Les interactions entre les membres d'une communauté microbienne peuvent être un moyen d'adaptation dans l'environnement. Parmi les nombreuses interactions qui ont lieu dans un écosystème et qui joue un rôle majeur sur la diversité et la dynamique des populations microbiennes est celui des virus procaryotes et leurs hôtes. Les virus peuvent également arbitrer le transfert de matériel génétique entre les procaryotes (transduction), qui pourrait être un mécanisme d'adaptation rapide. Afin de déterminer l'impact potentiel des virus et la transduction, nous avons besoin d'une meilleure compréhension de la dynamique des interactions entre virus et leurs hôtes dans l'environnement. Les données sur les virus de l'environnement sont rares, et les méthodes pour le suivi de leurs interactions avec les procaryotes sont nécessaires. Clustered regularly interspaced short palindromic repeats (CRISPRs), qui contiennent des séquences virales dans les génomes bactériens, pourraient aider à documenter l'histoire des interactions virus-hôte dans l'environnement. Ainsi, cette thèse vise à explorer les interactions virus-hôte dans un environnement donné à travers du séquences CRISPR.Les virus de la cryosphère sont considérés comme abondantes, très actif et avec de larges gammes d'hôtes. Ces caractéristiques pourraient faire de la transduction virale, un facteur clé pour l’adaptation microbienne dans ces environnements. Des métagénomes publics créés à partir des environnements avec une gamme de températures différents ont été examinés. De cette manière, certaines dynamiques d'interactions virus-hôte se sont révélées comme ayant une corrélation avec la température. Un flux de travail a ensuite été développé pour créer un réseau reliant les virus et leurs hôtes en utilisant des séquences CRISPR obtenus à partir de données métagénomiques de la glace des glaciers et du sol de l'Arctique. La création de réseaux d'infection à traves du CRISPRs a fourni une nouvelle perspective sur les interactions virus-hôte. En outre, nous avons cherché des événements de transduction dans les données métagénomiques par la recherche de séquences virales contenant de l'ADN microbien. L’analyse indiquée que les bactériophages du Ralstonia pourraient être des agents de transduction dans la glace des glaciers de l'Arctique. / Interactions between the members of a microbial community can be a means of adaptation in the environment. Among the many interactions that take place in an ecosystem and have been seen to play a major role on microbial diversity and population dynamics is that of prokaryotic viruses and their hosts. Viruses can also mediate the transfer of genetic material between prokaryotes (transduction), which could be a mechanism for rapid adaptation. In order to determine the potential impact of viruses and transduction, we need a better understanding of the dynamics of interactions between viruses and their hosts in the environment. Data on environmental viruses are scarce, and methods for tracking their interactions with prokaryotes are needed. Clustered regularly interspaced short palindromic repeats (CRISPRs), which contain viral sequences in bacterial genomes, might help document the history of virus-host interactions in the environment. Thus, this thesis aimed to explore virus-host interactions in a given environment through CRISPRs. Viruses in the cryosphere have been seen to be abundant, highly active and with broad host ranges. These characteristics could make viral transduction a key driver of adaptation in these environments. Public metagenomes created from environments over a range of temperatures were examined through sequence and CRISPR analysis. In this fashion, certain virus-host interaction dynamics were found to have a correlation with temperature. A workflow was then developed to create a network linking viruses and their hosts using CRISPR sequences obtained from metagenomic data from Arctic glacial ice and soil. The creation of CRISPR-based infection networks provided a new perspective on virus-host interactions in glacial ice. Moreover, we searched for transduction events in metagenomic data by looking for viral sequences containing microbial DNA. Further analysis of the viral sequences in the CRISPRs indicated that Ralstonia phages might be agents of transduction in Arctic glacial ice.

