World Journal of Microbiology and Biotechnology 8, 7-13
An effective automated glucose sensor for fermentation monitoring and control I. Queinnec,* C. Destruhaut, J.B. Pourciel and G. Goma An industrial glucose analyser was partnered to an automated injection s y s t e m to evaluate glucose in the culture medium of a bioreactor. This sensor has been validated on continuous cultures of Schizosaccharomycespombe and continuous and fed-batch cultures of Saccharomycescerevisiae. In addition to the advantage of a more accurate process monitoring, the main interest of this sensor deals with the control of the substrate concentration to a prespecified reference signal. Several experiments have been carried out first to validate the sensor, then to control the process evolution. Key words: Glucose analysis, process control, process monitoring, sensor.
Processes occurring in microbial cultures are complex and difficult to optimize and are essentially controlled by a biological strategy (medium, strain improvement, environmental conditions, etc.). Optimization and control of fermentation processes, however, require advanced control and modelling theories, and lead to an increasing search for suitable sensors for fermentation process control. This paper describes the use of a glucose analyser associated with an automated injection system in the development of an instrument for monitoring and control of the substrate concentration in the reactor. The sensor is interfaced to a microcomputer or a programmable logic computer which manages the measurement cycle. This sensor finds in this form its first utilization in the monitoring of fermentation processes (Bovee 1981, Queinnec & Pourciel 1989). It is also used for control of continuous, multistage or fed-batch fermentation processes (Chamilothoris et al. 1988; Vigi6 et al. 1991). Fermentation has been carried out with Saccharomyces cerevisiae and Schizosaccharomyces pombe. Different measurement frequencies have been selected depending on the process dynamics and characteristics.
The authors are with DGBA, INSA, UA CNRS 544, Centre de Transfert en Biotechnologie et Microbiologie, 54 Avenue de Rangueil, F-31077 Toulouse c6dex, France. Fax: 33 61 55 96 73. I. Queinnec and J.B. Pourciel are also affiliated to Laboratoire d'Automatique et d'Analyse des Systemes du C.N.R.S., and I. Queinnec also to GRECO-SARTA, 7, Avenue du Colonel Roche, 31077 Toulouse c~dex, France. * Corresponding author.
Materials
and Methods
Strains and Media Two strains were used in this work: Saccharomyces cerevisiae and Schizosaccharomyces pombe, the former in fed-batch, continuous and multistage cultures, the latter in continuous culture with cell re-cycle by cross-flow membrane filtration. The culture medium of S. cerevisiae and S. pombe had the composition given in Table 1 and Table 2, respectively. Glucose of different concentrations was used as carbon source. Culture Apparatus and Experimental Conditions For continuous cultures, a 2-litre fermenter equipped with magnetic agitator, temperature and pH controls was used. A level sensor maintained the fermenter at constant volume. Fresh medium was supplied by a pump working at computer controlled rates. The multistage reactor comprised four reactors of this type in series.
Table 1. Culture medium of
Saccharomyces
cerevisiae. KH2PO4
(NH4)2SO4 Sodium glutamate Na2HPO4.12H20 CaCI2 MgSO4.7H20 ZnSO4.7H20 (NH4)2FeSO4.6H20 + vitamins
3 g/I 3 g/I 1 g/I 3 g/I 0.25 g/I 0.25 g/I 5 mg/I 1.5 mg/I
9 1992Rapid Communications of Oxford Ltd
WorldJournalof Microbiologyand Biotechnalogy,!/ol8, 1992
7
L Queinnec et al. Table 2. Culture medium of Schizosaccharomyces
pombe. NH4CI K2HPO4 K2SO4 MgSO4.7H20 NaCI CaCla.2H20 MnSO4.H20 CuSO4.5H20 COC1.6H20
(NH4)6M07024 ZnSO. KI H3B03 + vitamins
1.15 g/I 0.75 g/I 0.5 g/I 0.25 g/I 0.2 g/I 0.15 g/I 4 mg/I 1 mg/I 0.4 mg/I 1 mg/I 4 mg/I 1 mg/I 1 mg/I
Cell re-cycle of S. pombe was achieved with a cross-flow membrane filtration. For fed-batch cultures, a 20-1itre fermenter was used. Fresh medium was supplied to the reactor when and as required. The control variable was the flow rate of nutrient calculated from the glucose concentration measurement. Stirrer speed, temperature and pH were monitored and controlled as given in Table 3.
