Optimal Feeding Trajectories Design for E. coli Fed-batch Fermentations

In this paper optimal control algorithms for two E. coli fed-batch fermentations are developed. Fed-batch fermentation processes of E. coli strain MC4110 and E. coli strain BL21(DE3)pPhyt109 are considered. Simple material balance models are used to describe the E. coli fermentation processes. The optimal feed rate control of a primary metabolite process is studied and a biomass production is used as an example. The optimization of the considered fed-batch fermentation processes is done using the calculus of variations to determine the optimal feed rate profiles. The problem is formulated as a free final time problem where the control objective is to maximize biomass at the end of the process. The obtained optimal feed rate profiles consist of sequences of maximum and minimum feed rates. The resulting profiles are used for optimization of the E. coli fed-batch fermentations. Presented simulations show a good efficiency of the developed optimal feed rate profiles

Specific growth rate regulation in a simulated fed-batch E. coli fermentation

The specific growth rate is one of the most important process variables characterizing the state of microorganisms during fermentations mainly because the biosynthesis of many products of interest is often related with the values assumed by this parameter. In the particular case of the fed-batch operation of Escherichia coli for the production of recombinant proteins, it is often argued that both pre- and the post-induction specific growth rates should be closely controlled in order to achieve maximum productivities on the desired recombinant protein. In this work a feedforward-feedback controller was developed with the purpose of regulating the global specific growth rate during a fed-batch fermentation of E. coli. The developed algorithm allows to maintain the cells in two different metabolic regimens (simultaneous oxidative and fermentative growth on glucose or oxidative growth on glucose), depending on the selected setpoint for the controlled variable. The pure open-loop version of the controller revealed a relatively poor performance when dealing with process noise. However, the introduction of on-line measurements of fermenter weight and biomass estimation obtained from asymptotic observers allowed a better approximation between the desired setpoints and the simulated values of the specific growth rates. Finally, the introduction of a proportional action in the controller equation allows an improved robustness against variations in model parameters.Fundação para a Ciência e a Tecnologia (FCT) - POCI/BIO/60139/2004

Multiple model approach to modelling of Escherichia coli fed-batch cultivation extracellular production of bacterial phytase

The paper presents the implementation of multiple model approach to modelling of Escherichia coli BL21(DE3)pPhyt109 fed-batch cultivation processes for an extracellular production of bacterial phytase. Due to the complex metabolic pathways of microorganisms, the accurate modelling of bioprocesses is rather difficult. Multiple model approach is an alternative concept which helps in modelling and control of complex processes. The main idea is the development of a model based on simple submodels for the purposes of further high quality process control. The presented simulations of E. coli fed-batch cultivation show how the process could be divided into different functional states and how the model parameters could be obtained easily using genetic algorithms. The obtained results and model verification demonstrate the effectiveness of the applied concept of multiple model approach and of the proposed identification scheme. © 2007 by Pontificia Universidad Católica de Valparaíso

Multiple model approach to modelling of Escherichia coli fed-batch cultivation extracellular production of bacterial phytase

The paper presents the implementation of multiple model approach to modelling of Escherichia coli BL21(DE3)pPhyt109 fed-batch cultivation processes for an extracellular production of bacterial phytase. Due to the complex metabolic pathways of microorganisms, the accurate modelling of bioprocesses is rather difficult. Multiple model approach is an alternative concept which helps in modelling and control of complex processes. The main idea is the development of a model based on simple submodels for the purposes of further high quality process control. The presented simulations of E. coli fed-batch cultivation show how the process could be divided into different functional states and how the model parameters could be obtained easily using genetic algorithms. The obtained results and model verification demonstrate the effectiveness of the applied concept of multiple model approach and of the proposed identification scheme

A feedforward-feedback substrate controller based on a Kalman filter for a fed-batch cultivation of Escherichia coli producing phytase

