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Bioreactor state estimation and control

Claire Komives

and Robert S Parker

Advanced control methods have been effectively employed for
industrial chemical processing for decades. Only recently,
however, have model-based strategies been implemented for
biological processes. Some notable advances include the
enhancement of metabolic ux models to describe the
dynamic behavior observed in biochemical reactors. The
combination of more than one type of model in a hybrid
form was shown to perform well for bioprocess control

Department of Chemical and Materials Engineering, San Jose State

University, 1 Washington Square, San Jose, CA 95192-0082, USA
Department of Chemical and Petroleum Engineering, 1249 Benedum
Hall, University of Pittsburgh, Pittsburgh, PA 15261, USA
Current Opinion in Biotechnology 2003, 14:468474
This review comes from a themed issue on
Biochemical engineering
Edited by Jeremy S Edwards and Kenneth J Kauffman
0958-1669/$ see front matter
2003 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2003.09.001
ANN articial neural network
CER carbon dioxide evolution rate
DO dissolved oxygen
FFN feed forward neural network
GA genetic algorithm
OUR oxygen uptake rate
PCA principal component analysis
RBFN radial basis function network
Bioprocess technology is currently employed for the
production of several commodity and ne chemicals.
Because of the complex nature of microorganism growth
and product formation in batch and fed-batch cultures,
which are often used in preference to continuous cultures,
the control of bioprocesses continues to present a chal-
lenge to chemical engineers. Extensive developments in
the area of process control have recently begun to impact
bioprocess development, but much work remains to be
done to couple model-based control methods to biochem-
ical reactor technology.
At the heart of bioprocess control is the ability to monitor
important process variables such as pH, temperature,
dissolved oxygen (DO), oxygen uptake rate (OUR) and
carbon dioxide evolution rate (CER). Although additional
measurements have become available recently [1], many
quantities of interest, such as intracellular product levels,
remain inaccessible. However, the inability to measure
certain variables need not keep the bioprocess engineer
from extracting valuable information from a reactor.
Advanced control systems typically employ mathematical
models that describe the process being controlled [2].
The use of a mathematical technique in conjunction with
the measurements can enable the estimation of para-
meters or process variables that cannot be directly mea-
sured. On-line estimation allows performance renement
and the ability to query the metabolic state of the cell and
reactor system. Figure 1 provides a qualitative description
of the relationship between the reliability of on-line
estimation and the complexity of the estimator. The
degree to which one can estimate the state of the system
is dependent on the availability of accurate measure-
ments of process variables such as pH, DO and off-gas
(the gas exiting a bioreactor) composition. More informa-
tive measurements, such as measurements of metabolic
products or intracellular metabolites, can provide a more
comprehensive description of the system. However, these
measurements often suffer from greater inaccuracy and
incomplete information. Thus, the improved perfor-
mance of the estimator is not guaranteed.
This paper will focus on methods to estimate parameters
and process variables that cannot be measured directly,
and the consequent use of these inferred measurements
for the control of bioprocesses. Recent reviews cover the
mathematical modeling aspects of this topic in greater
detail than space allows here [35,6

,79]. Some progress

in measurement methods that enable the estimation of
the metabolic state of the culture will also be discussed.
Emphasis has been placed on published work that incor-
porates experimental validation.
Empirical models
Models based on data-driven relationships can be used for
a variety of purposes. The mathematical structure of the
model and the incorporation of parameters to be esti-
mated can be designed by the user. Empirical models can
range from very simple, for the estimation of a single key
variable, to very complex, for the estimation and control
of the full process state. Recent articles have employed
empirical models ranging in complexity and scope.
The estimation of OUR and CER from measurements
of O
and CO
in the fermentor off-gas has become
commonplace. Several useful parameters and process
variables can be estimated and controlled on the basis
Current Opinion in Biotechnology 2003, 14:468474
of these measurements [10,11]. The approximation is
made that the concentrations of the gases measured
downstream at the monitor are equal to the partial pres-
sures in the fermentor head space; the incomplete mixing
in the head space and the time delay owing to the volume
ow in the tubing between the fermentor and the monitor
are ignored. To enable the kinetic analysis of bioreactors,
Wu and coworkers [12

