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C'est essentiellement sur les bactéries libres que portent les études récentes en écologie microbienne d'eau douce, et seulement quelques études ont concerné les communautés bactériennes attachées. Dans cette étude basée sur l'analyse des séquences du gène 16SARNr, la diversité des communautés bactériennes attachées ainsi que de la fraction libre a été étudiée sur deux systèmes d'eau douce ; un lac mésotrophe (le lac du Bourget) et un lac hypereutrophe (le lac Villerest). La diversité des Actinobacteria, Betaproteobacteria et Verrucomicrobia libres et attachées a été étudié en relation avec les variables environnementales, dans le lac du Bourget pendant deux périodes à dominances contrastées de phytoplancton. L'analyse des résultats a montré une différence au niveau phylogénétique entre les communautés bactériennes attachées et libres des trois groupes de bactéries étudiées. Le clade betaI, dominait les Betaproteobacteria des fractions libres et attachées, avec 57,8% de la totalité des unités taxinomiques opérationnelles (OTUs). Pour les Actinobacteria, le groupe d'acIV a été détecté comme le plus abondant, suivi par acI avec respectivement 45% et 25% du total des OTUs. De même, les groupes Verrucomicrobia d'eau douce, à savoir CREPA29, FukuN18, CL120-10 sont apparus comme les plus importants, avec 22,3%, 16,15% et 14,61% des OTUs respectivement. Cette étude a permis de définir 15 nouveaux clades putatifs représentant la diversité bactérienne d'eau douce des Betaproteobacteria (lbI-lbVIII), Actinobacteria (acLBI) et Verrucomicrobia (CRE-PA29, FukuS27, BourFI-BourFIV). Par ailleurs, 12 groupes représentant la diversité de phylogénétique des Betaproteobacteria, Actinobacteria et Verrucomicrobia contiennent exclusivement des OTUs de la fraction attachée. La dynamique saisonnière souligne les changements des phylotypes bactériens distincts pour les deux communautés attaché et libre. Les Actinobacteria dans la fraction attachée était associée avec la biomasse des Chrysophyceae et N-NO3, et les Betaproteobacteria avec la biomasse de Chlorophyceae et de la richesse du phytoplancton tandis que les Verrucomicrobia de cette même fraction ont semblé être principalement influencés par la richesse du phytoplancton, l′abondance des rotifères et les nutriments inorganiques (N-NO3, SiO2). D'autre part, dans les communautés libres, peu de clades d'actinobacterie dépendent des nutriments ou du phytoplancton, alors que les Betaproteobacteria et Verrucomicrobia ont été principalement associés avec les paramètres biologiques (i.e. phytoplancton et copépodes). Pendant le bloom des cyanobacteries (Microcystis sp.) dans le lac Villerest, les Betaproteobacteria, Gammaproteobacteria, Alphaproteobacteria, Bacteroidetes et Actinobacteria ont été détectés comme taxa dominants dans les banques de clones du gène 16S ARNr. Toutefois Verrucomicrobia et Deinococcus-Thermus sont apparus relativement moins abondants dans les deux fractions, tandis que,Gemmatimonadetes, Acidobacteria, Chloroflexi, Planctomycetes, Deltaproteobacteria, Firmicutes et Op11 sont apparus comme des phyla mineurs dans la banque de clones des communautés bactériennes attaché et libre. Les Betaproteobacteria (n=118) attachées sont apparus comme le groupe dominant, suivi par Gammaproteobacteria (n=74) et Bacteroidetes (n=52). L'analyse phylogénétique des séquences obtenues pour la banque de clone de la fraction libre a montré que la plupart des OTUs appartiennent à Betaproteobacteria (n=192), suivi par Bacteroidetes (n=132) et Actinobacteria (n=61). Tandis que les Gammaproteobacteria (n=42) et Alphaproteobacteria (n=42) sont présents dans des proportions égales dans la banque de clones du 16S ARNr libre. (...) / The free-living bacteria point of view dominates in recent research of freshwater microbial ecology, only a few studies have focused on attached bacterial communities. In present study, based on 16S rRNA gene sequences, diversity of attached and free-living bacterial community was investigated from two freshwater aquatic systems ; a mesotrophic lake Bourget and a hypereutrophic lake Villerest. The diversity of attached and free-living Actinobacteria, Betaproteobacteria and Verrucomicrobia, in relation to environmental variables was investigated from lake Bourget during two contrasting periods of phytoplankton dominance. Comparison analyses showed a phylogenetic difference between attached and free-living bacterial communities of all three studied bacterial groups. The betaI, appeared as most dominant among all clades representing phylogenetic diversity of freshwater Betaproteobacteria, for both attached and free-living fractions, contributing to 57.8% of of the total retrieved opertational taxonomic units (OTUs). For Actinobacteria, the acIV cluster was detected as dominant, followed by acI accounting for 