A predictive and feedback control algorithm maintains a constant glucose concentration in fed-batch fermentations

A combined predictive and feedback control algorithm based on measurements of the concentration of glucose on-line has been developed to control fed-batch fermentations of Escherichia coli. The predictive control algorithm was based on the on-line calculation of glucose demand by the culture and plotting a linear regression to the next datum point to obtain a predicted glucose demand. This provided a predictive "coarse" control for the glucose-based nutrient feed. A direct feedback control using a proportional controller, based on glucose measurements every 2 min, fine-tuned the feed rate. These combined control schemes were used to maintain glucose concentrations in fed-batch fermentations as tight as 0.49 +/- 0.04 g/liter during growth of E. coli to high cell densities.     

Use of multi-staining flow cytometry to characterise the physiological state of Escherichia coli W3110 in high cell density fed-batch cultures.

High cell density fed-batch fermentations of Escherichia coli W3110 have been carried out at specific growth rates of less than 0.3 h-1, to investigate the effect of glucose limitation on the physiological state of individual cells. After an initial exponential batch phase, the feed rate was held constant and a final dry cell weight of approximately 50 g per litre was achieved. The fermentations were monitored by mass spectrometry whilst measurements of pH, DOC, CFU/mL, TCN, OD500nm and residual glucose concentrations were made. Satisfactory and reproducible results were obtained. Flow cytometric analysis of cells in broth samples, based on either of two multi-staining protocols, revealed a progressive change in cell physiological state throughout the course of the fermentations. From these measurements it was concluded that the loss in reproductive viability towards the end of the fed-batch process is due to cell death and not due to the formation of a "viable but nonculturable state" as had previously been reported. Since the presence of a high proportion of dead or dying cells at any time during a fermentation has a detrimental effect on the synthesis of any desired product it is proposed that an on-line flow cytometric analysis and control strategy could be used as a means of increasing overall process efficiency.     

A probing feeding strategy for Escherichia coli cultures

A strain-independent feeding strategy for fed-batch cultures of Escherichia coli is presented. By superimposing short pulses in the glucose feed rate, on-line detection of acetate formation can be made using a standard dissolved oxygen sensor. A simple feedback algorithm is then used to adjust the feed rate to avoid acetate formation. The feasibility of the strategy is demonstrated by both simulation and experiments.     

Determination of the maximum specific uptake capacities for glucose and oxygen in glucose-limited fed-batch cultivations of Escherichia coli

A simple pulse-based method for the determination of the maximum uptake capacities for glucose and oxygen in glucose limited cultivations of E. coli is presented. The method does not depend on the time-consuming analysis of glucose or acetate, and therefore can be used to control the feed rate in glucose limited cultivations, such as fed-batch processes. The application of this method in fed-batch processes of E. coli showed that the uptake capacity for neither glucose nor oxygen is a constant parameter, as often is assumed in fed-batch models. The glucose uptake capacity decreased significantly when the specific growth rate decreased below 0.15 h(-1) and fell to about 0.6 mmol g(-1) h(-1) (mmol per g cell dry weight and hour) at the end of fed-batch fermentations, where specific growth rate was approximately 0.02 h(-1). The oxygen uptake capacity started to decrease somewhat earlier when specific growth rate declined below 0.25 h(-1) and was 5 mmol g(-1) h(-1) at the end of the fermentations. The behavior of both uptake systems is integrated in a dynamic model which allows a better fitting of experimental values for glucose in fed-batch processes in comparison to generally used unstructured kinetic models.     

Glucose-stat, a glucose-controlled continuous culture

A predictive and feedback proportional control algorithm, developed for fed-batch fermentations and described in a companion paper (G. L. Kleman, J. J. Chalmers, G. W. Luli, and W. R. Strohl, Appl. Environ. Microbiol. 57:910-917, 1991), was used in this work to control a continuous culture on the basis of the soluble-glucose concentration (called the glucose-stat). This glucose-controlled continuous-culture system was found to reach and maintain steady state for 11 to 24 residence times when four different background glucose concentrations (0.27, 0.50, 0.7, and 1.5 g/liter) were used. The predictive-plus-feedback control system yielded very tight control of the continuous nutristat cultures; glucose concentrations were maintained at the set points with less than 0.003 standard error. Acetate production by Escherichia coli B in glucose-stats was found not to be correlated with the level of steady-state soluble-glucose concentration.     

Glucose-stat, a glucose-controlled continuous culture.