Glucose Analysis Off-line measurements. Samples from fermentation broth were clarified by centrifugation (11,000 x 8 for 5 min) and diluted in distilled water before injection by a syringe (25/~1) into the glucose analyser (Yellow Springs Instrument, Yellow Springs, Ohio, USA). This analyser had a range of 0 to 2 g of glucose/L A glucose standard of 2 g/l was used for calibration (2 g/l corresponds to 200 mV). Automated measures. The automated system was built by association of the glucose analyser with a syringe. A set of pneumatic jacks around the syringe ensures sampling of the culture medium from a loop disposed on the culture vessel, injection into the glucose analyser and rinsing of the syringe before the next measurement. By first selecting the distance of syringe travel, the working scale of glucose concentrations to be measured can be set. The reduction of the volume to be injected represents an artificial dilution compared to the 25/21 of an off-line measurement. The choice of a good dilution level allows substrate concentrations to be measured up to 200 g/1 with good accuracy (see later). The
calibration was made with a glucose standard whose concentration depended on the glucose setpoint of the experiment. For fed-batch culture of S. cerevisiae and continuous culture of S. pombe, the calibration was 400 mV for 40 g/1. For continuous culture of S. cerevisiae, the calibration was 400 mV for 4 g/1. The active volume of the syringe was 1.5/~1 in the first case and 6/~1 in the second one.
Programmable Logic Computer The previously mentioned sensor is handled by a programmable logic computer (PLC) whose cycle ensures rinsing of the enzyme membrane (analyser digital input--CLEAR), rinsing of the syringe and injection of the sample (action on jacks) and control of the glucose measurement (digital input of the analyser). The duration of a measurement cycle depends on the enzyme analyser. As previously stated, the WAIT durations are approximately 60 s for the measurement and 40 s for the rinsing of the membrane. Moreover the PLC cycle sequentially executes a rinsing of enzyme membrane and syringe, the injection of the sample, the trigger action of the measurement (on the YSI) and rinsing of the membrane at the end of the measure. The minimal sampling period is then 2.5 min. This cycle is triggered by a microcomputer which also manages measurement acquisition (analogue input of the microcomputer). A PLC was used to control measurements in order to consider the whole glucose sensor system as a measurement equipment and, more especially, independently of the microcomputer. Meanwhile, the same cycle can be managed by a microcomputer. Micmcomputing System During experiments on fermentation processes, the plant was linked to a PC/AT compatible microcomputer, the main characteristics of which are described in Table 4, and supporting a multi-tasking environment that allowed real-time identification, monitoring and control subprograms written in turbo pascal to be simultaneously active. A RTI 815 board from Analog Devices'
Table 4. Main characlerislics of PC/AT compatible micro-computer.
RAM Hard disk Processor Graphic display MSDOS
1 Mbytes 20 Mbytes 80286 EGA 3.2
Table 3. Cultures' operating conditions. Continuous culture of Saccharomyces
cerevislae Active volume (I) Temperature (~ Stirrer speed (rev/min) pH Influent glocuse (g/h) Dilution rate (h -1) Flow rate (I/h)
1.34 30 200 3.8 160 0.00-0.12 -
* Total volume: plant + cross-flow membrane.
