>Magister Scientiae - MSc / Aims DNA metabarcoding has recently emerged as a valuable supplementary tool to ensure food authenticity within the global food market. However, it is widely known that highly processed food samples are one of DNA metabarcoding’s greatest shortfalls due to high DNA degradation, presence of PCR inhibitors and the incomplete removal of several undesirable compounds (such as polysaccharides) that makes the amplification of desired DNA challenging. This project has two main aims, the first of which was to determine and develop a cost and time effective DNA metabarcoding system that could successfully describe to species level the ingredient composition of highly processed vegetarian food products. The DNA metabarcoding system was thoroughly evaluated and tested by combining well-researched primers with varying concentrations into a multiplex reaction. The combination of plant and animal primers selected that yielded the best results were used to determine the species composition in the samples. The second aim is to determine the possible presence of meat contaminants within the highly processed vegetarian food samples. Numerous studies have shown that food adulteration is a wide-spread phenomenon throughout the world due to the economic gains it can provide. Animal primers were introduced into the multiplex reaction to aid in the identification of any meat products that could have been inserted into the vegetarian products to lower the overall cost to company. Methodology Thirty-two highly processed vegetarian food samples were collected in the Cape Town area from local and franchised supermarkets. DNA was extracted using the Chloroform/Isoamyl alcohol method best suited for plant-based samples followed by amplification of the following mini-barcoding regions: the mitochondrial 16S ribosomal rRNA, cytochrome B, tRNALeu – trnL – UAA intron and the ribosomal internal transcribed spacer region – ITS2 for plant and fungi identification. The PCR products were purified using the Qiaquick kit and library preparation and building was conducted using the TruSeq DNA PCR-free Library kit. Final purification was completed using AMPure XP kit and the pooled libraries were sequenced on an Illumina Miseq using 300bp paired-end run. Statistical and bioinformatic analysis on the NGS raw sequence reads was performed in R version 3.6.3. Results The results of the data analysis showed that the cytochrome B primer couldn’t detect any animal DNA in the vegetarian samples, however animal-derived sequences were detected in the positives present, validating the efficacy of the multiplex reaction. Mitochondrial 16S ribosomal rRNA was only able to detect plant-based DNA due to the structural homology between chloroplast and mitochondrial DNA. The fungal ribosomal internal transcribed spacer region – ITS2 detected sequences deriving from “Viridiplantae”. This result could have been due to the fungal and plant ribosomal internal transcribed spacer region – ITS2 sharing a reverse primer during amplification. The trnL region was able to detect the presence of undeclared coriander, mustard and wheat in 8 (29%), 6 (21%) and 5 (18%) samples respectively. Additionally, trnL was able to detect the presence of tobacco in 11 (35%) samples. This could have been due to cross-contamination between samples being co-extracted and amplified at the same time for separate studies. The PITS2 region was able to detect the presence of undeclared barley, mustard and wheat in 8 (25%), 4 (14%) and 4 (14%) samples respectively. Our results show the possibility of DNA metabarcoding for the authentication of a wide range of species present in highly processed vegetarian samples using a single assay. However, further optimization of the technique for the identification of both plant and animal species within vegetarian samples needs to be performed before the wide-spread implementation of this technology would be both feasible and viable. Eliminating primer biases, decreasing the risk of homology between different primers in the same assay as well as preventing the amplification of sequencing of undesirable DNA need to be further explored and ultimately mitigated before DNA metabarcoding can be widely seen as an effective and cost-effective method for authentication and food control.

