Tesis: "Soil metagenomic"

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Goode, Ann Marie Liles Mark Russell. "Polyketide synthase pathway discovery from soil metagenomic libraries". Auburn, Ala., 2009. http://hdl.handle.net/10415/1805.

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Shezi, Ntombifuthi. "Bio-prospecting a Soil Metagenomic Library for Carbohydrate Active Esterases". Thesis, Rhodes University, 2016. http://hdl.handle.net/10962/d1021266.

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Lignocellulosic biomass is a promising renewable resource on earth. Plant biomass contains fermentable sugars and other moieties that can be converted to biofuels or other chemicals. Enzymatic hydrolysis of these biopolymers is significant in the liberation of sugars for fermentation into desired products. Owing to its complex structure, synergistic action of enzymes is required for its degradation. Enzymes that are involved in biomass degradation include cellulases, hemicellulases and the accessory enzymes acetyl xylan esterases and ferulic acid esterases. Ferulic acid esterases (FAEs, EC 3.1.1.73), represent a subclass of carboxylester hydrolases (EC 3.1.1.-) that catalyse the release of hydroxycinnamic acids (such as ferulic acid, p-coumaric, ferulic, sinapic and caffeic acid) that are generally found esterified to polysaccharides, such as arabinoxylans. Hydroxycinnamic acids have widespread potential applications due to their antimicrobial, photoprotectant and antioxidant properties, as well as their use as flavour precursors. Therefore, this interesting group of FAEs has a potentially wide variety of applications in agriculture, food and pharmaceutical industries. In the search for novel biocatalysts, metagenomics is considered as an alternative approach to conventional microbe screening, therefore, searching for novel biocatalysts from a soil metagenome that harbours a unique diversity of biocatalyst is significant. The aim of this study was to extract DNA from soil associated with cattle manure and construct a soil metagenomic library using a fosmid based plasmid vector and subsequently functionally screen for ferulic acid esterases using ethyl ferulate as a model substrate. A total of 59 recombinant fosmids conferring ferulic acid esterase phenotypes were identified (Hit rate 1:3122) and the two fosmids that consistently showed high FAE activities were selected for further study. Following nucleotide sequencing and translational analysis, two fae encoding open reading frames (FAE9 and FAE27) of approximately 274 and 322 aa, respectively, were identified. The amino acid sequence of the two ORFs contained a classical conserved esterase/lipase G-x-S-x-G sequence motif. The two genes (fae9 and fae27) were successfully expressed in Escherichia coli BL21 (DE3) and the purified enzymes exhibited respective temperature optima of 50 °C and 40 °C, and respective pH optima of 6.0 and 7.0. Further biochemical characterisation showed that FAE9 and FAE27 have high substrate specificity, following the fact that EFA is the preferred substrate for FAE9 (kcat/Km value of 128 s−1.mM-1) and also the preferred substrate for FAE27 (kcat/Km value of 137 s−1.mM-1). This work proves that soil is a valuable environmental source for novel esterase screening through functional based metagenomic approach. Therefore, this method may be used to screen for other valuable enzymes from environmental sources using inexpensive natural sources to encourage the screening of specific enzymes. Biochemistry of the two isolated enzymes makes these enzymes to be useful in industrial applications due to broad substrate activity that could replace the specialised enzymes to complete plant biomass degradation.

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3

Spiegelman, Dan. "Exploring the fusion of metagenomic library and DNA microarray technologies". Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=98805.

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We explored the combination of metagenomic library and DNA microarray technologies into a single platform as a novel way to rapidly screen metagenomic libraries for genetic targets. In the "metagenomic microarray" system, metagenomic library clone DNA is printed on a microarray surface, and clones of interest are detected by hybridization to single-gene probes. This study represents the initial steps in the development of this technology. We constructed two 5,000-clone large-insert metagenomic libraries from two diesel-contaminated Arctic soil samples. We developed and optimized an automated fosmid purification protocol to rapidly-extract clone DNA in a high-throughput 96-well format. We then created a series of small prototype arrays to optimize various parameters of microarray printing and hybridization, to identify and resolve technical challenges, and to provide proof-of-principle of this novel application. Our results suggest that this method shows promise, but more experimentation must be done to establish the feasibility of this approach.

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Borsetto, Chiara. "Study and exploitation of diverse soil environments for novel natural product discovery using metagenomic approaches". Thesis, University of Warwick, 2017. http://wrap.warwick.ac.uk/97341/.

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Natural products with antimicrobial activity have played an important role in the treatment of infection since their discovery. The increasing emergence of pathogens resistant to multiple antibiotics has raised awareness of the urgent need for novel antibiotics. Soil microorganisms are the major source of antibiotics and Actinobacteria in particular have an impressive capacity for production of diverse bioactive secondary metabolites. However, culture-independent studies have shown a greater microbial diversity present in soil with potential for novel chemical structures and these can be explored further using metagenomic approaches capturing genes without the need to cultivate the host. Different metagenomic tools were used to study and explore microbial secondary metabolite diversity in soil. In particular, amplicon sequencing of 16S rRNA gene, NRPS and PKS biosynthetic genes allowed the identification of novel potential phylogenetic drivers of secondary metabolite diversity in the less characterized phyla Verrucomicrobia and Bacteroidetes and potential geographic hotspots harbouring unique biosynthetic diversity such as Antarctica and Cuba. The exploitation of these hotspots presented some bottlenecks in the form of DNA extraction efficiency, library creation, screening and heterologous expression. These were overcome by comparative analysis of different eDNA extraction methods to optimise fragment size and purity combined with development of new cloning tools for both DNA capture and expression. Modification of the microbial community through the amendment of the soil with chitin, highlighted the beneficial effect of microbial enrichment allowing a higher recovery of eDNA and higher detection of the biosynthetic gene of interest related to secondary metabolite production. Further additions were made to the metagenomic molecular toolbox in the form of BAC vectors (pBCaBAC and pBCkBAC) which were tested with suitable heterologous host systems (Streptomyces sp. and the engineered Pseudomonas putida species) potentially facilitating heterologous expression. In conclusion this is the first study to identify the drivers of microbial secondary metabolite diversity in situ and provided a comparative analysis of a range of diverse soil types. This approach paired with new developments in metagenomic technologies will make a substantial contribution to improving the likelihood for discovery and exploitation of new drugs for treating multi-resistant pathogenic bacteria.

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Andrews, Tucker. "Ecology Of Composted Bedded Pack And Its Impact On The Udder Microbiome With An Emphasis On Mastitis Epidemiology". ScholarWorks @ UVM, 2019. https://scholarworks.uvm.edu/graddis/989.

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Infections of the cow udder leading to mastitis and lower milk quality are a critical challenge facing northeast organic dairy farmers. Limited mastitis treatment options are available to organic producers and bedding systems impact cow health, including mastitis risk. Composted bedded pack, a system touted for increased cow comfort and well-being, allows stratified accumulation of bedding and manure in the barn. This method is gaining popularity among organic producers, yet little is known about the microbiota of the accumulated pack and its interaction with the cow mammary gland. An in-depth single farm study was conducted that surveyed bedded pack (microbiome and microarthropod community), dipteran vectors of bacterial mastitis pathogens, and the teat skin and teat cistern milk microbiomes. Comparisons were made with four additional farms utilizing bedded packs to test generality of results. Few fly pests were observed in the bedded pack. However, bedding on all farms was found to harbor the mesostigmatid mite genus Glyptholaspis, a well-established predator of nematodes and muscid fly larvae, suggesting that predators may suppress populations of biting flies in bedded pack barns. Additionally, the fungivorous genus Rhizoglyphus was commonly abundant in all farms, suggesting that the mite community regulates microbial activity at multiple trophic levels. High-throughput sequencing of universal marker genes for bacterial and fungal communities was used to characterize the skin and milk microbiome of cows with both a healthy and infected quarter on the case study farm, and the composted bedded pack of all five farms. The bedded pack microbiome varied with bedding material and management style; fungal taxa were primarily yeasts of the Ascomycota; all farms additionally contained anaerobic fungi associated with the bovine rumen. Common bacterial genera included Acinetobacter and Pseudomonas, both of which were also commonly observed on teat skin and in milk. The udder microbiome varied through time and between skin and milk. Both healthy and infected milk microbiomes reflected a diverse group of microbial DNA sequences. Health status of the quarter changed whether taxa were shared between the teat skin, milk, and bedding. Proportion of taxa shared between healthy milk and skin was stable while taxa shared in infected quarters varied widely. Taxa shared among all habitats included yeast genus Debaryomyces and bacteria Acinetobacter guillouiaea. Results support an ecological interpretation of both the udder and the bedded pack environment and support the notion that mastitis can be described as an imbalance of the healthy mammary gland microbiome. Future work might compare udder health between common bedding practices, investigating the impact of bedding on the microbiota of the mammary gland in the healthy and diseased state.

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Nesme, Joseph. "Characterization of antibiotic resistance genes abundance and diversity in soil bacteria by metagenomic approaches : what is the dissemination potential of the soil resistome?" Phd thesis, Ecole Centrale de Lyon, 2014. http://tel.archives-ouvertes.fr/tel-01068359.

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Environmental bacteria and especially soil bacteria are active producers of antibiotic molecules and most drugs used nowadays are isolated from saprophytic soil bacteria and these microorganisms have also evolved numerous resistance pathways leading to an arsenal of Antibiotic Resistance Genes Determinants (ARGD) known as the environmental resistome. A survey of ARGD prevalence is required in order to characterize this natural phenomenon with critical implications in our current infectious diseases management. In order to perform such analysis we compiled a set of 71 metagenomic datasets from various environmental origins: soils, oceans, lakes, human feces, indoor air, etc., and compared their sequences with a database of known antibiotic resistance gene determinants (ARGD). ARGD-annotated reads are found in every environment analyzed confirming their ubiquity. Soil is found to be the richest and shares a large part of ARGD with the human gut microbiome, indicating ARGD transfers between these environments. Experiments using qPCR and metagenomic DNA sequencing on soil samples from two sites with known and distinct antibiotic pollution history were conducted to understand how ARGD abundance and diversity in soil are affected when impacted by antibiotic molecules. The first site is a reference soil from a long-term experiment without history of antibiotic pollution (Rothamsted Park Grass, UK). Soil microcosms are setup with addition of either antibiotic-containg animal manure or pure molecules and incubated for 6 months to monitor changes in ARGD concentration following these perturbations. Our second study-site is a very remote settlement in French Guiana where antibiotics are available since recently and may have impacted the local soil microbial community. Soil samples are taken following a line-transect going from the village (antibiotic source) to 3km deep in the forest in a gradient of human-impact. Our results all confirm prevalence of ARGD in soil at significant abundance but also that ARGD distribution is more correlated to environmental factors such as soil type, microbial taxonomy composition or microcosms incubation conditions than antibiotic molecules exposure in both sites. Pathogens ARGD diversity is far lower than ARGD diversity found in the environment and not all the soil resistome is readily accessible for transfer. In order to characterize the soil mobile gene pool, a strategy is proposed to isolate specifically mobile DNA directly from the environment for sequencing purposes. Better knowledge on the microbial ecology factors limiting ARGD transfers to pathogens may greatly help us reduce the current threat on our limited medical antibiotic molecules resource.

