Microbial contribution to biofuels production

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

Fermentation of fruit juices by the osmotolerant yeast Candida magnoliae

This study focuses on the assessment of the fermentation conditions required to modulate the metabolic flux in the osmotolerant yeast Candida magnoliae and evaluate its potential to produce low-alcoholic and low-caloric fermented beverages. For that purpose, two strains, PYCC 2903 and PYCC 3191, were used and fermentation conditions as oxygenation, sugar concentration and the ratio of glucose to fructose were studied using synthetic culture media. Candida magnoliae PYCC 2903 was subsequently used to ferment real industrial fructose-rich substrates such as fruit juices. Sugar consumption profiles for C.magnoliae PYCC 2903 incubated aerobically in the presence of high fructose and glucose concentrations (15%, 10% and 5%) showed a selective utilization of fructose, denoting a preference for this sugar over glucose. The lower ratio between ethanol and sugar alcohols yield was obtained for both strains incubated under oxygen limitation simulating industrial fructose-rich substrates, confirming the ability of this yeast to direct fermentation towards alternative products. Enzymatic assays for hexokinase activity in terms of capacity and affinity for glucose and fructose were performed, aiming to elucidate its contribution to the fructophilic behaviour of this yeast. Enzymatic assays for both strains showed that the Vmax is two to threefold higher for fructose than for glucose but Km is also 10-20-fold higher for this sugar than for glucose. Hence, hexokinase kinetic properties do not explain fructophily in C.magnoliae. This indicates that fructose transport is probably determining in this respect, as observed for other fructophilic yeasts. Fruit juice fermentations with C.magnoliae PYCC 2903 revealed a potential for the production of beverages with interesting sensorial properties. Pear and peach fermentations exhibited the best results with the lowest ratio between ethanol and sugar alcohols yield and the most pleasant organoleptic features.

Consolidation and chromatic reintegration of historical renders with lime-based pozzolanic products

Historical renders are exposed to several degradation processes that can lead to a wide range of anomalies,such as scaling, detachments, and pulverization. Among the common anomalies, the loss of cohesion and of adhesion are usually identified as the most difficult to repair; these anomalies still need to be deeply studied to design compatible, durable, and sustainable conservation treatments. The restitution of render cohesion can be achieved using consolidating products. Nevertheless, repair treatments could induce aesthetic alterations, and, therefore, are usually followed by chromatic reintegration. This work aims to study the effectiveness of mineral products as consolidants for lime-based mortars and simultaneously as chromatic treatments for pigmented renders. The studied consolidating products are prepared by mixing air lime,metakaolin, water, and mineral pigments. The idea of these consolidating and coloring products rises from a traditional lime-based technique, the limewash, widely diffused in southern Europe and in the Mediterranean area. Consolidating products were applied and tested on lime-based mortar specimens with a low binder–aggregate ratio and therefore with reduced cohesion. A physico-mechanical, microstructural, and mineralogical characterization was performed on untreated and treated specimens, in order to evaluate the efficacy and durability of the treatments. Accelerated aging tests were also performed to assess consolidant durability, when subjected to aggressive conditions. Results showed that the consolidants tested are compatible, effective, and possess good durability.

Toxicity of carcinogenic and non-carcinogenic Polycyclic Aromatic Hydrocarbons and their mixtures to aquatic organisms under ecologically-relevant scenarios

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous priority pollutants that tend to be trapped in aquatic sediments due to their high hydrophobicity. Nonetheless, the differential toxicological effects and mechanisms between the various classes of PAHs and their mixtures, as they invariably occur in the environment, are scarcely known, especially under ecologically-relevant scenarios. This thesis aimed at establishing a bridge between the study of mechanistic pathways and environmental monitoring of carcinogenic and non-carcinogenic PAHs, by introducing ecological-relevance in the research with model PAHs. A first bioassay conducted in situ with the mussel Mytilus edulis demonstrated that, dredging operations in harbours increase PAH bioavailability, eliciting genotoxicity, and showed that established environmental guidelines underestimate risk. Subsequent ex situ bioassays were performed with the carcinogenic benzo[b]fluoranthene (B[b]F) and non-carcinogenic phenantrene (Phe), selected following preceding results, and revealed that low-moderate concentrations of these PAHs in spiked sediments induce genotoxic effects to the clam Ruditapes decussatus, therefore contradicting the general notion that bivalves are less sensitive to PAHs than vertebrates due to inefficient bioactivation. Also, it was demonstrated that passive samplers permit inferring on PAH bioavailability but not on bioaccumulation or toxic effects. On the other hand, sea basses (Dicentrarchus labrax), yielded a complex pattern of effects and responses, relatively to genotoxicity, oxidative stress and production of specific metabolites, especially when exposed to mixtures of the PAHs which led to additive, if not synergistic, effects. It was shown that Phe may elicit significant genotoxicity especially in presence of B[b]F, even though the low, albeit realistic, exposure concentrations diluted dose- and time-independent relationships. The present work demonstrated that environmental quality guidelines underestimate the effects of PAHs in realistic scenarios and showed that the significant genotoxic and histopathological effects caused by mixed PAHs may not be reflected by oxidative stress- or CYP-related biomarkers. Besides important findings on the metabolism of PAH mixtures, the work calls for the need to re-evaluate the criteria for assessing risk and for the disclosure of more efficient indicators of toxicological hazard.

