<p> Using soluble organic carbon in the form of glucose as a growth limiting nutrient, the kinetics of mixed microbial populations (mainly bacterial in content) were studied using completely mixed batch and continuous biochemical reactors, in order to determine if kinetic data obtained from these two processes is identical and reproducible. </p> <p> Significant differences were found in the metabolic activity of of bacteria growing in batch and continuous culture; also periods of continuous culture were found to alter the kinetics of subsequent batch cultures. Simultaneous batch experiments and consecutive batch experiments were found to be substantially reproducible with respect to kinetic data, but inconsistency was obtained in continuous culture kinetic data. The degree of dispersion of the bacteria was also found to be different in batch and continuous culture; continuous operation gave rise to dispersed growth of bacteria, whereas batch operation gave rise to flocculent bacterial growth. </p> / Thesis / Master of Engineering (MEngr)

The purpose of this study was to focus attention on the physiology of the bdellovibrio-host interaction and to determine the metabolic requirements for this reaction. Since bdellovibrio is an aerobic organism, direct measurements of respiration, turbidity, and viable cell counts are reliable indications of the metabolic activity of the cells. It was determined that the metabolic requirements for the parasitic interaction are constituents from either metabolically active host cells or cells which are capable of at least some metabolic activity. The nutritional requirements of host-independent bdellovibrios suspended in buffer are not met by the presence or absence of viable or nonviable Enterobacter aegnes. Unlike the HD bdellovibrios, the HI bdellovibrios lack the ability to make economical use of their self-digesting processes.

Studies using a rat model of prenatal protein malnutrition (PPM) followed by nutritional rehabilitation show that PPM produces changes in the brain and behavior that endure throughout adulthood. Early studies investigated the vulnerability of the hippocampus, a structure involved in learning and memory, and reported permanent anatomical, physiological, and functional alterations. However, PPM also produces deficits in attentional processes, suggesting vulnerability across a broader cortical network including the parahippocampal region (PHR) and the prefrontal cortex (PFC). This thesis investigates the anatomical, functional, and molecular alterations in these regions resulting from PPM. This was accomplished through 4 studies: 1) A quantitative assessment of the number of neurons in the PHR and in the PFC using design-based stereology; 2) An evaluation of the impact of the PPM on metabolic activity in the PFC using the metabolic marker 2-[14C]deoxyglucose (2DG); 3) The identification of specific neuronal subtypes differentially activated during restraint stress in the PPM network using double-labelling immunohistochemistry; 4) The quantification of mRNA and protein expression of KCNJ3 (GIRK1), a potassium channel involved in regulating neural excitability, using quantitative polymerase chain reaction and Western blot analysis. Results showed that: 1) Neuron number in the PFC is unchanged by PPM, but two subfields of the PHR, the presubiculum and medial entorhinal cortex, exhibit significantly lower numbers in PPM rats; 2) Metabolic activity in specific PFC regions associated with attention including the prelimbic, infralimbic, anterior cingulate, and orbitofrontal cortices was reduced relative to controls while other regions, such as the hippocampus, were unaffected; 3) Exposure to stress evokes a significant increase in the number of inhibitory interneurons that are activated in the PFC of PPM rats which could likely contribute to the observed overall reduction in PFC activity; 4) For the KCNJ3 channel, PPM induces lower levels of mRNA and protein expression in the PFC while levels in the hippocampus and brain stem/basal ganglia are unchanged. Together, these data show that PPM creates permanent anatomical, functional, and molecular alterations selective to specific subfields, cell types, and molecules leading to an imbalance between excitatory and inhibitory processes in the PHR-PFC network of adult rats.

