One of the challenges of biopharmaceutics research is correlating in vitro drug release information with the in vivo drug profiles often known as in vitro-in vivo correlations (IVIVC). These have huge cost savings in pharmaceutical industry. This dissertation describes the development of a novel in vitro dissolution model that has been designed to evaluate the in vitro release of poorly water-soluble model compounds so as to predict their in vivo behavior. The flow through apparatus (USP 4) has been coupled with the compendial dissolution apparatus (USP 2). A bi-phasic dissolution medium has been used to achieve sink conditions. The dissolved drug from the aqueous phase is continuously extracted into the organic phase of the biphasic dissolution medium, mimicking the dynamic process of in vivo absorption. The model can successfully discriminate between the bioequivalent and non-bioequivalent formulations and can be used to establish IVIVC. It was concluded that the model will serve as a surrogate for bioequivalence studies and to support biowaivers.

[pt] A injeção de água é o método mais utilizado para estender a vida produtiva de reservatórios de petróleo. No entanto, sua eficiência é limitada pela relação de mobilidade desfavorável entre a fase aquosa injetada e a fase oleosa deslocada. A heterogeneidade das formações agrava essa questão, direcionando a água através de caminhos preferenciais, resultando na retenção de óleo residual. Estudos recentes propõem emulsões de óleo-em-água como agentes de bloqueio para reduzir a mobilidade da fase aquosa. A redução da mobilidade associada à captura de gotas da fase dispersa leva a uma frente de deslocamento mais uniforme, aumentando a recuperação de óleo. Apesar dos avanços recentes na injeção de emulsões como método de recuperação avançada de petróleo (EOR), aspectos fundamentais do escoamento de emulsões óleo-em-água a nível microscópico e sua relação com a redução macroscópica na mobilidade da fase aquosa ainda necessitam de maior compreensão. Este estudo explora fatores que influenciam a eficácia de um processo de injeção de emulsão, incluindo o tamanho das gotas, a distribuição das gargantas de poros e a vazão de injeção, que influenciam diretamente na redução da mobilidade. Micromodelos bidimensionais foram empregados para visualizar a dinâmica de retenção e liberação de gotas, relacionando fenômenos em escala de poros à mobilidade da fase aquosa. Duas geometrias foram projetadas para este propósito. O micromodelo linear assegura um gradiente de pressão e uma velocidade constante ao longo de seu comprimento, enquanto a configuração radial avalia o desempenho da injeção de emulsão sob diferentes números de capilaridade. Nesta última configuração, a área de fluxo aumenta com o raio, reduzindo a velocidade do escoamento à medida que o fluido se afasta do ponto de injeção. Os resultados mostram que a redução da mobilidade pode ser controlada pelo número de capilaridade e pela distribuição do tamanho de gotas. Em números de capilaridade suficientemente altos, a diferença de pressão na maioria das gargantas de poro supera a pressão capilar, empurrando as gotas através das constrições. Nestes casos, a retenção de gotas é baixa e a redução da mobilidade é fraca. Por outro lado, em números de capilaridade baixos, a retenção de gotas é alta, causando uma redução significativa na mobilidade da fase aquosa, que é fortemente dependente da distribuição do tamanho de gotas. Além disso, no fluxo radial, o bloqueio de poros ocorre abaixo de um número de capilaridade crítico, onde a força capilar supera a pressão viscosa. O trabalho demonstra que a injeção de emulsão melhora a eficiência de deslocamento a nível microscópico, reduzindo a saturação residual de óleo. Os resultados podem orientar a seleção de características específicas de emulsões a serem injetadas em reservatórios com distribuições conhecidas de gargantas de poros, visando alcançar a necessária redução na mobilidade da fase aquosa e, consequentemente, incrementar a recuperação