O gênero Yersinia compreende 12 espécies. Y. enterocolitica, Y. pseudotuberculosis e Y. pestis são patógenos de vários animais, incluindo os humanos. Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. aleksiciae, Y. mollaretti e Y. rhodei são encontradas sobretudo no meio ambiente e alimentos e consideradas, usualmente, como bactérias oportunistas não-patogênicas e Y. ruckeri é um importante patógeno de peixes. Usualmente, as linhagens de Yersinia são classificadas em espécies de acordo com suas características bioquímicas. O Laboratório Nacional de Referência em Yersinia spp. outras que Y. pestis recebeu mais de 700 linhagens que foram identificadas bioquimicamente. Entretanto, sete linhagens de Yersinia não puderam ser identificadas pelos testes bioquímicos convencionais em nenhuma das espécies até o momento conhecidas e, por esse motivo, foram denominadas Yersinia atípicas. Os objetivos desse trabalho foram identificar as linhagens atípicas de Yersinia spp. em espécies por técnicas moleculares como Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR), Eletroforese em Campo Pulsado (PFGE), sequenciamento do gene 16S rRNA e Multilocus Sequencing Typing (MLST) e definir dentre as metodologias empregadas a que mais contribui para a identificação precisa dessas linhagens. Foi estudado um total de 59 linhagens de Yersinia spp., sendo 52 linhagens representantes das diferentes espécies do gênero e sete as linhagens bioquimicamente atípicas de Yersinia. As técnicas de ERIC-PCR, sequenciamento do gene 16S rRNA e o MLST foram eficientes na identificação molecular do gênero Yersinia, uma vez que conseguiram reunir todas as espécies em ramos espécie-específicos, com exceção de algumas linhagens de Y. frederiksenii e Y. kristensenii. A técnica de PFGE, pelo contrário, não agrupou as linhagens estudadas em clusters espécie-específicos. Os dados de ERIC-PCR, sequenciamento do gene 16S rRNA e MLST, sugerem que as linhagens atípicas FCF 229 e FCF 231 pertençam à espécie Y. ruckeri. Os dados de ERIC-PCR e MLST sugerem que a linhagem atípica FCF 487 pertença à espécie Y. enterocolitica. Ademais, os dados de ERIC-PCR, sequenciamento do gene 16S rRNA e MLST sugerem que as linhagens atípicas FCF 216, FCF 465, FCF 457 e FCF 494 pertençam a espécie Y. massiliensis. Os resultados obtidos nesse trabalho fornecem dados importantes para a caracterização molecular de linhagens bioquimicamente atípicas de Yersinia e contribuem para uma melhor descrição do gênero quanto a sua diversidade e reforçam o MLST como uma técnica confiável e reprodutível a ser usada na identificação de bactérias pertencentes a esse gênero, sendo dentre as metodologias utilizadas nesse estudo a mais indicada para tipagem molecular de yersiniae. / The genus Yersinia comprises 12 species. Y. enterocolitica, Y. pseudotuberculosis and Y. pestis are pathogens of various animals, including humans. Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. aleksiciae, Y. mollaretti and Y. rohdei have been mostly found in the environment and food sources and are commonly considered to be opportunistic nonpathogenic bacteria and Y. ruckeri is an important fish pathogen. Usually, Yersinia strains are classified into species according to their biochemical characteristics. The Brazilian Reference Center on Yersinia spp. other than Y. pestis received more than 700 strains that were biochemically identified. However, seven strains that were typed as Yersinia could not be biochemically identified in any one of the currently known Yersinia species and for this reason they were named as atypical strains. The aims of this work were to identify into species the atypical Yersinia strains using molecular techniques as Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR), Pulsed Field Gel Electrophoresis (PFGE), 16S rRNA gene sequencing and Multilocus Sequencing Typing (MLST) and to define which methodology better contribute to the identification of those strains. A total of 59 Yersinia spp. strains were studied, being 52 representative strains of the defined Yersinia species and seven atypical Yersinia strains. ERIC-PCR, 16S rRNA gene sequencing and MLST were efficient in molecular identifying the genera Yersinia once they grouped the strains into species-specific clusters, with exception of some Y. frederiksenii and Y. kristensenii strains. However, PFGE was not capable to cluster the defined Yersinia strains into species-specific clusters. The data obtained by ERIC-PCR, 16S rRNA gene sequencing and MLST suggest that the atypical strains FCF 229 and FCF 231 belong to Y. ruckeri species. The data obtained by ERIC-PCR and MLST suggest that FCF 487 belong to the Y. enterocolitica species. Additionally, ERIC-PCR, 16S rRNA gene sequencing and MLST suggest that the atypical strains FCF 216, FCF 465, FCF 457 and FCF 494 belong to the Y. massiliensis species. The results obtained provide important data for the molecular characterization of biochemically atypical strains and contribute for a better description of the genera regardless its diversity. Furthermore, the results reinforce MLST as a trustful and reproducible technique to be used in the identification of bacteria of this genus, being among the methodologies studied the most recommended one to molecular type yersiniae.

