Dissertations / Theses on the topic 'Microbial diversity'

Studies of subterranean ancient evaporites in different geographical and geological habitats around the world have revealed that these sites are populated by abundant populations of halophilic eubacteria. The diversity of these isolates was established by phenotypic, chemotaxonomic and phylogenetic analyses. The majority of the isolates were Gram-negatives (90%), the remainder being Gram-positives as judged by several different kinds of analyses. A numerical taxonomy study of the Gram-negative isolates revealed nine distinct phenons, whereas the Gram-positive isolates were represented by only two phenons. Several of the Gram-negative phenons were distinct from known halophiles included for comparison, confirmed by further chemotaxonomic and phylogenetic analysis. It is suggested that at least three new taxa, probably a genus level, are represented by some of these isolates. Other isolates were closely related to known representatives of the Halomonas group, which are widely distributed in a variety of hypersaline environments. A number of rare archaeal isolates from a particular alkaline hypersaline subterranean site were most closely related to the genus Natronobacterium on the basis of chemotaxonomic and phylogenetic analyses. There was less agreement between phenotypic, chemotaxonomic and phylogenetic analysis for representatives of the two Gram-positive phenons, although the centrotype of one phenon was most closely associated with the same genus Marinococcus and that of the other with the Bacillus spectrum.

Soil microbial biodiversity is an essential component of the natural ecosystem. Soil microbes work as decomposers contributing to soil nutrient cycling, primary production, and climate regulation. The heterogeneous edaphic properties lead to the diversity of microbial community structuring and functioning. This thesis investigates microbial community distributions and functions through vertical soil profiles, at the landscape level, and along regional transects. Vertically, soil microbial communities were depicted in soil profiles to a depth of 1 m using the concept of genosoils (soil formed and still under natural vegetation) and phenosoils (the same type of soil that has undergone cultivation). Bacteria community distribution in soil profiles differed by soil types but altered by soil forms. At the local scale, factors of land use and soil types on the microbial communities were evaluated in three soil depth layers through a survey across the Hunter Valley area in NSW, Australia. Topsoil microbial communities were generally regulated by land use, while the subsoil microbial communities were shaped by soil type. Additionally, microbial interactions reveal that soil protists regulate the bacterial and fungal diversity. At the regional scale, microbial functions were investigated across two ~1000 km transects which traversed significant temperature and/or rainfall gradients in NSW. Temperature and rainfall were important drivers of soil microbial functional groups. Paired (genosoils and phenosoils) samples showed that agriculture practices led to a significant shift in microbial functional groups related to particulate organic carbon (POC) degradation. Collectively, this thesis studied the factors of depth, soil type, land use, and environment for the underground microbial community, and demonstrated the significant role of soil biodiversity in the soil ecosystem, especially for soil carbon cycling.

Microbes are the most abundant and most diverse form of life on Earth, constituting the largest portion of the total biomass of the entire planet. They are present in every niche in nature, including very extreme environments, and they govern biogeochemical transformations in ecosystems. The human body is home to a diverse assemblage of microbial species as well. In fact, the number of microbial cells in the gastrointestinal tract, oral cavity, skin, airway passages and urogenital system is approximately an order of magnitude greater than the number of cells that make up the human body itself, and changes in the composition and relative abundance of these microbial communities are highly associated with intestinal and respiratory disorders and diseases of the skin and mucus membranes. In the early 1990's, cultivation-­‐independent methods, especially those based on PCR-­‐amplification and sequences of phylogenetically informative 16S rRNA genes, made it possible to assess the composition of microbial species in natural environments, advances in high-­‐throughput sequencing technologies in recent years have increased sequencing capacity and microbial detection by orders of magnitude. However, the effectiveness of current computational methods available to analyze the vast amounts of sequence data is poor and investigating the diversity within microbial communities remains challenging. In addition to offering an easy-­‐to-­‐use visualization and statistical analysis framework for microbial community analyses, the study described herein aims to present a biologically relevant computational approach for assessing microbial diversity at finer scales of microbial communities through nucleotide variation in 16S rRNA genes.

Microorganisms play a crucial role in supporting biodiversity, maintaining marine and terrestrial ecosystems at the crux of the nutrient cycle. They are the most diverse and abundant of all living creatures, yet little is understood about their distribution or their intimate relationship with the environment. Antarctic ecosystems are among the most simple on Earth; with basic trophic structuring and the absence of many taxonomic groups, they are also isolated geographically with small patchy areas of nutrient inputs. In this instance, Antarctica becomes a pristine laboratory to examine the ecological paradigms already applied to macro-organisms, to determine if common biological laws govern the distribution of biology globally. The decline of biodiversity with increasing latitude is one such observation in the distribution of macro-organisms. In this study, soil microbial community samples were retrieved over a latitude of 56 to 72 °S across the Antarctic Peninsula region. This is a region of special interest due to a rapidly warming climate with mean temperatures increasing at several times the rate of mean global warming. Sites were biologically and environmentally profiled and data used in a variety of multivariate analysis in order to identify spatial trends and infer mechanisms that may be driving Antarctic terrestrial food webs; or where this was not possible, the areas where focus was needed to increase the information profile to allow this. Results indicate a lack of linear latitudinal gradient in microbial diversity, but do show a correlation with environmental heterogeneity; analysis of site diversity identified a gradient between warmer wetter areas, and areas synonymous with cold desert environment at 66⁰S, supported by both phylum composition and indicative soil chemistry. This was confirmed through principal co-ordinates of neighbours’ matrices analysis (PCNM), with distinct regions of community composition being identified when viewed with respect to environmental variables. Considering an overview of diversity with respect to environmental variables provided additional structure to test hypotheses about nutrient webs through structural equation modelling (SEM), and inferred that areas of patchy nutrient input exist and by means of ornithogenic guano additions promote higher C and N availability, increasing microbial abundance and richness.

Thesis (M.S.)--University of North Carolina at Chapel Hill, 2009.
Title from electronic title page (viewed Oct. 5, 2009). "... in partial fulfillment of the requirements for the degree of Master of Science in the Department of Marine Sciences." Discipline: Marine Sciences; Department/School: Marine Sciences.

Caves are dark and oligotrophic habitats where chemotrophic microbial communities interact with the inorganic mineral rocks and cooperate organizing themselves in complex biological formations, which are visible in caves as biofilms, biodeposits or biospeleothems. In these environments, microorganisms contribute to the turnover of the matter and activate peculiar enzymatic reactions leading to the modification of the mineral rocks and to the production of metabolites with possible industrial and pharmaceutical interest. In this PhD thesis, various molecular and geomicrobiological approaches were used to investigate the microbial diversity and potential activities in different cave systems, i.e. the orthoquartzite cave Imawarì Yeuta, the sufidic cave Fetida and the ice cave Cenote Abyss. This is aimed at gathering indications on the possible interactions that support microbial growth and its impact in cave environments. As a result, microbial taxa and functions associated to light-independent chemolithotroph and heterotrophic activities were identified in the three caves, indicating the involvement of microorganisms in i) silica mobilization and amorphization processes and the formation of a novel type of silica-based stromatolite in Imawarì Yeuta Cave, ii) the formation of three types of biofilm/biodeposit involved in sulphur cycle and in the speleogenesis of Fetida Cave, iii) the development of biofilms and their maintenance under psychrophilic conditions in samples collected from ice in Cenote Abyss. Additionally, the metabolic potentials of around one hundred isolates derived from these cave systems were evaluated in terms on anti-microbial activity. The results pointed out that unexplored and oligotrophic caves are promising environments for novel bioactive molecules discovery.

Antarctica provides some of the most extreme environments on earth. Low temperatures, low water availability and nutrient deficiency are contributing factors to the limited colonisation of Antarctic biotopes, particularly in the continental Dry Valleys. The survival of microorganisms in this harsh continent provides the basis for the significance of this study. This study aimed to explore microbial phylotypic diversity across a 500 m altitudinal transect in the Miers Dry Valley, Ross Desert, East Antarctica. The study also attempted to infer from phylogenetic data, the possible presence of indicative phenotypes which might contribute to a functional microbial community.

>Magister Scientiae - MSc
Salt pans are a characteristic feature of many dry deserts. The microbial communities inhabiting salt pans are thought to be particularly complex and are generally dominated by halophilic microorganisms. Although saline pools are frequently found within the hyper-arid Namib Desert, the microbial communities of these saline sites have been scarcely investigated. The aim of the present study was to characterise the archaeal, bacterial and cyanobacterial diversity inhabiting these extreme saline pools using three culture independent molecular techniques (DGGE, T-RFLP and 16S rRNA clone libraries). The physiochemical results, mainly the conductivity readings recorded from the sampling sites, indicated that the Gobabeb (103.0mS/cm) region was less saline than the two Swakopmund [(Sps01) (150.0mS/cm) and Sps02 (180.0mS/cm)] sites. Results obtained from DGGE and T-RFLP data were in agreement for both bacterial and cyanobacterial analysis indicating that the Gobabeb site was more diverse than the two Swakopmund sites (Sps01 and Sps02). In comparison, the archaeal community profiles for DGGE and T-RFLP analysis were in agreement illustrating that the archaeal community were more abundant in the two extreme Swakopmund saline sites. Phylogenetic data obtained from 16S rRNA gene clone libraries identified halophilic phylotypes (Rhodothermaceae, Idiomarinaceae Puniceicoccaceae and Cyanobacteria/Chloroplast, Family VII) normally associated with salt rich sites. In addition, a large number of unclassified taxa were identified. To conclude, the study highlighted the presence of a rich microbial diversity present within the salt pans of the Namib Desert and establishes a platform for future investigations.
National Research Foundation

In marine hydrothermal sediments metal-rich, reduced fluids and oxidised seawater form steep geochemical gradients supporting abundant, but as yet poorly defined, chemosynthetic life and biogeochemical cycling. This project elucidates the functional diversity of chemosynthetic micro-organisms, and metabolic processes on the geochemistry of ferruginous (-50% Fe), arsenic-rich (-400 ppm As) hydrothermal sediment at Santorini, Greece (20-40°C, pH 6.0-6.3, Eh 0 to 155 mV). Culture and molecular-phylogenetic techniques revealed abundant chemosynthetic prokaryotes capable of transforming a range of chemical species common to marine and hydrothermal sediments via: anaerobic Fe(III), sulfate, nitrate and As(V) reduction, and nitrate-dependant Fe(lI) oxidation; micro-aerobic Fe(lI) monosulfide (FeS) oxidation; and aerobic As(lII) and free-sulfide oxidation. Geochemical analyses showed that oxidised species [Fe(III), As(V), sulfate and presumably Mn(IV)] dominated in the suboxic surface sediment layer (0-5 cm deep), but that reduced species [namely Fe(II), As(III), Mn(II)] increased in the lower suboxic-anoxic transition zone (5-20 em depth). From a biological perspective sulfate is the energetically least favourable of the oxidised species to be reduced, and sulfate concentrations were consistent throughout the portion of the sediment column analysed (0-30 em depth). The presence of a black precipitate (probably FeS) at the lower limit of the transition zone, however, did suggest at least a small amount of sulfate reduction was occurring. In contrast, no nitrate was detected indicating rapid reduction of this . energetically favourable compound at· the water-sediment interface. Microbiological and geochemical data, in combination, indicated that redox cycling of Fe, Mn and As were most likely key biogenic processes in biogeochemically stratifying the sediment, whereas S-cycling bacteria were comparatively minor contributors. Other principal findings included the cultivation of a novel neutrophilic Fe(II)-oxidiser related to marine microaerophilic Fe(II)oxidising bacterium Mariprofundus ferroxydans. This bacterium dominated the Fe(lIl) (oxy-)hydroxide-rich surface sediment, and most probably plays a significant role in catalysing suboxic Fe(lI) oxidation. Fe(II)-oxidising prokaryotes comprise a functional group not well understood in marine settings. Additionally, bacteria belonging to a ubiquitous, heterotrophic marine genus, Marinobacter, were shown to oxidise Fe(II). Of these, Marinobacter santoriniensis, was isolated and characterised, and also shown to both oxidise As(llI) and respire As(V), providing a rare example of a marine prokaryote capable of these functions. Futher to this, incubation experiments, conducted to establish the effect of microbial respiration on arsenic mobilisation, demonstrated that microbial Fe(lll) reduction largely controlled shifts in arsenic partitioning in the sediment. Specifically, results support other studies (mainly concerned with the toxic effects of arsenic) showing arsenic sequestration by Fe(II)-bearing minerals. This is contrary to the more common supposition of arsenic release to porewaters upon reductive dissolution of Fe(lIl) minerals. Overall, these results highlight the importance of microbial enzymatic processes in sediment geochemistry.

