La digestion anaérobie est un processus naturel de dégradation de la matière organique réalisé par l’action concertée de plusieurs populations microbiennes. Grâce au développement des techniques moléculaires et des projets de métagnéomique, notre connaissance de la diversité de ce monde microbien a été approfondie. Ainsi, de nombreuses nouvelles lignées bactériennes et archées ont été découvertes. L’association de ces techniques moléculaires nous a permis d’explorer et comparer la diversité des micro-organismes présents dans 3 digesteurs anaérobies des STEP Corbeil, Creil et Evry. Les analyses montrent des profils comparables pour Corbeil et Evry mais différents de ceux de Creil. Au sein du domaine Bacteria, les populations dominantes appartiendraient aux divisions des Firmicutes, Bacteroidetes, Proteobacteria, et Chloroflexi. Certaines populations minoritaires dans un digesteur deviennent dominantes dans l’autre (exemple des Spirochaetes à Corbeil). Concernant le domaine Archaea, la nouvelle lignée Arc I (ou WSA2) domine à Corbeil et à Evry. Celle ci n’a pas été détectée à Creil où les Methanosarcinales sont dominants. La découverte d’une nouvelle division candidate WWE3 par une approche metagénomique montre que les méthodes PCR-dépendantes reflètent une image infidèle de la diversité des écosystèmes. / Anaerobic digestion is a natural process, where, under anaerobic conditions, a consortium of microorganisms converts organic material into methane. The development of PCR-dependant techniques and also metagenomic approaches extended our view of population diversity of this consortium and permitted the discovery of novel bacterial and archaeal lineages. In this study, we analyse and compare the prokaryotic diversity of 3 anaerobic municipal sludge digesters: Corbeil, Creil and Evry. Phylogenetic analysis of 16S rDNA sequences shows similar profiles for Evry and Corbeil, while Creil is different. Within the bacterial domain, four predominant phylogenetic groups are detected: Bacteroidetes, Proteobacteria, Firmicutes and Chloroflexi. However, bacterial divisions which are minorities in some digesters, may dominate in others , such as Spirochaetales in Corbeil. Within the archaeal domain, the Arc I lineage (WSA2) is predominant in Evry and Corbeil digesters while it is absent in Creil, where Methanosarcinales is the dominant group. The discovery of the novel bacterial candidate division WWE3 by a metagenomic approach confirms that our view of the microbial diversity within these complex ecosystem remains incomplete.

Heat pasteurization is an effective water treatment to address the emerging plant pathogen issue associated with increased water recycling practices in the ornamental horticulture industry. The current protocol that recommends treating water at 95"C for 30 s, however, faces two major challenges: its energy cost and environmental footprint. We hypothesized that temperature required to inactivate major pathogens in re-circulated water may be substantially lowered from 95"C with extended exposure time. The goal of this study was to test this hypothesis and make this water decontamination technology economically more attractive while reducing its environmental impact. Specific objectives were to (1) examine the effect of water temperature on the survival of Phytophthora and bacterial species, two major groups of plant pathogens in water recycling systems, and (2) elucidate the underlying biological mechanisms by which plant pathogens are killed at those temperatures. Lab assays were performed to determine the survival of zoospores and chlamydospores of P. nicotianae, and oospores of P. pini as well as seven bacterial species after heat treatments at given periods of time. Greenhouse experiments were conducted to determine the applicability of the lab assay data to the real world using annual vinca (Catharanthus roseus) and P. nicotianae as a model system. The results of these studies indicated that the water temperature required to eliminate Phytophthora and bacterial species can be lowered to 48"C from 95"C if treatment time extends to 24 h. Two major steps were taken to elucidate the underlying biological mechanisms. Firstly, a scheme based on the DNA fingerprint and sequence analysis was developed for characterizing bacterial species in irrigation water, after comparing two typing strategies, three sample concentration methods, and evaluating conditions in denaturing gradient gel electrophoresis (DGGE) profiling. Bacterial species detected by culture-dependent and -independent strategies were rather different. The greater bacterial diversity was detected when water samples were concentrated by using both methods than centrifugation or filtration alone. As for DGGE profiling, 40 to 60% denaturant concentrations at 70 V for 16 h revealed the highest bacterial diversity. Secondly, water samples were taken from an irrigation reservoir in a local nursery and analyzed for bacterial diversity following heat treatments at 42 and 48"C. After these heat treatments "-proteobacteria, "-proteobacteria, and Firmicutes became dominant which presents a substantial shift of bacterial community structure compared to those in the control water at 25"C. Among the dominant in treated water were Bacillus, Pseudomonas, Paenibacillus, Brevibacillus, and Lysobacter species, which may have potential biocontrol activities against plant pathogens. This study provided the scientific basis for developing a more energy-efficient and environmentally sound heat pasteurization protocol for water decontamination. / Ph. D.

