Terra Preta Antropogênica (TPA) é um dos tipos de solos mais \"férteis\" do mundo. A TPA recebe esta denominação por ser encontrada em sítios arqueológicos onde viveram grupos pré-históricos. São pequenas faixas de solos que apresentam altas concentrações de nutrientes, matéria orgânica e encontram-se distribuídas aleatoriamente pela região Amazônica. A verdadeira origem destes solos ainda não está bem esclarecida. Devido à falta de informações sobre sua diversidade bacteriana, este trabalho estudou a diversidade bacteriana em amostras de solos TPA coletadas em duas regiões: Lagoa Balbina - sítio Terra preta (Amazônia Central- Amazonas) e Floresta Nacional de Caxiuanã - sítio arqueológico Mina I (Amazônia Oriental - Pará), através de técnicas moleculares independentes de cultivo. O DNA genômico total das amostras de solo foi extraído e usado como molde em uma reação de PCR utilizando oligonucleotídeos específicos do gene 16S rRNA para o Domínio Bactéria. O produto de PCR amplificado foi clonado no vetor pGEM-T e 980 clones foram selecionados e comparados com o banco de dados de 16S rRNA do RDP II e GenBank (NCBI-EUA). Os resultados apresentaram predominância com microrganismos não-conhecidos representando 41,6 % das seqüências de solo TPA- Balbina, 68,3 % das seqüências de ADJ-Balbina, 84,8% das seqüências de solo TPAMina e 47,7 % das seqüências de ADJ-Mina. O filo mais predominante nas amostras TPABalbina foi Firmicutes, representando 37,1% do total de seqüências analisadas. Os filos em destaque foram Proteobacteria (9,6%), seguidos de Verrucomicrobia (5,6%), Acidobacteria (2,5%), Gemmatimonadetes (2,5%), Actinobacteria (0,5%) e Nitrospira (0,5%). Por outro lado, em ADJ-Balbina destacaram-se os filos Proteobacteria 15,1%, Acidobacteria (12,5%), Firmicutes (2,3%), Nitrospira (1,1%) e Verrucomicrobia (0,8%). Em TPA-Mina, os filos apresentados foram Proteobacteria (6,5%), Acidobacteria (4,7%), Firmicutes (1,4%), Nitrospira (1,1%), Planctomycetes (1,1%) e Verrucomicrobia (0,4%). Contudo, na biblioteca ADJ-Mina verificou a presença dos filos Acidobacteria (27,2%), Proteobacteria (14,2%), Firmicutes (3,8%), Verrucomicrobia (3,8%), Nitrospira (1,3%), Planctomycetes (1,3%), Actinobacteria (0,4%) e Gemmatimonadetes (0,4%). O pH do solo pode ser um dos atributos do solo que pode ter influência direta na diversidade bacteriana dos solos estudados, assim como pode ter efeito uma floresta natural sobre as populações microbianas em seu solo, fato observado na adjacência do solo Terra Preta em Caxiuanã - PA. A estimativa da riqueza de UTOs pelo Bootstrap corroborou diretamente os valores de diversidade obtidos pelos índices de Simpson e Shannon. De um modo geral, uma maior probabilidade de ocorrência de UTOs únicas empregadas pelo estimador Jackknife se correlacionou com uma maior percentagem de baixas frequências de filotipos nas quatro bibliotecas. Os métodos não-paramétricos ACE e Chao1 para a estimativa da riqueza de UTOs também corroboraram com os valores obtidos com o estimador Jackknife. / Anthropogenic Dark Earth (ADE) is one of the most fertile soils in the world. ADE soils have received this nomination due to the pre-historical origin of these archaeological sites, established by pre-colombian populations. ADEs are small areas of soil which present high nutrient and organic matter contents and are randomly distributed throughout the Amazonian region. The true origin of these soils is not known yet. Due to the lack of information concerning the bacterial diversity, this work studied the bacterial diversity in ADE soils collected from two regions: Lagoa Balbina - site Terra Preta (Central Amazonia- Amazonas state) and National Forest of Caxiuanã - archaeological site Mina I (Oriental Amazonia - Pará state), using culture-independent molecular techniques. The total genomic DNAs extracted from the soil samples were used as templates in the PCR reactions using the universal primers for the 16S rRNA bacterial gene. The PCR-products were cloned into the pGEM-T vector and 980 clones were selected and searched using the GenBank (NCBI-USA) and the RDP II program. Data analyses indicated predominance of unknown microorganisms, representing 41.6 % among the sequences from ADE-Balbina, 68.3 % from Adjacent-Balbina, 84.8% from ADE-Mina and 47.7 % from Adjacent-Mina. The predominant phylum in ADEBalbina was Firmicutes, representing 37.1% of the total sequences from that site, followed by Proteobacteria (9.6%), Verrucomicrobia (5.6%) Acidobacteria (2,5%), Gemmatimonadetes (2,5%), Actinobacteria (0,5%) and Nitrospira (0.5%). On the other hand, in the adjacent soil ADJ-Balbina, the predominant phylla were Proteobacteria (15.1%), Acidobacteria (12.5%), Firmicutes (2.3%), Nitrospira (1.1%) and Verrucomicrobia (0.8%). In the Oriental Amazon, the prevalent phylla from the ADE-Mina soil were Proteobacteria (6.5%), Acidobacteria (4.7%), Firmicutes (1.4%), Nitrospira (1.1%), Planctomycetes (1.1%) and Verrucomicrobia (0.4%). In the ADJ-Mina verificou a presença dos filos Acidobacteria 27.2%, Proteobacteria 14.2%, Firmicutes 3.8%, Verrucomicrobia 3.8%, Nitrospira 1.3%, Planctomycetes 1.3%, Actinobacteria 0.4% e Gemmatimonadetes 0.4%. The soil pH may be of the soil attributes which may have directly influenced the bacterial diversity in those soils, as well as the above-ground vegetation from the natural forest in Caxiuanã-Pará. The estimates of the Operational Taxonomic Units (OTUs) richness using Bootstrap directly corroborated the diversity values obtained from the Simpson and Shannon indexes. Unique UTOs using Jackknife estimator were correlated with a higher percentage of the low frequencies of phylla in all the four clone libraries. The nonparametric ACE and Chao1 methods to estimate the OTUs richness also corroborated the Jackknife values.

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No. of bitstreams: 1 Tese - Marelis Margarita Ruiz.pdf: 48193021 bytes, checksum: 4e7e0fa3715216dbc1c8be5df2d8a197 (MD5) Previous issue date: 2014-02-02 / Não Informada / In the Amazon region, the Urucu Oil Province (Petrobras) has been disposing of hydrocarbon effluents into the surrounding aquatic environments since the beginning of its operations. The goal of this work was to analyze the bacterial biodiversity present in the dike of the effluents and in the stream before and after the disposal of hydrocarbon effluents. A study was conducted in two stages were carried out with the use of culture independent molecular techniques. The full genomic DNA of the sediment and water samples were extracted and used as templates in a PCR reaction with the use of specific oligonucleotides of the 16S rRNA gene bacteria domain. The PCR product was amplified and pyrosequenced, and the generated sequences were analyzed by the free software Mothur. The measurements of the physical-chemical and chromatographical parameters are within the measures established for these types of analyzed water bodies. In the first stage of the study, the taxonomic profiles of the samples have shown that the Proteobacteria phylum proved to be the most abundant together with the Deltaproteobacteria class and the gen known as Candidatus Solibacter. Similarly, in the second stage of the study, the Proteobacteria phylum proved to be the most abundant together with the Alphaproteobacteria class and the Geobacter gen predominant. Both genes are catalogued as bioremediators in contaminated environments, as well as 14% of the total genes. A large proportion of microorganisms were considered “Unclassified” (up to 30%). In first stage of the study the richness and the diversity of species in the Onça stream is greater following the disposal of hydrocarbon effluents, in second stage of the study the abundance and diversity of species in the natural stream (without a name in the region), is greater as compared to the same stream after the mixing of effluents. There is no significant statistical difference between the sample of the community in the natural stream and this effluent-mixed stream; and there is no difference in genetic structure among samples of each community analyzed, suggesting that the discharge of effluents in this body of water has no significant impact on the microbial biodiversity in this aquatic environment. This work, a pioneer in the taxonomic analysis of samples in dikes of effluents and streams with the disposal of effluents in the Urucu oil area, represents a challenge for understanding the relationship between the composition, abundance and diversity of microorganisms in this environment, and a rich source for the discovery of new taxonomic groups, as well as the huge potential for biotechnological exploration of such diversity. / Na região Amazônica, a Província Petrolífera de Urucu (Petrobras) realiza o despejo de efluentes de hidrocarbonetos em ambientes aquáticos ao redor, desde o início de suas operações. O objetivo deste trabalho foi analisar a biodiversidade bacteriana presente no dique de efluentes e no igarapé antes e depois do despejo de efluentes. Foi realizado um estudo em duas