering. DOI 10.1002/bit

Hypothesis: sulfur storage

(Lins and Farina, 1999; Shively, 2003)

(Bowden et al., 1991; Fischer et al., 1992;

Aggregation, incorrect folding,

Thiorhodaceae and other

, contains Fe3O4 or iron sulphides

Many (mainly recombinant)

Literature overview of intracellular particulate microbial products.

Type of inclusion body

listed of the various categories of particle mixtures that are

Spassov et al., 1996). For instance, both the substrate and

Biotechnology and Bioengineering, Vol. xx, No. x, month xx, xxxx

phase. In many cases, the surfaces of bioparticles contain

Van Hee et al.: Separation of Bioparticles from Particle Mixtures

Biotechnology and Bioengineering, Vol. xx, No. x, month xx, xxxx

0.55.0 (Agerkvist and Enfors, 1990; Bowden,

1,000-1,540 (Absolom et al., 1986; Bell et al., 1983; Jauregi et al.,

900-1,260 (Preusting et al., 1993; Taylor et al., 1986; Wong

1,0611,090 (Wong et al., 1997b)

1,040 (Lipschutz et al., 2000; Nikolai and Hu, 1992)

1,0801,120 (Erbeldinger et al., 1998; Wong et al., 1997b)

Fungi and algae

Whole cells bacterial

Table III. An overview of bioparticle characteristics.

311 (Weast et al., 1964; Chang, 1981)**

5.0 (Wangsa-Wirawan et al., 2001b)*

2.8 (Wangsa-Wirawan et al., 2001b)*

3.05.0 (Hayashi et al., 2001a,b)*

where c is the particle volume fraction, h the apparent

FORCES IN PARTICLEPARTICLE SEPARATION

External Field Forces

where a is the centrifugal acceleration (m/s ), Dr the density

where 1/k is the Debye decay length (m), e the elementary

where q is the particle charge (C), E the electrical field

Van Hee et al.: Separation of Bioparticles from Particle Mixtures

bacteria and red blood cells. Magnetotactic bacteria use

where w is the magnetic susceptibility of the particle (), H

magnetic field intensity. Nevertheless, if the saturation

Biotechnology and Bioengineering, Vol. xx, No. x, month xx, xxxx

interaction force can be approximated with the following

where z is the zeta-potential of the surface interacting

for h << dp << d; k dp >> 1 and

before, the Hamaker constant for bioparticles lies in the range

Interfacial Tension Force

where g is the interfacial tension (N/m) and y is the

Van Hee et al.: Separation of Bioparticles from Particle Mixtures

where the particle is pulled into. The increase in energy of

Lyddiatt, 1999) and other liquidliquid systems (Borbas

Biotechnology and Bioengineering, Vol. xx, No. x, month xx, xxxx

cases dependent on other forces and can only be calculated

where Fi are the driving forces and Fj are the dynamic

for 0:5 < Re < 6 103 :

At Reynolds numbers between 5 102 and 105 the drag