Arndt M, Kleist S, Miksch G, et al. A feedforward-feedback substrate controller based on a Kalman filter for a fed-batch cultivation of Escherichia coli producing phytase. COMPUTERS & CHEMICAL ENGINEERING. 2005;29(5):1113-1120.For the feeding-phase of a batch/fed-batch cultivation of a recombinant Escherichia coli producing extracellular phytase, a controller has been developed. Based on the estimated process variables by a Kalman filter, a feedforward-feedback controller has been implemented in order to maximize the phytase production and to minimize acetate production. The Kalman filter was used to reduce the noise of the glucose measurements and to estimate the biomass concentration, the substrate (glucose) concentration, the maximal growth rate as well as the reaction broth volume, which are used to calculate the feedforward controller contribution. A PI controller was applied to adjust the glucose concentration to the desired set point of 0.2 g/L. The secretion of phytase into the medium is increased at low glucose concentration whereas the acetate production is reduced, due to a low concentration avoiding significant overflow metabolism. The glucose concentration, as the sole measured variable used by the controller, is determined using flow injection analysis (FIA). The operation of the controller as well as its application to the E. coli cultivation is presented. The average on-line measured glucose concentration is 0.208 g/L with a standard deviation of 0.066 g/L. During the cultivation a fault occurred in the measurement system. The response of the controller system with respect to this fault is discussed in detail. Compared to a controller based on oxygen measurements, the yield of phytase is the same with the presented system. (c) 2004 Elsevier Ltd. All rights reserved

Process analytical technology in food biotechnology

Biotechnology is an area where precision and reproducibility are vital. This is due to the fact that products are often in form of food, pharmaceutical or cosmetic products and therefore very close to the human being. To avoid human error during the production or the evaluation of the quality of a product and to increase the optimal utilization of raw materials, a very high amount of automation is desired. Tools in the food and chemical industry that aim to reach this degree of higher automation are summarized in an initiative called Process Analytical Technology (PAT). Within the scope of the PAT, is to provide new measurement technologies for the purpose of closed loop control in biotechnological processes. These processes are the most demanding processes in regards of control issues due to their very often biological rate-determining component. Most important for an automation attempt is deep process knowledge, which can only be achieved via appropriate measurements. These measurements can either be carried out directly, measuring a crucial physical value, or if not accessible either due to the lack of technology or a complicated sample state, via a soft-sensor.Even after several years the ideal aim of the PAT initiative is not fully implemented in the industry and in many production processes. On the one hand a lot effort still needs to be put into the development of more general algorithms which are more easy to implement and especially more reliable. On the other hand, not all the available advances in this field are employed yet. The potential users seem to stick to approved methods and show certain reservations towards new technologies.Die Biotechnologie ist ein Wissenschaftsbereich, in dem hohe Genauigkeit und Wiederholbarkeit eine wichtige Rolle spielen. Dies ist der Tatsache geschuldet, dass die hergestellten Produkte sehr oft den Bereichen Nahrungsmitteln, Pharmazeutika oder Kosmetik angehöhren und daher besonders den Menschen beeinflussen. Um den menschlichen Fehler bei der Produktion zu vermeiden, die Qualität eines Produktes zu sichern und die optimale Verwertung der Rohmaterialen zu gewährleisten, wird ein besonders hohes Maß an Automation angestrebt. Die Werkzeuge, die in der Nahrungsmittel- und chemischen Industrie hierfür zum Einsatz kommen, werden in der Process Analytical Technology (PAT) Initiative zusammengefasst. Ziel der PAT ist die Entwicklung zuverlässiger neuer Methoden, um Prozesse zu beschreiben und eine automatische Regelungsstrategie zu realisieren. Biotechnologische Prozesse gehören hierbei zu den aufwändigsten Regelungsaufgaben, da in den meisten Fällen eine biologische Komponente der entscheidende Faktor ist. Entscheidend für eine erfolgreiche Regelungsstrategie ist ein hohes Maß an Prozessverständnis. Dieses kann entweder durch eine direkte Messung der entscheidenden physikalischen, chemischen oder biologischen Größen gewonnen werden oder durch einen SoftSensor. Zusammengefasst zeigt sich, dass das finale Ziel der PAT Initiative auch nach einigen Jahren des Propagierens weder komplett in der Industrie noch bei vielen Produktionsprozessen angekommen ist. Auf der einen Seite liegt dies mit Sicherheit an der Tatsache, dass noch viel Arbeit in die Generalisierung von Algorithmen gesteckt werden muss. Diese müsse einfacher zu implementieren und vor allem noch zuverlässiger in der Funktionsweise sein. Auf der anderen Seite wurden jedoch auch Algorithmen, Regelungsstrategien und eigne Ansätze für einen neuartigen Sensor sowie einen Soft-Sensors vorgestellt, die großes Potential zeigen. Nicht zuletzt müssen die möglichen Anwender neue Strategien einsetzen und Vorbehalte gegenüber unbekannten Technologien ablegen