Thesis (MSc (Microbiology))--Stellenbosch University, 2008.
The soil environment is thought to contain a lot of the earth’s undiscovered biodiversity. The aim of this study was to understand the extent of microbial diversity in the unique ecosystem of the Western Cape’s fynbos biome. It is known that many processes give rise to this immense microbial diversity in soil. In addition the aim was to link microbial diversity with the soils physio-chemical properties as well as the plant community’s structure. Molecular methods especially automated ribosomal intergenic spacer analysis (ARISA) was used in the study. The most important property of environmental DNA intended for molecular ecology studies and other downstream applications is purity from humic acids and phenolic compounds. These compounds act as PCR inhibitors and need to be removed during the DNA extraction protocol. The fist goal in the study was to develop an effective DNA extraction protocol by using cationic locculation of humic acids. The combination of cationic flocculation with CuCl2 and the addition of PVPP and KCl resulted in a high yield of DNA, suitable for PCR amplification with bacterial and fungal specific primers. Determining the reproducibility and accuracy of ARISA and ARISA-PCR was important because these factors have an important influence on the results and effectiveness of these techniques. Primer sets for automated ribosomal intergenic spacer analysis, ITS4/ITS5, were assessed for the characterization of the fungal communities in the fynbos soil. The primer set delivered reproducible ARISA profiles for the fungal community composition with little variation observed between ARISAPCR’s. ARISA proved useful for the assessment and comparison of fungal diversity in ecological samples. The soil community composition of both fungal and bacterial groups in the Sand fynbos was characterized. Soil from 4 different Sand fynbos sites was compared to investigate diversity of eubacterial and fungal groups at the local as well as a the landscape scale. A molecular approach was used for the isolation of total soil genetic DNA. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified when performing bacterial ARISA from total soil community DNA (BARISA). Correspondingly, the internal transcribed spacers, ITS1, ITS2 and the 5.8S rRNA gene from the fungal rRNA operon were amplified when undertaking fungal ARISA (F-ARISA). The community structure from different samples and sites were statistically analysed. ARISA data was used to evaluate different species accumulation and estimation models for fungal and bacterial communities and to predict the total community richness. Diversity, evenness and dominance were the microbial communities were used to describe the extent of microbial iversity of the fynbos soils. The spatial ordination of the bacterial and fungal species richness and diversity was considered by determining the species area relationship and beta diversity of both communities. The correlation between the soil physio-chemical properties was determined. The plant community structure data was correlated with the fungal and the bacterial community structure. The results indicated that bacterial species numbers and diversity were continually higher at the local scale. Fungi however showed higher species turnover at the landscape scale. Bacterial community structure showed stronger links to the plant community structure whereas the fungi community structure conformed to spatial separation patterns. To further investigate the diversity of soil microbes the potential of genus specific primes was investigated. The genus Penicillium is widespread in the soil environment and the extent of its diversity and distribution is however not. For this reason Penicillium was chosen as a model organism. To expand the insight into the diversity of Penicillium species in the fynbos soil ecosystem, a rapid group specific molecular approach would be useful. Penicillium specific primers targeting the 18S rRNA ITS gene region were evaluated. Fungal specific primers ITS4 and ITS5, targeting the internal transcribed region (ITS) were used to target Penicillium specific in the soil sample. Nested PCR, using primer Pen-10 and ITS5, was then utilized to target Penicillium species specifically. The discrimination of Penicillium species was possible due to length heterogeneity of this gene region. Eight different peaks was detected in the soil sample with ARISA and eight different species could be isolated on growth media. The technique proved useful for the detection and quantification of Penicillium species in the soil.

Thesis (Ph. D.)--Michigan State University. Dept. of Entomology and Program in Ecology, Evolutionary Biology and Behavior, 2008.
Title from PDF t.p. (viewed on July 7, 2009) Includes bibliographical references (p. 185-195). Also issued in print.

Els oceans conten aproximadament un total de 10^29 cèl·lules microbianes. Els bacteris marins són responsables de la major part de la respiració que es produeix en l’oceà i són essencials en els cicles biogeoquímics de la Terra. Estudiar la diversitat bacteriana dels ecosistemes marins i tenir accés als genomes mitjançant estudis dependents o independents de cultiu és important per desxifrar el potencial metabòlic dels bacteris marins. El cultius ens aporten informació sobre la fisiologia bacteriana, ecologia i contingut genòmic, però la majoria dels esforços en aïllar bacteris marins provenen de la zona fòtica de l’oceà, deixant les profunditats marines menys explorades. En aquesta tesi, tècniques estàndard de cultiu han permès crear una col·lecció marina de cultius de bacteris heterotròfics (MARINHET), composada per més de 2000 aïllats, recuperats de diverses regions oceanogràfiques, de diverses profunditats (superfície, mesopelàgic i batipelàgic) i cobrint diverses estacions i anys. El Capítol 1 descriu la seva taxonomia, diversitat filogenètica i biogeografia i revela que un 37% de les soques aïllades són 100% idèntiques en el gen ribosomal 16S (16S rRNA) entre la zona fòtica (superfície) i afòtica (mesopelàgic i batipelàgic). A més, hem identificat Alteromonas i Erythrobacter entre els gèneres marins heterotròfics més comuns que recuperem en cultiu usant un medi marí estàndard. Les tècniques tradicionals de cultiu generalment només recuperen una fracció petita de les comunitats bacterianes naturals, fenomen conegut com ‘la gran anomalia de recompte en placa’ i moltes de les soques que s’aïllen pertanyen a la biosfera rara. Tanmateix, no coneixem si aquests patrons, normalment descrits per als bacteris de la zona fòtica, també s’apliquen als bacteris de les profunditats. En el Capítol 2 he combinat els resultats obtinguts mitjançant tècniques dependents i independents de cultiu comparant les seqüències del 16S rRNA de la col·lecció MARINHET contra fragments de seqüenciació massiva del 16S rRNA, tant d’amplicons com de metagenomes, obtinguts de mostres globalment distribuïdes i de diferents profunditats. Trobem que una major proporció dels bacteris de l’oceà profund són cultivables i una fracció important dels aïllats té preferència a un estil de vida adherit a partícules. A més, confirmem que el dogma ‘menys de l’1% dels bacteris són cultivables’ ha de ser revisat ja que trobem variabilitat en les mostres de l’oceà profund, on fins a un 3% de les cèl·lules han sigut cultivades. Els aïllats bacterians són un excel·lent material per a aplicacions biotecnològiques com la bioremediació de zones marines contaminades. El mercuri és un metall pesat tòxic i la seva forma més perillosa, el metilmercuri (MeHg), es bioacumula a la cadena tròfica marina. No obstant això, es coneix molt poc la tolerància de bacteris marins enfront del mercuri o la fisiologia d’aquelles soques que codifiquen l’operó dels gens de resistència (operó mer). El Capítol 3 descriu els resultats del mapeig funcional dels gens merA i merB, clau en la detoxificació, en una fracció de la col·lecció MARINHET. Ens centrem en dos generes marins, amb un potencial genètic per a la degradació del mercuri conegut, com són Alteromonas i Marinobacter. Revelem que els gens merAB estan àmpliament distribuïts en diferents regions oceanogràfiques i en diverses profunditats. Addicionalment, hem seleccionat una soca d’ Alteromonas mediterranea per a futurs estudis de bioremediació degut a la seva alta tolerància i capacitat de degradació de diferents formes de mercuri.
Los océanos contienen aproximadamente un total de 10^29 células microbianas. Las bacterias marinas son responsables de la mayor parte de la respiración que se produce en el océano y son esenciales en los ciclos biogeoquímicos de la Tierra. Estudiar la diversidad bacteriana de los ecosistemas marinos y tener acceso a los genomas mediante estudios dependientes e independientes de cultivo es importante para descifrar el potencial metabólico de las bacterias marinas. Los cultivos nos aportan información sobre la fisiología bacteriana, ecología y contenido genómico, pero la mayoría de los esfuerzos en aislar bacteria marinas provienen de la zona fótica del océano, dejando las profundidades marinas menos exploradas. En esta tesis, técnicas estándar de cultivo han permitido crear una colección marina de bacterias heterótrofas (MARINHET), compuesta por más de 2000 aislados, recuperados de varias regiones oceanográficas, de varias profundidades (superficie, mesopelágico y batipelágico), y cubriendo varias estaciones y años. El Capítulo 1 describe su taxonomía, diversidad filogenética y biogeografía y revela que un 37% de las cepas son 100% idénticas en la secuencia parcial del gen ribosomal 16S (16S rRNA) entre la zona fótica (superficie) y afótica (mesopelágico y batipelágico). Además, hemos identificado Alteromonas y Erythrobacter entre los géneros marinos heterótrofos más comunes que recuperamos en cultivo usando un medio marino estándar. Las técnicas tradicionales de cultivo generalmente solo recuperan una fracción pequeña de las comunidades bacterianas naturales, fenómeno conocido como ‘la gran anomalía de recuento en placa’ y muchas de las cepas que se aíslan pertenecen a la biosfera rara. Sin embargo, no conocemos si estos patrones, normalmente descritos para las bacterias de superficie, también se aplican en las profundidades. En el Capítulo 2 he combinado resultados obtenidos mediante técnicas dependientes e independientes de cultivo comparando las secuencias del 16S rRNA de la colección MARINHET contra los fragmentos de secuenciación masiva del 16S rRNA (de amplicones y metagenomas), obtenidos de muestras globalmente distribuidas y de diferentes profundidades. Una mayor proporción de las bacterias del océano profundo son cultivables y una fracción importante de los aislados tiene preferencia a un estilo de vida adherido a partículas. Además, confirmamos que el dogma ‘menos del 1% de las bacterias son cultivables’ deber ser revisado ya que encontramos variabilidad en las muestras de profundidad, donde hasta un 3% de las células se han podido aislar. Los aislados bacterianos son un excelente material para aplicaciones biotecnológicas, como la biorremediación de zonas marinas contaminadas. El mercurio es un metal pesado tóxico y su forma más peligrosa, el metilmercurio (MeHg), se bioacumula en la cadena trófica marina. No obstante, se conoce muy poco la tolerancia de bacterias marinas frente al mercurio o la fisiológia de aquellas cepas que codifican los genes de resistencia (operón mer). El Capítulo 3 describe los resultados del mapeo funcional de los genes merA y merB, clave en la detoxificación, en una fracción de la colección MARINHET. Nos centramos en dos géneros marinos, con un potencial genético para la degradación del mercurio previamente descrito en la literatura, como son Alteromonas y Marinobacter. Desvelamos que los genes merAB están ampliamente distribuidos en diferentes regiones oceanográficas y en varias profundidades. Adicionalmente, hemos seleccionado una cepa de Alteromonas mediterranea para futuros estudios de biorremediación debido a su alta tolerancia y capacidad de degradación de diferentes formas de mercurio.
The world’s oceans sustain the life for an estimated total of 10^29 microbial cells. Marine bacteria are responsible for most part of the ocean respiration and are key in most biogeochemical cycles of the Earth. Accordingly, the study of the bacterial diversity present in different marine ecosystems is essential, and having access to their genomes through isolation or genomic centric studies is important to decipher their metabolic potential. Isolation of marine microorganisms is fundamental to gather information about their physiology, ecology and genomic content. To date, most of the bacterial isolation efforts have focused on the photic ocean leaving the deep ocean less explored. In this thesis, standard plating techniques allowed to create a marine culture collection of heterotrophic bacteria (MARINHET). More than 2000 isolates were retrieved from samples collected from a variety of oceanographic regions, from different depths including surface, mesopelagic and bathypelagic waters, and also covering different seasons and years. Chapter 1 describes the taxonomy, the phylogenetic diversity and the biogeography of culturable heterotrophic marine bacteria, and reveals that an important percentage of the strains (37%) are 100% identical in their partial 16S rRNA gene between photic and aphotic layers. In addition, we identified Alteromonas and Erythrobacter genera as the most frequently retrieved heterotrophic bacteria from the ocean in standard marine agar medium. It is a long-standing observation that traditional culture techniques only retrieve a small fraction of the microbial diversity found in natural environments including marine ecosystems, what is known as ‘the great plate count anomaly’. In addition, most of the retrieved isolates belong to the so-called rare biosphere. However, we do not know if these patterns, usually described for bacteria living in the photic ocean, also apply for the deep ocean bacteria. In Chapter 2 of this thesis, I combined results from culture-dependent and -independent techniques by comparing the 16S rRNA partial sequences of the MARINHET isolates with 16S rRNA amplicon Illumina TAGs (16S iTAGs) and metagenomic TAGs (miTAGs) from surface, mesopelagic and bathypelagic samples globally distributed. A high proportion of bacteria inhabiting the deep ocean could be retrieved by pure culture techniques and a significant fraction of the isolates preferred a lifestyle attached to particles. Additionally, I revised the axiom that ‘less than 1% of bacteria can be cultured’, finding variability between mesopelagic and bathypelagic samples, where up to 3% of the cells could be cultured. Bacterial isolates also represent a valuable genetic reservoir for biotechnology applications, such as bioremediation strategies of marine polluted environments. Mercury is one of the most toxic heavy metals in the planet and its most dangerous form, methylmercury (MeHg), is being bioaccumulated in the marine food web. However, little is known about the tolerance capacity and phenotypic characterization of marine bacteria codifying the mercury resistance operon (mer operon). Chapter 3 describes the functional screening of merA and merB genes, which are key in the mercury detoxification process, in well know marine genera with described genetic potential for mercury detoxification, such as Alteromonas and Marinobacter. I reported that the merAB genes from these two genera are widely distributed in different oceanographic regions and depths. In addition, I selected a promising candidate, phylogenetically affiliated to Alteromonas mediterranea, for future bioremediation studies due to its high tolerance and degradation ability of different mercury forms.
Universitat Autònoma de Barcelona. Programa de Doctorat en Microbiologia

