Cinética PEF

1.
12 Modeling and Simulation of PEF

Justus Knappert, Tobias J Horneber, Christopher McHardy, and Cornelia Rauh, Fachgebiet Lebensmittelbiotechnologie und
–prozesstechnik, Technische Universität Berlin, Berlin, Germany
© 2021 Elsevier Inc. All rights reserved.
1.12.1 Introduction 212

1.12.2 Background and Fundamentals 214
1.12.2.1 Governing Equations 214
1.12.2.2 Process Dependence of Fluid Properties 216
1.12.2.3 Kinetics of Cell Inactivation and Their Implementation in Numerical Simulations 217
1.12.2.4 Modeling Cell Inactivation on the Microscale 219
1.12.3 Case Studies of PEF Process Design and Scale-up by Means of Numerical Simulation 221
1.12.3.1 Case Study 1: Improvement of the Treatment Homogeneity 221
1.12.3.2 Case Study 2: Permeabilization of Microalgae by Pulsed Electric Field 226
1.12.3.2.1 Kinetic 226
1.12.3.2.2 Simulation 229
1.12.3.3 Case Study 3: Impact of Treatment Chamber Design on the Inactivation of Microalgae 233
1.12.4 Validation 238
1.12.5 Conclusion 238
References 239
Nomenclature
Latin Characters
a Cell radius ½m
al Surface of a lipid molecule ½m2
cp Heat capacity ½J kg1 K1
c Mass concentration ½kg m3
Cm Capacity of the cell membrane ½C
Dp Diffusion coefficient for pore radius ½m2 s1
dc Distance to the cell center ½m
e Internal energy ½J
E Electric field ½V m1
Ee Edge energy at the edge of a pore ½J
f Frequency ½s1
F Force density vector ½N m3
Fmax Max. Electric force for a transmembrane potential of 1 V ½N
gS Surface conductance ½m1
g Gravity ½ms2
Ip Electric current through pores ½A
Jfree Free charge ½As1
k Turbulent kinetic energy ½J kg1
ki;j ; ki;j;k ; ki;j;k;l Strength parameter for the force fields
kf 0 Pore formation rate ½s1
kB Boltzmann constant ½J K1
mi Mass of the ith atom ½kg
mI Mass of inactivated cells ½kg
m0 Initial mass of cells ½kg
n Flow behavior index ½
N Pore number density ½m2
Neq Equilibrium pore density ½m2
ncrit Critical number of pores ½
nP Number of pores ½
Innovative Food Processing Technologies, Volume 1 https://doi.org/10.1016/B978-0-12-815781-7.00020-2 211

p Pressure ½Pa
pi Momentum of the ith atom ½kgms1
q Heat flux vector ½Js1
RA Residual activity ½
rj Radius of a considered pore j ½m
rij Distance between the ith and the jth atom ½m
r Radius of a pore, corresponding to a specific energy barrier ½m
t; tt Time, treatment time ½s
T; Tave ; Tmax Temperature in Kelvin, average maximum ½K
u Velocity vector ½ms1
Up Velocity of the pore radii development ½ms1
U Time averaged velocity ½ms1
u’i Velocity fluctuation term ½ms1
vm Area averaged velocity ½ms1
vi Velocity of the ith atom ½ms1
Vep Characteristic voltage of electroporation ½V
Wspec Specific energy input ½kJ kg1
DWf Energy barrier for pore formation ½J
x Coordinate vector ½m
Greek Characters
a Angle to the surface normal ½
aW ; gW Scale and shape parameter for the Weibull distribution ½s1 ; ½
aK Creation rate coefficient ½m2 s1
aijkl Torsion angle between the ith, jth, kth and lth atom ½
bK Steric repulsion energy ½J
geff Effective surface tension of the cell membrane ½N m1
g_ Shear rate ½s1
d Kronecker delta tensor ½
εw ; εm Relative permittivity of the membrane and the water inside a pore ½F m1
ε Turbulent kinetic energy dissipation ½J kg1 s1
qijk Bend angle between the ith, jth and kth atom ½
kt Turbulent heat conductivity ½Wm1 K1
k Heat conductivity ½Wm1 K1
K Flow consistency index ½kg m1 s1
m Dynamic viscosity ½Pas
mr ; ms Relative viscosity/Viscosity of suspending medium ½Pas
½m Intrinsic viscosity ½
mt Turbulent eddy viscosity ½Pas
n Frequency of the molecule fluctuation ½s1
pI Inactivation source term ½kg s1 m3
pe Energy source term ½W m3
r Density ½kg m3
rJ Charge density ½As m3
s; si ; se Electric conductivity, internal and external of a cell ½S m1
sp Electric conductivity inside a pore ½S m1
s Stress tensor ½Pa
sP Pulse width ½s
s0 Yield stress ½Pa
F Electric potential ½V
DF0 Resting potential of a cell ½V
Fe Electric potential outside a cell ½V
Fm Transmembrane potential ½V
Ftot ; Fbonded ; Fnonbonded Total potential of a molecule and potential of bonding and nonbonding interactions ½J
Modeling and Simulation of PEF 213
Fmax Maximum volume fraction of suspended particles ½

Fn Volume fraction of suspended particles ½
u Vorticity ½s1
1.12.1 Introduction
Since the consumer demand for healthy and high-quality food is on the rise, new technologies are required to provide safe but nutri-
tional valuable and shelf-stable foods. Conventional technologies, such as thermal pasteurization, cannot meet these requirements
because many valuable nutrients are heat sensitive. Furthermore, their demand for energy is high which increases the overall
production costs. Other possibilities, such as chemical preservatives, are not wanted by consumers anymore and clean labels
with a short list of ingredients are trending. Thus, alternatives to thermal and chemical treatments are needed which lead to similar
or even better products with regard to taste, nutritional value and shelf life. The technology of pulsed electric fields (PEF) processing
can fit these requirements. The treatment effect is based on the exposure of matter to a pulsed electric field whose field strength is in
the order of several thousand volt per cm (kVcm1 ). Because the electric field is only active on a short time scale of micro- or nano-
seconds, which is considered as pulse, the temperature increase is moderate if the repetition rate or frequency of the pulsation is not
too high. Therefore, PEF can be considered as a non-thermal treatment. Nevertheless, the temperature increase due to the internal
energy generation by the electric field (Joule heating) has to be considered for a comprehensive process design. Besides the electric
field strength and the temperature, the treatment time is a major factor for a successful treatment.
The effect of PEF on biological matter takes place on a molecular level and the primary target of the treatment is a reduced integ-
rity of the cell membrane (Heinz et al., 2001). Even if the underlying mechanism is not fully understood, it is broadly accepted that
a difference of the electric potential across the cell membrane (transmembrane potential) is the driving force for the formation of
pores. Due to its lipophilic nature, the membrane can be regarded as a capacitor with a low electric conductance. Therefore, carriers
of opposite charges accumulate on the inside and the outside of the cell, respectively, which leads to an increase of the transmem-
brane potential. The transmembrane potential increases linearly with the strength of the electric field (Saulis, 2010) and above a crit-
ical value, the pores in the membrane start to form. This step is called electroporation or electropermeabilisation of the cell
membrane.
Other factors which affect the formation of pores are the pulse shape, the pulse duration and the pulse frequency, which together
define the total treatment time. Besides, parameters which describe the cell morphology affect the treatment, e.g., size or circularity
(Toepfl et al., 2014). For the simplest configuration of a PEF treatment chamber, which is the batch treatment chamber with parallel
plates and rectangular pulses, the total treatment time is the product of pulse duration and number of pulses. The electric field
strength is uniform across the treatment chamber for this configuration. Thus, the effect of the electric field strength on the electro-
poration of cellular material, like tissue or microorganisms, can be investigated and separated from other effects in this particular
case. However, continuous treatment chambers with a colinear electrode configuration are mainly used in practice since a high
throughput is essential for successful commercial applications. The advantage of a continuous treatment is related to the disadvan-
tage of inhomogeneous distributions of the electric field, temperature field and velocity field within the treatment chamber. This
makes the separation of the different effects on the treatment in experiments almost impossible. Furthermore, due to the strong
dependency of the mentioned physical fields on the treatment chamber design, it is hard to compare experimental results which
are obtained in different treatment chambers. This problem can be solved by numerical simulations, as they provide spatially
and temporally resolved information about the different fields.
Validated with experimental data, a comprehensive model of the PEF process can be used to scale treatments from a laboratory
scale to pilot or even industrial scale. A model can also be used to investigate the influence of the treatment parameters or the treat-
ment chamber design on the treatment homogeneity. Thus, numerical simulation of PEF is a powerful tool to save resources, espe-
cially time, labor, and material, which is important from an economical perspective.
From an engineering point of view, macroscopic effects like the velocity profile, the spatial distribution of the electric field, the
inactivation rate of microorganisms, or the average and peak temperatures are of interest. Since the underlying mechanism of the
treatment is the formation of pores, microscopic effects must be considered as well for a comprehensive model. Microscopic
phenomena like chemical reactions or the formation and evolution of pores take place on length scales of nanometers to microm-
eters and timescales of nanoseconds to microseconds. Before the manifold possibilities for the application of the numerical simu-
lation of PEF are discussed in section “Case Studies of PEF Process Design and Scale-up by means of Numerical Simulation”, the
theoretical background for modeling on a macroscopic scale is given in section “Background and Fundamentals”. This article
includes an overview of the relevant transport equations, which balance mass, momentum, internal energy, and free charges. It
is discussed how these equations are simplified to be in a good agreement with reality, while their solution remains possible
with an acceptable computational effort. Moreover, it is described how physical properties, like the temperature dependency of
material properties, the rheological properties of the fluid or the inactivation kinetic for microorganisms can be included within
a simulation. Nevertheless, also the knowledge about the microscopic effects, such as the size and number of pores is of interest
for technical applications like the extraction of valuable components or drying of tissue. Therefore, the modeling of microscopic
effects will also be discussed in section “Modeling Cell Inactivation on the Microscale”. Furthermore, a brief introduction to the
simulation of PEF on the molecular scale is given in this section.
As it was mentioned before, gaining local information about the physical quantities by experiments is often not possible due to
technical limitations of the available measurement techniques. This limits the experimental validation of numerical models. A brief
overview of the possibilities for the validation and their limitations are therefore discussed in section “Validation”.
1.12.2 Background and Fundamentals
The modeling of PEF on a macroscopic scale is based on balancing the quantities in question within a small control volume. In the
case of a continuous PEF process these are the conservative quantities mass, momentum, energy and free electric charges. The
balancing equation for each of these quantities describes the temporal change inside a control volume, which is given by the convec-
tive and diffusive transport through the surface of the volume and the generation or degeneration by a source or sink within the
volume, respectively. To solve these differential equations, appropriate boundary and initial conditions are needed in order to
get a closed system of equations. Since analytical solutions of the differential equations are often not available, numerical solutions
must be obtained. In the case of computational fluid dynamics (CFD), the finite element method or the finite volume method are
used, which transform the governing partial differential equations to a system of algebraic equations. These equations are solved
iteratively on a computer by minimizing the residual error which results from the approximation of the derivation terms of the
differential equations. Therefore, numerical simulations are not similar to reality, but if the residual error is small, the solution
approximates the formulated problem in a sufficient way. To get a stable approximate solution of the differential equations, the
domain has to be discretized within the spatial as well as the temporal dimensions. This step always contains a compromise
between the quality of the numerical solution and the required computational costs. Since special topics like meshing or consis-
tency, stability and convergence of the numerical method are outside the scope of this article, the interested reader is redirected
to the fundamental literature on CFD-simulation (Ferziger and Peric, 2008).
1.12.2.1 Governing Equations

The governing equations for the modeling of the PEF process are the balance equations of mass, momentum, internal energy and
free electric charges. This modeling approach is the common method of modeling PEF and was used in many publications (Fiala
et al., 2001; Wölken et al., 2017). The balance equations for mass, momentum and internal energy in cartesian coordinates can be
written as:
vr vruj
Mass : þ ¼0 (1)
vt vxj
vrui vrui uj sij