] developed a black-box transfer

function model and were able to identify the system
parameters from input-output data. They obtained excel-
lent agreement between the measured and estimated
values of the OUR and CER within seconds following
a system perturbation.
The black-box or input-output model is an important
type of empirical model. A black-box model is so-called
because the mathematical structure of this empirical
model is not based on rst-principles knowledge of the
process of interest. A popular black-box model in the
recent literature is the articial neural network (ANN)
[13]. The inputs to the model are process measurements.
The measurements are weighted individually or in
groups and then combined using a nonlinear activation
function at a node referred to as a neuron, named after
the nodes in the brain which combine information sent
from the natural senses. A process ANN model is often
composed of an input layer, an output layer, and one or
more hidden layers of nodes. The traditionally used
format of ANN is the feed forward neural network
(FFN). In this network, the outputs of one layer of nodes
serve as the inputs to the following nodes. Given a set of
process measurements, the outputs of the neural network
can be estimated parameter values or process variables.
The weights applied to the inputs in the model are
determined through the training process. To train the
ANN, complete process information, corresponding to
the neural network inputs and outputs, is required and
must be obtained from the data of a set of fermentation
runs. The set of process input and output measurement
values spanned by the experimental data is termed the
experimental space and the ANN will predict outputs
accurately within this range. Should a process modeled
by an ANN operate beyond the experimental space,
commonly referred to as an excursion, the ANN may
return inaccurate output estimates. This is a function of
the extrapolation properties of neural network systems,
which are known, in general, to be poor [13]. For this
reason, control software based on certain input-output
model structures, such as ANNs, is difcult to validate for
pharmaceutical processing.
Valdez-Castro and coworkers [14] used a recurrent train-
able neural network to estimate seven process variables
for a Bacillus thuringiensis fermentation for the production
of an insecticide protein. The control objective was to
maintain the feed rate at a limiting value to enable high
production of the protein product. The model architec-
ture contains an input layer, a hidden layer and an output
layer that back-propagates to the hidden layer. The
advantages of this approach are reported to be a faster
learning process and greater stability as compared with an
FFN. Given the data-intensive nature of ANN training,
and the extrapolation properties discussed above, the
recurrent neural network approach is likely to be superior
for bioreactor modeling and control.
A hybrid ANN can incorporate a priori knowledge of the
process along with the standard neuron-type nodes into
the neural network model in separate functional nodes, as
demonstrated by Fellner and Becker [15]. To incorporate
the process information, a differential equation for the
estimation of diacetyl is included at a functional node.
The estimation model incorporates measurements of pH,
temperature, specic gravity and turbidity, which can all
be measured on-line during beer fermentation. A descrip-
tion of the estimation method is described in a separate
paper [16]. Their approach demonstrated a 46%reduction
in necessary data for training of the model and elimination
of incorrect control decisions by the hybrid structure.
The reduction in the required training data produces a
signicant decrease in the cost and time lost to model
identication. Unfortunately, the ability of the reduced-
data hybrid ANN to extrapolate beyond the experimental
space was not explored.
Figure 1
Current Opinion in Biotechnology
Model/estimator complexity
Schematic describing the effects of model complexity on estimator
performance. The shaded regions represent potential bioreactor
measurements. As more detailed information becomes available,
theoretical estimator performance improves. Because of the uncertainty
and error that often exist in higher resolution measurements,
performance improvements are not guaranteed. Measurements include
dissolved oxygen (DO), pH, temperature (T), off-gas, substrate, biomass,
metabolic products and intracellular metabolites (IM).
Bioreactor state estimation and control Komives and Parker 469 Current Opinion in Biotechnology 2003, 14:468474
James and coworkers [17] compared six different methods
for estimating the biomass during the rst 10 h of fed-
batch Alcaligenes eutrophus fermentations. The FFN was
compared with a radial basis function network (RBFN) in
both the black-box and hybrid forms. Like the FFN, a
RBFN is a network of neurons consisting of an input
layer, a hidden layer or layers, and an output layer. Unlike
the FFN, the neurons of the hidden layer are governed by
radial basis functions, which are symmetric about a center
(e.g. Gaussians), and the network output is a weighted
sum of the outputs of the hidden layer(s). The key
difference in these networks is the choice of the threshold
function, sigmoidal for FFNs versus Gaussians or wave-
lets for the RBFN estimators. In addition, the FFN uses a
back-propagation approach for training, whereas the
RBFN does not. Both types of neural network model
