Biotechnology Biotechnol. J. 2014, 9, 727738 DOI 10.1002/biot.

201300236
Journal
www.biotechnology-journal.com

Perspective

Challenges in industrial fermentation technology research

Luca Riccardo Formenti1, Anders Nrregaard1, Andrijana Bolic1, Daniela Quintanilla Hernandez1,
Timo Hagemann1,2, Anna-Lena Heins3, Hilde Larsson1, Lisa Mears1, Miguel Mauricio-Iglesias1,
Ulrich Krhne1and Krist V. Gernaey1
1 Department of Chemical and Biochemical Engineering, Technical University of Denmark (DTU), Lyngby, Denmark
2 Novozymes A/S, Kalundborg, Denmark
3 Department of Systems Biology, Technical University of Denmark (DTU), Lyngby, Denmark

Industrial fermentation processes are increasingly popular, and are considered an important tech-
Received 03 FEB 2014
nological asset for reducing our dependence on chemicals and products produced from fossil Revised 01 APR 2014
fuels. However, despite their increasing popularity, fermentation processes have not yet reached Accepted 23 APR 2014
the same maturity as traditional chemical processes, particularly when it comes to using engi-
neering tools such as mathematical models and optimization techniques. This perspective starts
with a brief overview of these engineering tools. However, the main focus is on a description of
some of the most important engineering challenges: scaling up and scaling down fermentation
processes, the influence of morphology on broth rheology and mass transfer, and establishing
novel sensors to measure and control insightful process parameters. The greatest emphasis is on
the challenges posed by filamentous fungi, because of their wide applications as cell factories and
therefore their relevance in a White Biotechnology context. Computational fluid dynamics (CFD)
is introduced as a promising tool that can be used to support the scaling up and scaling down of
bioreactors, and for studying mixing and the potential occurrence of gradients in a tank.

Keywords: Computational fluid dynamics Fermentation process development Fungal morphology Modeling
Scale-down bioreactors

1 Introduction dependence on traditional products produced from fossil

Fermentation processes have been used for production
and conservation of food for thousands of years, and have
been increasingly used for the industrial production of bulk
chemicals, fine chemicals, and pharmaceuticals as well.
Nowadays, industrial biotechnology or white biotech-
nology is well accepted and considered to be one of the
important technologies that will help to reduce our
Correspondence: Prof. Krist V. Gernaey, Department of Chemical and
Biochemical Engineering, Technical University of Denmark (DTU),
Building 229, DK-2800 Lyngby, Denmark
E-mail: kvg@kt.dtu.dk
Abbreviations: CFD, computational fluid dynamics; CSTR, continuously
stirred tank reactor; DO, dissolved oxygen; GMP, good manufacturing prac-
tices; OTR, oxygen transfer rate; PID, proportional integral derivative; STR,
stirred tank reactor
fuels. Well-known examples of fermentation products are:
Organic acids produced by filamentous fungi, for exam-
ple citric acid production by Aspergillus niger [1].
1,3-propanediol produced by several species [2],
where the most well known is the 1,3-propanediol pro-
duction process established by DuPont, based on a
genetically modified Escherichia coli strain [3].
Antibiotics. Penicillin, the first antibiotic that was dis-
covered, is produced at large scale by Penicillium
chrysogenum [4].
The production of recombinant proteins such as
insulin by E. coli [5] and human interferon, the hepati-
tis B surface antigen and insulin by Saccharomyces
cerevisiae [6].
The start of the commercial operation of industrial
fermentation processes also meant that optimization of
fermentation processes has been a priority ever since.
Fermentation process optimization aims at reaching vol-
umetric production rates (=units of product formed per