With its unique consortium of microorganisms from all domains of life, termite gut is considered one of the most efficient lignocellulose degrading systems in nature. Recently, host diet and taxonomy as well as gut microenvironmental conditions have emerged as main factors shaping microbial communities in termite guts. The aim of this thesis was to investigate this highly efficient lignocellulolytic system at holobiont level, with a particular focus on gut microbiome function and composition in relation to the host diet. As a starting point, we optimised a complete framework for an accurate termite gut prokaryote-oriented metatranscriptomics, which was at the basis of all subsequent sequencing assay designs and analyses performed in the course of the work. Afterwards, we characterised the compositions and functions of biomass-degrading bacterial communities in guts of plant fibre- and soil-feeding higher termites, proving the existence of functional equivalence across microbial populations from different termite hosts. We also showed that each termite is a reservoir of unique microorganisms and their accompanying genes. We further extended above approach to metagenomics and bacterial genomes reconstruction and we applied it to explore the process of biomass digestion in the different sections of the highly compartmented gut of soil feeding Labiotermes labralis. We showed that primarily cellulolytic activity of the termite host was restricted to foregut and midgut, while bacterial contribution was most pronounced in P1 and P3 hindgut compartments and included activities targeting broad range of lignocellulose components. Finally, we investigated the adaptation of a laboratory-maintained grass-feeding higher termite colony of Cortaritermes spp. to Miscanthus diet at host and symbiont levels. A natural system of a termite gut was shown to progressively change in composition to yield a consortium of microbes specialised in degradation of a specific biomass. Overall, the integrative omics approach proposed here provide a framework for a better understanding of a complex lignocellulose degradation by a higher termite gut system and pave a road towards its future bioprospecting. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

<p>Bovine respiratory disease (BRD) is an ongoing health and economic issue in the dairy and beef cattle industry. Also, there are multiple risk factors that make an animal susceptible to BRD and it's diagnosis and treatment is a challenge for producers. Four bacterial species, <em>Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, </em>and<em> Mycoplasma bovis</em> have been associated with BRD mortalities. Hence, this study aims to characterize the cattle nasal microbiome as a potential additional diagnostic method to identify animals suspected to have a lung infection. Quantitative PCR and 16S rRNA gene sequencing were used to determine the bacterial load of these four bacterial pathogens in the nasal microbiome of apparently healthy (N=75) and (N=58) affected by BRD Holstein steers. We then sought to identify a value or equation that could be used to discriminate between BRD and healthy animals using a Linear Discriminant Model (LDA). Additionally, co-occurrence between commensal bacterial and BRD-pathogens were also identified. Cattle diagnosed with BRD presented lower richness, evenness and phylogenetic diversity than healthy pen-mates. Bacterial species and genera <em>Truperella pyrogenes </em>and <em>Bibersteina</em> were increased in the BRD group, and the species <em>Mycoplasma bovirhinis</em> and <em>Clostridium sensu stricto</em> increased in the healthy group. Prevalence of <em>H. somni </em>(98%)<em> </em>and <em>P. multocida </em>(97%) were the highest regardless of disease diagnosis in all the samples. Prevalence of <em>M. haemolytica </em>(81 vs. 61%) and<em> M. bovis </em>(74 vs. 50.7%) were higher in the BRD group. The bacterial density of <em>M. haemolytica</em> and<em> M. bovis </em>was also higher in the BRD group, whereas <em>Histophilus somni</em> was lower in the BRD group. Five different models were tested using LDA, and one model produced a sensitivity and specificity of 60% and 81% agreement with diagnosis based on animal symptoms. Co-occurrence analysis demonstrated that the nasal microbiome members are more likely to interact with each other than associations between BRD-pathogens and nasal microbiome members. This study offers insight into the BRD-pathogens prevalence and difference in nasal microbiome between healthy and BRD animals and provides a potential platform for future studies and potential pen-side diagnostic testing.</p>

