Development and optimization of a fermentation process for the production of 2,3-butanediol

The Ph.D. project aims to develop and optimize the fermentative production of diols from biowastes. In particular 2,3-butanediol (2,3-BDO), a promising platform chemical from which valuable derivatives can be produced. The biotechnological production of 2,3-BDO has been mainly studied with pathogenic bacteria by using pure sugars. However, the pathogenicity of such microorganisms, the high cost of sugars, and the not optimized fermentation conditions make this process not industrially competitive. During the first year, the microorganism Bacillus licheniformis ATCC-9789 was selected and its capability to ferment different sugars commonly occurring in biomass hydrolysates and biowastes was assessed. The selected microorganism is able to ferment hexoses and sucrose into 2,3-BDO. Furthermore, cheese whey could be potentially partially converted in 2,3-BDO, and molasses as well represent an interesting feedstock to produce 2,3-BDO with the selected strain. Since 2,3-BDO is produced preferentially under microaerobic conditions, the second year activities have been focused on the optimization of the oxygen supply conditions in batch fermentation mode using glucose as substrate. Experimentally, the application of a statistical design of the experiment allowed the identification of an optimal stirring/aeration combination, and the identification of a range of oxygen transfer rate values inside which the fermentation performances are maximized. The possibility of producing 2,3-BDO at high concentration and productivity was evaluated carrying out fed-batch experiments. Since complex media and the optimal oxygen supply conditions are both mandatory to obtain the best performances, different feed and oxygen supply strategies, were assayed. Another activity focused on the evaluation of alternative media instead of the complex media. Flasks and bioreactor experiments have been carried out using a mineral media, and meat and bone meal, whereas ,molasses and glucose were used as carbon sources. In conclusion, animal flour and molasses seem to be suitable alternatives to the commercial substrates to produce 2,3-butanediol

Digital Twins for Bioprocess Control Strategy Development and Realisation

New innovative Digital Twins can represent complex bioprocesses, including the biological, physico-chemical, and chemical reaction kinetics, as well as the mechanical and physical characteristics of the reactors and the involved peripherals. Digital Twins are an ideal tool for the rapid and cost-effective development, realisation and optimisation of control and automation strategies. They may be utilised for the development and implementation of conventional controllers (e.g. temperature, dissolved oxygen, etc.), as well as for advanced control strategies (e.g. control of substrate or metabolite concentrations, multivariable controls), and the development of complete bioprocess control. This chapter describes the requirements Digital Twins must fulfil to be used for bioprocess control strategy development, and implementation and gives an overview of research projects where Digital Twins or "early-stage" Digital Twins were used in this context. Furthermore, applications of Digital Twins for the academic education of future control and bioprocess engineers as well as for the training of future bioreactor operators will be described. Finally, a case study is presented, in which an "early-stage" Digital Twin was applied for the development of control strategies of the fed-batch cultivation of Saccharomyces cerevisiae. Development, realisation and optimisation of control strategies utilising Digital Twins

Development of advanced monitoring and control tools for rAAV production in the insect cell system

"Since the first publication introducing the concept in 1972, gene therapy has had a series of success stories and setbacks. However, the recent rise of awareness, public interest, promising results in clinical trials and recent market approvals indicate that gene therapy has come to stay. Currently there is a growing interest from the biopharmaceutical industry in gene and cell therapy, mostly using viral vectors. (...)

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

Bioprocess Systems Engineering Applications in Pharmaceutical Manufacturing

Biopharmaceutical and pharmaceutical manufacturing are strongly influenced by the process analytical technology initiative (PAT) and quality by design (QbD) methodologies, which are designed to enhance the understanding of more integrated processes. The major aim of this effort can be summarized as developing a mechanistic understanding of a wide range of process steps, including the development of technologies to perform online measurements and real-time control and optimization. Furthermore, minimization of the number of empirical experiments and the model-assisted exploration of the process design space are targeted. Even if tremendous progress has been achieved so far, there is still work to be carried out in order to realize the full potential of the process systems engineering toolbox. Within this reprint, an overview of cutting-edge developments of process systems engineering for biopharmaceutical and pharmaceutical manufacturing processes is given, including model-based process design, Digital Twins, computer-aided process understanding, process development and optimization, and monitoring and control of bioprocesses. The biopharmaceutical processes addressed focus on the manufacturing of biopharmaceuticals, mainly by Chinese hamster ovary (CHO) cells, as well as adeno-associated virus production and generation of cell spheroids for cell therapies

Procédés de culture cellulaire pour un système CHO inductible : cuvée-alimentée et perfusion