2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 727

 Biotechnology Biotechnol. J. 2014, 9, 727738
Journal
www.biotechnology-journal.com
unit reactor volume per time) that are as high as possible,
combined with achieving very high product titers. The
latter are crucial in achieving an economically feasible
process since high product titers will reduce the cost of
downstream processing. As a consequence of substrate
inhibition, a phenomenon that is common with many pro-
duction hosts, many commercial fermentation processes
are operated in fed-batch mode, where a limiting carbon
source substrate is dosed to the fermenter during the fed-
batch phase [7].
Optimization of fermentation processes is achieved
through a combination of strain improvements and
process improvements. Nowadays diverse and well-char-
acterized tools are available to achieve strain improve-
ments for many popular production hosts (e.g. [6, 8, 9]).
Process improvements can, for example, include changes
of the feeding strategy during the fermentation process,
manipulation of the media composition, or changes to the
pH set point, etc. For example, the lactic acid production
can be improved by selectively removing the product
from the fermentation broth, thereby reducing the effects
of product inhibition on the production strain at high
titers [10].
Interestingly, despite the fact that some fermenta-
tion processes have been in operation for several
decades, introducing a new or improved production
host in a production process is still done in a rather
empirical way, by performing experiments at bench-
scale and pilot-scale which yield a suitable production
process recipe that is subsequently transferred to full-
scale production fermenters, and further optimized
there if needed. In this respect, the introduction of the
process analytical technology (PAT) guidance [11] has in
general put more focus on the development of a more
detailed process understanding, also in the fermenta-
tion industry, but this has thus far not resulted in
improved scaling-up strategies. The use of advanced
analytical tools, such as near infrared (NIR) or Raman
spectroscopy, and the validation of relevant chemomet-
ric models in industrial-scale fermentation is far from
being fully developed, due to technical limitations of the
measurement equipment (spectrum of water is domi-
nating in NIR spectroscopy, and challenges with fluo-
rescent compounds in Raman spectroscopy), due to the
variability and heterogeneity of a fermentation broth
(i.e. complex media, cell morphology, and medium rhe-
ology), due to the interference of gas bubbles and bio-
mass particles with the liquid phase of aerobic fermen-
tations, and due to the required investment for such
expensive equipment compared to the low added value
of many fermentation products [1214].
The focus of this contribution is on some of the chal-
lenges related to achieving process improvements. First
of all, the paper provides an overview of engineering tools
which are available to the process scientist to facilitate
the search for potential process improvements. After-
728
www.biotecvisions.com
wards, the paper aims at discussing some of the major
issues that are standing in the way for a more general use
of these engineering tools, which in many cases are con-
sidered standard tools in other industries such as the
petrochemical industry. Note that we will purposely not
address the detailed biological aspects of industrial fer-
mentation, for example the issues related to genetic insta-
bility [15], population heterogeneity [16], or the modeling
of metabolism at single cells level [17]. We assume that
the environmental conditions put a selective pressure on
the phenotype of the cells, and therefore, by controlling
the macroscopic variables of a fermentation it is also
assumed to be possible to reduce and control the vari-
ability in the cellular population.
2 Engineering tools
for process improvements
In the search for fermentation process improvements, the
fermentation process scientist relies on the available
process knowledge to carefully select which steps are to
be taken or which modifications are to be made in order
to maximize the chance of success. In addition to that
process knowledge, the process engineer can rely on a
number of engineering tools to assist in the process opti-
mization task: statistical methods for design of experi-
ments, mathematical models, data pretreatment and data
mining tools, control algorithms, etc. Some of these tools
are briefly highlighted in this section.
2.1 Statistical design of experiments
Statistical design of experiments [18] plays an important
role in supporting the fermentation scientist in collecting
in a structured way a data set that is assumed to
include the effect of the main variables of interest
usually called process parameters on the fermentation
process performance. Albk et al. [19], for example, used
a full-factorial design consisting of two levels for three
process variables: Specific agitation power input
(1.5 and 15 kW/m3, respectively), aeration rate (96 and
320 NL/min, respectively), and headspace pressure
(0.1 and 1.3bar, respectively). The resulting data set sub-
sequently formed the basis for the development and vali-
dation of a mathematical model describing the produc-
tion of cellulosic enzymes by the filamentous fungus Tri-
choderma reesei. Media composition can be optimized in
a similar fashion using design of experiments, as for
example illustrated for optimization of phytase production
by Sporotrichum thermophile by Singh and Satyanara-
yana [20]. In general, the data resulting from a statistical
design of experiments form an essential source of infor-
mation feeding in to a typical process optimization task,
but can, for example, also form the basis for the develop-
ment of mathematical process models [19]. Finally, it is
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
 Biotechnology Biotechnol. J. 2014, 9, 727738
Journal
www.biotechnology-journal.com
also important to note that statistical design of experi-
ments combined with parallel cultivation techniques, for
example shake flasks or microtiter plates, can contribute
significantly to speeding up process development.
2.2 Mathematical models
Mathematical models of a fermentation process usually
consist of a combination of mechanistic and empirical
model elements. Mechanistic models increasingly
replace empirical models when more detailed process
knowledge becomes available. Empirical models as such
are powerful for data exploration (e.g. Kennedy and
Krouse [21]), since they can be used to extract relations in
a data set either historical process data or data resulting
from a dedicated statistical design of experiments with-
out requiring detailed knowledge of any underlying
mechanism. Especially in the case of process data, appro-
priate data pretreatment is essential before using the
data in the frame of data-driven or mechanistic model
building [22]. Once developed, a fermentation process
model can ideally predict performance of future produc-
tion facilities considering the scale of operation, the
process design and process conditions. For more details
on the state of the art within mathematical modeling of
fermentation processes, the review of Gernaey et al. [23]
can be consulted. A recent fermentation technology-relat-
ed example illustrating the practical use of mathematical
models demonstrated the prediction of enzyme produc-
tion by the filamentous fungus Aspergillus oryzae at pilot-
scale at different operating conditions [24]. The model
was developed in such a way that the central part of the
model the oxygen mass transfer model can be substi-
tuted for other applications. This was illustrated in prac-
tice as well, by applying the model to Trichoderma reesei
fermentations [19]. However, the limitation of the model
lies in the fact that the oxygen mass transfer correlation is
strain- and scale-specific; therefore, a practical limitation
is that a separate design of experiments needs to be car-
ried out every time a different process is introduced in
order to characterize the oxygen mass transfer correla-
tion.
2.3 Optimization algorithms
A mathematical optimization (mathematical program-
ming) algorithm is a useful add-on to a mathematical
model, and will attempt to select the best alternative from
a set of available alternatives. It usually relies on mini-
mization (or maximization) of an objective function.
Important applications of optimization algorithms in
industrial fermentation are parameter estimation and
process optimization. Parameter estimation is the task of
finding the values of model parameters so that the model
predictions best fit the available experimental data
usually expressed as minimizing the sum of squared dif-
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotecvisions.com
ferences between model prediction and experimental
data [25]. Since the fermentation models are usually non-
linear, parameter estimation is carried out by nonlinear
programming solvers (NLP). Popular solvers for least-
squares estimation are included in the softwares Matlab
(lsqnonlin), SAS (PROC NLP), Octave (sqp), etc.
The goal of process optimization is to change one or
several decision variables related to the design or the
operation of the fermentation in order to optimize a given
criterion (in general to maximize profit). In particular, the
research literature has focused on optimizing the feed
rate and composition in fed-batch fermentation as a
means to overcome substrate and product inhibition
while maximizing the conversion and production of the
fermenter, as reviewed by Smets et al. [26].
2.4 Uncertainty and sensitivity analysis
Uncertainty and sensitivity analysis are useful as model-
analysis tools [27]. In an uncertainty analysis, propagation
of the various sources of uncertainty e.g. in the model
parameters to the model outputs is studied. The sensi-
tivity analysis, on the other hand, aims at identifying and
quantifying the individual contributions of the uncertain
inputs to the output uncertainty. Local and global sensi-
tivity analysis methods are distinguished [28], where the
local methods aim at ranking the importance of the indi-
vidual parameters in a single specific combination of
parameters (one operating point), whereas the global
methods consider a much wider parameter space. Results
of a sensitivity analysis can be coupled back to experi-
ments and parameter estimation: the analysis can on the
one hand point toward which variables should be meas-
ured, for example when designing experiments which are
suited to generate data that allow estimation of specific
parameters. On the other hand, the sensitivity analysis
can result in a simplified parameter estimation problem
by pointing toward one or several parameters that are not
influential on the model output and should thus not be
estimated [29, 30].
2.5 Process control algorithms
In general, control is implemented to maintain a process
at the desired operating conditions safely and efficiently,
while satisfying environmental, economic and product
quality requirements. Control of fermentations deals with
complex dynamic behavior of microorganisms, signifi-
cant model mismatch, nonlinear and even inherently
unstable dynamics, scarce online measurements of a
number of representative variables, etc. [31]. Hence, to
date, the control systems applied to industrial fermenters
aim mainly at the regulation of the process, that is they
aim at keeping a certain control variable at a given set
point or trajectory despite disturbances. This is frequently
done by simple single inputsingle output (SISO) con-
729
 Biotechnology Biotechnol. J. 2014, 9, 727738
Journal
www.biotechnology-journal.com
trollers using the ubiquitous proportional integral deriva-
tive (PID) (most often only PI) algorithm [32].
2.6 Computational fluid dynamics
Computational fluid dynamics (CFD) is a powerful tool for
reactor design and process understanding, since it can be
used to model and predict important process parameters
such as mixing behavior, mass transfer, and energy input
requirements for different fermentation setups. CFD has
been employed for simulating fermentation systems on
many different scales, for example in order to model
gasliquid mass transfer in shake flasks [33], gas-liquid
dispersion in a 5L bioreactor [34] and gasliquid flow and
local mass transfer in reactors with volumes up to 200L
[35]. In [36], interfacial oxygen mass transfer and energy
dissipation is simulated and compared to experimental
data for a 10 mL cylindrical-shaped and paddle-stirred
reactor, designated for cultivation of filamentous microor-
ganisms. The mixing characteristics of the same system
have also been investigated and results are presented in
[37].
3 Major challenges
Despite the availability of an extended set of engineering
tools which was introduced in the previous section, a
large part of the engineering tools is not frequently used
in industry, or has not been exploited to its full potential.
In this section, we discuss a number of fermentation
process-related challenges that make it difficult for the
fermentation scientist to use specific engineering tools.
3.1 Scaling up
Scaling up a fermentation process serves the purpose of
transforming optimal operating conditions found in labo-
ratory- or pilot-scale bioreactors to the production-scale
bioreactor in order to reach maximal volumetric produc-
tivity of the full-scale process. However, several other
tasks need to be completed before scaling up a process:
screening and selection of the production strain, strain
improvements, manipulation of media composition, and
various process parameter optimizations [38]. All these
tasks are usually first performed as parallel microtiter
plate cultivations for screening purposes. Later on, when
the number of potential production strain candidates has
been narrowed down sufficiently, experiments are per-
formed in bench-scale cultivation equipment (stirred
bioreactors with a typical volume from 0.5 to 20L) for fur-
ther strain characterization and a first round of process
parameter optimization. Finally, further process parame-
ter optimization is done in pilot-scale equipment.
A major advantage of bench-scale cultivations is that
they can be run with different types of feeding schemes,
730
www.biotecvisions.com
and most types of standard sensor equipment can be
inserted in the bioreactor without any problem. A major
issue related to the use of bench-scale bioreactors as well
as the larger pilot-scale bioreactors (202000 L) is that
experiments at different scales are very difficult to com-
pare to larger size industrial bioreactors. Efficient and fast
scaling up of a process was described in detail by Schmidt
[39], and is even today one of the major challenges in the
fermentation industry. Indeed, scale-up is not done based
on a mathematical process model or by using design of
experiments, but is typically relying on a rather empirical
approach (e.g. relying on oxygen transfer correlations),
and is therefore still an important research topic. In prac-
tice, scaling up a process is often done iteratively, by com-
parison of results of pilot plant experiments with key data
obtained from the industrial-scale processes. If pilot plant
results can be reproduced at industrial scale, scale-up is
considered successful. If not, an additional round of pilot
plant experiments might be performed, for example to
investigate the effect of additional process parameters on
process performance.
The fact that most industrial-scale data are propri-
etary is a severe limitation. As a consequence, typical
data from process scale-up studies cannot be found as
part of the publicly available scientific literature, and can
thus not be used to extend the available knowledge about
this important engineering task. One should of course not
expect industrial partners to share their company secrets.
But somehow it must be clear that significant progress
with respect to understanding the scaling-up problem
can only be made through a close collaboration between
industry and academia: industry has the equipment and
the knowhow about how to run a full-scale fermentation,
whereas academia can contribute with expertise in, for
example, CFD (see also below) and detailed knowledge
related to the cellular behavior at large scale to develop a
better understanding of the major scaling-up challenges.
Due to the physical differences between scales, the
flow patterns in the bioreactors will differ. In bench-scale
bioreactors, perfect mixing of the broth can be assumed,
while in a large bioreactor the mixing time for achieving
95% homogeneity (95) can be in the range of minutes [40].
This can lead to oxygen starvation in the medium if the
degree of mixing is too low [41]. Furthermore, glucose,
pH, and temperature gradients are all likely to emerge in
production-scale fermentation [42, 43]. These hetero-
geneities in the environment of the organism will affect
biomass growth and are one of the primary concerns
when dealing with process scaling.
The Reynolds number is another way of clarifying the
differences in flow regime as it also changes between
scales. The same fermentation broth (i.e. same rheologi-
cal parameters) in bioreactors with equally scaled dimen-
sions and with the same specific power input will give a
Reynolds number in the turbulent flow regime in a pro-
duction-scale fermenter while in a bench-scale fermenter
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
 Biotechnology Biotechnol. J. 2014, 9, 727738
Journal
www.biotechnology-journal.com
Figure 1. The volumetric power input (kW/m3) plotted for different scales
of bioreactors while conserving oxygen transfer rate (mol/L/h) between
the processes. Both plots are calculated examples based on conserving
the OTR from large scale at smaller scales. Circles () represent water-like
broth, while squares () represent a highly viscous filamentous broth.
Information on the host organism is not available for proprietary reasons.
Reprinted with permission from Elsevier [44].
it will yield a Reynolds number in the transitional or lami-
nar flow regime [40, 44]. These differences in mixing prop-
erties will lead to differences in mass and heat transfer in
the processes [45]. Mass transfer of oxygen to the broth
and carbon dioxide removal are important for the cellular
response. The volumetric mass transfer coefficient of oxy-
gen (kLa) is often used as a critical parameter for scaling
purposes [46], meaning that scale-up then relies on main-
taining kLa constant across scales. Other important scal-
ing parameters include the oxygen transfer rate (OTR),
the volumetric power input (P/V) and the superficial gas
velocity (vg). It is important to mention that it is not pos-
sible to conserve all parameters across scales, and a suit-
able compromise has to be found. Figure1 shows the spe-
cific power consumption at different process scales with
a conserved OTR. As can be seen from Figure1, the pow-
er requirement for agitation in large vessels is lower than
that in small vessels, which is due to the effect of the high-
er back pressure and the increased pressure from the liq-
uid column that will facilitate oxygen mass transfer. In
order to achieve a high volumetric power input in small
bioreactors, the agitation speed has to be very high,
which leads to higher shear rates in the smaller vessels.
Fermentation broths of filamentous organisms can exhib-
it non-Newtonian flow behavior. The viscosity of non-
Newtonian broths is dependent on the shear rate and thus
the difference in shear rate across scales will lead to dif-
ferences in apparent viscosity [47]. This in turn affects kLa
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotecvisions.com
and explains why the mass transfer of filamentous broths
is limited in large scale, which can be observed as the
squares in Figure1.
3.2 Scaling down
Ideally, performing dedicated fermentation experiments
in large scale could avoid all problems related to scaling
up a process, but is not done in practice since it is pro-
hibitively expensive. This is not only due to the large con-
sumption of substrate and energy, but especially due to
the loss of production time, for example when an experi-
ment would result in off-spec product. In fermentation-
based production of pharmaceuticals, where good manu-
facturing practices (GMP) applies, performing experi-
ments in large scale is usually out of the question.
As a consequence, it is important to realize that scal-
ing down is also a major challenge in industrial fermenta-
tion technology. The overall goal of performing experi-
ments in small-scale equipment is to allow investigation of
the effect of process parameter changes on the process
performance under a set of conditions that are relevant for
production-scale reactors. Most biotechnological proc-
esses are indeed designed for existing equipment, that is
the full-scale fermentation tanks are installed and are then
used for several decades. As a consequence, the available
process equipment ranges are already known before a new
process is designed, and the bench-scale development
can thus be focused toward exploiting the potential for
maximizing volumetric productivity on condition that a
suitable down-scaled version of the full-scale reactor is
available, such that one is sure that the results obtained in
the down-scaled reactor are relevant for production scale.
With requirements for cost-effective, rapid process
development and optimization, good understanding of
critical process parameters, successful technology trans-
fer and fast time to the market, work at small scale
became critical for gaining initial knowledge and also a
potential bottleneck in further advancement. Hence, no
wonder that small-scale systems sometimes the term
ultra scale down is even used have received increased
attention in the last few decades compared to well-estab-
lished bench-scale stirred tank reactors (STR). The aim of
detailed studies of small-scale fermentation systems is on
the one hand to gain a better understanding of the engi-
neering fundamentals of small shaken or stirred systems
already in use while paying special attention to the possi-
bility for implementing improvements (microtiter plates
and shake flasks), and on the other hand to create com-
pletely new devices (microfluidic devices, microbioreac-
tors, and milliliter-scale stirred systems) that are consid-
ered to be suitable scale-down versions of larger bioreac-
tors. However, experience has shown that each small-
scale system brings benefits as well as problems during
process development across scales. Some of these sys-
tems are presented in Table1.
731
 Biotechnology Biotechnol. J. 2014, 9, 727738
Journal
www.biotechnology-journal.com www.biotecvisions.com