Brucelose na Província do Namibe, Angola, 2012 : prevalências humana (profissionais da pecuária) e animal, factores de risco, conhecimento e práticas

RESUMO - Enquadramento: A Brucelose é uma antropozoonose prevalente no Mundo e é uma das mais negligenciadas. A sua transmissão ao ser humano é directa e indirecta, e acontece por via de contacto com animal infectado, o consumo de leite e seus derivados não pasteurizados e a não observância de uso de equipamentos de protecção individual e colectiva, entre outros factores. O conhecimento da prevalência e incidência da brucelose animal e humana no Namibe, uma província de Angola, é muito escasso sendo poucos os estudos que evidenciam esta doença no seio dos profissionais da pecuária expostos: trabalhadores de matadouros, veterinários e criadores de gado. É assim pertinente, com base em estudos científicos específicos, caracterizar esta situação. Objectivos: Caracterizar os ambientes dos profissionais (matadouro, talhos e salas municipais de abate e explorações); estimar a seroprevalência da brucelose humana em profissionais da pecuária (trabalhadores de matadouros e criadores de gado bovino) na província do Namibe, Angola em 2012; determinar a associação da presença da brucelose humana com variáveis sócio-demográficas, de conhecimento, de práticas e de características das explorações; determinar a prevalência da Brucelose em animais e em explorações; caracterizar os factores associados à presença da Brucelose em explorações bovinas; caracterizar o conhecimento e práticas sobre a Brucelose dos profissionais da pecuária e analisar a relação entre as prevalências nas explorações (infectadas versus não infectadas) e nos criadores (infectados versus não infectados). Métodos e materiais: estudos observacional e transversal seroepidemiológico em 131 trabalhadores de talhos, salas de abate e matadouro e 192 criadores amostrados aleatoriamente em toda província do Namibe. Os dados foram obtidos através da colheita de sangue e da aplicação de um questionário. Os testes laboratoriais utilizados foram o Rosa de Bengala (RBT) e a Aglutinação Lenta em Tubos (SAT). O estudo de conhecimento foi principalmente centrado na pergunta “Já ouviu falar de Brucelose” e nas questões relativas ao nível de conhecimento e práticas (indicadores baseados nas percentagens de respostas correctas ou práticas adequadas) dos factores de risco da Brucelose. Também foram investigados 1344 animais (em 192 explorações) com recurso ao método de diagnóstico laboratorial RBT para análise de soro sanguíneo e, complementarmente, foi aplicado um questionário aos respectivos criadores. Em termos de análise estatística, para além da abordagem descritiva, foram utilizados os testes de Independência do Quiquadrado, Fisher, Teste não paramétrico de Mann-Whitney, Teste de correlação de Spearman. Adicionalmente, com base em modelos de regressão logística, foram determinados odds ratio e os respectivos intervalos de confiança utilizando um nível de significância de 5%. Resultados: os ambientes dos profissionais (matadouro, talhos e salas municipais de abate e explorações) não reuniram as condições higio-sanitárias definidas internacionalmente como adequadas. Nos profissionais a infecção geral ponderada da Brucelose foi de 15.56% (IC95% : 13.61-17.50), sendo 5.34% em trabalhadores e 16.66% (IC95% : 11.39-21.93) em criadores. A significância estatística foi observada entre a seroprevalência humana e a categoria (trabalhador e criador) (p< 0.001) e o nível de instrução (p= 0.032), início de actividade (p= 0.079) e local de serviço (p= 0.055). Num contexto multivariado o factor positivamente associado à brucelose em profissionais foi a categoria profissional (OR = 3.54, IC95%: 1.57-8.30, relativo aos criadores em relação a trabalhadores). As taxas gerais aparentes de prevalência em animais e explorações foram respectivamente de 14.96% (IC 95%, 12.97-17.19) e de 40.10% (IC 95%, 32.75-47.93). Encontrou-se uma correlação positiva moderada entre o número de animais infectados por exploração com a média do número de abortos na exploração = 0.531, p< 0.001). Em média os profissionais tiveram um conhecimento global muito insuficiente (16.1%), tendo os trabalhadores apresentado valores mais elevados que os criadores (20.2% e 13.8%), diferença não estatisticamente significativa (p= 0.170). As perguntas “o leite in natura é fervido antes do consumo humano?”, “contacto com materiais fetais animais?”, “contacto com aerossóis no local de trabalho?” e “já fez alguma vez o teste de Brucelose humana?” (relacionadas com práticas) e as perguntas “já ouviu falar da Brucelose?”, “Brucelose é doença zoonótica/só animal/só humana? e “como a Brucelose se transmite aos humanos?” apresentaram níveis médios de práticas adequadas e conhecimentos correctos inferiores a 20%. Nas explorações infectadas, 39% dos criadores foram positivos (infectados) e nas não infectadas apenas 1.7%. O risco de um criador ser infectado estando numa exploração infectada foi significativamente mais elevado (OR= 36, IC95%: 8.28-157.04). Conclusões: os ambientes dos profissionais (matadouros, salas municipais de abate e talhos e explorações) propiciam o risco à brucelose. O estudo permite aferir que a Brucelose humana em profissionais da pecuária e a Brucelose animal são prevalentes na província do Namibe. Os níveis de seroprevalência detectados são elevados comparandoos com outros encontrados em algumas localidades africanas que possuem condições similares às do Namibe. Perto de duas em cada cinco (40.10%) explorações estão infectadas por esta doença. O número de abortos (média) está claramente relacionado com as explorações infectadas. O conhecimento geral dos profissionais da pecuária sobre a Brucelose é muito insuficiente, tendo os trabalhadores mostrado um maior conhecimento em relação aos criadores, mas ambos com níveis alarmantes. Os criadores infectados estão relacionados com as explorações infectadas. Há necessidade de controlar a doença e de informar e educar os profissionais sobre a brucelose, sendo fundamental que os serviços provinciais de veterinária reforcem acções de divulgação e de fiscalização.