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Whissell, Gavin. "Merging metagenomic and microarray technologies to explore bacterial catabolic potential of Arctic soils". Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=98518.

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A novel approach for screening metagenomic libraries by merging both metagenomic and microarray platforms was developed and optimized. This high-throughput screening strategy termed "metagenomic microarrays" involved the construction of two Arctic soil large-insert libraries and the high density arraying of the clone plasmid DNA (~50 kb) onto glass slides. A standard alkaline lysis technique used for the purification of plasmid DNA was adapted and optimized to function efficiently in a 96-well format, providing an economically viable means of producing sufficient high-quality plasmid DNA for direct printing onto microarrays. The amounts of printed material and probe, required for maximal clone detection, were optimized. To examine catabolic clone detection libraries were first screened by PCR for catabolic genes of interest. Two PCR-positive clones were printed onto microarrays, and detection of these specific clones in the printed libraries was achieved using labeled probes produced from PCR fragments of known sequence. Also, hybridizations were performed using labeled PCR fragments derived from the amplification of a catabolic gene from the total community DNA. The ability of selected probes to specifically target clones of interest was demonstrated. This merger of metagenomics and microarray technologies has shown great promise as a tool for screening the natural microbial community for catabolic potential and could also be used to profile microbial diversity in different environments.

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NOVELLO, GIORGIA. "Exploring the microbiota of Vitis vinifera cv. Pinot Noir in two vineyards with different soil management: metagenomic and metaproteomic analysis". Doctoral thesis, Università del Piemonte Orientale, 2017. http://hdl.handle.net/11579/86922.

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Ortiz-Ortiz, Marianyoly. "Kartchner Caverns: Habitat Scale Community Diversity and Function in a Carbonate Cave". Diss., The University of Arizona, 2012. http://hdl.handle.net/10150/265356.

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This dissertation examines the microbial and functional diversity in Kartchner Caverns, a limestone cave in Arizona, USA. Kartchner is highly oligotrophic due to the lack of photosynthesis and the limited inputs of organic material from the surface. This characteristic poses a challenge for microbial life in the cave. The first objective of this work was to evaluate the bacterial richness, diversity and taxonomic composition of speleothems surfaces within Kartchner Caverns in order to gain insight into the distribution patterns associated with these communities. Secondly, the metabolic strategies used by cave communities to survive harsh cave conditions were investigated based on phylogenetic associations and metagenomics. Both objectives were directed toward answering the questions "who are there?" and "what are they doing?". The 454-pyrotag analysis of the V6 region of the 16S rRNA gene revealed an unexpectedly high bacterial diversity with each speleothem supporting a unique bacterial community profile. A focused study on one room of the cave revealed three community types: Type 1 was dominated by the phylum Proteobacteria; Type 2 by Actinobacteria; and Type 3 by Acidobacteria. Phylogenetic associations of the sequences generated by the 454 sequencing and by a Sanger clone library suggested cave microbial communities are supported by chemoautotrophic activities such as nitrite and iron oxidation. Results from the phylogenetic associations guided the metagenomic analysis which supports the presence of chemoautotrophic activities in the cave. Genes for two complete CO2 fixation mechanisms, the Calvin-Benson-Bashan and the rTCA cycles were identified in the cave metagenome, as well as genes for ammonia and nitrite oxidation. These genes are associated with both Bacteria and Archaea suggesting members of both domains are acting as primary producers in the cave ecosystem. Comparative analysis of cave samples to other environments suggests an overabundance of DNA repair mechanisms which could be potentially used by cave communities to overcome the toxicity due to high concentrations of calcium on the speleothem surfaces. This work provides the first comprehensive analysis of the microbial diversity and potential strategies used by microbial communities to survive under the extreme conditions found in a semi-arid limestone cave environment.

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Grisi, Teresa Cristina Soares de Lima. "Diversidade de Bacteria e Archaea do solo do Cariri paraibano e prospecção de celulases e xilanases em clones metagenômicos e isolados bacterianos". Universidade Federal da Paraíba, 2011. http://tede.biblioteca.ufpb.br:8080/handle/tede/342.

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Soil samples of native pasture (site A) and of soil cultivated with grass Paspalum conjugatum, Bergius (site B) collected from Caatinga vegetation in the semi-arid region in Paraíba state (07°23‟27 S 36°31‟58 W) were utilized for constructing four metagenomic libraries, aiming the evaluation of microbial diversity through amplification of gene 16S rRNA of domains Bacteria and Archaea. The metagenomic DNAs were extracted by utilizing FastDNA® SPIN Kit for Soil (BIO 101), which were amplified by PCR, by using universal primers 27F / 1525R (Bacteria) and 20F / 958R (Archaea). The purified fragments were linked to vector pGEM Teasy and transformed by thermal shock in chemically competent Escherichia coli DH10B. Transformants were cultivated in LB/Ampicillin medium (100 μM/ml), IPTG (800 μg/mL) and XGal (80 μg/mL) at 37ºC/18-20 h. A selection of 250 clones of each library was performed, sequenced and after discarding the low quality sequences and chimerics, 64 and 68 sequences were obtained (Bacteria) and 89 and 141 sequences (Archaea) from soils of sites A and B, respectively, which were compared to public bank of data RDB and NCBI (similarity >95%). In site A the phylum Acidobacteria (48.4%) was the most abundant, followed by phyla Bacteroidetes (10.9%), Proteobacteria (10.9%), and Firmicutes (6.3%). In site B Proteobacteria (45.6%) was the most abundant, followed by Firmicutes (10.3%), Acidobacteria (8.8%), Bacterioidetes (7.3%); and also Cyanobacteria (1.5%) and Planctomycetes (1.5%) which were not found in site A. Among the sequences obtained, 23.4% (site A) and 25.0% (site B) were not classified (similarity <95%). In the domain Archaea the phyla found were Euryarchaeota (3.4 and 45.4%) and Crenarchaeota (2.2 and 3.5%), in sites A and B, respectively; it should be observed that 94.4% and 51.1% of the sequences were not classified (similarity <95%), between sites A and B, respectively. Larger diversity (Shannon‟s índex), richness (Chao 1), and distribution (equity index) of communities were observed at species level, in the phyla Bacteria and Archaea, in both sites. The metagenomic libraries 16S rRNA of Bacteria and Archaea, when compared by using the LIBSHUFF program, differed significantly (p<0.0001). The results of the present study showed the occurrence of a great diversity of bacteria and archaea in that semi-arid environment, with peculiar features of elevated temperature and hydric limitations, emphasizing the possibility of investigations on search of new genes and/or microbial isolates with biotechnological potential.
Amostras do solo da pastagem nativa (sítio A) e sob cultivo do capim marrequinho (Paspalum conjugatum, Bergius) (sítio B), coletadas na região semi-árida do bioma Caatinga, Paraíba, (07°23‟27 S 36°31‟58 O), foram utilizadas para construção de quatro bibliotecas de clones metagenômicos, para avaliação da diversidade microbiana pela amplificação do gene 16S rRNA dos domínios Bacteria e Archaea. Os DNA metagenômicos foram extraídos utilizando FastDNA® SPIN Kit for Soil (BIO 101), os quais foram amplificados por PCR utilizando primers universais, 27F / 1525R (Bacteria) e 20F / 958R (Archaea). Os fragmentos purificados foram ligados ao vetor pGEM Teasy e transformados por choque térmico em Escherichia coli DH10B quimicamente competente. Os transformantes foram cultivados em meio Agar LB/Ampicilina (100 μ/mL), IPTG (800 μg/μL) e XGal (80 μg/μL), a 37ºC/18-20 h. Foram selecionados 250 clones de cada biblioteca os quais foram sequenciados e após descarte das sequências de baixa qualidade e quiméricas, foram obtidas 64 e 68, 89 e 141 sequências para Bacteria e Archaea, nos solos dos sítios A e B, respectivamente, as quais foram comparadas em banco de dados públicos RDB e NCBI (≥95% de similaridade). No sítio A o filo Acidobacteria (48,4%) foi o mais abundante, seguido dos filos Bacteroidetes (10,9%), Proteobacteria (10,9%), e Firmicutes (6,3%). No sítio B Proteobacteria (45,6%) foi o de maior destaque, seguido de Firmicutes (10,3%), Acidobacteria (8,8%), Bacterioidetes (7,3%); e ainda Cyanobacteria (1,5%) e Planctomycetes (1,5%), que não foram encontrados no sítio A. Entre as sequências geradas, 23,4% (sítio A) e 25,0% (sítio B) não foram classificadas (similaridade <95%). No domínio Archaea foram encontrados os filos Euryarchaeota (3,4 e 45,4%) e Crenarchaeota (2,2 e 3,5%), nos sítios A e B, respectivamente; destacando-se que 94,4% e 51,1% das sequências não foram classificadas (similaridade <95%), entre os sítios A e B, respectivamente. Uma maior diversidade (índice de Shannon), riqueza (índice Chao 1) e distribuição (índice de equidade) das comunidades foram observadas no nível de espécies, tanto para Bacteria como para Archaea, nos dois sítios. As bibliotecas de clones metagenômicos 16S rRNA de Bacteria e Archaea, quando comparadas, utilizando-se o programa LIBSHUFF, diferiram significativamente (p<0,0001). Os resultados desse estudo mostraram a ocorrência de uma grande diversidade de bactérias e arqueas, nesse tipo de ambiente pouco estudado e com características peculiares de temperatura elevada e limitações hídricas, com possibilidade de busca de novos genes e/ou isolados microbianos, com potencial biotecnológico.

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Araújo, Marcus Vinícius Forzani. "O impacto do manejo do cultivo de cana-de-açúcar (Saccharum sp.) e de pastagem (Brachiaria decumbens) na microbiota do solo". Universidade Federal de Goiás, 2017. http://repositorio.bc.ufg.br/tede/handle/tede/8291.

