Designing a Minimal-Knowledge Controller to Achieve Fast, Stable Growth for Recombinant Escherichia coli Cultures

The biopharmaceutical industry is constantly developing new recombinant Es-cherichia coli strains to bring new products to market. In early stages of development, small scale bioreactors are used to make the product and explore di˙erent growth pro-tocols. Researchers spend significant time finding a feed rate profile that will give fast growth and low byproduct accumulation. The objective for the controller pre-sented in this work is to achieve fast growth and low acetate accumulation for an E.coli fermentation. The controller does not rely on previous characterization data or models but on fundamental metabolic relationships between oxygen and glucose as dictated by the Crabtree e˙ect. The controller senses metabolic state using an on-line oxygen uptake rate (OUR) estimate and pushes the culture to the boundary of oxidative and overflow metabolism (BOOM). A simulated E.coli culture and biore-actor were constructed to test controller performance. Fermentation experiments compared the BOOM controller to an Exponential feed and a DO-stat controller. Using minimal knowledge about the strain, the BOOM controller kept an induced E.coli MG1655 pTVP1GFP strain growing near the boundary of oxidative and over-flow metabolism. The BOOM controller produced more recombinant protein than the Exponential feed controller and the DO-stat controller, even though the growth rate used by the Exponential feed controller was extensively researched by a previous group. In another fermentation, the temperature was lowered to incur a fast change in the E.coli metabolism. In all experiments, the BOOM controller demonstrated it could maintain fast growth and avoid inhibitory acetate concentrations while requir-ing minimal knowledge of theE.coli MG1655 pTVP1GFP strain. For laboratories which deal with many di˙erent strains and proteins, the BOOM controller would maximize protein production and speed up protocol development

Bioprocess Monitoring and Control

Process monitoring and control are fundamental to all processes; this holds especially for bioprocesses, due to their complex nature. Usually, bioprocesses deal with living cells, which have their own regulatory systems. It helps to adjust the cell to its environmental condition. This must not be the optimal condition that the cell needs to produce whatever is desired. Therefore, a close monitoring of the cell and its environment is essential to provide optimal conditions for production. Without measurement, no information of the current process state is obtained. In this book, methods and techniques are provided for the monitoring and control of bioprocesses. From new developments for sensors, the application of spectroscopy and modelling approaches, the estimation and observer implementation for ethanol production and the development and scale-up of various bioprocesses and their closed loop control information are presented. The processes discussed here are very diverse. The major applications are cultivation processes, where microorganisms were grown, but also an incubation process of bird’s eggs, as well as an indoor climate control for humans, will be discussed. Altogether, in 12 chapters, nine original research papers and three reviews are presented

Development of monitoring and control systems for biotechnological processes

The field of biotechnology represents an important research area that has gained increasing success in recent times. Characterized by the involvement of biological organisms in manufacturing processes, its areas of application are broad and include the pharmaceuticals, agri-food, energy, and even waste treatment. The implication of living microorganisms represents the common element in all bioprocesses. Cell cultivations is undoubtedly the key step that requires maintaining environmental conditions in precise and defined ranges, having a significant impact on the process yield and thus on the desired product quality. The apparatus in which this process occurs is the bioreactor. Unfortunately, monitoring and controlling these processes can be a challenging task because of the complexity of the cell growth phenomenon and the limited number of variables can be monitored in real-time. The thesis presented here focuses on the monitoring and control of biotechnological processes, more specifically in the production of bioethanol by fermentation of sugars using yeasts. The study conducted addresses several issues related to the monitoring and control of the bioreactor, in which the fermentation takes place. First, the topic concerning the lack of proper sensors capable of providing online measurements of key variables (biomass, substrate, product) is investigated. For this purpose, nonlinear estimation techniques are analyzed to reconstruct unmeasurable states. In particular, the geometric observer approach is applied to select the best estimation structure and then a comparison with the extended Kalman filter is reported. Both estimators proposed demonstrate good estimation capabilities as input model parameters vary. Guaranteeing the achievement of the desired ethanol composition is the main goal of bioreactor control. To this end, different control strategies, evaluated for three different scenarios, are analzyed. The results show that the MIMO system, together with an estimator for ethanol composition, ensure the compliance with product quality. After analyzing these difficulties through numeric simulations, this research work shifts to testing a specific biotechnological process such as manufacturing bioethanol from brewery’s spent grain (BSG) as renewable waste biomass. Both acid pre-treatment, which is necessary to release sugars, and fermentation are optimized. Results show that a glucose yield of 18.12 per 100 g of dried biomass is obtained when the pre-treatment step is performed under optimized conditions (0.37 M H2SO4, 10% S-L ratio). Regarding the fermentation, T=25°C, pH=4.5, and inoculum volume equal to 12.25% v/v are selected as the best condition, at which an ethanol yield of 82.67% evaluated with respect to theoretical one is obtained. As a final step, the use of Raman spectroscopy combined with chemometric techniques such as Partial Least Square (PLS) analysis is evaluated to develop an online sensor for fermentation process monitoring. The results show that the biomass type involved significantly affects the acquired spectra, making them noisy and difficult to interpret. This represents a nontrivial limitation of the applied methodology, for which more experimental data and more robust statistical techniques could be helpful