Soil microbial communities are affected extensively by addition of amendments to their environment. Of particular concern is the addition of poultry litter, which contains a substantial C, energy, and nutrient supply, but also antibiotic resistance genes (ARG), antimicrobials, and a multitude of microbial species. This project seeks to primarily assess if there is a change in bacterial community structure in response to poultry litter amendments to pasture land across geographically independent land across northern Georgia. It may be that changes in the relative abundance of bacterial communities also result in alteration in ARGs, and the community resistance to antibiotics (“resistome”) which in turn increases the potential threat of antibiotic resistance genes. While another part of this study will determine changes in integrons and specific ARGs, this project will focus on changes in bacterial communities and the potential functional changes in the community, which in turn have consequences for ARG levels and its horizontal transfer to various members of the soil community. Addition of waste from livestock is a historical method for increasing nutrients needed in the soil for the cultivation of crops, and in turn causes pronounced shifts in soil microbial communities due to the addition of large amounts of carbon, nutrients, foreign microbes, and other material. This study is unique because it utilizes a novel and relatively large landscape-scale to determine if there are discernable and repeatable patterns of bacterial community structure change in response to amendment regardless of exact soil type or source of chicken litter amendment. In the future, these data can also provide insight into the changes in the relative abundance antibiotic related genes associated with community change. / M.S. / Soil is complicated, both in terms of its physical makeup and the organisms that live inside of it. Predicting changes in soil based on the addition of foreign material such as chemicals or biological waste is not an easy process, and whether or not it is even possible to reliably predict those changes is a matter of some dispute. This study is designed to illustrate that such changes can in fact be reliably and consistently predicted even with regard to the addition of complicated materials to the soil. In this study, specifically, the material in question is chicken litter. A mix of the bedding and waste produced by chickens, litter is commonly handled by composting and is added to soil in farms as a fertilizer rich in organic matter. It is possible to point at specific elements of the soil such as the chemistry and bacteria and see how it is changed with the addition of chicken litter, which allows us to determine the nature and extent of the change that chicken litter has on soil. This study is conducted on a larger scale than similar experiments conducted in the past, making it apparent that these relationships exist on a repeated basis. It is the object of this study to pave the way and make it easier for scientists in the future to determine these relationships in other unique contexts.

To detect Post-kala-azar leishmaniasis (PKDL) cases, several molecular methods with promising diagnostic efficacy have been developed that involve complicated and expensive DNA extraction methods, thus limiting their application in resource-poor settings. As an alternative, we evaluated two rapid DNA extraction methods and determined their impact on the detection of the parasite DNA using our newly developed recombinase polymerase amplification (RPA) assay. Skin samples were collected from suspected PKDL cases following their diagnosis through national guidelines. The extracted DNA from three skin biopsy samples using three different extraction methods was subjected to RPA and qPCR. The qPCR and RPA assays exhibited highest sensitivities when reference DNA extraction method using Qiagen (Q) kit was followed. In contrast, the sensitivity of the RPA assay dropped to 76.7% and 63.3%, respectively, when the boil & spin (B&S) and SpeedXtract (SE) rapid extraction methods were performed. Despite this compromised sensitivity, the B&S-RPA technique yielded an excellent agreement with both Q-qPCR (k = 0.828) and Q-RPA (k = 0.831) techniques. As expected, the reference DNA extraction method was found to be superior in terms of diagnostic efficacy. Finally, to apply the rapid DNA extraction methods in resource-constrained settings, further methodological refinement is warranted to improve DNA yield and purity through rigorous experiments.

No description available.