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Characterized as extremely important, the soil is a complex environment and it shelters a great diversity of microorganisms. However, little is known about the diversity and ecology of the soil microbiota. Thus, the first part of this dissertation reviews the methodological evolution used to characterize the diversity and abundance of microorganisms found in soil. The second part consists of the application of two methodologies reviewed in the previous chapter, serial dilution and solid medium plating, to estimate free-living nitrogen fixing microorganisms, and fumigation-extraction to estimate soil microbial biomass (BMS). The last part employs the most modern microbial soil characterization technique, the metagenomics of 16S rRNA. Hence, our initial hypothesis was that sugarcane fields’ soils would have better soil microbiological indicators than grasslands’ soils. The results confirmed that the hypothesis was partially correct, and it was possible to find about 140% more free-living diazotrophic colony-forming units (CFUs) and a 17% richer alpha diversity in sugarcane fields’ soils than in grasslands’ soils. The beta diversity between sugarcane plantations and pastures presented clear differences. However, sugarcane fields’ soils obtained about 25% less BMS than grasslands’ soils. In relation to the bacterial phyla, the grasslands have more Actinobacteria, Chloroflexi and Planctomycetes and sugarcane fields have a greater number of TM7 and bacteria that were not identified, being Proteobacteria and Acidobacteria the dominating phyla in both types of soil. Although the results of nitrogen fixers and microbial biomass appear to be conflicting, it is an indication that the diazotrophic community undergoes with a diverse biotic and abiotic influences than the total community of soil microorganisms, and thus respond differently.
Caracterizado como de extrema importância, o solo é um ambiente complexo e que abriga uma grande diversidade de micro-organismos. Entretanto ainda pouco se sabe sobre a diversidade e ecologia da microbiota do solo. Deste modo, a primeira parte desta dissertação revisa a evolução metodológica empregada para caracterizar a diversidade e abundância dos micro-organismos encontrados no solo. A segunda parte consiste na aplicação de duas metodologias revisadas no capítulo anterior, a de diluição seriada e plaqueamento em meio sólido, para estimar micro-organismos fixadores de nitrogênio de vida-livre, e a fumigação-extração, para estimar a biomassa microbiana do solo (BMS). E a última parte emprega a técnica mais moderna de caracterização das comunidades microbianas de solo, a técnica de metagenômica de 16S rRNA. À vista disso, a nossa hipótese inicial era que solos de canavial teriam indicadores microbiológicos de solo melhores do que solos de pastagem. Os resultados comprovaram que a hipótese estava parcialmente correta, sendo possível encontrar cerca de 140% a mais de Unidades Formadoras de Colônias (UFCs) de diazotróficos de vida-livre e uma diversidade alfa 17% mais rica em solos de canaviais do que em solos de pastagens. A diversidade beta entre canaviais e pastagens apresentou diferenças nítidas. Entretanto, os solos de canaviais obtiveram cerca de 25% a menos de biomassa microbiana do solo do que solos de pastagens. Em relação aos filos bacterianos, os pastos possuem mais Actinobacteria, Chloroflexi e Planctomycetes e canaviais possuem maior número de TM7 e bactérias que não foram identificados, sendo Proteobacteria e Acidobacteria os filos dominantes nos dois tipos de solo. Apesar de parecerem conflitantes os resultados de fixadores de nitrogênio e biomassa microbiana, é um indicativo de que a comunidade de diazotróficos sofrem influências bióticas e abióticas diversas do que a comunidade total de micro-organismos do solo, e desta forma, respondem de forma diferente.

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Alvarez, Thabata Maria 1986. "Desenvolvimento de uma biblioteca de enzimas a partir de metagenoma de solo = Library generation for biomass conversion enzymes from soil metagenome". [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314206.

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Orientador: Fabio Marcio Squina
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Devido à necessidade do desenvolvimento de fontes de energias renováveis é de grande interesse a descoberta de novas enzimas envolvidas na desconstrução da parede celular vegetal para a produção de bicombustíveis. A metagenômica é uma poderosa ferramenta para a descoberta de novos genes em comunidades microbianas que não são passíveis de cultivo pelas técnicas tradicionais. Neste contexto, o objetivo desta tese foi o desenvolvimento de estratégias metagenômicas para prospecção de novas enzimas atuantes na degradação da biomassa vegetal no metagenoma de solo de canavial bem como a caracterização funcional das mesmas. A biblioteca metagenômica construída com DNA extraído de um consórcio microbiano especializado na degradação de bagaço de cana-de-açúcar explodido a vapor e deslignificado foi empregada nos experimentos de triagem funcional de alto desempenho. Como resultado, foram identificados três clones positivos com atividade celulolítica e dois clones com atividade xilanolítica. A análise dos insertos de cada um dos clones resultou na localização de ORFs cujas sequências de aminoácidos apresentaram identidade com domínios conservados de glicosil hidrolases da família 5 (celulases E-1 e E-2), família 6 (celulase E-3), família 10 (xilanase X-1) e família 16 (glicosil hidrolase X-2). A celulase E-1 apresentou em sua estrutura além do domínio catalítico, E-1 Cat, um domínio de ligação a carboidratos, denominado E-1 CBM, que não apresentou identidade de sequência com domínios conservados conhecidos. A análise funcional do E-1 CBM revelou tratar-se de um CBM específico para cadeias de glucano com grau de polimerização mínimo de cinco unidades de glicose. Ensaios de atividade enzimática em diferentes substratos mostraram que E-1 Cat atuou especificamente na hidrólise das ligações glicosídicas do tipo ß(1,4) entre resíduos de glicose. Os maiores valores de atividade enzimática foram obtidos em pH 7,0 e temperatura de 50ºC. Os parâmetros cinéticos calculados em CMC foram Km igual a 6,05 ± 0,37 mg/mL, Vmax de 42,51 ± 1,2 ?mol/min/mg e eficiência catalítica kcat/Km de 4,06 mL/mg/s. A enzima apresentou termoestabilidade a 40ºC por cinco horas. A atividade enzimática de E-1 Cat em celulose cristalina e bagaço de cana-de-açúcar explodido a vapor resultou na liberação de açúcares solúveis, evidenciando sua potencial aplicação em processos de conversão da biomassa vegetal. Ensaios de atividade em diferentes substratos mostraram que X-1 apresentou maior atividade enzimática em xilana não ramificada, nas condições de pH e temperatura de 6,0 e 45ºC, respectivamente. Os parâmetros cinéticos calculados utilizando como substrato xilana de madeira de faia foram Km de 2,18 ± 0,13 mg/mL, Vmax de 1.435 ± 30,4 ?mol/min/mg e kcat/Km de 496,32 mL/mg/s. Em relação à termoestabilidade, a enzima se manteve estável a 40ºC e 50ºC por seis horas. A hidrólise de substratos complexos com X-1 resultou na liberação de xilooligossacarídeos, xilobiose e xilose, que são compostos que apresentam potencial aplicação nas indústrias alimentícias e de biocombustíveis. Os resultados obtidos neste estudo validaram a abordagem metagenômica desenvolvida para a descoberta de novos genes codificantes para glicosil hidrolases. Além disso, a estratégia descrita nesta tese pode ser estendida para a descoberta de uma miríade de bioprodutos de interesse biotecnológico
Abstract: Due to the necessity of development of renewable sources of energy, it is of great interest the discovery of novel enzymes involved in plant cell wall deconstruction for biofuels production. Metagenomics is a powerful tool for the discovery of novel genes in microbial communities that are not liable to cultivation by traditional techniques. In this context, the aim of this thesis was the development of metagenomic strategies for prospection of novel enzymes involved in plant biomass degradation in sugarcane field soil metagenome and functional characterization of the identified enzymes. The metagenomic library constructed with DNA extracted from a microbial consortium specialized in degradation of steam exploded delignified sugarcane bagasse was used in the experiments of high-performance functional screening. As a result, we identified three positive clones with cellulolytic activity and two clones with xylanolytic activity. The analysis of the inserts from each clone resulted in the location of ORFs whose amino acid sequences showed identity to conserved domains of glycoside hydrolase family 5 (cellulases E-1 and E-2), family 6 (cellulase E-3), family 10 (xylanase X-1) and family 16 (glycoside hydrolase X-2). Cellulase E-1 exhibited in addition to the catalytic domain, E-1 Cat, a carbohydrate binding module, called E-1 CBM, which showed no sequence identity with known conserved domains. Functional analysis of E-1 CBM showed that it is a CBM specific for glucan chains with a degree of polymerization of at least five units of glucose. Assays with a set of different substrates revealed that E-1 Cat hydrolyzed specifically ß(1,4) glycoside bonds between glucose residues. The highest value of enzymatic activity was obtained at pH 7.0 and temperature of 50°C. The kinetic parameters Km, Vmax and catalytic efficiency kcat/Km calculated using CMC were 6.05 ± 0.37 mg/mL, 42.51 ± 1.2 ?mol/min/mg and 4.06 mL/mg/s, respectively. The enzyme showed thermal stability at 40°C for five hours. The enzymatic activity of E-1 Cat in crystalline cellulose and steam exploded sugarcane bagasse resulted in the release of soluble sugars, demonstrating its potential application in processes of biomass conversion. The xylanase X-1 showed higher enzyme activity in debranched xylan, in reactions conducted in pH 6.0 and temperature of 45°C. The kinetic parameters Km, Vmax and catalytic efficiency kcat/Km calculated using beechwood xylan were 2.18 ± 0.13 mg/mL, 1,435 ± 30.4 ?mol/min/mg and 496.32 mL/mg/s, respectively. In relation to thermal stability, the enzyme was stable at 40°C and 50°C for six hours. The hydrolysis of complex substrates resulted in the release of xylo-oligosaccharides, xylobiose and xylose, which are compounds that have potential application in food and biofuels industries. The results of this study validated the metagenomic approach developed for the discovery of novel genes coding for glycoside hydrolases. Moreover, the strategy described in this work can be extended to the discovery of a myriad of byproducts of biotechnological interest
Doutorado
Bioquimica
Doutora em Biologia Funcional e Molecular

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Braga, Lucas Palma Perez. "Disentangling the influence of earthworms on microbial communities in sugarcane rhizosphere". Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/64/64133/tde-26052017-100757/.