Table 1. Advantages and disadvantages of small-scale systems in correlation with scale-up/down [8892]

Small-scale systems Advantages Disadvantages

Microtiter plates Standardization and high-throughput Expensive (robotic hand)
(if automated robotic liquid-handling platform exist) Defined culture conditions
Not labor intensive Process control pH, DO
Evaporation, sterility
Shaking and surface aeration two main reaction
engineering differences compared to STR
Limited sampling end point measurement
Feeding capability (e.g. fed-batch)
Shake flasks Simple Defined culture conditions
Easy handling Process control pH, DO
Inexpensive Shaking and surface aeration two main reaction
Bigger volume compared to microtiter plate engineering differences compared to STR
Easy to maintain temperature Limited sampling end point measurement
Feeding capability (e.g. fed-batch)
Microfluidic devices, Low cost No standardization
microbioreactor Potentially high-throughput Small volume
Modification Evaporation
Not labor intensive disposable reactors Microfluidic connections
Different feeding strategies Analytical methods are not sufficiently developed
Better defined culture condition compared for such a small scale
to shake systems
Milliliter-scale Geometric similarity to STR Expensive
bioreactors Process control pH, DO Not standardized yet
Feeding capability Complex
High-throughput (if automated robotic liquid-handling
platform exist)
Sampling possibility

Despite the disadvantages, microtiter plates and
shake flasks are still very frequently used systems. Their
application allows to analyze different fermentation con-
ditions, such as growth rate, and to test the influence of
such a condition on the primary recovery of heterologous
proteins, as for example demonstrated for antibody frag-
ments expressed intracellularly in Escherichia coli [48].
Development of non-invasive optical sensing together
with adequate knowledge of engineering fundamentals
provided better process understanding and process mon-
itoring in these systems. Isett et al. [49] demonstrated
scalability from 24-well plate (46mL) to laboratory-scale
stirred tank (20L) using S. cerevisiae, while Islam et al.
[50] showed predictive scale-up from microwell plate-
(2mL) to laboratory- (7.5L) and pilot- (75L) scale using
E. coli. Both authors indicated that scale transfer from a
shaken to a stirred system is feasible if proper scaling-up
criteria are chosen [50].
Milliliter-scale stirred bioreactors are mostly devel-
oped with an idea to maintain geometrical similarities
with bench-scale reactors which according to us gives
them a leading position in application of existing scale-up
methodologies based on gassed power per unit volume;
agitator tip speed; constant dissolved oxygen tension
(DOT); oxygen mass transfer capacity (kLa); or mixing
time [51, 52]. A few commercial examples of milliliter-
scale stirred bioreactors are depicted in Table2 together
with commercial platforms that are employing orbital
shaking as a way of mixing.
An interesting and industrially very relevant trend
within scale-down reactors is the development of those
reactors consisting of two STRs connected to each other,
or consisting of an STR connected to a plug flow reactor
(PFR) [53]. The availability of two separate reactors with a
recycle in between allows investigating the effect of gra-
dients, for example of oxygen or substrate, in a well-con-
trolled environment. Such gradients do exist in large
scale, as for example documented in the study by Enfors
et al. [54], and are known to have an influence on produc-
tivity of the host organism in large scale. Neubauer and
Junne [55] have reviewed such multi-compartment scale-
down reactors.
3.3 Mass transfer, morphology, and rheology
Mass transfer is one of the crucial criteria for high biomass
systems as O2 and nutrients have to be distributed, and
possible toxic compounds, for example CO2, have to be