Die Rindertuberkulose ist eine chronische Erkrankung, die von Mycobacterium (M.) bovis und M. caprae, Mitgliedern des Mycobacterium-tuberculosis-Komplex (MTC), ausgelöst wird. Tuberkulose-Erregern werden sowohl mittels kultureller als auch molekulare Untersuchungsmethoden nachgewiesen. Ziel der vorliegenden Studie war es, die Sensitivität des DNA-Nachweises von Tuberkulose-Erregern zu steigern. Dafür wurden drei Fragestellung im Bereich der molekularen Mykobakterien-Diagnostik bearbeitet. I) Zur Verbesserung der Lyse der mykobakteriellen Zellwand als Voraussetzung für eine Zunahme der Freisetzung von DNA wurden im Vergleich zu einer standardisierten DNA-Isolierungsmethode vier verschiedene Lyseprotokolle (thermische, enzymatische, thermo-enzymatische und mechanische Lyse) entwickelt und mit M. bovis BCG durchgeführt. Die Verbesserung wurde anhand der cycle threshold (Ct)-Werte einer MTC-spezifischen Real-Time (rt) Polymerase-Kettenreaktion (PCR) geprüft. Zwei Lyseprotokolle (thermische und mechanische Lyse) wurden bei zehn Gewebeproben (Lymphknoten, Leber und Lunge) von zehn Tieren (acht Rinder, ein Lama und ein Luchs) mit nachgewiesener Tuberkulose ange-wendet. II) Ausserdem, wurde eine rt-PCR mit dem 16S rRNA Gen als Zielgen (16S-rt-PCR) für den direkten Nachweis von Erregern der Gattung Mycobacterium im Gewebe entwickelt. III) Ein neu entwickelter Spoligotyping-Microarray wurde mit der konventionellen Spoligoty-ping-Methode verglichen, um die neue Methode in Bezug die Sensitivität des Nachweises und des diskriminatorischen Potenzials direkt bei infizierten Gewebeproben zu analysieren. Bei der konventionellen Methode erfolgt die Hybridisierung des PCR-Produktes auf einer Nylon Membran, auf der spezifische Oligonukleotide fixiert sind. Bei der Microarray-Methode sind diese auf einem Microarray-Chip fixiert. Die Ergebnisse der Untersuchungen (I) zur Lyse der Zellwand bei M. bovis BCG zeigten, dass bei der mechanischen Lyse eine Zunahme um 14 % und bei der thermischen Lyse eine Zunahme an PCR-Produkt von 6 % im Vergleich zur Standardlyse erbrachte. Bei beiden Lyseprotokollen wurde eine statistische Signifikanz von α = 1 % (Mann-Whitney-Test) im Vergleich zur Standardlyse errechnet. Bei den tuberkulösen Gewebeproben wurde bei der mechanischen Lyse eine durchschnitliche Zunahme an PCR-Produkt um circa 9 % im Vergleich zur Standardlyse erzielt. Dieser Unterschied war jedoch auf Grund der geringen Probeanzahl nicht statistisch signifikant. II) Bei der Untersuchung von 43 Mykobakterien-Spezies, sechs Mitgliedern des MTC (unter anderen M. bovis BCG) und 37 Non Tuberculous Mycobacteria (NTM) Spezies, konnten alle mit der entwickelten Real-Time PCR (16S-rt-PCR) nachgewiesen werden. DNA-Extrakte von acht nicht zur Gattung Mycobacterium gehörenden Spezies wurden mit der 16S-rt-PCR nicht erfasst. Ein Erreger der Gattung Gordonia und ei-ner der Gattung Rhodococcus wurden auf Grund ihres engen Verwandtschaftsgrades jedoch ebenfalls mit der 16S-rt-PCR detektiert. Die oben erwähnten mittels MTC-spezifischer rt-PCR (Zielgen IS 1081) als infiziert identifizierten zehn Gewebeproben, wurden mittels 16S-rt-PCR untersucht. Die Ergebnisse zeigten, dass beiden rt-PCR Systeme eine vergleichbare Sensitivität aufwiesen. III) Bei dem Vergleich zwischen den Spoligotyping-Methoden zeigte sich die neue Methode um einen Faktor von 100 bei der M. bovis BCG-Reinkultur und um einen Fak-tor von 10 bei DNA-Extrakten aus tuberkulösen Gewebeproben sensitiver als die konventionelle Methode. Im Rahmen dieser Arbeit hat sich der Einsatz der mechanischen Lyse für die Verbesserung der Freisetzung von mykobakterieller DNA als routinefähig erwiesen. Die entwickelte 16S-rt-PCR erwies sich als brauchbare Methode für den Nachweis von Erregern der Gattung Mycobacterium. Die Microarray-Methode stellte sich wesentlich einfacher, sensitiver und schneller dar als die konventionelle Methode. Zusammenfassend kann gesagt werden, dass alle drei Ansätze dieser Arbeit einen Beitrag