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For the last 150 years many studies have shown the importance of earthworms for plant growth, but the exact mechanisms involved in the process are still poorly understood. Many important functions required for plant growth can be performed by soil microbes in the rhizosphere. To investigate earthworm influence on the rhizosphere microbial community, it was performed a macrocosm experiment with and without Pontoscolex corethrurus (EW+ and EW-, respectively) and followed various soil and rhizosphere processes for 217 days with sugarcane. In the second chapter of this thesis it was demonstrate that in EW+ treatments, N2O concentrations belowground (15 cm depth) and relative abundances of nitrous oxide genes (nosZ) were higher in bulk soil and rhizosphere, suggesting that soil microbes were able to consume earthworm-induced N2O. Shotgun sequencing (total DNA) revealed that around 70 microbial functions in bulk soil and rhizosphere differed between EW+ and EW- treatments. Overall, genes indicative of biosynthetic pathways and cell proliferation processes were enriched in EW+ treatments, suggesting a positive influence of worms. In EW+ rhizosphere, functions associated with plant-microbe symbiosis were enriched relative to EW- rhizosphere. Ecological networks inferred from the datasets revealed decreased niche diversification and increased keystone functions as an earthworm-derived effect. Plant biomass was improved in EW+ and worm population proliferated. Considering that earthworms contributed to with extra resources, it was evaluated in chapter three response of the soil resistome of sugarcane macrocosms under the influence of earthworms. Mechanisms of resistance against antimicrobial compounds appear to be an obligatory feature for the ecology and evolution of prokaryotic forms of life. However, most studies on resistance dynamics have been conducted in artificial conditions of anthropogenic inputs of antibiotics into very specific communities such as animal microbiomes. To resolve why and how resistance evolves, it is important to track antibiotics resistance genes (ARGs) (i.e., the resistome) in their natural hosts and understand their ecophysiological role in the environment. The results demonstrated that earthworms influenced changes of ARGs in bulk soil and rhizosphere. Negative correlations between ARGs and taxonomical changes were increased in EW+. Differential betweenness centrality (DBC=nBCEW+ - nBCEW-) values comparing the network models with and without earthworms showed earthworm presence changed the composition and the importance of the keystone members from the models. Redundancy analysis suggested that ARGs may be associated with microbial fitness, as the variance of relative abundance of members of the group Rhizobiales could be significantly explained by the variance of a specific gene responsible for one mechanism of tetracycline detoxification
Ao longo dos últimos 150 anos muitos estudos têm demonstrado a importância das minhocas para o crescimento de plantas. Porém o exato mecanismo envolvido neste processo ainda é muito pouco compreendido. Muitas funções importantes necessárias para o crescimento de plantas podem ser realizadas pela comunidade microbiana da rizosfera. Para investigar a influência das minhocas na comunidade microbiana da rizosfera, foi desenvolvido um experimento de macrocosmo com cana-de-açúcar com e sem Pontoscolex corethrurus (EW+ e EW-, respectivamente) seguindo diversos procedimentos por 217 dias. No Segundo capítulo da tese é demonstrado que no tratamento EW+, as concentrações de N2O dentro do solo (15 cm profundidade) e a abundância relativa dos genes óxido nitroso redutase (nosZ) foram elevadas no solo e na rizosfera, sugerindo que microrganismos do solo foram capazes de consumir a emissão de N2O induzida pelas minhocas. O sequenciamento do DNA total revelou que aproximadamente 70 funções microbianas no solo e na rizosfera apresentaram diferenças entre os tratamentos EW+ e EW-. No geral, genes associados a biossíntese e proliferação de células foram enriquecidos em EW+, sugerindo uma influencia positiva por parte das minhocas. Na rizosfera EW+, funções associadas a simbiose entre planta e microrganismos foram relativamente enriquecidas comparado com rizosfera EW-. Modelos de rede de interação ecológica revelam menor número de diversificação de nichos e aumento de funções importantes como um efeito derivado da influência das minhocas. A biomassa das plantas foi aumentada no tratamento EW+ e a população de minhocas proliferou. Considerando que as minhocas contribuíram com o aumento de nutrientes, foi avaliado no capítulo três a resposta do resistoma presente nas comunidades microbianas dos solos do experimento. Mecanismos de resistência contra compostos antimicrobianos parecem ser características obrigatórias para a ecologia e evolução de procariotos. Entretanto, a maior parte dos estudos sobre genes de resistência tem sido conduzida em condições artificiais utilizando fontes antropogênicas de antibióticos em comunidades microbianas muito específicas como por exemplo o microbioma animal. Para resolver por que e como a resistência evolui, é importante estudar genes de resistência a antibióticos (GRA) (i.e., resistoma) no seu ambiente natural e entender seu papel ecofisiologico no ambiente. Os resultados demonstraram que minhocas influenciaram a mudança na composição de GRA no solo e na rizosfera. Tratamentos EW+ apresentaram maior número de correlações negativas entre ARG e grupos taxonômicos. A medida de centralidade diferencial (DBC=nBCEW+ - nBCEW-) comparando os modelos de rede de interações obtidos mostrou que a composição e o nível de importância dos indivíduos mais influentes é alterado nos tratamentos EW+ comparado com EW-. Além disso, por meio de uma análise de redundância (RDA) foi demonstrado que as alterações na abundancia relativa de GRA podem ser explicadas pelas alterações verificadas em grupos taxonômicos

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Delmont, Tom. "Description des communautés microbiennes du sol par une approche métagénomique". Thesis, Ecully, Ecole centrale de Lyon, 2011. http://www.theses.fr/2011ECDL0048/document.

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Les données présentées dans ce manuscrit de thèse sont principalement basées sur l'analyse de séquences d'ADN extraites directement de l'environnement (et en particulier du sol) en les comparant aux données cumulées au fil des siècles sur les microorganismes cultivés en laboratoire. Les objectifs, difficultés, résultats et perspectives de cette approche, que l'on nomme « métagénomique », sont décrits. Un métagénome représente la diversité génétique d'un habitat défini (par exemple un sol du champ agricole) dans sa globalité. Ainsi, générer un métagénome a notamment pour but d’accéder à la diversité génétique de la majorité d'espèces récalcitrantes à la culture. Elle permet aussi d'estimer le potentiel fonctionnel d'un jeu de données métagénomiques représentant un écosystème, puis de le comparer à d'autres jeux de données, représentant quant à eux d'autres environnements. Enfin, un autre intérêt de cette approche est la possibilité, dans certains cas, d'assembler partiellement ou entièrement un métagénome, et ainsi de permettre la reconstruction de structures génétiques complexes, qu'elles représentent des plasmides ou des génomes, circulaires ou non. Ce manuscrit de thèse présente ces aspects de la métagénomique (extraction, séquençage, annotation, comparaison, assemblage) sur l'environnement sol, en se basant sur une prairie anglaise (Park Grass, Rothamsted Research) étudiée depuis plus de 150 ans. Les communautés microbiennes prédominantes ont été partiellement caractérisées, leur potentiel fonctionnel comparé à d'autre environnements majeurs de notre planète (océans, sédiments, neige, etc.). Parce que l'assemblage de ces données est très limité dû à une trop grande diversité, des expérimentations ont été faites en microcosmes afin de stimuler une partie de la communauté avant de générer de nouveaux jeux de données, hautement simplifiés. Cette approche a permis l'assemblage et l'étude structurelle de génomes correspondant à des espèces résistant à des conditions extrêmes (par exemple un apport considérable de mercure, ou de métaux lourds).Lorsque l'on regarde la métagénomique dans sa globalité, les perspectives apparaissent clairement : usant d'outils de plus en plus performants (séquençage, annotation, assemblage, etc.), les microbiologistes peuvent d'ores et déjà récolter le fruit de plus de trois milliards d'années d'évolution et d'adaptation microbienne
Microbial ecology is beginning to interact with metagenomics and many microbiologists are attracted to metagenomics in the hope of discovering novel relationships between microorganisms and/or confirming that work done on isolates applies to the remaining uncultured members of the different ecosystems. With a growing number of available metagenomic datasets, metagenomes can be intensively mined by microbial ecologists in search of previously undetected correlations (both structural and functional). Here, we provide a preliminary exploration of 77 publically available metagenomes corresponding to DNA samples extracted from oceans, atoll corals, deep oceans, Antarctic aquatic environments, Arctic snows, terrestrial environments (sediments, soils, sludges, microbial fuel cell anode biofilms, acid mine drainage biofilms), polluted air, and animal and human microbiomes (human feces, mouse and chicken cecum, and cow rumen). Results show well-defined environmental specificities that emphasize microbial adaptation and evolution capabilities. Unexpected observations were also made for several ecosystems, thus providing new hypotheses about the life style of their microbial communities. Available metagenomes are a gold mine of underexploited information that could be used to explore specific microbial structural and functional relationships. The statistical analysis provided here depends in part on replicates from the different ecosystems. With the continued emphasis on metagenomic sequencing, future analyses should support rigorous statistical treatment. This preliminary metagenomic decryption could represent a pilot-scale test for a future Earth microbiome global comparison

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Voget, Sonja. "Metagenomanalyse eines hydrolytischen Konsortiums Identifizierung und biochemische Charakterisierung von Polysaccharid-abbauenden Biokatalysatoren aus nicht kultivierten Mikroorganismen /". Doctoral thesis, [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=982145411.

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Bellinger, Christina G. "Commercial Soils as a Potential Vehicle for Antibiotic Resistance Transmission". The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1503298572132004.

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Hackman, Jacob James. "THE EFFECTS OF COVER CROPS ON THE SOIL MICROBIOME: A METAGENOMICS STUDY". OpenSIUC, 2018. https://opensiuc.lib.siu.edu/theses/2401.

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To our knowledge, this metagenomics study is the first of its kind to determine how cover crops and tillage management practices affect the soil microbiome in southern Illinois. Seven different cover crops were used over the course of two years from 2014 to 2015, and two different forms of tillage were used: Conventional Tillage (CT) and No-Tillage (NT). Four barcodes were used to generate libraries for the phylogenetic identification of fungi, bacteria, oomycetes, and fusaria: the ITS1, EF1a (Elongation Factor 1-a), and the V4 region of the 16s rRNA subunit. Targeted amplicon sequencing using 250 base pair Paired End (PE) reads yielded 14 x 106 base pair reads in total. Using these amplicons, we successfully unveiled the fungal and bacterial constituents of the studied field plots (database limitations considered) using the QIIME and NCBI Blast protocols. Specifically, this study had three goals 1) to determine if cover crops or tillage had a significant impact on the overall microbial diversity found in bulk soil samples taken from cover crop plots; 2) to determine if the incidence and abundance of individual bacterial or fungal taxa were affected by the cover crop or tillage treatment; 3) perform a bioinformatics methodology comparison for fungal identification using the ITS1 region between Qiime, and MEGAN protocols. Our results indicate many instances of cover crop or tillage interacting with one or more groupings of taxa. Significant whole community differences could be detected to the species (P=0.0335) and family (P=0.0001) taxonomic ranks of fungi using with the three most abundant families based on assigned reads being Mortierellaceae, Trichocomaceae, and Botryosphaeriaceae. Significant whole community interactions between tillage types and year at the level of phylum were observed between bacteria and archaea. Three main phyla constituting bacterial reads were Proteobacteria, Actinobacteria, and Acidobacteria. The primary driver in individual differences in bacterial populations appeared to be the year in which samples were taken either 2014 or 2015 (P=0.0001). This was attributed in part due to drastic fluctuations in weather from November 2014 to November 2015. Whole community differences and shifts could be observed based on cover crop down to the species level using both QIIME and NCBI BLAST protocols. The different dispersions and taxa found between cover crops imply that there is a relationship between certain organisms and the type of plant matter present. Tillage type, year, and cover crop were all found to have some degree of clustering based on reads taken from the four amplicons used. For comparison between NCBI and QIIME methodologies using the ITS1 region, the NCBI BLAST protocol provided the most overlap between taxa at the Order and Class taxonomic rankings. An upwards of 70% complementarity of taxa was found comparing the results after using the NCBI or the QIIME protocols. Whole community analysis using PERMANOVA revealed complementarity shifts based on treatment types when comparing both QIIME and NCBI protocols for taxonomic assignments visualized using PCoA plots. This comparison between the two methods for fungal community analysis using the ITS region, highlights the significant discrepancies as well as the complementarity of the two methodologies when analyzing fungal microbiomes.