Microorganisms are known to be agents involved in the decomposition of organic matter. However, little is known about the participation of the microbial communities during the decomposition of mammalian skeletal muscle tissue. This study investigates the capacity of the soil microbial community to adapt to the decomposition of skeletal muscle tissue in differing soils. This has implications for the study of mass graves and sites of repeated burial. A controlled laboratory experiment was designed to assess the adaptability of microbial communities present in three distinct soil types (sand, loamy sand and sandy clay loam) found near Perth, Western Australia. This experiment was split into two main stages. The initial decomposition stage involved the addition of porcine skeletal muscle tissue (SMT) (Sus scrofa) to each of the three soil types which were then left to decompose for a period of time. Controls were run in parallel, which had no porcine SMT present. The second decomposition stage involved a second addition of SMT to the soils obtained from the initial decomposition stage. Therefore, for each soil, SMT was either decomposed in the soil that had been pre-exposed to SMT or not. The rate of decomposition, microbial activity (CO2 respiration) and microbial biomass (substrate-induced respiration) were monitored during the second decomposition stage. The functional diversity of the microbial populations in the soil were assessed using Community-Level Physiological Profiling (CLPP). Across the three soil types, the re-introduction of SMT to the soil has led to its enhanced decomposition (measured by tissue mass loss and microbial activity) by the microbial communities. This microbial adaptation may have been facilitated by a functional change in the soil microbial communities.

The behaviour and impact of pharmaceuticals in the environment are still poorly understood. Pharmaceuticals are widely used and continually released into the environment causing increasing concerns about their impact on the environment beyond the intended human or veterinary use. Prescribed pharmaceuticals, typically, enter the environment either through excretion after human use or disposal of surplus medication. Sewage treatment plants do not completely remove pharmaceuticals and their metabolites and these have been detected in sewage treatment plant effluent and receiving waters. Land application of treated sewage effluent is widely practiced in New Zealand as an alternative to surface water discharge. Methods were developed to determine selected pharmaceuticals in environmental matrices such as sewage effluent, sewage solids, soil, and soil water. From these, pharmaceutical removal efficiencies were determined for three common sewage treatment processes; activated sludge, composting and land application of sewage effluent. The impacts of some common pharmaceuticals on soil microbial communities, together with the effect of prolonged exposure to sewage effluent on these communities were examined. Additionally, toxicity of sewage effluent, and toxicity mechanisms of specific pharmaceuticals were investigated using luminescent micro-organisms and lettuce seedlings. Pharmaceuticals were successfully detected in sewage effluent, sewage solids, compost, soil and soil water. The sewage treatment processes investigated, including land application, were able to remove or reduce pharmaceutical concentrations in sewage. In case of land application, volcanic soils were more efficient than sandy soils in pharmaceutical removal while irrigation rate and level of sewage pre-treatment also showed some effect on removal efficiency. Pharmaceuticals were not acutely toxic at environmental levels currently detected. Exposure of microbial communities to unnaturally high levels of pharmaceuticals did demonstrate that most pharmaceuticals were potentially able to induce stress in the microbial community although microbes were able to metabolise some of these pharmaceuticals. Twelve years of effluent irrigation resulted in microbial adaptation to aspirin, acetaminophen and tetracycline, indicating that these pharmaceuticals had an effect on microbial community. Presently, land application of treated sewage waste may be a suitable treatment for additional pharmaceutical removal provided that land application schemes are designed appropriately, and pharmaceutical accumulation in the soil is occasionally monitored. It would be prudent to recognise the potential risk that could be caused by chronic exposure to pharmaceuticals such that continued vigilance may lead to future indications of chronic effects at an early stage.