Optimal control of fed-batch fermentation processes

Optimisation of a fed-batch fermentation process typically uses the calculus of variations or Pontryagin's maximum principle to determine an optimal feed rate profile. This often results in a singular control problem and an open loop control structure. The singular feed rate is the optimal feed rate during the singular control period and is used to control the substrate concentration in the fermenter at an optimal level. This approach is supported by biological knowledge that biochemical reaction rates are controlled by the environmental conditions in the fermenter; in this case, the substrate concentration. Since an accurate neural net-based on-line estimation of the substrate concentration has recently become available and is currently employed in industry, we are therefore able to propose a method which makes use of this estimation. The proposed method divides the optimisation problem into two parts. First, an optimal substrate concentration profile which governs the biochemical reactions in the fermentation process is determined. Then a controller is designed to track the obtained optimal profile. Since the proposed method determines the optimal substrate concentration profile, the singular control problem is therefore avoided because the substrate concentration appears nonlinearly in the system equations. Also, the process is then operated in closed loop control of the substrate concentration. The proposed method is then called "closed loop optimal control". The proposed closed loop optimal control method is then compared with the open loop optimal feed rate profile method. The comparison simulations from both primary and secondary metabolite production processes show that both methods give similar performance in a case of perfect model while the closed loop optimal control provides better performance than the open loop method in a case of plant/model mismatch. The better performance of the closed loop optimal control is due to an ability to compensate for the modelling errors using feedback

Robust Nonlinear Model Predictive Control using Polynomial Chaos Expansions

The performance of model predictive controllers (MPCs) is largely dependent on the accuracy of the model predictions as compared to the actual plant outputs. Irrespective of the model used, first-principles (FP) or empirical, plant-model mismatch is unavoidable. Consequently, model based controllers must be robust to mismatch between the model predictions and the actual process behavior. Controllers that are not robust may result in poor closed loop response and even instability. Model uncertainty can generally be formulated into two broader forms, parametric uncertainty and unstructured uncertainty. Most of the current robust nonlinear MPC have been based on FP-model where only robustness to bounded disturbances rather than parametric uncertainty has been addressed. Systematically accounting for parametric uncertainty in the robust design has been difficult in FP-models due to varying forms in which uncertain parameters occur in the models. To address parametric uncertainty robustness tests based on Structured Singular Value (SSV) and Linear Matrix Inequalities (LMI) have been proposed previously, however these algorithms tend to be conservative because they consider worst-case scenarios and they are also computationally expensive. For instance the SSV calculation is NP-hard and as a result it is not suitable for fast computations. This provides motivation to work on robust control algorithms addressing both parametric and unstructured uncertainty with fast computation times. To facilitate the design of robust controllers which can be computed fast, empirical models are used in which parametric uncertainty is propagated using Polynomial Chaos Expansion (PCE) of parameters. PCE assists in speeding up the computations by providing an analytical expression for the L^2-norm of model predictions while also eliminating the need to design for the worst-case scenario which results in conservatism. Another way of speeding up computations in MPC algorithms is by grouping subsets of available the inputs and outputs into subsystems and by controlling each of the subsystems by MPC controllers of lower dimensions. This latter approach, referred in the literature as Distributed MPC, has been tackled by different strategies involving different degrees of coordination between subsystems but it has not been studied in terms of robustness to model error. Based on the above considerations the current work investigates different robustness aspects of predictive control algorithms for nonlinear processes with special emphasis on the following three situations, i) a nonlinear predictive control based on a Volterra series model where the uncertain parameters are formulated as PCE’s, ii) The application of a PCE-based approach to control and optimization of bioreactors where the model is based on dynamic flux metabolic models, and iii) A Robust Distributed MPC with a robust estimator that is needed to account for the interactions between sub-systems in distributed control