de óleo. / [en] Water injection is the most commonly used method for extending the productive life of oil reservoirs; however, its efficiency is limited by an unfavorable mobility ratio between the injected aqueous phase and the displaced oil phase. Reservoir heterogeneity exacerbates this issue, driving water through preferential flow paths with lower capillary resistance, leaving trapped oil behind. Recent studies propose oil-in-water emulsions as a pore-blocking agent to reduce aqueous phase mobility, leading to a more uniform displacement front and enhancing oil recovery. Despite recent developments in emulsion injection for enhanced oil recovery (EOR), fundamental aspects of the pore-scale dynamics of oil-in-water emulsion flow and its correlation with observed macroscopic mobility reduction remain not completely understood. This study explores key factors influencing the design of an effective emulsion injection process, including emulsion drop size, pore throat distribution, and injection flow rate, and their impact on the mobility reduction of the aqueous phase. Two-dimensional porous media micromodels were employed to visualize drop dynamics, examining how pore-scale phenomena affect aqueous phase mobility reduction. Two distinct geometries were designed for this purpose. The linear micromodel ensures a constant pressure gradient and flow velocity along its length, while the radial configuration assesses emulsion flooding performance under varying capillary numbers. In the latter configuration, the flow area increases with the radius, reducing the flow velocity as the fluid moves away from the injection point. Results show that mobility reduction can be finely controlled by the capillary number and the drop size distribution. At sufficiently high capillary numbers, the pressure difference in most pores is strong enough to overcome the capillary pressure needed to push a drop through the constriction; the number of trapped drops is relatively small, and mobility reduction is weak. Conversely, at low capillary numbers, the number of trapped drops is large; the mobility reduction is strong and dependent on the drop size distribution. Additionally, in radial flow, stronger pore-blocking occurs below a critical capillary number, where capillary resistance surpasses viscous pressure. Flow visualization demonstrates that emulsion flooding improves pore-level displacement efficiency, reducing residual oil saturation. These findings offer valuable insights into tailoring oil-in-water emulsions for injection into reservoirs with known pore throat distributions, aiming to achieve the necessary aqueous phase mobility reduction and consequently increase oil recovery factors.

(EN) The presented dissertation thesis summarizes the new results of electrochemical generation of volatile compounds usable in atomic spectral methods. The main aim of this work is to develop and to characterize new types of electrolytic flow-through cells and to examine their possibilities of determination of arsenic, selenium and antimony by using the electrochemical hydride generation technique coupled with atomic absorption spectrometry with a quartz tube atomizer. Individual electrolytic cells were designed and constructed to comply with two important requirements. The cathode chamber of the electrolytic cell should have a minimal volume and a high efficiency of analyte conversion to the volatile hydride. Constructed electrolytic cells are divided into the construction groups and described in the experimental part. Selenium was chosen as the first analyte. The relevant working parameters (such as type, concentration and flow rate of electrolytes, generation current and carrier gas flow rate) were optimized for each newly constructed electrolytic cell. Under the optimal working parameters, the basic characteristics of selenium determination were found out by using electrochemical hydride generation. The electrolytic cells were compared to each other and with the classical electrolytic cell...