Past studies characterizing the feline intestinal microflora have used traditional bacterial culture techniques. However, in recent years it has been recognized that the majority of intestinal bacteria are non cultivable. Therefore, the aim of this study was to describe the microflora along the intestinal tract in healthy cats using comparative 16S ribosomal DNA (16S rDNA) analysis. Intestinal content from the stomach, duodenum, jejunum, ileum, and colon was collected from 4 healthy cats and one specific pathogen free cat (SPF) and the bacterial composition was identified by direct sequencing of bacterial 16S rDNA amplicons. A predominant anaerobic microflora was observed in all evaluated segments of the intestine. Fourteen different bacterial orders were identified with the majority of all sequences classified in the class Clostridiales. Six different Clostridium clusters were identified with the majority of sequences affiliated with Clostridium cluster I. Comparative 16S rDNA analysis was also used to evaluate differences in the fecal microflora between healthy cats (n=6), cats with histopathologically confirmed inflammatory bowel disease (IBD; n=6), and cats with intestinal neoplasia (n=3). Compared to the IBD group, cats in the control group showed a significantly higher number of sequences classified as Firmicutes, Bacteroidetes, and Actinobacteria (p<0.0001). The control group had a significantly higher proportion of clones affiliated with Clostridium cluster XI, and a significantly lower proportion affiliated with cluster I (both p<0.0001). In the neoplasia group, the majority of sequences were classified in the phylum Firmicutes (97.9%) and clones were predominately affiliated with Clostridium clusters I and XI. These data indicate that the feline intestinal microflora is highly diverse and is comprised predominantly of anaerobic bacteria. Further studies are warranted to evaluate the clinical significance of the observed differences in intestinal microflora between healthy cats and cats with gastrointestinal disease.

Abstract This research used molecular biological techniques such as polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) to analyze the dynamic microbial community and biodiversity in the groundwater contaminated with petroleum-hydrocarbons. The 16S rDNA sequences from all water samples were compared with the sequences of relative bacteria in the Ribosomal Database Project Bank to construct a phylogenetic tree. The results allowed us to understand the composition of the microbial communities in the petroleum-hydrocarbon contaminated groundwater. In this study, groundwater samples taken from the Chinese Petroleum Corporation Kaohsiung Refinery (CPCKR), Chinese Petroleum Corporation at Ciaotou fuel Tank Farm (CPCCTF) and China Petrochemical Development Corporation at Kaohsiung Factory (CPDCKF) were analyzed. The contaminated sites at CPDCKR and CPCCTF are remeated by natural attenuation. While the CPDCKF site is remeated by an enhanced air sparging bioremediation. In CPDCKR, we found that the low polluted area contained the richest microbial community, followed by the non-polluted area, and the high polluted area. At the CPCCTF site, the microbial community in the non-polluted area was richer than the high-polluted area. Increased microbial populations and variation in microbial community have beenobserved in non-polluted, less polluted, and highly polluted areas. The microbial community showed a dynamic succession of complexity during the bioremediation process at the CPDCKF site. From the 16S rDNA sequence analysis, it is possible that all samples contained petroleum-hydrocarbon degrading bacteria. These petroleum-hydrocarbon degrading bacteria include Methylobacterium, Xanthobacter, Xanthomonas and Pseudomonas at CPCKR site, Flavobacterium at CPCCTF site, Nocardia, Pseudomonas, Rubrivivax, Methylobacterium, and Candida at CPDCKF site. This study also demonstrates that it is more economic and reliable of using molecular techuiques to analyze the groundwater. Thus, groundwater samples can be used to replace soil samples for future work.