8
Worm]ournalof Microbiologyand Biotechnology,Vol 8, 1992
Fed-batch culture of Saccharomyces
cerevisiae 6-16 30 250 3.8 160 0.0-2.0
Continuous culture of
Schtzosaccharomyces pombe 3* 30 400 3 500 0.0-2.0
Automated glucose sensor
Results and Discussion
analysis loop
Glucose Measurement Results Figure 2 shows automated and off-line analysis of the substrate concentration during a continuous culture of S. pombe with cell re-cycle by cross-flow membrane filtration (De Queiroz et aI. 1991). Automated measurements were carried out every 15 min for 150 h. Off-line measurements were carried out with a similar YSI analyser. This experiment considered different behaviours to be detected during a fermentation and from this it was concluded that: (a) the sensor behaves well, even after more than 600 injections (the enzyme membrane must then be changed which stops measurements for approximately 6 h), (b) the measurements are accurate for low concentrations (from 40 to 80 h) and for high concentrations (from 90 to 130 h) with a same injected volume, (c) it should also be added that the system detected the ruin of a steady state before the biologists. All the other figures presented in this paper correspond to continuous multistage or fed-batch cultures of S. cerevisiae for alcohol production, conducted in the aforementioned reactors. Figure 3 represents the evolution of automated and off-line glucose concentration measurements during a transient period of a multistage fermentation process (Vigi6 et al. 1991). In this case, the glucose sensor was part of an analysis loop which functioned for all the process; stages 2, 3 and 4 were selected sequentially by a flow multiplexer with a period of 20 min, thus setting a full sampling cycle for the process of 1 h. A similar behaviour of the sensor is stated in this case, with variations between the glucose
~{
y Figure 1. Control system of a fed-batch process: 1, fermenter; 2, feeding; 3, sugar analysis; 4, dilution loop; 5, programmable logic computer; 6, microcomputer; 7, log book printer.
family was used for the communications between the microcomputer and the process. The functions assumed by the software included control of the glucose sensor (PLC and glucose concentration addition), data acquisition (environment and state variables) and storage, fermentation monitoring through the editing of an experimental log book and graphic visualization and numerical application of the control algorithms and evaluation of the control signal. The whole monitoring system of a fed-batch process is shown in Figure 1.
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Figure 2. Automated and off-line measurements of the glucose concentration during a continuous culture of Schizosaccharornycespombe.
World Journal of Microbiology and Biotechnology, Vol 8, I992
9
L Queinnec et al.
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3. Automated (filled symbols) and off-line (open symbols) measurements of the glucose concentration during a transitory state of a multistage culture of Saccharomyces cerevisiae. Evolution in stages 2, 3 and 4. Control objective was the regulation of the substrate concentration in the process effluent (stage 4) by acting on feeding of stage 2. Figure
concentrations much more important. It appears that substrate concentration can be measured with an accuracy between 5 and 10% and even more accurately at low concentrations of carbohydrates. The precision of the first step is then 3 to 4%. Different types of faults can be detected. For example, a deterioration in the condition of the glucose sensor membrane can be induced by the ageing of the enzyme membrane, external temperature that is too high, or a decrease of the air pressure which becomes too low to move the pneumatic jacks. Bubbles due to the gas dissolved in the culture medium modify the sample volume which perverts the measurement. This problem is generally solved by use of a bubble extractor before the injection system. We have also noted a possible mechanical fragility due to severity and frequency of the injections. Although these faults rarely happen, the only real problem is that the enzyme membrane must be changed after more than 600 to 800 measurements. The problem of drift in calibration also has to be considered, but in practice only slight drifting has been detected and this is not a real drawback for the implementation of the automated analyser to control the process. The automatization of a YSI industrial analyser represents an effective good solution for automated glucose concentration measurement in a culture medium. It can be used not only for monitoring but especially for control of fermentation processes.
1{}
WorldJournalof Microbiologyand Biotechnology,Vol 8, 1992
Control of the Process
To control a process the variables, and in particular, in case of fermentation processes, the substrate concentration must be measured. In this way, the design of the glucose sensor represents an essential part of the control of continuous or fed-batch alcoholic fermentations of glucose by a yeast. The practical control objective is the regulation of the substrate concentration in the reactor. Theoretical and experimental studies concerning the application of modern adaptive techniques for the control of these processes from the measurement of the substrate concentration is receiving increasing attention. The control variables are the flow rate of flesh substrate in the case of fed-batch fermentation and dilution rate in the case of continuous fermentation. Figure 4a and b represents the evolution of the substrate concentration and of the control variable during a fed-batch fermentation. The set point is 60 g/1. A controller quotient type is applied to the bioreactor (Lakrori & Ch6ruy 1988; Queinnec I990). A similar controller is applied to a continuous fermentation process. Different set points are considered. Data from experiments are shown in Figure 5a and b (dilution rate and substrate concentration of the effluent). It is shown that from the measurement of the glucose concentration, it is very easy to implement controllers to regulate the substrate concentration from the flow rate or dilution rate.