732 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

 Biotechnology
Journal
www.biotechnology-journal.com
Table 2. Examples of commercially available high-throughput platforms for microbial fermentation and cell cultures
High-throughput platforms Working
volume
BioLector (m2p-labs) [93] 0.12 mL
2mag bioreactor 48 [94] 815 mL
Pall Micro 24 Reactor System [95, 96] 37 mL
AMBR 24 Reactor System (TAP) [97] 1015 mL
removed. High biomass is, of course, a relative term and
very much dependent on the specific cultivated organ-
ism. It is reported that mammalian cells can reach a con-
centration of about 10 g/L with 2 107 cells/mL [56], while
at least 50g/L [57] can be achieved with single cell organ-
isms.
Mass transfer is a phenomenon composed of different
sub-processes, which are strongly dependent on the vis-
cosity of the fermentation broth. The rheology of the broth
is thereby at the same time dependent on the biomass
concentration and the morphology of the cells used as
expression system [58]. It is the complex relation between
all these variables that makes scaling up/scaling down
difficult.
In order to have proper oxygen supply, stirring and aer-
ation are a prerequisite for almost all types of cells and
even for cell cultures [59]. However, agitation may also
lead to changes in morphology which in turn will affect
product formation [60, 61]. Therefore, with an aerated STR
setup the question of shear sensitivity of the employed
organisms arises, and there has been an ongoing discus-
sion around the topic.
Mammalian cells are supposed to be prone to higher
shear rates because of their size and the lack of a cell wall.
It is discussed that it is just a perception that originates
from experiments employing cell cultures immobilized on
cells on microcarriers with cells dying when they were
detached from them due to the hydrodynamic forces [56].
In fact, it is possible to run Chinese hamster ovary (CHO)
cells in a 2-L aerated STR with agitation rates up to
776RPM with just slightly reduced productivity for mon-
oclonal antibodies. This experiment was designed to
scale down a production process based on a local energy
dissipation rate of up to 1.015 W/kg and a tip speed of
2m/s [62].
Bacteria and yeast on the other hand appear to be
organisms that are more resistant to mechanical stresses,
and it has been suggested that their small size is the rea-
son for their resistance to shear stress: they are smaller
than the Kolmogorov micro-scale of turbulence [63].
Therefore, it is believed that the morphology is not affect-
ed by mechanical mixing. Nienow [64] reported no cell
damage by agitation based on flow cytometry. Neverthe-
less, they reported a reduction in biomass concentration,
but increase in viability when scaling up a process; this
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Biotechnol. J. 2014, 9, 727738
www.biotecvisions.com
Mode of operation Agitation type Parameters Parallelization
monitored
Batch, fed-batch Orbital shaking Biomass, NADH, 48/96
GFP, pH, DO, T
Batch, fed-batch Stirred pH, DO, OD, T 48
Batch, fed-batch Orbital shaking pH, DO, T 24
Batch, fed-batch Stirred pH, DO, T 24/48
was attributed to the poor homogeneities in industrial-
scale reactors [64].
The most challenging expression systems are the fila-
mentous microorganisms in terms of mass transfer and
rheology, since their morphology, both at the microscopic
and macroscopic level, will have a major influence on
mass transfer. At the microscopic level, the optimal mor-
phology for a given bioprocess varies and cannot be gen-
eralized, and relies on the desired product [65]. Also,
dependent on the fermentation and the employed strain,
pellet formation can occur. The pellet formation process
can start as early as aggregation of spores when inoculat-
ing the media and can be completed in a couple of min-
utes [66]. This makes the requirements for analysis
methods to document the change of morphology rather
demanding. A least, pelleted biomass is spherical and
almost non-transparent which could enable the utilization
of classic particle size analysis like laser diffraction [67]
and focused beam reflectance [66]. For tracking biomass
development, also for other organisms, different technolo-
gies like capacitance probes, multi-wavelength spec-
troscopy, scanning di-electric spectroscopy, turbidity
measurement and, of course image analysis (semi- and
automatic), are available [6870]. However, their applica-
bility remains limited due to the poor experience with the
use of the models developed on such data across scales or
processes. For example, Petersen et al. [71] developed a
multivariate partial least square (PLS) model, using laser
size distribution and biomass concentration to predict
rheological properties of filamentous fermentation broth;
nevertheless, their model was not applicable to different
scales or strains.
In some processes, a pellet-type morphology is pre-
ferred since it allows for simplified downstream process-
ing and yields a Newtonian fluid behavior of the medium
(macroscopic level), which results in low aeration and agi-
tation power input. However, the pelleted morphology
results in nutrient concentration gradients within the pel-
let. This situation is not observed in freely dispersed
mycelia, although gradients can occur in such a system
dependent on the mixing time of the reactor system, due
to their high broth viscosity [72]. Thus, freely dispersed
mycelia allow enhanced growth and production, which
has been attributed to the morphology having an influ-
ence on the production kinetics at the microscopic level,
733
 Biotechnology Biotechnol. J. 2014, 9, 727738
Journal
www.biotechnology-journal.com
for example higher enzyme secretion from a more dense-
ly branched mutant of A. oryzae [73].
So, the morphology of filamentous fungi is double
edged as the productivity as well as the fermentation con-
ditions can be affected by the outer appearance of the fun-
gus. The challenge is to separate the effects to be able to
definitely connect gains in productivity to the correct set-
ting: the balance between effects resulting from bulk gra-
dients in the fermentation media compared to gradients
inside the biomass, and between the effect of a higher
OTR at lower viscosities versus better growth/secretion of
proteins at higher biomass concentration. Inducing a shift
in morphology usually requires a disturbance of the sys-
tem/equilibrium in the form of adding particles, changing
pH and salt composition to start pellet formation from a
free mycelial growth or to employ N-starvation to achieve
the reverse effect. All of these methods have the potential
to harm productivity, making it at least difficult to com-
pare different morphologies directly.
Finally, one of the traditional problems when working
with fermentation broths non-Newtonian fluids espe-
cially for fungal fermentations is at which shear rate the
viscosity should be evaluated. Until now, it has not been
possible to estimate a reliable shear rate in the fermenta-
tion tank itself. Considering a typical STR, it is well known
that the shear rate is at its maximum at the agitator tip,
and it decreases when approaching the vessels walls;
therefore, the way of calculating shear rate can be
expressed as the maximum or the average shear rate.
However, hitherto it is not clear which shear rate is gov-
erning the mass transfer processes, and the way of calcu-
lating this shear rate is limited to the Metzner and Otto
correlation [74]. It should not be forgotten that this empir-
ical correlation was developed for Reynolds numbers in
the laminar and transitional regime. Thus its applicability
is limited to laboratory and pilot-scale fermenters, even
though it has been typically employed for calculating
shear rates in full-scale fermenters as well [44]. Therefore,
it still remains challenging to estimate/calculate a reliable
shear rate to evaluate the viscosity across scales. This
problem however is not typically experienced in bacteria
and yeast cultivation since they exhibit a Newtonian
behavior due to the spherical shape of their morphology
[75]. This assumption facilitates the work with these
types of microorganisms and it is one of the reasons why
many fermentation process optimization studies are
focused on filamentous fungi.
3.4 Measurements for control
The available sensors in industrial bioreactors are most
often limited to pH, dissolved oxygen (DO) and tempera-
ture sensors at a single location in vessels with large vol-
umes, often with concentration gradients [42]. These sen-
sors display an average value for the entire process which
can be correlated to the processes in the vessel, but the
734
www.biotecvisions.com
available information on the spatial heterogeneity in the
vessel is very limited.
A PID controller is the standard method used to keep
a controlled variable at or close to its set point or set point
trajectory. However, the downside of the PID controller is
that it cannot guarantee that the fermentation is operat-
ed optimally. The set points (which in batch or fed-batch
operation can be time-varying trajectories) which the
controlled variables must follow only result in an optimal
operation under certain nominal conditions. When distur-
bances such as changes in substrate quality or composi-
tion, moisture content, ambient temperature, etc. take
place, the set points should be modified accordingly,
which is often called supervision or optimization. In
industrial fermentations, supervision is difficult for the fol-
lowing reasons:
(i) The most common controlled variables, DO and pH,
are only indirectly linked to the optimal operation of a
fermenter, that is the operation that leads to the high-
est volumetric productivity. Important variables such
as substrate, product, or by-product concentration are
only sporadically monitored (and less frequently con-
trolled). Biomass concentration is seldom measured
online and it rarely plays a role in automatic control of
industrial-scale processes. Accurate online measure-
ment of biomass and substrate/product can be diffi-
cult in fermentations featuring a large number of com-
pounds, making pH and DO the most common vari-
ables for automatic control. However, optimal opera-
tion and fulfillment of quality constraints cannot be
guaranteed with pH and DO control only, thus
increasing the importance of advanced monitoring.
(ii) Models can be used to synthesize controllers that
operate close to optimal conditions and to determine
optimal trajectories for (fed-)batch operations. Despite
the advances in fermentation modeling, many indus-
trial operations lack sufficiently accurate models that
can be used for this purpose. For instance, Smets et al.
[26] reviewed optimal feed rate strategies and pointed
out how this approach is hampered in reality by mod-
el and sensor uncertainty; nevertheless, they derived
a number of near-optimal heuristic strategies, proving
that models are useful to gather a major insight in the
process behavior. Complementary approaches are
investigated to overcome these limitations: robust
control design with minimal modeling, and advanced
modeling based either on first-principles or the
so-called hybrid models (using both first-principles
and data-driven models).
It is also worth mentioning that one of the major limita-
tions in online measurements and the introduction of new
sensors for process monitoring originates from GMP
aspects, especially in the pharmaceutical biotechnology
industry. Fulfilling GMP regulations is not a trivial task,
and is a major bottleneck in process development and
optimization. A method to further improve data quality of
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
 Biotechnology Biotechnol. J. 2014, 9, 727738
Journal
www.biotechnology-journal.com www.biotecvisions.com

