Bioprocess control from a multivariate process trajectory

A multivariate bioprocess control approach, capable of tracking a pre-set process trajectory correlated to the biomass or product concentration in the bioprocess is described. The trajectory was either a latent variable derived from multivariate statistical process monitoring (MSPC) based on partial least squares (PLS) modeling, or the absolute value of the process variable. In the control algorithm the substrate feed pump rate was calculated from on-line analyzer data. The only parameters needed were the substrate feed concentration and the substrate yield of the growth-limiting substrate. On-line near-infrared spectroscopy data were used to demonstrate the performance of the control algorithm on an Escherichia coli fed-batch cultivation for tryptophan production. The controller showed good ability to track a defined biomass trajectory during varying process dynamics. The robustness of the control was high, despite significant external disturbances on the cultivation and control parameters.     

On-line HPLC-measurement and control of substrate in a continuously operated biological tank reactor

This paper presents a concept for the measurement and control of the substrate concentration in a continuously operated biological tank reactor (CBTR). The concept was successfully applied in the degradation of Naphthalenesulfonic acid by the culture Pseudomonas testosteroni. The measurement of the substrate concentration was carried out by means of an automated HPLC (high performance liquid chromatography) analysis of the contents of the reactor. The methods of cyclic sampling, sample filtration and evaluation of the chromatograms during the process control are explained. In addition the characteristics of the measuring system (linearity of measurement, noise disturbance data, sampling rates, transient behavior) are described in detail. The feed-back control of the substrate concentration was carried out by variations in the dilution rate with a constant substrate feed concentration. The control results were achieved with a controller which was specially developed to be used in connection with automated cyclic analysis processes. The control algorithm is explained and the conditions necessary for maintaining stability of the control loop are given.     

Adaptive predictive control of a multistage fermentation process

Theoretical and experimental studies concerning the application of modern adaptive techniques for the control of continuous alcoholic fermentation of glucose by a yeast strain conducted in a multistage reactor are reported. The practical control objective was the regulation of the substrate concentration in the process effluent. Mathematical expressions for the control scheme are given, with the underlying control engineering argumentation. Suitable feeding of sugar for stage 2 was controlled to minimize the effluent sugar concentration.     

Estimation and control techniques for continuous culture fermentation processes

This article presents results obtained when some modern estimation and control techniques are applied to a simulated fermentation process. The control structure uses a particular observer of the substrate concentration and assumes the biomass concentration is measurable. The overall structure has been tested for both external and parametric disturbances, with very good results.     

Metabolic control analysis. The effects of high enzyme concentrations

Differing views have been given in the literature as to whether the presence in a pathway of an enzyme at a concentration comparable to that of its substrate affects the values of control coefficients and the theorems of metabolic control analysis. Here we argue in favour of one of those views: that there is no effect unless the enzyme sequesters a substrate that contains a conserved moiety. In this particular case, we derive both a general criterion for estimating whether such an effect will be of a significant magnitude, and equations for determining the changes in the flux control coefficients. The nature of the phenomenom and the application of the equations are illustrated with a numerical simulation.     

On-line state estimation and control in glutamic acid production

An automatic computer control system for glutamic acid production has been developed. A method based on empirical reaction subspace and singular value decomposition was presented for on-line estimation of cell concentration, glutamic acid, and total sugar. The only on-line measured state variable was the oxygen uptake rate. The estimated cell concentration was used as an index for the addition of penicillin. The estimated total sugar concentration was used for system identification. Adaptive control was then applied to manipulate the substrate feeding rate. The total sugar concentration was maintained at a given value during the period of fed-batch culture for glutamic acid production.     