Biological wastewater treatment relies on the diverse and complex metabolic activities of bacteria to remove pollutants. Its success depends on the metabolic efficiency of the bacteria. Activated sludge models use parameters that attempt to depict bacterial growth and metabolic processes. However, current methods do not separate metabolic activity from growth and maintenance. As a result, activated sludge processes are misinterpreted or over-simplified. Alternative methods for gauging bacterial activity have been proposed and include the measurements of cellular derived compounds that relate specifically to energy cycling and include Nicotinamide Adenine Dinucleotide [NADH]. To date, NADH has been largely measured within activated sludge using commercial online fluorimeters with in situ probes. However, this current method provides a measure of the 'bulk' (raw) fluorescence within the system, resulting in difficulties when interpreting fluorescence data and poor sensitivity for detecting changes in intracellular [NADH]. This study has developed a more reliable method for estimating intracellular [NADH] and thus metabolic activity within activated sludge systems. Separating extracellular from intracellular [NADH] in samples was crucial because NADH was released and accumulates in the extracellular environment at a concentration of 200 ~M immediately following bacterial death or lysis. This concentration did not decline overtime. This not only caused high background fluorescence but also reduced the sensitivity of detection for changes in intracellular [NADH]. In particular, considerably higher [NADH] values to those from the extracellular suspensions were obtained following extraction of the intracellular material, suggesting that the cell membranes were not being penetrated by the excitable light source. Of the extraction procedures examined, filtration followed by extraction of the intracellular material with a hot Tris buffer was the most efficient and was recommended for accurate estimates of intracellular [NADH] in situ. In addition, standards were used to quantify NADH (moles per cell and/or unit volume) from unknown samples. The limits of detection were found to be 1.058 - 353 uM, whereas concentrations above 353 jAM self-quenched. Sample concentrations were always within these limits of detection. Hence, the sensitivity, reliability and experimental application of the original method was improved upon and able to be used for the direct measurement of microbial metabolic activity, something that has not been demonstrated before now. This study found that bacteria have between 106~ I 08 NADH molecules per cell depending on their metabolic state. A highly metabolically active bacterial cell had between 1O6~ tO7 NADH molecules, while a less active bacterial cell had between to7 -to8 NADH molecules. These measurements of metabolic activity were simultaneously monitored alongside other measures of bacterial growth, such as the incorporation of radiolabelled thymidine into DNA as a direct measure of DNA replication (new cell synthesis), the incorporation of radiolabelled leucine into protein as a direct measure of protein synthesis, oxygen uptake rates (OUR) as a direct measure of respiration, ATP as a measure of potential energy and dissolved organic carbon (DOC) as a measure of substrate assimilation. As OUR deceased, bacterial growth (using both the thymidine and leucine assays), specific [NADH] and specific [ATP] increased. High OUR and substrate oxidation rates simultaneous with low specific [NADH] indicated high rates of electron transport and thus efficient metabolic activity. Also, low OUR and substrate oxidation rates simultaneous with high specific [NADHI indicated inefficient rates of electron transport, therefore inhibiting oxidative phosphorylation (ATP production). A lack of oxygen as the terminal electron acceptor did not efficiently reoxidise NADH to NAD and resulted in an accumulation of NADH within the cell. Thus, a measure of low specific [NADHI was linked to the efficient rate of reoxidation of NADH to NAD* and reflects high metabolic efficiency. DNA and protein syntheses were coupled following substrate enrichment (glucose or acetate), indicating that bacteria were in balanced growth. However, DNA and protein syntheses became uncoupled once substrate was depleted, indicating unbalanced growth. An average Leu:TdR ratio of 7.4 was determined for activated sludge and was comparable to values published from marine systems. This ratio increased during log growth phase and decreased during stationary growth phases. Specific growth rates determined using the [3HITdR and [3H]Leu assay yielded values ranging from 2 - 10.5 d' and from 2.5 - 6 d1, respectively and were comparable to published values. Changes in OUR, NADH, ATE', DNA replication and protein synthesis were statistically ordinated using multidimensional scaling, and changes (in magnitude and direction) in bacterial metabolic activity were observed. Such methods enable the tracing of where bacteria divert their energies, such as to growth or maintenance and thus provide a greater understanding of bacterial behaviour in activated sludge. While studying anoxic and anaerobic conditions were beyond the scope of this work, the use of such methods to monitor bacterial metabolic activity under such conditions is warranted.

