Experimental design methodologies for the identification of Michaelis-Menten type kinetics

The main objective of this work was to investigate the application of experimental design techniques for the identification of Michaelis-Menten kinetic parameters. More specifically, this study attempts to elucidate the relative advantages/disadvantages of employing complex experimental design techniques in relation to equidistant sampling when applied to different reactor operation modes. All studies were supported by simulation data of a generic enzymatic process that obeys to the Michaelis-Menten kinetic equation. Different aspects were investigated, such as the influence of the reactor operation mode (batch, fed-batch with pulse wise feeding and fed-batch with continuous feeding) and the experimental design optimality criteria on the effectiveness of kinetic parameters identification. The following experimental design optimality criteria were investigated: 1) minimization of the sum of the diagonal of the Fisher information matrix (FIM) inverse (A-criterion), 2) maximization of the determinant of the FIM (D-criterion), 3) maximization of the smallest eigenvalue of the FIM (E-criterion) and 4) minimization of the quotient between the largest and the smallest eigenvalue (modified E-criterion). The comparison and assessment of the different methodologies was made on the basis of the Cramér-Rao lower bounds (CRLB) error in respect to the parameters vmax and Km of the Michaelis-Menten kinetic equation. In what concerns the reactor operation mode, it was concluded that fed-batch (pulses) is better than batch operation for parameter identification. When the former operation mode is adopted, the vmax CRLB error is lowered by 18.6 % while the Km CRLB error is lowered by 26.4 % when compared to the batch operation mode. Regarding the optimality criteria, the best method was the A-criterion, with an average vmax CRLB of 6.34 % and 5.27 %, for batch and fed-batch (pulses), respectively, while presenting a Km’s CRLB of 25.1 % and 18.1 %, for batch and fed-batch (pulses), respectively. As a general conclusion of the present study, it can be stated that experimental design is justified if the starting parameters CRLB errors are inferior to 19.5 % (vmax) and 45% (Km), for batch processes, and inferior to 42 % and to 50% for fed-batch (pulses) process. Otherwise equidistant sampling is a more rational decision. This conclusion clearly supports that, for fed-batch operation, the use of experimental design is likely to largely improve the identification of Michaelis-Menten kinetic parameters.

Contribuição para o estudo da influência do dióxido de carbono no processo de biodegradação (reactor fed-batch proporcional)

O reactor “Fed-batch” Proporcional utiliza o aumento de pressão que se verifica no interior do reactor, provocado pela acumulação do dióxido de carbono produzido no decurso da degradação aeróbia de um composto orgânico, para adicionar substrato ao reactor, sendo a alimentação proporcional à velocidade ou taxa de degradação de substrato. Nestas circunstâncias, e pretendendo-se avaliar da fiabilidade daquele tipo de reactor, era necessário verificar se a reacção biológica era perturbada pela acumulação de dióxido de carbono. Assim, o presente trabalho teve por objectivo estudar a influência do dióxido de carbono, dissolvido na solução de fermentação, no crescimento microbiano e no consumo de substrato, através da comparação do funcionamento, em paralelo, de dois reactores “fed-batch”, sendo um proporcional e outro aberto. Constatou-se que os valores das constantes cinéticas, taxa específica de consumo de substrato (qobs) e coeficiente de rendimento celular (Y(X/S)), determinados no reactor “Fed-batch” Proporcional e num reactor “Fed-batch” Aberto, operados em condições equivalentes, eram semelhantes. Os valores da taxa de crescimento específica (μobs) apresentam diferenças mais significativas, no entanto a maioria dos testes estatísticos não-paramétricos aplicados demonstraram que o conjunto de valores de cada reactor pertencem à mesma distribuição. A taxa de consumo de oxigénio (OUR), que reflecte a viabilidade da biomassa, é normalmente superior no reactor “Fed-batch” Aberto. Os resultados obtidos no presente estudo não evidenciaram efeitos inibidores, para a reacção biológica, provocados pelo dióxido de carbono dissolvido, ou pelos iões bicarbonato que se acumulam no reactor “Fed-batch” Proporcional.