Automated glucose sensor (a)
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Figure 4. (a) Automated (--) and off-line ([]) substrate concentration measurements during a fed-batch culture of Saccharomyces cerevisiae. Set point for the glucose concentration was 60 g/l. (b) Flow rate of nutrient during a fed-batch culture of Saccharomyces cerevisiae. Maximum flow rate was 2 I/h.
Conclusion Many different measurement systems have been developed for glucose determination (colorimetric methods, enzyme electrodes). In the last few years some authors have described on-line glucose biosensors (Chotani & Constantinides 1982; Lull et al. 1987; Schaffar & Wolfbeis 1990; Trojanowicz et al. 1990). All these systems have, however, used off-line measurements of glucose. The use of an automated injection system associated to a YSI analyser has
now been found to be useful for automated measurement of glucose. This sensor, in combination with a computering system is likely to be helpful in fermentation process control. The detectable glucose range covers the full concentration range to be expected in these fermentation processes. Moreover, it represents the basis for the application of modem automatic techniques for monitoring, control and optimization of these processes.
World Journal of Microbiology and Biotechnology, Vd 8, I992
11
I. Queinnec et al. (a)
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Figure 5. (a) Evolution of the automated substrate concentration during a continuous culture of Saccharomyces cerevisiae. Set point for the glucose concentration in the effluent changes from 4 to 2g/I at 9 h. (b) Evolution of the dilution rate during a continuous culture of
Saccharomyces cerevisiae.
References Bovee, J.P. 1981 Mod61isation, identification et premi6re approche de l'optimisation d'une unit6 pilote de fermentation alcoolique. Th6se de Docteur-lng6nieur de l'Universit6 Paul Sabatier, Toulouse, France. Chamilothoris, G., Renaud, P.Y., Sevely, Y. & Vigie, P. I988 Adaptive predictive control of a multistage fermentation process. International Journal of Control 48, 1089-1105. Chotani, G. & Constantinides, A. 1982 On-line glucose analyser for fermentation applications. Biotechnology & Bioengineering 24, 2743-3745. De Queiroz, H., Uribelarrea, J.L. & Pareilleux, A. I991 High cell density cultures of Schizosaccharomyces pombe in a cell-recycle
12
World Journal of Microbiology and Biofechnology, Vol 8, I992
reactor.
Growth kinetics and metabolic status.
Applied
Biochemistry and Biotechnology (in press). Lakrori, M. & Ch6ruy, A. 1988. A new nonlinear adaptive approach to automatic control of bioprocesses. In Computer Applications
in Fermentation Technology: Modelling and control of biotechnological processes, eds Fish, N.M., Fox, R.I. & Thomhill, N.F., pp. 341-348. London: Elsevier Applied Science. Luli, G.W., Schlasner, S.M., Ordaz, D.E., Mason, M. 8= Strohl, W.R. 1987 An automatic on-line glucose analyser for feed-back control of fedbatch growth of Escherichia coli. Biotechnology Techniques 1, 225-230. Queinnec, I. 1990 Automatisation d'un proc6d6 de fermentation semi-continu: Optimisation et Commande. Doctorat de l'Universit6 Paul Sabatier, Toulouse, France.
Automated glucose sensor Queinnec, I. & Pourciel, J.B. 1989 Real time control of a biotechnological process. In Thirty-ninth ISMM International Conference on Mini- and Micro-computers and their Applications, Zurich, 26-29 June, ed. Hamza, M.H., pp. 273-276. Anaheim, USA: Acta Press. Schaffar, B.P.H. & Wolfbeis, O.S. 1990 A fast responding fibre optic glucose biosensor based on an oxygen optrode. Biosensors & Bioelectronics 5, 137-I48.