Despite extensive preventative efforts, the problem of controlling infections associated with biomedical materials persists. Bacteria tend to colonize on biocompatible materials and form biofilms; thus, novel biomaterials with antibacterial properties are of great interest. In this thesis, titanium dioxide (TiO2)-associated photocatalysis under ultraviolet (UV) irradiation was investigated as a strategy for developing bioactivity and antibacterial properties on biomaterials. Although much of the work was specifically directed towards dental materials, the results presented are applicable to a wide range of biomaterial applications. Most of the experimental work in the thesis was based on a resin-TiO2 nanocomposite that was prepared by adding 20 wt% TiO2 nanoparticles to a resin-based polymer material. Tests showed that the addition of the nanoparticles endowed the adhesive material with photocatalytic activity without affecting the functional bonding strength. Subsequent studies indicated a number of additional beneficial properties associated with the nanocomposite that appear promising for biomaterial applications. For example, irradiation with UV light induced bioactivity on the otherwise non-bioactive nanocomposite; this was indicated by hydroxyapatite formation on the surface following soaking in Dulbecco’s phosphate-buffered saline. Under UV irradiation, the resin-TiO2 nanocomposite provided effective antibacterial action against both planktonic and biofilm bacteria. UV irradiation of the nanocomposite also provided a prolonged antibacterial effect that continued after removal of the UV light source. UV treatment also reduced bacterial adhesion to the resin-TiO2 surface. The mechanisms involved in the antibacterial effects of TiO2 photocatalysis were studied by investigating the specific contributions of the photocatalytic reaction products (the reactive oxygen species) and their disinfection kinetics. Methods of improving the viability analysis of bacteria subjected to photocatalysis were also developed.

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Ubiquitaires et très anciens, les communautés des tapis microbiens font preuve de capacités métaboliques et adaptatives très importantes. Situés en zone côtières, ces écosystèmes peuvent être soumis à des contaminations pétrolières. Dans ce contexte, cette étude vise d’une part à décrire la structure et le fonctionnement de tapis microbiens et d’autre part à comprendre l’impact d’une contamination pétrolière sur ces écosystèmes. Cette étude porte sur deux tapis microbiens de l’étang de Berre aux paramètres physico-Chimiques proches mais présentant des contaminations pétrolières contrastées. Le fonctionnement du système étant tributaire d’autres facteurs tels que la lumière et les saisons, les variations saisonnières et nycthémérales ont été prises en compte dans cette étude. Un accent particulier a également été porté sur le cycle du soufre de par son importance en milieu marin. Les résultats de cette étude mettent en évidence des structures de communautés différentes entre les deux tapis au niveau global, la séparation spatiale prévalant sur la séparation saisonnière. La fraction active de la communauté du site contaminé présente une évolution linéaire tandis que celle du site témoins suit pour sa part les variations saisonnières. Au niveau du site contaminé une augmentation de l’expression des gènes impliqués dans la dégradation des hydrocarbures couplée à une biodégradation des hydrocarbures suggère que le tapis contaminé est adapté à la contamination pétrolière. Malgré les différences de structures et d’activités de dégradation, des profils métaboliques très semblables sont cependant observables entre les deux tapis, avec des fonctions similaires laissant supposer une redondance fonctionnelle. Des variations saisonnières et nycthémérales ont également été observées avec notamment des Desulfobulbaceae plus abondantes au printemps et plus actives en journée. Des études culturales ont été réalisées en parallèle. Elles permettront d’appréhender de manière complémentaire la dynamique des communautés des sulfato-Réducteurs au sein du tapis et de mieux comprendre les variations mises en évidence dans cette étude. / Ubiquist and very ancient, the microbial mats communities demonstrate very important metabolic and adaptive capacities. Located in the coastal area, these ecosystems may be subject to oil contamination. In this context, the aim of this study is on one hand to describe the structure