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Abstract With the aid of a membrane introduction mass 2,3-BDL fermentation can be governed by the amount of
spectrometer (MIMS), the major product 2,3-butanediol supplied oxygen. Therefore, we correlated the measured
(2,3-BDL) as well as the other metabolites from the fer- product concentrations directly to the oxygen supply. Such
mentation carried by Klebsiella oxytoca can be measured an approach is somewhat different from the conventional
on-line simultaneously. A backpropagation neural net- methods. In general, glucose and cell mass are two im-
work (BPN) being recognized with superior mapping portant measurements for fermentation related studies.
ability was applied to this control study. This neural net- However, these two measurements were not used in this
work adaptive control differs from those conventional study. Only the measured data from the MIMS were re-
controls for fermentation systems in which the measure- quired so that the on-line system can be established. The
ments of cell mass and glucose are not included in the dissolved oxygen (DO) in the fermentation broth this re-
network model. It is only the measured product concen- ects the oxygen availability to the cells and hence also
trations from the MIMS that are involved. Oxygen com- affects the secretion of metabolites. The DO can be regu-
position was chosen to be the control variable for this lated by oxygen partial pressure (or oxygen composition)
fermentation system. Oxygen composition was directly as well as aeration rate. Therefore, to control the level of
correlated to the measured product concentrations in the DO in the fermentation broth this can be regulated by
controller model. A two-dimensional (number of input either oxygen composition or aeration rate or both. In this
nodes by number of data sets) moving window for on-line, study, oxygen composition was chosen as the control input
dynamic learning of this fermentation system was applied. variable. To regulate the oxygen composition by adjusting
The input nodes of the network were also properly se- the proportion of O2 and N2, larger range of variation
lected. Number of the training data sets for obtaining (0100%) can be obtained. If to control the DO by aeration
better control results was also determined empirically. rate, the oxygen composition can only be varied from
Two control structures for this 2,3-BDL fermentation are 021%. Since larger variation range for the control variable
discussed and compared in this work. The effect from is easier for observing the control effect, we thus chose
adding time delay element to the network controller was oxygen composition instead of aeration rate.
also investigated. It is rather difcult to nd a proper model for the entire
course of a fermentation process. Instead of a static model,
dynamic identication with adaptively adjusted parame-
1
ters was applied to the control system of this fermentation.
Introduction
The purpose of system identication is to search for a
2,3-Butanediol (2,3-BDL), the major product of the fer-
model which correlates the output (response) of a system
mentation by Klebsiella oxytoca, is produced at an optimal
to the control input command [4]. The control system can
fractional yield under a partially anaerobic condition [1].
then be performed by combining the process model from
Besides of 2,3-BDL, there are other metabolites such as
system identication with the controller model. Therefore,
acetate, ethanol, and acetoin secrete as the end products
a precise process model (or system identication) and a
from this fermentation. The on-line measurement of the
good controller model (control law) are two major com-
four products, acetic acid, acetoin, ethanol, and 2,3-BDL,
ponents for a control system. In general, the model from
was performed by a mass spectrometer modied by the
system identication is combined with the controller
insertion of a membrane probe [2]. The major metabolic
model to become a control system so that the future
pathway of this fermentation was shown in our previous
control input command can thus be predicted. However,
work [3]. The distribution of product formation during
the process model can also be merged with the controller
model to become only one model for a control system.
This model can be switched between the process model
and the controller model alternatively during the control
process. An inverse-type process model has been applied
Received: 27 August 1998
to adaptively identify a time-invariant nonlinear system
Mei-J. Syu (&), Cheng-L. Hou with unknown dynamics. The same inverse model was also
Department of Chemical Engineering, used as the controller for the system. However, the above
National Cheng Kung University, approaches were not used in the control system of this
Tainan, Taiwan 70101, R.O.C. study. Instead, we used two other control mechanisms that
Bioprocess Engineering 21 (1999)
will be described later in this article for this fermentation. command. In general, the proposed control can be
The identication and control of this fermentation was expressed as;
constructed so that the supplied oxygen composition was
only related to the measured product concentrations. ui f xi ; ui1 ; xdi1 ; 1
Backpropagation neural network was chosen to be the where ui is the predicted control input command with
method for the identication and control of this fermen- respect to xdi1 , the measured (or desired) x at time i+1;
tation system. The ability of neural networks on identify- xi xim ; . . . ; xi1 ; xi , the array of the measured system
ing bioprocesses has been demonstrated in previous work output (or response) up to time i; ui1 uin ; . . . ; ui1 ,
[3]. Hence, neural network dynamic identication and the array of the control input command up to time i. It
control would be further tested by the MIMS on-line should be noted that xi and ui1 may not be of the same
acquired fermentation data in this work. dimension.