RÉSUMÉ : La production de protéines thérapeutiques représente de nos jours l’un des marchés les plus fructueux de l’industrie pharmaceutique. Les rendements de production ont considérablement augmenté au cours de ces dernières décennies en raison des améliorations significatives apportées au niveau des procédés de culture et des systèmes d’expression. Les cellules de mammifères, qui sont aptes à réaliser des modifications post-traductionnelles essentielles aux fonctions biologiques de ces protéines, demeurent la plateforme d’expression de choix et les cellules CHO restent de loin les plus utilisées pour la production industrielle à grande échelle. Comme la production de protéines recombinantes est généralement non-associée à la croissance cellulaire, les recherches se sont penchées vers le développement de procédés bi-phasiques consistant à d’abord laisser croitre les cellules à haute densité, puis de ralentir ou de stopper la croissance pour favoriser l’expression du produit d’intérêt.----------ABSTRACT - Biotherapeutics represent today a fast-growing share of the global pharmaceutical market. Production yields have increased dramatically in recent decades due to significant improvements in bioprocess technologies and with the development of highly efficient expression systems. Mammalian cells remain the most widely-used expression platform, since they have the ability to perform post-translational modifications essential to the biological functions of recombinant proteins. CHO cells are the most commonly used host system for industrial biomanufacturing at large scale. Most often, the production of recombinant proteins is non-growth associated, which has led to the development of biphasic strategies whereby cells are rapidly grown to a high density followed by a production phase during which cell growth is stopped or reduced to increase the expression rate. In the same context, cell engineering has made it possible to regulate protein production with the development of inducible expression systems. Such systems offer the possibility to decouple the growth and production phases

Optimization of biohydrogen production inoculum development via hybrid pre-treatment techniques : semi pilot scale production assessment on agro waste (potato peels)

Doctor of Philosophy in Microbiology.The challenges of energy crisis and environmental pollution are vital issues hindering the global sustainable development as a result of over dependence on fossil fuels. These are driving the need to explore renewable and environmentally friendly energy sources. Biohydrogen has emerged as an eco-friendly renewable energy source and a suitable alternative to fossil fuels. However, the commercialization of biohydrogen energy is hindered by the high production cost and low yield which necessitates novel strategies for an economically feasible production. Some of these strategies include the development of stable inoculum, scale-up studies, and the utilization of renewable feedstock such as agro-food waste materials which are both abundant and sustainable. Inoculum pre-treatment is a vital aspect of hydrogen production technology as it contributes to the improvement of hydrogen yield. The inoculum pre-treatment method influences the community structure which in turn affects the microbial metabolism of hydrogen production. This study investigates novel inoculum development techniques and evaluates the feasibility of biohydrogen production from agro waste (potato peels). The linear and interactive effect of these techniques on inoculum efficiency as well as the key process parameters for hydrogen production from potato peels were modelled and optimized. Further assessment of the hydrogen production dynamics at the semi-pilot scale including the microbial community structure were investigated using the 16SrRNA gene clone library sequence analysis. A hybrid inoculum development technique of pH and Autoclave (PHA), pH and Heat shock (PHS) was modelled and optimized using the response surface methodology. The quadratic polynomial models had a coefficient of determination (R2) of 0.93 and 0.90 and the optimized pre-treatment conditions gave a 37.7% and 15.3% improvement on model predictions for PHA and PHS respectively. Maximum hydrogen yield of 1.19 mol H2/ mol glucose was obtained for PHA in a semi-pilot scale process. The interactive effect of a hybrid pH and microwave pre-treatment on mixed inoculum for biohydrogen production was investigated. The obtained model had a coefficient of determination (R2) of 0.87. Two semi pilot scale-up processes were carried out to assess the efficiency of the developed inoculum with and without pH control on biohydrogen production. A two fold increase in glucose utilization was obtained and a molar hydrogen yield of 2.07 mol H2/mol glucose under pH controlled fermentation compared to 1.78 mol H2/mol glucose without pH control. Methane production was not detected which suggests the effectiveness of the combined pre-treatment to enrich hydrogen producing bacteria. The developed inoculum was used to evaluate the feasibility of biohydrogen production from potato peels waste. The key process parameters of substrate concentration (g/L), nutrient supplementation (%), temperature (°C) and pH were modelled and optimized using the Artificial Neural Network (ANN) and Response surface methodology (RSM). The optimum conditions obtained were 50g/L of potato waste, 10% nutrients, 30°C and pH 6.5. A semi pilot production process under the optimized condition gave a hydrogen yield of 239.94mL/g TVS corresponding to a 28.5% improvement on hydrogen yield. Analysis of the microbial community structure showed the dominance of the genus Clostridium comprising of about 86% of the total microbial population including C. aminovalericum, C. intestinale, C. tertium, C. sartagofome, C. beijerinckii and C. butyricum in ascending order of predominance. Hydrogen consuming methanogens were not detected which further confirmed the efficiency of the hybrid inoculum pre-treatment. This study has highlighted the development of a novel hybrid inoculum pretreatment method to establish the requisite microbial community and to safeguard the stability of biohydrogen production. Furthermore, the potential of generating an economical feasible biohydrogen production process from potato waste was demonstrated in this work