45% and 25% of the total retrieved OTUs respectively. Similarly, freshwater Verrucomicrobia cluster namely, CRE-PA29, FukuN18, CL120-10 appeared as dominant, comprising 22.3%, 16.15% and 14.61% of the total retrieved OTUs respectively. This study allowed defining 15 new putative clades representing the freshwater bacterial divesity of Betaproteobacteria (lbI-lbVIII), Actinobacteria (acLBI) and Verrucomicrobia (CRE-PA29, FukuS27, BourFI-BourFIV). In addition, 12 clusters representing the phylogenetic diversity of Betaproteobacteria, Actinobacteria and Verrucomicrobia were exclusively comprised of OTUs from the attached fraction. The seasonal dynamics of environmental variables have been reflected as changes in distinct bacterial phylotypes for both attached and free-living communities. The attached bacterial communities of Actinobacteria showed affiliation with Chrysophyceae biomass and N-NO3, while attached Betaproteobacteria were affiliated with biomass of Chlorophyceae and phytoplankton richness. Similarly attached verrucomicrobial communities appeared to be mainly influenced by phytoplankton richness, rotifers abundances and inorganic nutrients (NNO3,SiO2). On the other hand, within free-living communities, few actinobacterial clades were found to be dependent on either nutrients or phytoplankton communities, whereas Betaproteobacteria and Verrucomicrobia were mainly associated with biological parameters (i.e. phytoplankton and copepods communities). In another study during a cyanobacterial (Microcystis sp.) bloom from lake Villerest, Betaproteobacteria, Gammaproteobacteria, Alphaproteobacteria, Bacteroidetes and Actinobacteria were detected as prevalent taxa among the 16S rRNA gene clone libraries, however, Verrucomicrobia and Deinococcus-Thermus appeared as comparatively less abundant bacterial groups in both fractions. Whereas, Gemmatimonadetes, Acidobacteria, Chloroflexi, Planctomycetes, Deltaproteobacteria, Firmicutes and Op11 were appeared as minor phyla in clone libraries of attached and free-living bacterial communities. For attached bacterial communities Betaproteobacteria (n=118) appeared as most dominant group, followed by Gammaproteobacteria (n=74) and Bacteroidetes (n=52). The phylogenetic analysis of the sequences obtained for the clone library from free-living fraction showed that most of the OTUs belonged to Betaproteobacteria (n=192) followed in decreasing order by Bacteroidetes (n=132) and Actinobacteria (n=61) whereas Gammaproteobacteria (n=42) and Alphaproteobacteria (n=42) appeared in equal proportion in free-living 16S rRNA clone libraries. (...)

Soil represents the largest global reservoir of microbial diversity for the discovery of novel genes and enzymes. Both stable-isotope probing (SIP) and metagenomics have been used to access uncultured microbial diversity, but few studies have combined these two methods for accessing the biotechnological potential of soil genetic diversity and fewer yet have employed functional metagenomics for recovering novel genes and enzymes for bioenergy or bioproduct applications. In this research, I demonstrate the power of combining functional metagenomics and SIP using multiple plant-derived carbon substrates and diverse soils for characterizing active soil bacterial communities and recovering glycosyl hydrolases based on gene expression. Three disparate Canadian soils (tundra, temperate rainforest and agricultural) were incubated with five native carbon (12C) or stable-isotope labelled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose and cellulose). Sampling at defined time intervals (one, three and six weeks) was followed by DNA extraction and cesium chloride density gradient ultracentrifugation. Denaturing gradient gel electrophoresis (DGGE) of all gradient fractions confirmed the recovery of labeled nucleic acids. Sequencing of original soil samples and labeled DNA fractions demonstrated unique heavy DNA patterns associated with all soils and substrates. Indicator species analysis revealed many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Salinibacterium (Actinobacteria), Devosia (Alphaproteobacteria), Telmatospirillum (Alphaproteobacteria), Phenylobacterium (Alphaproteobacteria) and Asticcacaulis (Alphaproteobacteria) were the bacterial ???indicator species??? for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (genus Phenylobacterium) were associated with metabolism of cellulose. Members of the Alphaproteobacteria were associated with the metabolism of arabinose and members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycosyl hydrolase gene representation within the pooled heavy DNA. By screening only 2876 inserts derived from the 13C-cellulose heavy DNA, stable-isotope probing and functional screens enabled the recovery of six clones with activity against carboxymethylcellulose and methylumbelliferone-based substrates.