Made available in DSpace on 2017-07-21T19:59:47Z (GMT). No. of bitstreams: 1 Stela Becher.pdf: 1500518 bytes, checksum: a9d6338bf319b6f8a5407fc7f6e00bdb (MD5) Previous issue date: 2009-09-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Mesotrione, 2-(4-Mesyl-2-nitrobenzoyl)-1,3-cyclohexanedione, is a trycetone herbicide chemical class, with high leaching potential, and competitively inhibit 4-hydroxyphenylpyruvate dioxygenase (HPPD), that converts tyrosine to plastoquinone and α-tocopherol, and was found in plants, fungus, and bacteria, including Acinetobacter. The aim of this work was to characterize pathways of Brazilian strains of Acinetobacter facing mesotrione. Acinetobacter calcoaceticus and Acinetobacter sp. were selected in up to 15.00 mM (resistance) and 0.03 mM (tolerance) concentrations of mesotrione and tested at 35 mM buffered solution in spectrophotometry until 60 hours, showing to be incapable to degrade mesotrione. Computer bacterial cells measures (ImageJ 1.37v) indicate starvation process, probably due to non use of mesotrione as carbon source, and this characteristic could be consider a marker for herbicide degradation. Mesotrione herbicide activity is major in N, and enhanced by S, and has a strong electron withdrawing. For this reason, we studied antioxidative stress enzymes catalase, superoxide dismutase and glutathione reductase, by SDS-PAGE, non-denaturatig PAGE and spectrophotometry. Acinetobacter calcoaceticus showed a higher response to oxidative stress than Acinetobacter sp., with more soluble proteins and lower number of electrophoresis bands isoforms (12 against 23), because its enzymes were selected for this herbicide and show better adaptation than Acinetobacter sp.. A microbial strategy of herbicide resistance/tolerance and specific oxidative stress raised by mesotrione herbicide could give additional non-degradating options to microorganisms, not necessarily leading to fast herbicide degradation, so determining the herbicide fate in soils and groundwater. / Mesotrione, 2 - (4-mesil-2-nitrobenzoil) -1,3-cicloexanodionas, é um herbicida da classe química tricetona, com alto potencial de lixiviação, inibe competitivamente a 4-hidroxifenilpiruvato dioxigenase (HPPD), que converte a tirosina em plastoquinona e α-tocoferol, e foi encontrado em plantas, fungos e bactérias, incluindo Acinetobacter. O objetivo deste trabalho foi caracterizar modos de adaptação de linhagens brasileiras de Acinetobacter ao mesotrione. Acinetobacter calcoaceticus e Acinetobacter sp. foram selecionadas em até 15.00 mM (resistência) e 0,03 mM (tolerância) de mesotrione e avaliadas para degradação em experimentos de até 60 horas a 35 mM do herbicida em espectrofotometria, mostrando-se incapazes de degradação. Medidas das células bacterianas em computador (ImageJ 1.37V) indicam processo de inanição, provavelmente devido à não utilização de mesotrione como fonte de carbono, e essa característica pode ser considerada como um marcador para a degradação do herbicida. A forte atividade oxidativa do herbicida mesotrione está vinculada ao radical N da molécula, e reforçada pelo radical S. Por este motivo, foi estudado o estresse oxidativo para as enzimas catalase, superóxido dismutase e glutationa redutase, por SDS-PAGE não denaturante, PAGE e espectrofotometria. Acinetobacter calcoaceticus mostrou uma maior resposta ao estresse oxidativo do que Acinetobacter sp., apresentando mais proteínas solúveis e menor número de isoformas de bandas em eletroforese (12 contra 23), provavelmente devido a suas enzimas terem sido selecionadas para este herbicida e mostraram melhor adaptação que Acinetobacter sp.. Estratégias de resistência microbiana ao herbicida e padrões específicos de enzimas anti-oxidativas poderiam dar opções não degradativas aos microorganismos, levando á manutenção de maiores quantidades herbicidas em solos e águas subterrâneas, e por mais tempo.

Les communautés microbiennes de sédiments de subsurface ont été décrites jusqu’à 1922 mbsf (meters below the seafloor) et pourraient représenter 0,6% de la biomasse totale. Largement incultivées, ces communautés comprennent des groupes endémiques aux environnements de subsurface et des généralistes retrouvés dans des environnements contrastés, appartenant aux 3 domaines du vivant (Bacteria, Eukarya and Archaea). Bien que jouant un rôle majeur dans les grands cycles géochimiques, l’écologie microbienne des sédiments de subsurface reste peu connue. Les conditions hostiles de ces sédiments contrastent avec la présence d’activité et de viabilité microbiennes. Dans ce contexte, de nombreuses questions sur les modes de vie et les métabolismes des microorganismes enfouis demeurent. L’objectif de cette thèse était de mieux comprendre quelles stratégies adaptatives pouvaient être mises en place par les communautés microbiennes de subsurface et de caractériser leur potentiel physiologique. Pour cela, 3 approches ont été utilisées.