Dans de nombreux pays, l’aquaculture de pectinidés dépend aujourd’hui du succès de la production contrôlée de juvéniles. Néanmoins, les fortes variations des taux d’éclosion des oeufs et de la survie larvaire, enregistrées à ce jour, rendent cette production imprévisible. Les élevages larvaires en flux ouvert de coquilles Saint-Jacques (Pecten maximus) ont été développés en Norvège et présentent des résultats prometteurs. Malheureusement, les rendements de production encore faibles et l’impossibilité de travailler à fortes densités restent un frein majeur au développement de cette technique. En France, une technique en flux-ouvert, en petit volume (5 L), et à forte densité (≤ 300 larves mL-1) a été développée pour les ostréidés. Des expériences préliminaires visant à décliner ce système d’élevage aux larves de P. maximus se sont avérées infructueuses : retard de croissance et forte mortalité en quelques jours. Il est reconnu que les larves de pectinidés doivent faire face à des contraintes diverses en écloserie : bactériologiques, physiologiques et environnementales. Elles sont également plus sensibles que les larves des autres espèces de bivalves comme par exemple l’huître japonaise (Crassostrea gigas). Il apparait donc nécessaire aujourd’hui d’identifier plus clairement l’origine des phénomènes perturbant le bon développement des larves en flux ouvert afin d’améliorer la qualité des élevages et les rendements larvaires. Grâce à l’étude et à la compréhension des mécanismes physiologiques impliqués dans la lutte contre le stress des larves de P. maximus en flux ouvert, ce projet de doctorat donne des clés permettant d’améliorer cette technique d’élevage tout en limitant l’utilisation de produits chimiques en milieu contrôlé. / In many countries, aquaculture of pectinids depends on the success of artificial spat production in hatchery. This production is always unpredictable due to the variability of hatching rate and larval survival. Flow-through larval rearing systems were developed in Norway for the King scallop Pecten maximus and showed promising results. Unfortunately the system needs to be optimized since the larval yields and the densities used are still relatively low. In France, a small-scale (5 L) and high-density (≤ 300 larva mL-1) flow-through larval rearing system was successfully developed for oysters. First trials in such system and in similar conditions with P.maximus failed as we registered slower growth and high mortality rate in only a few days. It is known that pectinids larvae are more sensitive to environmental conditions than the oyster Crassostrea gigas, for example.Nowadays, it is important to identify and to understand the phenomena disturbing larval development in flowthrough system to improve larval quality and production yields. This doctoral project provided some indications how improving P. maximus flowthrough rearing system while limiting the use of antibiotic through a better understanding the physiological mechanisms involved in the larval response to a stressful environment

Ziel der vorliegenden Arbeit war die Entwicklung einer SNP-Genotypisierungsmethode mit auf Mikroarrays immobilisierten PCR-Produkten. Für die Analyse wurde ein faseroptischer Affinitätssensor bzw. ein Durchfluss-Biochip-Scanner mit integrierter Fluoreszenzdetektion verwendet. An den immobilisierten Analyten (PCR-Produkten) wurde eine Fluoreszenzoligonukleotidsonde hybridisiert und anschließend die Dissoziation der Sonde im Fluss verfolgt. Die Diskriminierung von Wildtyp- und Mutanten-DNA erfolgte durch die kinetische Auswertung der Dissoziationskurven sowie durch die Analyse der Fluoreszenzintensität. <br> <br> Die Versuche am faseroptischen Affinitätssensor zeigten, dass DNA-DNA-Hybride sowohl von Oligonukleotiden als auch von PCR-Produkten ein typisches Dissoziationsverhalten aufweisen, wobei fehlgepaarte Hybride eine signifikant schnellere Dissoziation zeigen als perfekt passende Hybride. Dieser Geschwindigkeitsunterschied lässt sich durch den Vergleich der jeweiligen kinetischen Geschwindigkeitskonstanten kD quantitativ erfassen. </p> <p>Da die Kopplung des Analyten an der Chipoberfläche sowie die Hybridisierungs- und Dissoziationsparameter essentiell für die Methodenentwicklung war, wurden die Parameter für ein optimales Spotting und die Immobilisierung von PCR-Produkten ermittelt. Getestet wurden die affine Kopplung von biotinylierten PCR-Produkten an Streptavidin-, Avidin- und NeutrAvidin-Oberflächen sowie die kovalente Bindung von phosphorylierten Amplifikaten