online sensors without violating the GMP regulations is to

use a soft-sensor approach where already existing data is
interpreted with simultaneous computational analysis in
order to improve the quality and thus prediction of the
sensors [76]. In such a soft-sensor robust online measure-
ments, which are not subject to time delay, are used to
calculate the expected value of new parameters which are
desirable to control, such as biomass, specific growth
rate, or oxygen transfer capacity [77, 78]. Soft sensing can
also be a tool for industrial application as part of a frame-
work for quality by design (QbD) and PAT principles since
it provides a method for on line monitoring of the process
[79, 80].

4 Discussion
It is clear from the previous sections that the development
and scale-up of fermentation processes is of a very com-
plex nature. This is also an important reason explaining
why after so many years of use of continuously stirred
tank reactors (CSTR), this technology has so far not yet
changed substantially. Experience with such CSTRs has
been built up gradually, and gives the process scientist a
kind of a comfort zone when operating such equipment
Figure 2. Velocity vector profile of a full-scale reactor.
where the change of a single parameter in the system
might already have an impact on several output variables.
A second factor that is standing in the way for introduc-
ing significant changes to the reactor design is the cost sis of inhomogeneity of the concentrations of important
related to establishing a new full-scale fermenter, be it a variables such as the biomass concentration, substrate or
CSTR or any other configuration. product concentration. In Figs.2 and 3, the complex fluid
With the available knowledge on the occurrence of dynamic characteristics resulting from applying CFD are
gradients in industrial-scale CSTRs [42], and considering illustrated for two different reactors: (i) a full-scale CSTR
that it is very difficult to access experimental data char- (>150m3); and (ii) a miniaturized reactor (ca. 1mL). Both
acterizing the gradients occurring inside a large industri- numerically investigated mixing times have shown an
al-scale bioreactor, it becomes increasingly appealing to impressive convergence with the experimental mixing
model such systems. Until now, this has been especially time.
attractive for miniaturized systems, where laminar flow
conditions are often present, and therefore CFD methods
have a high predictive ability since no turbulence models
have to be applied. However, the prediction quality
becomes more and more reliable for laboratory, pilot and
full-scale fermentations as well, and hence we foresee that
such fluid dynamic models become increasingly used as
powerful tools for evaluation of mixing times, mass trans-
fer, shear stress levels, and dead volumes, to name a few
well-known challenges for the process scientist. Such
numerical investigations can thereby increasingly con-
tribute to the design of experiments, and will also help to
reduce the requested amount of experiments, since some
of the configurations evaluated with the CFD simulations
can be excluded beforehand from the experimental plan-
ning on the basis of the simulation result. This includes
obviously the geometric reactor design, but it is especial-
ly helpful when different operation patterns are investi-
gated like, for example fed-batch operations or the analy- Figure 3. Streamlines and concentration plot for a milliliter-scale reactor.