Agricultural management is essential for achieving optimum crop production and maintaining soil quality. Soil microorganisms are responsible for nutrient cycling and are an important consideration for effective soil management. The overall goal of the present research was to better understand microbial communities in agricultural soils as they relate to soil management practices. For this, we evaluated the differential impact of two contrasting drainage practices on microbial community composition and characterized active denitrifiers from selected agricultural sites. Field drainage is important for crop growth in arable soils. Controlled and uncontrolled tile drainage practices maintain water in the field or fully drain it, respectively. Because soil water content influences nutrient concentration, moisture, and oxygen availability, the effects of these two disparate practices on microbial community composition was compared in paired fields that had diverse land management histories. Libraries of the 16S rRNA gene were generated from DNA from 168 soil samples collected from eight fields during the 2012 growing season. Paired-end sequencing using next-generation sequencing was followed by read assembly and multivariate statistical analyses. Results showed that drainage practice exerted no measureable effect on the bacterial communities. However, bacterial communities were impacted by plant cultivar and applied fertilizer, in addition to sampled soil depth. Indicator species were only recovered for depth; plant cultivar or applied fertilizer type had no strong and specific indicator species. Among indicator species for soil depth (30-90 cm) were Chloroflexi (Anaerolineae), Betaproteobacteria (Janthinobacterium, Herminiimonas, Rhodoferax, Polaromonas), Deltaproteobacteria (Anaeromyxobacter, Geobacter), Alphaproteobacteria (Novosphingobium, Rhodobacter), and Actinobacteria (Promicromonospora). Denitrification in agricultural fields transforms nitrogen applied as fertilizer, reduces crop production, and emits N2O, which is a potent greenhouse gas. Agriculture is the highest anthropogenic source of N2O, which underlines the importance of understanding the microbiology of denitrification for reducing greenhouse gas emissions by altered management practices. Existing denitrifier probes and primers are biased due to their development based mostly on sequence information from cultured denitrifiers. To circumvent this limitation, this study investigated active and uncultivated denitrifiers from two agricultural sites in Ottawa, Ontario. Using DNA stable-isotope probing, we enriched nucleic acids from active soil denitrifiers by exposing intact replicate soil cores to NO3- and 13C6-glucose under anoxic conditions using flow-through reactors, with parallel native substrate controls. Spectrophotometric chemistry assays and gas chromatography confirmed active NO3- depletion and N2O production, respectively. Duplicate flow-through reactors were sacrificed after one and four week incubation periods to assess temporal changes due to food web dynamics. Soil DNA was extracted and processed by density gradient ultracentrifugation, followed by fractionation to separate DNA contributed by active denitrifiers (i.e., “heavy” DNA) from that of the background community (i.e., “light” DNA). Light and heavy DNA samples were analyzed by paired-end sequencing of 16S rRNA genes using next-generation sequencing. Multivariate statistics of assembled 16S rRNA genes confirmed unique taxonomic representation in heavy fractions from flow-through reactors fed 13C6-glucose, which exceeded any site-specific or temporal shifts in putative denitrifiers. Based on high relative abundance in heavy DNA, labelled taxa affiliated with the Betaproteobacteria (71%; Janthinobacterium, Acidovorax, Azoarcus, Dechloromonas), Alphaproteobacteria (8%; Rhizobium), Gammaproteobacteria (4%; Pseudomonas), and Actinobacteria (4%; Streptomycetaceae). Metagenomic DNA from the original soil and recovered heavy fractions were subjected to next-generation sequencing and the results demonstrated enrichment of denitrification genes with taxonomic affiliations to Brucella, Ralstonia, and Chromobacterium in heavy fractions of flow-through reactors fed 13C6-glucose. The vast majority of heavy-DNA-associated nitrite-reductase reads annotated to the copper-containing form (nirK), rather than the heme-containing enzyme (nirS). Analysis of recovered nirK genes demonstrated low sequence identity across common primer-binding sites used for the detection and quantification of soil denitrifiers, indicating that these active denitrifiers would not have been detected in molecular surveys of these same soils.