Model-Based State Estimation for Fault Detection under Disturbance

The measurement of process states is critical for process monitoring, advanced process control, and process optimization. For chemical processes where state information cannot be measured directly, techniques such as state estimation need to be developed. Model-based state estimation is one of the most widely applied methods for estimation of unmeasured states basing on a high-fidelity process model. However, certain disturbances or unknown inputs not considered by process models will generate model-plant mismatch. In this dissertation, different model-based process monitoring techniques are developed and applied for state estimation under uncertainty and disturbance. Case studies are performed to demonstrate the proposed methods. The first case study estimates leak location from a natural gas pipeline. Non-isothermal state equations are derived for natural gas pipeline flow processes. A dual unscented Kalman filter is used for parameter estimation and flow rate estimation. To deal with sudden process disturbance in the natural gas pipeline, an unknown input observer is designed. The proposed design implements a linear unknown input observer with time-delays that considers changes of temperature and pressure as unknown inputs and includes measurement noise in the process. Simulation of a natural gas pipeline with time-variant consumer usage is performed. New optimization method for detection of simultaneous multiple leaks from a natural gas pipeline is demonstrated. Leak locations are estimated by solving a global optimization problem. The global optimization problem contains constraints of linear and partial differential equations, integer variable, and continuous variable. An adaptive discretization approach is designed to search for the leak locations. In a following case study, a new design of a nonlinear unknown input observer is proposed and applied to estimate states in a bioreactor. The design of such an observer is provided, and sufficient and necessary conditions of the observer are discussed. Experimental studies of batch and fed-batch operation of a bioreactor are performed using Saccharomyces cerevisiae strain mutant SM14 to produce β-carotene. The state estimation of the process from the designed observer is demonstrated to alleviate the model-plant mismatch and is compared to the experimental measurements

Continuous Biochemical Processing: Investigating Novel Strategies to Produce Sustainable Fuels and Pharmaceuticals

Biochemical processing methods have been targeted as one of the potential renewable strategies for producing commodities currently dominated by the petrochemical industry. To design biochemical systems with the ability to compete with petrochemical facilities, inroads are needed to transition from traditional batch methods to continuous methods. Recent advancements in the areas of process systems and biochemical engineering have provided the tools necessary to study and design these continuous biochemical systems to maximize productivity and substrate utilization while reducing capital and operating costs. The first goal of this thesis is to propose a novel strategy for the continuous biochemical production of pharmaceuticals. The structural complexity of most pharmaceutical compounds makes chemical synthesis a difficult option, facilitating the need for their biological production. To this end, a continuous, multi-feed bioreactor system composed of multiple independently controlled feeds for substrate(s) and media is proposed to freely manipulate the bioreactor dilution rate and substrate concentrations. The optimal feed flow rates are determined through the solution to an optimal control problem where the kinetic models describing the time-variant system states are used as constraints. This new bioreactor paradigm is exemplified through the batch and continuous cultivation of β-carotene, a representative product of the mevalonate pathway, using Saccharomyces cerevisiae strain mutant SM14. The second goal of this thesis is to design continuous, biochemical processes capable of economically producing alternative liquid fuels. The large-scale, continuous production of ethanol via consolidated bioprocessing (CBP) is examined. Optimal process topologies for the CBP technology selected from a superstructure considering multiple biomass feeds, chosen from those available across the United States, and multiple prospective pretreatment technologies. Similarly, the production of butanol via acetone-butanol-ethanol (ABE) fermentation is explored using process intensification to improve process productivity and profitability. To overcome the inhibitory nature of the butanol product, the multi-feed bioreactor paradigm developed for pharmaceutical production is utilized with in situ gas stripping to simultaneously provide dilution effects and selectively remove the volatile ABE components. Optimal control and process synthesis techniques are utilized to determine the benefits of gas stripping and design a butanol production process guaranteed to be profitable

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

Dynamic surrogate modelling for multistep-ahead prediction of multivariate nonlinear chemical processes