Fifty two cellulolytic thermophilic microorganisms were analyzed for their physiological characterization and phylogenetic systematics. Based on 16S rDNA sequence analysis, 3 strains from DCB and 4 novel isolates from southern Taiwan are close related to the genera of Bacillus and Geobacillus respectively. Among 4 new Geobacillus strains, strain T4, a Gram negative, motile, aerobically growing sporulating rod, can secrete thermostable endoglucanase. When strain T4 was grown in CMC medium, the cellulolytic enzyme activity in culture supernatants was stable up to 70¢XC. Based on 16S rDNA sequence analysis, DNA G+C content, phenotypic and physiological characteristics, as well as DNA-DNA hybridization, strain T4 was classified as Geobacillus thermoleovorans T4 (DSM 14791 = CCRC 17200). Furthermore, a phylogenetic tree of 20 related microorganisms was also constructed based on their thermostable cellulase amino acid sequences. Our sequence analysis shows that cellulases belonging to the large family of glycoside hydrolases (GHs) can be divided into four subfamilies: TC-1 (GH family 12 group), TC-2 (bacterial group £L in which fungal species Thermoascus aurantiacus fits), TC-3 (bacterial group £L£L), and TC-4 (GH family 1 group). Together with the 16S rDNA sequence analysis of strain T4 and 10 related microorganisms, strain T4 has a close phylogenetic relationship with subfamily TC-4 but far from subfamily TC-1.

In this study, we had established a 16S rDNA-DGGE analys is system to detect the microbial community in petroleum polluted soil and assess the feasibility of using this system to monitor the bioremediation process. Three genomic DNA extracted methods, the KIT, the Bead-beating system, and the Freeze-thaw method were used to evaluate the DNA extraction efficiency and purity. These DNA samples were further tested by DGGE to analysis the microbial community in soil samples. The results showed that KIT method performed advantageous not only in the DNA extraction efficiency and purity, but also expressed the richest bacterial community in its PCR products. From the DGGE analysis, our data indicated that composition of bacterial community were different in the soil samples that were taken from the same site but at different time. This indicated that the species and number of microorganisms in a polluted soil were under a dynamic transition. The combination of DGGE and 16S rDNA gene sequence analysis system were also proven useful in identifying the predominant microbes in a soil sample and monitoring its bacterial community.

Thousands of parrots all over the world suffer from illnesses and medical complications that can result from interactions between their immune system and bacteria in their digestive tract. The aim of this master's thesis is to understand the link between symptoms of these medical issues, the composition of blood and gastrointestinal microbiota in parrots. Using the hematological methods, 198 blood samples representing 53 parrot species were analyzed. The composition of microbiome was defined by combination of a molecular approach using bacterial 16S rRNA gene sequencing in 132 fecal samples, 12 intestine samples, 228 cloacal swabs and 236 beak swabs representing in total 61 parrot species and a diagnostic approach by psittacine fecal Gram's stain method. Significant association of hematological parameters with individual, environmental and clinical factors was observed, as well as its considerable interspecific variability. Absolute heterophile and lymphocyte counts have been shown more useful for infectious and autoimmune disease monitoring than H/L ratio. Relative numbers of basophiles were the best indicator for behavioral disorders. In relation to hematological parameters, the effect of the bacterial family Flavobacteriaceae, as part of the oral microbiota, and the bacteria Escherichia or...