and functioning of microbial mats and on the other to understand the impact of oil contamination on these ecosystems. This study focused on two microbial mats from the Berre lagoon with close physical chemical parameters but with contrasted hydrocarbon contamination levels. The functioning of the system is dependent on other factors such as light and seasons, diurnal and seasonal variations were taken into account in this study. Special emphasis was placed on the sulfur cycle due to its importance in the marine environments. The results of this study highlighted different communities’ structures at the global level between both mats, the spatial variation prevailed on seasonal variation. The active part of the community from the contaminated site shows a linear trend while that one of the uncontaminated site follows the seasonal variations. The contaminated site shows genes involved in hydrocarbon degradation more expressed coupled to a hydrocarbon biodegradation suggesting that the contaminated mat is adapted to the petroleum contamination. Despite these differences in the structure and the degradation capacities, very similar metabolic profiles are observed between the two mats with similar functions, suggesting functional redundancy. Seasonal and diurnal variation was also observed, the Desulfobulbaceae were particularly more abundant in spring and more active during the day. A complementary cultural approach will allow to better understanding the dynamics of sulfate-Reducers communities in the mat and comprehending these variations.

Les compartiments géologiques profonds suscitent un intérêt grandissant dans la communauté scientifique depuis les 50 dernières années. Néanmoins, ces écosystèmes demeurent largement méconnus du fait de leur difficulté d’accès. Le forage profond réalisé par l’ANDRA dans le Bassin parisien en 2008 a offert une opportunité unique de les étudier. Dans ce cadre, cette thèse avait deux objectifs majeurs ; i) caractériser, d’un point de vue microbiologique, quatre formations sédimentaires terrestres triasiques situées entre 1700 et 2000 m de profondeur et ii) étudier les effets combinés des paramètres de température, pression et salinité ainsi que de leur interaction sur l’activité métabolique de procaryotes anaérobies afin de mieux appréhender leur comportement au cours d’un enfouissement géologique.Malgré la recherche de microorganisme par la réalisation d’une gamme de milieux de culture diversifiée, ciblant préférentiellement les types trophiques fréquemment rencontrés en subsurface (méthanogènes, fermentaires, réducteurs de composés soufrés), aucun microorganisme viable et cultivable n’ait été isolé. En parallèle, une approche moléculaire complémentaire, composée (i) de l’étude comparative de l’efficacité de différentes méthodes d’extraction directe d’ADN et (ii) de l’analyse de la diversité bactérienne par la réalisation d’inventaires moléculaires, par DGGE (Denaturing Gel Gradient Electrophoresis) et clonage, a été réalisée sur le coeur des carottes de roches, conservées à pression atmosphérique ou sous pression, dans leurs états initiaux et post-incubation. L’exploration de ces formations sédimentaires profondes a indiqué la présence d’une très faible biomasse et d’une biodiversité microbienne pauvre principalement composée de membres aérobies et mésophiles appartenant au domaine Bacteria. Cette communauté bactérienne inattendue car a priori peu adaptée aux conditions régnant in-situ, également retrouvée dans divers écosystèmes de subsurface ainsi que dans des biotopes extrêmes, pourrait provenir en partie d’une paléo-recharge de l’aquifère du Trias par des eaux froides dérivées de la fonte des glaces formées lors de la dernière glaciation du Pléistocène.Le second objectif a été abordé à travers l’élaboration d’un plan factoriel complet dans le but d’identifier les effets des paramètres sur les activités microbiennes. Ainsi, les activités métaboliques de huit souches microbiennes halophiles et thermo-tolérantes ont été mesurées sous trente conditions distinctes de température (40, 55 et 70°C), pression (1, 90 et 180 bars) et salinité (13, 50, 110, 180 et 260 g.l-1). Toutes les souches originaires d’environnements profonds se sont révélées être au minimum piézo-tolérantes et capables de maintenir leur activité métabolique sous pressions hydrostatiques. Les métabolismes fermentaires (Thermovirga lienii et Halothermothrix orenii) et thiosulfato-réducteurs (Petrotoga mexicana et Thermosipho japonicus) se sont avérés