Momentum in ith direction : þ ¼ Fi ði ˛ ½1; 2; 3Þ (2)
vt vxj
vre vreuj þ qj vui

Internal energy : þ ¼ rsij þ rpe (3)
vt vxj vxj
Here, r is the density of the fluid, t is the time, and u and x are the velocity and coordinate vectors, respectively. The indices i and j
represent the considered directions. The internal energy is indicated by the letter e. The internal diffusive transport of momentum is
represented by the stress tensor s. For the internal energy equation, q describes the transport of energy through heat conduction. On
the right-hand side of Eq. (2), F represents the force density vector which describes external fields influencing the fluid flow. A
simple example for this term is the gravitational force g in the direction of xi . The two terms on the right-hand side of Eq. (3)
are source terms for the internal energy. The first one describes the increase of the internal energy of the fluid through internal fric-
tion. The second one, represented by pe , is the internal energy production term. It describes the energy increase through an
exothermal reaction or, in the case of PEF, the energy input by an electric field. In cartesian coordinates, the system of the partial
differential Eqs. (1)–(3) provides five equations for the five unknown field variables, namely mass or density, the velocity compo-
nents in each spatial dimension and the internal energy. To obtain a closed and therefore solvable system of equations, one needs
further equations to describe all the components of s, q and pe . Diffusive terms for mass, momentum and energy can be described
by the laws of Fick, Newton or Fourier respectively. The choice of appropriate equations depends strongly on the considered
problem. For example, the material characteristics of the processed fluids can vary greatly in the field of food processing and
non-Newtonian flow behavior or temperature-dependency of the transport coefficients might be relevant. In order to reduce the
complexity of the system, several assumptions have to be made. Since the time scale of the electric field is very small in comparison
to the time scale of the flow field, the first assumption for the simulation of PEF is to assume a quasi-stationary electric field. That
means, the electric pulse is averaged over the time. The second assumption is the steady state for the flow field. Therefore, all time
derivatives within the balancing equations can be neglected. The third assumption is the incompressibility of the fluid. Therefore,
Eq. (1) simplifies to:
vuj
¼0 (4)
vxj
The assumption of incompressibility is valid for most liquids in flows which are characterized by a Mach numbers smaller than
0.3. In the case of a compressible fluid, the system of differential equations needs to be extended by a constitutive equation
describing the density of the fluid in dependency of the pressure. A detailed overview on the consideration of compressible fluids
in CFD models can be found in the book of Ferziger and Peric (Ferziger and Peric, 2008). Furthermore, the assumption of the
incompressibility leads to the following expression of the stress tensor si;j in Eq. (2):

vui vuj
si;j ¼ pdij þ m þ (5)
vxj vxi
Here, p is the static pressure, dij is the Kronecker delta and m is the dynamic viscosity of the liquid. In this case, the fluid is to be
considered as Newtonian. This is synonymic to a viscosity being independent of the shear stress. As mentioned above, in many
applications of food processing this assumption is invalid. For example, ketchup exhibits shear thinning behavior, which has a large
impact on the flow field within the treatment chamber and eventually on the treatment homogeneity. In such a case, the depen-
dency of the viscosity on the shear stress can be described by a suitable flow law, for example the Ostwald-de-Waele model or
the Hershel-Bulkley model. The force term F on the right-hand side of Eq. (3) can be used to consider the effect of external force
fields like gravity, magnetic or electric forces in the momentum balance equation. This is for example necessary if a multicomponent
fluid with charged particles needs to be modeled. In most cases, the assumption of uncharged particles is valid, and the force term
only represents the influence of the gravity, which is assumed to act in the negative of the z-direction in cartesian coordinates
(depending on the orientation of the domain). In this case, the balance equation for momentum can be written as:

vui vp v vui vuj
ruj þ m þ ¼ rFi ; F ¼ ð0; 0; gÞ (6)
vxj vxj vxj vxj vxi
The assumption of incompressibility leads also to a simplification of the balance equation for the internal energy. If no work is
done for a change of the volume, the first law of thermodynamics states that the internal energy can be expressed only by the thermal
energy. Therefore, Eq. (3) can be written as:

vT v vT
rui cp þ k ¼ pe (7)
vxi vxi vxi
Here, cp is the heat capacity of the fluid. In Eq. (7), the diffusive transport of thermal energy q is described by the Fourier law for
heat conduction. In this case, k represents the thermal conductivity of the fluid. Note that the first term of the right-hand side of Eq.
(3) is neglected in Eq. (7). This term represents the increase of the inner energy through viscous dissipation and must only be
considered for highly viscous fluids if internal friction is significant or if large velocity gradients occur. Since an electric field causes
an increase of the internal energy, these two fields must be coupled. Therefore, the source term pe in Eq. (7) is expressed by Joule
heating. In case of steady state simulation of a continuous treatment chamber, the source term must account for the fact that heating
occurs only during the pulse. In order to take the effective duty cycle of the pulse into account, the term has to be multiplied by the
pulse duration sP and the frequency f :
pe ¼ sE2 $sP f (8)
Here, s represents the electric conductivity and E the electric field strength. Lastly, an equation for the electric field itself is
needed. Following Meneses et al. (Meneses et al., 2011) it can be derived by the continuity equation for the free charge Jfree :
vrJ
þ V Jfree ¼ 0 (9)
vt
Here, rJ is the density of electric charges. If electrostatic conditions are assumed, no temporal change of the electric charges
occurs. Therefore, Eq. (9) reduces to:
free
vJi
V Jfree ¼ ¼0 (10)
vxi
Following Ohm’s law, the current density can be expressed with the electric field E and the electric conductivity s as the constant
of proportionality. Therefore, Eq. (10) becomes:
vsEi
¼0 (11)
vxi
Electrostatic fields are not only temporally constant but also free of rotation. Such a field can be described by the spatial gradient
of the electric potential F:
vsEi v vF
¼ s ¼0 (12)
vxi vxi vxi
If the electric conductivity is a constant, Eq. (12) becomes the Laplace equation:
v vF
¼ DF ¼ 0 (13)
vxi vxi
The Laplace equation is a partial differential equation of second order, which can be solved with a proper boundary condition.
The boundary condition can be either given by a Dirichlet boundary condition with a fixed value for F, or by a Neumann boundary
condition with a fixed value of the gradient at the boundary. In the case of a PEF treatment chamber, the electric field is built in the
insulator gap. Therefore, a suitable boundary condition at the electrodes and the insulator needs to be formulated. Typically, the
electrodes are modeled by assuming a fixed value for F at the boundary, which is the applied voltage at the high voltage electrode
and 0 Vat the grounding. As described by Meneses et al. (Meneses et al., 2011) and Wölken et al. (Wölken et al., 2017), the free
current at the other boundaries and therefore the gradient of the electric potential in normal direction n to the wall is set to zero:
En ¼ V n F ¼ 0 (14)
With the described set of differential equations (Eqs. (4), (6), (7) and (14)) and suitable boundary conditions, the PEF process can
be modeled and simulated. Since the inactivation of microorganisms is a common application of PEF the simulation of such
a process is of particular interest. This can be realized by a simple transport equation for the partial mass of microorganism cells c.
Here, the microorganisms are assumed to behave like passive tracers, which are homogenously distributed in the fluid. Due to the
small size of the microorganism cells, the convective mass transport is usually several orders of magnitude faster than the diffusive
one. Therefore, the transport equation for a mass concentration c of the considered microorganism can be described as:
vc
uj ¼ pI (15)
vxj
Here, pI represents a sink term, which accounts for the effect of the electric field on the microorganisms. In the case of micro-
organism inactivation, this term needs to be described by the time derivative of a kinetic function which models the inactivation
with respect to the treatment time t , the electric field Strength E and the treatment temperature T. A comprehensive literature review
on suitable kinetic models and their implementation in a CFD simulation of the PEF process will be given in section “Kinetics of
Cell Inactivation and their Implementation in Numerical Simulations”. Furthermore, a case study for the inactivation of micro-
algae by pulsed electric field is described in Case Study 2: Permeabilization of Microalgae by Pulsed Electric Field.
1.12.2.2 Process Dependence of Fluid Properties

Since material properties such as density, thermal and electric conductivity, viscosity and heat capacity depend on temperature, suit-
able equations which describe this dependency need to be considered. A detailed overview about this topic is given by Meneses
(Meneses et al., 2011) and Wölken (Wölken et al., 2017) and, therefore, a detailed list of existing equations shall not be part of
this article. Nevertheless, a brief instruction of important considerations that need to be done prior to the selection of model equa-
tions is given here. Because foods are usually complex multicomponent systems, theoretical models for material properties of
specific foods are hard to find. A common way to circumvent this issue is to fit empirical models to experimental data for the specific
food of interest. Caution is advised here, because many empirical models in the literature are only valid within a specific temper-
ature range. Therefore, basic knowledge about peak temperatures occurring during the process is important to avoid modeling
errors. Furthermore, some material properties depend much stronger on temperature than others. Due to the usual limitations
of the available computational capacity, it is always recommendable to keep the model as simple as possible. Wölken (Wölken
et al., 2017) emphasized that the density and the specific heat capacity of water vary less than 5% and 1%, respectively, within
a temperature range from 10 to 90 C. Thus, in cases were a water-like food is considered in simulations, these two material prop-
erties can be regarded as constant. In contrast, the dynamic viscosity, the electric conductivity and the thermal conductivity show
a significant temperature dependency. Therefore, model equations describing these properties as a function of temperature are rec-
ommended to use. Nevertheless, the decision if the temperature dependency of a material property should be considered for simu-
lations always depends on the type of the treated food.
Since the assumption of a single component food is often not valid, material properties need to be described by a combined
fractional model, which contains all equations for the description of the individual components. The calculation of the property
of the whole food can be done by using different models combining the properties of a pure substance. The simplest model is
the parallel model which sums up the property of every pure substance, weighted by its volume fraction. Further models exist,
such as the series model or the Krischer model, which are discussed by Sahin and Sumnu (Sahin and Sumnu, 2006). The choice
of the right model depends on the complexity of the food and the number of components or phases. Generally, special attention
should be paid to the modeling of material properties, since they couple the interaction of the different physical fields. The temper-
ature, for example, is affected by the electric field due to Joule heating and local peaks of the electric field lead to local temperature
peaks. Due to the thermal dependency of the electric conductivity, an increased electric current and therefore a higher energy input
occurs at spots with high temperature and, consequently, the temperature rises further. This self-enforcing process can lead to very
high local temperature peaks and in the end to a damage of the processed food and a reduced product quality. In order to detect
such effects in numerical simulations, the material properties must be carefully modeled.
In many cases, not only the temperature dependency of the material properties of the treated food is worth to be considered.
Since many foods show non-Newtonian behavior, the shear stress dependency of the fluid viscosity should be considered. To
find a model which describes this dependency, it is common to fit empirical models to experimental data. Buchmann et al. (Buch-
mann et al., 2018) used the Herschel-Bulkley equation to find an equation for the viscosity as a function of shear stress for an Arthro-
spira platensis suspension:
s ¼ s0 þ K g_ n (16)
Here, s is the shear stress, s0 is the yield stress, K is the flow consistency index, g_ is the shear rate and n is the flow behavior index.
The shear rate and the shear stress are linked with the fluid viscosity as a constant of proportionality. Therefore, Eq. (16) can be
rewritten as:
_ 1 þ Kjgj
m ¼ s0 jgj _ n1 ; jgj
_ g_ 0 (17)
Here, g_ 0 is the zero shear rate. Buchmann et al. (Buchmann et al., 2018) fitted Eq. (17) to their experimental data to obtain
values for the yield stress, flow consistency index, and flow behavior index. Since the flow behavior of a particle suspension nor-
mally depends on the particle concentration, they further used their rheological data to derive an equation describing the suspen-
sions viscosity as a function of the cell concentration by using the Krieger-Dougherty relation:

m Fn ½mFmax
mr ¼ ¼ 1 (18)
mS Fmax
Here, mr is the relative viscosity of the suspension, m represents the effective viscosity of the suspension, mS is the viscosity of the
suspending medium, Fmax is the maximum volume fraction, Fn is the volume fraction and ½m represents the intrinsic viscosity of the
suspended particles. Since Fmax is also a function of the shear stress and of the particle aspect ratio, Eq. (18) is only valid for a very
small range of shear stresses in which Fmax can be considered as constant. Further details on the modeling of Fmax as a function of
the aspect ratio of the particles are out of the scope of this article. For additional details, the contributions of Metzner (Metzner,
1985) or Pan (Pan, 1993) can be consulted. Nevertheless, for a fixed value for Fmax ¼ 0.43 for an Arthrospira platensis suspension,
Buchmann et al. (Buchmann et al., 2018) implemented the viscosity as a function of the cell concentration. Their simulated results
of the flow field in a PEF treatment chamber were in good agreement with experimental data, which were obtained by means of
Ultrasonic Doppler velocity profiling (UVP) for cell concentrations greater than 25 gL1 . According to their work, increasing the
cell or particle concentration is a good way to improve the treatment homogeneity. This is because of the characteristic velocity
profile of non-Newtonian fluids, which shows a more homogeneous velocity distribution across the radial coordinate in compar-
ison to Newtonian fluids. Therefore, the treatment time for cells in the center of the chamber is more similar to the one of cells
whose velocity is influenced by the proximity to the wall. Note that the presence of particles is considered just indirectly in this
approach by affecting the viscosity of the fluid. If the particles shall be simulated directly, several additional assumptions for the
particle-fluid and particle-particle interactions need to be made and an equation of motion for each particle needs to be solved.
This increases the computational time dramatically, even for relatively low particle concentrations. Therefore, this approach is
not suitable for dense particle suspensions but can be applied if a PEF process of larger particles such as tomatoes or potatoes shall
be simulated.
1.12.2.3 Kinetics of Cell Inactivation and Their Implementation in Numerical Simulations