Abstract The genetic diversity and identification of slow- and fast- growing soybean root nodule bacterial isolates from different agro-climatic regions in Mpumalanga, Limpopo and Gauteng Provinces of South Africa were evaluated. The 16S-rDNA-RFLP analysis of 100 rhizobial isolates and eight reference type strains placed the isolates into six major clusters, and revealed their site-dependent genomic diversity. Sequence analysis of single and concatenated housekeeping genes (atpD, glnII and gyrB), as well as the symbiotic gene nifH captured a considerably higher level of genetic diversity and indicated the dominance of Bradyrhizobium diazoefficiens and Bradyrhizobium japonicum in Mpumalanga, Limpopo and Gauteng Provinces. Gene sequence similarities of isolates with type strains of Bradyrhizobium ranged from 97.3 to 100% for the 16S rDNA, and 83.4 to 100% for the housekeeping genes. The glnII gene phylogeny showed discordance with the other genes, suggesting lateral gene transfer or recombination events. Concatenated gene sequence analysis showed that most of the isolates did not align with known type strains and might represent new species from South Africa. This underscores the high genetic variability associated with soybean Bradyrhizobium in South African soils, and the presence of an important reservoir of novel soybean-nodulating bradyrhizobia in the country. In this study, the grouping of isolates was influenced by site origin, with Group I isolates originating from Limpopo Province and Group II and III from Mpumlanga Province in the 16S rDNA-RFLP analysis.

Until recently it was assumed that the healthy mammalian lung did not harbour a microbiota, unlike other body sites. However, through the use of sequencing based technologies this has been shown to not be the case. Low biomass communities of microbes can be identified in the healthy lung and the lung microbiota in various diseases states has been shown to differ form these 'healthy' communities. The sheep respiratory microbiota is of interest from both an animal health perspective and due to the potential use of the sheep as a large animal model for studying the lung microbiota. In this thesis I seek to characterise the composition and variability of the sheep lung microbiota; the differences between the sheep upper and lower respiratory tract bacterial communities and to assess whether exhaled breath condensate collection can be used as a non-invasive lung microbiota sampling method. To study the bacterial communities present in samples I have used 16S rRNA gene sequencing and analysis. In Chapter 3 I examine the inter-individual and spatial variability present within the sheep lung microbiota. Protected specimen brushings were collected from three lung segments in six animals at three time-points. In a separate sheep a greater number of brushings was taken (n=16) in order to examine the amount of variability over a smaller spatial scale. I find that there can be large differences between the bacterial communities isolated from different locations within the lung, even over short distances. Samples also cluster by the sheep from which they were taken, indicating a host specific influence on the lung microbiota. In Chapter 4 I compare whole lung washes and oropharyngeal swabs from 40 lambs in order to examine the differences between the upper and lower respiratory tract microbiotas. I find that oropharyngeal swabs separate into rumen-like or upper respiratory tract-like bacterial communities. Despite the fact that in humans the upper and lower respiratory microbiotas have been shown to have similar compositions, the sheep lung microbiota samples in this study do not resemble either oropharyngeal samples or reagent only controls. In my first two results chapters, lung sampling methods were used which involved either anaesthesia combined with a bronchoscopic procedure (Chapter 3) or samples being taken from dead animals (Chapter 4). In Chapter 5 I assess whether there is a less invasive way of taking lung microbiota samples from a living individual, both to minimise the procedural stress on animals used as models and to increase the pool of potential volunteers for human lung microbiota studies. I compared samples taken via protected specimen brushings to samples taken via exhaled breath condensate collection, a less invasive sampling technique. I find that condensate samples contain less bacterial DNA and different bacteria than brushing samples, indicating that it is unlikely they could be used as a replacement for invasive sampling methods. In my final results chapter I compare the results across Chapters 3, 4 and 5 to identify bacteria which occur consistently in the sheep lung and could therefore potentially be described as core lung microbiota members. In conclusion, while I have found that there are large differences between the sheep lung microbiota and that which has previously been described in humans, the sheep can still be of use as a model in studies where these differences would not have a significant impact, such as in Chapter 5 of this thesis. I have identified several bacterial members of the core sheep lung microbiota which in future it would be interesting to better characterise and to assess whether they play a role in sheep health.