Understanding microbial evolution lies at the heart of microbiology and environmental sciences. Numerous studies have been dedicated to elucidating the underlying mechanisms that create microbial genetic diversity and adaptation. However, due to technical limitations such as the high level of uncultured cells in almost every natural habitat, most of current knowledge is primarily based on axenic cultures grown under laboratory conditions, which typically do not simulate well the natural environment. How well the knowledge from isolates translates to in-situ processes and natural microbial communities remains essentially speculative. The recent development of culture-independent genomic techniques (aka metagenomics) provides possibilities to bypass some of these limitations and provide new insights into microbial evolution in-situ. To date, most of metagenomic studies have been focused on a few reduced-diversity model communities, e.g., acid mine drainage. Highly complex communities such as those of soil and sediment habitats remain comparatively less understood. Furthermore, a great power of metagenomics, which has not been fully capitalized yet, is the ability to follow the evolution of natural microbial communities over time and environmental perturbations, i.e., times-series metagenomics. Although the recent developments in DNA sequencing technologies have enabled (inexpensive) time-series studies, the bioinformatics approaches to analyze the resulting data have clearly fallen behind. Taken together, to scale up metagenomics for complex community studies, three major challenges remain: 1) the difficulty to process and analyze massive short read sequencing data, often at the terabyte level; 2) the difficulty to effectively assemble genomes from complex metagenomes; and 3) the lack of methods for tracking genotypes and mutational events such as horizontal gene transfer (HGT) through time. Therefore, developing efficient bioinformatics approaches to address these challenges represents an important and timely issue. This thesis aimed to develop novel bioinformatics pipelines and algorithms for high performance computing, and, subsequently, apply these tools to natural microbial communities to generate quantitative insights into the relative importance of the molecular mechanisms creating or maintaining microbial diversity. The tools are not specific to a particular habitat or group of organisms and thus, can be broadly used to advance our understanding of microbial evolution in different settings. In particular, the comparative whole-genome analysis of 24 Escherichia isolates form various habitats, including human and non-human associated habitats such as freshwater ecosystems and beaches, showed that organisms with more similar ecologies tend to exchange more genes, which has important implications for the prokaryotic species concept. To more directly test these findings from isolates and quantify the patterns of genetic exchange among co-occurring populations, three years of time-series metagenomics data from planktonic samples from Lake Lanier (Atlanta, GA) were analyzed. For this, it was first important to develop bioinformatics algorithms to robustly assemble population genomes from complex community metagenomes, identify the phylogenetic affiliation of assembled genome and contig sequences, and detect horizontal gene transfer among these sequences. Using these novel algorithms, in situ bacterial lineage evolution was quantitatively assessed, especially with respect to whether or not ecologically distinct lineages evolve according to the recently proposed fragmented speciation model (Retchless and Lawrence, Science 2008). Evidence in support of this model was rarely observed. Instead, it appeared that rampant HGT disseminated ecologically important genes within the population, maintaining intra-population diversity. By expanding the previous approaches to include methods to assess differential gene abundance and selection pressure between samples, it was possible to quantify how soil microbial communities respond to a decade of warming by 2 0C, which simulated the predicted effects of climate change. It was found that the heated communities showed significant shifts in composition and predicted metabolism, reflecting the release of additional soil carbon compared to the unheated (control) communities, and these shifts were community-wide as opposed to being attributable to a few taxa. These findings indicated that the microbial communities of temperate grassland soils play important roles in mediating the feedback responses to climate change. Collectively, the findings presented here advance our understanding of the modes and tempo of microbial community adaptation to environmental perturbations and have important implications for better modeling the microbial diversity on the planet. The bioinformatics algorithms and approaches developed as part of this thesis are expected to facilitate future genomic and metagenomic studies across the fields of microbiology, ecology, evolution and engineering.

La fermentació alcohòlica la realitza una comunitat microbiana complexa, on els llevats vínics juguen un paper important. En els darrers anys, hi ha hagut un creixent interès per millorar complexitat del vi mitjançant fermentacions controlades quer utilitzaven no només S. cerevisiae sinó també soques seleccionades de llevats no-Saccharomyces. La investigació sobre la viabilitat dels llevats i les interaccions tenen un paper fonamental per entendre la diversitat de llevats en la fermentació mixta. En aquesta tesi, les tècniques independents de cultiu que es van aplicar per a l'anàlisi directa de mostres de vins inclouen la seqüenciació massiva, fluorescència per hibridació in situ (FISH) en combinació amb microscòpia i citometria de flux, RT-qPCR i EMA-DGGE. Aquestes tècniques independents de cultiu permeten una ràpida identificació i / o quantificació dels diferents llevats del vi. Aquestes tècniques han estat utilitzades en fermentacions espontànies a la regió del Priorat, i H. uvarum i Starm. bacillaris van ser les dues principals espècies de llevats no-Saccharomyces aïllades. H. uvarum o Starm. bacillaris perden gradualment la seva cultivabilitat quan els mostos es van inocular amb S. cerevisiae, però es podien quantificar cèl•lules de llevats en estat viable però no cultivable. La pèrdua de cultivabilitat dels no Saccharomyces va ser induïda principalment per alguns metabòlits secretats per S. cerevisiae, però els canvis en altres metabòlits principals també van influir. Aquesta interacció de llevat no Saccharomyces amb S. cerevisiae era específica d’espècie i soca.
La fermentación alcohólica es llevada a cabo por una comunidad microbiana compleja, donde las levaduras del vino juegan un papel importante. En los últimos años, ha habido un creciente interés para mejorar la complejidad del vino en fermentaciones controladas utilizando no sólo S. cerevisiae sino también algunas cepas seleccionadas de levaduras no-Saccharomyces. La investigación sobre la viabilidad de las levaduras y las interacciones tienen un papel fundamental para entender la diversidad de levaduras en fermentaciones mixtas. En esta tesis, se aplicaron técnicas independientes de cultivo para el análisis de muestras de vinos directa incluyendo la secuenciación masiva, fluorescencia por hibridación in situ (FISH) en combinación con microscopía y citometría de flujo, RT-qPCR y EMA-DGGE. Estas técnicas independientes de cultivo permiten una rápida identificación y / o cuantificación de las distintas levaduras del vino. Estas técnicas han sido utilizadas para el análisis de fermentaciones espontáneas en la región del Priorat, siendo las especies H. uvarum y Starm. bacillaris las dos principales especies de levaduras no-Saccharomyces. H. uvarum o Starm. bacillaris pierden gradualmente su cultivabilidad cuando los mostos se inocularon con S. cerevisiae, pero las levaduras se pudieron cuantificar en estado viable pero no cultivable. La pérdida de cultivabilidad de las especies no Saccharomyces fue inducida principalmente por algunos metabolitos secretados por S. cerevisiae, pero los cambios en otros metabolitos principales también influyen. Esta interacción entre levaduras no Saccharomyces con S. cerevisiae es especie y cepa dependiente.
Alcoholic fermentation is driven by complex microbial community, where wine yeasts play an important role. In recent years, there has been growing interest to enhance wine complexity by controlled fermentations using not only S. cerevisiae but also together with some selected non-Saccharomyces yeast strains. Research on yeast viability and interaction has a fundamental role to understand the diversity of yeast in mixed fermentations. In this thesis, culture-independent techniques were developed and applied for direct wine sample analysis including massive sequencing, fluorescence in situ hybridization (FISH) combined with microscopy and flow cytometry, RT-qPCR and EMA-DGGE. These culture-independent techniques enable fast identification and/or quantification of different wine yeasts. These techniques have been used during spontaneous fermentation in Priorat region, and H. uvarum and Starm. bacillaris where the two main non-Saccharomyces yeast species detected. H. uvarum or Starm. bacillaris gradually lost their culturability when musts were inoculated with S. cerevisiae, but quantifiable yeast cells existed in viable but non-culturable state. The culturability loss of non-Saccharomyces was mainly induced by some metabolites secreted from S. cerevisiae, but changes in other main metabolites also had some effect. This interaction of non-Saccharomyces yeast with S. cerevisiae showed the specificity of species and strains.

Microbial communities are recognized as major drivers of global biogeochemical processes. However, the genetic diversity and composition, as well as processes leading to the origin and diversification of these communities in space and time, are poorly understood. Character- ization of microbial communities using high-throughput sequencing of 16S tags shows that Operational Taxonomic Unit (OTU) abundances can be approximated by a gamma distribu- tion, which suggests structuring around small numbers of highly abundant OTUs and a large proportion of rare OTUs. The current methods used to characterize how communities are structured rely on multivariate statistics, which operate on pair-wise distance matrices. My analyses demonstrate that use of these methods, by reducing a highly-dimensional data set (tens of samples, thousands of OTUs), results in a significant loss of information. I demon- strate that, in some cases, up to 80% of the least abundant OTUs may be removed while still recovering the same community relationships; this indicates these metrics are biased toward the highly abundant OTUs. I also demonstrate that the observed patterns of OTU abundance detected from microbial communities can be robustly modeled using techniques similar to those used to model the presence and absence of genes in genome evolution. Using simulation studies, I show that general Markov models in a Bayesian inference framework out- perform traditional, multivariate ecological methods in recovering true community structure. Applying this new methodology to Atlantic Ocean communities uncovered a distance-decay effect which was not revealed by the traditional methods; applying to communities discov- ered on Hog Island point toward mechanisms of thicket establishment. Although the ocean data set operated on a much larger, continental scale, characterization of the sequence data generated from the nutrient-poor soil on Hog Island, a barrier island off the Virginia coast, allows for a better characterization of the processes affecting these communities on a much smaller scale. Finally, using 16S data from the Human Vaginal Microbiome Project, gener- ated here at VCU under the umbrella of the overall NIH HMP initiative, I give examples of the quality control, analysis and visualization pipeline that I developed to support the efforts of this project. In conclusion, my analyses of the metagenomic sequence data from bacterial communities sampled from different environments demonstrate that the proper identification of the biological processes influencing these communities requires the development and im- plementation of new statistical and computational methodologies that take advantage of the extensive amount of information generated in next-generation, high-throughput sequencing projects.

The main aim of this research project was to improve our understanding of the diversity, function and ecology of glacial microbiomes. Snow and ice algae are critical players in supraglacial habitats and form extensive blooms in spring and summer. Here I present results on the diversity and the function of snow and ice algae on 21 glaciers in 4 Arctic settings: Greenland, Iceland, Svalbard and Sweden. For the first time, I have evaluated the full microbial community composition (i.e., algae, bacteria, archaea) in the main supraglacial habitats, namely green snow, red snow, biofilms, dirty ice, and cryoconite holes. I have cross-correlated these data with metabolic analyses (i.e., metabolomics, pigments, fatty acids) and critical physico-chemical parameters. I found that snow and ice algae were the first communities to appear after the onset of melting and they showed positive net photosynthetic rates indicating accumulation of organic matter. Furthermore, for the first time I have described these communities in Iceland. My data reveal that red pigmented snow algae are cosmopolitan, and independent of location specific geochemical and mineralogical factors. Only six taxa made up >99% of the algal communities: two uncultured Chlamydomonadaceae, Chloromonas polyptera, Chloromonas nivalis, Chloromonas alpina and Raphidonema sempervirens. In contrast, the composition of green snow varied between the studied locations with higher relative abundance of Raphidonema sempervirens and Microglena sp. in Svalbard, and Chloromonas polyptera in Sweden. Furthermore, I show that green and red snow are not successive stages but two independent phenomena with different adaptation strategies. In all sites, bacteria were mostly represented by the phyla Bacteriodetes, Proteobacteria and Cyanobacteria. The bacterial community composition varied between the different habitats on the phylum level, whereas on the class level they also showed strong biogeography. Archaea showed overall low species diversity. The synthesis of pigments and fatty acids in snow and ice algae were mainly driven by nitrogen and less so by phosphorus limitation. This is especially important for pigments which cause a darkening of glacial surfaces. I show that snow and ice algae dramatically decrease surface albedo which will eventully result in higher melting rates of glaciers.