Photochemical degradation of triclosan: a comparison between different light sources

New emerging contaminants could represent a danger to the environment and Humanity with repercussions not yet known. One of the major worldwide pharmaceutical and personal care productions are antimicrobials products, triclosan, is an antimicrobial agent present in most products. Despite the high removal rate of triclosan present in wastewater treatments, triclosan levels are on the rise in the environment through disposal of wastewater effluent and use of sewage sludge in land application. Regulated in the EC/1272/2008 (annex VI, table 3.1), this compound is considered very toxic to aquatic life and it has been reported that photochemical transformation of triclosan produces dioxins. In the current work it was defined three objectives; determination of the most efficient process in triclosan degradation, recurring to photochemical degradation methods comparing different sources of light; identification of the main by-products formed during the degradation and the study of the influence of the Fenton and photo-Fenton reaction. Photochemical degradation methods such as: photocatalysis under florescent light (UV), photocatalysis under visible light (sunlight), photocatalysis under LEDs, photo-Fenton and Fenton reaction have been compared in this work. The degradation of triclosan was visualized through gas chromatography/mass spectrometry (GC/MS). In this study photo-Fenton reaction has successfully oxidized triclosan to H2O and CO2 without any by-products within 2 hours. Photocatalysis by titanium dioxide (TiO2) under LEDs was possible, having a degradation rate of 53% in an 8 hours essay. The degradation rate of the Fenton reaction, UV light and sunlight showed degradation between 90% and 95%. The results are reported to the data observed without statistic support, since this was not possible during the work period. Hydroquinone specie and 2,4-dichlorophenol by-products were identified in the first hour of photocatalysis by UV. A common compound, possibly identified has C7O4H , was present at the degradation by UV, sunlight and LEDs and was concluded to be a contaminant. In the future more studies in the use of LEDs should be undertaken given the advantages of long durability and low consumption of energy of these lamps and that due to their negative impact on the environment fluorescent lamps are being progressively made unavailable by governments, requiring new solutions to be found. Fenton and photo-Fenton reactions can also be costly processes given the expensive reagents used.