A predictive and feedback proportional control algorithm, developed for fed-batch fermentations and described in a companion paper (G. L. Kleman, J. J. Chalmers, G. W. Luli, and W. R. Strohl, Appl. Environ. Microbiol. 57:910-917, 1991), was used in this work to control a continuous culture on the basis of the soluble-glucose concentration (called the glucose-stat). This glucose-controlled continuous-culture system was found to reach and maintain steady state for 11 to 24 residence times when four different background glucose concentrations (0.27, 0.50, 0.7, and 1.5 g/liter) were used. The predictive-plus-feedback control system yielded very tight control of the continuous nutristat cultures; glucose concentrations were maintained at the set points with less than 0.003 standard error. Acetate production by Escherichia coli B in glucose-stats was found not to be correlated with the level of steady-state soluble-glucose concentration.     

Closed-loop control of fed-batch cultures of recombinant Escherichia coli using on-line HPLC

This article describes a fully automated system for the on-line monitoring and closed-loop control of a fed-batch fermentation of recombinant Escherichia coli, and presents two case studies of its used in limiting production of unwanted byproducts such as acetic in fed-batch fermentations. The system had two components. The first components, on-line monitoring, comprised an aseptic sampling device, a microcentrifuge, and HPLC System. These instruments removed a Sample from a fermentor, spun it at high speed to separate solid and liquid components, and then automatically injected the supernatant onto an HPLC column for analysis. The second component consisted of control algorithms programmed using the LabView visual programming environment in a control computer that was linked via a remote components were linked so that results from the on-line HPLC were captured and used by the control algorithm was designed to demonstrate coarse feedback control to confirm the operability of the controller. The second case study showed how the system could be used in a more sophisticated feedings strategy providing fine control and limiting acetate concentration to a low level throughout the fermentation. (c) 1994 John Wiley & Sons, Inc.     

Control of nitrate concentration in fermentations of Corynebacterium glutamicum

A nitrate control system has been devised for the maintenance of stable nitrate concentrations throughout fed-batch fermentations of Corynebacterium glutamicum. The feedback control system was based on the use of a nitrate-ion-selective electrode to directly monitor the nitrate levels in the fermentor and an automatic controller to activate a nitrate feed pump. The electrode which was used for controlling the nitrate level was stable through-out the fermentation period. The apparent maximum specific growth rate, biomass production, protein production, biomass yields on glucose and nitrate, and amino acid production were all optimal at approximately 50mM nitrate.     

Two-dimensional fluorescence spectroscopy: a novel approach for controlling fed-batch cultivations

In industrial fed-batch cultivations it is often necessary to control substrate concentrations at a low level to prevent the production of overflow metabolites and thus optimize the biomass yield. A new method for on-line monitoring and fed-batch control based on fluorescence measurements has been developed. Via instantaneous in situ measurements and multivariate data analysis a chemometric model has been established, which enables the rapid detection of ethanol production at aerobic Saccharomyces cerevisiae fed-batch cultivations. The glucose feed rate is controlled by predicting the metabolic state directly from the fluorescence intensities. Thus, ethanol production could be avoided completely while increasing the biomass yield accordingly. The robust instrumentation is suitable for industrial applications.     

An on-line adaptive glucose feeding system incorporating patterns recognition for glucose concentration control in glutamate fermentations

In glutamate fermentation, intermittent feeding is the most widely used glucose feed strategy. This feeding strategy causes severe fluctuations of glucose concentration and osmotic pressure in fermentation broth, which deteriorates the viability of the cell and reduces glutamate production in turn. In order to maintain glucose concentration at stable and constant levels, an on-line prediction and feedback control system based an empiric mass balance model was developed. However, the control system did not work properly and sometimes glucose concentration could even decline to 0 level (glucose exhaustion), as the model parameter varies in different runs. As a result, a novel model-based adaptive feedback control system incoporating with an artificial neural network (ANN) based pattern reconition unit for on-line diagnosizing the fault of glucose exhaustion was proposed and applied for glutamate fermentation. This adaptive control system could accurately detect glucose exhaustion when it occurs, and then immediately updates the control parameter based on some pre-defined rule. With the proposed control system, glucose was automatically fed, and its concentration could be maintained at desired levels constantly. As a result, glutamate concentration was 17 ~ 30% higher than that of the traditional fermentations using the intermittent glucose feed strategy.     

On-line estimation of the specific growth rate based on viable biomass measurements: experimental validation

The application of model based control techniques to biotechnological processes is often hampered due to the lack of reliable on-line sensors. This problem can be tackled by the application of software sensors, in which the available hardware measurements are combined with the model equations. The resulting estimates serve as additional measurements useful for process monitoring and control. In this paper, an observer based estimator for the specific growth rate based on on-line viable biomass measurements is studied. Several fed-batch experiments with baker's yeast in a stirred tank bioreactor illustrate the design, tuning, and implementation from a practical point of view. The main contributions of this paper are to illustrate (i) the implementation and validation of the presented algorithm in real-time, (ii) the use of an advanced on-line biomass measurement, and (iii) the design and tuning of the algorithm from a practical point of view. Real-time knowledge of the specific growth rate is important because it yields information on the viability of the cells and it can be used in real-time feedback control algorithms.     

