Biotechnology Advances 21 (2003) 417 430

www.elsevier.com/locate/biotechadv

Know-how and know-why in

biochemical engineering
U. von Stockar a,*, S. Valentinotti a, I. Marison a,
C. Cannizzaro a, C. Herwig b
a

Laboratory of Chemical and Biochemical Engineering, Swiss Federal Institute of Technology (EPFL),
CH-1015 Lausanne, Switzerland
b
Pharmaplan Engineering AG, Altkirchstrasse 8, CH-4054 Basel, Switzerland

Abstract
This contribution analyzes the position of biochemical engineering in general and bioprocess
engineering particularly in the force fields between fundamental science and applications, and
between academia and industry. By using culture technology as an example, it can be shown that
bioprocess engineering has moved slowly but steadily from an empirical art concerned with mainly
know-how to a science elucidating the know-why of culture behavior. Highly powerful monitoring
tools enable biochemical engineers to understand and explain quantitatively the activity of cellular
culture on a metabolic basis. Among these monitoring tools are not just semi-online analyses of
culture broth by HPLC, GC and FIA, but, increasingly, also noninvasive methods such as midrange
IR, Raman and capacitance spectroscopy, as well as online calorimetry. The detailed and quantitative
insight into the metabolome and the fluxome that bioprocess engineers are establishing offers an
unprecedented opportunity for building bridges between molecular biology and engineering
biosciences. Thus, one of the major tasks of biochemical engineering sciences is not developing new
know-how for industrial applications, but elucidating the know-why in biochemical engineering by
conducting research on the underlying scientific fundamentals.
D 2003 Elsevier Inc. All rights reserved.
Keywords: On-line monitoring; On-line midrange IR spectroscopy; On-line Raman spectroscopy; Capacitance
spectroscopy; Biochemical engineering sciences; Role of biochemical engineering; Scientific fundamentals of
biochemical engineering

* Corresponding author. Tel.: +41-21-693-31-91; fax: +41-21-693-36-80.

E-mail address: urs.vonstockar@epfl.ch (U. von Stockar).
0734-9750/03/$ - see front matter D 2003 Elsevier Inc. All rights reserved.
doi:10.1016/S0734-9750(03)00058-2

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1. Introduction
Biotechnology started out as a craft. For thousands of years, mankind exploited
empirical know-how handed down from generation to generation to produce bread, beer,
wine and the like. Throughout this long period, the evolution of production procedures
was so slow that it could hardly be perceived because new know-how had to be obtained
in a completely empirical way due to the total lack of know-why. This state of affairs only
started to change relatively recently when around 1700, Anton van Leeuwenhoek invented
the first optical microscope. The discovery of new and unsuspected microscopic life forms
ensuing from his invention marked the starting point of biology as a biotechnologyrelevant science. A steady development of biological knowledge and science followed,
pioneered by eminent personalities such as Louis Pasteur, Robert Koch, and Alexander
Fleming among many others.
In the last 30 years, the development of fundamental and molecular biology started to
accelerate in an exponential fashion due to breakthroughs such as genetic engineering,
hybridoma and animal cell culture technology, genomics and proteomics. As a result,
biotechnology today could not be further away from a craft; it has given rise to a high tech
industry par excellence. The biotechnology products sold on the market are often the result
of cutting-edge scientific research.
Due to the explosion-like development of fundamental biological sciences, biochemical
engineering finds itself in a very different situation. While biochemical engineering and
biotechnology designated quite similar fields 30 years ago in that they both meant the
science and art of using living cells and their constituents in order to obtain useful
industrial products or services, the term biotechnology has a much wider meaning today,
comprising all sorts of activities based on biological sciences and using some kind of
technical means, be it industrial development and production, applied or fundamental
research, or medical activities. This raises the intriguing question of whether biochemical
engineering is just the collection of know-how needed for industrial realization of
biotechnology results or whether it is a science in its own right that must be taught in
academia (Fig. 1). The question is especially pertinent for all process-relevant activities of

Fig. 1. Biochemical and chemical engineering in the force field between science and applications.