As already mentioned in section “Governing Equations”, modeling the inactivation of microorganisms is an important application
of PEF simulation, since an appropriate simulation is a powerful tool for process design and scale-up. The basis of a comprehensive
simulation of microbial inactivation is a kinetic equation describing the cell permeabilization as a function of treatment time, elec-
tric field strength and temperature. Therefore, the focus of this section is on existing kinetic models and how they need to be manip-
ulated in order to implement them in a numerical CFD simulation. All kinetic models have in common that the parameters of the
equation depend on different cell characteristics, for example the electric conductivity of the cytoplasm or of the cell membrane.
Since those characteristic cell properties are hard to acquire experimentally, it is necessary to determine these parameters by fitting
them to experimental data obtained for the perforation in dependency of at least the electric field strength, the treatment time and
the temperature. Also, other parameters such as the pH value can be considered in the experimental design. Therefore, it is crucial for
the accuracy and the validity of the model to ensure a proper experimental design and a precise measurement method to distinguish
between permeabilized and non-permeabilized cells. The detailed discussion of different experimental methods is out of the scope
of this contribution. It should only be mentioned here that the most common ways to detect inactivated cells are drop plating
method (see, for example (Heinz et al., 2003)) or staining the damaged cells with fluorescent dyes like propidium iodide (PI)
(see, for example (Knappert et al., 2020; Luengo et al., 2014; Kotnik et al., 2010; Arroyo et al., 2010)). If the kinetic model should
describe the inactivation as a function of the electric field, the temperature and the treatment time, a good control of those factors
during the calibration experiment is crucial for an accurate CFD model.
Several kinetic models for the inactivation of microorganisms or enzymes are reported in literature. Giner et al. (Giner et al.,
2005) investigated the impact of PEF on the reduction of pectinesterase activity and tested different models to describe their
data, including a first order decay model, Hülsheger’s model, Fermi’s model and a model based on the Weibull distribution. It was
shown that the Weibull equation described the experimental data best:

tt gW
RAt ¼ RA0 $exp (19)
aW
Here, RAt is the residual activity after the treatment time tt , RA0 is the residual activity at the beginning of the treatment, aW is the
scale parameter and gW is the shape parameter. These parameters can be obtained directly from experimental data. Also, the param-
eters can be fitted by a secondary model equation as a function of the electric field strength. In the work of Giner et al. (Giner et al.,
2005), aW showed an exponential decrease with increasing field strength whereas gW showed no significant dependency. Giner
et al. did not consider the treatment temperature within their model. Even if PEF is considered as a non-thermal technology, it
was demonstrated that the inlet temperature of the suspension has a significant impact on the inactivation rate of microorganism
(Heinz et al., 2003; Saldaña et al., 2011). Thus, this model should be extended by an additional secondary model equation
describing aW and possibly gW as a function of the treatment temperature. Another relevant effect on the inactivation kinetics could
be the pH value. Saldaña et al. (Saldaña et al., 2010) incorporated this effect by using a polynomial as a secondary model equation
for gW in order to describe this term as a function of the pH-value of the surrounding media. It is a task of the modeler to decide
which effects are relevant for the inactivation and to incorporate them into the kinetic model by searching suitable secondary
equations.
A kinetic model on basis of a Poisson-distribution is presented by Saulis and Venslauskas (Saulis and Venslauskas, 1993). It
describes the pore formation as a function of treatment time, electric field strength and treatment temperature:
nX

crit 1
kf 0 t n $exp kf 0 tt
Fp ðtÞ ¼ 1 (20)
n¼0
nP !
Here, kf 0 is the kinetic parameter, which needs to be modeled as a function of electric field strength and treatment temperature
by a secondary model equation. Fp ðtÞ represents the probability that a cell is perforated at given treatment parameters and increases
with the treatment time. In the case of a cell population, Fp can be considered as the fraction of perforated cells. The parameter nP
describes the number of pores and ncrit is the number of pores from which the cell can be considered as perforated. In the case of
ncrit ¼ 1, Eq. (20) simplifies to:

Fp ðtÞ ¼ 1 exp kf 0 tt (21)
Saulis and Venslauskas (Saulis and Venslauskas, 1993) further present a secondary model equation to describe kf 0 as a function
of the treatment temperature T and the electric Field strength E which describes the pore formation on a molecular level:
20 1 3
Z1 εw 2
2pna2 DWf ð0Þ εm 1 2 3
kf 0 ðE; TÞ ¼ exp exp4 @ pCm r A Eay DF0 5dy (22)
2kB T
$
al kb T 2
1
The underlying model assumption is that pores in the cell membrane are naturally formed and closed again. The action of the
electric field stabilizes the formed pores by increasing their size if a critical energy barrier is exceeded. The first exponential term on
the right-hand side of Eq. (22) describes the impact of the temperature on the pore formation with an Arrhenius approach. Here, al
is the surface of one lipid molecule, n is the frequency of the molecule fluctuation, DWf ð0Þ is the energy barrier at resting potential
which needs to be overcome for pore formation, kb is the Boltzmann constant. The second term contains an integral over the cell
surface and takes into account that the electroporation of the cell membrane is not uniform (Gabriel and Teissié, 1997). The param-
eter y ¼ cosðaÞ represents the position on the cell surface as a function of the normal surface angle a, a is the cell radius and DF0 is
the resting potential of the cell. Therefore, the term (32 Eay DF0 ) represents the transmembrane potential for a spherical cell as
a function of the position on the cell surface (Saulis, 2010). The parameter Cm is the capacity of the membrane and εw and εm stand
for the relative permittivity of the membrane and the water inside the pore, respectively. If these quantities are known, the model of
Saulis and Venslauskas allows the description of the pore formation on a microscopic scale. It is further possible to summarize all
quantities to a fitting parameter, which can be determined experimentally. With this approach, the inactivation can be described
only on a macroscopic level. However, it should be noted that the implementation of the complex integral Eq. (22) in numerical
simulations is not straightforward and the evolution of the integral in every volume element causes additional computational costs.
It is therefore recommendable to simplify Eq. (22) by suitable approximations.
If a suitable kinetic is found and the parameters are obtained by experiments, the kinetic model has to be implemented as a sink
term in a transport equation of the form of Eq. (15). According to Krauss et al. (Krauss et al., 2011), the source term of the transport
equation is simply the time derivate of the empirical kinetic model. In the case of a continuous treatment chamber, the treatment
time is expressed as the product of total time t, the pulse duration s and the frequency of the applied pulses f . Using the model of
Saulis and Venslauskas as an example, the time derivate of Eq. (21) is:
dFp
¼ kf 0 sf $exp kf 0 f sP t (23)
dt
Considering Fp as the mass fraction of perforated cells mI ðtÞ at given time, to the initial mass and the initial mass m0 is the sum of
perforated cells mI ðt0 Þ and unperforated cells mA ðt0 Þ at t ¼ 0, Eq. (23) can also be expressed as:
dmI
¼ ðmI ðt0 Þþ mA ðt0 ÞÞ$kf 0 sP f $exp kf 0 f s $ t (24)
dt
Lastly, the exponential term on the right side of this equation can be expressed as 1 Fp ðtÞ. Therefore, Eq. (15) becomes the final
form of a transport equation which describes the inactivation of a certain microorganism as a balance of mass:

vmI vmI mI ðtÞ
þ uj ¼ mo $kf 0 sf $ 1 (25)
vt vxj m0
Because the simulation of the inactivation of microorganisms is an essential part of this contribution, a detailed case study on the
example of the inactivation of the microalgae Chlorella vulgaris will be given in section “Case Study 2: Permeabilization of Micro-
algae by Pulsed Electric Field”. In this context, also a brief introduction of the experimental setup being used for obtaining cell
permeabilization kinetics is given.
1.12.2.4 Modeling Cell Inactivation on the Microscale