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Zanardo, Marina. "Soil microbial communities in environmental-agronomical context: quantitative analysis, metagenomics and signal exchange monitoring". Doctoral thesis, Università degli studi di Padova, 2014. http://hdl.handle.net/11577/3423522.

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Soil is one of the most challenging environments for microbiologists. The astounding number of microbial species living in soil makes it one of the most diverse and still mysterious environments known. The study of the soil nitrogen cycle is gaining in importance, as recently a strong land-use intensification and increase in N fertilization led to the rising of threats to environmental and human health. Moreover, major interest is given to the investigation of the structure of soil microbial communities, to their responses in the presence of external stimuli, and to their capacity to organize thanks to the exchange of specific signals dispersed in the environment. In this study, these aspects of soil microbial ecology were taken into account, and microbiology and molecular biology techniques were used to probe the bacterial community function and structure and to study the physical diffusion of signals emitted by certain bacteria. Real Time PCR analysis of functional genes of the N-cycle in different soils highlighted interesting differences and permitted to select the bacterial amoA and nosZ genes, for nitrification and denitrification, respectively, as useful indicators of soil health and functionality. Furthermore, a strict relationship between bacteria involved in nitrification and in denitrification was found in all the analysed soil samples, indicating that these microorganisms could be part of the same environmental niche, though being involved in opposite processes. Through T-RFLP experiments and a metagenomic analysis by amplicon sequencing performed in a 454 system (Roche), the effects of different fertilizers on an agricultural soil microbial community were investigated. In particular, it was possible to determine how bacterial communities changed over time in response to this kind of inputs, and to have a first insight into the relation among some bacterial phyla, which fluctuated in parallel or in opposition. Concerning microbial cell-to-cell signalling, in this work, efforts were made to define the consequences of the reflecting or adsorbing boundary conditions on the physical diffusion of AHL molecules, and therefore on bacterial intercellular communication, by combining physics and microbiology approaches. It was observed that the properties of the boundaries play a major critical role on the quorum sensing AHL concentration profiles around the cell environment.
Il suolo rappresenta uno degli ambienti più misteriosi e ricchi di biodiversità sulla Terra, e, nella maggior parte dei casi, i microrganismi adattati a vivere in questo ambiente non sono coltivabili e non sono stati ancora caratterizzati. Questo lavoro di tesi ha lo scopo di analizzare la complessità dell’ecologia microbica del suolo da due punti di vista diversi: da una parte si vuole ottimizzare e utilizzare tecniche di biologia molecolare per esaminare la struttura e la funzione delle comunità microbiche di diversi suoli, e dall’altra parte lo scopo è quello di studiare le forme di comunicazione tra i batteri, e i segnali che essi scambiano tra loro (Quorum Sensing). Di recente, molti ricercatori si sono dedicati allo studio dei batteri e degli archaea coinvolti nel ciclo dell’azoto, poiché il disequilibrio in questo processo, causato principalmente dall’uso eccessivo di fertilizzanti azotati e da altre pratiche agricole, è una minaccia per la salute dell’uomo, oltre che per l’ambiente. Inoltre, anche lo studio delle comunità microbiche del suolo sta assumendo sempre più importanza, in particolare per quanto riguarda la loro risposta in presenza di stimoli esterni, e la loro capacità di organizzarsi grazie allo scambio di segnali specifici emessi nell’ambiente dagli stessi microrganismi. L’analisi dei geni funzionali per il ciclo dell’azoto mediante esperimenti di Real Time PCR quantitativa in suoli di diversa provenienza o trattati in modi diversi ha permesso di evidenziare differenze significative e di selezionare i geni amoA batterico e nosZ, rispettivamente per la nitrificazione e per la denitrificazione, come possibili indicatori della qualità del suolo e della produttività in agricoltura. Questo tipo di analisi ha anche permesso di osservare come ci sia, in tutti i suoli analizzati, una correlazione positiva e statisticamente significativa tra i geni batterici per la nitrificazione e per la denitrificazione. Questo risultato potrebbe indicare che questi due gruppi di microrganismi sono in qualche modo legati, e che probabilmente vivono nella stessa nicchia ecologica. In questo lavoro, mediante esperimenti di T-RFLP e grazie all’uso della tecnologia di Next generation sequencing 454 (Roche), è stato possibile analizzare la struttura della comunità microbica in suoli trattati con fertilizzanti di diverso tipo, a due tempi di campionamento. Inoltre, sono state ricercate le correlazioni tra i phyla presenti, in modo da avere una prima idea di come questi varino anche in relazione alla variazione dell’abbondanza di altri gruppi batterici. Grazie a questo esame sono state trovate molte correlazioni forti e significative, che indicano come alcuni gruppi batterici siano strettamente in relazione tra loro e si influenzino fortemente. Per quanto riguarda lo studio dei segnali scambiati tra batteri presenti in una stessa comunità, e quindi del Quorum Sensing e del Diffusion Sensing, sono stati analizzati i meccanismi fisici di diffusione delle molecole quorum AHL in ambienti circoscritti, ed in particolare sono state definite le conseguenze delle condizioni al contorno, sia nel caso di confini di natura riflettente che di natura assorbente. Queste proprietà sono risultate giocare un ruolo fondamentale nel determinare i profili di concentrazione della molecola segnale AHL.

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Mendes, Lucas William. "Metagenome of Amazon forest conversion: impacts on soil-borne microbial diversity and functions". Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/64/64133/tde-11062014-144747/.

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The Amazon rainforest is considered the world\'s largest reservoir of plant and animal biodiversity, but in recent years has been subjected to high rates of deforestation for the conversion of native areas into agricultural fields and pasture. The understanding of the effects of land-use change on soil microbial communities is essential, taking into account the importance that these organisms play in the ecosystem. In this context, this thesis evaluated the effect of these changes on microorganism communities in soils under different land-use systems. In the first study, the microbial communities were analyzed using the nextgeneration sequencing Illumina Hiseq2000, considering samples from native forest, deforested area, agriculture and pasture. From the analysis of approximately 487 million sequences was possible to show that microbial communities respond differently in each landuse system, with changes in both taxonomic and functional diversity. Also, we suggested that ecosystem function in forest soils is maintained by the abundance of microorganisms, while in disturbed areas such functioning is maintained by high diversity and functional redundancy. In the second study, we assessed the extent to which a particular plant species, i.e. soybean, is able to select the microbial community that inhabits the rhizosphere. From the metagenomic sequencing by the 454 GS FLX Titanium platform we investigated the taxonomic and functional diversities of soil and rhizosphere communities associated to soybean, and also tested the validity of neutral and niche theories to explain rhizosphere community assembly process. The results suggest that soybean selects a specific microbial community inhabiting the rhizosphere based on functional traits, which may be related to benefits to the plant, such as growth promotion and nutrition. This process of selection follows largely the niche -based theory indicating the selection power of the plant and other environmental variables in shaping the microbial community both at the taxonomic and functional level. This thesis highlights the importance of microbial ecology studies in the context of the Amazon to a better understanding of the effects of deforestation on microorganisms, and provides information that can be suitable for future development of sustainable approaches for the ecosystem use
A floresta Amazônica é considerada o maior reservatório de biodiversidade vegetal e animal do mundo, porém, nos últimos anos tem sido submetida à altas taxas de desmatamento para a conversão de áreas de mata nativa em campos de agricultura e pastagem. A compreensão sobre os efeitos dessa mudança de uso da terra sobre as comunidades microbianas do solo é fundamental, levando-se em consideração a importância que esses organismos desempenham no ecossistema. Neste contexto, este trabalho de tese avaliou o efeito dessas mudanças sobre as comunidades de micro-organismos em solos sob diferentes sistemas de uso. No primeiro estudo, as comunidades microbianas foram analisadas por meio do sequenciamento de nova geração Illumina Hiseq2000, sendo consideradas amostras de áreas de floresta nativa, área desmatada, agricultura e pastagem. A partir da análise de aproximadamente 487 milhões de sequências foi possível mostrar que as comunidades microbianas respondem diferentemente em cada sistema de uso do solo, com alterações tanto na diversidade taxonômica quanto funcional. Também, sugere-se que o funcionamento do ecossistema em solos de floresta é mantido pela abundância dos micro-organismos presentes, enquanto nas áreas alteradas esse funcionamento é mantido pela alta diversidade e redundância funcional. No segundo estudo foi avaliado até que ponto uma espécie de planta, i.e. soja, é capaz de selecionar a comunidade habitante de sua rizosfera. A partir do sequenciamento metagenômico pela plataforma 454 GS FLX Titanium da Roche foi investigado a diversidade taxonômica e funcional das comunidades de solo e rizosfera associadas à soja, e testou-se a validade das teorias neutras e de nicho para explicar o processo de formação das comunidades microbianas. Os resultados sugerem que a soja seleciona uma comunidade específica que habita sua rizosfera com base em atributos funcionais, os quais podem estar relacionados com benefícios à planta, como promoção do crescimento e nutrição. Esse processo de seleção segue a teoria de nicho, indicando o poder de seleção da planta e de outras variáveis ambientais em moldar as comunidades microbianas tanto de forma taxonômica quanto funcional. Esta tese destaca a importância de estudos em ecologia microbiana no contexto da Amazônia para uma melhor compreensão dos efeitos do desmatamento sobre os microrganismos e disponibiliza informações que podem ser futuramente utilizados para o desenvolvimento de metodologias mais sustentáveis para o uso do ecossistema

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20

Sun, Shan. "Patterns, Processes And Models Of Microbial Recovery In A Chronosequence Following Reforestation Of Reclaimed Mine Soils". Diss., Virginia Tech, 2017. http://hdl.handle.net/10919/87754.