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419

biochemical engineering such as bioprocess development and design, bioprocess engineering, equipment design and engineering, and manufacturing.
The question is of utmost importance to academic biochemical engineers because many
university managers and research policymakers appear to be so impressed by the recent
successes of fundamental and molecular sciences that they tend to lose sight of engineering and biochemical engineering. At the same time, academic biochemical engineering
programs tend to shift their emphasis ever more from the process related to biomolecular
aspects of biochemical engineering (Chaudhuri, 1997). As a result, there is a trend in many
countries to outsource at least the bioprocess-related aspects of biochemical engineering
from academia to polytechnics or industries, a trend that was well characterized by
Wandrey (2001) in an editorial titled appropriately Bio- without Technology?

Fig. 2. Traditional brewingmodern monitoring. Reprinted with permission from Rose (1981), (Copyright: J.
Brenneis).

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The aim of this contribution is to analyze the position of biochemical engineering in

general and bioprocess engineering particularly in the force field between applications
and science by showing that it evolved gradually from a know-how-based type of
approach to a science elucidating more and more know-why.

2. The shift of bioprocess engineering from know-how to know-why

The evolution of biochemical engineering towards science may be illustrated using
culture technology as an example. Whereas the kettles used for beer brewing in Fig. 2 are
still very traditional, the figure also shows the presence of measuring probes and of a
modern control panel. The need to monitor and to control cultures has given rise to a more
widespread use of probes and control schemes in bioreactors as well. By developing and
applying more and more sophisticated online and at-line monitoring methods, biochemical
engineers obtain an ever more complete picture of why cultures behave as they do, which
enables them to develop new and more efficient know-how.
Table 1 shows a list of online and semi-online analysis methods that are available today
in many academic biochemical engineering laboratories. Amongst them are conventional
probes for pO2 and pH and off-gas analysis. In addition, a large number of metabolites
may nowadays be monitored by semi-online analysis based on repetitive sampling of the
culture at relatively high frequencies and on an analysis of the samples by GC or by FIA
(Herwig et al., 2001). Because taking large numbers of samples is often avoided in the
biological process industries, biochemical engineers have been looking for alternative
noninvasive methods to obtain the same type of information. One suitable method is the
use of online reaction calorimetry (von Stockar and Marison, 1991). By applying this
technique to a 300-l fermentation process, Voisard et al. (2002) have demonstrated very
clearly that calorimetric online measurements are a relatively simple and highly appropriate method to monitor industrial cultures online and also to control fed-batch processes.
Another class of online instruments that have recently generated a lot of interest are
spectroscopic measurements. Table 2 shows four types of spectroscopic online analyses
that have been investigated in the authors laboratory. Mid-infrared spectroscopy is able to
Table 1
Current on-line monitoring techniques for cellular cultures
Compound
Sensors
Off-gas
Semi-online analysis

H
O2
O2
CO2
EtOH
Acetic acid
Acetaldehyde
Glucose
Sucrose
EtOH
NH4+

Method
pH electrode
pO2 electrode
Paramagnetic
IR
GC
GC
GC
FIA
FIA
FIA
FIA

Detection limit

0.01%
0.01%
30 mg/l
30 mg/l
30 mg/l
10 mg/l
50 mg/l
50 mg/l
18 mg/l

Frequency
Continuous
Continuous
Continuous
Continuous
6/h
6/h
6/h
10/h
10/h
10/h
10/h

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Table 2
Spectroscopic techniques for on-line monitoring of cellular cultures
Analyte

Spectroscopy type

Detection limit

Frequency (min

Carbohydrates,
amino acids,
organic acids
Biomass
Carotenoids
Vitamins, cofactors