A afiliação genérica de Fischerella e Hapalosiphon é problemática devido à instabilidade dos caracteres morfológicos. Os gêneros Fischerella e Hapalosiphon são diferenciados pela presença de tricoma multisseriado e uni ou bisseriado, respectivamente. Porém, geneticamente esses caracteres não se mostraram diacríticos para diferenciar gêneros. Estudos moleculares de linhagens isoladas de ecossistemas brasileiros são escassos para Fischerella e inexistentes para Hapalosiphon. Neste estudo, oito linhagens de cianobactérias, pertencentes à família Hapalosiphonaceae, isoladas de água doce e solos brasileiros foram caracterizadas morfologicamente e geneticamente e analisadas para a produção de substâncias bioativas. As análises morfológicas identificaram cinco morfotípos de Fischerella (CENA19, CENA161, CENA212, CENA213, CENA214) e três de Hapalosiphon (CENA63, CENA71, CENA72). As análises filogenéticas do RNAr 16S usando neighbor-joining (NJ) e máxima verossimilhança (MV) colocaram todas as linhagens isoladas em um agrupamento com alto suporte (reamostragens de 99% NJ e MV) contendo membros da ordem Nostocales. Além disso, as linhagens de Fischerella selecionadas para o estudo agruparam-se em um clado interno com alto valor de reamostragem (100% NJ e 86% MV), com exceção da Fischerella CENA19. A posição dessa estirpe na árvore filogenética indica que necessita de revisão taxonômica. As linhagens de solo Hapalosiphon CENA71 e CENA72 também formaram um clado interno separado (99% NJ e 98% MV), mas a linhagem de água doce CENA63 foi colocada em um clado diferente (com valores de reamostragens de 99% NJ e MV), juntamente com linhagens do gênero Hapalosiphon e Westielopsis prolífica SAG 16.93, oriundas de solo. A comparação das análises filogenéticas individuais de regiões dos genes RNAr 16S, rpoC1, rbcL, tufA, e cpcBA-IGS das três linhagens de Hapalosiphon e de duas linhagens de Fischerella, CENA19 e CENA161, mostrou resultados incongruentes devido as diferentes taxas evolutivas desses genes. No entanto, a análise filogenética concatenada desses genes, mostrou que a Fischerella CENA19 agrupou com as duas linhagens de Hapalosiphon CENA71 e CENA72, com alto valor de reamostragem (100%), enquanto que a Fischerella CENA 161 e a Hapalosiphon CENA63 posicionaram-se cada uma em clados separados. Os resultados indicam que a nomenclatura das linhagens de cianobactérias da família Hapalosiphonaceae necessita de revisão. Os extratos intra e extracelulares das linhagens Fischerella sp. CENA161 e CENA19 e Hapalosiphon sp. CENA71 e CENA72 mostraram efeitos inibitórios no crescimento de bactérias patogênicas. As análises em espectrômetro de massas Q-TOF MS/MS indicaram a putativa presença de aeruginopeptina, cianopeptolina, fischerelina, aeruginosina, oscilapeptilida, microcistinas e ácido tumonóico nos extratos. No extrato intracelular da Fischerella sp. CENA161 identificou-se três ou quatro variantes de microcistinas, LR, LL, FR e/ou M(O)R. Fragmentos dos genes mcyA, mcyB, mcyC, mcyD, mcyE, mcyG e mcyI dessa linhagem foram seqüenciados. Nas duas análises filogenéticas realizadas com sequências de aminoácidos de McyE e sequências concatenadas de McyD, McyE e McyG, as enzimas da microcistina sintetase ficaram agrupadas de acordo com os gêneros de cianobactérias indicando um padrão de evolução / The generic affiliation of Fischerella and Hapalosiphon is problematic due to instability of morphological characters. The Fischerella and Hapalosiphon genera are differentiated by the presence of trichome multisseriate and uni or bisseriate, respectively. However, genetically these characters were not diacritical to distinguish genera. Molecular studies of strains isolated from Brazilian ecosystems are scarce for Fischerella and absent for Hapalosiphon. In this study, eight cyanobacterial strains, belonging to Hapalosiphonaceae family, isolated from Brazilian freshwater and soil were morphologically and genetically characterized and analyzed for bioactive compound productions. The morphological analyses identified five Fischerella (CENA19, CENA161, CENA212, CENA213, CENA214) and three Hapalosiphon (CENA63, CENA71, CENA72) morphotypes. The