mit der EDC/Methylimidazol-Methode. Die besten Ergebnisse sowohl in Spotform und -homogenität als auch im Signal/Rausch-Verhältnis wurden an NeutrAvidin-Oberflächen erreicht. </p> <p>Für die Etablierung der Mikroarray-Genotypisierungsmethode durch kinetische Analyse nach einem Hybridisierungsexperiment wurden Sondenlänge, Puffersystem, Spotting-Konzentration des Analyten sowie Temperatur optimiert. Das Analysensystem erlaubte es, PCR-Produkte mit einer Konzentration von 250 ng/µl in einem HEPES-EDTA-NaCl-Puffer auf mit NeutrAvidin beschichtete Glasträger zu spotten. In den anschließenden Hybridisierungs- und Dissoziationsexperimenten bei 30 °C konnte die Diskriminierung von homocygoter Wildtyp- und homocygoter Mutanten- sowie heterocygoter DNA am Beispiel von Oligonukleotid-Hybriden erreicht werden. </p> <p>In einer Gruppe von 24 homocygoten Patienten wurde ein Polymorphismus im SULT1A1-Gen analysiert. Sowohl durch kinetische Auswertung als auch mit der Analyse der Fluoreszenzintensität wurde der Genotyp der Proben identifiziert. Die Ergebnisse wurden mit dem Referenzverfahren, der Restriktionschnittstellenanalyse (PCR-RFLP) validiert. Lediglich ein Genotyp wurde falsch bestimmt, die Genauigkeit lag bei 96%. </p> <p>In einer Gruppe von 44 Patienten wurde der Genotyp eines SNP in der Adiponectin-Promotor-Region untersucht. Nach Vergleich der Analysenergebnisse mit denen eines Referenzverfahrens konnten lediglich 14 der untersuchten Genotypen bestätigt werden. Ursache für die unzureichende Genauigkeit der Methode war vor allem das schlechte Signal/Rausch-Verhältnis.</p> <p> Zusammenfassend kann gesagt werden, dass das in dieser Arbeit entwickelte Analysesystem für die Genotypisierung von Einzelpunktmutationen geeignet ist, homocygote Patientenproben zuverlässig zu analysieren. Prinzipiell ist das auch bei heterocygoter DNA möglich. Da nach aktuellem Kenntnisstand eine SNP-Analysemethode an immobilisierten PCR-Produkten noch nicht veröffentlicht wurde, stellt das hier entwickelte Verfahren eine Alternative zu bisher bekannten Mikroarray-Verfahren dar. Als besonders vorteilhaft erweist sich der reverse Ansatz der Methode. </p> <p>Der hier vorgestellte Ansatz ist eine kostengünstigere und weniger hoch dimensionierte Lösung für Fragestellungen beispielsweise in der Ernährungswissenschaft, bei denen meist eine mittlere Anzahl Patienten auf nur einige wenige SNPs zu untersuchen ist. Wenn es gelingt, durch die Weiterentwicklung der Hardware bzw. weiterer Optimierung, eine Verbesserung des Signal/Rausch-Verhältnisses und damit die Diskriminierung von heterocygoter DNA zu erreichen, kann diese Methode zukünftig bei der Analyse von mittelgroßen Patientengruppen alternativ zu anderen Genotypisierungsmethoden verwendet werden. / The aim of this thesis was the development of a SNP genotyping method involving PCR products immobilised on microarrays. For the analysis a fibre optic affinity biosensor and a flow-through biochip scanner were used. Fluorescent probes were hybridized with the immobilised PCR products. In order to start the dissociation process the surface was rinsed with buffer and the fluorescence intensity was measured. <br><br> Two different cases were studied: First, the full-matched DNA hybrid (wildtyp single strand with complementary wildtype single strand), second the mis-matched hybrid (wildtype single strand and mutant single strand). After determinating the reaction rates (kD) as kinetic parameter the kD values of both cases were compared. The experiments showed a significant difference in the kD value of the full- and the mis-match hybrids. Therefore, mutant and wildtype DNA were discriminated by kinetic analysis of the dissociation process and analysis of the fluorescence intensity. <br><br> To set up the complete analysis process the reaction parameters like coupling of the PCR products had to be optimised. Both affininty coupled (streptavidin, neutravidin, avidin - biotin) and covalent methods (EDC/methylimidazol) were carried out. Best results in spot homogeinity and spot appearance were obtained with coupling of biotinylated PCR products on neutravidin coated chip surfaces. Additionally, the length of the probe, the spotting concentration, the spotting buffer and the reaction temperature were optimised. In the optimised analysis PCR products (250 µg/µl) were spotted onto neutravidin coated surfaces. The hybridisation <br><br> and dissociation processes were carried out at 30°C. A HEPES-EDTA-NaCl buffer was used for spotting, diluting of the fluorescent probe and