Monitoring and characterization of yeasts behavior under fermentation processes using technometric approaches

Tese de doutoramento em Chemical and Biological EngineeringTechnometrics concerns on the development and use of statistical methods in different fields, such as biotechnological processes, in order to understand their multivariate and multidimensional complexity. Chemical changes occurring within these processes can be monitored using chemometric tools that combined with bioinformatic methodologies, can provide an enlarged overview of the process, enabling the unbiased study of metabolites and dynamic changes in response to the environmental conditions. For this purpose, different chemometric tools were used, namely relevant principal component analysis (RPCA), multi-way principal component analysis (MPCA), partial least squares logistic regression (PLSLOG) and unfolded partial least squares (U-PLS). Phenotypic and physiological behaviors of three different Saccharomyces cerevisiae strains, a laboratorial S288c, and two industrials CA11 and PE-2, were evaluated under different stress conditions. Toxic and inhibitory conditions were induced by introducing 1.0% (v/v) ethanol, 1-butanol, isopropanol, tert-Amyl alcohol, 0.2% (v/v) furfural and 0.5% (v/v) 5-hydroxymethylfurfural (5-HMF) in batch fermentations with YPD as culture medium. MPCA and PLS-LOG allowed to evidence the different behavior of S288c comparing to PE-2 and CA11, and a higher impact caused by 1-butanol, furfural and 5-HMF in phenotypic and physiological profiles. PE-2 revealed to be the most robust strain, quickly adapting to the environmental conditions, even under the highest stress conditions. It was also observed a correlation between the flocculation profile inhibition under those conditions, with an increased production of intracellular glycerol. This relationship was confirmed by PLS-LOG where intracellular glycerol and trehalose, as well as extracellular acetic acid production showed to be linked to the inhibition of CA11 cells flocculation. Metabolic changes occurring within CA11 and PE-2 fermentations in the presence of 1-butanol, furfural and 5-HMF were also evaluated, using RPCA. CA11 fermentations enhanced the production of ethanol, isovaleric acid and isoamyl acetate, whereas PE-2 favored the production of more aromatic compounds, such as esters - phenylethyl acetate, ethyl hexanoate, ethyl octanoate and ethyl dodecanoate. These results suggested that PE-2 is less susceptible to the stress effect of the three tested molecules. PLS-LOG models allowed the prediction (R2 =0.90) of the metabolic behavior of both strains during the fermentations: the presence of 1-butanol induced the production of esters ethyl acetate and isoamyl acetate (and its precursor, 3-methyl-1-butanol), as well as butyric acid (which encourages the use of both strains in bio-butanol production systems); CA11 and PE-2 synthesized furfuryl alcohol from furfural; the presence of furfural and 5-HMF induced the production and accumulation of fatty acids in the medium, to counterbalance the inhibitory effects. The impact of metabolic profile of S. cerevisiae PYCC 4653 on its antioxidant capacity, in synthetic grape juice supplemented with phenolics acids was assessed. A bioanalytical pipeline, combining electrochemical features with biochemical background was proposed, for biological systems fingerprinting and sample classification. The electrochemical profile, phenolic acids and the volatile fermentation fraction, were evaluated for 11 days, using cyclic voltammetry, target and non-target metabolic approaches, respectively. It was found that acetic acid, 2-phenylethanol and isoamyl acetate have a significative contribution for samples metabolic variability and the electrochemical features demonstrated redox-potential changes throughout the alcoholic fermentations, showing at the end, a similar pattern to normal wines. S. cerevisiae also showed the capacity of producing chlorogenic acid in the supplemented medium fermentation from simple precursors present in the minimal medium. The proposed bioanalytical