Aedes albopictus et Aedes aegypti sont des moustiques vecteurs d’arbovirus tels que le virus de la dengue et le virus du chikungunya. En plus des virus transmis, les moustiques hébergent également des bactéries dont certaines affectent la biologie des hôtes. Par exemple, la bactérie Wolbachia infecte naturellement Aedes albopictus. Comme chez la plupart des insectes, cette bactérie est un parasite qui manipule la reproduction du moustique et peut également interagir avec certains pathogènes, modifiant ainsi la transmission du pathogène par le moustique hôte. Hors Wolbachia, peu d’études ont été consacrées à l’étude des populations bactériennes hébergées par les moustiques du genre Aedes et aux interactions entre ces populations bactériennes et les arbovirus transmis. Dans ce contexte, le travail de cette thèse a consisté à caractériser la diversité des communautés bactériennes des moustiques du genre Aedes et à explorer l’interférence possible entre le compartiment bactérien et le virus chikungunya. L’utilisation de techniques telles que les isolements bactériens, l’amplification par PCR, la DGGE et l’hybridation in situ ont permis de détecter et localiser certaines bactéries présentes chez des populations naturelles et de laboratoires d’Aedes. Ces populations appartiennent aux Alpha, Beta et Gammaprotéobactéries ainsi qu’aux Firmicutes. L’étude de la dynamique des communautés bactériennes symbiotiques et de l’infection par le virus chikungunya chez Aedes albopictus par PCR quantitative et puces taxonomiques a révélé l’existence d’interactions entre les différents partenaires de ce système vectoriel / Aedes albopictus and Aedes aegypti transmit a large number of arboviruses, including dengue and chikungunya. In addition to viruses, mosquitoes harbour other symbionts that are able to affect its biology. For instance, the bacterium Wolbachia infects naturally Aedes albopictus. As for many insects, this bacterium is an obligate parasite that manipulates the host reproduction and can also interact with pathogens, modifying the transmission of the pathogens by the mosquitoes. Except Wolbachia, little is known about the bacteria associated with Aedes mosquitoes. First, we detected and localized bacteria in field-caught and laboratory populations of Aedes, using culture and non-culture methods including PCR, DGGE and in situ hybridization. The bacterial populations belonged to Alpha, Beta and Gammaproteobacteria as well as to Firmicutes. Then, the effects of chikungunya infection on Wolbachia and total bacterial community were measured using quantitative PCR and taxonomic microarrays. Results showed interactions between the different partners in this vectorial system

L’écosystème digestif est sous l’influence de facteurs abiotiques et biotiques qui déterminent son équilibre et par conséquent influencent la santé digestive de l'hôte. Chez le lapin en croissance, un déséquilibre de l’écosystème caecal est associé aux entéropathies, responsables de mortalités importantes en élevage. La compréhension des interactions biotope – biocénose digestive permettra la mise en oeuvre de stratégies pour garantir l'équilibre de cet écosystème. Ainsi, le rôle des facteurs biotiques de stabilité l’écosystème digestif, tels que des microorganismes exogènes probiotiques, fait actuellement l’objet de nombreuses études, mais leur mode d’action sur la biocénose et le biotope reste encore peu clair. L’objectif de notre travail est de contribuer à la compréhension du fonctionnement de l'écosystème caecal chez le jeune lapin, soumis ou non à un stress nutritionnel et en présence ou non d'une flore exogène ajoutée. Il s'agit aussi, de faire une approche comparative des effets d'un même probiotique (S. cerevisiae) dans cet écosystème et dans le rumen de la vache, pour mieux décrire les mécanismes d'action d'une levure probiotique sur les relations biocénose-biotope. Dans ce but, nous avons mis au point et validé pour le caecum du lapin la mesure du potentiel redox (Eh), pour mieux juger de l'état d'anaérobiose du biotope caecal. Nous avons également validé un nouvel indicateur de l’inflammation générale (haptoglobine sérique) en réponse à l'application d'un stress nutritionnel ou d'un état sanitaire déficient. Comparé au rumen, le biotope caecal est un milieu très anaérobie, puisque son potentiel redox moyen est de -220 mV et ne varie pas avec l’âge (de 28 à 64 jours). La biodiversité de la biocénose bactérienne caecale, calculée à partir de leur empreinte moléculaire (CE-SSCP) est en moyenne de 5,0 (indice de Simpson). Chez l'animal touché par un dysfonctionnement digestif, nous observons une élévation du niveau général de l'inflammation (+70% du taux d’haptoglobine sérique), associée à une chute de l'activité fermentaire caecal (-50%) et une hausse du pH (+ 0,7), mais qui n'est pas associée à des variations d'Eh caecal