142
Once a desired output is given, the control input com-
2 mand with respect to this desired output can then be
Backpropagation neural network computed in a straightforward manner. The neural net-
Backpropagation neural network (BPN) is a feedforward work prediction/control for this fermentation is thus
network with backward learning. It can identify different expressed in a general form as follows:
systems of different nonlinear characteristics with very
ui f ui 2; ui 1; Pi 1; Pi; P4 i 1 ;
high precision. BPN computation mainly includes two
phases; forward transferring of incoming information 2
during the rst phase and backward learning during the
where u(i) is the predicted oxygen composition with re-
second phase. The network computation is proceeded al-
spect to the measured data at time i + 1. u(i)2) and u(i)1)
ternatively between these two phases. Not only the theo-
are the oxygen composition supplied to the system at time
rem of backpropagation network was thoroughly studied,
i)1 and i, respectively. P(i) (Pj(i)), the array of the
there are also plenty of reports on applying backpropa-
selected measured product concentrations at time i, and
gation neural networks for different purposes of different
the notation j can be varied from 1 to 4. P(i)1) is dened
systems. Nevertheless, all the applications usually count on
as the array of the selected product concentrations from
its precise mapping ability. Glassey et al. [5] based ex-
the measurements at time i)1 accordingly. P4(i + 1) is the
periments on neural networks to carry out the design
2,3-BDL concentration measured at time i + 1, which, in
procedures for fermentation processes. Hence, the fer-
turn, will be the desired 2,3-BDL concentration (the target
mentation especially of the recombinant system was ef-
output) for the controller model.
ciently optimized through such a technique. Khalid et al.
Neural newtorks with adaptive learning ability in its
[6] applied neural network as an adaptive controller to
connecting weights can identify the correlation of desired
solve control problems. The neural network control results
system variables of different nonlinear systems. It was also
were compared to three other control algorithms: fuzzy
used as the controller for the study of this Klebsiella
logic control, generalized predictive control, and PID
oxytoca fermentation system. Neural networks have been
control. Backpropagation network was also applied to
applied to control different systems. Most systems being
texture analysis [7]. The analysis of variances by using
studied are robotic systems of purely physical response
neural networks for defect detection of texture surfaces
without chemical reaction. Although the nonlinearity of
can be made. Texture classication was also studied.
these systems may not be as high as those with chemical or
Backpropagation of neural network combined with fuzzy
biochemical reactions. Narendra and Mukhopadhyay [11]
sets and graph theories were used for the detection and
discussed the adaptive control of nonlinear multivariable
diagnosis of faults during process operation [8]. Assess-
systems by using backpropagation neural networks. Based
ment of potential hazards and operability problems in
on a backpropagation neural newtork, Khalid et al. [12]
design from these approaches were also discussed. The
provided a multilayered neural network control scheme on
backpropagation neural network with momentum term for
a MIMO control system of furnace. Yang and Linkens [13]
learning was analyzed [9]. The result is the conclusion that
used neural networks to control a CSTR with slow dy-
all the local minima of the sum of least square errors is
namics. Venugopal et al. [14] also presented an improved
stable. The transfer functions x=1 jxj and
direct control architecture for the backpropagation neural
sgn x x2 =1 x2 were proposed in our previous work
network on-line adaptive control of dynamic systems. Syu
[3] for backpropagation neural network study and signi-
and Chang [15] provided a recurrent backpropagation
cant results were achieved.
neural network for studying the dynamic control of the
penicillin acylase fermentation. Boskovic and Narendra
3 [16] also proposed a neural network controller for the fed-
Dynamic identification and control batch fermentation process. The control results from the
BPN was used to develop the dynamic identication/con- network controller were compared with the other four
trol system for this fermentation process. Prediction ca- different types of linear and nonlinear controllers and
pability performed by the neural networks has been better performance was concluded from the network
demonstrated10, which is a very important issue for con- controller. Oin et al. [17] used supervised network as a
trol. As a result, the BPN for identication/control was controller to the fermentation process which was carried
designed with the ability to predict the future control input by Bacillus thuringiensis.