in the hybrid form, incorporating a discretized mass
balance differential equation for the biomass, predicted
the biomass well. The hybrid models were superior to the
black-box models in their prediction with regards to the
computed sum of squares errors from a selected run. The
potential advantage of the RBFN model is reduced
sensitivity to sensor noise as compared with the FFN,
which can be signicant with bioprocess monitoring.
Mass balance models
Dynamic elemental mass balances are the traditional
chemical engineering approach to state estimation in
bioreactors. When many by-products are produced it is
difcult to account for all the species without a complex
monitoring system. As an alternative, it is possible to
avoid monitoring all the components by employing the
cybernetic modeling framework [18]. This approach uses
dynamic balances at the reactor scale and reasonable
assumptions regarding the regulatory structure of the
organism. Given the possibility of making accurate mea-
surements of bioreactor process variables, the possibility
of using mass balance models has a high chance of
success. For example, ow-injection analysis (FIA) can
be used to measure many compounds accurately in bior-
eactors, such as enzyme activity, biopolymer content
and turbidity. A sample can be automatically removed
by a sampling system and the analysis is performed
[8,19]. The information can then be used for the estima-
tion of concentrations in the bioreactor. Nilsson and
coworkers [20] controlled the feed rate in a yeast fer-
mentation on lignocellulosic hydrolysates by estimating
the sugar concentration in the bioreactor based on mea-
surements of CER and biomass. The biomass concentra-
tion was measured by FIA.
The ability to monitor the viable biomass concentration
in the bioreactor is more meaningful than the total
number of cells present for state estimation. Capacitance
can be measured from which the amount of viable bio-
mass can be estimated; a build-up of charge across cell
membranes of viable cells can be induced and measured
as the culture capacitance. Estimators for biomass based
on observer theory were developed by Bastin and
Dochain [4] and explored in a Candida utilis fermentation
by November and Van Impe [21]. The on-line measure-
ment of capacitance led to good estimates of the biomass
when appropriate tuning parameters and a lower bound
for the lowinitial values in the reactor were implemented.
Statistical estimation methods
Biomass measurements are frequently subject to experi-
mental error, and numerically differentiating this signal to
estimate specic growth rate amplies the error. The
accurate determination of specic growth rate enables
the calculation of kinetic parameters. Adata lter is able to
smooth out measurement errors and outliers. Neeleman
and van Boxtel [22] applied a backward and forward
extended Kalman lter to the off-line biomass mea-
surements to estimate the specic growth rate, which en-
abled the calculation of kinetic parameters. The use of
backward ltering to reduce estimator sensitivity to
initial condition effects combined with recursive forward
estimation of parameters provided superior performance
in biomass prediction for this case study.
Certain process characteristics, such as batch production,
lead to convenient estimator formulations. In the case of
batch processes, expected dynamic behavior can be
accounted for by enforcing smoothness constraints on
the estimated metabolite proles. Ethanol and residual
sugar concentrations in beer fermentations were recon-
ciled and consequently smoothed using a probabilistic
approach that enabled the calculation of rates with good
accuracy [23]. Experimental results validated the use of
smoothness constraints on the process dynamic response.
Parameters in the model were estimated through a novel
decomposition of the highly nonlinear optimization pro-
blem into a hierarchy, including an unconstrained non-
linear problem followed by four independent constrained
quadratic programming problems [23]. The result was a
stoichiometric model that successfully captured the
dynamic system response and provided estimates of
ethanol and sugar concentrations, and their corresponding
production and consumption rates; refractive index and
broth density were also estimated from on-line measure-
ments throughout the batch.
Database models
Database models have been explored recently as an
efcient approach to using historical data [23]. The
incorporation of information from fermentation runs into
a process model can be a daunting task in light of the
many components present in the broth. The choice of
critical variables can be simplied by using mathematical
tools designed to extract the most signicant variables or
sets of variables from a large dataset. By using these
critical variable sets, the modeling process becomes easier
without sacricing model quality.
470 Biochemical engineering
Current Opinion in Biotechnology 2003, 14:468474
Multiway principal component analysis (PCA) is an
approach to data reduction that enables the mathematical
classication and reduction of large datasets. The trans-
formation of seed fermentation data into principal com-
ponents enabled Cunha and coworkers [24] to improve
the prediction of nal process productivity as compared
with an analysis of the data only from the automated
main production fermentor. Principal components are
weighted sums of process measurements, or states, that
capture a percentage of the process variability using a