(1) Une approche culturale a permis de décrire 2 nouvelles espèces bactériennes sédimentaires (Halomonas lionensis, ungénéraliste versatile, et Phaeobacter leonis, une bactérie marine typique). L’étude de la résistance aux conditions de subsurface de ces deux espèces et de la bactérie Sunxiuqinia faeciviva, isolée à 247 mbsf, a ensuite été étudiée. (2) Par une étude de génomique comparée et structurale, la plasticité physiologique de H. lionensis a été analysée. (3) Enfin, le potentiel fonctionnel de communautés microbiennes enfouies à 31 et 136 mbsf dans le bassin de Canterbury a été étudié, en analysant les 2métagénomes correspondants. Les résultats culturaux et génomiques montrent que H. lionensis et S. faeciviva résistent mieux aux stress de subsurface que P. leonis et, dans le cas de H. lionensis, ceci impliquerait des propriétés physiologiques variées pouvant expliquer le succès écologique du genre Halomonas. Les données de métagénomique indiquent que les diversités phylogénétique et fonctionnelle de subsurface du bassin de Canterbury sont distinctes de celles d’environnements de surface et suggèrent que des métabolismes comme la fermentation, la méthanogenèse ou la β-oxydation pourraient être importants. La présence de gènes d’importance écologique et évolutive a permis d’émettre des hypothèses sur les modes de vie de ces microorganismes et des évènements de recombinaison génomique de groupes toujours incultivés ont aussi pu être décrits / Microbial communities inhabiting marine subsurface sediments were described up to 1922 mbsf (meters below the sea floor) andcould represent 0.6% of the total biomass. This microbial diversity, remaining elusive to cultivation, comprises groups specific to subsurface environments and groups of generalists found in contrasted habitats, all belonging to the 3 domains of life (Bacteria,Eukarya and Archaea). Although playing a major role in global geochemical cycles, the microbial ecology of the subseafloor remains largely unknown. The hostile conditions of subsurface sediments contrast with the descriptions of microbial activity andviability in the subseafloor. In this context, many questions related to the microbial physiology and the lifestyles of buried communities remain to be answered. The objective of this thesis was to better understand which adaptive strategies could be deployed by subseafloor microbial communities and to characterize their physiological potential. In that aim, 3 approaches were used.(1) A cultural approach enabled describing 2 novel sedimentary bacterial species (Halomonas lionensis, a versatile generalist and Phaeobacter leonis a typical marine bacterium). The survival of these 2 species to subseafloor conditions and of the subsurface bacteria Sunxiuqinia faeciviva, isolated at 247 mbsf, was then studied. (2) Using a structural and comparative genomic approach, the physiological plasticity of H. lionensis was investigated. (3) Finally, the functional potential of the microbial communities buried at 31 and 136 mbsf in the Canterbury Basin was analyzed, by studying the 2 corresponding metagenomes. Cultural and genomics results showed that H. lionensis and S. faeciviva are more resistant to subsurface constrains than P. leonis and, in the case of H. lionensis, this may involve various physiological properties, maybe explaining thee cological success of the genus Halomonas. Metagenomic data showed that the functional and the phylogenetic diversity of the subseafloor are distinct from the ones from surface environments and highlighted the importance of metabolic pathways like fermentation, methanogenesis and β-oxidation. Genes of ecological and evolutionary interests enabled speculating about lifestyles of buried microorganisms and analyses of genomic fragments highlighted recombination events of still uncultivated microbial groups

The behaviour and impact of pharmaceuticals in the environment are still poorly understood. Pharmaceuticals are widely used and continually released into the environment causing increasing concerns about their impact on the environment beyond the intended human or veterinary use. Prescribed pharmaceuticals, typically, enter the environment either through excretion after human use or disposal of surplus medication. Sewage treatment plants do not completely remove pharmaceuticals and their metabolites and these have been detected in sewage treatment plant effluent and receiving waters. Land application of treated sewage effluent is widely practiced in New Zealand as an alternative to surface water discharge. Methods were developed to determine selected pharmaceuticals in environmental matrices such as sewage effluent, sewage solids, soil, and soil water. From these, pharmaceutical removal efficiencies were determined for three common sewage treatment processes; activated sludge, composting and land application of sewage effluent. The impacts of some common pharmaceuticals on soil microbial communities, together with the effect of prolonged exposure to sewage effluent on these communities were examined. Additionally, toxicity of sewage effluent, and toxicity mechanisms of specific pharmaceuticals were investigated using luminescent micro-organisms and lettuce seedlings. Pharmaceuticals were successfully detected in sewage effluent, sewage solids, compost, soil and soil water. The sewage treatment processes investigated, including land application, were able to remove or reduce pharmaceutical concentrations in sewage. In case of land application, volcanic soils were more efficient than sandy soils in pharmaceutical removal while irrigation rate and level of sewage pre-treatment also showed some effect on removal efficiency. Pharmaceuticals were not acutely toxic at environmental levels currently detected. Exposure of microbial communities to unnaturally high levels of pharmaceuticals did demonstrate that most pharmaceuticals were potentially able to induce stress in the microbial community although microbes were able to metabolise some of these pharmaceuticals. Twelve years of effluent irrigation resulted in microbial adaptation to aspirin, acetaminophen and tetracycline, indicating that these pharmaceuticals had an effect on microbial community. Presently, land application of treated sewage waste may be a suitable treatment for additional pharmaceutical removal provided that land application schemes are designed appropriately, and pharmaceutical accumulation in the soil is occasionally monitored. It would be prudent to recognise the potential risk that could be caused by chronic exposure to pharmaceuticals such that continued vigilance may lead to future indications of chronic effects at an early stage.