ou de la diversité bactérienne. L'application d'un stress nutritionnel (déficience en fibres) entraîne chez le lapin une baisse de la concentration caecale en AGV totaux (-25%) et une hausse du pH (+0,1). Cependant, la déficience en fibres n’a pas d'effet marqué sur le Eh caecal, dont la moyenne est de -210 mV. De même, la diversité bactérienne n’est pas modifiée (5,3) par la réduction de la teneur en fibres de l’aliment et la similarité observée est de 76%. La teneur de fibres dans l’aliment n’influence pas non plus le niveau d'inflammation générale. L’apport de levures vivantes dans la ration du lapin tend à augmenter la diversité bactérienne (+10%), et peut élever le potentiel redox caecal de 25 mV caecal. Il n’affecte cependant pas la structure du microbiote bactérien caecal (similarité= 99%). Elle n’entraîne pas non plus de variation du taux d’haptoglobine. L'ingestion de levures vivantes a permis l’amélioration de la santé digestive par la réduction de la mortalité (jusqu'à -50%) pendant les périodes de forte mortalité où le taux d’haptoglobine sérique augmente d’environ 70%. L’effet de la levure observé ici dans le caecum du lapin diffère de celui observé dans le rumen de la vache, pour lequel on observe une baisse du potentiel redox et une hausse du pH, ce qui favoriserait l’activité des bactéries anaérobies strict transformant le lactate en propionate. La levure stabiliserait donc le biotope (pH, potentiel redox) qui favoriserait la croissance ou l’activité de certaines bactéries. Cette hypothèse reste encore à confirmer pour l'écosystème caecal du lapin, à l'aide méthode appropriées. / The digestive ecosystem is influenced by abiotics and biotics factors that determined its balance and consequently influenced the host digestive health. In the young rabbit, caecal ecosystem disorders are largely responsible for nonspecific enteropathies that cause livestock losses. Understanding biotope/biocenosis interrelationships would allow the development of new strategies that preserve the ecosystem balance. Thus, the role of biotic factors that stabilise the digestive ecosystem, such as probiotics is extensively studied, however their effects on biocenosis and biotope remain unclear. The aim of our work is to improve our understanding of the caecal ecosystem functioning, submitted or not to a nutritional stress and with or without addition of an exogenous flora. We also aimed to compare the effects of the same probiotic (S. cerevisiae) in the caecum and in the rumen (dairy cow), to improve our knowledge on the mechanisms of action of yeast probiotic on biocenosis and biotope. We have developed and validated the measure of redox potential in the caecum. We also validated for the growing rabbit, a new indicator of the general inflammation (haptoglobin) in response to the application of nutritional stress or under a deficient sanitary status. Compared to the rumen, the caecal biotope is very anaerobic, since its redox potential is meanly of -220mV, and do not vary with age (35-63d old). The biodiversity of the bacterial community in the caecum, calculated from fingerprint technique (SSCP), reached meanly 5.0 (Simpson index). In the rabbit having a digestive trouble, the seric haptoglobin concentration increased by 70%, while caecal fermentative activity dropped by 50%. In parallel, the caecal pH increased (+0,7 unit) whereas the redox potential and the bacterial diversity remain unaffected in the caecum. When the young rabbit is submitted to a nutritional stress (fibre deficiency), the caecal volatile fatty acids concentration dropped by 25%, while the pH increased by 0.1 unit. However, the fibre deficiency did not affect the caecal redox potential (meanly -210 mV). Similarly, the bacterial biodiversity in the caecum was not modified (5,3) according to dietary fibre intake, as well the bacterial community structure. Besides, the haptoglobin concentration remained similar with fibre intake. The live yeast added in the diet tended to increase the bacterial diversity (+10%), and could slightly increase the caecal Eh (+25 mV). Yeast have no effect on the structure of rabbit caecal microbiota (bacteria only), where the similarity is 99%. It does not change the serum haptoglobin level. In return, yeast addition improved the digestive health by reducing mortality rate by 50%, particularly during periods of high mortality, when the serum haptoglobin increased by 70%. The effect of yeast described in the rabbit caecum differed from that found for the cow rumen: yeast decreased the redox potential and increased the pH that favors the strict anaerobic bacterial activity. The live yeast thus would stabilise the biotope (pH, Eh) and would favor the growth and activity of specific bacteria. However, this hypothesis still remains to be confirmed for the rabbit caecal ecosystem, using pertinent methodology.