In many applications, PEF is used to enhance mass transfer. In this case, not only the knowledge about whether a cell is perforated or
not is important but also the knowledge about the numbers and size of the occurring pores. A model as proposed in section
“Kinetics of Cell Inactivation and their Implementation in Numerical Simulations” does not contain this information and there-
fore, additional simulation on a microscale can be carried out for a comprehensive process design. A detailed model which describes
the pore formation, the pore size distribution and the post-pulse evolution of the pores was proposed by Krassowska et al. (Kras-
sowska and Filev, 2007). It is based on two balance equations, which consider the electroporation of a single cell. The first balance
equation accounts for the number of pores and the second for the temporal evolution of the pore size distribution. In their model,
the electric potential inside and outside of a single cell are described by the Laplace equation similar to Eq. (13). The external electric
field is expressed through the electric potential Fe as a function of the time t, the distance to the center of the cell dc and the angle
with respect to the direction of the electric field Q:
Fe ðt; r; QÞ ¼ Edc cosðQÞasp ; dc / N (26)
Here, a is the radius of the cell and sp is the electric conductivity of the medium which fills the generated pores being a mixture of
the surrounding media and the cytoplasm of the cell.
The electric current density across the cell membrane is described by the temporal change of the transmembrane potential Fm ðt;
QÞ, the difference of the transmembrane potential to the resting potential of the cell Frest and the electric current through pores Ip .
The considered pores can either be protein channels as well as pores, which were induced by the electric field:
vF m
n $ðsi VFi Þ ¼ b
b n $ðse VFe Þ ¼ Cm þ gS ðFm Frest Þþ Ip (27)
vt
where Cm is the capacity of the membrane, gs is the surface conductance, si and se are the electric conductivity of the internal and the
external fluid, respectively, and b
n is the unit vector normal to the membrane surface. The temporal change of the pore number
density Nðt; QÞ can be described in dependency of the local transmembrane potential:
2
Fm
dN N
¼ aK e Vep 1 (28)
dt Neq ðFm Þ
The parameter aK is the creation rate coefficient, Vep is the characteristic voltage of electroporation which is given by Krassowska
et al. (Krassowska and Filev, 2007) as 0.258 V and Neq is the equilibrium pore number density for a given transmembrane potential.
If the pore number density is equal to the equilibrium pore number density, the right-hand side of Eq. (28) becomes zero and no
more pores are formed. On the other hand, the right-hand side of the equation becomes negative if the pulse is turned, since the
equilibrium pore number density is smaller than the actual pore number density. Therefore, Eq. (28) can also be used to describe
the resealing of the pores. Lastly, Krassowska provides a balance equation for the temporal change of the pore radius rj ðj ¼
1; 2.npores Þ:
( 4 )
drj Dp F2m Fmax r 1
¼ Up rj ; Fm ; geff ¼ rh 4bK 2pEe þ 2pgeff rj (29)
dt kB T 1 þ rj þr t
rj rj
Here, Up has the unit ½m s1 and represents the velocity of the pore radii development. Dp is the diffusion coefficient of the pore
radius, Fmax is the maximum electric force for Fm ¼ 1 V, rh and rt are constants, bK is the steric repulsion energy, r is the minimum
radius of a hydrophilic pore, Ee is the edge energy at the edge of a pore and geff is the effective surface tension, which is a function of
the total pore area. Values for the used constants and further equations describing Ip , Neq and geff are given in the paper of Kras-
sowska et al. (Krassowska and Filev, 2007). The results of this work revealed that the pore formation starts when a threshold of
the transmembrane potential of around 1 V was reached. Furthermore, it was shown that this threshold was reached at different
times at different positions on the cell membrane, depending on the orientation of the cell within the electric field. After the trans-
membrane potential has reached a maximum, it decreased again due to the increased electric current through the accrued pores. The
pore formation starts at the pole region of the cell and their border regions, while no pore formation was observed at the equator of
the cell. Although most pores were created at the poles of the cell, the largest pores occurred at the pole border region. Furthermore,
Krassowska et al. revealed that the pore diameter is a function of the treatment time. The maximum pore radius after 30 ms was only
90 nm whereas it was 419 nm after a treatment time of 1 ms. With regard to an application of PEF for mass transfer enhancement,
this result is very important. It shows that the simulation on a microscale can give important insights, which need to be incorporated
in macroscopic simulation as presented in section “Governing Equations”. Nevertheless, the model proposed by Krassowska et al.
cannot be implemented in a CFD simulation like the model which is described in section “Kinetics of Cell Inactivation and their
Implementation in Numerical Simulations” since the computational effort increases dramatically if more than one cell should be
modeled. Therefore, models of this type should be used in addition to a macroscopic CFD-simulation for a comprehensive process
design.
A way to describe the phenomenon of PEF on an even smaller scale than the one proposed by Krassowska et al. (Krassowska and
Filev, 2007) is a molecular dynamic simulation (MDS). By definition, the simulation describes the effect of the electric field on the
level of the molecules of a cell structure, for example the phospholipids of the cell membrane. Therefore, an MDS can be used to
perform mechanistic studies on a picometer scale with timesteps of femtoseconds. In MDS, the equations to solve are derived from
the classical equations of motion. The force which acts on the atoms is usually described by the potential energy of each atom. The
potential energy Ftot is then divided in non-bonded interactions Fnonbonded and bonded interactions Fbonded :
Ftot ¼ Fbonded þ Fnonbonded (30)
Nonbonded interactions are often described by a Lennard-Jones potential. The Lennard-Jones potential is a function of the
distance between two atoms and includes both, repulsive and attraction forces. The presence of electrostatic charges is described
by the Coulomb potential. The potential of bonded interactions can be described by the Hooke potential. For molecular systems,
the molecules are built out of site-site potentials describing the force of each atom, and their movement as a result of their inter-
actions. For a simple chain molecule, the intramolecular potential is described by the sum of the bonded and nonbonded potential.
The bonded potential can be further split into three terms describing the potential through the bond length Fbond , the bend angles
4bend angles and the torsion of this angle 4torsion angles respectively:
1X r 2
Fbond ¼ k rij req (31a)
2 bond ij
1 X rq 2
4bend angles ¼ k q qeq (32b)
2 bond ijk ijk
1 X X a m
4torsion angles¼ k 1 þ cos maijkl gm (33c)
2 torsion m ijkl
angles
Here, rij is the distance between the ith and the jth atom, qijk is the bend angle between the ith ; jth and kth atom and aijkl is the
torsion angle between the four atoms i; j; k and l. Usually, the Eqs. (31a), (32b) and (33c) are included in force field packages.
This package provides values for the various strength parameters k and the other constants in the model equations. Commonly
used force fields are AMBER (Assisted Model Building with Energy Refinement) or CHARMM (Chemistry at Harvard Macromolec-
ular Mechanics). These force fields are part of different open source software packages, which are available at no cost. After the mole-
cules of interest are described by the different potentials, the forces can be calculated with the derivative of the potentials over the
spatial coordinate ri . As mentioned before, the motion of atoms or molecules is described by Newton’s equation of motion:
dri pi
¼ ¼ vi (34)
dt mi
dpi
¼ fi (35)
dt
Here, pi is the momentum of the ith atom, mi is the mass and vi the velocity. The description of algorithms which can be applied
to solve this system of coupled ordinary differential equations are out of the scope of this article. A guideline on how to choose
proper boundary conditions and a detailed description of algorithms can be found in (Allen, 2004).
Regarding PEF, MDS provides a great mechanistical tool since the structure and composition of the cell membrane can be varied
in simulations. Therefore, the behavior of membranes with different structures or compositions being exposed to an electric field
can be investigated. Since a hydrophilic pore was never observed experimentally, MDS can be used to make the pore formation
process visible (Shillcock and Seifert, 1998; Delemotte and Tarek, 2012). The formation of a hydrophilic pore in response to an
external electric field was investigated by means of MDS by Böckmann et al. (Böckmann et al., 2008). Casciola et al. (Casciola
et al., 2014) used MDS to investigate the influence of different cholesterol levels within the cell membrane on pore formation.
They showed that the cholesterol molecules enhanced the membrane cohesion. Therefore, the threshold for electroporation was
higher for membranes containing cholesterol. Piggot (Piggot et al., 2011) showed by MDS that several lipid-lipid interactions stabi-
lize the membrane which leads to a different resistance of the cell membrane of E. coli and S. aureus due to the different composition
of their membranes. Polak (Polak et al., 2013) also investigated the influence of the lipid membrane composition. In contrast to
Piggot et al. they showed that also the hydrophobic tail of the lipid molecules has an influence on the pore formation. All the pre-
sented studies show the great potential of simulating PEF on the scale of single atoms or molecules. Since the complexity of the
models is often limited by computational resources, this field of PEF simulation might become increasingly relevant in future. Simu-
lations are typically restricted to several ten thousand atoms and physical times in the order of nanoseconds. Tarek et al. (Tarek,
2005) implemented 215 lipid molecules in addition to 6469 water molecules to simulate the membrane electroporation in the
time range of 5 to 10 nanoseconds. Since the available computational resources steadily increase more detailed models of the
cell membrane can be simulated in the future.
1.12.3 Case Studies of PEF Process Design and Scale-up by Means of Numerical Simulation
In this section, three case studies are presented which show the benefits of simulating the PEF treatment with regard to process
design and optimization. In the first study, the potential of CFD-simulations for optimizing the geometry of a PEF treatment
chamber is demonstrated. The second study presents a way to simulate the permeabilization of the microalgae Chlorella vulgaris,
on the basis of the approach described in section “Kinetics of Cell Inactivation and their Implementation in Numerical Simula-
tions”. In the third case study, this model is used to customize a treatment chamber for the treatment of microalgae.
1.12.3.1 Case Study 1: Improvement of the Treatment Homogeneity

In this section, a case study is presented which shows the influence of the treatment chamber geometry on the treatment homoge-
neity, which is expressed by the homogeneity of the electric, temperature and velocity fields. For this purpose, treatment chambers
on the industrial scale with a mass flow rate of m_ ¼ 5 t h1 were investigated. Two different treatment chambers were compared to
each other with regard to the achievable treatment homogeneity. The investigated chambers are depicted in Fig. 1. The left treatment
chamber represents an ordinary colinear treatment chamber without any modification. The right treatment chamber is equipped
with an additional displacer in the middle of the pipe (part number 6). Hereafter, this treatment chamber is referred to as modified
colinear treatment chamber.
Since the mass flow rate is very high, one should consider for the modeling if turbulence matters. Therefore, local Reynolds
numbers should be calculated at important points:
rvm di
Re ¼ (36)
m
Here, the magnitude of the Reynolds number was calculated at the inlet and within the two insulator gaps, which are charac-
terized by the local inner diameter di . The average velocity vm was expressed as the volume flow divided by the cross-sectional
area of the pipe at the points of interest. The calculated Reynolds numbers were 35367 for the inlet and 49121 and 38443 for
the insulator gap of the ordinary and the modified treatment chamber respectively. Therefore, the common k ε model was chosen
to describe turbulent effects in the investigated treatment chambers.
For the turbulent case, usually the basic conservation equation for mass, momentum and energy as described in section “Gov-
erning Equations”, are replaced by the so-called Reynolds-Averaged Navier-Stokes equations (RANS-equations). Within this
approach, the fluid velocity is decomposed into a temporally averaged component and velocity fluctuations around the average,
thus Uj ¼ Uj þ u0j . Here the first term Uj describes the time averaged value and u0 is a value for the fluctuation of the velocity. Consid-
ering a constant density r, the equation for mass conservation can be expressed as
Figure 1 Investigated treatment chambers in this case study. Left: ordinary colinear treatment chamber composed of two grounding electrodes (4),
one high voltage electrode (1) and two insulator rings (3). The inlet is on the left (1) and the outlet on the right side of the treatment chamber (5).
Right: modified colinear treatment chamber with a cigar shaped displacer (6) in the middle of the high voltage electrode (2) and the insulator rings
(3). The inner diameter dIE for ground and high voltage electrode for both chambers is 5 cm and the inner diameter for the insulator rings dII is
3 cm. The red rectangle indicates the section for which the contour plots of velocity, electric field strength and temperature in Fig. 2 are created.
vU j
r ¼0 (37)
vxj
It should be noted that the velocity fluctuations are equal to zero on average. For the turbulent case, the Navier-Stokes equation
for the momentum transport needs to be extended by the Reynolds Stress tensor ru0i u0j , which contains the non-vanishing average of
the product of the velocity fluctuations u0i and u0j . Therefore, the equation for momentum conservation can be written as:
! !
vUi U j vp v vUi vUj 0 0
r ¼ þ m þ rui uj (38)
vxj vxi vxi vxj vxi
Here, buoyancy and any other source terms for the momentum were neglected. Following the Boussinesq approximation for the
Reynolds stress tensor, the last term of Eq. (2) can be expressed as follows:
!
vUi vUj 2
ru0i u0j ¼ mt þ rdi;k k (39)
vxj vxi 3
Here, mt is the turbulent eddy viscosity, di;k is the Kronecker delta and k is the turbulent kinetic energy. The turbulent eddy
viscosity can further be described as
Cm rk2
mt ¼ (40)
ε
Cm is a constant which is usually set to 0.09 and ε is the dissipation rate of turbulent kinetic energy. In order to solve Eq. (2), two
additional balance equations for k and ε are needed. The temporal change for the turbulent kinetic energy k is described by the
following differential equation:

vrk v vk 2 vU i
¼ rDk þ Gk r k rε þ Sk (41)
vt vxj vxj 3 vxj
Here, Dk is a coefficient describing the diffusive transport of turbulent kinetic energy, Gk describes the impact of the buoyancy on
the turbulent kinetic energy and Sk is a general source term. The isotropic rate of dissipation ε is balanced by:

vrε v vε C1 Gε 2 vU i C2 rε2
¼ rDε þ C1 C3;RDT r ε þ SE (42)
vt vxj vxj k 3 vxj k
Here, C1 ¼ 1:44; C2 ¼ 1:92 and C3;RDT ¼ 0 are constants which can be found in the literature (Ferziger and Peric, 2008). Since ε
describes the dissipation of turbulent kinetic energy into thermal energy, this must be also reflected in the balance equation for the
internal energy:

vT v vT vU i T
rcp þ ðk þ kT Þ þ rcp ¼ pe (43)
vt vxj vxj vxj
The dissipation of turbulent kinetic energy is linked to Eq. (43) through the term kT which describes the turbulent heat conduc-
tivity and can be expressed as:
Cm rk2
kt ¼ cp mt ¼ cp $ (44)
ε
Here, cp is the heat capacity of the fluid. Since the presented turbulence model is isotropic, which means that all averaged quan-
tities are independent of the direction, wall-functions are needed to describe the quantities at the wall and in the turbulent boundary
layer which can be regarded as boundary conditions for the turbulent related quantities. Open source software such as OpenFOAM
provides sets of wall-functions from which the user can choose appropriate ones. The commercial software ANSYS (Canonsburg,
PA, USA) on the other hand does not provide this option and the wall functions are chosen automatically.
The electric field was modeled as described in section “Governing Equations” (see Eq. (14)). For the set of the presented equa-
tions, boundary conditions need to be set. The chosen boundary conditions for the presented case study are summarized in Table 1.
Simulations were performed with the open source software OpenFOAM.
Furthermore, values for the material properties are needed. In this case study, the heat capacity cp , the dynamic viscosity m, the
heat conductivity k, the density r and the electric conductivity s were considered as constants. The values are summarized in
Table 2.
To characterize the fields of temperature, velocity and electricity, the standard deviation XSD for these quantities were calculated
as described by Gerlach et al. (Gerlach et al., 2008):
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
u
u 1 X N
XSD ¼ t ðXi Xmean Þ2 dVi (46)
Vgap 1
Table 1 Boundary conditions as set for the CFD simulation in OpenFOAM. The near wall region was described by the standard wall functions of
OpenFOAM. Their definition can be obtained from the OpenFOAM user guide (OpenCFD Ltd, 2019). The turbulent kinetic energy at the inlet
was calculated from the mean inlet velocity U inlet under the assumption of a degree of turbulence I of 5%.
3 2
kinlet ¼ I 2 U Inlet (45)
2
Property P T U FE ε k mt at
m m2 m2
Inlet VP ¼ 0 283 ½K 0.7 s VFE ¼ 0 0,005 s3
0,001838 s2
0 0
Outlet P ¼ Pref ½bar VT ¼0 VU ¼ 0 VFE ¼ 0 Vε ¼ 0 Vk ¼ 0 Vmt ¼ 0 Vat ¼ 0
HVE VP ¼ 0 VT ¼0 0 15 [kV] ε- Wall function kq R- Wall function mt k -Wall function at - Wall function
Ground VP ¼ 0 VT ¼0 0 0 ε- Wall function kq R- Wall function mt k -Wall function at - Wall function
Insulator VP ¼ 0 VT ¼0 0 VFE ¼ 0 ε- Wall function kq R- Wall function mt k -Wall function at - Wall function
Table 2 Material properties and PEF-Parameters expect the applied voltage and the inlet velocity used for the CFD simulation.
Property Heat capacity Dynamic viscosity Heat conductivity Density Electric conductivity
Symbol cp m k r s
J kg J
kg S
Unit kg K ms smK m3 m
Value 4183 0.001 0.63 997 0.28
Here, Vgap is the volume of the insulator gap where the electric field is active. N is the number of finite volume elements within
this gap, Xi is the level of the considered quantity in each of these elements and dVi is the volume of the ith element. Xmean is the
average of the considered quantity within the insulator gap. It can be calculated as:
1 X N
Xmean ¼ Xi dVi (47)
Vgap 1
The volume of the treatment gap is the sum of all finite volume elements:
X
N
Vgap ¼ dVi (48)
1
Finally, a coefficient of variation can be defined to compare the homogeneity of the quantity X in different setups:
XSD
XCV ¼ (49)
Xmean
In this study X was the electric field strength E, the temperature T, or the velocity U respectively.
Simulation results for this case study are presented in Fig. 2. The plots show the section being indicated by the red rectangle in
Fig. 1. It can be seen from plot (A) that the velocity within the insulator gaps is higher for the modified treatment chamber. Never-
theless, the velocity distribution is more homogeneous and the boundary layer within the insulator gaps, as indicated by the blue
color in Fig. 2, was thinner for the modified treatment chamber as for the ordinary one. This is also expressed by the indicators USD ,
Umean and UCV , which were calculated with Eqs. (46), (47) and (49). The mean velocity for the ordinary treatment chamber inside
the insulator gaps was 1.25 ms1 and 1.24 ms1 for the first and the second insulator, respectively. For the modified treatment
chamber, the mean velocity was 1.33 ms1 and 1.34 ms1 for the first and the second insulator. The standard deviation for the
velocity was 0.28 ms1 and 0.34 ms1 for the ordinary treatment chamber and 0.26 ms1 and 0.30 ms1 for the modified treatment
chamber, respectively. Therefore, the coefficients of variation were lower for the modified treatment chamber (see also Table 3). This
indicates a more homogeneous velocity distribution inside the volume where the electric field is active. Consequently, the over-
processing of fluid elements which pass the treatment chamber close to the wall can be prevented. Another advantage of the velocity
field in the modified treatment chamber becomes clear in Fig. 3. As indicated by the streamlines, large recirculation areas occur
inside the high voltage electrode gap of the ordinary colinear treatment chamber. In contrast, similar recirculation zones are not
found in the modified colinear treatment chamber. Furthermore, a dead zone of the flow stream occurs inside the ordinary treat-
ment chamber behind the second insulator, which does not exist inside the modified treatment chamber.
Since the heating of the fluid is a function of the residence time, the displacer has a positive effect on the temperature peaks. As it
can be seen from Fig. 2 (C), the maximum temperature increase inside the high voltage electrode gap and behind the second insu-
lator was at least 10 K for the ordinary treatment chamber. Inside the modified treatment chamber, the maximum temperature
increase was around 5 K. This is a very important result of this case study since the enhancement of mass transfer for a subsequent
Figure 2 Contour plot for velocity field (A), the electric field (B) and the temperature field (C) for the modified treatment chamber with displacer
(upper row of each plot) and the ordinary colinear treatment chamber (lower row of each plot). The plots show a section of the treatment chamber
which is represented as a red square in Fig. 1.
Table 3 Comparative values for the electric field strength E, the temperature T and the velocity U calculated by Eqs. (46), (47) and (49) inside the first
and the second insulator gap (ISO 1 and ISO 2) for the ordinary and modified colinear treatment chamber.
Ordinary ISO 1 Ordinary ISO 2 Modified ISO 1 Modified ISO 2

Emax $ mV 5.18e05 5.18e05 5.46e05 5.22e05

Emean $ mV 3.60e05 3.60e05 3.66e05 3.67e05

V
ESD $ m 0.49e05 0.49e05 0.47e05 0.47e05
ECV $½ 0.135 0.136 0.128 0.128
Tmax $½K 313.27 305.55 293.86 298.13
Tmean $½K 284.84 287.18 284.38 284.88
TSD $½K 3.35 2.60 1.05 1.27
TCV $½ 0.01 0.01 0.004 0.004

h i
Umax $ ms 1.49 1.49 1.66 1.88
h i
Umean $ ms 1.25 1.24 1.33 1.34
h i
USD $ ms 0.28 0.34 0.26 0.30
UCV $½ 0.22 0.27 0.20 0.22
Figure 3 Contour plot and streamlines for the modified treatment chamber (upper) and the ordinary colinear treatment chamber (lower). The
section shows the high voltage electrode and both insulator gaps. Large areas of recirculation are revealed by the streamlines for the ordinary
colinear treatment chamber.
extraction of thermosensitive products is an important application of PEF. The effect of the displacer on the temperature can also be
expressed in terms of the mean temperature Tmean , the standard deviation TSD and the coefficient of variation TCV (see also Table 3).
Since these values are calculated for the insulator gaps, the effect of the displacer on them is not very high. This is caused by the fact
that the temperature increase is most important inside the gap of the high voltage electrode and behind the second insulator. Never-
theless, maximum temperature was 313.27 K and 305.5 K for the ordinary treatment chamber inside the first and the second
insulator respectively and 293.86 K and 298.13 K for the modified treatment chamber. This can be explained by the higher velocities
and the thinner boundary layer inside the insulator gaps of the treatment chamber with the displacer. Moreover, the standard devi-
ation for the temperature was 1.05 K and 1.27 K for the first and the second insulator in the modified treatment chamber, respec-
tively. On the contrary, the standard deviation was found to be 3.35 K and 2.60 K inside the insulators of the ordinary treatment
chamber, which indicates that the temperature distribution is more inhomogeneous.
One characteristic of colinear treatment chambers is the inhomogeneous distribution of the electric field. Since the electric field is
stronger at the wall of the insulator gap and weaker in the center of the chamber, the effect of over- and under-processing parts of the
product is further enhanced. Therefore, modifying the treatment chamber with the displacer also improves the homogeneity of the
electric field since the area with the lowest field strength is simply cut off. This leads to a slight improvement of the mean field
strength from 3.6*105 Vm1 in the ordinary treatment chamber to 3.7*105 Vm1 in the modified treatment chamber. Also, the
standard deviation was lower and, therefore, the coefficient of variation decreased from 0.135 for the ordinary treatment chamber
to 0.128 for the modified treatment chamber. Note that the temperature dependency of the electric conductivity was not considered
in this case study due to the large computational resources needed to compute the turbulent flow field. Therefore, the electric field
strength was almost the same in both insulator gaps of each treatment chamber. If the temperature dependency would be consid-
ered, the electric conductivity would increase in positive x direction due to the previously described temperature increase. This
would lead to a decreased strength of the electric field within the second insulator gap. As discussed before, the temperature increase
is more moderate in the second insulator gap of the modified treatment chamber in comparison to the ordinary one. Therefore, the
difference between the mean field strength between the two insulator gaps of the modified chamber would be smaller as in the
ordinary one, if the temperature dependency of the electric conductivity is considered.
The presented results show the potential of using CFD simulations for the optimization of a PEF treatment chamber. The modi-
fied treatment chamber has a better treatment homogeneity, which is mainly due to the improvement of the flow field. As a result,
the temperature increase inside the high voltage electrode gap and behind the second insulator is minimized. Furthermore, the elec-
tric field is more homogeneous because the volume with the lowest field strength is cut off by the displacer. If the temperature
dependency of the material properties would have been considered, the improvement of the electric field distribution would be
even more obvious. Nevertheless, the modified configuration has also disadvantages for some applications. For example, the
risk of clogging increases due to the smaller cross-sectional area. Therefore, the presented treatment chamber is not suitable for fluids
with a high particle density, for example mashes. Furthermore, the average field strength of around 3.6 kV cm1 in this case study is
not suitable for microbial inactivation. According to Saldaña et al. (Saldaña et al., 2011), the microbial inactivation in apple juice
requires an electric field strength of 25 kV cm1. Martínez et al. (Martínez et al., 2017) showed that at least 15 kV cm1 were neces-
sary to enhance the extraction of the blue pigment phycocyanin from the cyanobacteria Arthrospira platensis. Since the superiority of
the modified treatment chamber over the ordinary colinear treatment chamber was demonstrated within this case study, this treat-
ment chamber should be further optimized with regard to the electric field strength in a next step. Besides simply applying higher
voltages, which is limited by the pulse generator on the industrial scale, this can be done by varying the insulator length and diam-
eter (Gerlach et al., 2008). It was shown, that the mean electric field strength increases with a decreasing length and an increasing
radius respectively.
The presented case study also shows the potential of modifying the treatment chamber geometry. Thereby, the application for
which the treatment chamber will be used is very important and optimization must be carried out regarding the specific task, which
means that tailored treatment chambers should be designed for specific application. CFD simulation is a powerful tool for this
purpose and it can help to save labor and time expensive experiments.
1.12.3.2 Case Study 2: Permeabilization of Microalgae by Pulsed Electric Field

The second case study should serve as a guideline for the simulation of cell permeabilization. The focus is on modeling of cell per-
meabilization kinetics and their implementation in CFD simulations. For detailed information about experimental setups and the
discussion of the results, the interested reader is kindly directed to the original research (Knappert et al., 2020). The aim of this case
study is therefore to show how an inactivation kinetic can be fitted to experimental data and how it can be incorporated in a CFD
simulation of a PEF process. Therefore, an ordinary colinear treatment chamber with an inlet diameter of 6 mm and an insulator gap
with a diameter of 4 mm was considered. The general approach follows the one being described in section “Kinetics of Cell Inac-
tivation and their Implementation in Numerical Simulations” but several additional aspects must be considered which are out-
lined here. The most crucial step for fitting a kinetic model to experimental data is the exact knowledge of temperature, electric field
strength and treatment time. As it was shown in the previous section, the conditions in continuous treatment chambers are always
inhomogeneous to a certain degree. To achieve an accurate model for the inactivation under industrial conditions, it is therefore
very important to distinguish between the effects of the different parameters on the permeabilization. Thus, the independent param-
eters must be accurately controlled in calibration experiments.
1.12.3.2.1 Kinetic
For this case study, experimental data for the calibration of a kinetic model was generated in electroporation cuvettes. These treat-
ment chambers were chosen because the parallel plate configuration provides the most homogeneous field distribution. The gap
distance in the cuvettes was 4 mm and the electric field strength was calculated from the applied voltage, which was measured
directly at the treatment chamber with a high voltage probe. The whole plant was placed in a temperature-controlled atmosphere
Table 4 Treatment parameters for the batch treatment of the microalgae Chlorella vulgaris. The treatment was done in an electroporation cuvette
with a gap distance of 4 mm. The electric field strength was calculated from the applied voltage, which was measured at the treatment
chamber with a high voltage probe.
Applied Voltage 3 kV 4 kV 6 kV 9 kV 12 kV
Electric field strength kV cm1 6.5 9 13.5 20 27

Temperature ½ C 20 30 40 20 30 40 20 30 40 20 30 40 20 30 40
Number of pulses 0, 1, 2, 4, 8, 16, 32, 64
Pulse duration ½ms 4.0
Dry matter ½g l1 1
to ensure the right treatment temperature. The investigated microorganism was the green microalgae Chlorella vulgaris, which was
cultivated in a bubble column photobioreactor under continuous illumination with constant light intensity. The microalgae
suspension was preheated at least for 15 minutes before the PEF treatment. The applied treatment parameters are summarized
in Table 4. In order to minimize the increase of the temperature during the treatment, a pulse frequency of 1 Hz was chosen.
The treatment with every parameter set was conducted in duplicate, the one with 64 pulses in triplicate. The degree of permeabilized
cells after the treatment was measured by means of flow cytometry (BD Accuri C6). Therefore, the strong hydrophilic dye propidium
iodide (PI) was used. This dye cannot penetrate an intact cell membrane and therefore, a stained cell can be considered as perme-
abilizated or perforated. The intensity of the PI signal was measured on the FL2-filter (585/40 nm) of the flow cytometer. The frac-
tion of the cell population with a stronger signal on the FL2-Filter than a previously determined threshold was considered as the
fraction of perforated cells Fp . An untreated sample was stained to evaluate the degree of permeabilized cells before the PEF treat-
ment. The experimental data is the basis for the calibration of a kinetic model for the inactivation of C. vulgaris, which can be imple-
mented in a CFD simulation.
The basis for the implementation of the permeabilization of Chlorella vulgaris within the numeric CFD simulation was Eq. (15).
As described in section “Kinetics of Cell Inactivation and their Implementation in Numerical Simulations”, an expression for the
source term needs to be found. In the first step, the experimental data for Fp were fitted with the following primary kinetic model:

Fp ¼ 1 exp kf ;0 np sP $Fp;max (50)
This model is based on the kinetic presented by Saulis and Venslauskas (Saulis and Venslauskas, 1993) (see Eq. (21)). In Eq.
(50), the treatment time t was replaced by the product of the pulse number np and the pulse duration s. The fit was obtained using
a solver from the commercial software package Matlab (The Mathworks, Natick, MA, USA). It was reached by minimizing the
absolute error ε between the measured fraction of perforated cells Fp:exp and the predicted Fp;Sim with an iterated combination of
the fitting parameters. Fitting parameters were the kinetic constant kf ;0 and the maximum fraction of perforated cells Fp;max . The
latter was introduced since the fraction of perforated cells did not reach one at the lower treatment intensities, which was also
the case within the work of Saulis and Venslauskas. The experimental results and the best fits are presented in Fig. 4.
In the next step, secondary model equations for the fitting parameters kf ;0 and Fp;max were developed. Both of the chosen
secondary model equations are describing the fitting parameters as a function of the electric field strength E and the treatment
temperature T. Sadik et al. (Sadik et al., 2014) showed that the viability of mouse fibroblast cells decreased linearly with the treat-
ment time and quadratically with the electric field strength. A similar relation was considered for the equation for kf ;0 :

Bc ½K
kf ;0 ¼ Ac V 2 m2 s2 $exp $E2 $sP (51)
T
Similar to the model equation for kf ;0 as proposed by Saulis and Venslauskas, the treatment temperature was considered with an
Arrhenius-like term in Eq. (51). The fitting parameters Ac and Bc are listed in Table 5.
The predicted values of kf ;0 (kf ;model ) according to Eq. (51) are plotted versus the kf ;0 obtained by the best fit of the experimental
data (kf ;experimental ) in the left plot of Fig. 5. As indicated by the angle bisector, the model for kf ;0 predicts the values in good agree-
ment with the experimental data. Furthermore, a secondary model equation for Fp;max needs to be found. Here, the values obtained
by the best fit were first fitted isothermally as a function of the electric field strength because the experimental data indicated no
temperature effect:

E kS
Fp;max ¼ 1 exp (52)
lS
The fitting parameter lS and kS were described with respect to the temperature:

lS ¼ 6:68$109 Vm1 $exp 0:0201 K1 $ T (53)

kS ¼ 0:0742 K1 $T þ 26:51 (54)
Figure 4 Fraction of perforated cells (Fp ) as a function of electric field strength, treatment time and treatment temperature. The Upper diagram
shows the results at 40 C, the middle at 30 C and the lower diagram at 20 C. The different field strengths are indicated by different symbols:
kV (stars), 9 kV cm1 (filled circles), 13:5 kV cm1 (empty circles), 20 kV cm1 (squares), 27 kV cm1 (diamonds). Error bars indicate the
6:5 cm
standard deviation. The dashed lines are the best fits obtained for Eq. (50).
Table 5 Fitting parameters for Eq. (51).

2 2 2
Ac ½V m s Bc ½K
3:92:107 7:28:103
The complete kinetic model for the permeabilization of the microalgae Chlorella vulgaris contains Eqs. (50–54). In general,
a complete kinetic model always consists of a primary model equation which describes the experimental data and several secondary
model equations, one for each fitting parameter. In some cases, such as the presented one, the model can be extended by tertiary
equations, which describe fitting parameter of the secondary models. For PEF, the secondary model equations should describe the
Figure 5 Predicted values of the secondary model equation for kf ;0 (see Eq. 51) and Fp;max (see Eq. (52)) versus the fitting parameters obtained by
fitting the experimental results with Eq. (50) (considered as kf ;experiment and Fp;max ; experiment ).
main fitting parameters as a function of the electric field strength and the temperature since these are the most important factors for
the PEF treatment. Nevertheless, depending on the application, other factors can be included, for example the pH value of the fluid.
As described in section “Kinetics of Cell Inactivation and their Implementation in Numerical Simulations” the kinetic model
needs to be transferred in an expression for the source term of Eq. (15) in order to consider the inactivation kinetics in a CFD simu-
lation. Therefore, the temporal derivative of Eq. (50) needs to be calculated:

dFp d 1 exp kf ;0 sP ft Fp;max
¼ ¼ Fp;max kf ;0 sP f exp kf ;0 sP ft (55)
dt dt
Here, the treatment time is expressed as the product of the pulse with s, the pulse frequency f and the physical time t. The expo-
nential term on the right-hand side of Eq. (55) can be expressed as:

Fp ðtÞ
exp kf ;0 sP ft ¼ 1
Fp;max
Furthermore, Fp ðtÞ can also be expressed as the ratio of the mass of perforated cells at given time mperforated ðtÞ and the mass m0 of
cells at t0 . With this, Eq. (55) can be rewritten as:

vmperforated mperforatedðtÞ
¼ m0 kf ;0 sP f Fp;max (56)
vt m0
This equation has the same form as Eq. (25) in section “Kinetics of Cell Inactivation and their Implementation in Numerical
Simulations” and can be implemented as a source term pI in a general transport equation as given by Eq. (15) in section “Govern-
ing Equations”.
1.12.3.2.2 Simulation
The goal of the case study is the simulation of the inactivation of Chlorella vulgaris cells in an ordinary continuous treatment
chamber. The simulated geometry is presented in the left plot of Fig. 1 with different dimensioning. For the conductance of the
case study, the commercial software package Ansys CFX was used. The equations of the kinetic model (Fp ; Fp;max and kf ;0 ) were
implemented via user defined expressions and the derived source term for the transport equation of the perforated Chlorella cells
was included within a subdomain of the model. Note, that in this approach the cells of Chlorella vulgaris were modeled as inactive
tracers. This is reasonable because a one-way coupling can be safely assumed for a low-concentrated (1 g/L) flowing suspension of
microorganisms with the size of Chlorella (diameter 2–5 mm) (Michaelides et al., 2016). One-way coupling means that the presence
of the cells does not affect the flow and no inertial effect of the cells must be taken into account. Therefore, no interactions of the
cells or between cells and the surrounding fluid were considered in the simulations. Furthermore, the sedimentation of cells can be
neglected for similar reasons. Besides, the residence time of the cells inside the treatment chamber is less than a second, which is too
short for sedimentation. The flow field, the internal energy and the electric field were modeled as described in section “Governing
Equations”. The algae suspension was considered to have similar material properties as water. All material properties were modeled
with temperature dependency. The model equations for the temperature dependency are given in Table 6.
Table 6 Temperature dependency of the material properties of the fluid. The subscript C describes the temperature in degree Celsius, the subscript
K stands for Kelvin.
Parameter Symbol Equation

3
Densitya r½kg m 1000:22 þ 1:0205$102 $Tc 5:8149$103 $Tc2 þ
1:496$105 $Tc3
Heat capacitya cp ½kJ kg1 K1 4176:2 0:0909:Tc þ 5:4731:103 :Tc2
Thermal conductivitya k½W m1 K1 0:57109 þ 1:7625:103 :Tc 6:7036:106 :Tc 2
247:8 ½K
Dynamic viscositya m ½kg m1 s1 2:414:105 :10TK 140 ½K
s ½S m1
A
Electric conductivityb 0:889:10B :sref
A 1:37023:ðTc 20 CÞþ 8:36:104 :ðTc 20 CÞ2
B 109 þ Tc
a, Wölken et al. (2017), b, Atkins and de Paula (2006).
The boundary conditions were similar to the first case study in this article (see section “Case Study 1: Improvement of the Treat-
ment Homogeneity”), except from the once for turbulence, which is irrelevant for the present case. A parabolic profile for the
velocity in the main flow direction with respect to the volume flow was assumed at the inlet:
r 2
V_
ux ¼ 2$ 2 $ 1 (57)
pR R
A parameter study for the effect of different treatment conditions in terms of volume flow, inlet temperature, applied voltage and
pulse frequency on the cell permeabilization was conducted and therefore, the fixed value boundary conditions were varied from
case to case at three levels each. The levels for the fixed boundary conditions and the frequency are given in Table 7. The specific
parameter combinations where chosen by means of the response surface methodology. The pulse width was kept constant at
s ¼ 5 ms. The design of experiments led to 30 design points in total, whereby the repetitions were neglected because no variance
occurs in numerical simulations with similar settings.
After the implementation of the model, the domain was meshed by using the software Ansys mesh. A grid convergence study was
conducted to minimize the numerical error. Therefore, the quantities of the velocity, temperature, mass fraction of perforated cells
and the electric potential were evaluated at several points within the treatment chamber as a function of the grid refinement level.
The relative error from the finer to the coarser grid was calculated for all the mentioned quantities. If this error was below 3% for all
quantities, the numerical error was considered to be reasonably small. It should be noted that a grid convergence study is a crucial
step for obtaining accurate results from the numerical simulation. Further information about this topic can be found in (Steffen
et al., 1995) or (Roy, 2003). The final grid was refined at zones where high gradients of the different fields are expected. This ensures
the convergence of the transport equations. The z-y-plane of the created grid is presented in Fig. 6. A higher grid resolution was
chosen at the wall, to meet the no-slip boundary condition, which causes a high velocity gradient toward the wall, inside the insu-
lators gaps and behind the insulators to ensure the correct solution of the transport equation for the chlorella mass fraction and the
velocity related quantities, respectively.
The fraction of perforated cells (Fp ) is plotted in Fig. 7 exemplarily for one set of the parameter study (A). Compared to the
contour plot of the velocity and the temperature (plot B and C respectively), the areas with the highest rate of inactivation are
congruent to the areas of the highest temperature increase and the slowest velocity. In many cases, the maximum temperature
was above 55 C which was reported by Postma et al. (Postma et al., 2016) as the critical temperature at which autolysis of Chlorella
vulgaris starts. Note, that such an effect on the cell viability is not considered by the kinetic model. Since PEF is generally considered
as a non-thermal treatment, these temperature hotspots should be avoided. The temperature increase at this point is directly linked
to the recirculation zones, occurring behind the insulator gap (see case study presented in section “Case Study 1: Improvement of
the Treatment Homogeneity”). This again demonstrates the need of special tailored treatment chambers for any specific application
of PEF. In the case of microalgae treatment, the mass transfer enhancement is the desired effect. Since many cell metabolites are
thermosensitive, this result is of special interest.
Table 7 Variation of the boundary conditions for the numerical design of experiments.