neighbor-Joining (NJ) and maximum likelihood (ML) phylogenetic analyses of 16S rRNA placed all isolated strains in high supported (99% NJ and ML of bootstrap) cluster containing members of the order Nostocales. Furthermore, the Fischerella strains studied were grouped in an internal clade with high bootstrap value (100% NJ and 86% ML), with exception of Fischerella CENA19. The position of this strain in the phylogenetic tree indicates that it needs taxonomical revision. The soil Hapalosiphon strains CENA71 and CENA72 also formed a separated tight internal clade (99% NJ and 98% ML), but the freshwater strain CENA63 was placed in a different clade (99% NJ and ML of bootstrap value) together with Hapalosiphon strains genera and Westielopsis prolifica SAG 16.93, originated from soil. The comparison of the phylogenetic analyses of individual regions of the genes 16S rRNA, rpoC1, rbcL, tufA, and cpcBA-IGS from the three Hapalosiphon strains and the two Fischerella strains CENA19 and CENA161 showed incongruent results due to different evolutionary rates of these genes. However, the concatenated phylogenetic analysis of these genes, showed that Fischerella CENA19 grouped with the two Hapalosiphon strains CENA71 and CENA72 with high bootstrap value (100%), while Fischerella CENA 161 and Hapalosiphon CENA63 were positionated each one in separate clades. The results indicate that the nomenclature of cyanobacterial strains from the family Hapalosiphonaceae needs revision. The intra and extracellular extracts of the Fischerella sp. strains CENA161 and CENA19 and Hapalosiphon sp. strains CENA71 and CENA72 showed inhibitory effects on the growth of pathogenic bacteria. The analysis in the mass spectrometer Q-TOF MS/MS indicated the presence of aeruginopeptin, cyanopeptolin, fischerellin, aeruginosin, oscillapeptilide, microcystins and tumonoic acid in the extracts. In the intracellular extracts of Fischerella sp. CENA161, three or four variants of microcystins, LR, LL, FR and/or M(O)R, were identified. Fragments of genes mcyA, mcyB, mcyC, mcyD, mcyE, mcyG and mcyI of this strain were sequenced. In both phylogenetic analyses performed with amino acid sequences of McyE and concatenated sequences of McyD, McyE and McyG, the microcystin synthetase enzymes were grouped according to the cyanobacterial genera, indicating a pattern of evolution

O presente trabalho teve por objetivo avaliar a eficiência de remoção do surfactante aniônico alquilbenzeno linear sulfonado (LAS) em reator anaeróbio de leito granular expandido - EGSB (1,5 litros) com recirculação e alimentação com meio mineral. Além de caracterizar filogeneticamente a diversidade de bactérias na presença do surfactante. O sistema foi operado em condição mesofílica em 4 etapas: (I), (II) e (IV) com TDH de 32 horas, e (III) com TDH de 26 horas. Em todas as etapas a DQO foi em média de 609 \'+ OU -\' 137 mg/L e 14 \'+ OU -\' 1,71 mg/L de LAS afluente. As maiores remoções de LAS foram verificada nas etapas II e IV, com valores de 73,6 \'+ OU -\' 5,6% e 63,6 \'+ OU -\' 6,17%, respectivamente de. Na etapa III essa remoção foi de 47,8 \'+ OU -\' 6,2%. Por meio do balanço de massa constatou-se que 56,6% do total de LAS adicionado foram removidos compreendendo 48,4% por biodegradação e 8,2% por adsorção. A remoção de matéria orgânica não foi afetada com a adição do LAS e nem pela exposição prolongada a esse surfactante. Entretanto, a estrutura do grânulo foi comprometida quando da adição do surfactante, observado pelo aumento da concentração de sólidos totais efluente de 0,049 g/L na etapa I (sem LAS), 0,128 g/L na etapa II, 0,064 g/L na etapa III e 0,038 g/L na etapa IV, quando da adição de 14 \'+ OU -\' 1,71 mg LAS/L. Além disso, foi notada diminuição do diâmetro médio dos grânulos no decorrer da operação do reator de 0,36 cm nas etapas I e III para 0,34 cm na etapa IV. Por meio da técnica de tubos múltiplos (NMP) foi constatado aumento das bactérias anaeróbias totais e diminuição das arqueias metanogênicas, em função do tempo de operação do reator. As bactérias redutoras de ferro representaram 8% da biomassa anaeróbia na etapa IV. Por meio do seqüenciamento