Polymerase chain reaction (PCR) is a method used in many research protocols to amplify a small amount of a short segment of DNA to millions of copies. PCR is used for many taxonomic studies, as well as for some medical diagnostic procedures. Through PCR, short DNA primers bind to the template DNA to allow the thermostable DNA polymerase to copy the DNA. Often, researchers create universal primers to target a conserved region of DNA in multiple species, for example, the 16S rRNA gene in bacteria. The problem with these universal primers is that they do not always perfectly match the target DNA. The mismatch primers can still bind to the template, but could affect the efficiency of the PCR amplification. The effect of mismatch primers on the efficiency of the amplification in PCR is the focus of this thesis. Four forward primers with various mismatch overhangs were generated and incorporated into a DNA template through an initial PCR. These primers contained the binding region complementary to the V3/V4 region of the 16S rRNA bacterial gene. Further quantitative PCR (qPCR) reactions were run on these newly-made templates using two sets of primers complementary to the 16S rRNA gene region – one with ambiguous base pairs, one with unambiguous base pairs. The qPCR amplification curves, the Cq values, and the initial concentrations of DNA products (seed values) were analyzed and compared. The results showed differences in the Cq values and seed values between the reactions containing mismatches and those not containing mismatches. Other variables including annealing temperature, addition of Illumina sequencing tails to the primers, and initial primer concentration were also tested to determine if these variables had an effect on the amplification. The results from these reactions using different variables were inconclusive.

As soluções convencionais de tratamento biológico de corantes azo são baseadas em processos anaeróbio-aeróbios ocorrendo em unidades distintas. Este estudo avaliou o uso da aeração intermitente para a descoloração do Direct Black 22 (DB22) e biodegradação simultânea de subprodutos tóxicos no mesmo compartimento reacional. Os microrganismos utilizados foram obtidos de florestas tropicais e previamente adaptados a concentrações crescentes de DB22 (10-32,5 mg.L-1) em meio de cultivo. Os efeitos da concentração inicial de glicose (1 &#8211; 2 &#8211; 3 g.L-1) e aeração intermitente (0 &#8211; 4 &#8211; 8 h.d-1) sobre a descoloração, constante de descoloração e remoção de demanda química de oxigênio (DQO) foram investigados por planejamento fatorial e análise de superfície de resposta. Os testes foram conduzidos com água residuária (AR) que simulava a composição dos efluentes de lavanderias têxteis do agreste pernambucano. Os resultados demonstraram que a descoloração no longo prazo não foi inibida para ciclos de aeração de até 4 h.d-1, embora menores velocidades de remoção de cor tenham sido obtidas nestas condições. Os efeitos negativos da aeração foram significativamente reduzidos pelo aumento da concentração de glicose na AR. Ademais, a remoção de DQO foi potencializada com o aumento da frequência de aeração. Após descoloração do DB22 nos ensaios não aerados, verificou-se a formação de picos de absorbância relacionados à presença de aminas aromáticas ou outros intermediários da descoloração redutiva; o que não ocorreu nos experimentos aerados. Estes fatores resultaram em menor toxicidade à Daphnia magna em experimento modelo com nível intermediário de aeração. O sequenciamento do gene 16S rRNA na plataforma Illumina HiSeq revelou a presença de gêneros de bactérias conhecidos por produzirem enzimas envolvidas na biodegradação do azo. Observou-se, ainda, uma correlação positiva entre diversidade microbiana e eficiência de descoloração. Os resultados sugerem que a estratégia de aeração intermitente, corretamente implementada, pode melhorar a performance do tratamento biológico de efluentes têxteis que contém azo-corantes. / Conventional technologies for biological treatment of azo dyes are based on anaerobic-aerobic processes taking place into distinct units. This study evaluated the use of intermittent aeration strategy for decolorization of the Direct Black 22 (DB22) and simultaneous biodegradation of metabolites. Microorganisms were obtained from tropical forests and previously acclimated to increasing concentrations of DB22 (10-32.5 mg.L-1) in growth medium. Effects of initial glucose concentration (1 &#8211; 2 &#8211; 3 g.L-1) and intermittent aeration (0 &#8211; 4 &#8211; 8 cycles.d-1) on response variables decolorization, decolorization rate, and removal of chemical oxygen demand (COD) were investigated using factorial design and response surface analysis. Tests were conducted using a wastewater that simulated the composition of textile laundry effluents from a region with harsh climate in the state of Pernambuco, known as agreste pernambucano. Results showed long-term decolorization was not impaired for up to 4 cycles.d-1 of aeration, although a decrease in color removal velocities was observed in these experiments. Negative impacts of aeration were significantly reduced by increasing initial glucose concentration. Moreover, COD removal was enhanced with increased aerations levels. After DB22 degradation in non-aerated batches, the formation of absorbance peaks associated with aromatic amines and other byproducts of reductive decolorization was observed; which did not occur in the aerated experiments. These resulted in lower toxicity to Daphnia magna in model experiment using intermediate level of aeration. 16S rRNA gene sequencing on the Illumina HiSeq platform revealed the presence of several bacteria known to produce enzymes involved in azo compounds degradation. Furthermore, a positive correlation between microbial diversity and decolorization efficiency was observed. Results suggest intermittent aeration strategy can enhance biological treatment of textile effluents containing azo dyes when correctly implemented.