Orientador: Valéria Maia Merzel / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T08:23:29Z (GMT). No. of bitstreams: 1 GonzalesLimache_ElmerErasmo_M.pdf: 2196065 bytes, checksum: 46f92b130d253afebe31e1385f5a430b (MD5) Previous issue date: 2015 / Resumo: A água residuária da extração de petróleo é altamente salina e contém uma mistura complexa de hidrocarbonetos, muitos dos quais são altamente tóxicos. Com o aumento da preocupação em preservar o meio ambiente, as tecnologias baseadas em processos biológicos para o tratamento das águas de efluentes estão recebendo atenção especial por serem mais econômicas e versáteis. No entanto, tais processos são influenciados por fatores, tais como: pH, temperatura, salinidade e pressão. Condições extremas destes fatores podem matar ou inibir espécies que não estejam adaptadas. O presente trabalho teve como objetivos identificar e caracterizar 141 bactérias isoladas de águas de produção e lodos ativados do Terminal Marítimo Almirante Barroso (TEBAR-SP) utilizando técnicas de taxonomia molecular, e avaliar o potencial de biodegradação frente a distintos hidrocarbonetos do petróleo, sob condições de hipersalinidade. O DNA genômico extraído de todos os isolados foi utilizado em reação de PCR para amplificação do gene que codifica para o RNAr 16S. O sequenciamento e a análise filogenética do gene RNAr 16S revelaram que estes isolados pertenciam a 20 gêneros distintos, Idiomarina (48), Halomonas (32), Marinobacter (20), Modicisalibacter (3), Arhodomonas (2), Vibrio (1), Pseudoalteromonas (1), Shewanella (1), Alcanivorax (3), Nitratireductor (2), Marispirillum (2), Thioclava (1), Martelella (2), Pusillimonas (2), Brevibacterium (6), Dietzia (3), Rhodococcus (2), Bacillus (2), Staphylococcus (2) e Salegentibacter (4). O método de tipagem molecular RAPD (Random Amplified Polymorphic DNA) foi utilizado para diferenciar os isolados em nível infra-específico, permitindo identificar 49 perfis genéticos diferentes entre os 141 isolados. Os resultados obtidos no presente estudo demonstraram uma alta diversidade taxonômica da fração cultivada da comunidade de bactérias aeróbias presente em águas de produção e lodos ativados, assim como uma alta capacidade dos isolados para suportar concentrações elevadas de salinidade. A habilidade dos isolados para degradar hidrocarbonetos sob condições de hipersalinidade foi avaliada por cromatografia gasosa. Os resultados evidenciaram a preferência das bactérias pela biodegradação de hidrocarbonetos aromáticos / Abstract: The wastewater from oil extraction is highly saline and contains a complex mixture of hydrocarbons, many of which are highly toxic. With the increasing concern to preserve the environment, technologies based on biological processes for the treatment of wastewater are receiving special attention, since they are more economical and versatile, however, such processes are influenced by factors such as pH, temperature, salinity and pressure. Extreme conditions of these factors can kill or inhibit species not adapted to such environments. This study aimed to identify and characterize 141 bacteria isolated from production water and activated sludge derived from Ferry Terminal Almirante Barroso (TEBAR-SP) using molecular taxonomy techniques, and to evaluate the potential for biodegradation of distinct oil hydrocarbons under hypersaline conditions. Genomic DNA extracted from all isolates were used in PCR reactions for amplification of the gene coding for 16S rRNA. The sequencing and phylogenetic analysis of the 16S rRNA gene revealed that these isolates belonged to the following 20 different genera: Idiomarina (48), Halomonas (32), Marinobacter (20), Modicisalibacter (3), Arhodomonas (2), Vibrio (1), Pseudoalteromonas (1), Shewanella (1), Alcanivorax (3), Nitratireductor (2), Marispirillum (2), Thioclava (1), Martelella (2), Pusillimonas (2), Brevibacterium (6), Dietzia (3), Rhodococcus (2), Bacillus (2), Staphylococcus (2) and Salegentibacter (4). The molecular typing method RAPD (Random Amplified Polymorphic DNA) was used to differentiate isolates at the infra-specific level, allowing the identification of 49 different genetic profiles out of 141 isolates. The results obtained in this study demonstrated a high taxonomic diversity of the cultivated fraction of the aerobic bacterial community present in production waters and activated sludge, as well as a high capacity of the individual members to support high levels of salinity. The ability of the isolates to degrade hydrocarbons under hypersaline conditions was evaluated by gas chromatography. The results showed the preference of bacteria by biodegradation aromatic hydrocarbon compounds / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

A Terra Preta Antropogênica (TPA) tem essa denominação porque é encontrada em sítios arqueológicos, onde viveram grupos pré-históricos e é considerada um dos solos mais férteis do mundo. Nela é encontrada grande quantidade de material deixado por grupos indígenas como fragmentos cerâmicos, artefatos líticos, e especialmente carvão pirogênico. Estudos realizados com o carvão pirogênico verificaram que ele aumenta a capacidade de trocas catiônicas nesses solos. Por meio de microscopia de fluorencência, foi observada a presença de microrganismos habitando esse carvão, no entanto, não se sabe quais seriam. Devido à falta de informações sobre a diversidade bacteriana nessas estruturas, este trabalho estudou a diversidade bacteriana em amostras de carvão pirogênico de solos TPA coletadas nos sítios Lagoa Balbina (Amazônia Central- Amazonas) e Mina I (Amazônia Oriental - Pará), através de técnicas moleculares independentes de cultivo. O estudo visou também comparar essa diversidade com a encontrada no solo de onde carvão foi isolado. As estruturas de carvão foram separadas fisicamente dos solos e seu DNA genômico total extraído e usado como molde em reação de PCR utilizando oligonucleotídeos do gene 16S rRNA para o Domínio Bacteria. O produto da PCR foi clonado em vetor e os clones foram sequenciados e comparados com o banco de dados de 16S rRNA do RDPX. Com a construção das bibliotecas de clones do gene 16S rRNA a partir das amostras de carvão pirogênico observou-se que existe maior número de bactérias desconhecidas no carvão pirogênico do que no solo onde ele foi isolado. Acidobacteria foi o filo predominante nas bibliotecas de carvão pirogênico das duas localidades de estudo, assim como na biblioteca do solo do sítio Mina I. Já na biblioteca do solo do sítio Lagoa Balbina houve predominância do filo Firmicutes. Por meio do método de rarefação foi