Dynamics and control of continuous microbial propagators to subject substrate inhibition

Inhibitory substrate levels are common in industrial fermentations and in biological waste-water treatment of many industrial wastes. Continuous microbial cultures are unstable to certain disturbances, such as shock loading by inhibitory substrates. Two feedback proportional control strategies are analyzed and compared for a simple model culture assumed represent able by the culture concentrations of biomass and a single rate-limiting and growth-limiting nutrient (substrate). One control strategy, the well known turbidostat, consists of adjusting culture holding time (e.g., by flow rate adjustment) in response to deviations in turbidity or some other measure of culture biomass concentration. The other control strategy is to adjust holding time in response to deviations in limiting nutrient concentrations in the culture. This second control strategy, termed the nutristat, can be superior to the turbidostat in many applications. The sign and magnitude of the dimensionless group {(X /YD )[dμ/dS]s }, is shown to be an important determinant, in the behavior of the open loop and the two closed loop processes. This characteristic group is positive when the specific growth rate is increased by increases in the nutrient concentration, zero when the growth rate is unaffected by the nutrient concentration, and negative in the presence of nutrient or substrate inhibition. The effects of process modifications and of modeling assumptions on the control of the process are discussed and more sophisticated control schemes are also proposed.     

Knowledge based control applied to fixed bed pulsed bioreactors

In this work, an expert system was developed and applied for on-line control and supervision of ethanolic fermentation by immobilized Saccharomyces cerevisiae in a fixed-bed pulsed bioreactor of 1.2 l of working volume. A number of experiments with different substrate concentrations (75, 100, 150 and 200 g/l) and hydraulic residence times (2.4, 1.2 and 0.8 h) were carried out. Knowledge-based computer-aided supervision of this process involves accurate on-line measurement of the relevant process variables (temperature, pH, flow rate, carbon dioxide production, etc.). Carbon dioxide production was used for the estimation of the ethanol productivity. The analysis of the measured data allowed to detect states or trends that may be indicative of process or system failures, providing advices and/or alarms. The results showed the reliability of the control system. In previous works, it was proven that pulsing the feed stream highly improves the productivity of fermentation processes carried out in fixed-bed bioreactors [14, 15, 16]. The amplitude and frequency of the pulsation, which is a key factor in the performance of a pulsed feed bioreactor [13], was selected by the control system by using an algorithm allowing the ethanol productivity to be optimized. The pulsation frequency which maximizes the ethanol productivity, presents a high dependency on the hydraulic residence time and the feeding substrate concentration. When increasing the substrate concentration the optimum pulsation frequency also increases; when increasing the hydraulic residence time the optimum pulsation frequency decreases.     

Model for the feedback control system of bacterial growth. II. Growth in continuous culture

A mathematical model is developed that describes substrate limited bacterial growth in a continuous culture and that is based upon the conceptual framework elaborated in a previous paper for describing the feedback control system of cell growth [S. Bleecken, (1988). J. theor. Biol. 133, 37.] Central to the theory are the ideas that the limiting substrate is converted into low molecular weight building blocks of macromolecular synthesis which again are converted into biomass (RNA and protein) and that the rates of RNA and protein synthesis are controlled by the intracellular concentration of building blocks. It is shown that a continuous culture can be simulated by two interconnected feedback control systems the actuating signals of which are limiting substrate concentration and the intracellular concentration of building blocks, respectively. Three types of steady-states are found to appear in a continuous culture, besides the well-known stable steady-state of the whole culture there exist two batchlike steady-states of the biotic part of the culture which are metastable. The model is used to analyse the steady-states and their stability properties as well as the dynamic responses of biomass, RNA, protein, building block and substrate concentrations to changes in environmental conditions. Especially the inoculation of a continuous culture and the effects of step changes in dilution rate, inlet substrate concentration and growth temperature are studied in detail. Relations between the growth behaviour of a single cell and that of a continuous culture are derived. The RNA to protein ratio is introduced as a rough measure of the physiological state of cells and it is shown that a cell reacts to environmental changes with a simple pattern of basic responses in growth rate and physiological state. There are reasons to assume that the model presented is the minimal version of a structured model of bacterial growth and represents an optimum compromise between biological relevance and mathematical practicability.     