2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 735

 Biotechnology Biotechnol. J. 2014, 9, 727738
Journal
www.biotechnology-journal.com
Specifically with respect to filamentous fungi fermen-
tations, CFD models are used in order to understand the
impact of shear stress on the morphology of the organism
as well as for the mass transfer from the bulk solution to the
organisms [8183]. But the real potential of CFD models is,
according to us, the coupling of CFD models with biologi-
cal kinetic reaction models. Such kinetic models are avail-
able for several commonly used production hosts for per-
fectly mixed reactors, for example at bench-scale, and can
thereby be readily implemented in CFD models. One major
advantage of CFD is that the model parameters identified
at laboratory-scale in well-mixed reactors can be more
readily translated to large scale, since the parameters do
not have to be retuned in order to compensate for inade-
quate description of reactor hydrodynamics, as is the case
when using a standard system of ordinary differential
equations to describe biological phenomena in a large
tank. In this way it is also possible to switch desired reac-
tions on and off to investigate their impact on concentra-
tion dynamics, and even more complex models as, for
example population balance models (PBM) have already
been implemented [84, 85]. Such models might yield better
insight into the impact of spatial inhomogeneity on the
individual cell [17, 84] and on the fermentation perform-
ance, and could thereby contribute to improved under-
standing of the processes, also at industrial scale, and
hence also to more efficient operation and improved design
of new processes. Furthermore, theoretical numerical
investigations can be implemented and yield new ideas for
optimized reactor configurations. This was, for example
demonstrated by Schpper et al. [86] where a cultivation of
surface adhered yeast cells was optimized in a microbiore-
actor setup to yield a considerably improved product for-
mation rate. Furthermore, different model hypotheses can
be implemented and tested in simulation before seeking
experimentally validation, as shown by Krhne et al. [87],
where the impact of shear stress on a tissue engineering
cultivation in a micro-scaled scaffold has been studied.
5 Conclusions
The use of common engineering tools, such as mathe-
matical models and optimization techniques, in fermen-
tation process development and optimization is seriously
challenged by our lack of understanding about scaling
up/scaling down fermentation processes and phenomena
such as the influence of morphology on broth rheology
and mass transfer. Furthermore, establishing suitable
online measurements for control is not that straightfor-
ward either. CFD is now reaching a level of maturity such
that CFD can start supporting the process scientist in
addressing some of the engineering challenges, that is
CFD can be increasingly used to test new ideas, both with
respect to reactor operation and design, and to support
scaling up and scaling down bioreactors.
736
www.biotecvisions.com
The authors acknowledge financial support of the follow-
ing organizations: The Danish Council for Strategic
Research, project Towards robust fermentation process-
es by targeting population heterogeneity at microscale
(project number 0603-00203B). The Novo Nordisk Foun-
dation, project Exploring biochemical process perform-
ance limits through topology optimization. Region
Zealand, the European Regional Development Fund
(ERDF), CAPNOVA, CP Kelco, DONG Energy, Novo
Nordisk, and Novozymes for funding the BIOPRO project
(www.biopro.nu).
The authors declare no financial or commercial conflict of
interest.
6 References
[1] Shu, P., Johnson, M. J., Citric acid. J. Ind. Eng. Chem. 1948, 40,
12021205.
[2] Biebl, H., Menzel, K., Zeng, A.-P., Deckwer, W.-D., Microbial produc-
tion of 1,3-propanediol. Appl. Microbiol. Biotechnol. 1999, 52, 289
297.
[3] Adkesson, D. M., Alsop, A. W., Ames, T. T., Chu, L. A., et al., Purifi-
cation of biological-produced 1,3-propanediol. International Patent
WO2004101479, 2004.
[4] Moyer, A. J., Method for production of penicillin. US patent 2442141,
1948.
[5] Johnson, I. S., Human insulin from recombinant DNA technology.
Science 1983, 219, 632637.
[6] Ostergaard, S., Olsson, L., Nielsen, J., Metabolic engineering of Sac-
charomyces cerevisiae. Microbiol. Mol. Biol. Rev. 2000, 64, 3450.
[7] Lee, J., Lee, S. Y., Park, S., Middelberg, A. P. J., Control of fed-batch
fermentations. Biotechnol. Adv. 1999, 17, 2948.
[8] Ltke-Eversloh, T., Bahl, H., Metabolic engineering of Clostridium
acetobutylicum: Recent advances to improve butanol production.
Curr. Opin. Biotechnol. 2011, 22, 634647.
[9] Woolston, B. M., Edgar, S., Stephanopoulos, G., Metabolic engineer-
ing: Past, and future. Annu. Rev. Chem. Biomol. Eng. 2013, 4, 259
288.
[10] Schmidt, A. S., Garde, A., Klinke, H. B., Thomsen, A. B., et al., Lac-
tic acid production from wheat straw: Effect of pretreatment condi-
tions. In: Proceedings of the First World Conference and Exhibition
on Biomass for Energy and Industry. Sevilla, Spain 2000.
[11] U.S. Department of Health and Human Services, Food and Drug
Administration 2004: Guidance for industry: PAT a framework for
innovative pharmaceutical development, manufacturing and quali-
ty assurance http://www.fda.gov/down loads/Drugs/GuidanceCom-
plianceRegulatoryInformation/Guidances/ucm070305.pdf
[12] Gernaey, K. V., Bolic, A., Svanholm, B., PAT tools for fermentation
processes, Chem. Today 2012, 30, 3843.
[13] Cervera, A. E., Petersen, N., Eliasson Lantz, A., Larsen, A., Gernaey,
K. V., Application of near-infrared spectroscopy for monitoring and
control of cell culture and fermentation. Biotechnol. Prog. 2009, 25,
15611581.
[14] Scarff, M., Arnold, S. A., Harvey, L. M., McNeil, B., Near infrared
spectroscopy for bioprocess monitoring and control: Current status
and future trends. Crit. Rev. Biotechnol. 2006, 26, 1739.
[15] Sipiczki, M., Diversity, variability and fast adaptive evolution of the
wine yeast (Saccharomyces cerevisiae) genome a review. Ann.
Microbiol. 2011, 61, 8593.
[16] Lencastre Fernandes, R., Nierychlo, M., Lundin, L., Pedersen, A. E.,
et al., Experimental methods and modeling techniques for descrip-
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
 Biotechnology Biotechnol. J. 2014, 9, 727738
Journal
www.biotechnology-journal.com
tion of cell population heterogeneity. Biotechnol. Adv. 2011, 29,
575599.
[17] Lapin, A., Klann, M., Reuss, M., Multi-scale spatio-temporal model-
ing: Lifelines of microorganisms in bioreactors and tracking mole-
cules in cells. Adv. Biochem. Eng. 2010, 121, 2343.
[18] Mandenius, C.-F., Brundin, A., Bioprocess optimization using
design-of-experiments methodology. Biotechnol. Prog. 2008, 24,
11911203.
[19] Albk, M. O., Gernaey, K. V., Hansen, M. S., Stocks, S. M., Evalua-
tion of the energy efficiency of enzyme fermentation by mechanis-
tic modeling. Biotechnol. Bioeng. 2012, 109, 950961.
[20] Singh, B., Satyanarayana, T., Improved phytase production by a
thermophilic mould Sporotrichum thermophile in submerged fer-
mentation due to statistical optimization. Bioresour. Technol. 2008,
99, 824830.
[21] Kennedy, M., Krouse, D., Strategies for improving fermentation
medium performance: A review. J. Ind. Microbiol. Biotechnol. 1999,
23, 456475.
[22] Montague, G. A., Martin, E. G., OMalley, C. J., Forecasting for fer-
mentation operational decision making. Biotechnol. Prog. 2008, 24,
10331041.
[23] Gernaey, K. V., Lantz, E. A., Tufvesson, P., Woodley, J. M., Sin, G.,
Application of mechanistic models to fermentation and biocatalysis
for next generation processes. Trends Biotechnol. 2010, 28, 346354.
[24] Albk, M. O., Gernaey, K. V., Hansen, M. S., Stocks, S. M., Modeling
enzyme production with Aspergillus oryzae in pilot scale vessels
with different agitation, aeration and agitator types. Biotechnol. Bio-
eng. 2011, 108, 18281840.
[25] Rivera, E. C., Costa, A. C., Atala, D. I. P., Maugeri, F., et al., Evalua-
tion of optimization techniques for parameter estimation: Applica-
tion to ethanol fermentation considering the effect of temperature.
Proc. Biochem. 2006, 41, 16826187.
[26] Smets, I., Claes, J., November, E., Bastin, G., van Impe, J., Optimal
adaptive control of (bio)chemical reactors: Past, present and future.
J. Process Contr. 2004, 14, 795805.
[27] Sin, G., Gernaey, K. V., Lantz, E. A., Good modelling practice (GMoP)
for PAT applications: Propagation of input uncertainty and sensitiv-
ity analysis. Biotechnol. Prog. 2009, 25, 10431053.
[28] Saltelli, A., Ratto, M., Andres, T., Campolongo, F., et al., Global Sen-
sitivity Analysis: The Primer, John Wiley & Sons 2008.
[29] Kurtanjek, Z., Optimal nonsingular control of fed-Batch fermenta-
tion. Biotechnol. Bioeng. 1991, 37, 814823.
[30] Park, S., Ramirez, W. F., Optimal production of secreted protein in
fed-batch reactors. AIChE J. 1988, 34, 15501558.
[31] Bogle, I. D. L., Cockshott, A. R., Bulmer, M., Thornhill, N., et al.,
A process systems engineering view of biochemical process opera-
tions. Comput. Chem. Eng. 1996, 20, 943949.
[32] Lee, J., Lee, S.-Y., Park, S., Middelberg, A. P. J., Control of fed-batch
fermentations. Biotechnol. Adv. 1999, 17, 2948.
[33] Zhang, H., Williams-Dalson, W., Keshavarz-Moore, E., Shamlou, P. A.,
Computational-fluid-dynamics (CFD) analysis of mixing and gas-
liquid mass transfer in shake flasks. Biotechnol. Appl. Biochem.
2005, 41, 18.
[34] Kerdouss, F., Bannari, A., Proulx, P., Bannari, R., et al., Two-phase
mass transfer coefficient prediction in stirred vessel with a CFD
model. Comput. Chem. Eng. 2008, 32, 19431955.
[35] Moilanen, P., Modelling gasliquid flow and local mass transfer in
stirred tanks. PhD thesis, Helsinki University of Technology, Finland,
2009, ISBN 978-951-22-9788-7.
[36] Brning, S., Weuster-Botz, D., CFD analysis of interphase mass
transfer and energy dissipation in a milliliter-scale stirred-tank reac-
tor for filamentous microorganisms. Chem. Eng. Res. Design 2014,
92, 240248.
[37] Brning, S., Stromungssimulation als Werkzeug zur Bioreaktor-
charakterisierung, PhD Thesis, Technische Universitt Mnchen,
Germany, 2012 [in German].
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotecvisions.com
[38] Posch, A. E., Herwig, C., Spadiut, O., Science-based bioprocess
design for filamentous fungi. Trends Biotechnol. 2013, 31, 3744.
[39] Schmidt, F. R., Optimization and scale up of industrial fermentation
processes. Appl. Microbiol. Biotechnol. 2005, 68, 425435.
[40] Gabelle, J. C., Augier, F., Carvalho, A., Rousset, R., Morchain, J.,
Effect of tank size on kLa and mixing time in aerated stirred reactors
with non-Newtonian fluids. Can. J. Chem. Eng. 2012, 89, 11391152.
[41] Junker, B. H., Scale-up methodologies for Escherichia coli and yeast
fermentation processes. J. Biosci. Bioeng. 2004, 97, 347364.