zur Verbesserung der molekularen Labordiagnostik der Tuberkulose leisten. / Bovine tuberculosis is a chronic disease, that results from infection of Mycobacterium (M.) bovis and M. caprae, members of Mycobacterium tuberculosis complex (MTC), respectively. The laboratory diagnosis of bovine tuberculosis is possible with culture as well as considerate fast molecular methods. The aim of this study was to improve the sensitivity of DNA detec-tion of tuberculosis-causing pathogens. Therefore, three different issues were addressed in the complex molecular procedure targeted. I) four different lytic protocols (thermal, enzymatic, thermo-enzymatic and mechanical lysis) were developed and compared to a standardized DNA isolation protocol that was performed on pure culture of Mycobacterium (M.) bovis BCG in order to improve the mycobacterial cell wall lysis leading to an increase of DNA release. The efficiencies of the lysis protocols were assessed by the resulting cycle threshold (Ct) values of a MTC-specific real time (rt) Polymerase Chain Reaction (PCR). Two lysis protocols (thermal and mechanical) were selected to fur-ther testing of ten tuberculosis-infected tissue samples (lymph nodes, liver and lungs) from ten animals (eight cattle, one lama and one lynx). II) In addition, a real time PCR using the 16S rRNA gene as target sequence was developed, which is also suitable to detect pathogens of Genus Mycobacterium on tissue samples. III) A comparison of a newly developed microarray and the conventional spoligotyping method was realised, to analyse the applicability of the microarray in relation of the sensitivity of the method and to analyse discriminatory potential directly from infected tissue samples. During the conventional spoligotyping method, the PCR product is hybridized with specific oligonucleotides, fixed on a nylon membrane. Using the newly developed method these oligonucleotides are fixed on a microarray-chip. The results I) of the mycobacterial cell wall lysis experiment with pure culture of M. bovis BCG showed an increase of 14 % by using mechanical lysis and an increase by 6 % of the PCR product by using thermal lysis compared to the standard protocol. Using the mechanical lysis as well as the thermal lysis a statistically significant (α = 1 % (Mann-Whitney-Test)) im-provement compared to the standard lysis was achieved. Mechanical lysis was performed on tuberculous tissue samples and the results of the lysis were improved by 9 % compared to the standard lysis. However, the difference between mechanical and standard lysis was not statistically significant due the small sample number. II) Forty-three different mycobacterial species, six members of MTC (among them M. bovis BCG) and 37 Non Tuberculous Mycobacteria (NTM), were detected using the newly developed real time PCR (16S-rt-PCR). Eight non-mycobacterial species were not detected using this rt-PCR, whereas one of genus Rhodococcus and one of genus Gordonia were detected by the 16S-rt-PCR due to their close genetic similarity to the genus Mycobacterium. The ten tuberculosis-infected tissue samples (see above) testing positive using a MTC-specific real time rt-PCR (target gene IS 1081) were sub-jected to the 16S-rt-PCR. Both rt-PCR systems showed a comparable sensitivity. III) By comparing the two spoligotyping-methods, the ArrayStrip™–format method was more sensitive than the conventional method by a factor of 100 applied to pure culture and by a factor of 10 when applied to DNA extracts from infected tissue samples. In conclusion, the mechanical lysis proved to be a practical method to liberate mycobacterial DNA. The newly developed rt-PCR was suitable to detect members of the genus Mycobacterium. The spoligotyping ArrayStrip™–format method appeared to be substantially easier to perform, more sensitive, and less time-consuming than the conventional method. The three methods described were suitable to improve the molecular laboratory diagnosis of tuberculosis.