La metagenomica ha permesso lo studio della diversità degli organismi non coltivabili e il loro ruolo nei vari ecosistemi tra cui suoli e sedimenti marini. acque interne e di oceano aperto. L’analisi della diversità attraverso gli approcci metagenomici è basata sull’estrazione del DNA genomico assumendo che questo sia completamente associato a biomassa vivente. Tuttavia, recenti studi hanno dimostrato che il metagenoma completo di ogni campione ambientale è costituito da pool differenti, tra cui: ,quello dei virus, quello associato a biomassa microbica e quello associato a biomassa non vivente (i.e., DNA extracellulare). Le procedure comunemente utilizzate per isolare il DNA da campioni ambientali non discriminano fra i diversi pool di DNA, influenzando quindi i risultati delle indagini effettuate. I viromi sono metagenomi contenenti DNA virale. I virus non sono solo le più abbondanti entità biologiche negli oceani del mondo, ma attraverso le loro infezioni controllano sia l'abbondanza e la diversità procariotica, sia cicli biogeochimici importanti a livello dell’ecosistema marino. L’approccio metagenomico applicato alla componente virale dei sistemi marini ha permesso di scoprire che i virus potenti possono essere importanti agenti di trasferimento genico: infatti , attraverso la ricombinazione e integrazione, i virus possono sia prelevare porzioni di genomi dei loro ospiti sia trasferirli ad altri ospiti. Nonostante la loro importanza, la diversità virale negli ecosistemi bentonici marini profondi è ancora del tutto sconosciuta e la metagenomica sembra essere l'approccio più efficace per analizzarla. Finora, diversi strumenti bioinformatici sono stati utilizzati per analizzare le sequenze virali in campioni ambientali, ma la maggior parte di questi strumenti non sono stati specificamente progettati per l'analisi delle sequenze virali e confronti per verificare la loro validità non esistono o sono troppo limitati. In questo studio abbiamo sviluppato una procedura specifica per il recupero selettivo del DNA virale da sedimenti marini profondi. Il DNA virale è stato sequenziato, attraverso tecniche di pirosequenziamento, e analizzato confrontando tre pipeline di annotazione di sequenze metagenomiche (MG-RAST, VMGAP, MetaVir). Questo al fine di testare la loro efficienza nell'analisi della diversità virale, sia utilizzando i dati di sequenziamento sia quelli derivanti da metagenomi virali ottenuti in silico a partire da sequenze depositate nelle banche dati. Queste analisi indicano che la diversità tassonomica e funzionale dei virus varia in funzione della pipeline utilizzata. MetaVir è risultata la pipeline più affidabile per l’annotazione tassonomica della diversità virale. Tuttavia, tale pipeline, non essendo stata progettata per annotazioni di tipo funzionale, deve essere necessariamente integrata con altre come VMGAP. Pertanto, questo studio evidenzia la necessità di sviluppare una piattaforma bioinformatica completa per un’annotazione efficiente sia tassonomica sia funzionale, al fine di .far luce sull'enorme diversità genetica contenuta nei virus presenti nel più grande ecosistema della Terra. La diversità tassonomica virale è stata esplorata in campioni di sedimento marino raccolti in differenti aree oceaniche del Globo, da 2000 a 10000 m di profondità. I risultati di questa analisi hanno permesso di evidenziare, per la prima volta, che la diversità virale negli ecosistemi bentonici profondi non solo è molto elevata, ma anche che alcune famiglie virali sono molto diffuse, nonostante le differenze ambientali ed ecologiche degli ecosistemi analizzati. Le similarità tra i campioni analizzati in questo studio e la maggior parte dei viromi ad oggi pubblicati suggeriscono che diversi fattori contribuiscono a modellare la diversità dell’assemblage virale. In aggiunta, tutti i viromi presentano un’elevata diversità funzionale putativa e contengono anche funzioni derivanti dai loro ospiti, tra cui funzioni metaboliche chiave. I metagenomi microbici sono costituiti da DNA associato a biomassa vivente, che nei sedimenti marini profondi è rappresentata prevalentemente da procarioti (Batteri e Archaea). La possibilità di studiare le comunità microbiche attraverso gli approcci metagenomici ha permesso di capire meglio il loro ruolo nell'ambiente marino anche negli ambienti marini profondi, che sono molto difficili da raggiungere, consentendo anche la scoperta di nuovi enzimi e vie metaboliche, spesso utili per applicazioni industriali o biotecnologiche. Il DNA extracellulare ha un ruolo chiave negli ecosistemi marini, sia come fonte di nutrienti che come fonte di geni. Può essere sia rilasciato dalla comunità procariotica durante la crescita sia attraverso la lisi cellulare (a causa di infezione virale o per morte cellulare naturale). Sedimenti e suoli possono anche preservare questo DNA rilasciato, che può rimanere adeso alle particelle minerali e organiche. Questo pool extracellulare conservato può essere incorporato da cellule naturalmente competenti, che possono andare incontro a processi di trasformazione naturale. In questo studio l'analisi contestuale dei metagenomi microbici ed extracellulari nei diversi ecosistemi bentonici profondi, non solo ha fornito informazioni riguardanti la composizione specifica di ciascun pool, ma ha anche rivelato che il DNA extracellulare contiene una diversità genetica elevata che ad oggi non era mai stata considerata. Tale diversità genetica costituisce una frazione rilevante della diversità genetica associata all’intero metagenoma. La maggior parte della diversità genetica del DNA totale è rappresentata da geni afferenti al DNA extracellulare e, in media, circa il 50% delle «specie» contenute nel DNA extracellulare è condiviso con il microbioma. Inoltre, dal confronto tra tutti i metagenomi risulta che sequenze virali sono presenti non solo, come atteso, nei viromi ma anche nei microbiomi e nei metagenomi extracellulari. L’analisi bioinformatica comparativa tra tutti i metagenomi ha rivelato la presenza di funzioni putative coinvolte in differenti processi di trasferimento genico orizzontale. Di particolare rilevanza sono le funzioni relative all’uptake del DNA e la sua mobilizzazione e quelle relative ad elementi genetici mobili come i gene transfer agents (GTAs) ed i profagi. Esperimenti di laboratorio condotti su sedimenti marini profondi dimostrano anche che il DNA extracellulare può essere una risorsa genetica importante per la comunità microbica dal momento che fino al 6% delle cellule procariotiche risultano essere competenti e in grado di acquisire nuove funzioni. Nel loro insieme, questi risultati suggeriscono che gli ecosistemi bentonici profondi hanno un elevato potenziale di trasferimento genico che può avvenire attraverso meccanismi multipli.
Metagenomics has allowed the study of the diversity of organisms which can’t be cultivated and their role in various ecosystems, including soils and marine sediments, inland waters and the open ocean. The analysis of diversity through metagenomic approaches is based on the extraction of genomic DNA, assuming that it is completely associated with living biomass. However, recent studies have shown that the whole metagenome of each environmental sample is composed of different pools, including that of viruses, that associated with microbial biomass and that associated with non-living biomass (i.e, extracellular DNA). The procedures commonly used to isolate DNA from environmental samples do not discriminate between the various pools of DNA, thus affecting the results of investigations carried out. Viromes are metagenomes containing viral DNA. Viruses are not only the most abundant biological entities in the World’s oceans, but through their infection they control both prokaryotic abundance and diversity and important biogeochemical cycles of the marine ecosystem. The metagenomic approach applied to the viral component of marine systems has led to the discovery that viruses can be important agents of gene transfer: in fact, through recombination and integration viruses can either excide portions of the genomes of their hosts and transfer them to other hosts. Despite their importance, viral diversity in the deep-sea benthic ecosystems is still completely unknown and metagenomics seems to be the most effective approach to analyze it. So far, several bioinformatics tools were used to analyze viral sequences in environmental samples, but most of these tools have not been specifically designed for the analysis of viral sequences and comparisons to test for their validity do not exist or are too limited. In this study, we developed a specific procedure for the selective recovery of viral DNA from deepsea sediments. Viral DNA was sequenced, through pyrosequencing techniques, and analyzed comparing three annotation pipelines for metagenomic sequences (MG-RAST, VMGAP, MetaVir). To test their efficiency in the analysis of viral diversity we used both the sequencing data derived from viral metagenomes and those obtained in silico from sequences deposited in public databases. These analyses indicate that the taxonomic and functional diversity of viruses varies depending on the pipeline used. MetaVir proved to be the most reliable pipeline for the annotation of viral taxonomic diversity. However, since this pipeline was not designed for functional annotations of viral sequences, its results must necessarily be integrated with those obtained by other pipelines such as VMGAP. Therefore, this study highlights the need to develop a comprehensive bioinformatics platform for efficient functional and taxonomic annotation of viromes to shed light on the enormous genetic diversity contained in the viruses present in the largest ecosystem on Earth. The viral taxonomic diversity has been explored in marine sediment samples collected in different ocean areas of the globe, ranging from 2000 to 10000 m depth. The results of this analysis have revealed, for the first time, that viral diversity in benthic deep-sea ecosystems, is not only very high but also that some viral families are widespread, despite the environmental and ecological differences in the ecosystems analyzed. The similarity between the samples analyzed in this study and the majority of viromes published to date suggests that several factors contribute to shape the diversity of the viral assemblage. In addition, all viromes have a high functional diversity and also contain putative functions derived from their hosts, including key metabolic functions. The microbial metagenomes are made of DNA associated with living biomass and in deep-sea sediments they are mainly represented by prokaryotes (Bacteria and Archaea). The possibility of studying the microbial communities through metagenomic approaches has allowed us to better understand their role in the marine environment in deep marine environments, which are very difficult to reach, enabling the discovery of new enzymes and metabolic pathways, often useful for industrial or biotechnological applications. The extracellular DNA plays a key role in marine ecosystems, both as a source of nutrients and as a source of genes. It can be released from prokaryotic community during growth and through cell lysis (due to viral infection or to natural cell death). Sediments and soils can also preserve this released DNA, which can be adsorbed onto mineral and organic particles. This preserved extracellular pool can be incorporated into naturally-competent cells, which may undergo natural transformation processes. In this study, the contextual analysis of extracellular and microbial metagenomes in different benthic deep-sea ecosystems, has not only provided information on the specific composition of each pool, but also has revealed that the extracellular DNA contains a high genetic diversity, which to date has never been considered. This genetic diversity represents a major fraction of the genetic diversity associated with the whole metagenome. Most of the genetic diversity of the total DNA is represented by genes related to the extracellular DNA and, on average, about 50 % of the "species" contained in the extracellular DNA is shared with the microbiome. Moreover, the comparison among all the metagenomes showed that viral sequences are present not only in viromes, as expected, but also in microbiomes and extracellular metagenomes. The comparative bioinformatic analysis between all metagenomes has revealed the presence of putative functions involved in different processes of horizontal gene transfer. Of particular relevance are the functions for DNA uptake and mobilization and those related to mobile genetic elements, such as gene transfer agents (GTAs) and prophages. Laboratory experiments conducted in deep-sea sediments also show that the extracellular DNA may be an important genetic resource for the microbial community, since up to 6% of prokaryotic cells appear to be competent and able to acquire new functions. Taken together, these results suggest that deep benthic ecosystems have a high potential for gene transfer that can occur through multiple mechanisms.

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CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico
The Amazon, which owns the largest fauna and flora in the world presents unparalleled wealth of biological diversity, however, keep intact this megadiversity requires scientific and technological knowledge. In this context, there is an important biotechnological tool that is the bioremediation of impacted environments with petroleum hydrocarbons and derivatives. In the present study samples of microbial communities of fungi and bacteria associated with aquatic macrophytes Eichhornia crassipes (Mart.) Solms, Ichnanthus calvescens Döll and Cyperus ligularis L., were researched. These host plants common at Rio Negro were collected in contaminated environments by oil and oil products at Waterway Station from Manaus. From the species selected, 155 bacterial strains were isolated, being 97 epiphytic and 58 endophytic, and 54 fungi strains, being 30 epiphytic and 24 endophytic. Selective media were used for isolation of microorganisms such as BH liquid medium (Bushhnell Haas) plus oil. The oil and diesel used are from the Base Oil Urucu, Amazonas. The biodegradability tests were performed on selective medium (BH), with the addition of oil as the carbon source known as Medium I This test was repeated with the addition to the medium I the redox indicator 2,6-dichlorophenol indophenol (DCPIP), called medium II. After the evaluationof microbial isolates were selected 6 bacteria and 7 fungi. Molecular identification of bacteria was performed by the 16S ribosomal DNA region and revealed the presence of Bacillus pumilus (endophytic / epiphytic), Lysinibacillus fusiform (epiphytic), Pseudomonas aeruginosa (epiphytic) and Acinetobacter junii (epiphytic/epiphytic). For molecular identification of fungi was performed by the ITS1 and ITS2 region and revealed the presence of Curvularia trifolii (epiphytic), Curvularia clavata (endophytic / epiphytic), Gibberella intermedia (epiphytic/endophytic), Phoma herbarum (epiphytic) and Dothideomycetes sp (epiphytic). These microorganisms were selected for composition of microbial consortium that were used for hydrocarbon biodegradation tests. The measurement of biodegradation of oil and diesel activities was estimated by chromatography and mass spectrometry. In tests we used water of Rio Negro with the aim of approaching research the environment that are being studied. Degradation of hydrocarbons by consortia of fungi and bacteria had significant average values (98.7 to 100%), but did not show any statistical difference between the degradation of the control containing water of the Rio Negro (97.3%). In the experiment with the mixed consortium (FB), there were significant differences, because although the control containing water of the Rio Negro has promoted degradation of diesel by wild microbiota (81.7%), this degradation was lower and statistically different from the mixed consortium (97,5%). Analysis were carried out for degradation of the compounds naphthalene and phenanthrene of diesel by consortia . It was observed that phenanthrene was the best that has been degraded by the mixed consortium (F / B), however the naphthalene was better degraded by the control containing only water from the Rio Negro, highlighting the potential of wild microorganisms that deserve attention in future research, the isolation of these ones in waters from Rio Negro. In the experimental design with the consortia, the results showed that mixed consortia (FB) have potential for use in future bioremediation.
A Amazônia, detentora da maior fauna e flora do mundo apresenta riqueza inigualável de diversidade biológica, entretanto, manter intacta essa megadiversidade requer conhecimentos científicos e tecnológicos. Nesse contexto, situa-se uma importante ferramenta biotecnológica que é a biorremediação de ambientes impactados por hidrocarbonetos de petróleo e derivados. No presente trabalho realizou-se estudo de amostras das comunidades microbianas de fungos e bactérias associadas às macrófitas aquáticas Eichornia crassipes (Mart.) Solms, Ichnanthus calvescens Döll e Cyperus ligularis L. Essas plantas hospedeiras comuns nas águas do rio Negro foram coletadas em ambientes contaminados por petróleo e derivados na Estação Hidroviária de Manaus. Das espécies vegetais selecionadas foram isoladas 155 linhagens de bactérias, sendo 97 epifíticas e 58 endofíticas e 54 cepas de fungos, sendo 30 epifíticos e 24 endofíticos. Foram empregados meio seletivo para isolamento dos microrganismos tal como meio liquido BH (Bushhnell Haas) acrescido de petróleo. O petróleo e o diesel utilizados foram provenientes da Base Petrolífera de Urucu, Amazonas. Os ensaios de biodegradabilidade foram realizados em meio seletivo (BH), com a adição de petróleo como fonte de carbono denominado Meio I. Este ensaio foi repetido com a adição ao meio I do indicador redox 2,6-diclorofenol indofenol (DCPIP), denominado meio II. Após a avaliação dos isolados microbianos foram selecionados 6 bactérias e 7 fungos. A identificação molecular das bactérias foi realizada por meio da região do DNA ribossomal 16S e revelou a presença de Bacillus pumilus (Endofítica/epifítica), Lysinibacillus fusiformes (Epifítica), Pseudomonas aeruginosa (Epifítica) e Acinetobacter junii (Epifítica/epifítica). A identificação molecular dos fungos foi realizada por meio da região TS1 e ITS2 e revelou a presença das seguintes espécies: Curvularia trifolii (epifítica), Curvularia clavata (endofítica/epifítica), Gibberella intermedia (epifítica/endofitica), Phoma herbarum (epifítica) e Dothideomycetes sp (epifítica). Estes microrganismos foram selecionados para composição do consórcio microbianos que foram utilizados em ensaios de biodegradação de hidrocarbonetos. A mensuração das atividades de biodegradação de petróleo e diesel foi estimada por cromatografia e espectrometria de massa. Nos ensaios utilizou-se água do rio Negro com o objetivo de aproximar a pesquisa ao ambiente de estudo. A degradação dos hidrocarbonetos pelos consócios de fungos e bactérias apresentaram médias significativas (98,7-100%), mas não apresentaram diferenças estatísticas entre a degradação do controle contendo água do rio Negro (97,3%). No experimento com o consórcio misto (F/B), houve diferenças significativas, pois embora o controle contendo água do rio Negro tenha promovido degradação do diesel pela microbiota selvagem (81,7%), esta degradação foi inferior e diferente estatisticamente do consórcio misto (97,5%). Foram realizadas análises de degradação dos compostos naftaleno e fenantreno do óleo diesel pelos consórcios Observou-se que o composto fenantreno foi o que melhor foi degradado pelo consórcio misto (F/B). Entretanto, o naftaleno foi melhor degradado pelo controle contendo somente água do rio Negro, destacando o potencial dos microrganismos selvagens que merecem atenção nas futuras pesquisas, no isolamento destes em águas do rio Negro. O índice de toxicidade dos extratos microbianos foram avaliados como toxicidade moderada para o consórcio misto (F/B), já para o consórcio de fungos e consórcios de bactérias não apresentou toxicidade. No planejamento experimental com os consórcios, os resultados obtidos demonstraram que consórcios mistos (F/B) apresentaramm potencial para uso em futuros processos de biorremediação.