rinsing the microarray surface. A fluorescent probe was used with 13 nucleotides in length. The mis- or full-matching base indicating the polymorphism was located in the center position of the probe. <br><br> The analysis system was tested with the genomic DNA of a group of 24 homocygote individuals with a SNP in the SULT1A1 gene region. The hybridisation and dissociation processes were carried out and the reaction rates were determinated. Subsequently after the analysis in the flow-through biochip scanner the fluorescence intensity of the <br><br> spots were measured. The results showed very good comparability with results of a PCR-RFLP analysis (one false genotype). Additionally, a group of 44 heterocygote DNA samples with one SNP in the adiponectin promotor region were also genotyped. Compared to a reference method only 14 genotypes were correctly determined. This was mostly due to a low signal-noise-ratio and needs to be further investigated. <br><br> Besides the problem in analysing heterocygote DNA samples the developed analysis system is very useful for genotyping SNP in homocygote DNA samples. The successful analysis of heterocygote sample is principally possible and with further investigations/optimisation, a better analysis should be possible. <br><br> The most important advantage of the developed method is the reverse approach of binding PCR products at the surface instead of oligonucleotides. This allows the parallel genotyping of several individuals. Other advantages include low costs and medium sized dimensions in terms of throughput.

Ziel der Arbeit war eine möglichst umfassende Analyse der Nahrungsbedingungen von D. galeata unter Berücksichtigung der damit verbundenen Strategien der Daphnien zur Optimierung des Nahrungserwerbs, um den Einfluß dieser Faktoren bezüglich des Auftretens einer Sommerdepression der Daphnien zu beurteilen. Aufbauend auf den bereits vorhandenen Kenntnissen aus der Literatur sollte dazu im Rahmen der Arbeit festgestellt werden, ob morphologische Veränderungen des Filtrationsapparates der Daphnien (Ausbildung großer, hydrodynamisch dichter Filter), die als Reaktion auf die geringen Nahrungsmengen im Klarwasserstadium auftreten, bei einer drastischen Erhöhung der Nahrungskonzentration zu einer &amp;quot;Überlastung&amp;quot; der Filterbeine der Daphnien, zu hohen Energieausgaben und schließlich zu erhöhter Mortalität (Sommerdepression) führen können. Hauptziel war dabei die Klärung der Frage, ob bottom-up gesteuerte schnelle Veränderungen im Nahrungsangebot, wie sie für geringe bottom-up Limitation (Hypertrophie) typisch sind, eine erhöhte Mortalität bei Daphnien hervorrufen können. Die Beantwortung dieser Fragestellung ist wichtig, um die Effizienz der top-down Steuerung (Biomanipulation) als ökotechnologische Maßnahme zur Gewässersanierung zu erhöhen. Dazu wurden in der biomanipulierten Talsperre Bautzen in drei Untersuchungsjahren (1997 - 1999) wichtige bottom-up wirkende Steuerfaktoren der Daphnienentwicklung sowie morphologische Charakteristika der Filterkämme und physiologische Leistungskriterien von D. galeata vor und während der Zeit des potentiellen Auftretens einer Sommerdepression untersucht. Aus den Ergebnissen ließ sich ableiten, daß das Zusammenspiel der vielfältigen Anpassungsmöglichkeiten der Daphnien in einem bestimmten Variationsbereich der Steuerfaktoren eine erfolgreiche Strategie zur Optimierung der Nahrungsaufnahme darstellen kann. Werden jedoch bestimmte Schwellenwerte über- oder unterschritten, ist eine Kompensation durch die Anpassungsstrategien nicht oder nur bedingt möglich. Dabei kann durch verschiedene Ursachen der gleiche Effekt (Mortalität adulter Daphnien) auftreten, was die Interpretation erschwert. Dieser Effekt wird besonders bei adulten Daphnien durch die gegenüber juvenilen Daphnien höheren Filterwiderstände und die damit verringerte effektive Energieaufnahme begünstigt. Erst wenn ungünstige Nahrungsbedingungen und die am Ende der Klarwasserphase beobachteten ungünstigen Konstellationen der Filterkämme zusammentreffen (timing), tritt die erhöhte Mortalität adulter Daphnien auf. Die Ergebnisse der Untersuchungen unterstreichen damit die Bedeutung von Ganzsee-Experimenten und die möglichst simultane Erfassung verschiedener Zustandsgrößen, um eine umfassende Kausalanalyse der Sommerdepression der Daphnien zu ermöglichen, zu der die vorliegende Arbeit einen Beitrag liefert. Für den Erfolg der Biomanipulation konnte daher geschlußfolgert werden, daß eine nachhaltige Wirkung nur dann erreicht werden