da região 16S do RNAr para o domínio Bacteria da biomassa da extremidade superior do reator e da biomassa do leito, foi verificado semelhança com os seguintes filos Proteobacteria, Firmicutes e Synergistetes. Notou-se diferença significativa entre as bibliotecas de clones para essas duas amostras. / This study aimed to evaluate the efficiency of removal of linear alkylbenzene sulfonate (LAS) in expanded bed reactor (1.5 liters) using granular sludge (EGSB) with recirculation and feed with mineral medium modified. The system was operated at mesophilic condition in four stages: (I) (II) and (IV) with HRT of 32 hours, and (III) with HRT of 26 hours. At all stages the COD averaged 609 \'+ OR -\' 137 mg/L and 14 \'+ OR -\' 1.71 mg/L LAS influent. The higher removals of LAS were found in stages II and IV, respectively, 73.6 \'+ OR -\' 5.6% and 63.6 \'+ OR -\' 6.17%. In stage III this removal was 47.8 \'+ OR -\' 6.2%. Through mass balance was found that 56.6% of total LAS added were removed by biodegradation comprising 48.4% and 8.2% by adsorption. The organic matter removal was not affected by the addition of LAS and not by prolonged exposure to this surfactant. However, the granule structure was compromised after the addition of surfactant, the observed increase in effluent total solids concentration of 0.049 g/L in stage I (no LAS), 0.128 g/L in stage II, 0.064 g/L in stage III and 0.038 g/L in stage IV when adding 14 \'+ OR -\' 1.71 mg/L. Furthermore, it was noticed significant decrease in mean diameter of the granules during the operation of the reactor of 0.36 cm in stages I and III to 0.34 cm in stage IV. Through the multiple tube method (MPN) was found to increase the total anaerobic bacteria and methanogenic archaea decreased depending on the time of reactor operation. Iron-reducing bacteria accounted for 8% of anaerobic bacteria total in step IV. By sequencing the 16S rRNA for the domain Bacteria biomass from the upper end of the reactor and the biomass of the bed, was found similar to the following phyla Proteobacteria, Firmicutes and Synergistetes. Significant difference was noted between the clone libraries for these two samples.

Diverse and ecologically important microbial communities (microbiomes) are symbiotic within marine sponges. In this study, the microbiome of Amphimedon compressa from three sample locations (Broward and Dade Counties, Southeast Florida, USA and the Southern Caribbean, Bocas del Toro, Panama) is characterized using 16S rRNA Illumina sequencing. The predominant taxa are Proteobacteria and Cyanobacteria, as expected for Low Microbial Abundance sponges, accounting for over 53% of the total microbiome community. The numbers of Operational Taxonomic Units (OTUs) decrease from Broward County (2,900) to Dade County (2,300) and then Bocas del Toro (1,200). The correlates to a decreasing north-south gradient of sponge microbiome richness and diversity. Sponge microbiome richness and Alpha diversity are nearly identical from the two closest locations (37 km), both in Southeast Florida (Tukey HSD/ANOVA; p=0.999). However Panama sponge microbiome richness and Alpha diversity are distinctly lower, with the primary driver being distance, ~1,850 km from Southeast Florida. Abiotic factors driving this trend of decreased richness and diversity include increased temperature, and deceased salinity in relation to precipitation-based seasons. Sponge microbiome Beta diversity as determined by Bray-Curtis Dissimilarity and Non-Metric Multidimensional Scaling documents the clustering of Panama samples as distinct from the Broward and Dade County samples. In a seasonal comparison, Broward County sponge microbiome richness (p=0.026, r2=0.92) and Alpha diversity (p=0.007, r2=0.98) are significantly different, documenting robust effects of temperature. This comparison confirms lowest microbiome OTU diversity in the season with highest precipitation and highest temperatures of 29.8 °C. These results are consistent with prior studies that report decreasing microbiome OTU richness and diversity under conditions of environmental stress such as decreased salinity and increased temperatures.