Little is known about how the leaf bacterial community is affected by the seed microbiota at different stages of plant development. The bacterial populations of spinach seed and leaves after germination were compared using DGGE, to assess bacterial community richness, and real-time PCR to compare the abundance of select phyla (total bacteria, Actinobacteria, Bacteroidetes, Firmicutes, α-Proteobacteria and β- Proteobacteria). To determine the effect of environment, the plants were grown in the field and growth chambers. Vertical transmission of bacterial community members was evident; the developmental stage of the plant affected the richness and abundance of select bacterial phyla. The bacterial richness of plants grown in the two environments was not affected. However, overall numbers of bacteria increased in field grown samples in comparison to those produced in growth chambers during development. A statistically significant interaction was seen between growth stage and environment with each of the selected phyla. Populations on cotyledons were smaller than mature leaves, but were not significantly different than the 3-4 leaf stage plants. The culturable populations of bacteria on seeds (~5 log CFU/g) were significantly smaller than determined using real time PCR (~7 log copies). Of these bacteria cultured from spinach seeds, isolates belonging to the genera Pantoea were found to inhibit growth of E. coli O157:H7 in vitro. This study highlights the importance of vertical transmission on the bacterial community of plants and suggests the importance of developing strategies to influence these communities on seed to control human and plant pathogens on the leaf surface.

Master of Science in Life Sciences

La generació d’aigua residual és una conseqüència inevitable de les activitats humanes. Aquestes activitats modifiquen les característiques de les aigües de consum, contaminant-les i invalidant la seva posterior aplicació per altres usos. Abocar aquestes aigües sense depurar ocasiona danys, a vegades irreversibles, al medi ambient. D’altra banda, l’abocament d’aigües no depurades suposa riscos per la salut pública. Per aquesta raó, la depuració de les aigües residuals esdevé una necessitat important a la nostra societat. Les estacions depuradores d’aigües residuals (EDARs) constitueixen un dels processos biotecnològics més importants utilitzats arreu del món per depurar aigües residuals municipals i industrials. Des d’un punt de vista biològic, els microorganismes juguen un paper important en la depuració de les aigües residuals ja que són els principals responsables de l’eliminació de la matèria orgànica i dels nutrients d’aquestes aigües. D’altra banda, la reutilització de l’aigua s’està convertint en un aspecte cada vegada més important de la gestió sostenible dels recursos hídrics. En aquesta tesi s’ha estudiat la composició de les comunitats microbianes en diferents ambients relacionats amb la depuració d’aigües residuals i la seva reutilització. Per un costat, s’han estudiat i comparat les comunitats microbianes trobades en processos convencionals de depuració amb dos sistemes atípics de depuració d’aigua residual (un fang activat provinent d’una EDAR que processa aigua de mar i uns aiguamolls construïts). Per un altre, s’han determinat les diferències en el perfil microbià d’una aigua depurada versus una aigua dolça natural, amb la intenció de trobar un indicador alternatiu de qualitat de l’aigua. Finalment, s’ha analitzat la població microbiana associada amb els problemes derivats de la reutilització d’aigua. Les mostres del fang activat marí van ser analitzades utilitzant una aproximació completa per l’anàlisi de rRNA (electroforesi en gel de gradient desnaturalitzant, genoteca i hibridació in situ), obtenint-se una imatge de la composició de la comunitat completament diferent a la d’un fang activat convencional, amb la particularitat que es va trobar una presència molt baixa del grup Betaproteobacteria. A més, la detecció del gen funcional amoA va corroborar la presencia d’oxidadors d’amoni, que probablement eren de generes diferents als descrits en la literatura. Per un altra banda, les comunitats microbianes trobades en aiguamolls construïts experimentals pertanyien a espècies relacionades amb l’eliminació de matèria orgànica. L’estudi dels perfils d’aquestes comunitats va permetre determinar l’efecte del tipus de planta, el disseny hidràulic i la càrrega de matèria orgànica d’aquests sistemes en la seva composició. Comparant els nostres sistemes atípics de depuració amb els sistemes convencionals, es va poder concloure que cada sistema té la seva comunitat microbiana específica, adaptada a les seves condicions ambientals, duent a terme les mateixes funcions però amb diferents grups implicats. Tanmateix, l’examen dels perfils de diversitat microbiana d’aigües depurades provinents de EDARs i la seva comparació amb els perfils d’aigües naturals no contaminades mostrava que els efluents de depuradora tenen una empremta comú, que difereix de la d’una aigua natural. Segons això, tenint en compte els grups taxonòmics trobats més abundants en aquest tipus d’aigües, proposem l’ús de la ratio Bacteroidetes, Gammaproteobacteria i Nitrospira / Betaproteobacteria com a indicador alternatiu de qualitat ecològica de l’aigua. Pel que fa a la reutilització d’aigües, es va estudiar un exemple d’un problema de formació de biofilms derivat de l’ús d’aigua regenerada en un sistema de reg per goteig. En aquests biofilms la comunitat microbiana estava principalment composada per microorganismes termòfils i esporulats, la qual cosa suggereix que les altes temperatures dins de la infrastructura de reg van seleccionar aquestes poblacions. Per tant, sembla ser que les condicions en el sistema de reg poden ser més importants que la composició de la pròpia aigua regenerada.
Wastewater generation is an inevitable consequence of human activities. These activities modify the characteristics of the original waters, contaminating them and invalidating subsequent application for other uses. Dumping of raw sewage causes damage, sometimes irreversible, to the environment. On the other hand, the discharge of untreated sewage poses risks to public health. Thus, wastewater treatment becomes an important need in our society. Wastewater treatment plants constitute one of the most important biotechnological processes used worldwide to treat municipal and industrial sewage. From a biological point of view, microorganisms play an important role in wastewater treatment since they are the main responsible of the remove of organic matter and nutrients from this sewage. Furthermore, water reuse is becoming increasingly important as a component of sustainable management of water resources. In this thesis we investigated the composition of microbial communities in different environments related to wastewater treatment and reuse. First, we studied and compared the microbial communities found in conventional processes with two atypical treatment systems (activated sludge from a seawater-processing EDAR and constructed wetlands). On the other hand, we determined how different was the microbial profile of a treated wastewater versus a natural freshwater, and tried to find out an alternative indicator of water quality. Finally, we examined the microbial population associated with the problems arising from water reuse. Samples of a marine activated sludge were analyzed by using the full-cycle rRNA approach (denaturing gradient gel electrophoresis, clone libraries and in situ hybridization), obtaining a picture of the composition of the community completely different from a conventional activated sludge, with particularly a low presence of Betaproteobacteria. In addition, detection of the functional gene amoA corroborated the presence of ammonia oxidizers, corresponding probably to different genera from those described in the literature. On the other hand, the microbial communities found in experimental constructed wetlands belonged to species related with organic matter removal. The study of the profiles of these microbial communities allowed determining the effect of the type of plant, hydraulic design and organic matter load of these systems in their composition. Comparing our atypical treatment systems with conventional ones, we can conclude that each system has its specific microbial community, adapted to its environmental conditions, with the same functions being carried out by different groups. Besides, examination of microbial diversity profiles of treated wastewater from different EDARs and comparison with natural non-contaminated water showed that sewage effluents have a common fingerprint, which differs from natural water. Thus, taking into account the most abundant groups found in these types of waters, we proposed the use of the Bacteroidetes, Gammaproteobacteria and Nitrospira / Betaproteobacteria ratio as an alternative indicator of ecological water quality. Concerning water reuse, we focused on an example of a biofouling problem derived from the use of reclaimed water in a drip irrigation system. In these biofilms the microbial community was mainly composed of thermophilic and sporulated microorganisms, suggesting that high temperatures within the irrigation infrastructure selected these populations. Thus, it seemed that the conditions in the system may be more important than the composition of reclaimed water itself.

Esta Tesis doctoral comprenden una revisión de las poblaciones termófilas que habitan en algunos Hot Spots de diversidad de América, como lo son el bosque lluvioso Valdiviano en la Patagonia, o los Bosques Nubosos de Costa Rica. También se incluyen ecosistemas extremos como el desierto de Atacama y la Isla Decepción en la Antártica. En este trabajo de tesis se caracterizó la estructura de las comunidades bacterianas en tapetes microbianos de varias termas terrestres diferentes mediante métodos de ecología molecular microbiana, y se midió la relación de los cambios de diversidad con respecto a la temperatura a diferentes escalas espaciales. El filo Cyanobacteria fue identificado como uno de los mayores componentes de la comunidad bacteriana, del que no se observaron diferencias sustanciales entre muestras obtenidas en invierno y verano. Se detectó una similitud significativa entre las termas que comparten ambientes semejantes a su alrededor. A pequeña escala (cm), se determinó que también miembros del filo Chloroflexi dominaban en ciertas zonas del tapete, y se observó una diferenciación por temperatura entre Chloroflexi y Cyanobacterias. En este sentido, se detectaron cambios significativos de diversidad entre muestras en un mismo tapete microbiano, y al mismo tiempo, se detectó una alta similitud en la estructura de las comunidades dentro de algunos “parches” en los tapetes. Posteriormente se detectó la presencia de varios grupos heterotróficos como Bacteroidetes; Thermales; y Proteobacteria, entre otros menos abundantes. No se detectó una relación entre riqueza bacteriana y latitud, ya que las muestras más cercanas a la línea del Ecuador no mostraron mayor diversidad o riqueza que las más cercanas al Polo Sur. La temperatura exhibió una relación negativa significativa con respecto a la riqueza bacteriana independientemente de la latitud geográfica. Finalmente, las muestras dentro de una escala local, regional y global (hasta 1000 m2) mostraron una disminución significativa de la diversidad a medida que la distancia geográfica y la temperatura se incrementaban. A nivel continental, estas relaciones no fueron significativas.
The results presented in this PhD thesis include a description on the thermophilic populations thriving in biodiversity hot spots such as the Valdivian Rain Forest in Patagonia, and Cloud Forests in Costa Rica, as well as the Atacama Desert and Deception Island in the South Shetland Archipelago in Antarctica. The bacterial community structure of several hot spring microbial mats was characterized by means of molecular microbial ecology methods, and the relationship between diversity and temperature was measured at different spatial scales. Phylum Cyanobacteria was identified as the major component of the microbial community by at least three different sequencing methods with different taxonomic resolution, and a deeper diversity analysis on this group showed no substantial differences between samples taken in winter and summer, although significant similarities were observed among hot springs with common surrounding environments. The horizontal microheterogeneity of microbial mats at a centímeter scale also revealed Chloroflexi as dominant phylum together with Cyanobacteria, and a temperature differentiation was observed between both potential primary producers. Significant diversity shifts were detected among closely situates samples in single mats, but at the same time, samples within mat patches showed a high community structure similarity. A deeper characterization of the bacterial community structure revealed the presence of several heterotrophic groups such as Cytophagia and Sphingobacteria; Thermales; and Alpha, Beta, and Gammaproteobacteria; among others. When compared with other thermophilic microbial mats along a latitudinal gradient, no relationship was detected between richness and latitude, as samples closer to the equator were not more diverse or harbored more richness than samples closer to the pole. Temperature, on the other hand, influenced significantly the bacterial richness. Finally, samples at local, regional and global scales (up to 1000 m2) showed a decrease in similitude as geographic distance, but not at the continental scale.