etapas, aplicando técnicas moleculares independentes de cultivo. O DNA genômico total das amostras de sedimento e água foi extraído e usado como molde em uma reação de PCR utilizando oligonucleotídeos específicos do gene 16S rRNA para o domínio Bactéria. O produto de PCR foi amplificado e pirosequenciado, e as sequências geradas foram analisadas pelo programa livre Mothur. As medidas dos parâmetros físico-químicos e cromatográficos estão dentro dos valores estabelecidos para os tipos de corpos de água analisados. Na primeira etapa do estudo, os perfis taxonômicos das amostras mostraram que o filo Proteobacteria foi o mais abundante com a classe Deltaproteobacteria e gênero conhecido o Candidatus Solibacter. Assim mesmo, na segunda etapa do estudo, o filo Proteobacteria foi o mais abundante; porém, com a classe Alphaproteobacteria e o gênero Geobacter como predominante. Ambos gêneros são catalogados como biorremediadores de ambientes contaminados, assim como 14% dos gêneros totais. Uma grande parte de microrganismos foram considerados “Não Classificados” (até 30%). Na primeira etapa do estudo a riqueza e diversidade de espécies no Igarapé da Onça, é maior depois do despejo de efluentes de hidrocarboneto, e na segunda etapa do estudo a riqueza e diversidade de espécies no igarapé natural (sem nome na região), é maior com relação a este mesmo igarapé depois da mistura com efluentes. Não existe diferença estatística significativa entre a comunidade do igarapé natural e entre este igarapé misturado com efluentes; e não existe diferença na estrutura genética entre as amostras de cada comunidade analisada, indicando que a descarga de efluentes neste corpo de água não tem impacto significativo na biodiversidade microbiana deste ambiente aquático. Este trabalho, pioneiro em análise taxonômica de amostras de um dique de efluentes e os igarapés com despejo de efluentes na área petrolífera de Urucu, representa uma oportunidade para a compreensão da relação entre a composição, abundância e diversidade dos microrganismos neste ambiente, e uma fonte rica para descoberta de novos grupos taxonômicos, assim como um enorme potencial para exploração biotecnológica desta diversidade.

Abstract The aim of this work was to assess and improve the enrichment cultivation of food-contaminating bacteria prior to detection by means of RNA-based sandwich hybridization assay (SHA). The examples of beer-spoiling lactic acid bacteria (LAB) and food-borne Salmonella Typhimurium were selected based on their relevance in Finnish food industry. Also universal challenges affecting on the selection of the enrichment cultivation procedure are discussed, including some potential possibilities for improved enrichment cultivation. The results of this study may therefore be used for the assessment of the efficiency of bacterial cultivation in other applications. The evaluation of the enrichment cultivation procedures prior to SHA lead to following conclusions: i) the enrichment cultivation procedure is necessary prior to rRNA-based SHA, and it directly influences the accuracy of SHA; ii) the improvement of the enrichment cultivation may allow faster recovery and growth of bacteria; iii) the improved recovery of bacteria can be achieved by reducing environmental stress factors in the enrichment culture; and iv) the growth of bacteria may be accelerated by assuring the selectivity of medium and allowing accessibility to growth factors. Several growth factors were studied by means of full factorial design and response surface modeling. Measured cell densities, as well as predicted lag-times and maximum growth rates in the bacterial cultures were used as responses. The results show that small shifts in the cultivation conditions extend the lag-time and decrease the growth rate of both LAB and Salmonella. Besides adjusting the temperature and pH, the growth of LAB was facilitated by reducing osmotic and oxidative stresses in the enrichment medium. In this study, a novel enzyme controlled glucose delivery system was used for the first time in the enrichment cultivation of food-contaminating bacteria. The glucose delivery system improved the growth of LAB in single strain cultures and in actual brewing process samples. The recovery of injured Salmonella was also enhanced by using the glucose delivery system together with selective siderophore ferrioxamine E, both in terms of reduced lag-times and increased growth rates. Based on the SHA, the adjusted BPW broth enhanced the molecular detection of heat-injured Salmonella in meat.