A abundância e diversidade dos microrganismos do solo podem ser influenciadas por grande número de fatores, associados, principalmente, ao tipo de solo, sua cobertura e uso. A microbiota do solo apresenta grande importância pelos processos desempenhados pelos seus organismos, essenciais para todos os ecossistemas terrestres. Apesar da grande complexidade associada a esses estudos, os fatores que influenciam as comunidades microbianas podem ser melhor elucidados pela pesquisa com grupos específicos. Nesse sentido, o filo bacteriano Verrucomicrobia, grupo ubíquo em solos, apresenta elevada abundância em diferentes ambientes, o que sugere sua grande importância ecológica. Mas, pela dificuldade de isolamento de seus organismos, ainda pouco se sabe a respeito da ecologia do filo. O presente trabalho foi desenvolvido com o objetivo de analisar os efeitos do uso do solo e da rizosfera de cana-de-açúcar nas comunidades de Bacteria e do filo Verrucomicrobia. Com essa finalidade, amostras de solo foram coletadas em áreas sob diferentes usos em uma usina sucroalcooleira na cidade de Piracicaba (SP). As amostras foram utilizadas em dois estudos distintos. No primeiro, foram analisadas as comunidades microbianas presentes nas amostras de solo obtidas na coleta das áreas de estudo e, no segundo, retiradas de um experimento controlado conduzido em estufa para analisar o efeito do uso do solo e, principalmente, da rizosfera nas mesmas. As comunidades foram avaliadas quanto a sua abundância, pela técnica de qPCR, e estrutura, pela técnica de T-RFLP. No primeiro estudo, a diversidade do filo também foi acessada pelo desenvolvimento de bibliotecas do seu gene 16S rRNA. Os resultados dos estudos indicam que a comunidade bacteriana e, principalmente, do filo foram afetadas pelo uso do solo e pelo manejo adotado em cada área, o que evidencia a importância de sistemas conservacionistas. A análise das bibliotecas demonstrou que o filo Verrucomicrobia apresentou maior diversidade na mata nativa do que nos canaviais. Adicionalmente, a incidência e a abundância das subdivisões do grupo foram alteradas de acordo com o uso do solo. As comunidades também foram influenciadas pela rizosfera em sua estrutura e abundância, resultados de grande interesse para o estudo do filo, pois poucos trabalhos analisaram o efeito rizosférico em seus organismos. Além disso, a sua elevada abundância encontrada no estudo, que tem sido comumente subestimada, ressalta a importância de trabalhos com foco específico em grupos de interesse / The abundance and diversity of soil microbial communities can be influenced by many factors, mainly associated with soil type, its coverage and use. The soil microbiota has great importance due to the processes performed by its organisms, essential for all terrestrial ecosystems. Despite the great complexity associated with these studies, the factors that affect the microbial communities can be better explained through the research of specific groups. In this sense, the bacterial phylum Verrucomicrobia, a ubiquitous soil group, presents high abundance in different environments, which suggests its great ecological importance. But due to the difficulty of isolating its organisms, little is known about the ecology of the phylum. The present work was developed with the objective of analyzing the effect of land use changes and sugarcane rhizosphere on the structure and abundance of Bacteria and Verrucomicrobia communities. For this purpose, soil samples were collected in areas under different land uses in a sugarcane mill in Piracicaba (SP). The samples were used in two separate studies. In the first, the microbial communities present in soil samples obtained in the sampling areas were analyzed and, in the second, taken from a controlled experiment conducted in a greenhouse to analyze the effect of land use and, especially, the rhizosphere on the communities. The communities were evaluated for their abundance, by the qPCR technique, and structure, by T-RFLP. In the first study, the phylum diversity was also accessed with the development of 16S rRNA gene libraries. The results from the studies indicate that bacterial and, especially, the phylum community were affected by land use and the management adopted in each area, which evidences the importance of conservationist systems. The analysis of the clone library demonstrated that the phylum Verrucomicrobia presented greater diversity in native vegetation than in the sugarcane fields. Additionally, the incidence and abundance of the group subdivisions have been changed in accordance with land use. The structure and abundance of the communities were also influenced by the rhizosphere, results of great interest to the reserach of the phylum, since few studies have analyzed the rhizosphere effect on its organisms. Furthermore, the high abundance of the phylum found in the study, which has been commonly underestimated, emphasizes the importance of research with specific focus on groups of interest

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.