Mid-IR with ATR

100 500 mg/l

0.33

Dielectric
Raman
Fluorescence

105 cells/l
1 mg/l

2
0.1
1

measure the concentration of a number of different metabolites simultaneously at high

frequency (Doak and Phillips, 1999; Fayolle et al., 2000; Pollard et al., 2001; Sivakesava
et al., 2001). Because the spectra of the metabolites of interest normally overlap, it is
necessary to work with so-called multivariate calibration models (Martens and Naes,
1988). Fig. 3 shows that if this model is correctly constructed, it is possible to follow
online quite a number of different metabolites (Kornmann et al., submitted for publication). The graph also demonstrates the reliability of the respective signals during spiking of
the culture with important metabolites, such as ethanol or acetic acid. The respective
signals reproduced the peaks in a faithful way, but the spiking did not disturb other
unrelated signals, thus showing the correctness of the calibration.
Biomass cannot be seen with infrared spectroscopy. One way to follow cell concentration is to use dielectric or capacitance spectroscopy (Kell et al., 1990; Olsson and
Nielsen, 1997). Cannizzaro et al. (in press a,b) show that this technique is especially
promising if exploited as a real spectroscopy. Systematically scanning over a range of
frequencies while doing the measurements and using multidimensional calibration models,
they were able to follow not only the cell counts, but also the viability and even to get
online information on the size of the cells.

Fig. 3. Monitoring four metabolites simultaneously by IR spectroscopy during a culture of Gluconoacetobacter

xylinus. Keys: measured concentrations (g/l) of fructose (E), acetate (D), ethanol (z), and gluconacetan ( ).
Lines: predictions by IR spectroscopy. At 28 h, a pulse of ethanol was administered that was rapidly metabolized
into acetate, which was consumed in turn. Reprinted with permission from Kornmann et al. (submitted for
publication).

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Another interesting form of spectroscopy is Raman spectroscopy, which picks up

conjugated double bonds occurring typically in the membranes. It is thus possible to
measure online the concentration of carotenoids with a high detection sensitivity (Weesie
et al., 1999; Cannizzaro et al., in press a,b).
The online analytical tools just described were applied to a culture undergoing standard
transient experiments (Fig. 4). In standard transient experiments on continuous cultures
growing initially at steady state, the metabolism is challenged by forcing the culture
through a transient either by applying a pulse to the culture by a shift-up or shift-down of
the dilution rate or a substrate feeding rate. Such standard transient experiments are a
highly powerful way to investigate the metabolism of a culture (Duboc et al., 1998;
Herwig et al., 2001). It is possible to obtain fundamental and qualitative insight into
physiology and metabolism, and quantitative data on the kinetics of regulation. It has also
been shown that such experiments permit rapid and effective strain and mutant characterization and yield quantitative data for mathematical models, for rapid and rational
bioprocess development, and for control (Duboc et al., 1998). Fig. 4 shows the transients
that occur if a culture of Saccharomyces cerevisiae growing at a subcritical dilution rate is
subjected to a shift-up to a higher dilution rate but which is still subcritical. This means
that both values of the dilution rate will allow continuous cultures of S. cerevisiae to grow
by a completely oxidative metabolism, i.e., by pure respiration. One could thus expect that
the signals simply evolve in an exponential way from a lower level to a higher level
because of the shift-up. However, Fig. 4 clearly shows that a small overshoot of glucose
occurs and that the other concentrations and signals show pronounced excursions with
overshoots. The fact that the carbon evolution rate increases much faster than the oxygen
uptake rate clearly indicates a transient excursion into reductive metabolism for many

Fig. 4. Shift-up in dilution rate from 0.07 h 1 to 0.22 h 1 in a culture of S. cerevisiae. Glucose (z), ammonium
( ), biomass (o), specific CO2 evolution (E) and oxygen consumption (  ) rates. Reprinted with permission
from Herwig and von Stockar (2003).