A study of the microbial diversity in sediments of the Great Berg River estuary is carried out using modern molecular phylogenetic methods. The aim of this study was to determine the effect of (pollution by) the effluents of the fish industry on the composition of the microbial community in the sediments. The diversity in microbial groups of sediment samples that received wastewater from the local fishing industry was investigated by a PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) approach and compared to an unaffected site.

Our understanding of microbial evolution is largely dependent on available genomic data of diverse organisms. Yet, genome-sequencing efforts have mostly ignored the diverse uncultivable majority in favor of cultivable and sociologically relevant organisms. In this thesis, I have applied and developed cultivation independent methods to explore microbial diversity and obtain genomic data in an unbiased manner. The obtained genomes were then used to study the evolution of mitochondria, Rickettsiales and Haloarchaea. Metagenomic binning of oceanic samples recovered draft genomes for thirteen novel Alphaproteobacteria-related lineages. Phylogenomics analyses utilizing the improved taxon sample suggested that mitochondria are not related to Rickettsiales but rather evolved from a proteobacterial lineage closely related to all sampled alphaproteobacteria. Single-cell genomics and metagenomics of lake and oceanic samples, respectively, identified previously unobserved Rickettsiales-related lineages. They branched early relative to characterized Rickettsiales and encoded flagellar genes, a feature once thought absent in this order. Flagella are most likely an ancestral feature, and were independently lost during Rickettsiales diversification. In addition, preliminary analyses suggest that ATP/ADP translocase, the marker for energy parasitism, was acquired after the acquisition of type IV secretion systems during the emergence of the Rickettsiales. Further exploration of the oceanic samples yielded the first draft genomes of Marine Group IV archaea, the closest known relatives of the Haloarchaea. The halophilic and generally aerobic Haloarchaea are thought to have evolved from an anaerobic methanogenic ancestor. The MG-IV genomes allowed us to study this enigmatic evolutionary transition. Preliminary ancestral reconstruction analyses suggest a gradual loss of methanogenesis and adaptation to an aerobic lifestyle, respectively. The thesis further presents a new amplicon sequencing method that captures near full-length 16S and 23S rRNA genes of environmental prokaryotes. The method exploits PacBio's long read technology and the frequent proximity of these genes in prokaryotic genomes. Compared to traditional partial 16S amplicon sequencing, our method classifies environmental lineages that are distantly related to reference taxa more confidently. In conclusion, this thesis provides new insights into the origins of mitochondria, Rickettsiales and Haloarchaea and illustrates the power of cultivation independent methods with respect to the study of microbial evolution.

Interactions between plant roots and soil microorganisms in the rhizosphere are critical for plant growth. However, understanding of precisely how root exudates influence the diversity and activity of rhizosphere microorganisms is limited. The main objective of this study was to investigate the effect of radiata pine (Pinus radiata) root exudates on rhizosphere soil microbial communities, with an emphasis on the role of low molecular weight organic anions. The study involved the development and validation of new methods for investigating rhizosphere processes in a purpose-built facility. This included development of an in situ sampling technique using an anion exchange membrane strip to collect a range of organic anions exuded from radiata pine roots grown in large-scale rhizotrons. These included tartarate, quinate, formate, malate, malonate, shikimate, lactate, acetate, maleate, citrate, succinate and fumarate. Soil microbial activity and diversity were determined using dehydrogenase activity and denaturing gradient gel electrophoresis. Links between organic anions in root exudates and rhizosphere soil microbial community structures were investigated by comparing wild type and genetically modified radiata pine trees which were grown in rhizotrons for 10 months. As expected, there was considerable temporal and spatial variability in the amounts and composition of organic anions collected, and there were no consistent or significant differences determined between the two tree lines. Significant differences in rhizosphere microbial communities were detected between wild type and genetically modified pine trees; however, they were inconsistent throughout the experiment. The shifts in microbial communities could have been related to changes in exudate production and composition. Based on results from the main rhizotron experiment, a microcosm study was carried out to investigate the influence of selected pine root exudate sugars (glucose, sucrose and fructose) and organic anions (quinate, lactate and maleate) on soil microbial activity and diversity. Soil microbial activity increased up to 3-fold in all of the sugar and organic anion treatments compared to the control, except for a mixture of sugars and maleate where it decreased. The corresponding impacts on soil microbial diversity were assessed using denaturing gradient gel electrophoresis and 16S rRNA phylochips. Addition of the exudate compounds had a dramatic impact on the composition and diversity of the soil microbial community. A large number of bacterial taxa (88 to 1043) responded positively to the presence of exudate compounds, although some taxa (12 to 24) responded negatively. Organic anions had a greater impact on microbial communities than sugars, which indicated that they may have important roles in rhizosphere ecology of radiata pine. In addition, a diverse range of potentially beneficial bacterial taxa were detected in soil amended with organic anions, indicating specific regulation of rhizosphere microbial communities by root exudates. This project highlighted the considerable challenges and difficulties involved in detailed investigation of in situ rhizosphere processes. Nonetheless, the findings of this study represent a significant contribution to advancing understanding of relationships between root exudates and soil microbial diversity, which will be further enhanced by refinement and application of the specific methodologies and techniques developed.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Civil and Environmental Engineering, 2004.
Includes bibliographical references (p. 102-103).
(cont.) competitive mechanisms are too weak to purge diversity from within them.
Molecular surveys have revealed that microbial communities are extraordinarily diverse. Yet, two important questions remain unanswered: how many bacterial types co-exist, and do such types form phylogenetically discrete units of potential ecological relevance? This thesis explores these questions by investigating bacterial diversity in two complex marine communities (coastal bacterioplankton and sediment sulfate-reducing bacteria) by (i) comprehensive analysis of large 16S rRNA clone libraries, and (ii) refinement and application of parametric diversity estimators. Identification and correction of sequence artifacts demonstrated their potentially significant contribution to diversity estimates. Still, hundreds of unique rRNA sequences (ribotypes) were detected in the corrected libraries, and extrapolation to community diversity with commonly used non-parametric diversity estimators suggested at least thousands of co-existing ribotypes in the two communities. However, close inspection revealed that the non-parametric estimators likely lead to underestimation of ribotype diversity in the clone libraries. Thus, an improved parametric method was developed and shown to closely fit the data. The extrapolated total ribotype diversity in the sample by the improved method was up to one order of magnitude higher than estimated with common non-parametric approaches. Most significantly, the compensation for artifacts and improved estimation revealed that the vast majority of ribotypes fall into microdiverse clusters containing <1% sequence divergence. It is proposed that the observed microdiverse clusters form important units of differentiation in microbial communities. They are hypothesized to arise by selective sweeps and contain high diversity because
by Vanja Klepac-Ceraj.
Ph.D.

Emerging infectious diseases are major contributors to morbidity and mortality. To prevent such diseases from occurring in the future it is important to understand pathogen evolution and emergence. Unfortunately, we know little about the virosphere outside of clinically significant viruses, leaving the bulk of viral diversity unexplored. A key aim of my thesis was to reveal more of the unknown diversity of viruses, particularly in under-studied animal hosts. To this end I employed bulk RNA sequencing (“meta-transcriptomics”) to identify novel viruses in a range of hosts, including native Australian wildlife and invertebrate species, as well as from mining databases of short-read archives. The novel viruses discovered were associated with an array of viral families, including the Flaviviridae, Parvoviridae, Circoviridae, Nudiviridae, Polyomaviridae, and Herpesviridae, in turn expanding our knowledge of the diversity and evolutionary history of these families. I similarly used meta-transcriptomics to document the presence of viral, bacterial and eukaryotic parasite sequences in commonly used laboratory reagents. Additionally, I explored the evolutionary history of two important members of the family Poxviridae: variola virus and the vaccinia virus. Accordingly, I described the historical context of smallpox, with an emphasis on the initial outbreak in Australia, and used ancient DNA techniques to reveal the origins and evolutionary history of the poxviruses used in early vaccination campaigns. Broadly, this thesis has expanded our knowledge of the diversity of viruses and revealed the evolutionary history of viral families that have a major impact on human and animal health. By increasing our knowledge of viral diversity, my work provides important new insights into their ecology and evolution, particularly the transmission of viruses to new host species that underpins disease emergence.