possível constatar uma menor riqueza de UTOs nas comunidades bacterianas presentes nas estruturas de carvão pirogênico quando comparado à riqueza de UTOs das comunidades bacterianas cujo habitat é o solo. Mas quando se compara a riqueza de UTOs entre as estruturas de carvão isoladas das duas localidades, observa-se que a riqueza é maior no sítio Mina I. Os valores obtidos com os índices de diversidade revelaram menor diversidade de UTOs nas bibliotecas obtidas para o carvão pirogênico das duas regiões estudadas se comparado dos valores para as bibliotecas obtidas do solo da mesma região. Os valores obtidos com os métodos não paramétricos revelaram maior riqueza de UTOs para as bibliotecas do carvão do sítio Mina I e solo TPA do sítio Balbina. A análise da PCA revelou que as bibliotecas do sítio Balbina mostraram-se altamente similares. Em adição, a análise com S-Libshuff, verificou que todas as bibliotecas comparadas são significativamente diferentes quanto à composição das comunidades bacterianas. O carvão pirogênico não é uma estrutura inerte, pois é capaz de ser habitado por diferentes bactérias e a sua estrutura da comunidade bacteriana é diferente daquela de onde ele foi segregado / Anthropogenic Dark Earth (ADE) has this denomination because it is found at archeological sites, where prehistoric groups lived, and it is considered one of the most fertile soils of the world. In this soil a great amount of material left by indigenous groups was found as ceramic fragments, lithic workmanships, and especially pyrogenic black carbon. Studies accomplished with the pyrogenic black carbon verified that it increases the capacity of cationic changes in soils. Through fluorescence microscopy, the presence of microorganisms was observed inhabiting that black carbon, however, this community is still unknown,due to the lack of information about the bacterial diversity in those structures.This work studied the bacterial diversity in samples of pyrogenic black carbon of ADE soils, collected at the sites Lagoa Balbina (Central Amazon) and Mina I (Oriental Amazon), through molecular techniques independent of cultivation. The study also sought to compare that diversity with the one of the soil where black carbon was isolated. The structures of black carbon were separate physically from the soils and total genomic DNA was extracted and used as template in a PCR reaction, using primers of the 16S rRNA gene for the Bacteria Domain. The PCR product was used for construction of clone libraries and the clones were sequenced and compared with the 16S rRNA of RDPX database. The 16S rRNA gene clone libraries from the samples of pyrogenic black carbon, it shown that is a larger number of unknown bacteria in the black carbon than in the soil where it was isolated. Acidobacteria was the predominant phylum in the pyrogenic black carbon libraries from the both studied places, as well as in the soil library from Mina I site. However in the library Lagoa Balbina site there was predominance of the phylum Firmicutes. Through the rarefaction method it was possible to verify a smaller richness of OTUs in the bacterial communities presents in the pyrogenic black carbon structures when compared to the OTUs richness of the bacterial soil communities.But, when the OTUs richness is compared among the isolated structures of pyrogenic black carbon of the two places, it is observed that the richness is higher in the Mina I site. The values from diversity indexes revealed smaller diversity of OTUs in the pyrogenic black carbon libraries when compared with the soil libraries for the two studied areas. The obtained values with the nonparametric methods revealed larger OTUs richness in the black carbon library of Mina I site and in the ADE soil library of the Balbina site. The PCA analysis showed that the libraries of the site Balbina site were highly similar. In addition, the analysis with S-Libshuff verified that all of the compared libraries were significantly different in bacterial communities composition. The pyrogenic black carbon is not an inert structure, once it is capable of being inhabited by different bacteria, and its bacterial community structure is different from that one where is was segregated

Unraveling soil microbial ecology is essential for improving sustainable agricultural productivity. Community-based studies revolutionized this field in the last decades, but much is yet to be disclosed. This thesis proposed an approach to increase the resolution of such studies by combining 16S rDNA high-throughput sequencing and population genomics, aiming to further explore the differences pointed by community analyses, as well as to overcome the limitations of using operational taxonomic units (OTUs) as ecological entities, and to introduce the evolutionary thinking in microbial ecology. Our main goal was to understand the features that make bacteria able to colonize sugarcane rhizosphere or live saprophytically in bulk soil. Rhizosphere and bulk soil are contrasting habitats for microbial life as they are highly distinct in its physical, chemical and consequently biological characteristics. Our results indicated that sugarcane shapes the rhizosphere microbiome and metabolism of D-galacturonic acid is a key function for colonizing this niche. Among the taxa prevailing in the rhizosphere, Pseudomonas genus was targeted for a more detailed study considering its known attributes for plant growth promotion. Seventy-six fluorescent Pseudomonas spp. were isolated and submitted to whole genome sequencing (WGS). A comparative genomic analysis was performed between populations from rhizosphere and bulk soil. Phylogenetic analyses classified the isolates in the P. fluorescens (57) or P. putida (19) groups. Twelve putative new species and two new proposed P. fluorescens subgroups were found in the prospected tropical soil. Comparative genomics revealed that phosphatases or xylose-utilization genes were significantly enriched in the rhizosphere and bulk soil populations of the P. fluorescens group, respectively. D-galactonate catabolism was higher in the rhizosphere population of the P. putida group based on both genotypic and phenotypic results. Growth in D-xylose was further explored using genetic modified strains and confirmed that this sugar is more used by members of the bulk soil than the rhizosphere population of the P. fluorescens group, a pattern also observed in the bulk soil microbiome. In summary, these findings constitute a step forward in understanding the ecology of rhizosphere and bulk soil bacteria, by overcoming some limitations of