pipeline proved to be a very efficient strategy for fingerprinting biological systems, by integration of the information from different chemical detectors. Finally, a non-targeted high-throughput metabolomics pipeline combining GC-MS data preprocessing with multivariate analysis, was developed and integrated in new “in-house” software, called XMetabolomics (developed during this thesis). The pipeline was built to enhance the identification of key metabolites involved in the process, through the exploration of the temporal relationships between interesting metabolites related to a chemical phenomenon. It was applied to a Port wine “forced aging” process under different oxygen saturation regimes. RPCA showed that the use of extreme oxygen saturation and high temperatures during Port wine aging induced the occurrence of chemical reactions undesirable for the aromatic profile, affecting the quality of the final product. Under those conditions an increased production of dioxane and dioxolane isomers and furfural was observed, leading to excessive degradation of the wine aromatic profile, color and taste. The production of dioxane isomer was highly correlated with the production of dioxolane isomer, benzaldehyde, sotolon, and many other metabolites whose identification could be of great interest for their contribution for the final aromatic profile of the Port wine. In sum, during this thesis, the potential of the use of chemometrics and bioinformatics approaches was explored in the characterization (by RPCA and MPCA), classification and prediction (by PLS-LOG and UPLS, respectively) of physiological, phenotypic and metabolic changes in bioprocesses as an adaptation response to environmental conditions. The joint effect of distinct variables (measured using HPLC, GCFID, GC-MS and cyclic voltammetry) in multivariate data analysis allowed enhancing the knowledge about chemical and biochemical dynamics in biotechnological processes.A tecnometria consiste no desenvolvimento e uso de métodos estatísticos em diferentes áreas, tais como processos biotecnológicos, de modo a compreender a sua complexidade multivariada e multidimensional. As alterações químicas que ocorrem nestes processos podem ser monitorizadas utilizando ferramentas de quimiometria que, associadas a métodos de bioinformática, podem proporcionar uma visão alargada do processo e logo, o estudo equitativo dos metabolitos e as alterações dinâmicas em resposta às condições ambientais. Ao longo deste trabalho, diferentes ferramentas de quimiometria foram utilizadas, nomeadamente, relevant principal component analysis (RPCA), multi-way principal component analysis (MPCA), partial least squares logistic regression (PLS-LOG) e unfolded partial least squares (U-PLS). Foi efetuado o estudo de comportamentos fenotípicos e fisiológicos de três estirpes diferentes de Saccharomyces cerevisiae, uma laboratorial, S288c, e duas industriais, CA11 e PE -2, sob diferentes condições de stress. Foram adicionadas moléculas tóxicas e inibitórias no meio YPD, nomeadamente, 1,0% (v/v) de etanol, 1-butanol, isopropanol e 2-metil-2-butanol, 0,2 % (v/v) de furfural e 0,5 % (v/v) de 5-hidroximetil- furfural (5-HMF). O MPCA e o PLS-LOG evidenciaram o diferente comportamento da estirpe S288c em relação à CA11 e PE-2, e um maior impacto causado pelo 1-butanol, furfural e 5-HMF nos perfis fenotípicos e fisiológicos. A PE-2 revelou ser a estirpe mais robusta e a que melhor se adaptou às condições ambientais impostas, mesmo sob as mais severas. Observou-se uma correlação entre a inibição do perfil de floculação nestas condições, com um aumento da produção de glicerol intracelular. Esta relação foi confirmada utilizando o PLS-LOG onde a produção de glicerol e trealose intracelulares, bem como de ácido acético extracelular mostraram estar associadas ao fenómeno de inibição da floculação das células da CA11. As alterações metabólicas que ocorrem nas fermentações utilizando a CA11 e PE- 2 na presença de 1- butanol, furfural e 5- HMF também foram avaliadas por RPCA. Enquanto a estirpe CA11 favoreceu a produção de etanol, ácido