Challenges in mercury speciation and fractionation in soil and sediment

This investigation focused on the development, test and validation of methodologies for mercury fractionation and speciation in soil and sediment. After an exhaustive review of the literature, several methods were chosen and tested in well characterised soil and sediment samples. Sequential extraction procedures that divide mercury fractions according to their mobility and potential availability in the environment were investigated. The efficiency of different solvents for fractionation of mercury was evaluated, as well as the adequacy of different analytical instruments for quantification of mercury in the extracts. Kinetic experiments to establish the equilibrium time for mercury release from soil or sediment were also performed. It was found that in the studied areas, only a very small percentage of mercury is present as mobile species and that mobility is associated to higher aluminium and manganese contents, and that high contents of organic matter and sulfur result in mercury tightly bound to the matrix. Sandy soils tend to release mercury faster that clayey soils, and therefore, texture of soil or sediment has a strong influence on the mobility of mercury. It was also understood that analytical techniques for quantification of mercury need to be further developed, with lower quantification limits, particularly for mercury quantification of less concentrated fractions: water-soluble e exchangeable. Although the results provided a better understanding of the distribution of mercury in the sample, the complexity of the procedure limits its applicability and robustness. A proficiency-testing scheme targeting total mercury determination in soil, sediment, fish and human hair was organised in order to evaluate the consistency of results obtained by different laboratories, applying their routine methods to the same test samples. Additionally, single extractions by 1 mol L-1 ammonium acetate solution, 0.1 mol L-1 HCl and 0.1 mol L-1 CaCl2, as well as extraction of the organometallic fraction were proposed for soil; the last was also suggested for sediment and fish. This study was important to update the knowledge on analytical techniques that are being used for mercury quantification, the associated problems and sources of error, and to improve and standardize mercury extraction techniques, as well as to implement effective strategies for quality control in mercury determination. A different, “non chemical-like” method for mercury species identification was developed, optimised and validated, based on the thermo-desorption of the different mercury species. Compared to conventional extraction procedures, this method has advantages: it requires little to no sample treatment; a complete identification of species present is obtained in less than two hours; mercury losses are almost neglectable; can be considered “clean”, as no residues are produced; the worldwide comparison of results obtained is easier and reliable, an important step towards the validation of the method. Therefore, the main deliverables of this PhD thesis are an improved knowledge on analytical procedures for identification and quantification of mercury species in soils and sediments, as well as a better understanding of the factors controlling the behaviour of mercury in these matrices.

Extracção supercrítica de óleo de grainha de uva combinada com pré-tratamentos enzimáticos