kann, wenn zusätzlich zur top-down Steuerung des Nahrungsnetzes gleichzeitig eine erfolgreiche Kontrolle der bottom-up Prozesse im Gewässer realisiert wird, indem starke Schwankungen von bottom-up Faktoren vermieden werden und damit das System insgesamt stabilisiert wird. / The occurrence of midsummer declines of daphnids is often related to poor food conditions or fish predation. In this three-year-study on the midsummer decline of Daphnia galeata field investigations in the highly eutrophic biomanipulated Bautzen reservoir were combined with flow-through experiments in the laboratory. The following hypothesis was tested: The inability of adult Daphnia to reduce the filtering area of their feeding appendages in response to fast increasing food levels after a clear-water phase enhances adult mortality. During the clear-water phase daphnids develop large and dense filtercombs to optimise their filter feeding. When food concentrations increase, however, this adaptation to low-food situation may be inappropriate due to too high energy consumtion. As adult daphnids cannot reduce the size of their feeding appendages, the filtration process may be hindered when seston concentrations rise, resulting in reduced fitness and increasing adult mortality. Indeed, adult survival in the laboratory (predation excluded) drastically decreased exactly at that time when the Daphnia galeata population in Bautzen reservoir declined. However, enhanced mortality was found irrespective of different food conditions offered during the experiments. It is concluded that increased mortality of adult daphnids is not an direct effect of their inability to adapt the morphology of their filtercombs to high seston concentrations. Furthermore daphnids are able to compensate unfavorable food conditions within a certain range by different kinds of adaptation mechanisms. Therefore the timing of the changes of food conditions and the adaptations of daphnids is most important to the efficiency of energy gain of the daphnids. Concerning the success of biomanipulation it is concluded that in addition to the regulation of top-down mechanisms there is an urgent need for control of bottom-up processes in order to stabilize the ecosystem.

Non-overflow flow-through rockfill structures are river engineering elements used to attenuate and delay inflow hydrographs. They represent expedient places to deposit rather enormous quantities of waste rock at mountainous mine sites. Their application has become so common that matters of safety regarding their design have been laid out in Section 8.5 of the Canadian Dam Safety Guidelines (CDA 2007). The research described herein was directed at investigating the different aspects of the hydraulics of these flow-through rockfill structures. In order to assess the potential for an unraveling failure of flow-through rockfill dams, a systematic study of the hydraulic design of these structures was conducted and the non-linear nature of flow through these structures was dealt with using a p-LaPlacian-like partial differential equation. Subsequently, factors of safety against this type of failure are presented for a range of downstream slopes, thus showing the unsafe combinations of embankment slope and particle diameter. Three different index gradients within the toe of such structures were investigated. In this regard, the gradient most suitable for independently computing the height of the point of first flow emergence on the downstream face is examined and a method for independently computing the variation in hydraulic head within that vertical (which allows for the toe of the structure to be isolated) is presented. An additional gradient that allows for the independent estimation of the default tailwater depth is proposed. In order to provide better tools to assess the behavior of these embankments at the toe, laboratory and analytical studies were undertaken. In this regard, the hydraulics associated with the zone of the downstream toe were studied. The depth variation of the seepage-face was computationally modeled, and two approaches for solving the spatially varied flow (SVF) condition problem within the toe region undertaken. The results show that a dual linear variation in depth can be used to good accuracy, without inducing any unrealistic exit gradients in the zone of primary concern with respect to unraveling. It is hoped that these techniques and computational tools provided herein will aid in facilitating the design and assessment of these flow-through rockfill structures.