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Soil microbial communities mediate important ecological processes and play essential roles in biogeochemical cycling. Ecosystem disturbances such as surface mining significantly alter soil microbial communities, which could lead to changes or impairment of ecosystem functions. Reforestation procedures were designed to accelerate the reestablishment of plant community and the recovery of the forest ecosystem after reclamation. However, the microbial recovery during reforestation has not been well studied even though this information is essential for evaluating ecosystem restoration success. In addition, the similar starting conditions of mining sites of different ages facilitate a chronosequence approach for studying decades-long microbial community change, which could help generalize theories about ecosystem succession. In this study, the recovery of microbial communities in a chronosequence of reclaimed mine sites spanning 30 years post reforestation along with unmined reference sites was analyzed using next-generation sequencing to characterize soil-microbial abundance, richness, taxonomic composition, interaction patterns and functional genes. Generally, microbial succession followed a trajectory along the chronosequence age, with communities becoming more similar to reference sites with increasing age. However, two major branches of soil microbiota, bacteria and fungi, showed some contrasting dynamics during ecosystem recovery, which are likely related to the difference in their growth rates, tolerance to environmental change and relationships with plants. For example, bacterial communities displayed more intra-annual variability and more complex co-occurrence networks than did fungi. A transition from copiotrophs to oligotrophs during succession, suggested by taxonomic composition shifts, indicated that the nutrient availability is one important factor driving microbial succession. This theory was also supported by metagenomic analysis of the functional genes. For example, the increased abundance of genes involved in virulence, defense and stress response along ages indicated increased competition between microorganisms, which is likely related to a decrease of available nutrients. Metagenomic analysis also revealed that lower relative abundances of methanotrophs and methane monooxygenase at previously-mined sites compared with unmined sites, which supports previous observations that ecological function of methane sink provided by many forest soils has not recovered after 30 years. Because of the difficulty identifying in situ functional mechanisms that link soil microorganisms with environmental change, modeling can be a valuable tool to infer those relationships of microbial communities. However, the extremely high richness of soil microbial communities can result in extremely complicated models that are difficult to interpret. Furthermore, uncertainty about the coherence of ecological function at high microbial taxonomic levels, grouping operational taxonomic units (OTUs) based on phylogenetic linkages can mask trends and relationships of some important OTUs. To investigate other ways to simplify soil microbiome data for modeling, I used co-occurrence patterns of bacterial OTUs to construct functional groups. The resulting groups performed better at characterizing age-related microbial community dynamics and predicted community structures and environmental factors with lower error.
PHD

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21

Adesioye, Fiyinfoluwa Adenike. "Structural and functional characterization of a novel acetyl xylan esterase from a desert soil metagenome". Thesis, University of Pretoria, 2017. http://hdl.handle.net/2263/65881.

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A Namib Desert soil hypolith metagenomic dataset was screened, in silico, for novel acetyl xylan esterase (AcXE) - encoding genes. AcXEs hydrolyse ester bonds to liberate acetic acid in acetylated polymeric xylan and xylooligosaccharides during bioconversion of lignocellulosic biomass for sustainable biofuel production. One of the identified genes (NaMet1) was synthesized, cloned and expressed to produce a ~36 kDa protein. This protein, NaM1, was confirmed to be functional and was purified and characterized. NaM1, a carbohydrate esterase (CE) 7 enzyme, was optimally active on para-nitrophenol acetate at pH 8.5 and 30 oC, and remained active in up to 5 M NaCl and 65% DMSO. The specific activity and catalytic efficiency were 488.9 Umg-1 and 3.26x106 M-1s-1, respectively. NaM1 deacetylated para-nitrophenol acetate and butyrate, 7-aminocephalosporanic acid and acetylated xylan. Most investigations of CE7 esterases have been carried out using structural information from thermostable members of this family and little is known about thermolabile members. A 2.03 Å crystal structure of native NaM1, the first CE structure of metagenomic origin to be submitted to the Protein data bank, was solved. The structure was compared with those of thermostable CE7 enzymes and used to study the thermal stability determinants of this enzyme family. This comparison showed strong structural conservation between both enzyme types and suggested that differences in several key residues, as well as, packing within the core, were responsible for thermal stability. Directed evolution (DE) of NaM1 yielded thermostable variants, including a variant with 10oC improved stability. Analyses of the kinetic and putative structural characteristics of selected variants in comparison with those of the wild-type provided insights to the role of residues influencing the thermal stability, substrate specificity and activity of NaM1. A single substitution was found to expand acyl moiety specificity and improve both thermal stability and activity of NaM1. Knowledge of key residues identified during NaM1 DE is useful for the future engineering of CE7 and ?/? hydrolase enzymes in order to improve catalytic turnover, substrate specificity and thermal stability.
Thesis (PhD)--University of Pretoria, 2017.
Genetics
PhD
Unrestricted

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22

Anderson, Dominique Elizabeth. "Gene Discovery in Antarctic Dry Valley Soils". Thesis, University of the Western Cape, 2008. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_6598_1265941858.

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The metagenomic approach to gene discovery circumvents conventional gene and gene product acquisition by exploiting the uncultured majority of microorganisms in the environment. It was demonstrated in this study that metagenomic methods are suitable for gene mining in extreme environments that harbor very high levels of unculturable microorganisms. DNA was extracted from Antarctic mineral soil samples taken from the Miers Valley, Antarctica. The metagenomic DNA was also used to construct a fosmid library comprising over 7900 clones with an average insert size of 29 kb. PCR amplification using bacterial and archaeal 16S rRNA gene specific primers and subsequent denaturing gradient gel electrophoresis (DGGE) of bacterial 16S rDNA amplicons showed that a small percentage of bacterial diversity (>
1%) was captured in the metagenomic fosmid library. Activity-based screening for lipase and esterase genes using a tributyrin plate assay yielded twelve positive clones. LD1, a putative, novel cold-active GDSL lipase/esterase was identified and sequenced. The C-terminal domain of the ORF was found to be an autotransporter similar to those associated with type V secretion systems in Gram negative bacteria. Sub-cloning of the gene resulted in lipolytic activity in E. coli. Preliminary enzyme assays have determined that LD1 hydrolyses p-nitrophenyl esters with chain lengths shorter than C10, an indication that the enzyme is an esterase. Complete purification and characterisation of this enzyme is subject to further study.

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23

Wilhelm, Roland Conrad. "Deciphering decomposition and the effects of disturbance in forest soil microbial communities with metagenomics and stable isotope probing". Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/59430.

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Forest industries are expected to bolster the renewable resource economy, but must contend with ecological challenges in maintaining the long-term fertility of forest plantation soils, and technological challenges in converting forest biomass into industrially relevant sources of carbon and energy. This thesis advances research related to both, first by describing the broad changes in soil microbial communities in the decades following timber harvesting, their implications for soil processes and the influence of biomass retention for mitigation (Chapter 3) and, second, by conducting the first comprehensive culture-independent survey of lignocellulolytic organisms in forest soils to expand knowledge of their diversity and catabolic capabilities (Chapter 4). Analysis of over 1,300 bacterial (16S rRNA gene) and fungal (ITS region) pyrotag libraries demonstrated consistent changes in microbial communities at harvested sites across North America, such as i) the increase of desiccation- and heat-tolerant organisms, ii) the general decline of ectomycorrhizal (EM) fungi with a rise of select EM genera (Suillus and Thelephora), iii) the moderation of population shifts by organic matter retention and iv) changes in the functional character of harvested soils, including reduced methanotrophic populations and cellulolytic activity. Biogeographical differences in community structure revealed the potential for variation in the impacts of harvesting. Overall, a number of taxonomic groups were identified that may be important indicators for assessing the long-term impact of timber harvesting. Stable isotope probing revealed the degradation of model hemicellulose, cellulose and lignin substrates by specialized taxa, active on a sole substrate, and groups capable of degrading all three plant polymers, such as members of Burkholderiales and Caulobacteraceae. Bacterial lignin-degraders were more active than fungi in soil microcosms, represented by taxa with characterized lignolytic capability (Sphingobacteriaceae and Sphingomonadaceae) and novel taxa, such as members of Elusimicrobia and Acidobacteria. Differences in lignocellulolytic populations were observed among ecozones and soil layers. Mineral soils harboured a greater proportion of poorly characterized functional taxa and represent reservoirs of unexplored catabolic diversity. Metagenome assembly was ~3 to 20-fold higher as a result of SIP, providing a trove of sequence data containing carbohydrate- and lignin-active enzymes from lignolytic and cellulolytic taxa for future characterization.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate

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24

Trubl, Gareth. "Pioneering Soil Viromics to Elucidate Viral Impacts on Soil Ecosystem Services". The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1543425468999981.

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Hariharan, Janani. "Predictive Functional Profiling of Soil Microbes under Different Tillages and Crop Rotations in Ohio". The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1435856176.

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Alahmad, Abdelrahman. "La métagénomique, un outil pertinent pour évaluer l'impact de différentes pratiques agricoles sur les communautés microbiennes du sol". Thesis, Amiens, 2017. http://www.theses.fr/2017AMIE0038.