isovalérico e acetato de isoamilo, a PE-2 levou à produção de outros compostos aromáticos, tais como o acetato de feniletilo, etil hexanoato, octanoato e dodecanoato ao longo das fermentações. Estes resultados reforçam que a PE-2 é menos suscetível ao efeito stressante dessas moléculas. Os modelos PLS-LOG permitiram prever (R2 = 0,90) o comportamento metabólico de ambas as estirpes, durante as fermentações: a presença de 1-butanol induziu a produção de ésteres de acetato de etilo e acetato de isoamilo (e o seu precursor, 3-metil -1- butanol), bem como o ácido butírico (encorajando a utilização de ambas as estirpes em sistemas de produção de bio-butanol); as estirpes CA11 e PE-2 sintetizaram álcool furfurílico a partir de furfural; a presença de furfural e 5- HMF induziu a produção e acumulação de ácidos gordos, de forma a contrabalançar os efeitos inibitórios na obtenção de energia para as células, metabolizando ácidos gordos no meio. O impacto do perfil metabólico da S. cerevisiae PYCC 4653 sobre a capacidade antioxidante foi avaliado, em fermentações utilizando sumo de uva sintético suplementadas com ácidos fenólicos. Foi apresentada uma metodologia bio-analítica (combinando os perfis eletroquímico e bioquímico) para a caracterização do comportamento da levedura em resposta às perturbações impostas. O perfil eletroquímico, os ácidos fenólicos e a fração volátil das fermentações, foram avaliados durante 11 dias, utilizando a voltametria cíclica, e abordagens metabólicas supervisionadas e não supervisionadas. Verificou-se que o ácido acético, 2- feniletanol e o acetato de isoamilo têm uma contribuição significativa na variabilidade metabólica e as características electroquímicas revelaram as alterações do potencial redox durante as fermentações. O perfil eletroquímico da fermentação alcoólica mostrou, no final, um padrão semelhante ao dos vinhos reais. A S. cerevisiae também mostrou a capacidade de produzir ácido clorogénico, no meio de fermentação suplementado a partir de precursores simples, presentes no meio mínimo. A metodologia proposta provou ser uma estratégia eficiente na caracterização de fenómenos biológicos e químicos, através da integração da informação de vários detetores químicos. Por fim, uma metodologia de processamento metabólico não-direcionado e de alto-débito, combinando o pré-processamento dos dados de GC-MS com a análise multivariada, foi desenvolvida e integrada num novo software, denominado X-Metabolomics também desenvolvido no decorrer desta tese. A metodologia foi construída para melhorar a identificação dos metabolitos-chave envolvidos no processo biotecnológico, através da exploração das relações temporais entre os metabólitos interessantes relacionados ao mesmo fenómeno químico. Esta foi aplicada a um processo de “envelhecimento forçado” de vinho do Porto, sob diferentes regimes de saturação de oxigénio. O RPCA mostrou que a utilização da saturação extrema de oxigénio e de temperaturas elevadas durante o envelhecimento do vinho do Porto induziu a ocorrência de reações químicas indesejáveis para o perfil aromático, que afetam a qualidade do produto final. Nestas condições, foi observado um aumento da produção de isómeros de dioxano e dioxolano e furfural, que levaram a uma degradação excessiva do perfil aromático, cor e sabor do vinho. A produção do isómero de dioxano está altamente correlacionada com a produção de um isómero dioxolano, benzaldeído, sotolon, e muitos outros metabolitos, cuja identificação poderia ser de grande interesse pela sua contribuição para o perfil aromático final do vinho do Porto. Em suma, durante esta tese, foi explorado o potencial da utilização de abordagens de tecnometria, incluindo métodos de quimiometria e bioinformática, na caracterização (por RPCA e MPCA), classificação e previsão (por PLS-LOG e U-PLS respetivamente) das alterações fisiológicas, fenotípicas e metabólicas em bioprocessos, em resposta às condições ambientais. O efeito conjunto de distintas variáveis na análise multivariada, permitiu ampliar o conhecimento acerca das dinâmicas químicas e bioquímicas em processos biotecnológicos