community-based analyses and showing genomic differences between bacterial populations of these habitats. / Desvendar a ecologia microbiana do solo é essencial para aumentar a produtividade agrícola sustentável. Estudos baseados em comunidades revolucionaram esse campo nas últimas décadas, mas ainda há muito a ser revelado. Esta tese propôs uma abordagem para aumentar a resolução desses estudos, combinando sequenciamento em larga escala de rDNA 16S e genômica populacional, com o objetivo de explorar mais a fundo as diferenças apontadas por análises de comunidades, assim como superar as limitações do uso de unidades taxonômicas operacionais (UTOs) como entidades ecológicas e introduzir o pensamento evolutivo na ecologia microbiana. Nossa principal meta foi entender as características que tornam as bactérias hábeis em colonizar a rizosfera de cana-de-açúcar ou viver no solo saprofiticamente. Rizosfera e solo são hábitats contrastantes para a vida microbiana, já que são altamente distintos em suas características físicas, químicas e, consequentemente, biológicas. Nossos resultados indicaram que a cana-de-açúcar modifica o microbioma da rizosfera e o metabolismo do ácido D-galacturônico é uma função chave para colonizar este nicho. Dentre os táxons que prevalecem na rizosfera, o gênero Pseudomonas foi escolhido para um estudo mais detalhado, considerando os seus atributos de promoção de crescimento de plantas. Setenta e seis Pseudomonas spp. fluorescentes foram isoladas e submetidas ao sequenciamento do genoma. Uma análise de genômica comparativa foi realizada entre as populações obtidas do solo e rizosfera. As análises filogenéticas classificaram os isolados nos grupos P. fluorescens (57) ou P. putida (19). Doze prováveis novas espécies e dois novos subgrupos propostos de P. fluorescens foram encontrados no solo tropical prospectado. A genômica comparativa revelou que genes de fosfatases e de uso de xilose foram significativamente enriquecidos nas populações da rizosfera e solo do grupo P. fluorescens, respectivamente. O catabolismo do ácido D-galactônico foi maior na população da rizosfera do grupo P. putida, baseado tanto em resultados genotípicos quanto fenotípicos. O crescimento em D-xilose foi mais explorado usando linhagens geneticamente modificadas e confirmou que este açúcar é mais utilizado por membros da população do solo do que da rizosfera no grupo P. fluorescens, um padrão também observado no microbioma do solo. Em resumo, essas descobertas constituem um passo adiante no entendimento da ecologia bacteriana do solo e rizosfera, por superar algumas limitações de análises de comunidades e mostrar diferenças genômicas entre populações bacterianas destes hábitats.

Les folates sont des vitamines indispensables à tous les âges, particulièrement pendant la grossesse et l’enfance, étant donné leur fonction dans la division cellulaire. Le régime alimentaire en Afrique est essentiellement basé sur les céréales, consommées toujours après transformation. La fermentation est l’un des moyens de transformation des céréales pouvant augmenter les folates dans les aliments. L’objectif de ce travail était d’utiliser la fermentation pour augmenter les ingérés en folates des populations africaines via la consommation d’aliments céréaliers fermentés. Sept aliments céréaliers fermentés couramment consommés en Afrique de l’Ouest ont été investigués. La plupart des aliments avaient des teneurs en folates (1,8–31,3 µg/100g matière fraîche) inférieures à celles des céréales de départ (13,8-73,4 µg/100g matière fraîche). Des pertes en folates ont lieu au cours de certaines étapes de procédés dont le décorticage, le séchage, le trempage, le broyage et la filtration. Toutefois, la fermentation a permis une augmentation de la teneur en folates dans certains aliments. La bioaccessibilité des folates, évaluée à l'aide d'un modèle de digestion statique in vitro, variait de 23% à 81%. Elle était influencée par la matrice alimentaire et la stabilité des folates au cours de la digestion. Il a été calculé qu’au maximum 8% des besoins journaliers en folates des jeunes enfants consommant l’un des aliments étudiés pourraient être couverts. Des essais d’inoculation de bouillies fermentés à base de mil avec des souches de bactéries lactiques sélectionnées pour leurs propriétés nutritionnelles (synthèse de folates, hydrolyse de l’amidon) permettaient d’augmenter significativement les teneurs en folates (jusqu’à 8,7 µg/100 g matière fraîche) par rapport à leur équivalent préparées de manière traditionnelle (2,5-5,4 µg/100 g matière fraîche). L’inoculation par un pied-de-cuve provenant d’une fermentation spontanée permettait aussi une augmentation significative des teneurs folates (jusqu’à 7,4 µg/100 g matière fraîche). La caractérisation de la diversité bactérienne de 7 aliments céréaliers fermentés du Burkina Faso, d’Ethiopie et de la Finlande montrait que les bactéries lactiques du genre Lactobacillus, Enterococcus, Weissella, Pediococcus, Lactococcus et Streptococcus étaient les principaux acteurs de la fermentation de ces aliments. Toutefois, une présence non négligeable d’autres microorganismes potentiellement pathogènes des genres Bacillus, Pseudomonas, Staphylococcus, Erwinia, Klebsiella, Escherichia et Acinetobacter a été identifiée. Cette contamination était liée à certaines étapes du procédé de transformation des céréales dont le stockage et broyage dans les moulins publics. Ces microorganismes pathogènes étaient réduits par fermentation et finalement éliminés après l'étape de cuisson. / Folates represent an essential vitamin in the human diet at all ages, particularly during pregnancy and infancy, as it is required for the production of new cells. In many African countries, the main staple foods are based on cereals, which are always consumed after processing. Fermentation is one of the processing, which could increase folate contents in foods. The objective of this work was to increase folates intake of African people through the consumption of cereal-based fermented foods using fermentation. Seven types of cereal-based fermented foods (CBFF), commonly consumed in West Africa, were investigated in this study. Total folate content of cereal-based fermented ranged between 1.8 and 31.3 µg/100g fresh weight, and was almost always lower than in the raw material (13.8-73.4 µg/100g fresh weight). Folate losses occurred during some processing steps like debranning, soaking and drying steps. However, fermentation was able to increase the folate content in some CBFF. Folate bioaccessibility was assessed using a static in vitro digestion model, and