signicantly reduced number of process variables. How-
ever, it was suggested by Takors and coworkers [25] that
relatively large errors would make multivariate PCA
unsuitable for use along the course of a fermentation
run. The identication of critical process parameters for
the development of the process model can also be facili-
tated using information from fermentation runs by per-
forming a decision tree analysis [26]. Like PCA, this
approach begins with the historical fermentation data
from several runs and identies the signicant variables
for effective analysis and modeling of the process by an
automated objective method. Decision tree analysis is
able to incorporate both continuous variables, such as
consumption rates, as well as categorical variables such as
fermentor number and inoculation time. The net result of
both approaches is to reduce the dataset while maintain-
ing the requisite information to classify the outputs.
Optimization algorithms
The use of optimization algorithms to assist in driving
the process towards the production objective is an entic-
ing application of on-line estimation [9]. Here, the focus
shifts from the ability to measure or estimate a parameter
or process variable to the opportunity to improve process
performance using these measurements and estimates.
Ideally, a process should be operated at the optimum
condition, as dened by the operating objective(s), such
as maximum productivity and minimized environmental
impact. To employ an optimization algorithm, a process
model is required. Furthermore, it must be recognized
that the optimum calculated from the model and the
experimentally observed optimum may differ owing
to the presence of process-model mismatch. Some ap-
proaches to estimation and optimization are presented
below. Although the results are often promising, the
mathematical and/or computational burden in employ-
ing these techniques is higher than many of the other
methods discussed in this work.
Neural networks can be used in both estimation and
process optimization. Becker and coworkers [27] applied
an FFN for the control and optimization of a pilot-scale
beer fermentation. Diacetyl is a principal avoring com-
ponent in beer and must be maintained below 0.15 ppm
for product quality; however, there are no cost-effective
ways to measure the diacetyl concentration during the
run. To account for the dynamics, a special neuron, called
a nite impulse response neuron, was incorporated into
the neural network. This special neuron allows for a one-
step-ahead prediction, and measurements during the run
can be used as input data to adapt the prediction along the
course of the fermentation. This dynamic neural network
serves as the process model and allows the optimization to
be performed through the application of variational cal-
culus. The inputs to the model are values of the diacetyl
measured on-line (via a software sensor), pH, tempera-
ture, specic gravity and time. Comparisons of the one-
step-ahead predictions of diacetyl and specic gravity
with the on-line measurements were excellent, and the
optimization of the process enabled a reduction in process
time of 10% averaged over four fermentation runs.
The search for an optimum process condition when a
process model is linear is relatively straightforward. Bio-
logical processes, however, are highly non-linear. Genetic
algorithms (GAs) are optimization routines that operate in
a similar manner to natural genetic selection and which
can be used with nonlinear process models (for more
details see [28]). Na and coworkers [29] employed GAs
to optimize the fed-batch growth of S. cerevisiae on glu-
cose. The approach proved a success for optimizing and
controlling the bioreactor and estimating reactor para-
meters on-line. The strategy for optimizing the process
was to calculate a sequence of feed ow rates and the
corresponding times to switch between these ow rates.
In addition, another GA was used to update the produc-
tion rate and yield parameters on-line. Although generally
successful as nonlinear optimizers, GAs are computation-
ally demanding. In fact, in the study by Na and colleagues
[29] the time taken for the algorithms to compute the next
manipulated variable move was about 25 min, including
the parameter update step.
Chiou and Wang [30] used a hybrid differential evolution
(HDE) algorithm as an approach to state estimation.
Differential evolution and GAs are both types of evolu-
tionary algorithms that are appropriate to use when little
or no a priori knowledge of the process is available,
although the degree of mathematical complexity is high.
In this work, the addition of acceleration and migration
operations enabled faster convergence to the optimum
conditions. The model was tested with an E. coli fer-
mentation for the production of a recombinant protein.
The steady-state performance of the estimator was quite
good. Furthermore, the estimator qualitatively captured
the dynamic behavior, although quantitative estimation
error remained. Whether the dynamic error is the fault of
the estimation algorithmor the proposed model structure
is uncertain.
Another strategy proposed for optimizing a nonlinear
process is the use of Pontryagins maximum principle
[31]. This method involves an off-line analytical solution,
which returns a manipulated variable trajectory, such as a
Bioreactor state estimation and control Komives and Parker 471 Current Opinion in Biotechnology 2003, 14:468474
feed-rate prole, that can be implemented on-line.
Levisauskas and colleagues [32