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Fig. 5. Shift-up in dilution rate from 0.07 h 1 to 0.22 h 1 in a culture of S. cerevisiae. Ethanol (n), acetic acid
(+), respiratory quotient (w). Reprinted with permission from Herwig and von Stockar (2003).

hours. Fig. 5 demonstrates that reductive overflow metabolites such as ethanol and acetic
acid are secreted in the fermentation broth and disappear again after a number of hours.
In order to obtain more insight and to understand why the culture behaves as shown in
Figs. 4 and 5, it is useful to consider the rates at which a given metabolite is consumed or
produced by the cells rather than the concentration profiles. The concentration data may be

Fig. 6. Bioreactor with system boundary for computing consumption and production rates.

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Fig. 7. Opening the black box: metabolic flux analysis (MFA) allows an estimation of intrinsic metabolic reaction
rates.

transformed into rates online by performing material balances over the whole fermenter
(Fig. 6) and by correctly accounting for the accumulation effects inside the fermenter
(Herwig et al., 2001). Even more insight into the culture behavior may be obtained by
interpreting it in terms of intrinsic metabolic rates rather than exterior consumption and
production rates (Fig. 7). This type of analysis permits a partial opening of the black box
of the cell and an online observation of the metabolic activity going on inside. For this
purpose, it is necessary to draw a very simple metabolic map and then to calculate the rates
rj of the metabolic reactions from the exterior conversion rates ri by online metabolic flux
analysis. Fig. 8 shows the simplified metabolic map that was drawn for the shift-up
experiment just discussed. As depicted, the metabolism was represented in an extremely
simple way by one anabolic reaction ( qan), one catabolic reaction representing glycolysis
( qcat), two reactions representing oxidative ( qcatox) and reductive ( qcatred) catabolism of

Fig. 8. Simple online metabolic flux model for aerobic culture on glucose for yeast. Reprinted with permission
from Herwig and von Stockar (2003).

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pyruvate, oxidative phosphorylation ( qoxphos), and the production of ethanol ( qEtOH) and
of acetic acid ( qNAc). The volumetric rates rj of these reactions may be computed online
(Herwig and von Stockar, 2002a,b), so that a quantitative picture of the metabolic activity
of the culture is obtained in real time.
Fig. 9 shows this picture as a function of time in terms of specific rates qj, which were
obtained off-line using biomass concentration data. Although the glucose uptake rate
increases dramatically fast after shift-up (not shown), it cannot quite cope with the increase
in glucose feeding rate, thus explaining the small glucose-concentration peak in the broth.
Due to the dramatic glucose uptake rate just after the shift-up, catabolism is also seen to
increase enormously during almost an hour ( qcat, Fig. 9a). Oxidative catabolism through
the TCA cycle qcatox, as well as oxidative phosphorylation qoxphos jump to a higher value
immediately after shift-up, but as this value is insufficient to match the dramatic increase
of qcat, a large fraction of pyruvate overflows into reductive metabolism ( qcatred). There

Fig. 9. Intrinsic metabolic rates as a function of time during transient growth. (a) qcat (bold black line), qcatox
(black line), qoxphos (fine black line), qcatred (bold gray line); (b) qcatred (bold black line), qEtOH (black line), qHAc
(gray line).

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seems to exist another bottleneck for qEtOH, which cannot cope with qcatred, as shown in
Fig. 9b. The difference thus overflows into acetic acid ( qHac).
Over time, qcatox and qoxphos increase exponentially to meet the oxidative demand of
qcat (Fig. 9a). This reduces the overflow reactions such that first qHac and then qEtOH
come to a halt. At that point, which is reached 6 h after the start of the experiment, qcatox
and qoxphos are strong enough to handle the glucose influx oxidatively, and now
correspond to their new steady state values. Nevertheless, they increase further, making
qcatred, qHac and qEtOH negative. The disappearance of these two metabolites from the
culture is thus not only due to washout, but also to actual consumption by the culture. As
soon as the last traces of ethanol have disappeared from the fermentation broth at about
9.5 h, qcatox and qoxphos fall back to their steady-state values. Fig. 9 illustrates that using
modern analytical online tools, biochemical engineers are in the position of getting a fair
amount of insight into the metabolic behavior of their cultures right during the
fermentation process.