Trojanowicz, M., Matuszewski, W. & Podsiadla, M. 1990 Enzyme entrapped polypyrrole modified electrode for flow-injection detection glucose. Biosensors & Bioelectronics 5, 149--156. Vigi4, P., Dahhou, B., Queinnec, I., Lakori, M., Cheruy, A. & Pourciel, J.B. 199I Control of substrate concentration in a continuous bioprocess. Bioprocess Engineering, in press. (Received in revised form 5 June 1991; accepted 7 June 1991)
Wodd]oumalof Microbiologt/and Biotechnoloooy,Vol S, 1992
13
An effective automated glucose sensor for fermentation monitoring and control I. Queinnec,* C. Destruhaut, J.B. Pourciel and G. Goma An industrial glucose analyser was partnered to an automated injection s y s t e m to evaluate glucose in the culture medium of a bioreactor. This sensor has been validated on continuous cultures of Schizosaccharomycespombe and continuous and fed-batch cultures of Saccharomycescerevisiae. In addition to the advantage of a more accurate process monitoring, the main interest of this sensor deals with the control of the substrate concentration to a prespecified reference signal. Several experiments have been carried out first to validate the sensor, then to control the process evolution. Key words: Glucose analysis, process control, process monitoring, sensor.
Processes occurring in microbial cultures are complex and difficult to optimize and are essentially controlled by a biological strategy (medium, strain improvement, environmental conditions, etc.). Optimization and control of fermentation processes, however, require advanced control and modelling theories, and lead to an increasing search for suitable sensors for fermentation process control. This paper describes the use of a glucose analyser associated with an automated injection system in the development of an instrument for monitoring and control of the substrate concentration in the reactor. The sensor is interfaced to a microcomputer or a programmable logic computer which manages the measurement cycle. This sensor finds in this form its first utilization in the monitoring of fermentation processes (Bovee 1981, Queinnec & Pourciel 1989). It is also used for control of continuous, multistage or fed-batch fermentation processes (Chamilothoris et al. 1988; Vigi6 et al. 1991). Fermentation has been carried out with Saccharomyces cerevisiae and Schizosaccharomyces pombe. Different measurement frequencies have been selected depending on the process dynamics and characteristics.
The authors are with DGBA, INSA, UA CNRS 544, Centre de Transfert en Biotechnologie et Microbiologie, 54 Avenue de Rangueil, F-31077 Toulouse c6dex, France. Fax: 33 61 55 96 73. I. Queinnec and J.B. Pourciel are also affiliated to Laboratoire d'Automatique et d'Analyse des Systemes du C.N.R.S., and I. Queinnec also to GRECO-SARTA, 7, Avenue du Colonel Roche, 31077 Toulouse c~dex, France. * Corresponding author.
Materials
and Methods
Strains and Media Two strains were used in this work: Saccharomyces cerevisiae and Schizosaccharomyces pombe, the former in fed-batch, continuous and multistage cultures, the latter in continuous culture with cell re-cycle by cross-flow membrane filtration. The culture medium of S. cerevisiae and S. pombe had the composition given in Table 1 and Table 2, respectively. Glucose of different concentrations was used as carbon source. Culture Apparatus and Experimental Conditions For continuous cultures, a 2-litre fermenter equipped with magnetic agitator, temperature and pH controls was used. A level sensor maintained the fermenter at constant volume. Fresh medium was supplied by a pump working at computer controlled rates. The multistage reactor comprised four reactors of this type in series.
Table 1. Culture medium of
Saccharomyces
cerevisiae. KH2PO4
(NH4)2SO4 Sodium glutamate Na2HPO4.12H20 CaCI2 MgSO4.7H20 ZnSO4.7H20 (NH4)2FeSO4.6H20 + vitamins
3 g/I 3 g/I 1 g/I 3 g/I 0.25 g/I 0.25 g/I 5 mg/I 1.5 mg/I
9 1992Rapid Communications of Oxford Ltd
WorldJournalof Microbiologyand Biotechnalogy,!/ol8, 1992
7
L Queinnec et al. Table 2. Culture medium of Schizosaccharomyces
pombe. NH4CI K2HPO4 K2SO4 MgSO4.7H20 NaCI CaCla.2H20 MnSO4.H20 CuSO4.5H20 COC1.6H20
(NH4)6M07024 ZnSO. KI H3B03 + vitamins
1.15 g/I 0.75 g/I 0.5 g/I 0.25 g/I 0.2 g/I 0.15 g/I 4 mg/I 1 mg/I 0.4 mg/I 1 mg/I 4 mg/I 1 mg/I 1 mg/I
Cell re-cycle of S. pombe was achieved with a cross-flow membrane filtration. For fed-batch cultures, a 20-1itre fermenter was used. Fresh medium was supplied to the reactor when and as required. The control variable was the flow rate of nutrient calculated from the glucose concentration measurement. Stirrer speed, temperature and pH were monitored and controlled as given in Table 3.