Saccharomyces cerevisiae oxidative response evaluation by cyclic voltammetry and gas chromatography−mass spectrometry

This study is focused on the evaluation of the impact of Saccharomyces cerevisiae metabolism in the profile of compounds with antioxidant capacity in a synthetic wine during fermentation. A bioanalytical pipeline, which allows for biological systems fingerprinting and sample classification by combining electrochemical features with biochemical background, is proposed. To achieve this objective, alcoholic fermentations of a minimal medium supplemented with phenolic acids were evaluated daily during 11 days, for electrochemical profile, phenolic acids, and the volatile fermentation fraction, using cyclic voltametry, high-performance liquid chromatography-diode array detection, and headspace/solid-phase microextraction/gas chromatography-mass spectrometry (target and nontarget approaches), respectively. It was found that acetic acid, 2-phenylethanol, and isoamyl acetate are compounds with a significative contribution for samples metabolic variability, and the electrochemical features demonstrated redox-potential changes throughout the alcoholic fermentations, showing at the end a similar pattern to normal wines. Moreover, S. cerevisiae had the capacity of producing chlorogenic acid in the supplemented medium fermentation from simple precursors present in the minimal medium.C.C.C. (SFRH/BD/46737/2008) gratefully acknowledges the Fundacao para a Ciencia e Tecnologia (FCT) for her Doctoral grant. This research was funded by the projects: "Open-Microbio (PTDC/BIO/69310/2006)-Framework for Computational Simulation of Cellular Communities during BioProcess Engineering" (FCT), "A Metrica do Vinho: Elucidar o Padrao Molecular Volatile Responsavel pelo aroma "Tipo-Vinho" Fundamental para a Definicao da Qualidade" (PTDC/AGR-ALI/121062/2010), and partially supported by CBMA, IBB, and ESB/UCP plurianual funds through the POS-Conheci-mento Program that includes FEDER funds

Microbial co-culturing strategies for fructo-oligosaccharide production

Fructo-oligosaccharide (FOS) mixtures produced by fermentation contain large amounts of non-prebiotic sugars. Here we propose a mixed culture of Aureobasidium pullulans and Saccharomyces cerevisiae cells to produce FOS and consume the small saccharides simultaneously, thereby increasing FOS purity in the mixture. The use of immobilised A. pullulans in co-culture with encapsulated S. cerevisiae, inoculated after 10h fermentation, enhanced FOS production in a 5L bioreactor. Using this strategy, a maximal FOS concentration of 119g L1, and yield of 0.59gFOS gsucrose1, were obtained after 20h fermentation, increasing FOS productivity from about 4.9 to 5.9gFOS L1h1 compared to a control fermentation of immobilized A. pullulans in monoculture. In addition, the encapsulated S. cerevisiae cells were able to decrease the glucose in the medium to about 7.6% (w/w) after 63h fermentation. This provided a final fermentation mixture with 2.0% (w/w) sucrose and a FOS purity of over 67.0% (w/w). Moreover, a concentration of up to 58.0g L1 of ethanol was obtained through the enzymatic transformation of glucose. The resulting pre-purified FOS mixture could improve the separation and purification of FOS in downstream treatments, such as simulated moving bed chromatography.Cristiana C. Castro gratefully acknowledges the financial support from the F.R.S.-FNRS, the Belgium National Fund for Scientific Research (PDR: T.0196.13). Clarisse Nobre acknowledges the Portuguese Foundation for Science and Technology (FCT) for the postdoctoral grant (reference SFRH/BPD/87498/2012) and the strategic funding of the UID/BIO/04469/2013 unit, Project ColOsH 02/SAICT/ 2017 (POCI-01-0145-FEDER-030071), COMPETE 2020 (POCI-01-0145FEDER-006684), and BioTecNorte operation (NORTE-01-0145-FEDER000004), funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Sensitivity analysis and reduction of a dynamic model of a bioproduction of fructo-oligosaccharides

Starting from a relatively detailed model of a bioprocess producing fructo-oligosaccharides, a set of experimental data collected in batch and fed-batch experiments is exploited to estimate the unknown model parameters. The original model includes the growth of the fungus Aureobasidium pullulans which produces the enzymes responsible for the hydrolysis and transfructosylation reactions, and as such contains 25 kinetic parameters and 16 pseudo-stoichiometric coefficients, which are not uniquely identifiable with the data at hand. The aim of this study is, therefore, to show how sensitivity analysis and quantitative indicators based on the Fisher information matrix can be used to reduce the detailed model to a practically identifiable model. Parametric sensitivity analysis can indeed be used to progressively simplify the model to a representation involving 15 kinetic parameters and 8 pseudo-stoichiometric coefficients. The reduced model provides satisfactory prediction and can be convincingly cross validated.The authors thank the financial support from the F.R.S.-FNRS, the Belgium National Fund for the Scientific Research (Research Project 24643.08). C. Nobre thanks the Fundação para a Ciência e Tecnologia for the strategic funding of UID/BIO/04469 /2013 unit.info:eu-repo/semantics/publishedVersio