Syftet med litteraturstudien är att sammanfatta vilken gensekvens som används vid genetisk barcoding av växter och hur väl metoden i fråga tillämpas i tre biologiska yrkesområden: dietanalyser i ekologin, analys av pollensporer i forensisk biologi samt analys av uråldrigt DNA (ancient DNA) i paleontologin. Vidare var det även av intresse att se hur genetisk barcoding kan användas i den gymnasiala undervisningen och hur väl den passar in med de svenska styrdokumenten för skolan. Hur elever har gynnats av den valda metoden samt vilka begränsningar som har uppstått har också berörts. Litteraturstudien baseras på vetenskapliga artiklar som har sökts fram med de nedan listade nyckelorden. Resultaten visar att en kombination av gensekvenser, däribland rbcL, matK, trnH-psbA och ITS, fungerar bäst vid identifiering av växter. I dagsläget är genetisk barcoding fortfarande i utvecklingsfasen, där metoden begränsas av antalet referenssekvenser i databaserna, vilket gör det svårt att utesluta morfologiska identifieringsmetoder i de tre yrkesområdena. Vid användning av barcoding i den gymnasiala undervisningen visar det sig att det stämmer väl överens med de svenska styrdokumenten och ökar elevers intresse för de naturvetenskapliga ämnena, då de kan bidra med värdefull forskning genom tillägg av referenssekvenser i databaserna. De största begränsningarna är att det blir ett stort arbetslass för läraren, att läraren i fråga måste vara bekväm med de olika laboratiska momenten och att skolan ska ha tillgång till nödvändig apparatur. / The purpose of the literature study is to conclude which gene sequences are being used in DNA barcoding on plants and how the method in question is being used in three different biological occupations: diet analysis in ecology, analysis of pollen in forensics and analysis of ancient DNA (aDNA) in paleontology. Further it was also of interest to study how DNA barcoding can be used in high school settings and how the method correlates with the Swedish curriculum. How pupils have benefited from the chosen method and what limitations have arisen have also been touched upon. This literature study is based on scientific articles that have been sought with the keywords listed below. The results show that a combination of gene sequences, including rbcL, matK, trnH-psbA and ITS, works best in plant identification. At present, genetic barcoding is still in the developmental phase, where the method is limited by the number of reference sequences in the databases, which makes it difficult to exclude morphological-based methods in the three occupational fields. When using barcoding in upper secondary education it turns out that it’s in good agreement with the Swedish curriculum and increases the students' interest in the scientific subjects, since they can contribute with genuine research when adding reference sequences in the databases. The main limitations are the workload for the teacher, the teacher in question must be comfortable with the different laboratory steps and that the school must have access to necessary equipment.

No description available.

Le microbiote bactérien cuticulaire des fourmis (Hymenoptera : Formicidae) est connu pour avoir un rôle défensif chez ces insectes sociaux, notamment chez les fourmis attines (Formicidae : Attini) grâce l’utilisation de molécules antimicrobiennes produites par des actinobactéries cuticulaires. Dans le cadre de cette thèse, nous avons étudié le microbiote bactérien des fourmis de Guyane en utilisant différentes approches en chimie des produits naturels et en écologie moléculaire. Le premier chapitre décrit l’isolement, l’identification, la culture et l’évaluation biologique de 43 actinobactéries cuticulaires de fourmis de Guyane. Les tests d’antagonismes des souches isolées et l’activité antibiotique des extraits de culture contre des micro-organismes pathogènes humains sont présentés ainsi que l’identification d’un dipeptide cyclique (Cyclo(LPro-LPhe)) antimicrobien qui a été isolé à partir d’une souche proche de Streptomyces thioluteus. Par ailleurs, la mise en œuvre de réseaux moléculaires appliqués à une analyse par UPLC/MS/MS de cocultures d’actinobactéries a permis d’explorer la diversité des métabolites produits dans ces conditions. Le deuxième chapitre présente une étude méthodologique pour comparer quatre méthodes d’extraction d’ADN, en termes de richesse et de composition du microbiote bactérien cuticulaire, par séquençage