ranged from 23% to 81%. The bioaccessible folate content was influenced by total folate content, the structure of food matrices that modulated folate release, and folate stability during digestion process. Calculations of the contributions of CBFF to the reference nutrient intake for folate showed that folate intakes from these foods would cover a maximum of 8% of the folate requirements for young children. Porridges prepared with starter cultures of lactic acid bacteria selected for the nutritional properties (folate synthesis, starch hydrolysis) had significantly higher folate contents (up to 8.7 µg/100 g fresh matter) than the porridge prepared using the traditional process (2.5-5.4 µg/100 g fresh matter). Back slopping using an inoculum from a spontaneous fermentation also enabled an interesting increase in folate contents (up to 7.4 µg/100 g fresh matter). The bacterial diversity of seven cereal-based fermented foods from Burkina Faso, Ethiopia and Finland were assessed using 16S rRNA amplicon sequencing. Lactic acid bacteria genus, including Lactobacillus, Enterococcus, Weissella, Pediococcus, Lactococcus and Streptococcus were the main bacteria present in cereal-based fermented foods. Several potentially pathogenic bacteria, namely, Bacillus, Pseudomonas, Staphylococcus, Erwinia, Escherichia, Klebsiella and Acinetobacter were also found in some intermediary products resulting from storage and wet milling. These microorganisms were reduced by fermentation and finally removed after the cooking step.

Unraveling soil microbial ecology is essential for improving sustainable agricultural productivity. Community-based studies revolutionized this field in the last decades, but much is yet to be disclosed. This thesis proposed an approach to increase the resolution of such studies by combining 16S rDNA high-throughput sequencing and population genomics, aiming to further explore the differences pointed by community analyses, as well as to overcome the limitations of using operational taxonomic units (OTUs) as ecological entities, and to introduce the evolutionary thinking in microbial ecology. Our main goal was to understand the features that make bacteria able to colonize sugarcane rhizosphere or live saprophytically in bulk soil. Rhizosphere and bulk soil are contrasting habitats for microbial life as they are highly distinct in its physical, chemical and consequently biological characteristics. Our results indicated that sugarcane shapes the rhizosphere microbiome and metabolism of D-galacturonic acid is a key function for colonizing this niche. Among the taxa prevailing in the rhizosphere, Pseudomonas genus was targeted for a more detailed study considering its known attributes for plant growth promotion. Seventy-six fluorescent Pseudomonas spp. were isolated and submitted to whole genome sequencing (WGS). A comparative genomic analysis was performed between populations from rhizosphere and bulk soil. Phylogenetic analyses classified the isolates in the P. fluorescens (57) or P. putida (19) groups. Twelve putative new species and two new proposed P. fluorescens subgroups were found in the prospected tropical soil. Comparative genomics revealed that phosphatases or xylose-utilization genes were significantly enriched in the rhizosphere and bulk soil populations of the P. fluorescens group, respectively. D-galactonate catabolism was higher in the rhizosphere population of the P. putida group based on both genotypic and phenotypic results. Growth in D-xylose was further explored using genetic modified strains and confirmed that this sugar is more used by members of the bulk soil than the rhizosphere population of the P. fluorescens group, a pattern also observed in the bulk soil microbiome. In summary, these findings constitute a step forward in understanding the ecology of rhizosphere and bulk soil bacteria, by overcoming some limitations of community-based analyses and showing genomic differences between bacterial populations of these habitats. / Desvendar a ecologia microbiana do solo é essencial para aumentar a produtividade agrícola sustentável. Estudos baseados em comunidades revolucionaram esse campo nas últimas décadas, mas ainda há muito a ser revelado. Esta tese propôs uma abordagem para aumentar a resolução desses estudos, combinando sequenciamento em larga escala de rDNA 16S e genômica populacional, com o objetivo de explorar mais a fundo as diferenças apontadas por análises de comunidades, assim como superar as limitações do uso de unidades taxonômicas operacionais (UTOs) como entidades ecológicas e introduzir o pensamento evolutivo na ecologia microbiana. Nossa principal meta foi entender as características que tornam as bactérias hábeis em colonizar a rizosfera de cana-de-açúcar ou viver no solo saprofiticamente. Rizosfera e solo são hábitats contrastantes para a vida microbiana, já que são altamente distintos em suas características físicas, químicas e, consequentemente, biológicas. Nossos resultados indicaram que a cana-de-açúcar modifica o microbioma da rizosfera e o metabolismo do ácido D-galacturônico é uma função chave para colonizar este nicho. Dentre os táxons que prevalecem na rizosfera, o gênero Pseudomonas foi escolhido para um estudo mais detalhado, considerando os seus atributos de promoção de crescimento de plantas. Setenta e seis Pseudomonas spp. fluorescentes foram isoladas e submetidas ao sequenciamento do genoma. Uma análise de genômica comparativa foi realizada entre as populações obtidas do solo e rizosfera. As análises filogenéticas classificaram os isolados nos grupos P. fluorescens (57) ou P. putida (19). Doze prováveis novas espécies e dois novos subgrupos propostos de P. fluorescens foram encontrados no solo tropical prospectado. A genômica comparativa revelou que genes de fosfatases e de uso de xilose foram significativamente enriquecidos nas populações da rizosfera e solo do grupo P. fluorescens, respectivamente. O catabolismo do ácido D-galactônico foi maior na população da rizosfera do grupo P. putida, baseado tanto em resultados genotípicos quanto fenotípicos. O crescimento em D-xilose foi mais explorado usando linhagens geneticamente modificadas e confirmou que este açúcar é mais utilizado por membros da população do solo do que da rizosfera no grupo P. fluorescens, um padrão também observado no microbioma do solo. Em resumo, essas descobertas constituem um passo adiante no entendimento da ecologia bacteriana do solo e rizosfera, por superar algumas limitações de análises de comunidades e mostrar diferenças genômicas entre populações bacterianas destes hábitats.