Coral diseases were first noted in the 1960s and 1970s and have had major impacts globally on coral reef community structures. In the Caribbean, a major outbreak of white band disease has been considered responsible for the drastic decline of Caribbean Acroporids since the 1970s. In addition to white band disease, another more recently described condition known as rapid tissue loss (RTL) has had major impacts on Acropora cervicornis populations, specifically offshore Broward County Southeast Florida. While these diseases have contributed to the population decline, determining their etiologies has been elusive. Coral diseases have been characterized by shifts in their microbial counterparts within many levels of the coral host. While some coral diseases have had specific pathogens identified, research has not been able to determine pathogens for most. Evidence points toward bacterial causes for many diseases, but due to the complexity of the coral holobiont and the interaction with the environment, elucidating the causes has proven difficult. Many studies have examined the microbiomes of specific diseases and determined some potential pathogens or at least taxa playing important roles in the disease, although none have looked at RTL. Recognizing the local affect of RTL on A. cervicornis, this study set out to gain a baseline understanding of the healthy and RTL affected microbiome of A. cervicornis. 16S rRNA gene sequencing was used to examine the microbiome of completely healthy colonies, healthy regions of diseased colonies, and the disease margin of diseased colonies. Analysis of four microbial diversity metrics revealed marked increases in diversity with respect to declining health states. Additionally, community dissimilarity analysis and analysis of differentially abundant taxa exhibited distinct microbial community structures due to coral health. Several highly abundant (Rickettsiales, Rhodobacteraceae) and a few low abundance (Bdellovibrionales) taxa were identified as primary drivers of the differences. Additionally, Piscirickettsiaceae, a known fish pathogen, was consistently associated with RTL and warrants further investigation. All of the taxa identified with in RTL have been associated with other Acroporid and non-Acroporid diseases throughout the Caribbean and the rest of the world. The consistent IV association of similar taxa for coral diseases around the world, including those found in this study, supports the recent ideas of non-specific primary pathogens. While most disease studies, coral and otherwise, aim to determine a single pathogen for a single disease, this study and others suggest there could be a multitude of organisms responsible for the disease. Therefore understanding the interactions of the coral holobiont and the environment is important to understanding coral disease. While this study reveals significant changes in the bacterial community associated with RTL as well as some potential pathogens, the relationships appear complex and perhaps at a functional level rather than merely taxonomic. Furthermore, this study did not examine viruses, fungi, or protists, which could be possible pathogens. Therefore, to further develop an understanding of RTL and many other coral diseases it will be necessary to consider additional none-bacterial members of the holobiont as well as the bacterial functions and taxa coupled with the roles of environmental factors.

O presente trabalho teve como objetivos caracterizar uma coleção de 98 bactérias isoladas de amostras de petróleo e água de formação de reservatórios da Bacia de Campos (RJ) utilizando técnicas de taxonomia molecular e avaliar o potencial de degradação de biomarcadores do petróleo. O sequenciamento e análise filogenética do gene RNAr 16S revelaram Bacillus firmus, megaterium, pumilus, sphaericus, simplex, cereus/B. thuringiensis Marinobacter lutaoensis, Halomonas shengliensis/H. alimentaria/ H.campisalis, Citreicella thiooxidans, Stenotrophomonas maltophilia, Achromobacter xylosoxidans, Micrococcus luteus, Kocuria rosea, Streptomyces alboniger/S. chartreusis /S. moderatus, Staphylococcus hominis e Staphylococcus pasteuri/S. warneri. Os resultados evidenciaram a preferência pela biotransformação do ácido nonadecanóico e esqualano. A caracterização da microbiota presente nos reservatórios e avaliação do potencial de biodegradação pode contribuir para fornecer subsídios para estudos futuros sobre os mecanismos biológicos responsáveis pela biodegradação do petróleo. / This study is aimed to characterize a collection of 98 bacteria isolated from oil and formation water samples derived from reservoirs of the Campos Basin (RJ) using molecular biology-based techniques and to evaluate the degradation potential of petroleum biomarkers. Further sequencing and phylogenetic analysis of 16S rRNA genes revealed species of Bacillus firmus, megaterium, pumilus, sphaericus, simplex, cereus/thuringiensis, Marinobacter lutaoensis, Halomonas shengliensis/H. alimentaria/H. campisalis, Citreicella thiooxidans, Stenotrophomonas maltophilia, Achromobacter xylosoxidans, Micrococcus luteus, Kocuria rosea, Streptomyces alboniger/S. chartreusis/S. moderatus, Staphylococcus hominis and Staphylococcus pasteuri/S. warneri. The results showed the preference of bacteria for the biotransformation of nonadecanoic acid and squalane. The characterization of the microbiota associated to reservoirs and the evaluation of their biodegradation potential may provide subsidies for future studies about the biological mechanisms responsible for petroleum biodegradation.