Numerical Method to Predict Void Formation during The Liquid Composite Molding Process

Void formation during the injection phase of the liquid composite molding process can be explained as a consequence of the non-uniformity of the flow front progression. This is due to the dual porosity within the fiber perform (spacing between the fiber tows is much larger than between the fibers within in a tow) and therefore the best explanation can be provided by a mesolevel analysis, where the characteristic dimension is given by the fiber tow diameter of the order of millimeters. In mesolevel analysis, liquid impregnation along two different scales; inside fiber tows and within the open spaces between the fiber tows must be considered and the coupling between the flow regimes must be addressed. In such cases, it is extremely important to account correctly for the surface tension effects, which can be modeled as capillary pressure applied at the flow front. Numerical implementation of such boundary conditions leads to illposing of the problem, in terms of the weak classical as well as stabilized formulation. As a consequence, there is an error in mass conservation accumulated especially along the free flow front. A numerical procedure was formulated and is implemented in an existing Free Boundary Program to reduce this error significantly.

Application of the EcoBlock method to eco-design - electric hand dryers versus paper towels

Sustainable Construction, Materials and Practice, p. 426-432

Preparation of gas-filled porous microparticles (GPPs) and microbubbles (MBs) by PGSS method

Dissertation to obtain the Master Degree in Biotechnology

The sum-of-the-parts method: An international application

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Finance from the NOVA – School of Business and Economics

What is the best accounting method for financial assets?

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Finance from the NOVA – School of Business and Economics

Speed control of induction machine based on direct torque control method

Dissertação para obtenção do Grau de Mestre em Engenharia Electrotécnica e de Computadores

Food waste valorization through the production of polyhydroxyalkanoates by mixed microbial cultures

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

A nanopore-based stochastic detection method: Single molecule characterisation of nanoparticles using ()- hemolysin

Dissertation presented to obtain the Ph.D degree in Chemistry.

Almirante: Evaluating the board game: Board game, startup, discounted cash flow, venture capital method, real options

This thesis evaluates a start-up company (Jogos Almirante Lda) whose single asset is a board game named Almirante. It aims to conclude whether it makes sense to create a company or just earn copyrights. The thesis analyzes the board game’s market, as part of the general toy’s market, from which some data exists: European countries as well as the USA. In this work it is analyzed the several ways to finance a start-up company and then present an overview of the valuation of the Jogos Almirante based on three different methods: Discounted Cash Flow, Venture Capital Method and Real Options.

Development of process control strategies exploiting knowledge from systems biology: application to MDCK suspension cells

Madine Darby Canine Kidney (MDCK) cell lines have been extensively evaluated for their potential as host cells for influenza vaccine production. Recent studies allowed the cultivation of these cells in a fully defined medium and in suspension. However, reaching high cell densities in animal cell cultures still remains a challenge. To address this shortcoming, a combined methodology allied with knowledge from systems biology was reported to study the impact of the cell environment on the flux distribution. An optimization of the medium composition was proposed for both a batch and a continuous system in order to reach higher cell densities. To obtain insight into the metabolic activity of these cells, a detailed metabolic model previously developed by Wahl A. et. al was used. The experimental data of four cultivations of MDCK suspension cells, grown under different conditions and used in this work came from the Max Planck Institute, Magdeburg, Germany. Classical metabolic flux analysis (MFA) was used to estimate the intracellular flux distribution of each cultivation and then combined with partial least squares (PLS) method to establish a link between the estimated metabolic state and the cell environment. The validation of the MFA model was made and its consistency checked. The resulted PLS model explained almost 70% of the variance present in the flux distribution. The medium optimization for the continuous system and for the batch system resulted in higher biomass growth rates than the ones obtained experimentally, 0.034 h-1 and 0.030 h-1, respectively, thus reducing in almost 10 hours the duplication time. Additionally, the optimal medium obtained for the continuous system almost did not consider pyruvate. Overall the proposed methodology seems to be effective and both proposed medium optimizations seem to be promising to reach high cell densities.