Glucose Analysis Off-line measurements. Samples from fermentation broth were clarified by centrifugation (11,000 x 8 for 5 min) and diluted in distilled water before injection by a syringe (25/~1) into the glucose analyser (Yellow Springs Instrument, Yellow Springs, Ohio, USA). This analyser had a range of 0 to 2 g of glucose/L A glucose standard of 2 g/l was used for calibration (2 g/l corresponds to 200 mV). Automated measures. The automated system was built by association of the glucose analyser with a syringe. A set of pneumatic jacks around the syringe ensures sampling of the culture medium from a loop disposed on the culture vessel, injection into the glucose analyser and rinsing of the syringe before the next measurement. By first selecting the distance of syringe travel, the working scale of glucose concentrations to be measured can be set. The reduction of the volume to be injected represents an artificial dilution compared to the 25/21 of an off-line measurement. The choice of a good dilution level allows substrate concentrations to be measured up to 200 g/1 with good accuracy (see later). The
calibration was made with a glucose standard whose concentration depended on the glucose setpoint of the experiment. For fed-batch culture of S. cerevisiae and continuous culture of S. pombe, the calibration was 400 mV for 40 g/1. For continuous culture of S. cerevisiae, the calibration was 400 mV for 4 g/1. The active volume of the syringe was 1.5/~1 in the first case and 6/~1 in the second one.
Programmable Logic Computer The previously mentioned sensor is handled by a programmable logic computer (PLC) whose cycle ensures rinsing of the enzyme membrane (analyser digital input--CLEAR), rinsing of the syringe and injection of the sample (action on jacks) and control of the glucose measurement (digital input of the analyser). The duration of a measurement cycle depends on the enzyme analyser. As previously stated, the WAIT durations are approximately 60 s for the measurement and 40 s for the rinsing of the membrane. Moreover the PLC cycle sequentially executes a rinsing of enzyme membrane and syringe, the injection of the sample, the trigger action of the measurement (on the YSI) and rinsing of the membrane at the end of the measure. The minimal sampling period is then 2.5 min. This cycle is triggered by a microcomputer which also manages measurement acquisition (analogue input of the microcomputer). A PLC was used to control measurements in order to consider the whole glucose sensor system as a measurement equipment and, more especially, independently of the microcomputer. Meanwhile, the same cycle can be managed by a microcomputer. Micmcomputing System During experiments on fermentation processes, the plant was linked to a PC/AT compatible microcomputer, the main characteristics of which are described in Table 4, and supporting a multi-tasking environment that allowed real-time identification, monitoring and control subprograms written in turbo pascal to be simultaneously active. A RTI 815 board from Analog Devices'
Table 4. Main characlerislics of PC/AT compatible micro-computer.
RAM Hard disk Processor Graphic display MSDOS
1 Mbytes 20 Mbytes 80286 EGA 3.2
Table 3. Cultures' operating conditions. Continuous culture of Saccharomyces
cerevislae Active volume (I) Temperature (~ Stirrer speed (rev/min) pH Influent glocuse (g/h) Dilution rate (h -1) Flow rate (I/h)
1.34 30 200 3.8 160 0.00-0.12 -
* Total volume: plant + cross-flow membrane.