I microrganismi svolgono un ruolo fondamentale nella regolazione dei vari ecosistemi, rappresentando la maggior parte delle specie sulla Terra. Lo scopo di questa tesi di dottorato è stato quello di migliorare le conoscenze sulla diversità microbica e sulle dinamiche delle comunità microbiche in varie condizioni negli habitat in diversi processi biotecnologici nell ambito ambientale e industriale, nonché sulle interazioni microbiche utilizzando metodologie classiche e molecolari. Nella prima parte è stata valutata l'efficienza dei letti biologici (biobed) in area Mediterranea nella degradazione dei fungicidi generalmente utilizzati nei vigneti valutando gli effetti sulla comunità microbica presente in questo ecosistema. I biobed sono sistemi biologici sviluppati nei paesi UE per proteggere i corpi idrici da contaminazione dei pesticidi a livello aziendale. I risultati hanno mostrato che il biobed ha una buona capacità di degradare i pesticidi. Infatti, alla fine dell'esperimento (112 giorni), la maggior parte dei pesticidi era quasi completamente degradata. Elettroforesi su gel in gradiente denaturante (DGGE) ha mostrato una variazione evidente nella diversità microbica dopo l'aggiunta dei fungicidi. Tuttavia, alla fine del processo di degradazione, nessun cambiamento significativo nella composizione della comunità microbica è stato rilevato. In questo caratteristico substrato utilizzato per allestire il biobed, i lieviti ed i funghi filamentosi ascomiceti sembrano essere i microrganismi maggiormente coinvolti nella attività degradativa. Nella seconda parte di questa tesi di dottorato è stato indagato il possibile impiego del glicerolo grezzo come ammendante nel suolo. Il glicerolo grezzo è il principale sottoprodotto della produzione di biodiesel. L'industria del biodiesel è in rapida espansione negli ultimi anni e sta creando un eccesso di glicerolo grezzo come sottoprodotto. In questo contesto, quindi dobbiamo valutare usi alternativi per questo sottoprodotto. Considerando che il glicerolo grezzo è ricco in composti organici, è stato valutato l'effetto di diverse dosi di glicerolo sulla diversità microbica e sulle proprietà chimiche e biochimiche del terreno agricolo allo scopo di valutare il possibile uso agronomico del glicerolo grezzo, senza una sua ulteriore purificazione. I risultati dei metodi coltura dipendenti hanno mostrato che l'aggiunta di glicerolo grezzo ha stimolato la crescita di batteri e funghi coltivabili, mentre l'analisi DGGE ha mostrato che il glicerolo aggiunto aumenta la diversità batterica nel terreno. L’attività microbica in tutti i suoli trattati ha raggiunto uno stato stazionario in termini di attività respiratoria quotidiana indicando un adattamento positivo della biomassa microbica. Questa indagine iniziale ha mostrato che il glicerolo grezzo non sembra avere effetti negativi sul suolo, ma piuttosto favorisce lo sviluppo delle comunità microbiche e incrementa la diversità microbica. Nella terza parte di questa tesi, è stata valutata l'influenza dei diversi trattamenti antifungini su lieviti che naturalmente colonizzano la superficie della bacca d’uva in un vigneto biologico (prodotti a base di rame/zolfo) e un vigneto convenzionale (fungicidi di uso comune). È stata condotta l’analisi quantitativa e qualitativa di lieviti presenti sulla superficie della bacca e nel succo d'uva durante il processo fermentativo, utilizzando i metodi coltura-dipendenti ed indipendenti. La quantità di lieviti che sono risultati presenti corrisponde al numero generalmente riportato per le uve mature, con un maggiore livello di colonizzazione nei campioni convenzionali rispetto a quelli biologici. L’isolamento diretto e l’analisi della DGGE dei campioni di uva alla fase iniziale di fermentazione hanno mostrato una più bassa diversità di lieviti nel vigneto biologico rispetto a quello convenzionale. Nei vigneti biologici e convenzionali, i lieviti dominanti erano Candida zemplinina e Hanseniaspora uvarum, rispettivamente, le specie tipiche per le superfici di bacche d'uve mature. Saccharomyces cerevisiae è stato isolato usando i metodi tradizionali e la DGGE solo alla fine della fermentazione (dopo autoarricchimento), mostrando livelli più bassi nei campioni biologici. Inoltre, S. cerevisiae ha mostrato una più bassa diversità intraspecifica nei campioni organici (due genotipi), in confronto con i campioni convenzionali (sei genotipi). Complessivamente, questi risultati mostrano che i trattamenti di rame e zolfo hanno una maggiore influenza negativa sui livelli quantitativi e sulla diversità della comunità lievitiforme che colonizza la bacca d’uva (incluso S. cerevisiae), in confronto con fungicidi utilizzati per i trattamenti convenzionali. Nella quarta parte della tesi, l'attenzione è stata focalizzata sulle interazioni tra lieviti durante la fermentazione vinaria. Il controllo di questo processo, da tempo si effettua attraverso l’uso di colture selezionate di S. cerevisiae. Per migliorare la qualità e complessità del vino, recentemente, è stato proposto l’uso di fermentazioni miste controllate, con colture selezionate di S. cerevisiae e non-Saccharomyces. Lo scopo di questa indagine è stato quello di studiare l'influenza di Starmerella bombicola sul ceppo di S. cerevisiae a livello di espressione genica e di'attività enzimatica della piruvato decarbossilasi (Pdc1) e dell’alcool deidrogenasi (Adh1), i due enzimi chiave della fermentazione alcolica. Per fare ciò sono state utilizzate cellule immobilizzate di S. bombicola in fermentazione mista. I risultati hanno confermato un aumento di velocità di fermentazione, un consumo combinato di glucosio e fruttosio, un aumento di produzione del glicerolo, una riduzione della produzione del’etanolo e la modifica di sottoprodotti della fermentazione. Inoltre, S. bombicola in fermentazione mista ha mostrato una forte influenza sulla fermentazione alcolica di S. cerevisiae. Infatti, l’attività di Pdc1 in fermentazione mista è stata inferiore a quella mostrata in coltura pura, mentre l’attività di Adh1 ha mostrato un comportamento opposto. L'espressione dei geni PDC1 e ADH1 era altamente indotta nella fase iniziale di fermentazione ed è stata più evidente nella cultura mista. L'influenza di S. bombicola sembra essere molto complessa e non limitata ad un effetto additivo sulla composizione analitica del vino, ma la sua presenza ha causato modificazioni metaboliche durante la fermentazione di S. cerevisiae determinando variazioni dell'espressione genica e dell'attività enzimatica della piruvato decarbossilasi e alcol deidrogenasi.
Microorganisms play a central role in the regulation of ecosystem processes, and they comprise the vast majority of species on Earth. The aim of this PhD thesis was to improve our knowledge on microbial diversity and dynamics of microbial communities in various conditions and ecological habitats related to environmental and industrial biotechnological processes as well as on the microbial interactions by using molecular and classic methods. In the first part of the present thesis it was evaluated the efficiency of the Mediterranean biomixture (the main part of a biobed) in the degradation of fungicides generally used to control pests in vineyards and to assess their effects on microbial community. Biobeds are biological systems developed all over EU countries to protect water-bodies from pesticide contamination at farm level. Results showed that the biomixture had a good capability of degrading pesticides. Indeed, at the end of the experiment (112 days), the concentration of most of the pesticides was close to complete degradation. Denaturing gradient gel electrophoresis (DGGE) analysis showed an evident modification of microbial diversity after the addition of fungicides. However, at the end of degradation process, no significant changes in the composition of microbial community were seen. In the specific substrate used in the biomixture, both yeast flora and ascomycete filamentous fungi seemed to be involved in the degradation activity. In the second part of this PhD thesis it was investigated on the possible use of crude glycerol as a soil amendment. Crude glycerol is a principal by-product of biodiesel production. As the biodiesel industry is rapidly expanding over the last years, a glut of crude glycerol is being created, so there is an urgent need to find alternative uses for this by-product. Considering that crude glycerol is rich in organic compounds, a laboratory experiment was carried out to study the effect of different glycerol amounts on microbial diversity and chemical and biochemical properties of agricultural soil in order to assess the possible agronomical use of crude glycerol without further purification. Results of culture dependent methods showed that the addition of crude glycerol has stimulated the growth of cultivable bacteria and fungi, while DGGE analysis demonstrated that the added glycerol increase bacterial diversity in the soil. Microbial activity in all treated soils reached a steady-state condition in terms of daily respiration activity indicating a positive adaptation of the microbial biomass. This initial investigation showed that the crude glycerol does not seem to have negative effects on the soil but rather causes the development of microbial communities and increases microbial diversity. In the third part of this thesis, the yeast communities colonising grape berry surfaces were evaluated for the influence of fungicide treatments in an organic vineyard (copper/sulphur-based products) and a conventional vineyard (commonly used fungicides). Analysis of yeast abundance and diversity was carried out on grape berries and the grape juice during fermentation, using culture-dependent and -independent approaches. Yeast abundance was as generally reported for mature grapes, with higher counts from grapes treated with conventional fungicides. Direct isolation and DGGE analysis of initial grape samples showed less yeast species diversity in the organic vineyard compared with the conventional vineyard. In the organic and conventional vineyards, the dominant yeasts were Candida zemplinina and Hanseniaspora uvarum, respectively, typical species that colonise surfaces of mature grape berries. Saccharomyces cerevisiae was isolated by traditional methods and detected by DGGE only at the end of fermentation, with lower levels in the organic samples. Moreover, S. cerevisiae showed less intraspecific diversity in the organic samples (two genotypes), in comparison with the conventional samples (six genotypes). Altogether, these results show that the copper and sulphur treatments have greater negative influences on abundance and diversity of grape berry yeast communities (included S. cerevisiae), in comparison with fungicides used for conventional treatments. In the fourth part of the thesis, the attention was focused on the yeast interactions during wine fermentation. The use of a multistarter fermentation process with S. cerevisiae and non Saccharomyces wine yeasts has been proposed to simulate natural must fermentation and to confer greater complexity and specificity to wine. The aim of this work was to study the influence of Starmerella bombicola on S. cerevisiae gene expression and enzymatic activity of pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1), the two key enzymes of the alcoholic fermentation pathway. The presence of S. bombicola immobilized cells in a mixed fermentation trial confirmed an increase in fermentation rate, a combined consumption of glucose and fructose, an increase in glycerol and a reduction in the production of ethanol as well as a modification in the fermentation byproducts. Also the alcoholic fermentation of S. cerevisiae was influenced by S. bombicola immobilized cells. Indeed, Pdc1 activity in mixed fermentation was lower than that exhibited in pure culture while Adh1 activity showed an opposite behavior. The expression of both PDC1 and ADH1 genes was highly induced at the initial phase of fermentation which was more evident in mixed culture. S. bombicola immobilized cells greatly affected the fermentation behavior of S. cerevisiae and the analytical composition of wine. The influence of S. bombicola was not limited to a simple additive contribution but its presence caused metabolic modifications during S. cerevisiae fermentation causing variation in the gene expression and enzymatic activity of alcohol deydrogenase and pyruvate decarboxylase.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Polybia ignobilis é uma vespa social que apresenta uma alimentação variável e inespecífica, consumindo outros insetos, pedaços de animais mortos, néctar, polpa de madeira e água. Dentro do ninho ocorre a trofalaxia, onde o alimento é distribuído entre os indivíduos da colônia através de secreção labial, podendo ocorrer entre adulto e larva ou apenas entre adultos, propiciando a dispersão de micro-organismos. A associação entre leveduras e insetos pode ser considerada transitória, onde os insetos podem atuar como vetores na dispersão desses micro-organismos sem benefício nutricional; por outro lado, também pode ser considerada como uma associação benéfica, onde o inseto utiliza as leveduras como complemento alimentar. O presente estudo teve como objetivos: caracterizar a comunidade de leveduras associadas à P. ignobilis; verificar se há relação entre a distribuição das leveduras nas diferentes castas da vespa e contribuir com a formação de um banco temático de leveduras isoladas de insetos sociais. Indivíduos do ninho de P. ignobilis e amostras de mel foram coletados no campus da UNESP - Rio Claro. O isolamento e identificação das estirpes foi feita de acordo com a metodologia clássica descrita em YARROW (1998). Foram isoladas 167 estirpes, sendo 59 do primeiro ninho e 108 do segundo. Após uma seleção baseada em análises morfológicas, bioquímicas e perfil de bandas de microsatélites (MSP-PCR) estirpes representativas de cada grupo, bem como as estirpes únicas foram identificadas pelo sequênciamento da região D1/D2 do rDNA 26S. Os resultados revelaram a prevalência no primeiro ninho dos gêneros Candida, Cryptococcus, Hanseniaspora e Rhodotorula, compreendendo 45% do total das estirpes, sendo as espécies mais freqüentes Candida azyma, Candida chrysomelidarum, Cryptococcus liquefaciens e Rhodotorula mucilaginosa. No segundo ninho, as espécies...
Polybia ignobilis is a social wasp that features a variable and unspecific alimentation, eats other insects, bits of dead animals, nectar, wood pulp and water. Inside the nest occurs trofalaxis, where food is distributed among individuals of the colony through labial secretion, it can occur between adult and larva or adults only, being vectors in the dispersion of microorganisms. The association between yeasts and insects can be considered temporary, where the insects serve as vectors in the dispersal of these micro-organisms without nutritional benefit; on the other hand, can also be regarded as a beneficial association, where the insect uses the yeasts as a food supplement. This present study aimed to characterize the yeast community associated with P. ignobilis; verify whether exists a relation between the distribution of yeasts in different varieties of wasp and contribute to the formation of a thematic database of yeasts isolated from social insects. Individuals from the nest of P. ignobilis and honey samples were collected on the campus of UNESP - Rio Claro. The isolation and identification of strains was made according to classical methodology described in YARROW (1998). We isolated 167 strains, 59 and 108 of the first and second nest. After a selection based on morphological, biochemical and microsatellite band profiles (MSP-PCR) representative strains of each group as well as unique strains were identified by sequencing the D1/D2 region of 26S rDNA. The results revealed the prevalence in the first nest of Candida, Cryptococcus, Hanseniaspora and Rhodotorula, comprising 45% of all strains, being the most frequent species Candida azyma, Candida chrysomelidarum, Cryptococcus liquefaciens and Rhodotorula mucilaginosa. In the second nest of the species that prevailed were Aureobasidium pullulans, Meyerozyma guillermondii. Some strains may constitute new... (Complete abstract click electronic access below)

Leafy greens like lettuce and spinach are a common vehicle for foodborne illness in United States. It is unknown if native plant epiphytic bacteria may play a role in the establishment of enteric pathogens on leaf surfaces. The objective of this study was to characterize the bacterial communities of fresh and packaged spinach leaves and to explore interactions with E. coli O157:H7. We assessed the bacterial diversity present on the spinach leaf surfaces and how parameters such as spinach cultivar, field conditions, post-harvest operations and the presence of E. coli O157:H7 affected its diversity. Differences in bacterial population size and species richness were associated with differences in plant topography; flat leaves had smaller bacterial populations than savoy leaves, which correlated with larger number of stomata and trichomes in savoy leaves. During spinach growing season shifts in environmental conditions affected richness and population size of the spinach bacterial community. Decreases in the overall soil and ambient temperature and increased rainfall decreased richness and bacterial population size. Fresh spinach richness and composition assessed by parallel pyrosequencing of 16S rRNA elucidated 600 operational taxonomic units, with 11 different bacterial phyla. During postharvest operations diversity indexes and evenness tended to decrease, likely attributed to storage at low temperature and time of storage (4°C and 10°C), that promoted the dominance of g-Proteobacteria. Bacteria isolated from fresh spinach elicited growth inhibition of E. coli O157:H7 in vitro, which was associated with nutrient competition. In contrast growth enhancement produced by epiphytes was associated to low correlations in carbon source utilization and the ability of E. coli O157:H7 to rapidly utilize carbon resources. In packaged spinach, E. coli O157:H7 altered the composition of the bacterial community and its growth was promoted on packaged spinach when a disinfection and temperature abuse occurred, removal of the epiphytic bacteria resulted in significant increases in numbers of E. coli O157:H7 at 10°C and was associated with increased expression of E. coli O157:H7 virulence and stress response genes. The large diversity present on the surface of spinach leaves significantly impacted the ecology of enteric pathogens like E. coli O157:H7 on the phyllosphere.
Ph. D.

Soil communities are regarded as among the most complex and diverse assemblages of microorganisms with estimated bacterial numbers in the order of 10â?¹ cells.gâ?»¹
. Studies on extreme soils however, have reported lower cell densities, supporting the perception that the so-called extreme environments exhibit low species diversity. To assess the extent of microbial diversity within an extreme environment, the mineral soils of the Dry Valleys, Ross Dependency, Eastern Antarctica were investigated using 16S rDNA analysis.