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Selon les projections démographiques de la FAO, la population mondiale atteindra 9 milliards de personnes d'ici 2050. Cette augmentation sera associée à une demande accrue de produits agricoles et à une augmentation de la production de déchets. Par conséquent, des approches alternatives dans les pratiques agricoles, tels que l'utilisation permanente de la couverture végétale et/ou l'application de boues d'épuration, sont envisagées pour répondre aux exigences mondiales et préserver l'environnement. Ces nouvelles pratiques pourraient influencer le fonctionnement et les propriétés du sol et des organismes microbiens présents dans cet environnement. Par conséquent, passer de l'agriculture intensive à une agriculture écologiquement intensive pourrait entraîner des modifications de la biodiversité des sols. En utilisant différents systèmes expérimentaux permettant une comparaison entre différentes pratiques agricoles, des études de la diversité microbienne taxonomique et fonctionnelle du sol (bactéries et champignons) ont été entreprises. La diversité taxonomique des organismes microbiens a été obtenue par séquençage à haut débit des régions hypervariables des gènes codant l'ARN16S et l'ITS1. Nous avons évalué les rôles écologiques des microorganismes du sol en utilisant des identifications taxonomiques, puis des études permettant d'examiner leur physiologie et leurs fonctions par rapport à différentes propriétés physicochimiques du sol. Nous avons constaté que la fertilisation azotée avait une incidence négative sur la diversité microbienne du sol et modifié leur fonctionnalité. Ces effets peuvent être modulés par l'utilisation de PPC ou l'application de boues. Ces travaux indiquent que les pratiques agricoles conventionnelles ont un impact sur la biodiversité microbienne du sol et peuvent être remplacées par des pratiques agricoles plus respectueuses de l'environnement afin de préserver l'écosystème et ses services
According to demographic projections, world population will reach 9 billion people by 2050. This increase will be associated with higher demand of agricultural products and an increase in wastes production. Therefore, alternative approaches in agricultural practices; such as permanent plant cover usage and/or sewage sludge application, are envisaged to meet global demands and preserve the environment. These new practices could therefore influence the properties of the soil and its functioning. Therefore moving from intensive to ecologically intensive agriculture could lead to modifications in soil biodiversity. Using different experimental systems allowing comparison between different agricultural practices, studies of the taxonomic and functional soil microbial diversity (bacteria and fungi) had been undertaken. This was achieved by next generation high-throughput sequencing of the hypervariable regions of the genes encoding RNA16S and ITS1. Sequencing was performed using an Illumina platform and the sequences obtained were analyzed using various bioinformatic tools. We inferred the ecological roles of soil micro-organisms by using taxonomic identifications, moving on to the examination of their physiology and functions in comparison with different soil physiochemical properties. We found that nitrogen fertilization negatively impacted the soil microbial diversity and altered their functionality. These negative effects have been modulated by the PPC usage or SS application. Proving that conventional agricultural practices effects the soil biodiversity and can be replaced by ecofriendly farming applications in order to preserve the ecosystem and its services

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Saurey, Sabrina Deni. "Resource Legacies and Priming Regulate Microbial Communities in Antarctica's Dry Valleys". BYU ScholarsArchive, 2013. https://scholarsarchive.byu.edu/etd/4051.

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Multiple mechanisms control bacterial community structure but two in particular, the "legacy" of past environmental conditions, and the "priming" of bacteria to respond to seasonal or reoccurring fluctuations in resources, have the potential to determine both bacterial communities, as well as, temporal shifts in active bacterial taxa. To begin to evaluate the legacy effects of resources on microbial communities, we added four limiting resources annually (i.e., water only; C-mannitol + water; N-NH4NO3 + water; and C, N + water) and measured shifts in bacterial community composition after seven years in a cold desert ecosystem in the McMurdo Dry Valleys, Antarctica. Further, to investigate the ecological significance of priming, we conducted a series of stable isotope probing experiments (i.e., 18O-DNA SIP with 18O-labeled water, 13C-DNA SIP with 13C-labeled mannitol, 15N-DNA with 15N- NH4NO3, and a combined C and N SIP) and characterized the responding (i.e., isotopically labeled) and seed bank (i.e., unlabeled) bacterial communities. We performed each of the SIPs in soil microcosms corresponding to a single resource manipulation (e.g., 13C-labeled mannitol in C addition soils). We hypothesized that all long-term additions of nutrients and water will lead to a distinct bacterial community—a legacy effect due to the nutrient and water impoverished state of Antarctica soils. We also hypothesized that the stronger the legacy effects demonstrated by a specific community the more adapted or primed bacterial species will be to take advantage of the resource and respond. As hypothesized, resource additions created distinct bacterial legacy but to different degrees among the treatments. The extent of the resource legacy effects was greatest in the CN, intermediate in water and N, and lowest in C communities relative to the control communities, suggesting that C induced changes in communities were intensified by tandem N additions and that water alone created a more distinct legacy than water and C additions combined. Contrary to our hypothesis, the stronger the legacy effects, the less adapted or primed the community was to take advantage of resource additions. For example, the CN treatment that induced the greatest effect on bacterial communities had the lowest number of species (20.9%) in common between the responding and seed bank communities. This inverse relationship may be due to only two species (i.e., Arthrobacter, Actinobacteria and Massilia, Betaproteobacteria) really being primed to take advantage of CN and these species constituting over 75% of the seed bank community. Water, N, and C additions had similar levels of priming with 38.4%, 41.4%, and 36.3% of the responding species being present in the seed bank community, respectively. But of these three treatments, only the priming with water resulted in a unique responding community, suggesting that water, a universal bacterial resource, was enough to prime bacteria. Furthermore, water generates the most diverse responding community of all the resources with stemming from all of the fourteen dominant phyla. We did find patterns of ecological coherence among the responders, especially in the major responders (i.e., responders that increased in relative recovery by at least ten-fold). These responders were predominantly found in only three phyla (i.e., Actinobacteria, Bacteriodetes, and Gammaproteobacteria) regardless of resource addition. Alternatively minor responders (i.e., responders that increased in relative recovery at least two-fold) were contained in fourteen different phyla with specific taxa stimulated by CN (i.e., Betaproteobacteria) and N and water (i.e., Deltaproteobacteria). Further, resource additions elicited responses from 37% of bacterial species with species specializing on a specific resource (e.g., Chloroflexi) or being a generalist (e.g., Planctomycetes and Gammaproteobacteria). Our results offer the first direct links between legacy and priming effects on bacterial community composition and demonstrate that these mechanisms are not always complimentary leading to the formation of similar communities but may both be essential to maintain the high levels of bacterial diversity. Further, all resources produced elicited responders that were either specialists of generalists demonstrating that even bacteria in the extreme environment of Antarctica respond to pulses of resources.

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Pacchioni, Ralfo Goes. "Metagen?mica comparativa de solo de regi?es de mata atl?ntica e caatinga do estado do Rio Grande do Norte - Brasil". Universidade Federal do Rio Grande do Norte, 2010. http://repositorio.ufrn.br:8080/jspui/handle/123456789/12572.

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Made available in DSpace on 2014-12-17T14:03:36Z (GMT). No. of bitstreams: 1 RalfoGP_DISSERT_partes_autorizadas.pdf: 608925 bytes, checksum: dbfcce5de284f89c5cdaee179cdd9bc2 (MD5) Previous issue date: 2010-12-02
Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico
The microorganisms play very important roles in maintaining ecosystems, which explains the enormous interest in understanding the relationship between these organisms as well as between them and the environment. It is estimated that the total number of prokaryotic cells on Earth is between 4 and 6 x 1030, constituting an enormous biological and genetic pool to be explored. Although currently only 1% of all this wealth can be cultivated by standard laboratory techniques, metagenomic tools allow access to the genomic potential of environmental samples in a independent culture manner, and in combination with third generation sequencing technologies, the samples coverage become even greater. Soils, in particular, are the major reservoirs of this diversity, and many important environments around us, as the Brazilian biomes Caatinga and Atlantic Forest, are poorly studied. Thus, the genetic material from environmental soil samples of Caatinga and Atlantic Forest biomes were extracted by direct techniques, pyrosequenced, and the sequences generated were analyzed by bioinformatics programs (MEGAN MG-RAST and WEBCarma). Taxonomic comparative profiles of the samples showed that the phyla Proteobacteria, Actinobacteria, Acidobacteria and Planctomycetes were the most representative. In addition, fungi of the phylum Ascomycota were identified predominantly in the soil sample from the Atlantic Forest. Metabolic profiles showed that despite the existence of environmental differences, sequences from both samples were similarly placed in the various functional subsystems, indicating no specific habitat functions. This work, a pioneer in taxonomic and metabolic comparative analysis of soil samples from Brazilian biomes, contributes to the knowledge of these complex environmental systems, so far little explored
Os microorganismos desempenham importantes fun??es na manuten??o dos ecossistemas, o que explica o enorme interesse em compreender as rela??es existentes entre estes organismos, bem como entre eles e o meio. Estima-se que o n?mero total de c?lulas procari?ticas na Terra seja entre 4 e 6 x 1030, constituindo um enorme pool biol?gico e gen?tico a ser explorado. Apesar de atualmente apenas 1% de toda essa riqueza poder ser cultivada por t?cnicas laboratoriais padr?o, ferramentas metagen?micas permitem o acesso ao potencial gen?mico de amostras ambientais de forma independente de cultivo, e em associa??o com tecnologias de sequenciamento da terceira gera??o, a cobertura amostral se torna ainda maior. Solos, em particular, s?o os maiores reservat?rios dessa diversidade, e muitos ambientes importantes ao nosso redor, como os biomas brasileiros Caatinga e Mata Atl?ntica, s?o pouco estudados. Sendo assim, o material gen?tico ambiental de amostras de solo dos biomas Caatinga e Mata Atl?ntica foi extra?do atrav?s de t?cnicas diretas, pirosequenciado, e as seq??ncias geradas foram analisadas atrav?s de programas de bioinform?tica (MEGAN, MG-RAST e WEBCarma). Perfis taxon?micos comparativos das amostras mostraram que os filos Proteobacteria, Actinobacteria, Acidobacteria e Planctomycetes foram os mais representativos. Em adi??o, fungos do filo Ascomycota foram identificados predominantemente na amostra de solo de Mata Atl?ntica. Perfis metab?licos mostraram que, apesar da exist?ncia de diferen?as ambientais, sequ?ncias de ambas as amostras foram inseridas similarmente nos diversos subsistemas funcionais, n?o indicando fun??es habitat espec?ficas. Este trabalho, pioneiro em an?lises taxon?micas e metab?licas comparativas de amostras de solo de biomas brasileiros, contribui para o conhecimento destes sistemas ambientais complexos, at? ent?o pouco explorados

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Wu, Xiaofen. "Structure and function of microbial communities in acid sulfate soil and the terrestrial deep biosphere". Doctoral thesis, Linnéuniversitetet, Institutionen för biologi och miljö (BOM), 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-52538.