[42] Larsson, G., Trnkvist, M., Wernersson, E. S., Trgard, C., et al., Sub-
strate gradients in bioreactors: Origin and consequences. Bio-
process. Eng. 1996, 14, 281289.
[43] Oosterhuis, N. M. G., Kossen, N. W. F., Dissolved oxygen concentra-
tion profiles in a production scale bioreactor. Biotechnol. Bioeng.
1984, 26, 546550.
[44] Stocks, S. M., Industrial enzyme production: Process scale up/scale
down. in: McNeil, B., Archer, D., Giavasis, I., Harvey, L. (Eds.), Micro-
bial Production of Food Ingredients, Enzymes and Nutraceuticals,
Woodhead Publishing 2013, pp. 144172.
[45] Pollard, D. J., Kirschner, T. F., Hunt, G. R., Tong, I.-T., et al., Scale up
of a viscous fungal fermentation: Application of scale-up criteria
with regime analysis and operating boundary conditions. Biotech-
nol. Bioeng. 2007, 96, 307317.
[46] Reuss, M., Oxygen transfer and mixing: Scale-up implications. in:
Rehm, H. J., Reed, G. (Eds.), Biotechnology, Vol. 3, 2nd Edn., VCH
1993, pp. 185213.
[47] Meerman, H. J., Kelley, A. S., Ward, M., Advances in protein expres-
sion in filamentous fungi. in: Baneyx, F. (Ed.), Protein Expression
Technologies. Current Status and Future Trends, 1st Edn., Horizon
Bioscience 2004, pp. 345385.
[48] Li, Q., Mannall, G. J., Ali, S., Hoare, M., An ultra scale-down
approach to study the interaction of fermentation, homogenization,
and centrifugation for antibody fragment recovery from recombi-
nant E. coli. Biotechnol. Bioeng. 2013, 110, 21502160.
[49] Isett, K., George, H., Herber, W., Amanullah, A., Twenty-four-well
plate miniature bioreactor high-throughput system: Assessment for
microbial cultivations. Biotechnol. Bioeng. 2007, 98, 10171028.
[50] Islam, R. S., Tisi, D., Levy, M. S., Lye, G. J., Scale-up of Escherichia
coli growth and recombinant protein expression conditions from
microwell to laboratory and pilot scale based on matched kLa.
Biotechnol. Bioeng. 2008, 99, 11281139.
[51] Betts, J. I., Baganz, F., Miniature bioreactors: Current practices and
future opportunities, Microb. Cell Fact. 2006, 5, 21.
[52] Hortsch, R., Weuster-Botz, D., Milliliter-scale stirred tank reactors for
the cultivation of microorganisms. Adv. Appl. Microbiol. 2010, 73,
6182.
[53] Papagianni, M., Mattey, M., Kristiansen, B., Design of a tubular loop
bioreactor for scale-up and scale-down of fermentation processes.
Biotechnol. Prog. 2003, 19, 14981504.
[54] Enfors, S. O., Jahic, M., Rozkov, A., Xu, B., et al., Physiological
responses to mixing in large scale bioreactors. J. Biotechnol. 2001,
85, 175185.
[55] Neubauer, P., Junne, S., Scale-down simulators for metabolic analy-
sis of large-scale bioprocesses. Curr. Opin. Biotechnol. 2010, 21, 114
121.
[56] Hu, W., Berdugo, C., Chalmers, J. J., The potential damage to animal
cell cultures of industrial relevance: Current understanding.
Cytotechnology 2011, 63, 445460.
[57] Salehmin, M. N. I., Annuar, M. S. M., Chisti, Y., High cell density fed-
batch fermentations for lipase production: Feeding strategies and
oxygen transfer. Bioproc. Biosyst. Eng. 2013, 36, 15271543.
[58] Cascaval, D., Oniscu, C., Galaction, A., Rheology of fermentation
broths 2. Influence of the rheological behavior on biotechnological
processes. Rev. Roum. Chim. 2003, 48, 339356.
[59] Ma, N., Mollet, M., Chalmers, J. J., Aeration, mixing and hydrody-
namics in bioreactors, in: Ozturk, S., Hu, W. (Eds.), Cell Culture Tech-
737
 Biotechnology Biotechnol. J. 2014, 9, 727738
Journal
www.biotechnology-journal.com
nology for Pharmaceutical and Cell-Based Therapies, Taylor & Fran-
cis 2006.
[60] Smith, J. J., Lilly, M. D., Fox, R. I., The effect of agitation on the mor-
phology and penicillin production of Penicillium chrysogenum.
Biotechnol. Bioeng. 1990, 35, 10111023.
[61] Jsten, P., Paul, G. C., Nienow, A. W., Thomas, C. R., Dependence of
mycelial morphology on impeller type and agitation intensity.
Biotechnol. Bioeng. 1996, 52, 672684.
[62] Sieck, J. B., Cordes, T., Budach, W. E., Rhiel, M. H., et al., Develop-
ment of a scale-down model of hydrodynamic stress to study the per-
formance of an industrial CHO cell line under simulated production
scale bioreactor conditions. J. Biotechnol. 2012, 164, 4149.
[63] Nienow, A. W., McLeod, G., Hewitt, C. J., Studies supporting the use
of mechanical mixing in large scale beer fermentations. Biotechnol.
Lett. 2010, 32, 623633.
[64] Nienow, A. W., Scale-up considerations based on studies at the
bench scale in stirred bioreactors. J. Chem. Eng. Jap. 2009, 42, 789
796.
[65] Gibbs, P. A., Seviour, R. J., Schmid, F., Growth of filamentous fungi
in submerged culture: Problems and possible solutions. Crit. Rev.
Biotechnol. 2000, 20, 1748.
[66] Grimm, L. H., Kelly, S., Hengstler, J., Gbel, A., et al., Kinetic studies
on the aggregation of Aspergillus niger conidia. Biotechnol. Bioeng.
2004, 87, 213218.
[67] Rnnest, N. P., Stocks, S. M., Lantz, A. E., Gernaey, K. V., Compari-
son of laser diffraction and image analysis for measurement of Strep-
tomyces coelicolor cell clumps and pellets. Biotechnol. Lett. 2012, 34,
14651473.
[68] Barry, D. J., Williams, G. A., Microscopic characterisation of fila-
mentous microbes: Towards fully automated morphological quan-
tification through image analysis, J. Microsc. 2011, 244, 120.
[69] Petersen Rnnest, N., Stocksm, S. M., Lantzm, A. E., Gernaey, K. V.,
Introducing process analytical technology (PAT) in filamentous cul-
tivation process development: Comparison of advanced online sen-
sors for biomass measurement. J. Ind. Microbiol. Biotechnol. 2011,
38, 16791690.
[70] Krull, R., Wucherpfening, T., Esfandabadi, M. E., Walisko, R., et al.,
Characterization and control of fungal morphology for improved pro-
duction performance in biotechnology. J. Biotechnol. 2013, 163,
112123.
[71] Petersen, N., Stocks, S., Gernaey, K. V., Multivariate models for pre-
diction of rheological characteristics of filamentous fermentation
broth from size distribution. Biotechnol. Bioeng. 2008, 100, 6171.
[72] Riley, G. L., Tucker, K. G., Paul, G. C., Thomas, C. R., Effect of bio-
mass concentration and mycelial morphology on fermentation broth
rheology. Biotechnol. Bioeng. 2000, 68, 160172.
[73] Spohr, A., Dam-Mikkelsen, C., Carlsen, M., Nielsen, J., et al., On-line
study of fungal morphology during submerged growth in a small
flow-through cell. Biotechnol. Bioeng. 1998, 58, 541553.
[74] Metzner, A. B., Otto, R. E., Agitation of non-Newtonian fluids. AIChE
J. 1957, 3, 310.
[75] Oniscu, C., Galacton, A., Cascaval, D., Rheology of fermentation
broths 2. Rheological behaviors and influence factors. Rev. Roum.
Chim. 2003, 48, 91110.
[76] Chruy, A., Software sensors in bioprocess engineering, J. Biotech-
nol. 1997, 52, 193199.
[77] Luttmann, R., Bracewell, D. G., Cornelissen, G., Gernaey, K. V., et al.,
Soft sensors in bioprocessing: A status report and recommenda-
tions. Biotechnol. J. 2012, 7, 10401048.
[78] Sundstrm, H., Enfors, S.-O., Software sensors for fermentation
processes. Bioproc. Biosyst. Eng. 2008, 31, 145152.
738
www.biotecvisions.com
[79] Sagmeister, P., Wechselberger, P., Jazini, M., Meitz, A., et al., Soft
sensor assisted dynamic bioprocess control: Efficient tools for bio-
process development. Chem. Eng. Sci. 2013, 96, 190198.
[80] Montague, G. A., Morris, A. J., Ward, A. C., Fermentation monitor-
ing and control: A perspective. Biotechnol. Genet. Eng. Rev. 1989, 7,
147188.
[81] Yu, L., Chao, Y., Wensel, P., Shulin, C., Hydrodynamic and kinetic
study of cellulase production by Trichoderma reesei with pellet mor-
phology. Biotechnol. Bioeng. 2012, 109, 17551768.
[82] Krull, R., Cordes, C., Horn, H., Kampen, I., et al., Morphology of fila-
mentous fungi: Linking cellular biology to process engineering using
Aspergillus niger. Adv. Biochem. Eng. Biotechnol. 2010, 121, 121.
[83] Nez-Ramrez, D. M., Valencia-Lpez, J. J., Calderas, F., Sols-Soto,
A., et al., Mixing analysis for a fermentation broth of the fungus
Beauveria bassiana under different hydrodynamic conditions in a
bioreactor. Chem. Eng. Technol. 2012, 35, 19541961.
[84] Lencastre Fernandes, R., Krhne, U., Nopens, I., Jensen, A. D., Ger-
naey, K. V., Multi-scale modeling for prediction of distributed cellu-
lar properties in response to substrate spatial gradients in a contin-
uously run microreactor. Comp. Aid. Chem. Eng. 2012, 31, 545549.
[85] Lencastre Fernandes, R., Carlquist, M., Lundin, L., Heins, A.-L., et
al., Cell mass and cell cycle dynamics of an asynchronous budding
yeast population: Experimental observations, flow cytometry data
analysis, and multi-scale modeling. Biotechnol. Bioeng. 2013, 110,
812826.
[86] Schpper, D., Lencastre Fernandes, R., Lantz, A. E., Okkels, F., et al.,
Topology optimized microbioreactors. Biotechnol. Bioeng. 2011, 108,
786796.
[87] Krhne, U., Wendt, D., Martin, I., Juhl, M., et al., A transient 3D-CFD
model incorporating biological processes for use in tissue engineer-
ing. Micro Nanosyst. 2010, 2, 249260.
[88] Burke, F., Scale up and scale down of fermentation processes, in:
McNneil, B., Harvey, L. M. (Eds.), Practical Fermentation Technolo-
gy, John Wiley & Sons 2008, pp. 231269.
[89] Bchs, J., Introduction to advantages and problems of shaken cul-
tures. Biochem. Eng. J. 2001, 7, 9198.
[90] Bareither, R., Pollard, D. A., Review of advanced small-scale parallel
bioreactor technology for accelerated process development: Current
state and future need. Biotechnol. Prog. 2011, 27, 214.
[91] Weuster-Botz, D., Hekmat, H., Puskeiler, R., Franco-Lara, E.,
Enabling technologies: Fermentation and downstream processing.
Adv. Biochem. Eng. Biotechnol. 2007, 105, 205247.
[92] Schpper, D., Alam, M. N. H. Z., Szita, N., Eliasson Lantz, A., Ger-
naey, K. V., Application of microbioreactors in fermentation process
development: A review. Anal. Bioanal. Chem. 2009, 395, 679695.
[93] Funke, M., Buchenauer, A., Schnakenberg, U., Mokwa, W., et al.,
Microfluidic biolector microfluidic bioprocess control in microtiter
plates. Biotechnol. Bioeng. 2010, 107, 497505.
[94] Puskeiler, R., Kaufmann, K., Weuster-Botz, D., Development, paral-
lelization, and automation of a gas-inducing milliliter-scale bioreac-
tor for high-throughput bioprocess design (HTBD). Biotechnol. Bio-
eng. 2005, 89, 512523.
[95] Chen, A., Chitta, R., Chang, D., Amanullah, A., Twenty-four well
plate miniature bioreactor system as a scale-down model for cell cul-
ture process development. Biotechnol. Bioeng. 2009, 102, 148160.
[96] Isett, K., George, H., Herber, W., Amanullah, A., Twenty-four-well
plate miniature bioreactor high-throughput system: Assessment for
microbial cultivations. Biotechnol. Bioeng. 2007, 98, 10171028.
[97] Moses, S., Manahan, M., Ambrogelly, A., Ling, W. L. W., Assessment
of AMBR TM as a model for high-throughput cell culture process
development strategy. Adv. Biosci. Biotechnol. 2012, 3, 918927.
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
 ISSN 1860-6768 BJIOAM 9 (6) 711862 (2014) Vol. 9 June 2014