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No. of bitstreams: 1 buch_b_dr_sjrp.pdf: 6058711 bytes, checksum: 91a5b502646692883f387f5ae5a634f6 (MD5) Previous issue date: 2018-12-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As cianobactérias são importantes componentes das comunidades aquáticas em diversos ecossistemas, graças a longa história evolutiva do grupo que desenvolveu diversas adaptações fisiológicas e citológicas que permitiram a sua dominância ao redor do globo. As cianobactérias são também os bactérias fotossintetizantes com a morfologia mais diversificada e, embora essa qualidade tenha sido extensivamente utilizada pelos cientistas para a delimitação dos táxons e reconstrução da história evolutiva, vem perdendo cada vez mais espaço para o uso de marcadores moleculares, os quais são capazes de inferir relações filogenéticas mais robustas e que mais proximamente refletem o percurso evolutivo traçado por esses microrganismos. Desse modo, o objetivo da realização deste estudo foi caracterizar taxonomicamente populações de cianobactérias bentônicas de ambientes lênticos da região noroeste do estado de São Paulo, utilizando uma abordagem polifásica, por meio do uso do gene rRNA 16S e da estrutura secundária do ITS 16S-23S, além de considerar aspectos morfológicos e ecológicos. Como resultado deste trabalho, 41 populações de cianobactérias bentônicas foram avaliadas, sendo alocadas em 15 gêneros distribuídos em 11 famílias e cinco ordens. A ordem Oscillatoriales foi a mais representativa entre as populações estudadas (68,3%, 28 populações), seguida pela ordem Synechococcales (19,5%, 8 populações). A análise filogenética do gene RNAr 16S foi capaz de revelar a presença de táxons crípticos, que embora apresentem morfologia correspondente com táxons já descritos, formaram clados separados, indicando se tratarem de táxons ainda não conhecidos, e esse foi o caso de 13 populações aqui estudadas. Parte dos táxons crípticos foi trabalhada em maior profundidade, resultando em três manuscritos apresentados na forma de capítulos que correspondem à descrição dos novos gêneros e espécies Koinonema pervagatum (Capítulo III) e Blennothricopsis periphytica (Capítulo IV), além da descrição de três novas espécies para o gênero Phormidium (Capítulo V), com o registro da primeira espécie bentônica produtora de microcistina para o estado de São Paulo. Embora a prospecção dos genótipos tóxicos, utilizando marcadores específicos para os genes mcyE e sxtA responsáveis pela síntese de microcistinas e saxitoxinas, respectivamente, tenha revelado apenas uma linhagem tóxica, esse resultado é positivo do ponto de vista de impactos relacionados à presença de cianobactérias em corpos d’água para uso público. Entretanto, mostra a importância dos estudos de prospecção de toxinas em cianobactérias bentônicas no Brasil, ainda pouco explorados. / Cyanobacteria are important components of aquatic communities in different ecosystems, thanks to its long evolutionary history that provided several physiological and ecological adaptations, allowing them to spread around the globe. Cyanobacteria are also the most morpological diversified photossintetic bacterial group and, despite many taxonomists have extensively used this character to delimit taxa and to reconstruct their evolutionary history, it has been losing its prominence to molecular markers, which are most suited to infer robust phylogenetic relationships that properly reflect the evolutionary path followed by these organisms. Therefore, the aim of the study was to taxonomically characterize benthic cyanobacterial populations in lentic habitats from the Northwest region of São Paulo state, using a polyphasic approach, through the phylogenetic analysis of the 16S rRNA gene and the 16S 23S ITS secondary structure, aside from morphological and ecological aspects. As result, 41 benthic cyanobacterial populations were evaluated and assigned to 15 genera distributed in 11 families and five orders. The Oscillatoriales order was the most representative among all (69.3%, 28 populations), followed by the Synechococcales (19.5%, eight populations). The 16S rRNA phylogenetic analysis revealed the presence of cryptotaxa, which, despite morphologically ressembling previously described taxa, formed separated clades, suggesting not yet acknowledged taxa. That was the case of 13 populations studied. Some of these cryptotaxa were deeper evaluated in three manuscripts presented here as chapters and described as the new genera and species Koinonema pervagatum (Chapter III) and Blennothricopsis periphytica (Chapter IV), the description of three new species from the Phormidium genus (Chapter V) and the first record of a benthic microcystin producing cyanobacteria in the São Paulo state. Although the toxic genotypes prospection using specific molecular markers for the mcyE and sxtA genes, responsible for microcystin and saxitoxin production, respectively, revealed only one toxic strain, it demonstrates a positive result regarding the impact caused by toxic cyanobacterial strains in waters for public use worldwide. However, it highlights the importance of studies on potentially toxic benthic cyanobacterial communities, still little explored in Brazil.