haut débit à partir des espèces Atta cephalotes et Pseudomyrmex penetrator. Les résultats du métabarcoding ADN mettent en lumière deux méthodes d’extraction et révèlent des différences inter- et intraspécifiques dans la composition des communautés bactériennes cuticulaires. Enfin, le chapitre trois décrit la composition du microbiote bactérien cuticulaire des espèces Camponotus femoratus et Crematogaster levior dans les jardins de fourmis. Les résultats soulignent l’acquisition d’une partie du microbiote dans l’environnement. En parallèle l’analyse métabolomique des cuticules montre à contrario une plus grande spécificité liée à l’espèce de fourmi. Les recherches futures axées sur les stratégies d’analyses statistiques combinant le métabarcoding et la métabolomique sont discutées. / The bacterial microbiota of ants (Hymenoptera: Formicidae) is known to have a defensive role in social insects, particularly for leaf-cutting ants (Formicidae: Attini) due to the use of antimicrobial molecules produced by cuticular actinobacteria. In this thesis, we studied the bacterial microbiota of ants in French Guyana using different approaches based on natural products chemistry and molecular ecology. The first chapter describes the isolation, identification, culture and biological evaluations of 43 cuticular actinobacteria. Antagonism bioassays of isolated strains and antibiotic activities of the culture extracts against human pathogens are presented as well as the identification of an antimicrobial cyclic dipeptide (Cyclo (LPro-LPhe)) isolated from a strain close to Streptomyces thioluteus. Moreover, the implementation of molecular networks applied to UPLC/MS/MS analysis of actinobacterial cocultures allowed us to explore the diversity of metabolites produced under these conditions. The second chapter presents a methodological study to evaluate the capacity of four DNA extraction methods, in terms of richness and composition of the cuticular bacterial microbiota, in high-throughput sequencing from Atta cephalotes and Pseudomyrmex penetrator. The results of metabarcoding highlight two methods of extraction and reveal inter- and intraspecific differences in the composition of cuticular bacterial communities. Finally, chapter three describes the composition of the cuticular bacterial microbiota of Camponotus femoratus and Crematogaster levior in ant garden and the results reveal the acquisition in the environment of a part of the microbiota. In parallel, metabolomic analyses of ant’s cuticle show, on the contrary, a greater specificity related to the ant species. Future researches focusing on statistical analysis strategies combining metabarcoding and metabolomics data are discussed.

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.

As cyanobacteria can be found in a wide variety of environments and can cause great damage to ecosystems, human health, and economy, it is important to monitor and understand their behaviour. In this case, the study aims to understand the spatial variations of cyanobacteria communities in the Gulf of Bothnia, by analysing environmental factors that affect them. I hypothesize that the variations from north to south and coast to offshore of nutrients such as phosphorus and nitrogen promote the occurrence of different cyanobacteria orders, i.e. Chroococcales, Oscillatoriales, and Nostocales. The methods used were 16S amplicon DNA sequencing and the traditional microscopy method. Both showed similar results: Nostocales seem to predominate south-offshore zones and Oscillatoriales coast-north stations. Nevertheless, contrary to what was hypothesized on the beginning, Chroococcales were widely distributed between the stations. Both analysis methods used have limitations and strengths, thus, they should complement each other. Moreover, the DNA method must be implemented in cyanobacterial monitoring for the Baltic Sea. On the other hand, it can be said that environmental variables such as nutrients, jointly with other parameters, such as salinity, pH, humic substances and DOC (dissolved organic carbon), influence cyanobacteria communities’ composition. Overall, the environmental factors in the Baltic Sea give cyanobacteria the perfect conditions for living and develop. Therefore, it is important to implement water management and monitoring plans, as temperatures are predicted to rise globally with climate change, thus, cyanobacterial blooms are also predicted to be more common.