A produção de etanol nas destilarias brasileiras é baseada na atividade fermentativa da levedura Saccharomyces cerevisiae que utiliza o caldo da cana-de-açúcar e/ou o melaço como substrato. Bactérias contaminantes da fermentação alcoólica competem com as leveduras pelos açúcares, afetando o rendimento do sistema produtivo e, consequentemente, causando perdas econômicas significativas às usinas. Biofilmes formados nos tanques de fermentação alcoólica agem como reservatórios de bactérias, contribuindo para contaminações persistentes e de difícil controle. Os biofilmes proporcionam aos seus habitantes certo grau de proteção contra diversas ameaças do meio, incluindo a ação dos antibióticos. Desta forma, o conhecimento da comunidade bacteriana dos biofilmes é fundamental para as medidas que visam o controle das contaminações na produção do bioetanol. No primeiro estudo, a composição e dinâmica da comunidade bacteriana foram determinadas pela análise de sequências do gene 16S rRNA de biofilmes com diferentes períodos de crescimento, correspondentes aos estágios iniciais de estabelecimento destes biofilmes dentro dos tanques de fermentação alcóolica Os resultados mostraram que estas comunidades foram compostas predominantemente pelas bactérias ácido-lácticas (LAB), com destaque para o gênero Lactobacillus. A visualização da estrutura dos biofilmes por microscopia eletrônica de varredura evidenciou que estes são formados por bactérias e leveduras (biofilmes mistos). No segundo estudo, a suscetibilidade aos antimicrobianos (monensina, virginiamicina e beta-ácido derivado do lúpulo) e a capacidade de formação de biofilmes em culturas puras foram avaliadas para isolados de Lactobacillus spp. provenientes de biofilmes (células sésseis) e de vinho bruto (células planctônicas) coletados dos tanques de fermentação. A partir dos resultados foi possível observar que as diferenças na suscetibilidade aos antimicrobianos e na habilidade de formar biofilmes foram estirpe-dependentes e que, em alguns casos, o perfil apresentado para algumas espécies mostrou-se relacionado à fonte de isolamento. Este foi o primeiro estudo sobre biofilmes contaminantes da fermentação alcoólica, em escala industrial, para a produção de etanol a partir da cana-de-açúcar / Bioethanol production in Brazilian distilleries is based on fermentative activity of the yeast Saccharomyces cerevisiae which uses sugarcane juice and/or molasses as a substrate. Bacterial contaminants of alcoholic fermentation compete with yeasts for sugars, affecting ethanol yield and consequently causing relevant economic losses to the fuel ethanol industry. Biofilms formed into fermentors act as bacterial reservoirs, contributing to persistent contaminations that are difficult to control. Biofilms provide a certain degree of protection for their inhabitants against some environmental threats, including antibiotics. Thus, understanding bacterial community within biofilms is essential for actions to control contaminations in bioethanol production. In the first study, composition and dynamic of bacterial community were determined by 16S rRNA gene sequences analysis of biofilms with different growth periods, corresponding to initial stages of biofilm establishment in fermentation tanks. Results showed that these communities were dominated by lactic acid bacteria (LAB), mainly of the genus Lactobacillus. Visualization of biofilm structure by scanning electron microscopy revealed a mixed-species biofilm composed by bacteria and yeasts. In the second study, susceptibility to antimicrobials (monensina, virginiamicina and beta-acids from hops) and capacity to form biofilm in pure culture were evaluated for Lactobacillus spp. isolated from biofilms (sessile cells) and wine (planktonic cells) collected from fermentors. The results showed that differences in the susceptibility to antimicrobials and the ability to form biofilms were strain-specific and, in certain cases, the response of some species was related to the isolation source. This was the first investigation of contaminant biofilms from sugarcane-based alcoholic fermentation on an industrial scale