Support in decision-making under uncertainty: a project risk assessment tool development and supplementary advances

A foremost dispute that persists on the contemporary world’s agenda is change. The on-going social/technological/economic changes create a competitive and challenging environment for companies to endure. To benefit from these changes, world economies partially depend on emerging Small and Medium Enterprises (SMEs) and their adaptability skills, and subsequently the development of an integrated capability to innovate has become the prime strategy for most of SMEs to subsist and grow. However, innovation and change are always somewhat bonded to an inherent risk development, which subsequently brings on the necessity of a revision of risk management approaches in innovative processes, whose importance SMEs tend to disregard. Additionally, little efforts have been made to improve and create empirical models, metrics and tools to assist SMEs managing latent risks in their innovative projects. This work seeks to present and discuss a solution to support SMEs in engaging on systematic risk management practices, which consists on an integrated risk assessment and response support web-based tool - Spotrisk® - designed for SMEs. On the other hand, an inherent subjectivity is linked with risk management and identification processes, due to uncertainty trait of its nature, for each individual perceives situations according to his own idiosyncrasy, which brings complications in normalizing risk profiles and procedures. This essay aims to bring insights concerning the support in decision-making processes under uncertainty, by addressing issues related with the risk behavior character among individuals. To address such issues, subjects of neuroscience or psychology are explored and models to identify such character are proposed, as well as models to improve presented tool. This work attempts to go beyond the restrictive aim of endeavoring on technical improvement dissertation, and in embraces an exploratory conceptualization concerning micro, small and medium businesses’ traits regarding risk characters and project risk assessment tools.

Attractiveness of European masters under Bologna constraints: The South American case

This paper studies strategies to attract students from outside Europe to European preexperience masters. We characterize the value added by such masters through interviews with key players at the universities and multinational recruiting corporations. We considered a strategy for segmenting international students in the US and extended it to the European market. We have analyzed data from international applications to Nova SBE as a proxy for applications in European institutions. Based on that analysis we conclude with recommendations to attract suitable candidates from outside Europe. In particular we also provided three different solutions to attract students from the southern hemisphere: we conclude that European institutions should (a) increase the spring semester intake, (b) provide bridging courses for some students, or (c) could place some accepted candidates in internships before starting classes.

Fiscal policy under financial stress: A threshold VAR approach

The purpose of the project is to measure the impact of fiscal policy on the Portuguese GDP and how it may vary according to the state of the financial market. A Threshold VAR model is presented in which the two regimes are found using a financial stress index that divides the economy into a situation of financial stress and financial stability.

Prospective system analysis of stationary battery systems under the frame of Constructive Technology Assessment

Based on the report for the unit “Project IV” of the PhD programme on Technology Assessment under the supervision of Dr.-Ing. Marcel Weil and Prof. Dr. António Brandão Moniz. The report was presented and discussed at the Doctorate Conference on Technologogy Assessment in July 2013 at the University Nova Lisboa, Caparica campus.

Structured products in the current low interest rate environment

Directed Research Internship

Evaluating the potential of yeast strains to produce added value products for the food and/or pharmaceutical industries

This study focus in the valorization of the apple pomace with the main goal of obtaining added value products. For that, hot compressed water technology was used for the extraction of phenolic compounds and hydrolysis of polysaccharides presents in the lignocellulosic structure of apple pomace to obtain simple sugars. The sugars have been utilized as alternative carbon source for growth, lipid accumulation and carotenoids production by five different yeast Yarrowia lipolytica, Rhodotorula mucilaginosa, Rhodotorula glutinis, Rhodosporidium babjevae and Rhodosporidium toruloides. Hydrolysis experiments were carried out with constant pressure of 100 bar, flow rate of 2mL/min and temperatures between 50°C and 250°C. The amount of total sugars present in apple pomace hydrolysates showed maximum values for the hydrolysis temperatures of 110°C and 190°C. In fact, these temperatures revealed the best results regarding the monosaccharides quantities. The amount of 5-HMF and furfural in each hydrolysate varied through the different temperatures. Maximum values for 5-HMF were obtained with 170°C, while furfural showed to be maximum at 210°C. Extraction of phenolic compounds were performed in simultaneously with hydrolysis reactions. Total phenolic compounds (TPC) increased along the temperature, however with small variations between 170°C and 250°C. Hydrolysates were then used as alternative carbon source to yeast growth. R. mucilaginosa shows the highest optical density, with the hydrolysate obtained at 130°C. Carotenoids produced by these yeast scored a total of 7.02μg carotenoids/g cell dry weight, while for the control assay, the same yeast scored 9.31μg caratonoides/g cell dry weight. β-carotene was quantified by HPLC, were 33% of the carotenoid production by R. mucilaginosa with hydrolysate as carbon source, corresponded to β-caroteno.