Neste trabalho estudou-se a extracção supercrítica do óleo de grainha de uva, usando dióxido de carbono, e combinou-se este processo com um prétratamento enzimático da semente para aumentar o rendimento global da extracção. A qualidade dos extractos obtidos foi avaliada pelo seu conteúdo em triacilglicerídeos, perfil de ácidos gordos e capacidade antioxidante. Realizaram-se também alguns estudos exploratórios sobre a aplicação de um pré-tratamento de alta pressão (HPP) à grainha da uva. Adicionalmente, efectuou-se o estudo da extracção, fraccionamento e caracterização estrutural das procianidinas da grainha da uva, bem como a avaliação da sua capacidade antioxidante. A extracção de procianidinas da grainha da uva foi efectuada sequencialmente com metanol e acetona/água, tendo sido posteriormente fraccionadas por adição sucessiva de misturas metanol/clorofórmio progressivamente mais concentradas em clorofórmio. A caracterização das procianidinas foi feita por HPLC-UV e LC–MS, antes e depois de sujeitar as amostras a uma tiólise, e também por ESI-MS e ESI-MS/MS. Este estudo permitiu reportar, pela primeira vez, a ocorrência de procianidinas do tipo-A galoiladas na grainha da uva. Os resultados de HPLC-UV permitiram determinar o grau médio de polimerização das procianidinas e a sua composição monomérica em (+)- catequina, (-)-epicatequina e (-)-epicatequina-O-galato. Mostrou-se que a (+)- catequina é o flavan-3-ol terminal mais abundante e a (-)-epicatequina predomina largamente como unidade de extensão. No caso de procianidinas do tipo A, a ligação interflavânica C2-C7 encontra-se essencialmente nas unidades terminais. O grau médio de polimerização das diversas fracções varia entre 1.0 e 10.8. A sua capacidade antioxidante, medida pelo método espectrofotométrico de DPPH•, mostrou-se ser equivalente à de uma amostra comercial de (+)-catequina usada como referência. A partir dos graus médios de polimerização experimentais e das análises de FTIR das fracções correspondentes foi possível obter um modelo preditivo O-PLS com apenas uma variável latente. O pré-tratamento enzimático justificou-se pelo conhecimento existente acerca do uso de enzimas específicas que destroem parcialmente as paredes celulares. Atendendo à composição das paredes celulares da grainha da uva preparou-se uma suspensão contendo protease, xilanase, pectinase e celulase. Para determinar as condições experimentais do pré-tratamento que maximizam o rendimento da extracção, estudou-se o efeito do tempo de reacção, temperatura, pH, diâmetro médio das partículas de grainha moída e a concentração das enzimas. Os incrementos do rendimento da extracção de óleo observados atingiram 163.2%. O estudo da extracção supercrítica (SFE) do óleo da grainha de uva tratada e não-tratada permitiu obter as curvas de extracção correspondentes, bem com analisar a influência das condições operatórias sobre o seu andamento. Montou-se uma instalação laboratorial onde se realizaram experiências com dióxido de carbono a 160, 180, 200 e 220 bar e temperaturas de 313.15 e 323.15 K. Os rendimentos obtidos por SFE foram semelhantes aos de Soxhlet com n-hexano. As curvas de extracção medidas compreendem um primeiro período de extracção, onde se remove cerca de 92-97% do óleo disponível, e um segundo período, essencialmente difusional, com pouco impacto no rendimento final. Os vários extractos recolhidos e o óleo global obtido foram caracterizados para avaliar a sua qualidade e relacioná-la com as condições operatórias de SFE. Determinaram-se o conteúdo total em triacilglicerídeos, o seu perfil de ácidos gordos e a capacidade antioxidante (AOC). Os resultados mostraram que a AOC aumenta com a elevação da pressão e, acentuadamente, com o acréscimo da temperatura. Ao longo da curva de extracção, a AOC é mais pronunciada nos extractos iniciais, nomeadamente nos primeiros 30 a 40% da extracção. A modelação efectuada considerou que o óleo extractável se reparte entre células rompidas, predominantes na periferia da semente, e células intactas, mais interiores. Admitiu-se que o transporte de massa ocorre em série, i.e. das células intactas para as rompidas e destas para o solvente; mostrou-se que a dispersão axial era desprezável. Os balanços materiais à fase fluida e aos volumes de células rompidas e intactas, combinados com os fluxos interno, externo e a relação de equilíbrio foram resolvidos numericamente pelo método das linhas combinado com diferenças finitas atrasadas. O modelo reproduziu bem as curvas experimentais e permitiu simular curvas de eluição e os três perfis de concentração no leito.

Structural characterization and biological activity of polysaccharides from infusions used in the popular tradition