particulièrement bien adaptées, d’un point de vue métabolique, aux hautes pressions, les plus hautes activités ayant été détectées sous pression. Certaines souches ont montré une résistance accrue aux hautes températures sous pression (Petrotoga mexicana). Toutefois une résistance variable à la salinité dans les différentes conditions de température et de pression a été observée pour chacune des souches, suggérant que certains mécanismes de résistance contre la pression osmotique seraient également efficaces pour lutter contre les températures et les pressions hydrostatiques élevées.Ce travail souligne que l’étude des écosystèmes terrestres profonds d’un point de vue microbiologique ne doit pas se restreindre à la recherche et à l’analyse de la diversité présente. L’étude des activités métaboliques de souches de subsurface en conditions profondes ouvre la voie à une meilleure compréhension des rôles joués par les communautés microbiennes en milieu extrême. / Over the past 50 years, the scientific community has shown a growing interest for deep geological compartments. However, these ecosystems remain largely unknown due to their inaccessibility. The aim of the present thesis was double; the first aim was to characterize, from a microbiological perspective, four terrestrial Triassic sedimentary formations located between 1700 and 2000 m depth in the Parisian Basin and collected by the ANDRA during a deep drilling campaign in 2008, and the second aim was to study the combined effects of temperature, pressure and salinity on the metabolic activity of anaerobic prokaryotes in order to predict their reaction to geological burial.Incubations in a large variety of media were carried out in order to stimulate the growth of the main trophic types found in such environments such as methanogens, fermenters and bacteria reducing sulphur compounds, however, no viable and cultivable microorganisms could be isolated. In parallel, a molecular approach was used to i) compare the efficacy of several DNA extractions methods and ii) analyse the bacterial diversity, using DGGE (Denaturing Gel Gradient Electrophoresis) and cloning, present in rock inner cores conserved either at atmospheric pressure or under pressure, in their initial states and following incubations in various media. The genetic exploration of these samples revealed a very low biomass and a poor diversity composed mainly of aerobic and mesophilic members of the Bacteria domain, a priori unadapted to such a deep, hot, saline and anoxic environment. This unexpected microbial community also found in many subsurface ecosystems as well as in extreme ecosystems could have partially originated from a paleorecharge of the Trias aquifer with cold waters coming from the melting of ice formed during the last Pleistocene glaciation.The second objective was to study the combined effects of temperature (40, 55 and 70°C), pressure (1, 90 and 180 bars) and salinity (13, 50, 110, 180 and 260 g.l-1) on the metabolic activity of anaerobic prokaryotes. In order to identify effects of parameters on microbial activities, a complete factorial plan was constructed from the metabolic activities measured for eight halophile and thermo-tolerant bacterial strains exposed to 30 distinct temperature, pressure and salinity conditions. All the strains issued from deep environments were at the least piezo-tolerant and capable of maintaining their activity under hydrostatic pressures. The fermenting (Thermovirga lienii and Halothermothrix orenii) and thiosulfate reducing strains (Petrotoga mexicana and Thermiosipho japonicus) were particularly well adapted, from a metabolic point of view, to high pressures; indeed the highest activities were measured under pressure. Also, several strains (such as Petrotoga mexicana) showed an increased resistance to high temperatures under pressure. However, resistance to an increase in salinity was variable for each strain under the different temperature and pressure conditions. This suggests that the resistance mechanisms for osmotic pressure also enable resistance to high temperatures and hydrostatic pressures.This work underlines that the microbiological characterization of deep terrestrial ecosystems must not be limited to the search and analyses of the existing diversity. Moreover, such upstream studies of the metabolic activities of subsurface bacterial strains in deep terrestrial conditions are a necessary beginning towards understanding the role of microbial communities in extreme environments.