M-J. Syu, C-L. Hou: Neural network predictive control by MIMS
In this work, neural networks provided a dynamic prevented standard solutions from entering the return line
learning and prediction process that moved along the time to the fermentor. In addition, in the FIA module was the
sequence batchwise. In other words, a scheme of two- necessary ow network to carry out reactions of the
dimensional moving window (number of input nodes by sample stream with a reagent stream for derivation,
the number of training data) was proposed for reading in acidication or dilution. The sampling schedule was pre-
new data while forgetting part of the old data. Training cisely controlled to load and inject samples and standards
data provided in such a mode can save computation time alternatively. The schedule was arranged as: load sample
by removing previous unnecessary data while carrying out ! inject sample ! load standard ! inject standard.
the on-line control system. At the same time. precise
identication of the system characteristics most closest to 4.2
the next control point must be satised. Therefore, the MIMS/Fermentation system
143
control action can be efciently predicted from the net- The set-up of the whole MIMS/fermentation system is
work controller and sent to the system in time. When the shown in Fig. 1. A 2 l fermentor (Microgen, New Bruns-
control time is not yet due, the neural network process wick Scientic, Edison, NJ) was controlled at the agitation
model reads in a certain amount of data and is then speed of 600 rpm. During the fermentation, temperature
trained to obtain precise mapping of the system behavior. and pH were controlled at 37 C and 5.7, respectively. Air
During the control phase, the network controller predicts a was supplied to the fermentor through a 0.2 lm m air lter
control action. By switching between the learning phase disk. A 10%(v/v) antifoam solution, polydimethylsiloxane
and the predicting phase, the control system can thus be was added to the fermentor as needed.
proceeded.
4.3
4 Gas mixer
Materials and methods A gas mixer was constructed for the control of inlet gas
ow rate and gas composition by mixing N2 and air. The
4.1 main components of this unit were two mass ow con-
Membrane introduction mass spectrometry (MIMS) trollers (Model 1259 C, MKS Instruments, Andover, MA).
The MIMS included a Finnigan TSQ mass spectrometer Each ow controller consisted of an accurate mass ow
modied by the insertion of a dimethylvinylsilicone meter coupled to a control valve. It can receive an input
polymer membrane probe, data acquisition system, and signal of 010 V which was linearly equivalent to the ow
control. It can allow the selective passing and monitoring rates of 04/3 vvm. Inlet gas ow rate can be varied by
of four major products, acetic acid, ethanol, acetoin, and providing different voltages to each mass ow controller.
2,3-BDL from the fermentation of Klebsiella oxytoca. The The composition of the inlet gas was regulated as well.
Finnigan TSQ mass spectrometer included INCOS data The oxygen composition was controlled by a D/A board,
acquisition. The INCOS system was connected to a PC DDA-06 (MetaByte Corp., Taunton, MA). The D/A board
which controlled the ow injection device, the gas mixer was inserted in Keithley 500 A control unit. DDA-06 has
and the chemical ionization (CI) reagent gas line valve of the specication of 6 independent, equal-function chan-
the MIMS. A tangential ow device, a lter acquisition nels; and 12-bit resolution. The output range was selected
module, and a ow injection analysis unit were also in- as 010 V, which was the full input range for adjusting the
cluded in the whole on-line monitoring system. N2 and air ports on the gas mixer. The correlation of
A tangential ow device was used to circulate the fer- voltage and oxygen composition from DDA-06 board must
mentation broth which was free from cell mass. The l- be precisely calibrated with the supplied calibration/in-
tered broth was injected and transferred to the MIMS stallation program before use. With this on-line system,
through a lter acquisition module (FAM, Waters, Milford, the oxygen composition control input command can thus
MA). The other main component of the FAM was a six- be computed according to the control algorithm. The ox-
port HPLC sampling valve (Rheodyne, Berkeley, CA). In ygen composition control input command was then con-
the FAM, a 250 ll loop was lled with ltered sample broth verted to the voltage mode through DDA-06 and sent to
or standard, which was controlled by a switching valve. adjust the gas mixer.
The sample broth or standard was then injected into a
constantly owing water stream by which the plug ow 4.4
was transported to the membrane probe. The extra cell- Microorganism
free broth was pumped back to the fermentor. The broth The bacterial cell line used in this work was Klebsiella
circulation rate was maintained at about 1.0 ml/min. The oxytoca (ATCC 13882). The cells were kept at )60 C in a
FAM was to control the alternative transportation of the 10% glycerol based medium. They were grown on agar
injected sample and the standard solution to the mem- slants with 10 g=l glucose.