AD is regarded as a sustainable technology that could assist the UK Government meet internationally agreed GHG emission targets by 2050. However, the mature status of the technology is based on expensive systems that rely on high energy feedstock to be profitable. Meanwhile, the natural biodegradation of cow slurry is a recognised contributor to climate change despite having a relatively low CH4 potential because of the large volumes produced. Economic mixing is essential to the cost-effectiveness of farm AD but techniques applied are not always appropriate as slurry is a shear thinning thixotropic Herschel-Bulkley fluid and therefore challenging to mix. The apparent viscosity of slurry and the shear stress induced was most influenced by solids content (exponential change) followed by temperature (linear). Most shear thinning occurred before a rising shear rate of 20s-1 was achieved with the fluid acting near-Newtonian above. Thixotropic recovery occurred within 1 hour of resting. Rheological values were also much higher than previously reported. Highest CH4 production occurred in the first 10 days of the batch process using a range of mixing regimes with different shear rates and rest periods. During fed-batch operations, changing shear rate had a minimal effect on CH4 production using a 30-day HRT whereas shorter rest periods increased production. Specific CH4 production rate was highest when feeding and mixing coincided. However, when HRT was reduced (OLR increased) the CH4 produced by all mixed regimes significantly increased with highest values being achieved using high intensity mixing rested for short periods. Lower HRTs also requires smaller digesters. Parasitic mixing energy invariably had the most influence on net energy production. Signs of instability were evident after 20 days using the low HRT. Significant microbial adaptation was also observed as the experiments progressed. The research outcomes demonstrate that mixing regime and HRT can be managed to maximise net energy production whilst reducing capital expenditure.

Inspired by Gould, who claimed life would be arriving at a different outcome each time it were allowed to run from the same beginning, I have attempted to determine the repeatability of the adaptive course of one Pseudomonas fluorescens lineage. In addition, my study aimed to establish whether the likelihood of parallel evolution of the two synonymous single-nucleotide substitutions was contingent upon a prior motility-impairing deletion or a prior increase in fitness. Further, the study was designed to provide empirical data addressing the long-standing question of the effect of starting fitness on the ensuing rate of adaptation. Although no exact replay of the initial evolutionary trajectory was observed, I have demonstrated that gtsB, but not gtsC gene, is likely to be a mutational hotspot under the low glucose with a recovery of two undescribed mutations in gtsB. My data are consistent with a notion that substitutions in gtsB may be contingent upon Δ35kB(fliJ-PFLU4466) motility-impairing deletion, but not the fitness increase associated with it. Finally, the features of the adaptive landscape of P. fluorescens in the minimal glucose provide languid support for Fisher’s hypothesis of a decrease in adaptation rate with the rise in the starting fitness. Taken together, these original results reinforce the non-negligible role of history in shaping the outcomes of biological evolution and call for caution in attempting a formulation of rigid predictive models of evolutionary change. Inspiré par les travaux de Stephen J. Gould qui affirmait que la vie sur terre arriverait à une forme différente si elle repartait à zéro, je présente ici mes travaux où je teste la reproductibilité du cours adaptatif d’une lignée expérimentale de Pseudomonas fluorescens. L’objectif de cette étude était de déterminer si la probabilité que deux mutations synonymes évoluent en parallèle est affectée par la présence d’une délétion affectant la motilité de la bactérie ou de l’augmentation de la valeur sélective de celle-ci. De plus, le design expérimental de cette étude permet de tester si la valeur sélective initiale d’une population affecte le taux d’adaptation de cette même population. Bien d’une reproductibilité exacte du cours adaptatif initial