A abundância e diversidade dos microrganismos do solo podem ser influenciadas por grande número de fatores, associados, principalmente, ao tipo de solo, sua cobertura e uso. A microbiota do solo apresenta grande importância pelos processos desempenhados pelos seus organismos, essenciais para todos os ecossistemas terrestres. Apesar da grande complexidade associada a esses estudos, os fatores que influenciam as comunidades microbianas podem ser melhor elucidados pela pesquisa com grupos específicos. Nesse sentido, o filo bacteriano Verrucomicrobia, grupo ubíquo em solos, apresenta elevada abundância em diferentes ambientes, o que sugere sua grande importância ecológica. Mas, pela dificuldade de isolamento de seus organismos, ainda pouco se sabe a respeito da ecologia do filo. O presente trabalho foi desenvolvido com o objetivo de analisar os efeitos do uso do solo e da rizosfera de cana-de-açúcar nas comunidades de Bacteria e do filo Verrucomicrobia. Com essa finalidade, amostras de solo foram coletadas em áreas sob diferentes usos em uma usina sucroalcooleira na cidade de Piracicaba (SP). As amostras foram utilizadas em dois estudos distintos. No primeiro, foram analisadas as comunidades microbianas presentes nas amostras de solo obtidas na coleta das áreas de estudo e, no segundo, retiradas de um experimento controlado conduzido em estufa para analisar o efeito do uso do solo e, principalmente, da rizosfera nas mesmas. As comunidades foram avaliadas quanto a sua abundância, pela técnica de qPCR, e estrutura, pela técnica de T-RFLP. No primeiro estudo, a diversidade do filo também foi acessada pelo desenvolvimento de bibliotecas do seu gene 16S rRNA. Os resultados dos estudos indicam que a comunidade bacteriana e, principalmente, do filo foram afetadas pelo uso do solo e pelo manejo adotado em cada área, o que evidencia a importância de sistemas conservacionistas. A análise das bibliotecas demonstrou que o filo Verrucomicrobia apresentou maior diversidade na mata nativa do que nos canaviais. Adicionalmente, a incidência e a abundância das subdivisões do grupo foram alteradas de acordo com o uso do solo. As comunidades também foram influenciadas pela rizosfera em sua estrutura e abundância, resultados de grande interesse para o estudo do filo, pois poucos trabalhos analisaram o efeito rizosférico em seus organismos. Além disso, a sua elevada abundância encontrada no estudo, que tem sido comumente subestimada, ressalta a importância de trabalhos com foco específico em grupos de interesse / The abundance and diversity of soil microbial communities can be influenced by many factors, mainly associated with soil type, its coverage and use. The soil microbiota has great importance due to the processes performed by its organisms, essential for all terrestrial ecosystems. Despite the great complexity associated with these studies, the factors that affect the microbial communities can be better explained through the research of specific groups. In this sense, the bacterial phylum Verrucomicrobia, a ubiquitous soil group, presents high abundance in different environments, which suggests its great ecological importance. But due to the difficulty of isolating its organisms, little is known about the ecology of the phylum. The present work was developed with the objective of analyzing the effect of land use changes and sugarcane rhizosphere on the structure and abundance of Bacteria and Verrucomicrobia communities. For this purpose, soil samples were collected in areas under different land uses in a sugarcane mill in Piracicaba (SP). The samples were used in two separate studies. In the first, the microbial communities present in soil samples obtained in the sampling areas were analyzed and, in the second, taken from a controlled experiment conducted in a greenhouse to analyze the effect of land use and, especially, the rhizosphere on the communities. The communities were evaluated for their abundance, by the qPCR technique, and structure, by T-RFLP. In the first study, the phylum diversity was also accessed with the development of 16S rRNA gene libraries. The results from the studies indicate that bacterial and, especially, the phylum community were affected by land use and the management adopted in each area, which evidences the importance of conservationist systems. The analysis of the clone library demonstrated that the phylum Verrucomicrobia presented greater diversity in native vegetation than in the sugarcane fields. Additionally, the incidence and abundance of the group subdivisions have been changed in accordance with land use. The structure and abundance of the communities were also influenced by the rhizosphere, results of great interest to the reserach of the phylum, since few studies have analyzed the rhizosphere effect on its organisms. Furthermore, the high abundance of the phylum found in the study, which has been commonly underestimated, emphasizes the importance of research with specific focus on groups of interest

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. (...)