brane probe through a switching valve. The standard so-
lution was made of 1:0 g=l acetic acid, 0:5 g=l acetoin, 4.5
0:5 g=l ethanol, and 2:0 g=l 2,3-BDL. Growth medium and inoculum
A ow injection analysis (FIA) device was used to K. oxytoca was cultured on PA medium added with trace
control the sampling schedule of the FAM to deliver the metals [18]. Glucose was used as the only limiting sub-
sample and/or standard solution to the membrane probe. strate. 5 ml of the concentrated 200x solution of trace
Within the FIA module was an arrangement of valves that metals was added per liter of the PA medium. The culture
Bioprocess Engineering 21 (1999)
144
broth was incubated at 37 C with the shaker's speed of Springs, OH). The instrument was calibrated with a stan-
220 rpm in a oor shaker (Model G24, New Brunswick dard glucose solution with the concentration of 2:0 g=l.
Scientic, Edison, NJ). The cells were pre-cultivated twice Sample readings in the unit of mg/dl were available di-
and lasted about 10 hours for each pre-culture before in- rectly from the digital recording on the instrument.
oculated into the fermentor so that the cells can be adapted
to the fermentation environment within a short time.
5
Results and discussion
4.6
Cell mass and glucose measurements 5.1
A spectrophotometer (Coleman model 55, Perkin-Elmer, MIMS on-line monitoring fermentation data
Maywood, IL) was used to measure the absorbance of the Four major products of this fermentation, ethanol, acetic
samples at a wavelength of 540 nm. Dry cell weight is di- acid, acetoin, and 2,3-BDL can be measured on-line from
rectly proportional to absorbance in the range of 00.3. the mass spectrometer modied by the insertion of a
The correlation was found to be 0.32 g dry cell weight per membrane probe. The schedule for each measurement
unit absorbance. Samples were analyzed for glucose by a from the FIA/MIMS device required approximately 6
glucose analyzer (model 27, YSI Company, Inc., Yellow minutes. The measured data acquired from a batch of
M-J. Syu, C-L. Hou: Neural network predictive control by MIMS
145
density. At the production phase, to stimulate the forma- tion system. Hence, the neural network should include the
tion of the secondary metabolite 2,3-BDL, oxygen com- measurements of the previous times. Three different types
position was regulated decreasingly. The fermentation of transfer functions were chosen for studies. They were all
experiment was carried out with a predetermined oxygen of sigmoid shape but with different degrees of nonlinear-
supply schedule, therefore, the success of this neural net- ity. The nonlinearity is 2=1 ex 1 >sgnx x2 =1
work of predicted control further implies the feasibility of x2 > x=1 jxj. Both the functions of x=1 jxj and
the neural network control with this oxygen input sched- sgnx x2 =1 x2 were proposed and have been suc-
ule. cessfully performed in previous work. The input nodes
The moving mode of dynamic identication and pre- including the information from time t)1 was used at the
diction is applied in this work. In each training of the initial stage of this study as a primary result from previous
neural network for precise identication, several data sets work.
146
were provided as a training batch. When the training was The input nodes were studied from using different
nished, then the learned network was used to predict the transfer functions. The results are shown in Figs. 3(a)(c).
output node which is the control input command to the It can be concluded from these gures that to include both
on-line fermentation system for next control interval. For the time delay and the oxygen control variable in the
next training, in order to keep a xed and efcient com- network nodes did not improve much of the results on the
putation time, instead of a convergence criterion, the prediction of next oxygen control command from the
training number was set to be the same. Each prediction transfer function of either sgnx x2 =1 x2 or 2=1
was switched on after the training of the data sets. The ex 1. However, the prediction results were rather dif-
neural network with input nodes including the informa- ferent from the function of x=1 jxj. If both gures were
tion related to the current time and previous times as examined, it can be found that the error growing behavior
appeared in our previous work3 was used in this study. were all similar. At rst, the errors maintained small,
The response of product formation to the regulation of starting from different times, the errors grew fast. The best
oxygen composition was also slow. Concluded from the result obtained from the function of x=1 jxj as shown
above, time delay can be expected to exist in a fermenta- in Fig. 3(b) was caused from the time for fast growth of
5.4
Number of data sets in a training batch
The second network control scheme was used to study the
effect of the number of training data sets on the prediction
results during the dynamic learning phase. 5, 10, and 15
data sets respectively as a training batch were chosen for
this study with the results shown in Figs. 7(a) and 7(b). It
seems that the number of data sets contained in each
training batch did not show much difference in the pre-
diction performance. However, with 5 data sets, the results
seem slightly better. Hence, we may conclude that the
Fig. 4. The rst neural network control system number of training data sets did not have signicant effect
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