ne fut pas observée, je démontre que le gène gtsB est probablement un « hotspot »mutationnel permettant l’adaptation à de bas niveau de glucose, ayant trouvé deux mutations dans ce site; alors que le gène gtsC ne l’est pas. Mes données sont également conséquentes avec le fait que les mutation dans le gène gtsB dépendent de l’effet de la délétion Δ35kB(fliJ-PFLU4466) affectant la motilité de la bactérie, mais non de l’augmentation de la valeur sélective qui y est associée. Finalement, la forme du plateau adaptative associé à de bas niveaux de glucose chez P. fluorescens supporte l’hypothèse émise par Fisher qui stipule que le taux d’adaptation d’un organisme diminue avec la valeur sélective initiale qui y est associée. L’ensemble de ces résultats supporte le rôle non-négligeable de l’histoire de vie d’une population en ce qui attrait à l’évolution future de cette même population. Aussi, ces résultats appelle à la prudence quand vient le temps de formuler des modèles prédictifs des changements évolutifs d’une population.

Inspired by Gould, who claimed life would be arriving at a different outcome each time it were allowed to run from the same beginning, I have attempted to determine the repeatability of the adaptive course of one Pseudomonas fluorescens lineage. In addition, my study aimed to establish whether the likelihood of parallel evolution of the two synonymous single-nucleotide substitutions was contingent upon a prior motility-impairing deletion or a prior increase in fitness. Further, the study was designed to provide empirical data addressing the long-standing question of the effect of starting fitness on the ensuing rate of adaptation. Although no exact replay of the initial evolutionary trajectory was observed, I have demonstrated that gtsB, but not gtsC gene, is likely to be a mutational hotspot under the low glucose with a recovery of two undescribed mutations in gtsB. My data are consistent with a notion that substitutions in gtsB may be contingent upon Δ35kB(fliJ-PFLU4466) motility-impairing deletion, but not the fitness increase associated with it. Finally, the features of the adaptive landscape of P. fluorescens in the minimal glucose provide languid support for Fisher’s hypothesis of a decrease in adaptation rate with the rise in the starting fitness. Taken together, these original results reinforce the non-negligible role of history in shaping the outcomes of biological evolution and call for caution in attempting a formulation of rigid predictive models of evolutionary change. Inspiré par les travaux de Stephen J. Gould qui affirmait que la vie sur terre arriverait à une forme différente si elle repartait à zéro, je présente ici mes travaux où je teste la reproductibilité du cours adaptatif d’une lignée expérimentale de Pseudomonas fluorescens. L’objectif de cette étude était de déterminer si la probabilité que deux mutations synonymes évoluent en parallèle est affectée par la présence d’une délétion affectant la motilité de la bactérie ou de l’augmentation de la valeur sélective de celle-ci. De plus, le design expérimental de cette étude permet de tester si la valeur sélective initiale d’une population affecte le taux d’adaptation de cette même population. Bien d’une reproductibilité exacte du cours adaptatif initial ne fut pas observée, je démontre que le gène gtsB est probablement un « hotspot »mutationnel permettant l’adaptation à de bas niveau de glucose, ayant trouvé deux mutations dans ce site; alors que le gène gtsC ne l’est pas. Mes données sont également conséquentes avec le fait que les mutation dans le gène gtsB dépendent de l’effet de la délétion Δ35kB(fliJ-PFLU4466) affectant la motilité de la bactérie, mais non de l’augmentation de la valeur sélective qui y est associée. Finalement, la forme du plateau adaptative associé à de bas niveaux de glucose chez P. fluorescens supporte l’hypothèse émise par Fisher qui stipule que le taux d’adaptation d’un organisme diminue avec la valeur sélective initiale qui y est associée. L’ensemble de ces résultats supporte le rôle non-négligeable de l’histoire de vie d’une population en ce qui attrait à l’évolution future de cette même population. Aussi, ces résultats appelle à la prudence quand vient le temps de formuler des modèles prédictifs des changements évolutifs d’une population.