Fed-batch cultivations for high-yield production of tissue engineering related bio-molecules

[no abstract

Real-time monitoring and control of the specific growth rate in yeast fed-batch cultures based on process analytical technology tools such as biocalorimetry or spectroscopy

Key features of bioprocesses, such as product quantity and quality, but also cell physiology can be related to the growth characteristics of the organism under study. The specific growth rate, a key variable, cannot be measured directly, but might be estimated and inferred from other measurable variables such as biomass, substrate or product concentrations. The present thesis reviews techniques for real-time estimation and control of the specific growth rate in microbial fed-batch cultures by focusing on its importance in the development of processes for the production of high-value products such as recombinant proteins. Existing models and monitoring techniques are discussed before comparing two particular approaches, developed within the scope of this thesis, to estimate the biomass concentration and the specific growth rate of yeast cells in real-time, based on spectroscopic methods on the one hand and on heat flow measurements on the other. Particular emphasis is given to changes that need to be undertaken when adapting the initial strategy, developed for a process with Kluyveromyces marxianus, to different type of yeast cells such as Candida utilis or Pichia pastoris or Saccharomyces cerevisiae. For both control strategies, controller errors of less than 20 % were achieved, allowing ton control the specific growth rate of the four different yeast strains at a constant setpoint

Optimização de estratégias de alimentação para identificação de parâmetros de um modelo de E. coli. utilização do modelo em monitorização e controlo