The use of plants with medicinal purposes is an ancient practice still very common in developing regions, and is rapidly spreading in industrialized countries. This fact is evidenced by the large number of ethnobotanical studies found in the literature referring that these plants are often used as decoctions and infusions. In most studies the reported biological activities are attributed to the presence of phenolic compounds, due to their antioxidant properties, and to polysaccharides, with its anti-tumoral properties. In “Trás-os-Montes” region, some of the most popular infusions used by the popular medicine are prepared with the dried leaves of Fraxinus angustifolia, the dried shoots of Mentha suaveolens, and the dried inflorescences of Pterospartum tridentatum. However, there are no studies about the polysaccharides present in these infusions. Thus, through the structural characterization of the polysaccharides present in the infusions of F. angustifolia, M. suaveolens, and P. tridentatum, the present PhD thesis intends to evaluate the possible relation between polysaccharides and the immunostimulatory activity that these infusions might present. In a preliminary phase, infusions of F. angustifolia were prepared according to the popular tradition, and it was observed that the obtained water soluble material contained approximately 85% of material non-retained in C18 cartridges, with hydrophilic characteristics, with the remaining 15% comprising retained-material with hydrophobic characteristics. It was also shown that the infusions only contained between 2 and 4% of high molecular weight material (HMWM), which comprised approximately 30% of carbohydrate material. Sugar and methylation analysis of the HMWM suggested the presence of pectic polysaccharides, together with type II arabinogalactans, mannans, and xyloglucans. However, the amount of material obtained is to low for the fractionation, and structural analysis of the polysaccharides present. The 4 h decoction, divided in two periods of 2 h, with water renewal, allowed to increase the HMWM yield, relatively to the infusions traditional infusions. It was also observed that the decoction also allowed to increase the HMWM proportion of carbohydrate material, due to an increase in the proportion of uronic acid present, although the neutral sugar residues seemed to be detected in similar proportions. Therefore, in all the experiments subsequently performed, the HMWM used was obtained through the decoction of F. angustifolia dried leaves, M. suaveolens dried shoots, and P. tridentatum dried inflorescences. x After the fractionation, through ethanol precipitation, and anion exchange chromatography, of the polysaccharides from the HMWM obtained by the decoction of the vegetable material of the distinct studied plants, it was observed the presence of high proportions of pectic polysaccharides, containing type I arabinogalactans, together with minor proportions of type II arabinogalactans, mannans, and xyloglucans. The presence of pectic polysaccharides in the extracts from F. angustifolia was also evidenced through endo-polygalacturonase treatment, and ESI-MS and ESI-MS/MS experiments. The detection of linked pentose and uronic acid residues, also seemed to suggest the presence of xylogalacturonan domains in the pectic polysaccharides from F. angustifolia. The extracts from F. angustifolia dried leaves also contained type II arabinogalactans that exhibited a higher structural diversity than those detected in the M. suaveolens, and P. tridentatum extracts, particularly in the substitution degree of the galactan backbone, and in the extension of the (1→5)-Araf side chains. Moreover, for all the plants studied, it was also observed that the type II arabinogalactans, extracted during the 2nd 2h of the extraction process, exhibited a substitution degree of the galactan backbone higher than those extracted during the 1st 2h. The extracts from P. tridentatum dried inflorescences contained higher proportions of mannans, and also of xyloglucans, both presenting a substitution degree higher than those, which were detected in lower proportion in the extracts of F. angustifolia and M. suaveolens. Through ESI-MS and ESI-MS/MS it was possible to evidence that the mannans present in the extracts of P. tridentatum presented acetyl groups on the O-2 of the mannosyl residues. It was also evidenced that the P. tridentatum mannans were more extensively acetylated than the mannans detected in the coffee infusion, LBG, and other non-conventional mannan sources. Moreover, it was detected the presence of oligosaccharides comprising hexose residues linked to non acetylated pentose residues, suggesting the possible presence of arabinose residues in the mannans from P. tridentatum extracts. The immunostimulatory activity of three fractions isolated from the extracts of F. angustifolia, M. suaveolens, and P. tridentatum, was tested and an increase in the NO production by macrophages, without compromising their cellular viability, was observed. The type I, and type II arabinogalactans detected in the extracts from F. angustifolia, and M. suaveolens seem to have contributed for the observed immunostimulatory activity. For the fraction from P. tridentatum, the mannans acetylation, and the presence of type I, and type II arabinogalactans seemed to contribute for the macrophage immunostimulatory activity observed. The possible presence of storage xyloglucans from the inflorescences seeds, also seems to have contributed for the immunostimulatory activity registered when the macrophages were stimulated with higher extract concentrations. The results obtained allow to conclude that the extracts of F. angustifolia dried leaves, M. suaveolens dried shoots, and P. tridentatum dried inflorescences contained high proportions of pectic polysaccharides, exhibiting type I arabinogalactans, together with other polysaccharides, such as type II arabinogalactans, mannans, and xyloglucans. This polysaccharide mixture seems to have contributed to the immunostimulatory activity of fractions isolated from the extracts of the studied plants. Therefore, as the same type of polysaccharides seem to be present in the decoctions and in the infusions, it seems possible that the polysaccharides might contribute for the therapeutic properties frequently associated by the popular tradition to the infusions of these plants.

Relações petrogénicas entre o complexo gnáissico migmático e o granito de Banabuiú (Nordeste do Brasil)