Book of abstracts of the 10th International Chemical and Biological Engineering Conference: CHEMPOR 2008

This book contains the extended abstracts presented at the 10th International Chemical and Biological Engineering Conference - CHEMPOR 2008, held in Braga, Portugal, over 3 days, from the 4th to the 6th of September, 2008. Previous editions took place in Lisboa (1975, 1889, 1998), Braga (1978), Póvoa de Varzim (1981), Coimbra (1985, 2005), Porto (1993), and Aveiro (2001). The conference was jointly organized by the University of Minho, “Ordem dos Engenheiros”, and the IBB - Institute for Biotechnology and Bioengineering with the usual support of the “Sociedade Portuguesa de Química” and, by the first time, of the “Sociedade Portuguesa de Biotecnologia”. Thirty years elapsed since CHEMPOR was held at the University of Minho, organized by T.R. Bott, D. Allen, A. Bridgwater, J.J.B. Romero, L.J.S. Soares and J.D.R.S. Pinheiro. We are fortunate to have Profs. Bott, Soares and Pinheiro in the Honor Committee of this 10th edition, under the high Patronage of his Excellency the President of the Portuguese Republic, Prof. Aníbal Cavaco Silva. The opening ceremony will confer Prof. Bott with a “Long Term Achievement” award acknowledging the important contribution Prof. Bott brought along more than 30 years to the development of the Chemical Engineering science, to the launch of CHEMPOR series and specially to the University of Minho. Prof. Bott’s inaugural lecture will address the importance of effective energy management in processing operations, particularly in the effectiveness of heat recovery and the associated reduction in greenhouse gas emission from combustion processes. The CHEMPOR series traditionally brings together both young and established researchers and end users to discuss recent developments in different areas of Chemical Engineering. The scope of this edition is broadening out by including the Biological Engineering research. One of the major core areas of the conference program is life quality, due to the importance that Chemical and Biological Engineering plays in this area. “Integration of Life Sciences & Engineering” and “Sustainable Process-Product Development through Green Chemistry” are two of the leading themes with papers addressing such important issues. This is complemented with additional leading themes including “Advancing the Chemical and Biological Engineering Fundamentals”, “Multi-Scale and/or Multi-Disciplinary Approach to Process-Product Innovation”, “Systematic Methods and Tools for Managing the Complexity”, and “Educating Chemical and Biological Engineers for Coming Challenges” which define the extended abstracts arrangements along this book. A total of 516 extended abstracts are included in the book, consisting of 7 invited lecturers, 15 keynote, 105 short oral presentations given in 5 parallel sessions, along with 6 slots for viewing 389 poster presentations. Full papers are jointly included in the companion Proceedings in CD-ROM. All papers have been reviewed and we are grateful to the members of scientific and organizing committees for their evaluations. It was an intensive task since 610 submitted abstracts from 45 countries were received. It has been an honor for us to contribute to setting up CHEMPOR 2008 during almost two years. We wish to thank the authors who have contributed to yield a high scientific standard to the program. We are thankful to the sponsors who have contributed decisively to this event. We also extend our gratefulness to all those who, through their dedicated efforts, have assisted us in this task. On behalf of the Scientific and Organizing Committees we wish you that together with an interesting reading, the scientific program and the social moments organized will be memorable for all.Fundação para a Ciência e a Tecnologia (FCT

Design and Evaluation of an Optimal Controller for Simultaneous Saccharification and Fermentation Process

Ethanol from corn is produced using dry grind corn process in which simultaneous saccharification and fermentation (SSF) is one of the most critical unit operations. In this work an optimal controller based on a previously validated SSF model was developed by formulating the SSF process as a Bolza problem and using gradient descent methods. Validation experiments were performed to evaluate the performance of optimal controller under different process disturbances that are likely to occur in practice. Use of optimal control algorithm for the SSF process resulted in lower peak glucose concentration, similar ethanol yields (13.38 +/- 0.36% v/v and 13.50 +/- 0.15% v/v for optimally controlled and baseline experiments, respectively). Optimal controller improved final ethanol concentrations as compared to process without optimal controller under conditions of temperature (13.35 +/- 1.28 and 12.52 +/- 1.19% v/v for optimal and no optimal control, respectively) and pH disturbances (12.65 +/- 0.74 and 11.86 +/- 0.49% v/v for optimal and no optimal control, respectively). Cost savings due to lower enzyme usage and reduced cooling requirement were estimated to be up to $1 million for a 151 million L/yr (40 million gal/yr) dry grind plant.Keywords: Cybernetic model, SSF process, Process disturbances, Saccharomyces cerevisiae, Optimal controller, Gradient descent, Dry grind corn ethano