L'utilisation des terres (UT) vers des pratiques agricoles non durables amplifie la dégradation et l'érosion des sols, et la perte de leur diversité microbienne. Cependant, les impacts du changement d'UT sur la structure de la communauté microbienne dans les cours d'eau adjacent restent mal compris, en particulier dans les écosystèmes tropicaux. Grâce à des expériences contrôlées et à des études in situ, j'ai évalué comment différentes pratiques agricoles et d'UT, via des processus hydrologiques, affectent la quantité et la qualité de la matière organique dissoute dans le cours d'eau et la structure de la communauté microbienne associée. Les résultats de ce travail montrent l'importance de considérer à la fois l’UT passée et présente avec les processus hydrologiques lors de l'évaluation de la diversité microbienne et des capacités métaboliques des cours d’eau. Alors que les expériences dans des conditions contrôlées (micro- et mésocosmes) ont permis de distinguer l'importance relative des ruissèlements d’eau de surface sur la structure bactérienne du milieu aquatique, l'approche in situ a permis de donner une vision intégrée de ces processus à l'échelle du bassin. Cela a mis en évidence la nécessité d'utiliser des pratiques de gestion durable d'UT si nous souhaitons atténuer les impacts sur les systèmes aquatiques en aval / Land use (LU) change towards non-sustainable agricultural practices enhances soil degradation, erosion, and the loss of soil microbial diversity. However, the impacts of LU change on in-stream microbial community structure remain poorly understood, particularly in tropical ecosystems. Through controlled experiments and in situ investigations, I assessed how different LU and agricultural practices, via hydrological processes, affect the quantity and quality of stream dissolved organic matter and associated microbial community structure. The results of this work show the importance of considering both past and present LU along with hydrological processes when assessing stream microbial diversity and metabolic capacities. While the experiments in controlled conditions (micro- and mesocosms) allowed disentangling the relative importance of direct overland flow and soil community on stream bacterial structure, the in situ approach gave an integrated view of these processes at the basin scale. This emphasizes the need to use sustainable LU management practices if we wish to mitigate off-site impacts on downstream aquatic systems

Les milieux oligotrophes sont pauvres en éléments nutritifs. En utilisant la technologie de séquençage à haut débit, on a étudié la diversité bactérienne dans trois environnements oligotrophes différents, y compris A. sâbles du désert, B. sâbles dans les tempêtes de l'Asie et C. l’eau et biofilms dans les réseaux de distribution d'eau potable.A. Le désert représente 30% de la surface de la terre. Les conditions de vie dans ces environnements sont un réel défi pour les micro-organismes à cause de nombreux facteurs limitants : peu d’eau et/ou de carbone disponible, une variation importante de température et une forte exposition aux irradiations UV. Le but de cette recherche est donc d’étudier la diversité bactérienne à la surface du sable du désert Taklemaken et du désert de Gobi en utilisant la technologie de séquençage à haut débit. Nos résultats ont révélé une grande diversité bactérienne dans le sol du désert comparable à d'autres types de sols. En outre, nous avons observé une corrélation positive entre la richesse bactérienne et le rapport C/N du sol.B. Les tempêtes de sable d'Asie se produisent presque toujours au printemps, elles sont générées dans les régions arides d'Asie telles que le désert Taklamaken et le désert de Gobi. L'arrivée des tempêtes de sable pourrait largement modifier l'environnement de l'air dans ces régions sous l’effet du vent, surtout dans les villes asiatiques qui sont le plus souvent touchées. Nos travaux visent à étudier la modification de la composition et la diversité des bactéries associées aux particules au moment de tempête de sable en Asie par la technologie de séquençage à haut débit. Nos résultats ont démontré que les compositions des bactéries associées aux particules sont modifiées pendant les tempêtes, en particulier, la proportion des Proteobacteria qui augmentent les jours de tempête. Nous avons signalé neuf genres bactériens détectés en plus pendant les jours de tempêtes, cela nécessite des études plus approfondies.C. Après avoir analysé la population bactérienne dans les tempêtes de sable, et celles des déserts, nous poursuivons notre objectif de recherche à un environnement aquatique. Nous avons suivi le flux d'eau provenant de l'usine d'Orly (DW-A) à l'entrée du réservoir (DW-B), et à la sortie du réservoir (DW-C). Nous avons constaté une forte variation de la communauté bactérienne, dans DW-A et DW-B, les bactéries prédominantes appartiennent aux populations des Betaproteobacteria, puis nous avons observé une conversion vers la population de Alphaproteobacteria dans DW-C. Le DW-C a montré une forte similitude avec un échantillon de biofilm (BF-C), ce qui suggère l'effet important du biofilm sur la modification des communautés bactériennes dans l'eau lors de la distribution. / Oligotrophic ecosystems can be loosely defined as environments that