Protease production by Bacillus subtilis in oxygen-controlled,glucose fed-batch fermentations

Summary An open-loop, on-off control system using the dissolved oxygen level to control a glucose feed was used in a study of growth and production of protease by Bacillus subtilis CNIB 8054. With this system, both glucose and oxygen were controlled at low concentrations. In batch fermentations, protease activity in the fermentation broth was maximum when growth had stopped. During oxygen-controlled, glucose fed-batch fermentations, growth and the production of protease activity continued during glucose feeding. Oxygen-controlled, glucose fed-batch fermentations produced more protease activity than batch fermentations, depending upon the set point for dissolved oxygen. These results indicate that control of glucose and oxygen concentrations can result in improvements in protease production.     

On-line detection of acetate formation in Escherichia coli cultures using dissolved oxygen responses to feed transients.

Recombinant protein production in Escherichia coli can be significantly reduced by acetate accumulation. It is demonstrated that acetate production can be detected on-line with a standard dissolved oxygen sensor by superimposing short pulses to the substrate feed rate. Assuming that acetate formation is linked to a respiratory limitation, a model for dissolved oxygen responses to transients in substrate feed rate is derived. The model predicts a clear change in the character of the transient response when acetate formation starts. The predicted effect was verified in fed-batch cultivations of E. coli TOPP1 and E. coli BL21(DE3), both before and after induction of recombinant protein production. It was also observed that the critical specific glucose uptake rate, at which acetate formation starts, was significantly decreased after induction. On-line detection of acetate formation with a standard sensor opens up new possibilities for feedback control of substrate feeding.     

Control of continuous fed-batch fermentation process using neural network based model predictive controller

Cell growth and metabolite production greatly depend on the feeding of the nutrients in fed-batch fermentations. A strategy for controlling the glucose feed rate in fed-batch baker’s yeast fermentation and a novel controller was studied. The difference between the specific carbon dioxide evolution rate and oxygen uptake rate (Q _c − Q _o) was used as controller variable. The controller evaluated was neural network based model predictive controller and optimizer. The performance of the controller was evaluated by the set point tracking. Results showed good performance of the controller.     

Evaluation of spectrofluorometry as a tool for estimation in fed-batch fermentations

Native culture fluorescence was investigated as an additional source of information for predicting biomass and glucose concentrations in a fed-batch fermentation of Alcaligenes eutrophus. Partial least squares (PLS) regression and a feed forward neural network (FFNN) coupled with principle component analysis (PCA) were each used to model the kinetics of the fermentation. Data from three fermentations was combined to form a training set for model calibration and data from a fourth fermentation was used as the testing set. The fluorescent soft-sensors were compared with a previously developed feed forward neural network soft-sensor model which used oxygen uptake rate (OUR), carbon dioxide evolution rate (CER), aeration rate, feed rate, and fermentor volume to estimate biomass and glucose concentrations. The best model performance for predicting both biomass and glucose concentrations was achieved using the native fluorescence-based models. Real data predictions of the biomass concentration in the testing set were obtained using both the PLS and FFNN PCA modeling utilizing fluorescence measurements plus the rate of change of the fluorescence measurements. Accurate predictions of the glucose concentration in the testing set were obtained using the FFNN PCA modeling technique utilizing the rate of change of the fluorescence measurements. Substrate exhaustion was indicated qualitatively by a first-order PLS model utilizing the rate of change of fluorescence measurements. These results indicate that native culture fluorescence shows promise for providing additional valuable information to enhance predictive modeling which cannot be extracted from other easily acquired measurements.     