3. Know-why in biochemical engineering and molecular biosciences

Fig. 10 puts the observation of the previous section into a wider perspective. It
shows a schematic view of a single living cell with its genome, proteome, metabolome
and fluxome. Biochemical engineers are most interested of course in the lower part of
the figure. They have to be able to predict which sort of substrate is consumed at
what rate and internally transformed into what interesting product under which
conditions. But by developing tools that enable them to obtain insight into the

Fig. 10. Towards an understanding of the cell at the molecular level.

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internal metabolic activity and also by fundamental research on metabolomics, or

fluxomics, performed in several laboratories in Europe, biochemical engineers already
developed quite extensive quantitative understanding of the inner workings of the
cellular metabolism.
Biomolecular sciences, on the other hand, work more from the top downward on this
scheme. Starting from molecular biology, they have developed an impressive array of
highly efficient tools for large-scale genome analysis. New tools are being developed in
the present postgenomic era that will allow them to investigate also the proteome.
Functional genomics and proteomics, based on advances in microarray techniques, on
capillary electrophoresis, on 2-D gel electrophoresis, on mass spectrometry and on an
enormous effort in bioinformatics, now permit genome-wide exploration of transcriptional
and expression profiles (Ryu and Nam, 2000; Ye et al., 2001; Shoemaker and Linsley,
2002; Panda et al., in press). There is little doubt that these tools will allow biomolecular
sciences to predict relevant features also of the metabolome and of the fluxome. It is thus
certainly possible that in the future there will be a coalescence of the research efforts by
biochemical engineers, working from the bottom upwards in Fig. 10, and of biomolecular
scientists, working from the top downwards.
Biochemical engineers are already involved in many activities at the frontier between
biomolecular sciences and biochemical engineering, and are undoubtedly playing a key
role in bringing about the coalescence referred to above. Methods such as oligonucleotide-directed mutagenesis, DNA shuffling, combinatorial chemistry and peptidomimetics
are harnessed by biochemical engineers for rapid and efficient discovery of new drugs
(Weng and De Lisi, 2002; Ryu and Nam, 2000). Together with protein engineers, they
also form the basis for developing more efficient biocatalysts for biomolecule production processes. Biochemical engineers are, however, also involved in building bridges
from biomolecular sciences all the way to bioprocess engineering. The challenge in
predicting the phenotype and the metabolic capacities and behaviour in a quantitative
way exceeds very clearly the current potential of a purely molecular approach due to
the phenomenal complexity of cells and biology. The fact that this endeavour requires
considering cells and organisms as hugely complex systems (Palsson, 2000) explains
why many biochemical engineers are active in metabolic pathway analysis, metabolic
engineering and systems biology. Their engineering background enables them to tackle
complex systems and they are able to draw from nonmolecular fundamentals of
engineering sciences. Once the frontiers between biomolecular sciences and bioprocess
engineering have been bridged, biochemical engineers will be able to predict the
behavior of the cultures quantitatively under actual application conditions, e.g., in
bioreactors, directly from the molecular knowledge of the genome. It is obvious that
such an understanding of the cell at a molecular level would enable biochemical
engineers to develop their bioprocesses, biosystems, applications and new know-how
much more efficiently and rationally, based not on empirism, but on fundamental knowwhy. The opportunity that now exists to build bridges from bioengineering to the
biomolecular sciences makes biochemical engineering a particularly exciting subject
today.
Based on these statements, Fig. 11 positions biochemical engineering in the force
fields between science and application (vertical axis) and between academia and industry

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Fig. 11. Biochemical engineering between sciences and applications.