] used this approach

to maximize protein production in E. coli with a fed-
batch bioreactor. Parameters for a mass balance model of
the process that incorporated cell maintenance require-
ments were determined from three initial experiments
using an evolutionary algorithm [33]. The fermentation
included both the biomass growth phase and the protein
production phase, during which time the biomass growth
slowed but was not halted. During the protein produc-
tion phase, the authors measured higher total protein
production at lower specic growth rates. The quantita-
tive relationship between protein production and growth
rates was incorporated into the model. The resulting
optimization scheme returned the optimal time for
induction and the feed rates that maximized biomass
production in the rst phase and protein production
at the end of batch. With careful experimentation and
a well-designed mass balance model, this approach
resulted in a doubling of the protein production with
only four fermentation experiments.
Mahadevan and Doyle [34] studied the optimization of
recombinant protein production in a fed-batch bioreac-
tor using tools from nonlinear analysis [35]. The princi-
ple advantage of their strategy is to reduce on-line
computation time by input elimination, which uses an
off-line mathematical transformation to simplify the on-
line problem. The solution successfully estimates, and
compensates for, parametric drift in the model. Although
this approach demonstrates the capability of involved
mathematical approaches in bioreactor control and esti-
mation, the cost is often increased off-line complexity or
on-line calculation.
Metabolic ux models
Metabolic ux models describe the relative utilization
of the metabolic pathways in a whole-cell biocatalyst.
Originally, the use of these models required the system
tobeat steadystate. Thetermpseudo-steady-stateallowed
a broader use of these models for approximation of meta-
bolic activity in a dynamic setting. The papers described in
this section touch on improvements in the application of
metabolic ux models for dynamic modeling. Further-
more, it is suggested that this model class may soon be
used for on-line bioreactor state estimation and control.
The use of
C labeling for the measurement of meta-
bolites for metabolic ux analysis has been described [36].
El Massaoudi and coworkers [37,38] showed the feasi-
bility of using this technique in a large fermentor (300 L)
by running a parallel, small reactor that they called the
sensor reactor. Fresh production broth from the large
reactor enters the sensor reactor every 20 min for
labeling and the data are generated for the metabolic ux
analysis. The study establishes the feasibility of this
parallel reactor system for fast data collection that mirrors
the conditions in the large reactor. A future paper will
describe the system for process control.
Sainz and coworkers [39] presented a metabolic ux
model for yeast combined with a dynamic process model
to mathematically represent batch wine fermentation.
The ux model did not contain dynamic terms, but the
model was used with a linear optimization routine to
determine the uxes present with the objective to max-
imize growth and glucose uptake. The uxes determined
were then fed to differential equations for the description
of the process. The combined models were able to
represent the dynamics quite well. Data for the validation
of their model was taken from the literature.
Dynamics of a bioreactor can be studied experimentally
by perturbing a steady state. Herwig and coworkers [40]
developed a dynamic metabolic ux model for use with
transient on-line analysis. To continuously check the
data consistency, a simple statistical evaluation of the
data was performed as originally described in Wang and
Stephanopoulos [41]. Although they have not demon-
strated their approach for on-line estimation or process
control, the effective use of a metabolic ux model under
transient conditions shows promise for non-steady state
metabolic estimation.
Mahadevan and coworkers [42

] combined optimization
with a dynamic metabolic ux model to describe the
diauxic growth of E. coli. The dynamic model is able
to determine the rate of change of the ux constraints.
Unlike the traditional static ux balance, their strategy
enabled the prediction of the reuptake of acetate after
glucose exhaustion in the broth. In the absence of kinetic
parameters, this approach shows great potential for bio-
reactor state estimation.
Advances in monitoring for state estimation
New techniques for process monitoring offer the poten-
tial for considerable insight into the properties of the
broth and cellular dynamics during the course of a fer-
mentation run. Although extensive programs have been
developed in this area, only two techniques will be
described here. Comprehensive reviews can be found
in the recent literature [1,8].
Mid-infrared spectroscopy offers in situ multicomponent
monitoring that suits the requirements for stability, relia-
bility, and extended monitoring time. In addition, no
recirculation loop outside the reactor is required [43

Calorimetry is another technique that can involve mea-
surements of both the reactor power input as well as the
generation of metabolic heat. Because of the high surface-
to-volume ratio in small reactors, the technique of calori-
metry has more potential at the large scale than in small-
scale bioreactors. Voisard and colleagues [44] developed
and tested a calorimeter system for a 300 L fermentor.
472 Biochemical engineering
Current Opinion in Biotechnology 2003, 14:468474
This method shows potential for an effective and low-cost
method to determine the metabolic state of the cells.
Progress has been made in coupling advanced modeling
and control methods to bioreactor technology. Papers de-
scribed here demonstrate excellent agreement between
experimental data and both estimated variables as well as
model trajectories. The incorporation of a priori process
knowledge with neural networks demonstrated shorter
training times for control and estimation applications, as
well as reduced sensitivity to sensor noise over traditional
neural networks. The application of a transfer function or
a Kalman lter to process measurements was shown to
signicantly improve the quality of the dynamic process
model established with the measurements. Evolutionary
algorithms also show much potential for bioreactor opti-
mization and control strategies. Novel approaches with
database models showed that satisfactory models can
be developed with a reduced set of model components.
The models and techniques employed in estimation will
continue to increase in resolution and complexity, respec-
tively, but only as the sensor technology allows mean-
ingful measurements to be collected.
References and recommended reading
Papers of particular interest, published within the annual period of
review, have been highlighted as:

of special interest

of outstanding interest
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