Estuaries are the transition between freshwater and marine environments, and regulate the delivery of riverine fluxes to the oceans. The Humber estuary (UK) is considered a major source of nutrients to the North Sea. It is a highly turbid and dynamic macrotidal estuary that receives contaminated fluxes from agriculture, urbanisation, industry and historical mining activities. The chemistry of the river water and the sediments is modified within the estuarine continuum due to mixing. Sediments are subjected to resuspension periodically (on a tidal cycle timescale) and occasionally or seasonally (due to extreme rainfall or flooding episodes), which triggers a series of redox processes that control nutrient and pollutant cycling. During simulated sediment resuspension in aerated conditions, the release of accumulated reduced substrates (ammonium, manganese, iron, sulphur) and trace metals were reversed within relatively short timescales, which is important when assessing the environmental consequences of different resuspension episodes. However, the position in the salinity gradient was the dominant control on sediment geochemistry since a transition from the inner estuary (Mn/Fe-dominated redox chemistry) to the outer estuary (Fe/S-dominated redox chemistry) was observed. To better understand the role of the benthic biogeochemical denitrification processes in the nitrogen cycling, nitrate-dependent oxidation was also investigated in microcosm experiments. The same transition was observed in the nitrate reduction coupled with the oxidation of different inorganic species from the inner to the outer estuary. In this oxidation scenario there was also evidence of trace metal mobilisation. Due to the greater availability of electron donors in the mudflats of the outer estuary, they showed the greatest potential for denitrification and therefore are considered a relevant nitrogen sink in the Humber estuary. Furthermore, in this context of highly spatiotemporal variability, benthic microbial diversity showed a decreasing trend with increasing salinity, but sediment mixing and transport and the presence of strong redox transitions were also environmental parameters shaping the microbial communities in the Humber sediments.

A synergistic relationship between soil diversity (pedodiversity) and soil microbial diversity (biodiversity) seems axiomatic. Soil microbes contribute with biogeochemical cycles on which rely soil services (e.g. food production) and; vice-versa the soil matrix provides the living conditions that structure its microbial communities. A better insight would enable us to quantify/qualify and so sustain, protect, and improve those processes underpinned by soil microorganisms. We hypothesize that the structural patterns of soil microbes rely on multivariate soil units and gradients (e.g. soil horizons) instead of single discrete ‘factors’ (soil pH) and that the microbial patterns can become a well-defined property of determined soils. We began exploring this biotic-abiotic dynamic by modeling soil microbial α-diversity using two orthogonal transects (~900 km each) across NSW. Soils were sampled from paired conserved and disturbed ecosystems. Soil biophysicochemistry was characterized using 16SrDNA/ITS metabarcoding and pedometric approaches. Soil microbial patterns and physicochemical attributes were assessed using linear and non-linear relationships (bootstraped regression trees models) whose output enabled the microbial mapping at 1km across NSW. These maps showed a higher diversity of soil microbes in western than eastern NSW. Despite this gradient, fungi and archaea were respectively lower and higher in Vertosols, whereas bacteria distribution was less clear. Our results suggested that microbial structural patterns relate to most pedological attributes and, the extent of this relationship varies according to the structural parameters analyzed (taxa composition, abundance, diversity metric). Therefore, microbial patterns are more consistent with grouped features defining soil gradients (soil types) rather than on individual soil properties. These conclusions will be supported by analyzing microbial and pedological dissimilarities (β-diversity) in a further research.

Investigations seeking definition of the microbial diversity present in the human oral cavity have been underway for many years. These investigations seek to define the normal microflora of the oral cavity, as well as the stability of the microflora. Predominantly, these kinds of studies have focused on the differences in microbial carriage between healthy and diseased oral cavities and have identified several species which may be causative and promote oral diseases such as periodontitis and halitosis. Less is known about how co-habitants of a healthy oral cavity interact and how those interactions are mediated and controlled. The genes that may be involved in microbial interactions and their regulation and roles within response pathways in oral microbes is an interesting area of research to pursue, as it could give valuable insight into the changing genetic and potentially physiological state of oral microbes. An evaluation of oral microbial diversity performed during this study by Restriction Fragment Length Polymorphism (RFLP) and Fatty Acid Methyl Ester (FAME) profiling analyses carried out on whole mouth rinse samples identified many bacterial genera from six phyla. These genera included Streptococcus, Gemella, Veillonella, Eubacterium, Porphyromonas, Neisseria, Rothia and Prevotella. The three prevalent genera, as defined by the number of times observed from mouth rinse samples, were Streptococcus, Neisseria and Veillonella. Solid-phase co-culture experiments using isolates of the three prevalent genera revealed a strong interaction between Streptococcus and Neisseria, with Streptococcus exerting a negative effect on Neisserial growth. Liquid phase co-culture experiments between these two genera confirmed initial observations and it was hypothesised that the interaction was mediated by toxic hydrogen peroxide produced by Streptococcus. Further experiments showed that Neisseria were protected from Streptococcal killing upon addition of exogenous catalase, confirming that the interaction was mediated by hydrogen peroxide. The roles of the key genes of the oxidative stress response of Neisseria were investigated by assessing the interactions between Streptococcus pneumoniae and several mutant Neisseria meningitidis, defective in one or more genes of the oxidate stress response. These experiments resulted in observations differing to those previously published, particularly in relation to the prx gene (encoding the peroxidase peroxiredoxin), which lead to the proposal of multiple pathways of stress response existing in N. meningitidis. It was observed that N. meningitidis survival was increased in the absence of the bcp gene (encoding the peroxidase bacterioferritin co-migratory protein) and it was hypothesised that this was due to an up-regulation of other peroxidases in this mutant. Real-Time PCR (RT-PCR) experiments showed increased expression of prx in a bcp-deficient N. meningitidis mutant in the presence of hydrogen peroxide, but this up-regulation was only marginal in the un-stressed mutant strain. Furthermore, it was shown that in the presence of high-levels of hydrogen peroxide (1 mM), N. meningitidis and the various mutant strains examined, behaved as previously observed. This gives rise to the notion that the hydrogen peroxide levels produced in co-culture with S. pneumoniae are not high enough to induce the typical oxidative stress response in N. meningitidis, indicating that another pathway(s) must exist in order to deal with low / intermediate-level hydrogen peroxide / organic peroxides experienced during co-colonisation of the oral cavity.

Alluvial aquifers are important sources of drinking water for Eastern Iowans. However, alluvial aquifers are particularly susceptible to contamination from surface activities due to their shallow depth, permeable material, and close connection to surface waters. Domestic wells and monitoring wells located in alluvial aquifers within the Eastern Iowa Basins study unit, which covers 19,500 square miles, were sampled. The study unit includes the Wapsipinicon, Cedar, Iowa, and Skunk River basins, all of which drain to the Mississippi River. During the summer of 2017, a sampling effort of twelve domestic, twenty-four monitoring, and two municipal wells within the Eastern Iowa Basins study unit was conducted. Water quality data, including dissolved oxygen, nitrate, ammonia, dissolved organic carbon, iron, and sulfate concentrations, was collected. Additionally, microbial DNA samples were collected via filtration of alluvial groundwater. We tested the hypotheses that microbial species richness would decrease with increasing NO3--N concentrations and that differences in groundwater chemistry would be associated with differences in microbial community taxonomy. Overall, the current state of microbial populations in alluvial aquifers was studied, where the risk of groundwater contamination is high.

Microbial mats exist in freshwater pools atop the Ward Hunt and Markham ice shelves in the Canadian high Arctic. In this study, culture-dependent and culture-independent techniques were used to describe the microbial diversity and activity of these mat communities.Bacterial and archaeal 16S rRNA gene clone libraries were constructed from Markham and Ward Hunt mat samples. Bacterial libraries from both mats had high diversity, though the Markham library appeared more diverse than the Ward Hunt library. Over 95% of sequences in both bacterial libraries, and all isolates from both mats, grouped within the phyla Bacteroidetes, Proteobacteria, and Actinobacteria. Archaeal diversity appeared low in both mats. Only one phylotype, potentially representing a novel Euryarchaeota, was observed in both archaeal libraries. Populations in both communities showed activity at subzero temperatures, with growth of isolates at -5°C and detectable metabolic activity at -10°C, measured by radiorespiration assays of mat microcosms.

Vitis vinifera subsp. vinifera L., les principales espèces de raisins sont cultivées pour la production de fruits et la production de vin dans le monde est un hôte naturel d'une grande variété de micro-organismes procaryotes et eucaryotes qui interagissent avec la vigne, ayant des effets bénéfiques ou phytopathogènes. Ils pourraient également jouer un rôle majeur dans le rendement des fruits, la qualité du raisin, la protection des plantes et, finalement, dans le modèle de la fermentation du raisin et la production de vin. La phyllosphère (constituée des parties aériennes de la plante) est l'un des habitats microbiens les plus répandus sur terre et est un milieu assez négligé, en particulier dans les vignes et de nombreuses questions liées à cet habitat microbien sont toujours sans réponse.Cette thèse est un effort pour répondre à une question fondamentale en écologie microbienne: quels sont les facteurs qui déterminent le microbiome dans la phyllosphère de la vigne? Les communautés microbiennes de la phyllosphère (PMCs) vivent à l'interface plante-climat et sa capacité à s'établir, prospérer et se reproduire sur la surface des feuilles ou des fruits dépend de plusieurs caractéristiques fonctionnelles microbiennes, comme la capacité de se fixer sur la cuticule et d'utiliser la foliaire. nutriments ainsi que les conditions climatiques dominantes comme la température, l'humidité de l'air et la pluie. La chimie des feuilles ou des fruits, la physiologie et la structure morphologique diffèrent selon le génotype et l'espèce puisque tous ces traits ont une base génétique, et cette variation peut mener à une combinaison différente d'assemblage de PMC parmi les génotypes de plantes. Ainsi, le premier objectif de notre travail était d'évaluer les impacts des cultivars de vigne (variétés de Vitis vinifera L) et des espèces de vigne (espèces Vitis entièrement différentes) sur l'assemblage du microbiome dans la phyllosphère à un endroit géographique particulier (pour minimiser les effets environnementaux) . Plus tard, les impacts de certains cultivars et terroirs de vigne commercialement importants (représentés par trois zones climatiques françaises) ont également été évalués et comparés. Les impacts de la saison et des organes extérieurs de la plante (feuilles et baies) sur la structuration des taxons microbiens dans la phyllosphère ont également été évalués et présentés dans ce travail. De plus, des impacts spécifiques à l'espèce sur le microbiome de la phyllosphère ont également été testés et représentés.Dans l'ensemble, notre étude a évalué et comparé les nombreuses facettes des facteurs qui peuvent influencer structure du microbiome dans la phyllosphère avec un accent particulier sur la pression de sélection relative exercée par le génotype de la vigne et son interaction avec différentes conditions climatiques (ou terroir), ce qui peut améliorer nos chances de trouver des gènes contrôlant les PMCs sur la phyllosphère. les gènes sont réellement importants dans des environnements réalistes et probablement ces gènes nous donneraient de nouvelles idées pour la sélection de nouveaux cépages sains présentant de meilleurs caractères sur leur phyllosphère. De plus, considérant que les PMC végétales jouent un rôle crucial dans la santé et la forme des plantes car elles peuvent moduler la susceptibilité foliaire aux infections, cette étude pourrait également être utile pour développer des méthodes de biocontrôle innovantes et naturelles ou phytostimulation contre les pathogènes de la vigne. de variétés résistantes innovantes
Vitis vinifera subsp. vinifera L., the main grape species are grown for fruit and wine production over the world is a natural host of a wide variety of prokaryotic and eukaryotic microorganisms that interact with grapevine, having either beneficial or phytopathogenic effects. They could also play a major role in fruit yield, grape quality, plant protection and, ultimately, in the pattern of grape fermentation and wine production. Phyllosphere (consists of the aerial parts of the plant) is one of the most prevalent microbial habitats on earth and is quite a neglected milieu, especially in grapevines and many questions related to this microbial habitat, are still unanswered.This thesis is an effort to answer a very fundamental question in microbial ecology- what are the drivers that shape the microbiome in the grapevine's phyllosphere? The phyllosphere microbial communities (PMCs) live at the plant-climate interface and its ability to establish, thrive and reproduce on the leaf or fruit surface depends on several microbial functional traits, such as the ability to attach to the cuticle and to use the foliar nutrients as well as well as to the prevailing climatic conditions like temperature, air humidity and rain. Leaf or fruit chemistry, physiology, and morphological structure differ among plant genotype and species as all these traits have a genetic basis, and this variation may lead to a different combination of PMCs assemblage among plant genotypes. Hence, the first objective of our work was to assess the impacts of grapevine cultivars (varieties of Vitis vinifera L) and grapevine species (entirely different Vitis species) on microbiome assemblage in the phyllosphere at a particular geographic location (to minimize the environmental effects). Later on, impacts of some commercially important grapevine cultivars and terroirs (represented by three French climate zones) were also assessed and compared. Impacts of the season and exterior plant organs (leaf and berries) on microbial taxa structuring in the phyllosphere was also assessed and presented in this work. Furthermore, species-specific impacts on phyllosphere microbiome were also tested and represented.Overall our study assessed and compared the many facets of the factors that may influence themicrobiome structure in the phyllosphere with a special focus on relative selection pressure exerted by grapevine genotype and its interaction with different climatic conditions (or terroir), which may improve our chances to find genes that controls PMCs on phyllosphere, and simultaneously increase our confidence that those genes are actually important in realistic environments and probably those genes would give us new insights for breeding new and healthy grape varieties displaying better traits on their phyllosphere. Moreover, considering that the plant PMCs plays a crucial role in plant health and fitness as it can modulate leaf susceptibility to infection, this study could also be helpful to develop innovative and natural biocontrol methods or phytostimulation against grapevine pathogens or rethink breeding schemes for the creation of innovative resistant varieties