Ľudské telo je prostredím pre život neuveriteľného množstva mikróbov. Niektoré z nich môžu spôsobovať rôzne choroby, ale ďalšie, napríklad črevný mikrobióm, sú pre život a zdravie človeka nepostrádateľné. Nanešťastie, črevný mikrobióm nie je detailne preštudovaný, pretože obsahuje tisíce rôznych druhov baktérií, z ktorých väčšina sa nedá kultivovať v laboratórnych podmienkach. Riešením tohto problému sú nové rýchle metódy sekvenovania v kombináciou s bioinformatickými nástrojmi na výpočet funkčného profilu baktérií vo vzorke. V tejto práci si predstavíme existujúce nástroje predpovedajúce funkčný profil, a následne navrhneme nový nástroj, ktorý môže implementovať konsenzus nad výsledkami existujúcich nástrojov, alebo sa môže jednať o úplne nový nástroj.

The aim of this work is to critically review the anatomical and morphological characters being currently used in the determination of Central European species of the genus Anisus and to confront them with molecular characters. For the molecular analysis mitochondrial genes for 16S rRNA and cytochrome c oxidase - subunit I (COI) were used. DNA analysis showed that known species occuring in the Czech Republic form well distinguishable genetic lines. Subsequent revisions of the anatomical characters of these lines have proven that these characters are due to their variability not suitable for determination, especially for the differentiating of the species A. spirorbis and A. leucostoma. The conchological characters have proven more suitable, especially the ratio of the size of the last and penultimate whorl. With the help of this character, we can safely determine a population within which there are transitional forms in other morphological and anatomical characters.

The main objective of this study was the characterization of surface associated microbial communities in breweries. In addition, the beer-spoiling potential of isolated strains and biofilm samples was investigated. Some studies reported the identity of cultivatable organisms from industrial plants. However, there were no data available about the composition of biofilm communities from these habitats for cultivation-independent techniques. Consequently, the fatty acid methyl esters (FAMEs) analysis, the fluorescence in situ hybridization (FISH) and the construction and investigation of 16S rRNA gene clone libraries were applied to reveal the structure of these communities. All of these methods have different advantages and therefore, they complement each other to get a more reliable picture of the biofilm communities. The cultivation method was included in this study because it enables a verification of results from other studies. Furthermore, the obtained strains are genuine brewery isolates and can be used for physiological tests. Isolates were obtained from seven different sample sites (Chapter 1 and 5). They were identified and affiliated to 25 different genera. Some of these strains were inoculated in beer but none of them was able to grow in it (Chapter 1 and 5). However, these strains can still be harmful for the industry, e.g. if they are able to form biofilms. This aspect was investigated by analyzing the potential of the isolates to produce acyl-homoserine lactones (AHLs) (Chapter 6). These quorum sensing mediating molecules are involved in the maturation process of biofilms. Indeed, some strains were found to secrete these autoinducer molecules, they mainly belonged to the genus Pseudomonas. An abundant proportion among the isolates was constituted by members of the Enterobacteriaceae (Chapter 7). In the beginning of this study, there was a minor suspicion concerning their beer-spoiling potential. Indeed, all isolated Enterobacteriaceae were found to be able to multiply in non-alcoholic beer under access of oxygen but they represented no risk for filled beer. The beer-spoiling potential of biofilm communities was investigated by inoculating them in beer (Chapter 3). These enrichments allowed the detection of minor proportions of beer-spoiling organisms. About 25% of the biofilms contained microorganisms which were able to multiply in beer with 4.8% of ethanol (v/v). The absence of anaerobic beer-spoiling bacteria in most of the biofilms was confirmed by using specific FISH probes for Pectinatus and Megasphaera cells (Chapter 9). However, Pectinatus cells constituted one of the most abundant groups in two biofilm communities. These samples clearly demonstrated that brewery biofilms can become hazardous