Interdependence between cooperativity and control coefficients

Influence of the concentration of internal metabolites on the control coefficient (defined as fractional change in flux per fractional change in enzyme activity) and regulatory properties of a given enzyme have been studied theoretically using a cyclic model of three enzymes. This model is useful to investigate the properties of the flux control coefficient for an enzyme following different rate equations. Enzymes can have high or low values of control coefficient irrespective of the type of kinetic equation, but the results obtained show that the sensitivity of these values to substrate variations is strongly dependent on its rate equation. These results help identify which kinetic equation allows the best control of a given metabolic pathway. These results have been applied to the purine nucleotide cycle. It is demonstrated that the best control of the cycle is reached when the irreversible reaction catalyzed by AMP deaminase follows a rate law that corresponds to a rational function of 2:2 degree with respect to AMP concentration.     

Fed-batch culture for L-lysine production via on-line state estimation and control

Computer application for fed-batch culture of Brevibacterium flavum for L-lysine production has been developed. The organisms are auxotrophic mutants for L-homoserine and are resistant to S-(2-aminoethyl)-L-cysteine. Adaptive control is applied for substrate addition. The sugar concentration is estimated using online respiratory measurement. During the period of fed-batch culture, the total sugar concentration is maintained at a given value. The cultivation strategy results in high productivity and high conversion yield.     

On the optimal control of fed-batch reactors with substrate-inhibited kinetics

The optimal feed rate profiles, for fed-batch fermentation that maximizes the biomass production and accounts for time, are analyzed. The solution can be found only if the final arc of the optimal control is a batch arc, since in this case the final concentrations of substrate and biomass can be determined by ulterior conditions on the mass balance and on the final growth rate of biomass and thus it is possible to solve the resulting time optimal problem by using Green's theorem. This evidences the "turnpike property" of the solution, which tries to spend the maximum time on or at least near the singular arc along which the substrate concentration is maintained constant. The optimality of the final batch arc is related to the time operational cost in the performance index. The sequence of the control depends on the initial conditions for which six different regions, with the respective patterns, have been identified, in case the performance index allows the control sequence to have a final batch.     

A simple method to monitor and control methanol feeding of Pichia pastoris fermentations using mid-IR spectroscopy

Mid-infrared FTIR spectroscopy is an efficient tool for the monitoring of bioprocesses, since it is fast and able to detect many compounds simultaneously. However, complex and time-consuming calibration procedures are still required, and have inhibited the spreading of these instruments. A simple and quick method to calibrate a FTIR instrument was developed for the control of fed-batch fermentations of the methylotrophic yeast Pichia pastoris. Based on the assumptions that (1) only substrate concentration may change significantly during a fed-batch process and (2) absorbance can be considered as proportional to concentration, a linear two-point calibration was implemented. Long-term instability of the instrument had to be addressed in order to get accurate results: two fixed points, on both sides of substrate absorbance peak, were used to perform on-line a linear correction of the signal drift. Fed-batch experiments at constant methanol (substrate) concentration ranging from 0.8 to 15gl(-1) were carried out. Off-line HPLC control analysis showed a good agreement with on-line FTIR data, with standard error of prediction values < 0.12gl(-1). Even though methanol acts both as carbon source and inducer of protein expression, no significant effect was observed on the level of protein expression in the recombinant strain used.     