Production of polyhydroxyalkanoates from oil-containing substrates

Different oil-containing substrates, namely, used cooking oil (UCO), fatty acids-byproduct from biodiesel production (FAB) and olive oil deodorizer distillate (OODD) were tested as inexpensive carbon sources for the production of polyhydroxyalkanoates (PHA) using twelve bacterial strains, in batch experiments. The OODD and FAB were exploited for the first time as alternative substrates for PHA production. Among the tested bacterial strains, Cupriavidus necator and Pseudomonas resinovorans exhibited the most promising results, producing poly-3-hydroxybutyrate, P(3HB), form UCO and OODD and mcl-PHA mainly composed of 3-hydroxyoctanoate (3HO) and 3-hydroxydecanoate (3HD) monomers from OODD, respectively. Afterwards, these bacterial strains were cultivated in bioreactor. C. necator were cultivated in bioreactor using UCO as carbon source. Different feeding strategies were tested for the bioreactor cultivation of C. necator, namely, batch, exponential feeding and DO-stat mode. The highest overall PHA productivity (12.6±0.78 g L-1 day-1) was obtained using DO-stat mode. Apparently, the different feeding regimes had no impact on polymer thermal properties. However, differences in polymer‟s molecular mass distribution were observed. C. necator was also tested in batch and fed-batch modes using a different type of oil-containing substrate, extracted from spent coffee grounds (SCG) by super critical carbon dioxide (sc-CO2). Under fed-batch mode (DO-stat), the overall PHA productivity were 4.7 g L-1 day-1 with a storage yield of 0.77 g g-1. Results showed that SCG can be a bioresource for production of PHA with interesting properties. Furthermore, P. resinovorans was cultivated using OODD as substrate in bioreactor under fed-batch mode (pulse feeding regime). The polymer was highly amorphous, as shown by its low crystallinity of 6±0.2%, with low melting and glass transition temperatures of 36±1.2 and -16±0.8 ºC, respectively. Due to its sticky behavior at room temperature, adhesiveness and mechanical properties were also studied. Its shear bond strength for wood (67±9.4 kPa) and glass (65±7.3 kPa) suggests it may be used for the development of biobased glues. Bioreactor operation and monitoring with oil-containing substrates is very challenging, since this substrate is water immiscible. Thus, near-infrared spectroscopy (NIR) was implemented for online monitoring of the C. necator cultivation with UCO, using a transflectance probe. Partial least squares (PLS) regression was applied to relate NIR spectra with biomass, UCO and PHA concentrations in the broth. The NIR predictions were compared with values obtained by offline reference methods. Prediction errors to these parameters were 1.18 g L-1, 2.37 g L-1 and 1.58 g L-1 for biomass, UCO and PHA, respectively, which indicates the suitability of the NIR spectroscopy method for online monitoring and as a method to assist bioreactor control. UCO and OODD are low cost substrates with potential to be used in PHA batch and fed-batch production. The use of NIR in this bioprocess also opened an opportunity for optimization and control of PHA production process.

Cell functional enviromics: applications to Pichia pastoris

The present PhD thesis develops the cell functional enviromics (CFE) method to investigate the relationship between environment and cellular physiology. CFE may be defined as the envirome-wide cellular function reconstruction through the collection and systems-level analysis of dynamic envirome data. Throughout the thesis, CFE is illustrated by two main applications to cultures of a constitutive P. pastoris X33 strain expressing a scFv antibody fragment. The first application addresses the challenge of culture media development. A dataset was built from 26 shake flask experiments, with variations in trace elements concentrations and basal medium dilution based on the standard BSM+PTM1. Protein yield showed high sensitivity to culture medium variations, while biomass was essentially determined by BSM dilution. High scFv yield was associated with high overall metabolic fluxes through central carbon pathways concomitantly with a relative shift of carbon flux from biosynthetic towards energy-generating pathways. CFE identified three cellular functions (growth, energy generation and by-product formation) that together described 98.8% of the variance in observed fluxes. Analyses of how medium factors relate to identified cellular functions showed iron and manganese at concentrations close to PTM1 inhibit overall metabolic activity. The second application addresses bioreactor operation. Pilot 50 L fed-batch cultivations, followed by 1H-NMR exometabolite profiling, allowed the acquisition of data for 21 environmental factors over time. CFE identified five major metabolic pathway groups that are frequently activated by the environment. The resulting functional enviromics map may serve as template for future optimization of media composition and feeding strategies for Pichia pastoris. The present PhD thesis is a step forward towards establishing the foundations of CFE that is still at its infancy. The methods developed herein are a contribution for changing the culture media and process development paradigm towards a holistic and systematic discipline in the future.

Lag-user method: Using laggards to leverage user innovation

Laggards are the last users to adopt a product. Prior literature on user-led innovation ignores laggards’ impact on innovation. In this paper, we develop the Lag-User Method, through which laggards can generate new ideas. Through six studies with 62 teams in three countries, we apply the method to different technologies and services and present our findings to executives to get managerial insights. Findings reveal that laggards who generate new ideas (lag-users) have different perceptions of user-friendly products and different unfulfilled needs. They prefer simple products. We propose that by involving lag-users in NPD, firms can improve the effectiveness of NPD.