Doutoramento em Engenharia Química e BiológicaOs principais objectivos desta tese são: o desenho óptimo de experiências para a identificação de coeficientes de rendimento de um modelo não estruturado de um processo de fermentação semicontínua de Escherichia coli; a verificação experimental das trajectórias de alimentação obtidas por simulação; o desenvolvimento de estratégias de monitorização avançada para a estimação em linha de variáveis de estado e parâmetros cinéticos; e por fim o desenvolvimento de uma lei de controlo adaptativo para controlar a taxa específica de crescimento, com base em estratégias de alimentação de substrato com vista à maximização do crescimento e/ou produção. São apresentadas metodologias para o desenho óptimo de experiências, que visam a optimização da riqueza informativa das mesmas, quantificada por índices relativos à Matriz de Informação de Fisher. Embora, o modelo utilizado para descrever a fermentação semi-contínua de E. coli não esteja ainda optimizado em termos cinéticos e de algumas dificuldades encontradas na implementação prática dos resultados obtidos por simulação para o desenho óptimo de experiências, a qualidade da estimativa dos parâmetros, especialmente os do regime respirativo, é promissora. A incerteza das estimativas foi avaliada através de índices relacionados com o modelo de regressão linear múltipla, índices relativos à matriz de Fisher e pelo desenho das correspondentes elipses dos desvios. Os desvios associados a cada coeficiente mostram que ainda não foram encontrados os melhores valores. Procedeu-se também à investigação do papel do modelo dinâmico geral no desenho de sensores por programação. Foram aplicados três observadores – observador estendido de Kalman, observador assimptótico e observador por intervalo – para estimar a concentração de biomassa, tendo sido avaliado e comparado o seu desempenho bem como a sua flexibilidade. Os observadores estudados mostraram-se robustos, apresentando comportamentos complementares. Os observadores assimptóticos apresentam, em geral, um melhor desempenho que os observadores estendidos de Kalman. Os observadores por intervalo apresentam vantagens em termos de implementação prática, apresentando-se bastante promissores embora a sua validação experimental seja necessária. É apresentada uma lei de controlo adaptativo com modelo de referência que se traduz num controlo por antecipação/retroacção cuja acção de retroacção é do tipo PI, para controlar a taxa específica de crescimento. A robustez do algoritmo de controlo foi estudada por simulação numérica gerando dados “pseudo reais”, por aplicação de um ruído branco às variáveis medidas em linha, por alteração do valor de referência, por alteração do valor da concentração da glucose na alimentação e variando os valores nominais dos parâmetros do modelo. O estudo realizado permite concluir que a resposta do controlador é em geral satisfatória, sendo capaz de manter o valor da taxa específica de crescimento na vizinhança do valor de referência pretendido e inferior a um valor que conduz à formação de acetato, revestindo-se este facto de grande importância numa situação real, em especial, numa fermentação cujo objectivo seja a produção, nomeadamente de proteínas recombinadas. Foram ainda, analisados diferentes métodos de sintonização dos parâmetros do controlador, podendo concluir-se que, em geral, o método de sintonização automática com recurso à regra de adaptação dos parâmetros em função do erro relativo do controlador foi o que apresentou um melhor desempenho global. Este mecanismo de sintonização automática demonstrou capacidade para melhorar o desempenho do controlador ajustando continuamente os seus parâmetros.The main objectives of this thesis are: the optimal experiment design for yield coefficients estimation in an unstructured growth model for Escherichia coli fed-batch fermentation; the experimental validation of the simulated feed trajectories; the development of advanced monitoring strategies for the on-line estimation of state variables and kinetic parameters; and at last the development of an adaptive control law, based on optimal substrate feed strategies in order to increase the growth and/or the production. Methodologies for the optimal experimental design are presented, in order to optimise the richness of data coming out from experiments, quantified by indexes based on the Fisher Information Matrix. Although the model used to describe the E. coli fed-batch fermentation is not optimised from the kinetic properties point of view and the fact that some difficulties were encountered in practical implementation of the simulated results obtained with the optimal experimental design, the estimated parameter quality, especially for the oxidative regimen, is promising. The estimation uncertainty was evaluated by means of indexes related with multiple linear regression model, indexes related to the Fisher matrix as well as by the construction of the related deviation ellipses. The deviations associated to each coefficient show that the best values were not yet found. The role of the general dynamical model was also investigated in which concerns the design of state observers, also called software sensors. The performance of three observer classes was compared: Kalman extended observer, assimptotic observer and interval observer. The studied observers showed good performance and robustness, being complementary of each other. Assimptotic observers showed, in general, a better performance than the Kalman extended observer. Interval observers presented advantages concerning practical implementation, showing a promising behaviour although experimental validation is needed. A model reference adaptive control law is presented and can be interpreted as a PI like feedforward/feedback controller, for specific growth rate control. Algorithm robustness was studied using “pseudo real” data obtained by numerical simulation, by applying a white noise to the on-line measured variables, by modifying the set-point value, by changing the glucose concentration value of the feed rate and varying the nominal model parameter value. The study made allowed to conclude that the controller response is, generally, satisfactory being able to keep the specific growth rate value in the proximity of the desired set-point and lower than the value that permits acetate formation, which is of major importance namely for real cases, specially, in a fermentation which objective was the production of recombinant proteins. Different tuning devices for controller parameters were analysed being the better performance achieved by the automatic tuning method with an adaptation rate as a function of the controller relative error. This automatic tuning mechanism was able to improve the controller performance adjusting continuously its parameters