8
Worm]ournalof Microbiologyand Biotechnology,Vol 8, 1992
Fed-batch culture of Saccharomyces
cerevisiae 6-16 30 250 3.8 160 0.0-2.0
Continuous culture of
Schtzosaccharomyces pombe 3* 30 400 3 500 0.0-2.0
Automated glucose sensor
Results and Discussion
analysis loop
Glucose Measurement Results Figure 2 shows automated and off-line analysis of the substrate concentration during a continuous culture of S. pombe with cell re-cycle by cross-flow membrane filtration (De Queiroz et aI. 1991). Automated measurements were carried out every 15 min for 150 h. Off-line measurements were carried out with a similar YSI analyser. This experiment considered different behaviours to be detected during a fermentation and from this it was concluded that: (a) the sensor behaves well, even after more than 600 injections (the enzyme membrane must then be changed which stops measurements for approximately 6 h), (b) the measurements are accurate for low concentrations (from 40 to 80 h) and for high concentrations (from 90 to 130 h) with a same injected volume, (c) it should also be added that the system detected the ruin of a steady state before the biologists. All the other figures presented in this paper correspond to continuous multistage or fed-batch cultures of S. cerevisiae for alcohol production, conducted in the aforementioned reactors. Figure 3 represents the evolution of automated and off-line glucose concentration measurements during a transient period of a multistage fermentation process (Vigi6 et al. 1991). In this case, the glucose sensor was part of an analysis loop which functioned for all the process; stages 2, 3 and 4 were selected sequentially by a flow multiplexer with a period of 20 min, thus setting a full sampling cycle for the process of 1 h. A similar behaviour of the sensor is stated in this case, with variations between the glucose
~{
y Figure 1. Control system of a fed-batch process: 1, fermenter; 2, feeding; 3, sugar analysis; 4, dilution loop; 5, programmable logic computer; 6, microcomputer; 7, log book printer.
family was used for the communications between the microcomputer and the process. The functions assumed by the software included control of the glucose sensor (PLC and glucose concentration addition), data acquisition (environment and state variables) and storage, fermentation monitoring through the editing of an experimental log book and graphic visualization and numerical application of the control algorithms and evaluation of the control signal. The whole monitoring system of a fed-batch process is shown in Figure 1.
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Figure 2. Automated and off-line measurements of the glucose concentration during a continuous culture of Schizosaccharornycespombe.
World Journal of Microbiology and Biotechnology, Vol 8, I992
9
L Queinnec et al.
s (M)
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3. Automated (filled symbols) and off-line (open symbols) measurements of the glucose concentration during a transitory state of a multistage culture of Saccharomyces cerevisiae. Evolution in stages 2, 3 and 4. Control objective was the regulation of the substrate concentration in the process effluent (stage 4) by acting on feeding of stage 2. Figure
concentrations much more important. It appears that substrate concentration can be measured with an accuracy between 5 and 10% and even more accurately at low concentrations of carbohydrates. The precision of the first step is then 3 to 4%. Different types of faults can be detected. For example, a deterioration in the condition of the glucose sensor membrane can be induced by the ageing of the enzyme membrane, external temperature that is too high, or a decrease of the air pressure which becomes too low to move the pneumatic jacks. Bubbles due to the gas dissolved in the culture medium modify the sample volume which perverts the measurement. This problem is generally solved by use of a bubble extractor before the injection system. We have also noted a possible mechanical fragility due to severity and frequency of the injections. Although these faults rarely happen, the only real problem is that the enzyme membrane must be changed after more than 600 to 800 measurements. The problem of drift in calibration also has to be considered, but in practice only slight drifting has been detected and this is not a real drawback for the implementation of the automated analyser to control the process. The automatization of a YSI industrial analyser represents an effective good solution for automated glucose concentration measurement in a culture medium. It can be used not only for monitoring but especially for control of fermentation processes.
1{}
WorldJournalof Microbiologyand Biotechnology,Vol 8, 1992
Control of the Process
To control a process the variables, and in particular, in case of fermentation processes, the substrate concentration must be measured. In this way, the design of the glucose sensor represents an essential part of the control of continuous or fed-batch alcoholic fermentations of glucose by a yeast. The practical control objective is the regulation of the substrate concentration in the reactor. Theoretical and experimental studies concerning the application of modern adaptive techniques for the control of these processes from the measurement of the substrate concentration is receiving increasing attention. The control variables are the flow rate of flesh substrate in the case of fed-batch fermentation and dilution rate in the case of continuous fermentation. Figure 4a and b represents the evolution of the substrate concentration and of the control variable during a fed-batch fermentation. The set point is 60 g/1. A controller quotient type is applied to the bioreactor (Lakrori & Ch6ruy 1988; Queinnec I990). A similar controller is applied to a continuous fermentation process. Different set points are considered. Data from experiments are shown in Figure 5a and b (dilution rate and substrate concentration of the effluent). It is shown that from the measurement of the glucose concentration, it is very easy to implement controllers to regulate the substrate concentration from the flow rate or dilution rate.