Les hydrocarbures aromatiques polycycliques (HAP) sont des composés ubiquitaires issus de la combustion incomplète de matières organiques. Ils sont à l'origine de pollutions de l'environnement, surtout liées à l'exploitation des produits pétroliers, car ce sont des composés toxiques pour les êtres vivants et pour l'homme en particulier. De nombreuses bactéries capables de dégrader les HAP ont été isolées et étudiées, mais celles qui les dégradent in situ sont mal connues, car moins de 5% des bactéries du sol sont cultivables en laboratoire. Le premier objectif de cette étude était d'identifier les bactéries qui dégradent les HAP dans le sol par des méthodes moléculaires indépendantes de la culture. A cette fin, une stratégie de marquage isotopique in situ a été mise en œuvre qui repose sur l'utilisation du phénanthrène, un HAP à trois cycles, dans lequel l'isotope naturel du carbone a été remplacé par le 13C. Cette molécule a été introduite comme traceur dans des microcosmes contenant du sol provenant d'un bassin de rétention des eaux de ruissellement d'autoroute. Les bactéries ayant incorporé le 13C ont ensuite été identifiées par séquençage des gènes d'ARNr 16S amplifiés à partir de l'ADN marqué extrait du sol. Les résultats montrent que des Betaprotéobactéries peu étudiées à ce jour, appartenant aux genres Acidovorax, Rhodoferax, Hydrogenophaga et Thiobacillus, ainsi que des Rhodocyclaceae, étaient les principaux acteurs de la dégradation du phénanthrène. La prépondérance des Betaprotéobactéries a été établie par des mesures de PCR quantitative. Une analyse dynamique de la diversité bactérienne a montré que celle-ci changeait en fonction de la biodisponibilité du phénanthrène. En outre, la diversité d'arène-dioxygénases impliquées dans la dégradation des HAP a été explorée sur le plan phylogénétique et fonctionnel. Nous avons ainsi détecté des séquences nouvelles, pour la plupart apparentées à des dioxygénases de Sphingomonadales et de Burkholderiales. Grâce à la construction et l'expression d'enzymes hybrides, il a été possible, pour la première fois, d'associer une activité catalytique d'oxydation des HAP à des séquences partielles de gènes, amplifiées à partir de l'ADN du sol. Les résultats obtenus et les outils mis au point dans cette étude pourront servir à développer des méthodes de diagnostic et de suivi de biodégradation de polluants, par exemple dans le cadre d'opérations de bioremédiation de sites pollués par les HAP. / Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous compounds produced by incomplete combustion of organic matter. They are a source of environmental pollution, especially associated to oil product exploitation, and represent a threat for living organisms including human beings because of their toxicity. Many bacteria capable of degrading PAHs have been isolated and studied. However, since less than 5% of soil bacteria can be cultivated in the laboratory, bacterial species able to degrade PAHs in situ have been poorly studied. The first goal of this study was to identify bacteria that degrade PAHs in soil using culture-independent molecular methods. To this end, a strategy known a stable isotope probing has been implemented based on the use of phenanthrene, a three rings PAH, in which the natural isotope of carbon was replaced by 13C. This molecule has been introduced as a tracer in microcosms containing soil from a constructed wetlands collecting contaminated water from highway runoff. Bacteria having incorporated the 13C were then identified by 16S rRNA gene sequence analysis after PCR amplification from labeled genomic DNA extracted from soil. The results show that so far little studied Betaproteobacteria, belonging to the genera Acidovorax, Rhodoferax, Hydrogenophaga and Thiobacillus, as well as Rhodocyclaceae, were the key players in phenanthrene degradation. Predominance of Betaprotéobactéries was established thanks to quantitative PCR measurements. A dynamic analysis of bacterial diversity also showed that the community structure of degraders depended on phenanthrene bioavailability. In addition, the phylogenetic diversity of ring-hydroxylating dioxygenases, enzymes involved in the first step of PAH degradation, has been explored. We detected new sequences, mostly related to dioxygenases from Sphingomonadales and Burkholderiales. For the first time, we were able to associate a catalytic activity for oxidation of PAHs to partial gene sequences amplified from soil DNA, by constructing hybrid enzymes and assaying their activity The results obtained and the tools implemented in this study may be used to develop methods for the diagnostic and monitoring of pollutant biodegradation in processes such as bioremediation of PAHs contaminated sites.