Agricultural management is essential for achieving optimum crop production and maintaining soil quality. Soil microorganisms are responsible for nutrient cycling and are an important consideration for effective soil management. The overall goal of the present research was to better understand microbial communities in agricultural soils as they relate to soil management practices. For this, we evaluated the differential impact of two contrasting drainage practices on microbial community composition and characterized active denitrifiers from selected agricultural sites. Field drainage is important for crop growth in arable soils. Controlled and uncontrolled tile drainage practices maintain water in the field or fully drain it, respectively. Because soil water content influences nutrient concentration, moisture, and oxygen availability, the effects of these two disparate practices on microbial community composition was compared in paired fields that had diverse land management histories. Libraries of the 16S rRNA gene were generated from DNA from 168 soil samples collected from eight fields during the 2012 growing season. Paired-end sequencing using next-generation sequencing was followed by read assembly and multivariate statistical analyses. Results showed that drainage practice exerted no measureable effect on the bacterial communities. However, bacterial communities were impacted by plant cultivar and applied fertilizer, in addition to sampled soil depth. Indicator species were only recovered for depth; plant cultivar or applied fertilizer type had no strong and specific indicator species. Among indicator species for soil depth (30-90 cm) were Chloroflexi (Anaerolineae), Betaproteobacteria (Janthinobacterium, Herminiimonas, Rhodoferax, Polaromonas), Deltaproteobacteria (Anaeromyxobacter, Geobacter), Alphaproteobacteria (Novosphingobium, Rhodobacter), and Actinobacteria (Promicromonospora). Denitrification in agricultural fields transforms nitrogen applied as fertilizer, reduces crop production, and emits N2O, which is a potent greenhouse gas. Agriculture is the highest anthropogenic source of N2O, which underlines the importance of understanding the microbiology of denitrification for reducing greenhouse gas emissions by altered management practices. Existing denitrifier probes and primers are biased due to their development based mostly on sequence information from cultured denitrifiers. To circumvent this limitation, this study investigated active and uncultivated denitrifiers from two agricultural sites in Ottawa, Ontario. Using DNA stable-isotope probing, we enriched nucleic acids from active soil denitrifiers by exposing intact replicate soil cores to NO3- and 13C6-glucose under anoxic conditions using flow-through reactors, with parallel native substrate controls. Spectrophotometric chemistry assays and gas chromatography confirmed active NO3- depletion and N2O production, respectively. Duplicate flow-through reactors were sacrificed after one and four week incubation periods to assess temporal changes due to food web dynamics. Soil DNA was extracted and processed by density gradient ultracentrifugation, followed by fractionation to separate DNA contributed by active denitrifiers (i.e., “heavy” DNA) from that of the background community (i.e., “light” DNA). Light and heavy DNA samples were analyzed by paired-end sequencing of 16S rRNA genes using next-generation sequencing. Multivariate statistics of assembled 16S rRNA genes confirmed unique taxonomic representation in heavy fractions from flow-through reactors fed 13C6-glucose, which exceeded any site-specific or temporal shifts in putative denitrifiers. Based on high relative abundance in heavy DNA, labelled taxa affiliated with the Betaproteobacteria (71%; Janthinobacterium, Acidovorax, Azoarcus, Dechloromonas), Alphaproteobacteria (8%; Rhizobium), Gammaproteobacteria (4%; Pseudomonas), and Actinobacteria (4%; Streptomycetaceae). Metagenomic DNA from the original soil and recovered heavy fractions were subjected to next-generation sequencing and the results demonstrated enrichment of denitrification genes with taxonomic affiliations to Brucella, Ralstonia, and Chromobacterium in heavy fractions of flow-through reactors fed 13C6-glucose. The vast majority of heavy-DNA-associated nitrite-reductase reads annotated to the copper-containing form (nirK), rather than the heme-containing enzyme (nirS). Analysis of recovered nirK genes demonstrated low sequence identity across common primer-binding sites used for the detection and quantification of soil denitrifiers, indicating that these active denitrifiers would not have been detected in molecular surveys of these same soils.