On-line monitoring and controlling system for fermentation processes

A personal computer-based on-line monitoring and controlling system was developed for the fermentation of microorganism. The on-line HPLC system for the analysis of glucose and ethanol in the fermentation broth was connected to the fermenter via an auto-sampling equipment, which could perform the pipetting, filtration and dilution of the sample and final injection onto the HPLC through automation based on a programmed procedure. The A/D and D/A interfaces were equipped in order to process the signals from electrodes and from the detector of HPLC, and to direct the feed pumps, the motor of stirrer and gas flow-rate controller. The software that supervised the control of the stirring speed, gas flow-rate, pH value, feed flow-rate of medium, and the on-line measurement of glucose and ethanol concentration was programmed by using Microsoft Visual Basic under Microsoft Windows. The signal for chromatographic peaks from on-line HPLC was well captured and processed using an RC filter and a smoothing algorithm. This monitoring and control system was demonstrated to be effective in the ethanol fermentation of Zymomonas mobilis operated in both batch and fed-batch modes. In addition to substrate and product concentrations determined by on-line HPLC, the biomass concentration in Z. mobilis fermentation could also be on-line estimated by using the pH control and an implemented software sensor. The substrate concentration profile in the fed-back fermentation followed well the set point profile due to the fed-back action of feed flow-rate control.     

Influence of bioreactor scale and complex medium on probing control of glucose feeding in cultivations of recombinant strains of Escherichia coli

The objective of this work was to evaluate the performance of a feedback glucose control strategy (the probing strategy) in production relevant bioreactors with complex and mineral media. Experimental results from fed-batch cultivations with two recombinant Escherichia coli constructs expressing two different human therapeutic proteins were used to assess the performance and limitations of the glucose probing technique. Even though the performance of the probing strategy was affected by scale and complex media, this methodology rapidly identified a glucose feed protocol similar to an experimentally derived feed regime. This methodology may serve as a powerful tool for industrial process development and in optimization of glucose feed regimes when transferring process technology from one bioreactor system to another.     

Process control for enhanced L-phenylalanine production using different recombinant Escherichia coli strains

A novel fed-batch approach for the production of L-phenylalanine (L-Phe) with recombinant E. coli is presented concerning the on-line control of the key fermentation parameters glucose and tyrosine. Two different production strains possessing either the tyrosine feedback resistant aroF(fbr) (encoding tyrosine feedback resistant DAHP-synthase (3-desoxy-D-arabino-heptusonate-7-phosphate)) or the wild-type aroF(wt) were used as model systems to elucidate the necessity of finding an individual process optimum for each genotype. With the aid of tyrosine control, wild-type aroF(wt) could be used for L-Phe production achieving higher final L-Phe titers (34 g/L) than the aroF(fbr) strain (28 g/L) and providing higher DAHP-synthase activities. With on-line glucose control, an optimum glucose concentration of 5 g/L could be identified that allowed a sufficient carbon supply for L-Phe production while at the same time an overflow metabolism leading to acetate by-product formation was avoided. The process approach is suitable for other production strains not only in lab-scale but also in pilot-scale bioreactors.     

Advanced control of glutathione fermentation process

A study was performed to understand the fermentation process for production of glutathione fermentation (GSH) with an improved strain of baker's yeast. Simultaneous utilization of sugar and ethanol has been found to be a key factor in the industrial process to produce GSH using Saccharomyces cerevisiae KY6186. Based on this observation, the optimal sugar feed profile for the fed-batch operation has been determined. A feedforward/feedback control system was developed to regulate the sugar feed rate so as to maximize GSH production yields. Using the feedforward/feedback control system and the on-line data of oxygen and ethanol concentration in exhaust gas, the successful scaleup to the production level was accomplished. An average of 40% improvement of glutathione production compared to a conventionally programmed control of exponential fed-batch operation was found in the new process. (c) 1992 John Wiley & Sons, Inc.     

Evaluation of the GFP signal and its aptitude for novel on-line monitoring strategies of recombinant fermentation processes

A high number of economically important recombinant proteins are produced in Escherichia coli based host/vector systems. The major obstacle for improving current processes is a lack of appropriate on-line in situ methods for the monitoring of metabolic burden and critical state variables. Here, a pre-evaluation of the reporter green fluorescent protein (GFP) was undertaken to assess its use as a reporter of stress associated promoter regulation. The investigation of GFP and its blue fluorescent variant BFP was done in model fermentations using E. coli HMS 174(DE3)/pET11 aGFPmut3.1 and E. coli HMS174(DE3)/pET1aBFP host/vector systems cultured in fed-batch and chemostat regime. Our results prove the suitability of the fluorescent reporter proteins for the design of new strategies of on-line bioprocess monitoring. GFPmut3.1 variant can be detected after a short lag-phase of only 10 min, it shows a high fluorescence yield in relation to the amount of reporter protein, a good signal to noise ratio and a low detection limit. The fluorescence-signal and the amount of fluorescent protein, determined by ELISA, showed a close correlation in all fermentations performed. A combination of reporter technology with state of the art sensors helps to develop new strategies for efficient on-line monitoring needed for industrial process optimisation. The development of efficient monitoring will contribute to advanced control of recombinant protein production and accelerate the development of optimised production processes.