Valorization of agroindustrial waste

The main objective of this work is the valorization of residues from agro-industry giving them an added value. The valorization was performed by using a "green" and sustainable solvent - supercritical fluid, in this case carbon dioxide. Two residues and one biomass were used to produce two different final products, thereby emphasizing the versatility of the waste recovery - spent coffee grounds and microalgae Chlorella protothecoides to produce biodiesel, and tomato pomace to extract carotenoids. In the first part of this work it was demonstrated the possibility to obtain a conversion of coffee spent grounds oil into biodiesel, through an enzymatic transesterification reaction, of 98.01% with the following operating conditions: molar ratio oil:methanol 1:24, residence time 0.8 min, pressure 25 MPa, temperature 313,15K. In this first phase, it was also used the microalgae Chlorella protothecoides, a biomass, to produce biodiesel and favorable results were obtained with this green process compared with a traditional process - basic catalysis / acid. In the second part of this work, by an extraction with supercritical CO2 it was obtained 3.38% oil from tomato pomace under the following conditions: pressure 35.1 MPa, temperature 313,15K. It was found that this oil contains various carotenoids: β-carotene, lutein and lycopene. The latter is present in larger amount.

“Private label products: A category analysis”

The presented dissertation was developed within a partnership between Nova School of Business and Economics and the Portuguese retailer Sonae MC. The main objective of the study was to develop an analysis for the confectionary category to identify potential development opportunities for new Private Label products. In order to do so, the starting point was to understand how the confectionery market was behaving, followed by and understanding of Continente’s performance in that market. Aiming to point out development opportunities, the analysis was split between the subcategories – Chocolate, Chewing Gums and Sweets. The Subcategory performance was assessed in terms of sales, number of SKU’s, Private Label weight and it market position in terms of share. For the potential development opportunities a comparison between the top selling Branded Product and the competitors’ position was developed, in order to establish a reasonable size and retail price for such products. Key Word: Private Label, Branded Products, Continente, Sonae MC, Retail, SKU’s, Sales, Price, Market Share,

Optimal design and operation of compact simulated moving bed processes for enantioseparations

Simulated moving bed (SMB) chromatography is attracting more and more attention since it is a powerful technique for complex separation tasks. Nowadays, more than 60% of preparative SMB units are installed in the pharmaceutical and in the food in- dustry [SDI, Preparative and Process Liquid Chromatography: The Future of Process Separations, International Strategic Directions, Los Angeles, USA, 2002. http://www. strategicdirections.com]. Chromatography is the method of choice in these ¯elds, be- cause often pharmaceuticals and ¯ne-chemicals have physico-chemical properties which di®er little from those of the by-products, and they may be thermally instable. In these cases, standard separation techniques as distillation and extraction are not applicable. The noteworthiness of preparative chromatography, particulary SMB process, as a sep- aration and puri¯cation process in the above mentioned industries has been increasing, due to its °exibility, energy e±ciency and higher product purity performance. Consequently, a new SMB paradigm is requested by the large number of potential small- scale applications of the SMB technology, which exploits the °exibility and versatility of the technology. In this new SMB paradigm, a number of possibilities for improving SMB performance through variation of parameters during a switching interval, are pushing the trend toward the use of units with smaller number of columns because less stationary phase is used and the setup is more economical. This is especially important for the phar- maceutical industry, where SMBs are seen as multipurpose units that can be applied to di®erent separations in all stages of the drug-development cycle. In order to reduce the experimental e®ort and accordingly the coast associated with the development of separation processes, simulation models are intensively used. One impor- tant aspect in this context refers to the determination of the adsorption isotherms in SMB chromatography, where separations are usually carried out under strongly nonlinear conditions in order to achieve higher productivities. The accurate determination of the competitive adsorption equilibrium of the enantiomeric species is thus of fundamental importance to allow computer-assisted optimization or process scale-up. Two major SMB operating problems are apparent at production scale: the assessment of product quality and the maintenance of long-term stable and controlled operation. Constraints regarding product purity, dictated by pharmaceutical and food regulatory organizations, have drastically increased the demand for product quality control. The strict imposed regulations are increasing the need for developing optically pure drugs.(...)