for the quality of the product. The acetic acid bacteria were supposed to be abundant brewery biofilm organisms. This was not confirmed by any method used (Chapter 8). Instead, FISH signals were found for many other taxa in considerable proportions, e.g. communities from the conveyors consisted of members of the Eukarya, Archaea, Alpha-, Beta-, Gammaproteobacteria, Cytophaga-Flavobacteria, Planctomycetales, Actinobacteria and Firmicutes (Chapter 1). Such diverse communities were also evidenced for three other biofilms analyzed by FISH (Chapter 2 and 9). Whereas the FISH technique allows the specific detection of single cells, the FAME analysis targets all organisms present, except the Archaea. The fatty acid profiles of 78 biofilms indicated significant differences between the communities, even between those which were exposed to similar conditions. In addition, repeated sampling of identical sites revealed a temporal variability of the microbial communities (Chapter 3). Characteristical fatty acids of beer-spoiling bacteria were almost absent. Typical fatty acids of Eukarya dominated nearly half of all biofilms. The high proportions of Eukarya in some biofilms was not confirmed, as these samples were also investigated by FISH. This divergence was found to be due to the higher biomass of eukaryotic cells compared to bacterial cells (Chapter 3). As some wild yeast strains were isolated and characterized, they are a potential source of these fatty acids. In contrast to the revealed bacterial diversity, most of the isolated yeasts were assigned to Saccharomyces or Candida spp. (Chapter 4). The Saccharomyces spp. showed a high beer-spoiling potential and many Candida species were able to form biofilms. The construction of 16S rRNA gene clone libraries and the analysis of the clones with amplified ribosomal DNA restriction analysis (ARDRA) was performed with two biofilm communities (Chapter 2). Clones with identical ARDRA patterns were grouped and some representatives were identified by sequencing. These clone sequences were affiliated to 30 different genera, most of which were members of the Alpha- and Gammaproteobacteria and the Bacteroidetes. In addition, some clone sequences were assigned to uncultured organisms. Despite of the presence of 53 and 59 different ARDRA patterns in the two clone libraries, respectively, they had only four patterns in common. This result underlined the differences in the microbial composition of these communities. In conclusion, breweries represent a habitat with high cleaning and disinfecting pressure, which might have selected for a limited number of more resistant or adopted species. Instead, the community structures of biofilms in industrial environments were found to be diverse and variable in their compositions.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20953号 / 理博第4405号 / 新制||理||1633(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 中野 伸一, 教授 木庭 啓介, 教授 中川 尚史 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

The use of the herbicide glyphosate and its formulations on protein-rich feedstuff for cattle leads to a considerable intake of glyphosate into the rumen of the animals, where glyphosate may potentially impair the 5-enolpyruvylshikimate-3-phosphate pathway of the commensal microbiota, which could cause dysbiosis or proliferation of pathogenic microorganisms. Here, we evaluated the effects of pure glyphosate and the formulations Durano TF and Roundup® LB plus in different concentrations on the fermentation pattern, community composition and metabolic activity of the rumen microbiota using the Rumen Simulation Technique (RUSITEC). Application of the compounds in three concentrations (0.1mg/l, 1.0mg/l or 10mg/l, n=4 each) for 9days did not affect fermentation parameters such as pH, redox potential, NH3-N concentration and production of short-chain fatty acids compared to a control group. Microbial protein synthesis and the degradation of different feed fractions did not vary among the treatments. None of the used compounds or concentrations did affect the microbial diversity or abundance of microbial taxa. Metaproteomics revealed that the present metabolic pathways including the shikimate pathway were not affected by addition of glyphosate, Durano TF or Roundup® LB plus. In conclusion, neither pure glyphosate, nor its formulations Durano TF and Roundup® LB plus did affect the bacterial communities of the rumen.

No description available.

No description available.