Level Voltage ½kV Temperature ½K Volume flow ml
min Frequency ½Hz
1 7 293 100 100

2 11 298 150 150
3 15 303 200 200
Figure 6 Meshing of the colinear treatment chamber as used in the presented case study. The section shows the central z-y- plane. The grid was
refined at the wall near regions by using prism layers to resolve the velocity gradient toward the wall. The remaining domain was meshed by using
polyhedrons. A higher resolution was chosen inside and after the insulators to ensure the correct solution of the transport equation for the Chlorella
mass fraction and the velocity related quantities.
In Fig. 8, the results for the fraction of perforated cells Fp for all tested parameter sets are plotted versus the electric energy input,
which can be calculated as:
Z
sP f
wspec ¼ sðTÞE2 dV (58)
m_
Here V is the volume of the treatment chamber and m_ is the mass flow rate. The results show that for a complete perforation of
the cell population a minimum energy input of at least 64.64 kJ kg1 is necessary, which is around three times higher than the
energy input which as necessary for a full perforation in the batch cuvette experiments (19.6 kJ kg1). From the batch experiments,
which were used to calibrate the kinetic model, it can be seen that an electric field strength of 20 kV cm1 was needed to perforate
100% of the cell population (compare to Fig. 4). In this case study, the maximum field strength in the continuous chamber was
higher than this critical value (compare plot D, Fig. 7), but only at the edges of the insulators, which is typical for a colinear treat-
ment chamber (compare also to Fig. 2). The average residence time, calculated from the fraction of the volume of the treatment zone
and the volume flow, was found to be 0.062 s, 0.0417 s and 0.0313 s, depending on the flow rate. At a frequency of 200 Hz, a liquid
volume was exposed to 12.4 pulses at the lowest flow rate. This corresponds to an average treatment time of 0.062 ms for a pulse
width of 5 ms. Referring to Fig. 4, this should ensure a sufficient electropermeabilization, but as mentioned before, only a small
fraction of the suspension passes the areas with the highest field strength. For cells passing the insulators in the center of the
pipe, the field strength was not high enough for a complete permeabilization. Furthermore, the treatment time is smaller in the
center due to the higher velocity. Therefore, an over-processing occurs in the wall near regions whereas the fluid in the center of
the flow is potentially undertreated. On average, a reduced permeabilization efficiency can be observed for the whole treatment
process. As already mentioned, a complete perforation of the cell population was possible with the chosen parameter level. For
example, at a voltage of 15 kV an inlet temperature of 303 K, a frequency of 200 Hz and a volume flow rate of 100 ml min1,
Fp corresponds to a value of 0.99. Nevertheless, the outlet temperature was 321 K and the maximum temperature inside the treat-
ment chamber was 367.3 K. In this case, a temperature-related damage on desired products cannot be excluded. Therefore, a higher
degree of perforated cells cannot be reached by simply applying a higher voltage, since this might cause a decrease of the product
recovery. However, the presented case study demonstrates that numerical simulation can be a powerful tool to understand the
complex interaction between the flow field, the temperature and the electric field and their effect on biological material.
From the left plot of Fig. 8, a certain scatter of the results can be seen. Since the velocity or the mass flow and the pulse frequency
are represented in the equation for the specific electric energy input, it is obvious that this scatter must be caused by the different
inlet temperatures. On the right graph of Fig. 8, the fraction of perforated cells is plotted versus the total energy input. This includes
an estimation of the energy input which is needed for the prewarming of the fluid. It is calculated as:
wtotal ¼ wspec þ cp DT (59)
Here, DT was calculated between the inlet temperature and the reference temperature of 20 C. The heat capacity was taken from
data from the literature for the average temperature inside the preheater. It can be seen from the right plot of Fig. 8, that the fluc-
tuation disappears if the additional energy input through preheating is considered. It can be observed that the overall energy input
for a full perforation is more or less similar for all inlet temperature. Therefore, a mild preheating might be a good option to
improve the cell permeabilization of microalgae in case of technical limitations of the pulse generator. On the other hand, caution
is advised since increasing the inlet temperature might lead to the damage of thermosensitive cell valuables.
At this point, it should be mentioned, that the presented case study only demonstrates a valid setup for the permeabilization of
microalgae with the focus on mass transfer enhancement. For other applications, such as microbial inactivation, the optimal param-
eter set might be different since in such cases the focus is food safety and the level of the peak temperatures is of less importance.
Furthermore, the presented method for the detection of perforated cells with PI is not suitable to detect a 5 log inactivation, since the
accuracy of the staining is around 0.1% which is sufficient if the application is not inactivation but mass transfer enhancement. In
such cases, a different technique such as plate count should be chosen to calibrate the kinetic model. On the other hand, one should
keep in mind that the level of the accepted numerical error limits the prediction of microbial inactivation over several log cycles.
Figure 7 Contour plots of the fraction of perforated cells (Fp ) (A), flow velocity (B), temperature (C) and the electric field strength (D), respectively.
The plots show the middle plane of the colinear treatment chamber. The flow is from left to right. The specific energy input wspec was 58.62
kJ kg1 ðV ¼ 15 kV; Tinlet ¼ 298 K; V_ ¼ 150 ml min1 Þ: Note that the plots depict not the entire simulated domain, since the parts not shown
contain no addition relevant information. From Knappert et al. (2020).
Furthermore, the presented model is only valid for a specific strain, cultured under specific conditions. Therefore, a kinetic model
should be calibrated to the species or strain which is used for the specific application.
In this case study, a kinetic model for microalgae was presented but the method can be applied to any kind of microorganism
which is important for the food industry if a suitable method for the detection of the membrane perforation is available. First, the
experiments for the calibration of the kinetic model are related to a great effort in time and labor. But once a model is available, it
Figure 8 Results from the numerical parameter study with the parameter sets as presented in Table 7. (A) Fp plotted versus the electric energy
input. (B) Fp plotted versus the total energy input calculated with Eq. (59). From Knappert et al. (2020).
can be used for the investigation of the treatment homogeneity in any treatment chamber geometry within the calibrated parameter
range. Therefore, the presented method provides a powerful tool for the optimization of PEF treatment chambers, since the effect of
changing field distributions and parameter sets can be seen directly. Time-consuming experiments can be reduced, and the devel-
opment of new treatment chamber geometries is facilitated. Furthermore, it can be used as a tool for the scale-up of PEF processes
(see, (Knappert et al., 2020)), because the impact of changing geometries on the cell perforation can be investigated. In the next case
study, the advantage of this tool will be demonstrated by changing the treatment chamber design but keeping the applied treatment
parameters constant.
1.12.3.3 Case Study 3: Impact of Treatment Chamber Design on the Inactivation of Microalgae
In this case study, a treatment chamber with a modified inlet is presented, as it was investigated before by Schottroff et al. (Schottroff
et al., 2020). The fluid flows into the domain through two pipes, which were drilled into the first insulator. The inlet is characterized
by a fixed diameter and two entrance angles aI and bI . The grounding was realized by a ring electrode. The fluid domain of the
treatment chamber is illustrated from the side view in Fig. 9 and from the front view (in positive direction of x) in Fig. 10. The
dimensions of the treatment chamber and the tested entrance angles of the inlet pipes are summarized in Table 8. The aim of
the study is to investigate how the modified inlet changes the flow conditions in the chamber in order to increase the flow vorticity.
This leads to a harmonization of the residence time in the different parts of the treatment chamber. Thus, the treatment homoge-
neity is improved due to the induced transverse motion of the fluid elements with respect to the main flow direction. The effect of
the design on the treatment is validated with the model of microalgae inactivation as presented in section “Case Study 2:
Figure 9 Side view of the modified colinear treatment chamber: The inlets (1 & 2) are drilled into the first insulator (3). The entrance angle of the
inlets bI was variable within the presented case study. The high voltage electrode (HVE, 4), the second insulator (5), the second grounding (6) and
the outlet of the treatment chamber are configurated in the same way as usual for colinear treatment chambers. The letters di are indicating the
diameter of the different compartments (i ¼ 1,2,3.7) and Li their length. The values of di and Li are given in Table 8.
Figure 10 Front view (in x-direction) of the modified colinear treatment chamber. Here, 3 and 0 are insulators and 8 is the grounding. 9 and 10 are
the pipes of the inlet. The angle aI was varied in the presented case study. The letters di are indicating the diameter of the different compartments
and Li their length. The values of di and Li are given in Table 8.
Table 8 Values for the material properties as set in the presented case study.
Property Value Unit
Density r 999:99 kg m3

Dynamic viscosity m 1:00 mPas
Heat capacity cp 4184:52 J kg1 K1
Thermal conductivity k 599:35,103 W m1 K1
Electric conductivity s 0:0073K1 $ ðT 273:15KÞþ 0:1874 S m1
Permeabilization of Microalgae by Pulsed Electric Field”. The flow field and the electric field was modeled as described in section
"Governing Equations”. The absolute value of the flow vorticity was calculated as:
u ¼ jV uj (60)
Here, u represents the velocity vector. In order to investigate the geometry effects on mixing, the average vorticity magnitude was
calculated according to Eq. (47) for certain regions of interest such as the treatment zones within the chamber. Therefore, the re-
ported values of u refer to the averaged absolute vorticity in the following. If the value of this quantity is high, the flow can be
considered as well mixed. As a second quantity of interest, the specific energy input was calculated by means of Eq. (58).
The fraction of perforated cells Fp was modeled with Eq. (50), as presented in section “Case Study 2: Permeabilization of Micro-
algae by Pulsed Electric Field”. In order to evaluate the success of the treatment, the residual activity of the microalgae was calcu-
lated as the average over the outlet area:
RA ¼ 1 F p;out (61)
The boundary conditions in this case study were of the same type as in the study presented in section “Case Study 2: Permeabi-
lization of Microalgae by Pulsed Electric Field”. The velocity normal to the inlet was set to u ¼ 0:45 m s1 , the temperature at the
inlet was TInlet ¼ 293:15K, the frequency f ¼ 105½Hz, pulse width s ¼ 3½ms and the applied voltage at the high voltage electrode
was U ¼ 20 kV. For the material properties, fixed values were assumed for the density r, the dynamic viscosity m, the heat capacity cp
and the thermal conductivity k. The electric conductivity s was implemented as a function of the temperature. The function and the
values for the other material properties are given in Table 9.
Within the first part of the presented case study, the angle bI was kept constant at 95 . For details the interested reader is redir-
ected to the original research (Schottroff et al., 2020). The simulation results are presented in Table 10. According to the presented
results, the average vorticity was the highest in the first insulator (zone 1) for an angle aI ¼ 65 . Nevertheless, the energy input was
the lowest (wspec ¼ 101 kJ kg1 Þ for this configuration. This can be explained by the high attenuation of the vorticity along the treat-
ment chamber which is expressed by the difference of the vorticity between the first and the second insulator Du. Here, the config-
uration with the angle aI ¼ 61 showed the best results. This means that the swirling flow can be maintained for a longer period,
which is reflected by a higher vorticity in the second insulator (u ¼ 434:16 s1 ). In contrast, the lowest vorticity in the second insu-
lator occurred at angle aI ¼ 65 . Generally, it was observed that Du shows a linear correlation with the average vorticity in the first
isolator (R2 ¼ 0:9946). The higher the vorticity in the first insulator, the lower it was in the second. This also corresponds to the
estimated pressure loss, which increases with vorticity in the first insulator and was therefore the lowest for the configuration with
Table 9 Values for the dimensions of the modified colinear treatment chamber as depicted in Figs. 9 and 10. The angles a and b were varied in the
presented case study. The cross marker indicates cases which were simulated whereas the black filled cells indicates cases which were not
simulated due to manufacturing limitations.
Dimension do ; d1;2 d3;5 d7 L L3 L4 L5 L6 L9 ; L10
Value [mm] 2.0 4.0 6.0 73.5 3.5 35.0 5.0 30 5