Optimal nonsingular control of fed-batch fermentation

Presented is a new simple method for multidimensional optimization of fed-batch fermentations based on the use of the orthogonal collocation technique. Considered is the problem of determination of optimal programs for fermentor temperature, substrate concentration in feed, feeding profile, and process duration. By reformulation of the state and control variables is obtained a nonsingular form of the optimization problem which has considerable advantage over the singular case since a complicated procedure for determination of switching times for feeding is avoided. The approximation of the state variables by Lagrange polynomials enables simple incorporation of split boundary conditions in the approximation, and the use of orthogonal collocations provides stability for integration of state and costate variables. The interpolation points are selected to obtain highest accuracy for approximation of the objective functional by the Radau-Lobatto formula. The control variables are determined by optimization of the Hamiltonian at the collocation points with the DFP method. Constraints are imposed on state and control variables.The method is applied for a homogeneous model of fermentation with volume, substrate, biomass, and product concentrations as the state variables. Computer study shows considerable simplicity of the method, its high accuracy for low order of approximation, and efficient convergence.     

Advanced monitoring and control of pharmaceutical production processes with Pichia pastoris by using Raman spectroscopy and multivariate calibration methods

This contribution includes an investigation of the applicability of Raman spectroscopy as a PAT analyzer in cyclic production processes of a potential Malaria vaccine with Pichia pastoris. In a feasibility study, Partial Least Squares Regression (PLSR) models were created off‐line for cell density and concentrations of glycerol, methanol, ammonia and total secreted protein. Relative cross validation errors RMSE_cvrel range from 2.87% (glycerol) to 11.0% (ammonia). In the following, on‐line bioprocess monitoring was tested for cell density and glycerol concentration. By using the nonlinear Support Vector Regression (SVR) method instead of PLSR, the error RMSE_Prel for cell density was reduced from 5.01 to 2.94%. The high potential of Raman spectroscopy in combination with multivariate calibration methods was demonstrated by the implementation of a closed loop control for glycerol concentration using PLSR. The strong nonlinear behavior of exponentially increasing control disturbances was met with a feed‐forward control and adaptive correction of control parameters. In general the control procedure works very well for low cell densities. Unfortunately, PLSR models for glycerol concentration are strongly influenced by a correlation with the cell density. This leads to a failure in substrate prediction, which in turn prevents substrate control at cell densities above 16 g/L.     

Substrate concentration control by dialysis in fermentations of Escherichia coli

A substrate control system based upon dialysis culture techniques has been developed. In fermentations of Escherichia coli where phosphate or sulfate concentrations were controlled, the relation between the apparent specific growth rate, mu, and the phosphate concentration followed Monod kinetics, while the relation between mu and sulfate showed a sharp optimun. The pH of fermentations of E. coli was controlled by dialysis culture techniques without the fluctuations in pH associated with control by direct addition of acid or base.     

Closed-loop control of bacterial high-cell-density fed-batch cultures: production of mcl-PHAs by Pseudomonas putida KT2442 under single-substrate and cofeeding conditions.

Pseudomonas putida KT2442 is able to accumulate medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHAs) as intracellular inclusions on a variety of fatty acids and many other carbon sources. Some of these substrates, such as octanoic acid, alkenoic acids, and halogenated derivatives, are toxic when present in excess. Efficient production of mcl-PHAs on such toxic substrates therefore requires control of the carbon source concentration in the supernatant. In this study, we develop a closed-loop control system based on on-line gas chromatography to maintain continuously fed substrates at desired levels. We used the graphical programming environment LABVIEW to set up a flexible process control system that allows users to perform supervisory process control and permits remote access to the fermentation system over the Internet. Single-substrate supernatant concentration in a high-cell-density fed-batch fermentation process was controlled by a proportional (P) controller (P = 50%) acting on the substrate pump feed rate. Na-octanoate concentrations oscillated around the setpoint of 10 mM and could be maintained between 0 and 25 mM at substrate uptake rates as high as 90 mmol L(-1) h(-1). Under cofeeding conditions Na-10-undecenoate and Na-octanoate could be individually controlled at 2.5 mM and 9 mM, respectively, by applying a proportional integral (PI) controller for each substrate. The resulting copolymer contained 43.5 mol% unsaturated monomers and reflected the ratio of 10-undecenoate in the feed. It was suggested that both substrates were consumed at similar rates. These results show that this control system is suitable for avoiding substrate toxicity and supplying carbon substrates for growth and mcl-PHA accumulation.     