Systems & Synthetic Biology

Special Issue: Industrial Biotechnology. This Special Issue, edited by Jarka Glassey and Marcel Ottens, contains
Nanobiotech Medicine

6/2014

articles from the 2nd European Conference on Applied Biotechnology (ECAB2) in The Hague, The Netherlands,
Industrial Biotechnology
Fermentation technology
Biorefinery
Antibody production

April 2013. The cover image shows a tubular photobioreactor for cultivation of microalgae. It is the most common
large-scale reactor type for production of high and medium algae products. The picture is provided by Clemens
Posten (KIT, Karlsruhe, Germany), co-author of the Review on Biorefinery of microalgae opportunities and
constraints for different production scenarios.
www.biotechnology-journal.com
http://dx.doi.org/10.1002/biot.201300142

Biotechnology Journal list of articles published in the June 2014 issue.

Editorial: Industrial biotechnology Technologies Research Article

and methods for rapid process development Aerobic expression of Vitreoscilla hemoglobin efficiently
Jarka Glassey and Marcel Ottens reduces overflow metabolism in Escherichia coli
http://dx.doi.org/10.1002/biot.201400304 Tania E. Pablos, Juan Carlos Sigala, Sylvie Le Borgne
and Alvaro R. Lara
Perspective http://dx.doi.org/10.1002/biot.201300388
Hybrid modeling for quality by design and PAT benefits
and challenges of applications in biopharmaceutical industry Technical Report
Moritz von Stosch, Steven Davy, Kjell Francois, Vytautas Prediction of IgG1 aggregation in solution
Galvanauskas, Jan-Martijn Hamelink, Andreas Luebbert, Frida Ojala, Marcus Degerman, Thomas Budde Hansen,
Martin Mayer, Rui Oliveira, Ronan OKennedy, Paul Rice Ernst Broberg Hansen and Bernt Nilsson
and Jarka Glassey http://dx.doi.org/10.1002/biot.201400018
http://dx.doi.org/10.1002/biot.201300385
Technical Report
Perspective Robust, microfabricated culture devices with improved
Challenges in industrial fermentation technology research control over the soluble microenvironment for the culture
Luca Riccardo Formenti, Anders Nrregaard, Andrijana of embryonic stem cells
Bolic, Daniela Quintanilla Hernandez, Timo Hagemann, Rhys J. Macown, Farlan S. Veraitch and Nicolas Szita
Anna-Lena Heins, Hilde Larsson, Lisa Mears, Miguel http://dx.doi.org/10.1002/biot.201300245
Mauricio-Iglesias, Ulrich Krhne and Krist V. Gernaey
Technical Report
http://dx.doi.org/10.1002/biot.201300236
Enzymatic hydration activity assessed by selective spectro-
Review photometric detection of alcohols: A novel screening assay
Biorefinery of microalgae opportunities and constraints using oleate hydratase as a model enzyme
for different production scenarios Aida Hiseni, Rosario Medici, Isabel W. C. E. Arends
Ioanna Hariskos and Clemens Posten and Linda G. Otten
http://dx.doi.org/10.1002/biot.201300142 http://dx.doi.org/10.1002/biot.201300412

Research Article Regular Articles

Assessing the environmental sustainability of ethanol
from integrated biorefineries Review
Temitope Falano, Harish K. Jeswani and Adisa Azapagic Biocatalytic reduction of carboxylic acids
http://dx.doi.org/10.1002/biot.201300246 Kamila Napora-Wijata, Gernot A. Strohmeier
and Margit Winkler
Research Article
http://dx.doi.org/10.1002/biot.201400012
Economics of recombinant antibody production processes
at various scales: Industry-standard compared to continuous Research Article
precipitation In vivo and in vitro activity of an immunoglobulin Fc fragment
Nikolaus Hammerschmidt, Anne Tscheliessnig, (Fcab) with engineered Her-2/neu binding sites
Ralf Sommer, Bernhard Helk and Alois Jungbauer Max Woisetschlger, Bernhard Antes, Radha Borrowdale,
http://dx.doi.org/10.1002/biot.201300480 Susanne Wiederkum, Manuela Kainer, Herta Steinkellner
Gordana Wozniak-Knopp, Kevin Moulder, Florian Rker
Research Article
and Geert C. Mudde
iOD907, the first genome-scale metabolic model for the milk
yeast Kluyveromyces lactis http://dx.doi.org/10.1002/biot.201300387
Oscar Dias, Rui Pereira, Andreas K. Gombert, Eugnio C.
Research Article
Ferreira and Isabel Rocha
Microscopic monitoring provides information on structure
http://dx.doi.org/10.1002/biot.201300242 and properties during biocatalyst immobilization
Sarka Bidmanova, Eva Hrdlickova, Josef Jaros, Ladislav
Ilkovics, Ales Hampl, Jiri Damborsky and Zbynek Prokop
http://dx.doi.org/10.1002/biot.201300049

2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.biotechnology-journal.com