Angle aI ½
90 85 80 75 70 65 61

bI ½ 95 X x x X X x x
100 X X x x
105 x x
115 x
125 x x x
Table 10 Characteristics of the flow and treatment conditions in modified colinear treatment chamber (vortex) and the standard configuration
(colinear). Zone 1 and Zone 2 stand for the first and the second insulator respectively. Results from Schottroff et al. (2020).
Design Angle aI ½ w spec ½kJ kg1 DT ½K u½s1 Zone 1 u½s1 Zone 2 Du½s1 Dp½Pa
Vortex 90 103.51 24.06 1170.41 331.03 839.38 324.142
85 103.27 24.23 1184.92 319.473 865.447 326.013
80 102.87 24.41 1211.37 302.013 909.357 331.107
75 102.79 24.05 1222.3 287.414 934.886 341.66
70 102.56 24.47 1249.92 266.761 983.159 353.855
65 101.00 24.73 1320.12 260.208 1059.912 367.052
61 105.03 24.84 1002.55 434.163 568.387 296.311
Colinear – 76.64 21.75 177.16 206.03 28.87 327.54
angle aI ¼ 61 . The specific energy input wspec was significantly higher for all the tested configurations of the vortex chamber (101
105:03 kJ kg1 ), compared to the standard colinear configuration (76:64 kJ kg1 ). This can be explained by the more homogenous
temperature distribution insight the modified treatment chamber, as it increases the electrical conductivity in the treatment zone
and therefore the energy input in areas of high electric field strength (see also Fig. 11). The temperature inside the insulator gap
was the highest for aI ¼ 61 with 308.88 K. Compared to the other angles, this increase is moderate since the minimum temper-
ature was achieved for aI ¼ 65 with 307.82 K. The overall temperature increase at the outlet was around 24 K for all inlet config-
urations, which is below the critical value of 55 C at which autolysis of Chlorella vulgaris cells starts (Postma et al., 2016). The first
part of the presented case study indicates that the modified treatment chamber is superior to the standard colinear configuration for
all tested angles. For further investigation, the angle a ¼ 61 was chosen for testing the perforation of the microalgae since it unifies
the highest specific energy input, the lowest loss in vorticity and the lowest pressure loss, which are both important parameters for
applications at larger scales.
In a second step, also the angle b was varied. For the aI ¼ 61 the maximum value for bI was 100 due to manufacturing limi-
tations. Within these geometric boundary conditions (indicated by the x’s in Table 9) random combinations of aI and bI were
chosen. However, no clear correlations were found since not all angle combinations could be realized due to manufactural restric-
tions. In general it can be said that dead zones behind the insulators can be prevented by choosing the combination of aI and bI in
a way that the inflows of both inlets are directed toward each other, what leads to a high vorticity. Nevertheless, clear dependencies
of the vorticity from the angle bI were not seen. This indicates a more complex impact on the flow conditions as it was observed for
the angle aI . Therefore, this should be part of future investigations for a better understanding of the complex flow inside the modi-
fied treatment chamber.
In the last step of this case study, the modified treatment chamber with the angle combination of aI ¼ 61 and bI ¼ 95 was
compared with an ordinary colinear treatment chamber with regard to the perforation of the microalgae Chlorella vulgaris as
described in section “Case Study 2: Permeabilization of Microalgae by Pulsed Electric Field”. The inner diameter for the electrodes
is 6 mm and 4 mm for the insulator. The contour plots for the residual activity RA are shown in Fig. 12 for the colinear treatment
chamber and the modified treatment chamber, respectively. It is easy to see, that the ordinary treatment chamber performs worse
than the modified one. For the ordinary treatment chamber, the average RA at the outlet was 0.480 compared to 0.340 for the modi-
fied one. This corresponds to an additional reduction of RA of 29.17% simply by optimization of the treatment chamber geometry.
The better treatment conditions inside the modified treatment chamber are also expressed by the specific energy input wspec . For the
ordinary treatment chamber the specific energy input was 60:8 kJ kg1 compared to 83:08 kJ kg1 for the modified one. This can be
explained by a less homogeneous distribution of the residence time inside the treatment zone in case of the ordinary treatment
Figure 11 Comparison of the colinear (left) and the vortex (right, a ¼ 61 ) treatment chambers, including velocity streamlines (A), with colors
indicating the fluid velocity, temperature distribution (B), als well as electric field strength (C). Assumed processing conditions: m_ ¼ 10:2 kg h1 ,
Tin ¼ 293 ½K, f ¼ 105 Hz, s ¼ 3 ms. All plots show the x-z-plane at the center of the treatment chamber. From Schottroff et al. (2020).
chamber, which is also expressed by the low vorticity (see Table 10). This means that almost no movement transversely to the main
flow direction occurs and therefore, the mixing of the fluid is not as pronounced in comparison to the treatment chamber with the
modified inlet. The poor mixing for the ordinary treatment chamber is also illustrated by the streamline plots in Fig. 11, which
shows streamlines in the modified and the ordinary treatment chambers. The streamlines are parallel for the ordinary treatment
chamber what means that a fluid element in the center of the flow field stays in the center of the flow field. In contrast, a strong
mixing in the z and y direction takes place in the modified treatment chamber.
As opposed to the first presented case study in this chapter, here the flow field was manipulated by changing the inlet geometry.
The presented results show that the treatment homogeneity and therefore the success of the PEF treatment is improved by optimi-
zation of the inflow conditions. This is mainly based on a higher vorticity, which causes a more homogeneous distribution of the
residence time within the treatment zones. Therefore, all fluid elements are treated more equally due to the induced movement in
the radial directions. It should be kept in mind that the average temperature inside the treatment zones is slightly higher due to the
longer residence time. For thermosensitive foods, for example milk or juice, this should be considered. The case study demonstrates
again that the geometry of the PEF treatment chamber should be developed in dependence of the specific application. Optimizing
a treatment chamber for all possible applications is not possible. In the end, the best treatment chamber for a specific application is
a compromise between the best flow conditions, the temperature increase and the electric field distribution. Therefore, it could be
possible that one of the presented entrance angle combinations is better for a specific application as the suggested set of aI ¼ 61
and bI ¼ 95 . This shows that the decision for the best treatment conditions cannot be made alone based on CFD simulations.
However, CFD provides a powerful tool to design an optimized PEF process for the application in question. This tool is even
more powerful, if a suited kinetic of inactivation is included, which makes the impact on the treatment directly visible. Here, an
inactivation kinetic for microalgae was used as an example but also a kinetic for the inactivation of other microorganisms or
enzymes, as it was done previously in (Schottroff et al., 2020), can be chosen.
For a further optimization of the inflow conditions, other angle combinations can be chosen. Furthermore, the two inlets could
be varied non-symmetrically. This should be done under consideration of a specific constraint of the acceptable maximum and
average temperatures, which again depend on the application. In the presented case study, water-like behavior of the fluid was
assumed. But many foods have a higher viscosity and often, they show non-Newtonian behavior. In these cases, the flow field
changes significantly, and it is an open question if the influence of the vorticity is the same. Lastly, a scale-up should be done of
Figure 12 Contour plot of the residual activity RA on the middle plane of a colinear treatment chamber (A) and a colinear treatment chamber with
modified inlet and grounding (B). The letters indicate the compartments of the treatment chamber: Inlet (1), High voltage electrode (HVE, 2),
Insulators (3), Grounding (4) and Outlet (5). Treatment conditions were the same for both plots (Voltage U ¼ 20 kV, Temperature T ¼ 293.15 K,
velocity u ¼ 0.4 m s1 ).
Figure 13 A) Results for the fraction of perforated cells as predicted by the numerical simulation according to section “Case Study 2:
Permeabilization of Microalgae by Pulsed Electric Field” versus the fraction of permeabilized cells as measured in the validation experiment. (B)
Results for the temperature increase as predicted by the same numerical simulation versus the temperature increase as measured at the outlet in the
validation experiment. The dashed line represents the bisector. From Knappert et al. (2020).
the presented geometry. On a larger scale, the flow conditions at the inflow and inside the treatment chamber are probably different
and therefore, the presented optimal parameters are not valid anymore. Since turbulence plays a role for higher mass flows and
larger geometries (see section “Case Study 1: Improvement of the Treatment Homogeneity”), this could also have an effect on
the best parameter set.
1.12.4 Validation
A model of a PEF process is just as good as it describes the reality. Therefore, the experimental validation is a crucial step of modeling
and simulation. Since the electric energy input is proportional to the temperature increase, the most common way of validating
a PEF model is the measurement of the temperature. For this, it is important which quantities are compared. If the temperature
is measured at the outlet, the averaged outlet temperature of the simulation should be taken as the comparative quantity. The vali-
dation of the temperature increase for the case study presented in section “Case Study 2: Permeabilization of Microalgae by Pulsed
Electric Field” is shown in plot B of Fig. 13. It should be mentioned here, that this method of validation can cause some errors since
the measurements of the temperature are often conducted several centimeters behind the treatment chamber. Therefore, the exact
position of the temperature measurement should be mentioned in relation to the treatment chamber. If a direct measurement of the
temperature is not possible, the temperature increase can also be calculated by the specific energy input, which can be derived from
measurements of the applied voltage and the electric current. Nevertheless, the measurement of the global temperature increase is
not suitable to evaluate the effect of local temperature hotspots inside the treatment chamber. By using fiber optic probes, it is
possible to measure the temperature inside the treatment chamber, either inside the insulator gap or inside the high voltage and
the grounding electrode gap respectively (Schroeder et al., 2009; Buckow et al., 2010). If this method is applied, it should be consid-
ered, that the probe can influence the velocity field and therefore, the temperature distribution inside the treatment chamber. This
problem can be avoided if the probe is included within the simulation. Nevertheless, errors can occur since it is difficult to place the
probe on an exact position inside the treatment chambers (Schroeder et al., 2009). Furthermore, this method is only practicable for
treatment chambers with a larger inner diameter. Since the model of PEF described in section “Background and Fundamentals”
simulates the steady state, it is crucial to wait for constant values for a proper data acquirement.
The proposed model of the inactivation of the microalgae Chlorella vulgaris provides another tool to validate the simulation
model of the PEF process. Since the degree of permeabilization is easy to detect by the proposed flow cytometric method, the addi-
tional simulation of the inactivation provides another advantage compared to the simulation of the PEF process alone. A validation
experiment was conducted for the case study presented in section “Case Study 2: Permeabilization of Microalgae by Pulsed Elec-
tric Field”. Some parameter sets from the numerical parameter study where chosen for experiments in a colinear treatment chamber
with the same dimensions as simulated. The results of this experiment are presented in Fig. 13 (Plot A). It can be seen that the model
predicts the experimental data reasonably well. It shall be mentioned that the difference between the CFD simulation and the exper-
iment increased with the specific energy input, especially at higher inlet temperatures. One possible explanation for this derivation
are local temperature hotspots which are outside the calibrated temperature range of the model. In those cases, the temperature
effect might be underestimated by the model. Further, one point clearly derivates from the experimental data. Since the temperature
increase for this case matches well with the simulation, it is likely that the deviation is caused by an experimental error. Nevertheless,
the results show that the proposed model is valid in the investigated parameter range and that it can be used as a tool for the opti-
mization of PEF treatment chambers or for the upscaling of the whole process. It should be considered that the validity of the model
can be lost if another strain is used or if the microalgae are cultivated under different conditions. For a comprehensive model, it is
therefore recommendable to have more kinetic models which cover the variety of the used microorganism strains.
In the end, modeling is always a simplification of the reality and therefore, differences between the model and the reality are
inevitable. It is part of the model validation that a trained person decides if these differences are critical for the specific application
or not.
1.12.5 Conclusion
Modeling of the PEF process on difference scales was addressed in this chapter. The advantages of the simulation on different length
scales were pointed out. In section “Governing Equations”, the governing equation for the modeling of a continuous PEF process
were introduced and common simplifications were discussed. The modeling of material parameters was discussed in section
“Process Dependence of Fluid Properties” and a review of suitable kinetic models for the modeling of the cell perforation was
given in section “Kinetics of Cell Inactivation and their Implementation in Numerical Simulations”. A brief introduction of
the modeling of cell perforation on the length scale of a single cell and single molecules was given in section “Modeling Cell Inac-
tivation on the Microscale”.
Within the case studies in chapter 3, the advantage of numerical CFD simulations as a tool for optimization and scale-up was
demonstrated. A valid model for the inactivation of the microalgae Chlorella vulgaris was proposed, which further extends the field of
application of numerical simulations of PEF. This model reveals that ordinary treatment chambers are often not suitable for the
treatment of thermosensitive valuables. On the other hand, it was demonstrated that the effect of the electric field on the microbial
inactivation is often overestimated in experiments since additional effects due to the temperature increase at high treatment
intensities cannot be neglected. It was further shown that this problem can be overcome by the optimization of the PEF treatment
chamber geometry. It was pointed out, that the validity of the simulation needs to be proven by experiments. Since the mechanism
of PEF is still not well-understood, the treatment is often a case to case study which is related to a high effort in time and labor. The
approaches presented in the case studies can help to reduce the number of experiments which are needed to design a new process.
Therefore, planning of a new process becomes easier, less time consuming and cheaper which can eventually help to open the PEF
technology for a broader field of commercial applications.
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1.

12 Modeling and Simulation of PEF

1.12.1 Introduction 212

Innovative Food Processing Technologies, Volume 1 https://doi.org/10.1016/B978-0-12-815781-7.00020-2 211

Fmax Maximum volume fraction of suspended particles ½

1.12.2 Background and Fundamentals

1.12.2.1 Governing Equations

vrui vrui uj sij

vre vreuj þ qj vui

1.12.2.2 Process Dependence of Fluid Properties

1.12.2.3 Kinetics of Cell Inactivation and Their Implementation in Numerical Simulations

1.12.2.4 Modeling Cell Inactivation on the Microscale

1.12.3.1 Case Study 1: Improvement of the Treatment Homogeneity

Value 4183 0.001 0.63 997 0.28

Ordinary ISO 1 Ordinary ISO 2 Modified ISO 1 Modified ISO 2

ECV $½ 0.135 0.136 0.128 0.128

Tmax $½K 313.27 305.55 293.86 298.13

Tmean $½K 284.84 287.18 284.38 284.88

TSD $½K 3.35 2.60 1.05 1.27

TCV $½ 0.01 0.01 0.004 0.004

UCV $½ 0.22 0.27 0.20 0.22

1.12.3.2 Case Study 2: Permeabilization of Microalgae by Pulsed Electric Field

Electric ﬁeld strength kV cm1 6.5 9 13.5 20 27

Table 5 Fitting parameters for Eq. (51).

Parameter Symbol Equation

a, Wölken et al. (2017), b, Atkins and de Paula (2006).

1 7 293 100 100

Property Value Unit

Density r 999:99 kg m3

Dimension do ; d1;2 d3;5 d7 L L3 L4 L5 L6 L9 ; L10

Value [mm] 2.0 4.0 6.0 73.5 3.5 35.0 5.0 30 5

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