Plug flow simulating and completely mixed reactors with a premixing tank,in the control of filamentous bulking

Summary A sequence of a substrate rich and substrate starvation phase (feast and famine strategy) is created in both compartmentalized reactors and reactors with premixing tanks. This condition is known to be essential in the control of bulking. With a synthetic waste water containing glucose a reactor with 12 compartments was effective in the control of filamentous bulking. With a dairy industrial waste water, containing a slowly biodegradable COD-fraction, this reactor could not suppress the growth of filamentous bacteria. With dairy and brewery waste water, reactors with premixing tanks were used to create a more pronounced exogenous (substrate rich) phase. Using three or more premixing tanks filamentous bulking could be controlled. During the exogenous phase the floc-forming microorganisms, having a higher substrate uptake rate, can take up the largest amount of substrate and can survive better during the endogenous phase.Substrate concentration and respiration rate profiles were studied and the concentration of the reserve materials was monitored.For each type of waste water the sludge loading and the fraction of readily available COD will determine the system's design.     

Experimental determination of control by the H+-ATPase inEscherichia coli

Strains carrying deletions in theatp genes, encoding the H⁺-ATPase, were unable to grow on nonfermentable substrates such as succinate, whereas with glucose as the substrate the growth rate of anatp deletion mutant was surprisingly high (some 75–80% of wild-type growth rate). The rate of glucose and oxygen consumption of these mutants was increased compared to the wild-type rates. In order to analyze the importance of the H⁺-ATPase at its physiological level, the cellular concentration of H⁺-ATPase was modulated around the wild-type level, using genetically manipulated strains. The control coefficient by the H⁺-ATPase with respect to growth rate and catabolic fluxes was measured. Control on growth rate was absent at the wild-type concentration of H⁺-ATPase, independent of whether the substrate for growth was glucose or succinate. Control by the H⁺-ATPase on the catabolic fluxes, including respiration, was negative at the wild-type H⁺-ATPase level. Moreover, the turnover number of the individual H⁺-ATPase enzymes increased as the H⁺-ATPase concentration was lowered. The negative control by the H⁺-ATPase on catabolism may thus be involved in a homeostatic control of ATP synthesis and, to some extent, explain the zero control by the H⁺-ATPase onE. coli growth rate.     

Metabolic control analysis of glycerol synthesis in Saccharomyces cerevisiae

Glycerol, a major by-product of ethanol fermentation by Saccharomyces cerevisiae, is of significant importance to the wine, beer, and ethanol production industries. To gain a clearer understanding of and to quantify the extent to which parameters of the pathway affect glycerol flux in S. cerevisiae, a kinetic model of the glycerol synthesis pathway has been constructed. Kinetic parameters were collected from published values. Maximal enzyme activities and intracellular effector concentrations were determined experimentally. The model was validated by comparing experimental results on the rate of glycerol production to the rate calculated by the model. Values calculated by the model agreed well with those measured in independent experiments. The model also mimics the changes in the rate of glycerol synthesis at different phases of growth. Metabolic control analysis values calculated by the model indicate that the NAD(+)-dependent glycerol 3-phosphate dehydrogenase-catalyzed reaction has a flux control coefficient (C(J)v1) of approximately 0.85 and exercises the majority of the control of flux through the pathway. Response coefficients of parameter metabolites indicate that flux through the pathway is most responsive to dihydroxyacetone phosphate concentration (R(J)DHAP= 0.48 to 0.69), followed by ATP concentration (R(J)ATP = -0.21 to -0.50). Interestingly, the pathway responds weakly to NADH concentration (R(J)NADH = 0.03 to 0.08). The model indicates that the best strategy to increase flux through the pathway is not to increase enzyme activity, substrate concentration, or coenzyme concentration alone but to increase all of these parameters in conjunction with each other.