Neste trabalho é apresentada uma nova modelagem matemática para a descrição do escoamento de um líquido incompressível através de um meio poroso rígido homogêneo e isotrópico, a partir do ponto de vista da Teoria Contínua de Misturas. O fenômeno é tratado como o movimento de uma mistura composta por três constituintes contínuos: o primeiro representando a matriz porosa, o segundo representando o líquido e o terceiro representando um gás de baixíssima densidade. O modelo proposto possibilita uma descrição matemática realista do fenômeno de transição insaturado/saturado a partir de uma combinação entre um sistema de equações diferenciais parciais e uma desigualdade. A desigualdade representa uma limitação geométrica oriunda da incompressibilidade do líquido e da rigidez do meio poroso. Alguns casos particulares são simulados e os resultados comparados com resultados clássicos, mostrando as consequências de não levar em conta as restrições inerentes ao problema. / This work is concerned with a new mathematical modelling for describing the flow of an incompressible fluid (a liquid) through a rigid, homogeneous and isotropic porous medium, from a Continuum Mixtures point of view. The phenomenon is regarded as the motion of a mixture composed by three overlaping continuous constituents: the first one representing the porous matrix, the second one representing the liquid and the third one representing a (very) low density gas. The proposed mathematical modelling allows a realistic mathematical description for the unsaturated/saturated transition process by means of a combination between a system of partial differential equations and an inequality. This inequality represents a geometrical constraint arising from the liquid incompressibility merged with the porous matrix rigidity. The simulation of some interesting particular cases is carried out presenting a comparison between the obtained results and the classical ones, showing the consequences of disregarding the constraints associated to the phenomenon.

Neste trabalho é apresentada uma nova modelagem matemática para a descrição do escoamento de um líquido incompressível através de um meio poroso rígido homogêneo e isotrópico, a partir do ponto de vista da Teoria Contínua de Misturas. O fenômeno é tratado como o movimento de uma mistura composta por três constituintes contínuos: o primeiro representando a matriz porosa, o segundo representando o líquido e o terceiro representando um gás de baixíssima densidade. O modelo proposto possibilita uma descrição matemática realista do fenômeno de transição insaturado/saturado a partir de uma combinação entre um sistema de equações diferenciais parciais e uma desigualdade. A desigualdade representa uma limitação geométrica oriunda da incompressibilidade do líquido e da rigidez do meio poroso. Alguns casos particulares são simulados e os resultados comparados com resultados clássicos, mostrando as consequências de não levar em conta as restrições inerentes ao problema. / This work is concerned with a new mathematical modelling for describing the flow of an incompressible fluid (a liquid) through a rigid, homogeneous and isotropic porous medium, from a Continuum Mixtures point of view. The phenomenon is regarded as the motion of a mixture composed by three overlaping continuous constituents: the first one representing the porous matrix, the second one representing the liquid and the third one representing a (very) low density gas. The proposed mathematical modelling allows a realistic mathematical description for the unsaturated/saturated transition process by means of a combination between a system of partial differential equations and an inequality. This inequality represents a geometrical constraint arising from the liquid incompressibility merged with the porous matrix rigidity. The simulation of some interesting particular cases is carried out presenting a comparison between the obtained results and the classical ones, showing the consequences of disregarding the constraints associated to the phenomenon.