(horizontal axis). In this schematic drawing, biochemical engineering denotes not only
the biomolecular aspects of the field, but explicitly also the nonmolecular aspects
including bioprocess engineering. Industry is clearly interested in real-world applications. It usually has its own science programs that feed scientific knowledge into the
development of applications. Academia, on the other hand, is mainly concerned with
basic and molecular sciences, but it also continuously produces new ideas, new
molecules and new know-what for applications. These are then transferred into industry
and into the real world through technology transfer. This transfer process is often much
longer and more complicated than what scientists only familiar with basic and molecular
science imagine. That is exactly why engineers are needed in the process. Their typical
systemic and multidisciplinary engineering approach and profound knowledge of what is
needed in the real world and on the market enables engineers to catalyze the transition
of discoveries from basic and molecular science into real-world applications. This can of
course be done in a more or less empirical way, or in a scientific way. An example is
bioprocess development in the biotechnology industry, which is often done under huge
time pressures and is constrained by complex regulatory issues. Therefore, there may not
always be the time to optimize bioprocesses in a scientific and rational way. This has led
to the paradox situation that biotechnology offers high-tech products at the cutting edge
of science but tends to produce them using very traditional process technology that has
not changed much in the last 30 years or so. An example is erythropoietin production by
Amgen, which was scaled up simply by multiplying a relatively inefficient laboratory
culture technique, namely roller bottles.
However, if biotechnology as a whole, including engineering activities such as
bioprocess development, is to move away from an art and become a thoroughly scientific
endeavor, then engineering activities must themselves be based on scientific fundamentals
and on solid know-why and not only on empirical know-how.
Although trivial to engineers, many scientists are not aware that the scientific basis
underlying biochemical engineering activities is quite different from molecular and
fundamental sciences. It is much wider, and may or may not, or may not yet, have a
molecular basis. The opportunity existing today to build bridges between bioengineering

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and biomolecular sciences, and thus to construct a molecular basis, is one of the
particularly fascinating challenges that lie ahead.
This is also the reason for the need for strong academic programs in biochemical
engineering sciences. The main task of academic biochemical engineering is not primarily
to develop new know-how for industrial applications, but to elucidate the know-why by
conducting research on the scientific fundamentals underlying biochemical engineering
activities. Although thoroughly scientific, this type of research is not usually conducted by
fundamental scientists for two reasons. First, todays competitive climate in academic
research forces fundamental scientists to pursue topics that are recognized as hot in
fundamental sciences and does little to motivate them to deviate into engineering issues. In
addition, it takes a good understanding of research needs in the real world of industrial
environment to define meaningful research programs. Conducting research into the
fundamentals of engineering science is thus usually performed by researchers with a
strong engineering background.
Similar remarks hold for teaching. Although biomolecular aspects will increase in
importance in engineering education, the nonmolecular engineering sciences and methods
remain crucial for biochemical engineers, who will have to elucidate our comprehension of
biology as a complex system. Equally important is a certain knowledge of industrial
practices and needs if young biochemical engineers want to fulfill their role of catalyzing
the transition from molecular discoveries to real-world applications. Only the existence of
strong academic curricula in engineering, emphasizing both modern biomolecular and
nonmolecular engineering sciences, will make sure that an adequate number of chemical
and biochemical engineers are trained who will be able to tackle empirical and traditional
know-how in industry from a scientific point of view and who will establish a rational and
scientific basis for industrial procedures, thus making empirical and trial-and-error-based
search for know-how obsolete.

4. Conclusions
The role of biochemical engineering sciences is to provide a sound scientific basis for
the engineering activities, i.e., a sound basis of know-why. The aim of this quest is to
provide tools for rational, rapid and efficient development of processes, products and
services, i.e., for the efficient development of novel know-how.
The mission of chemical and biochemical engineering alike could be formulated as the
advancement of the scientific fundamentals of engineering for a rational, sustainable and
safe approach to the development of products, processes and services in the chemical,
pharmaceutical and life science industries. These scientific fundamentals, underlying both
chemical and biochemical engineering, must become increasingly molecular in nature.
However, in part due to the phenomenal complexity of the biological world and real-world
systems, they are wider than the molecular sciences and must comprise the principles of
nonmolecular engineering sciences. It is precisely one of the fascinating challenges of
todays biochemical engineers to not only elucidate the scientific basis of their engineering
profession, but in doing so also to build the necessary bridges between engineering and
biomolecular sciences.

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