Cross Species Analysis and Comparison of Tumors in Dogs and Cats, by Age, Sex, Topography and Main Morphologies. Data from Vet-OncoNet

The animal cancer burden is essential for the translational value of companion animals in comparative oncology. The present work aims to describe, analyze, and compare frequencies and associations of tumors in dogs and cats based on the Animal Cancer Registry created by Vet-OncoNet. With 9079 registries, regarding 2019 and 2020, 81% (n = 7355) belonged to dogs. In comparison, cats have a general one-year right advance in the mean age of cancer diagnosis compared to dogs. The multivariate topography group analysis shows a distinct pattern between the two species: dogs have higher odds of cancer in the genito-urinary system, spleen, soft tissue tumors and skin, while cats show higher odds for tumors in the eyes, digestive organs, nasal cavity, lymph nodes, bones and mammary glands. Regarding morphologies, dogs are overrepresented in mast cell tumors (MCT), melanomas, and hemangiosarcomas. While cats are overrepresented in fibrosarcomas, lymphomas (T and B-cell), in malignant mammary tumors, and squamous cell carcinoma (SCC). Females have greater odds only in the mammary gland, with males having greater odds in six of twelve topographies. This study is the first outcome of continuous animal cancer registration studies in Portugal

Gene expression profile during coffee fruit development and identification of candidate markers for phenological stages

The objective of this work was to identify genes that could be used as suitable markers for molecular recognition of phenological stages during coffee (Coffea arabica) fruit development. Four cultivars were evaluated as to their differential expression of genes associated to fruit development and maturation processes. Gene expression was characterized by both semi-quantitative and quantitative RT-PCR, in fruit harvested at seven different developmental stages, during three different seasons. No size polymorphisms or differential expression were observed among the cultivars for the evaluated genes; however, distinct expression profiles along fruit development were determined for each gene. Four out of the 28 evaluated genes exhibited a regular expression profile in all cultivars and harvest seasons, and, therefore, they were validated as candidate phenological markers of coffee fruit. The gene α-galactosidase can be used as a marker of green stage, caffeine synthase as a marker of transition to green and yellowish-green stages, and isocitrate lyase and ethylene receptor 3 as markers of late maturation

Adherence to treatment in allergic rhinitis using mobile technology. The MASK Study

Background: Mobile technology may help to better understand the adherence to treatment. MASK-rhinitis (Mobile Airways Sentinel NetworK for allergic rhinitis) is a patient-centred ICT system. A mobile phone app (the Allergy Diary) central to MASK is available in 22 countries. Objectives: To assess the adherence to treatment in allergic rhinitis patients using the Allergy Diary App. Methods: An observational cross-sectional study was carried out on all users who filled in the Allergy Diary from 1 January 2016 to 1 August 2017. Secondary adherence was assessed by using the modified Medication Possession Ratio (MPR) and the Proportion of days covered (PDC) approach. Results: A total of 12143 users were registered. A total of 6949 users reported at least one VAS data recording. Among them, 1887 users reported >= 7 VAS data. About 1195 subjects were included in the analysis of adherence. One hundred and thirty-six (11.28%) users were adherent (MPR >= 70% and PDC = 70% and PDC = 1.50) and 176 (14.60%) were switchers. On the other hand, 832 (69.05%) users were non-adherent to medications (MPR Conclusion and clinical relevance: Adherence to treatment is low. The relative efficacy of continuous vs on-demand treatment for allergic rhinitis symptoms is still a matter of debate. This study shows an approach for measuring retrospective adherence based on a mobile app. This also represents a novel approach for analysing medication-taking behaviour in a real-world setting.Peer reviewe