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Figure 4. (a) Automated (--) and off-line ([]) substrate concentration measurements during a fed-batch culture of Saccharomyces cerevisiae. Set point for the glucose concentration was 60 g/l. (b) Flow rate of nutrient during a fed-batch culture of Saccharomyces cerevisiae. Maximum flow rate was 2 I/h.
Conclusion Many different measurement systems have been developed for glucose determination (colorimetric methods, enzyme electrodes). In the last few years some authors have described on-line glucose biosensors (Chotani & Constantinides 1982; Lull et al. 1987; Schaffar & Wolfbeis 1990; Trojanowicz et al. 1990). All these systems have, however, used off-line measurements of glucose. The use of an automated injection system associated to a YSI analyser has
now been found to be useful for automated measurement of glucose. This sensor, in combination with a computering system is likely to be helpful in fermentation process control. The detectable glucose range covers the full concentration range to be expected in these fermentation processes. Moreover, it represents the basis for the application of modem automatic techniques for monitoring, control and optimization of these processes.
World Journal of Microbiology and Biotechnology, Vd 8, I992
11
I. Queinnec et al. (a)
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Figure 5. (a) Evolution of the automated substrate concentration during a continuous culture of Saccharomyces cerevisiae. Set point for the glucose concentration in the effluent changes from 4 to 2g/I at 9 h. (b) Evolution of the dilution rate during a continuous culture of
Saccharomyces cerevisiae.
References Bovee, J.P. 1981 Mod61isation, identification et premi6re approche de l'optimisation d'une unit6 pilote de fermentation alcoolique. Th6se de Docteur-lng6nieur de l'Universit6 Paul Sabatier, Toulouse, France. Chamilothoris, G., Renaud, P.Y., Sevely, Y. & Vigie, P. I988 Adaptive predictive control of a multistage fermentation process. International Journal of Control 48, 1089-1105. Chotani, G. & Constantinides, A. 1982 On-line glucose analyser for fermentation applications. Biotechnology & Bioengineering 24, 2743-3745. De Queiroz, H., Uribelarrea, J.L. & Pareilleux, A. I991 High cell density cultures of Schizosaccharomyces pombe in a cell-recycle
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World Journal of Microbiology and Biofechnology, Vol 8, I992
reactor.
Growth kinetics and metabolic status.
Applied
Biochemistry and Biotechnology (in press). Lakrori, M. & Ch6ruy, A. 1988. A new nonlinear adaptive approach to automatic control of bioprocesses. In Computer Applications
in Fermentation Technology: Modelling and control of biotechnological processes, eds Fish, N.M., Fox, R.I. & Thomhill, N.F., pp. 341-348. London: Elsevier Applied Science. Luli, G.W., Schlasner, S.M., Ordaz, D.E., Mason, M. 8= Strohl, W.R. 1987 An automatic on-line glucose analyser for feed-back control of fedbatch growth of Escherichia coli. Biotechnology Techniques 1, 225-230. Queinnec, I. 1990 Automatisation d'un proc6d6 de fermentation semi-continu: Optimisation et Commande. Doctorat de l'Universit6 Paul Sabatier, Toulouse, France.
Automated glucose sensor Queinnec, I. & Pourciel, J.B. 1989 Real time control of a biotechnological process. In Thirty-ninth ISMM International Conference on Mini- and Micro-computers and their Applications, Zurich, 26-29 June, ed. Hamza, M.H., pp. 273-276. Anaheim, USA: Acta Press. Schaffar, B.P.H. & Wolfbeis, O.S. 1990 A fast responding fibre optic glucose biosensor based on an oxygen optrode. Biosensors & Bioelectronics 5, 137-I48.
Trojanowicz, M., Matuszewski, W. & Podsiadla, M. 1990 Enzyme entrapped polypyrrole modified electrode for flow-injection detection glucose. Biosensors & Bioelectronics 5, 149--156. Vigi4, P., Dahhou, B., Queinnec, I., Lakori, M., Cheruy, A. & Pourciel, J.B. 199I Control of substrate concentration in a continuous bioprocess. Bioprocess Engineering, in press. (Received in revised form 5 June 1991; accepted 7 June 1991)
Wodd]oumalof Microbiologt/and Biotechnoloooy,Vol S, 1992
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