exhibit low ambient nutrient levels. During my thesis, I used 454 DNA pyrosequencing of partial 16S rDNA to explore the bacterial diversity in three different oligotrophic environments, including A. surface desert soil, B. Asian sandstorm dust and C. a section of the city of Paris’s drinking water distribution system.A. Arid regions represent nearly 30% of the Earth’s terrestrial surface. The living conditions at the surface of deserts are a challenge for microorganisms, as there is little available water and/or carbon, a very large range of temperatures and high exposure to UV irradiation from the Sun. In surface sand samples from two large Asian deserts, unexpectedly large bacterial diversity residing was revealed. Sequences belonging to the Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria phyla were the most abundant. An increase in phylotype numbers with increasing C/N ratio was noted, suggesting a possible role in the bacterial richness of these desert sand environments.B. Desert sandstorms are a meteorological phenomenon which have been postulated affect the Earth's climate and public health. We examined the particle-associated (dust and sand-associated) bacterial populations of atmospheric sand in the absence (as control) and presence of sandstorms in five Asian cities. Greater than 90% of the sequences can be classified as representing bacteria belonging to four phyla: Proteobacteria, Bacteriodetes, Actinobacteria and Firmicutes. Principal component analyses showed that the sandstorm-associated bacterial populations were clustered by sampling year, rather than location. Members belonging to nine bacterial genera (Massilia, Planococcus, Carnobacterium, Planomicrobium, Pontibacter, Pedobacter, Lysobacter, Sanguibacter, Ohtaekwangia) were observed to increase in sand-associated samples from sandstorms, versus the controls. C. We characterized the bacterial communities in three water and three biofilm samples from one part of the Parisian drinking water distribution system. A dramatic change in bacterial population in the water during flow through the distribution system from the water treatment plant to the exit from the reservoir was found. The richness of the bacterial population was reduced from the water treatment plant to the reservoir (from 336 to 165 OTUs for water samples leaving the reservoir and from 947 to 275 for biofilm samples in the network). Several OTUs belonging to pathogenic genera were detected in our samples, mostly in the biofilm samples, thus suggesting that the biofilms may be an important source of bacteria during water distribution to the consumers.

Oligotrophic ecosystems can be loosely defined as environments that exhibit low ambient nutrient levels. During my thesis, I used 454 DNA pyrosequencing of partial 16S rDNA to explore the bacterial diversity in three different oligotrophic environments, including A. surface desert soil, B. Asian sandstorm dust and C. a section of the city of Paris's drinking water distribution system.A. Arid regions represent nearly 30% of the Earth's terrestrial surface. The living conditions at the surface of deserts are a challenge for microorganisms, as there is little available water and/or carbon, a very large range of temperatures and high exposure to UV irradiation from the Sun. In surface sand samples from two large Asian deserts, unexpectedly large bacterial diversity residing was revealed. Sequences belonging to the Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria phyla were the most abundant. An increase in phylotype numbers with increasing C/N ratio was noted, suggesting a possible role in the bacterial richness of these desert sand environments.B. Desert sandstorms are a meteorological phenomenon which have been postulated affect the Earth's climate and public health. We examined the particle-associated (dust and sand-associated) bacterial populations of atmospheric sand in the absence (as control) and presence of sandstorms in five Asian cities. Greater than 90% of the sequences can be classified as representing bacteria belonging to four phyla: Proteobacteria, Bacteriodetes, Actinobacteria and Firmicutes. Principal component analyses showed that the sandstorm-associated bacterial populations were clustered by sampling year, rather than location. Members belonging to nine bacterial genera (Massilia, Planococcus, Carnobacterium, Planomicrobium, Pontibacter, Pedobacter, Lysobacter, Sanguibacter, Ohtaekwangia) were observed to increase in sand-associated samples from sandstorms, versus the controls. C. We characterized the bacterial communities in three water and three biofilm samples from one part of the Parisian drinking water distribution system. A dramatic change in bacterial population in the water during flow through the distribution system from the water treatment plant to the exit from the reservoir was found. The richness of the bacterial population was reduced from the water treatment plant to the reservoir (from 336 to 165 OTUs for water samples leaving the reservoir and from 947 to 275 for biofilm samples in the network). Several OTUs belonging to pathogenic genera were detected in our samples, mostly in the biofilm samples, thus suggesting that the biofilms may be an important source of bacteria during water distribution to the consumers.