This work proposes a methodology for multivariate dynamic modeling and multistep-ahead prediction of nonlinear systems using surrogate models for the application to nonlinear chemical processes. The methodology provides a systematic and robust procedure for the development of data-driven dynamic models capable of predicting the process outputs over long time horizons. It is based on using surrogate models to construct several nonlinear autoregressive exogenous models (NARX) with each one approximating the future behavior of one process output as a function of the current and previous process inputs and outputs. The developed dynamic models are employed in a recursive schema to predict the process future outputs over several time steps (multistep-ahead prediction). The methodology is able to manage two different scenarios: (1) one in which a set of input–output signals collected from the process is only available for training and (2) another in which a mathematical model of the process is available and can be used to generate specific datasets for training. With respect to the latter, the proposed methodology includes a specific procedure for the selection of training data in dynamic modeling based on design of computer experiment (DOCE) techniques. The proposed methodology is applied to case studies from the process industry presented in the literature. The results show very high prediction accuracies over long time horizons. Also, owing to the flexibility, robustness, and computational efficiency of surrogate modeling, the methodology allows dealing with a wide range of situations, which would be difficult to address using first-principles models.Peer ReviewedPostprint (author's final draft

Modélisation et commande robuste des systèmes biologiques : exemple de la production d’acide lactique en fermenteur industriel

This PhD thesis focuses on the optimization of the bioprocess of lactic acid production from wheat flour. Indeed, lactic acid has received much attention for the production of PLA (Poly Lactic Acid), a biopolymer, since different inexpensive raw material such as wheat flour are now used as carbon source for its production. This work was performed in three main steps. In the first step, an innovative wheat transformation process is proposed, whose main steps are the following: a liquefaction followed by a simultaneous saccharification, proteins hydrolysis (SSPH) and and a final simultaneous saccharification, proteins hydrolysis and fermentation (SSPHF). Secondly, the modeling of the SSPHF (limiting step) in a continuous bioreactor is considered. The determination and validation of model parameters is performed by means of experimental campaigns in a 5 L bioreactor.In the last step, the development of control strategies to maintain the process at its optimal operating point is considered. To do so, due to the absence of sensors for real-time measurement of the concentrations of key variables of the bioreactor, estimators of these concentrations and of the lactic acid production rate are first developed. Then, control strategies for regulating the lactic acid concentration at its optimal value are designed and compared in simulation. An adaptive control combining a state feedback linearizing control and an estimator of the lactic acid production rate is finally chosen to be experimentally validated on an instrumented reactor. This strategy showed good robustness features with respect to modeling mismatches and was able during experiments to increase twice the lactic acid productivity.Cette thèse de doctorat porte sur l’optimisation du bioprocédé de production d’acide lactique à partir de la farine de blé. L'acide lactique s’avère en effet de plus en plus attractif pour la production de PLA (acide poly lactique), un bio polymère, d’autant plus que différentes matières premières peu coûteuses comme la farine de blé sont désormais utilisées comme sources de carbone pour sa production. Cette thèse comprend trois parties principales. Une première partie propose pour l’optimisation du procédé de transformation du blé un schéma innovant composé de trois étapes successives : une liquéfaction, suivi d’une étape de saccharification et hydrolyse des protéines simultanées (SSPH) et une étape finale de saccharification, hydrolyse des protéines et fermentation simultanées (SSPHF). La deuxième partie s’intéresse à la modélisation de l’étape SSPHF (étape limitante) dans un bioréacteur continu. La détermination des paramètres du modèle ainsi que leur validation sont réalisées à l’aide de campagnes d’essais sur un bioréacteur de 5 L.Enfin, la dernière partie développe la mise en oeuvre de stratégies de commande permettant de maintenir le bioprocédé à son point optimal de fonctionnement. Pour ce faire, du fait de l’absence de capteurs pour la mesure en temps réel des concentrations des variables clé dans le bioréacteur, des estimateurs de ces concentrations ainsi que du taux de production en acide lactique sont tout d’abord élaborés. Des stratégies de commande régulant la concentration d’acide lactique à sa valeur optimale sont ensuite synthétisées et comparées en simulation. Une commande adaptative combinant une commande linéarisante par retour d’état et un estimateur du taux de production en acide lactique est finalement retenue et validée expérimentalement sur un réacteur instrumenté. Cette dernière s’est avérée robuste vis-à-vis des erreurs de modélisation et a permis lors des expériences de doubler la productivité de l’acide lactique