A região de Banabuiú situa-se no Domínio Ceará Central, na porção setentrional da Província Borborema, um dos cinturões orogénicos formados durante o evento Brasiliano/Pan-Africano no final do Neoproterozóico. As duas unidades litológicas principais presentes na área são: o complexo gnáissicomigmatítico (metapelitos e metagrauvaques) e granitóides brasilianos. Para além das formações paraderivadas, no substrato da região também foi identificado um conjunto de rochas ortoderivadas, até então não individualizado na cartografia existente. Tanto a sequência paraderivada, como os materiais ortoderivados, foram intensamente afectados por metamorfismo regional da fácies granulítica durante a Orogenia Brasiliana, que atingiu as condições de fusão parcial, gerando migmatitos com um amplo espectro de morfologias. Estes migmatitos apresentam estruturas dominantemente estromáticas, embora localmente se tenham identificado também corpos irregulares de diatexitos de tipo“schlieren”, “schollen” e “maciço (s.s)”, indicando que o processo de migmatização culminou com a produção de maiores quantidades de fundido. Em termos tectónicos, o basamento da região regista os efeitos de três fases de deformação, embora as estruturas concordantes à D3 sejam dominantes e obliterem, em muitos casos, as anisotropias anteriores. A maior parte dos fundidos anatécticos parece ter sido produzida durante tectónica transcorrente D3. No entanto, as condições metamórficas para o início da fusão parcial parece ter sido atingidas antes, durante a D2, já que também existem leucossomas, embora em proporções reduzidas, associados com as estruturas desta fase. A grande quantidade de volumes de leucossomas / veios leucocráticos encontrados na região, está relacionada com a actuação da zona de cisalhamento de Orós e parece corresponder a fundidos anatécticos gerados em níveis mais profundos que foram injectados nas sequências orto- e para-derivadas, devido a notória escassez de leucossomas “in situ” nestas rochas. A presença de fluidos aquosos injectados no complexo migmatítico de Banabuiú terá proporcionado a re-hidratação e retrogradação das rochas hospedeiras, evidenciada, essencialmente, pela presença de moscovite tardia, amplamente distribuída nos metassedimentos e ortognaisses, sobretudo nas zonas próximas aos leucossomas e veios leucocráticos. Dados isotópicos apontam que as rochas da região de Banabuiú apresentam valores fortemente negativos de εNdt e positivos de εSrt sugerindo um significativo envolvimento de materiais supracrustais do grupo Acopiara na formação do complexo migmatítico e na petrogénese do maciço granítico de Banabuiú e o marcado fraccionamento isotópico Sm-Nd observado nalguns dos leucossomas analisados indica que os líquidos anatécticos que lhes deram origem resultaram de processos de fusão em desequilíbrio, em condições anidras, e foram rapidamente extraídos da área-fonte, comprovando o carácter alóctone dos veios leucocráticos intercalados nos ortognaisses e paragnaisses de Banabuiú.

Development of a sustainable method for the extraction of lycopene and β-carotene from food wastes

The main objective of the present work is the study of a profitable process not only in the extraction and selective separation of lycopene and β-carotene, two compounds present in tomato, but also in its potential application to food industry wastes. This is one of the industries that produce larger amounts of wastes, which are rich in high value biomolecules with great economic interest. However, the conventional methods used to extract this kind of compounds are expensive which limits their application at large scale. Lycopene and βcarotene are carotenoids with high commercial value, known for their antioxidant activity and benefits to human health. Their biggest source is tomato, one of the world’s most consumed fruits, reason for which large quantities of waste is produced. This work focuses on the study of diverse solvents with a high potential to extract carotenoids from tomato, as well as the search for more environmentally benign solvents than those currently used to extract lycopene and β-carotene from biomass. Additionally, special attention was paid to the creation of a continuous process that would allow the fractionation of the compounds for further purification. Thus, the present work started with the extraction of both carotenoids using a wide range of solvents, namely, organic solvents, conventional salts, ionic liquids, polymers and surfactants. In this stage, each solvent was evaluated in what regards their capacity of extraction as well as their penetration ability in biomass. The results collected showed that an adequate selection of the solvents may lead to the complete extraction of both carotenoids in one single step, particularly acetone and tetrahydrofuran were the most effective ones. However, the general low penetration capacity of salts, ionic liquids, polymers and surfactants makes these solvents ineffective in the solid-liquid extraction process. As the organic solvents showed the highest capacity to extract lycopene and βcarotene, in particular tetrahydrofuran and acetone, the latter solvent used in the development process of fractionation, using to this by strategic use of solvents. This step was only successfully developed through the manipulation of the solubility of each compound in ethanol and n-hexane. The results confirmed the possibility of fractionating the target compounds using the correct addition order of the solvents. Approximately, 39 % of the β-carotene was dissolved in ethanol and about 64 % of lycopene was dissolved in n-hexane, thus indicating their separation for two different solvents which shows the selective character of the developed process without any prior stage optimization. This study revealed that the use of organic solvents leads to selective extraction of lycopene and β-carotene, allowing diminishing the numerous stages involved in conventional methods. At the end, it was possible to idealize a sustainable and of high industrial relevance integrated process, nevertheless existing the need for additional optimization studies in the future.