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This thesis describes the use of different DNA sequencing technologies to investigate the structure and function of microbial communities in two extreme environments, boreal acid sulfate soil and the terrestrial deep biosphere. The first of the two investigated environments was soils containing un-oxidized metal sulfides that are termed ‘potential acid sulfate soil’ (PASS) materials. If these materials are exposed to atmospheric oxygen by either natural phenomena (e.g., land uplift) or human activities (e.g., drainage) then the metal sulfides become oxidized and the PASS becomes acidic and is defined as an ‘acid sulfate soil’ (ASS). The resulting acid and metal release from metal sulfide oxidation can lead to severe environmental damage. Although acidophilic microorganisms capable of catalyzing acid and metal release have been identified from many sulfide mineral containing environments, the microbial community of boreal PASSs/ASSs remains unclear. This study investigated the physicochemical and microbial characteristics of PASSs and ASSs from the Risöfladan experimental field in Vasa, Finland. Sanger sequencing of 16S rRNA gene sequences of microorganisms present in the PASSs and ASSs were mostly assigned to acidophilic species and environmental clones previously identified from acid- and metal-contaminated environments. Enrichment cultures inoculated from the ASS demonstrated that the acidophilic microorganisms were responsible for catalyzing acid and metal release from PASSs/ASSs. Lastly, the study investigated how to mitigate metal sulfide oxidation and the concomitant formation of sulfuric acid by treating ASSs in situ with CaCO3 or Ca(OH)2 suspensions. The DNA sequencing still identified acidophilic microorganisms after the chemical treatments. However, the increased pH during and after treatment suggested that the activity of the acidophiles might be inhibited. This study was the first to identify the microbial community present in boreal PASSs/ASSs and suggested that treatment with basic compounds may inhibit microbial catalysis of metal sulfide dissolution. The second studied environment was the deep, dark terrestrial subsurface that is suggested to be both extremely stable and highly oligotrophic. Despite the scarcity of carbon and energy sources, the deep biosphere is estimated to constitute up to 20% of the total biomass on earth and thus, represents the largest microbial ecosystem. However, due to the difficulties of accessing this environment and our inability to cultivate the indigenous microbial populations, details of the diversity and metabolism of these communities remain largely unexplored. This study was carried out at Äspö Hard Rock Laboratory, Sweden and utilized second-generation sequencing to identify the taxonomic composition and genetic potential of planktonic and biofilm populations. Community DNA sequencing of planktonic cells from three water types at varied age and depth (‘modern marine’, ‘undefined mixed’, and ‘old saline’) showed the existence of ultra-small cells capable of passing through a 0.22 μm filter that were phylogenetically distinct communities from the >0.22 μm fraction. The reduced cell size and/or genome size suggested a potential adaptation to the oligotrophic environment in the terrestrial deep biosphere. The identified planktonic communities were dominated by Proteobacteria, Candidate divisions, unclassified archaea, and unclassified bacteria. Functional analysis of the assembled genomes showed that the planktonic population from the shallow modern marine water demonstrated a predominantly anaerobic and heterotrophic lifestyle. In contrast, the deeper, old saline water was more closely aligned with the hypothesis of a hydrogen-driven deep biosphere. Metagenomic analysis of subsurface biofilms from ‘modern marine’ and ‘old saline’ water types suggested only a subset of populations were involved in initial biofilm formation. The identified biofilm populations from both water types were distinct from the planktonic community and were suggested to be dominated by hydrogen fed, chemolithoautotrophic and diazotrophic populations.

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30

Fogler, Kendall Wilson. "Effect of Soil Amendments from Antibiotic Treated Cows on Antibiotic Resistant Bacteria and Genes Recovered from the Surfaces of Lettuce and Radishes: Field Study". Thesis, Virginia Tech, 2018. http://hdl.handle.net/10919/92587.

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Cattle are commonly treated with antibiotics that may survive digestion and promote antibiotic resistance when manure or composted manure is used as a soil amendment for crop production. This study was conducted to determine the effects of antibiotic administration and soil amendment practices on microbial diversity and antibiotic resistance of bacteria recovered from the surfaces of lettuce and radishes grown using recommended application rates. Vegetables were planted in field plots amended with raw manure from antibiotic-treated dairy cows, composted-manure from cows with different histories of antibiotic administration, or a chemical fertilizer control (12 plots, n=3). Culture-based methods, 16SrDNA amplicon sequencing, qPCR and shot-gun metagenomics were utilized to profile bacteria and characterize the different gene markers for antibiotic resistance. Culture-based methodologies revealed that lettuce grown in soils amended with BSAs had significantly larger clindamycin resistant populations compared to control conditions. Growth in BSAs was associated with significant changes to the bacterial community composition of radish and lettuce. Total sul1 copies were 160X more abundant on lettuce grown in manure and total tet(W) copies were 30X more abundant on radishes grown in manure. Analysis of shotgun metagenomic data revealed that lettuce grown in manure-amended soils possessed resistance genes for three additional antibiotic classes compared to other treatments. This study demonstrates that raw, antibiotic-exposed manure may alter microbiota and the antibiotic resistance genes present on vegetables. Proper composting of BSAs as recommended by the U.S. Department of Agriculture and Environmental Protection Agency is recommended to mitigate the spread of resistance to vegetable surfaces.
MSLFS

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31

Castillo, Villamizar Genis Andrés [Verfasser], Rolf [Akademischer Betreuer] Daniel, Rolf [Gutachter] Daniel y Michael [Gutachter] Hoppert. "Discovery and Functional Characterization of Novel Soil-metagenome Derived Phosphatases / Genis Andrés Castillo Villamizar ; Gutachter: Rolf Daniel, Michael Hoppert ; Betreuer: Rolf Daniel". Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2019. http://d-nb.info/1191479668/34.

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32

Will, Christiane Verfasser], Rolf [Akademischer Betreuer] [Daniel y Michael [Akademischer Betreuer] Hoppert. "Assessment of the functional diversity of soil microbial communities in the German Biodiversity Exploratories by metagenomics / Christiane Will. Gutachter: Rolf Daniel ; Michael Hoppert. Betreuer: Rolf Daniel". Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2011. http://d-nb.info/1042528373/34.

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33

Lucas, Lyle. "Post-Fire response of botanical and microbial communities in the succulent Karoo". University of the Western Cape, 2018. http://hdl.handle.net/11394/6428.

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Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv & Cons Biol)
Fire as a form of disturbance is unique in the way it impacts upon the environment, acting like a herbivore with a ubiquitous appetite. Consuming both dead and living material, converting complex organic molecules into organic and mineral products, which return to the soil. The role of disturbance has long been considered a driver of diversity within Mediterranean type ecosystems. Recently the interest in soil microbes has been piqued, as the importance thereof has been emphasised, particularly their role in nutrient cycling and the chelation of essential plant nutrients. The occurrence of fire results in several environmental and ecological impacts on soil, as well as the dynamics of the microbial populations present. This study explores the impact of fire as a disturbance on the plant and bulk soil microbial communities of the Succulent Karoo. This was achieved through two sub-studies, in which three different states were studied: unburnt, 7-year and 2-year post-fire. Today microbial profiles are also used as indicators of disturbance, thus many techniques exploring microbial community composition are available.

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34

Tatsumi, Chikae. "Nitrogen cycling driven by soil microbial communities in exotic black locust plantations and native oak forests in the drylands of East Asia". Kyoto University, 2020. http://hdl.handle.net/2433/253313.

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Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第22477号
農博第2381号
新制||農||1074(附属図書館)
学位論文||R2||N5257(農学部図書室)
京都大学大学院農学研究科森林科学専攻
(主査)准教授舘野隆之輔, 教授北島薫, 教授德地直子
学位規則第4条第1項該当

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35

Beaulne, Jean-Sébastien. "Analyse spatiale et multi-échelle de la distribution des bactéries dans le sol et les sédiments". Thesis, Ecully, Ecole centrale de Lyon, 2015. http://www.theses.fr/2015ECDL0037/document.

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Les bactéries ont colonisé toutes les niches écologiques de la planète. Plus précisément, les sols sont l’hôte de la plus grande biodiversité terrestre, la faune microbienne. Cette grande diversité de bactéries et leur relative ubiquité rendent difficile l’identification des variables contrôlant la distribution spatiale des bactéries vivant dans le sol. Comme les bactéries du sol jouent un rôle important dans les grands cycles biogéochimiques globaux, il est important de mieux comprendre les variables qui peuvent influencer la composition bactérienne des sols. Dans cette thèse, nous émettons l'hypothèse que l'hétérogénéité de la composition de la communauté bactérienne apparaît à la même échelle spatiale que l'hétérogénéité des propriétés physico-chimiques du sol. Afin de comprendre la relation entre la composition bactérienne des sols (à l’échelle d’une carotte de sol jusqu’à l’échelle d’une région entière du nord de la France) et les paramètres physico-chimiques du sol à différentes échelles spatiales, nous allons utiliser une approche intégrant des données issues d’analyses SIG (Système d’Information Géographique), d’analyses physico-chimiques du sol et d’analyses des communautés bactériennes du sol. A travers une suite de trois expérimentations, nous allons répondre à trois questions: Est-ce qu’une pression environnementale uniforme à une plus grande échelle (cm) peut atténuer l’hétérogénéité microbienne à micro-échelle? Est-ce que les variables ayant une distribution spatiale suivant un gradient géographique sont des variables structurant fortement la distribution spatiale des bactéries à l’échelle de ce même gradient? Est-ce que certains bio-indicateurs à grandes échelles peuvent intégrer des groupes de variables pour modéliser la distribution des bactéries pour une région entière ?
The bacteria have colonized all the niches of the planet. Specifically, soils are home of the largest terrestrial biodiversity, microbial fauna. This great diversity of bacteria and their relative ubiquity make it difficult to idendified variables driving the spatial distribution of bacteria living in the soil. As soil bacteria play a significant role in the main global biogeochemical cycles, it is important to better understand the variables that can influence bacterial composition of soils. In this thesis, we hypothesize that heterogeneity of the bacterial community composition appears at the same scale level as the heterogeneity of soil physicochemical properties. In order to understand the relationship of bacterial composition of soils (from core experiment to field study in large region in the northern France) and soil factors at different spatial scales, we will use an approach coupling GIS tools, soil physico-chemical analysis and 16S rRNA gene NGS. With Three set of experiment we will answer three questions: Can a uniform environmental pressure at a larger scale (cm) overcome microbial micro-scale heterogeneity? Are geographical gradients strong drivers of the microbial community structure at the scale of the gradient? Do large-scale geographical features that integrate groups of parameters model the differences in microbial community structure for an entire region?

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36

Buckley, Elan. "Change in the Structure of Soil Microbial Communities in Response to Waste Amendments". Thesis, Virginia Tech, 2020. http://hdl.handle.net/10919/101499.

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Soil microbial communities are affected extensively by addition of amendments to their environment. Of particular concern is the addition of poultry litter, which contains a substantial C, energy, and nutrient supply, but also antibiotic resistance genes (ARG), antimicrobials, and a multitude of microbial species. This project seeks to primarily assess if there is a change in bacterial community structure in response to poultry litter amendments to pasture land across geographically independent land across northern Georgia. It may be that changes in the relative abundance of bacterial communities also result in alteration in ARGs, and the community resistance to antibiotics (“resistome”) which in turn increases the potential threat of antibiotic resistance genes. While another part of this study will determine changes in integrons and specific ARGs, this project will focus on changes in bacterial communities and the potential functional changes in the community, which in turn have consequences for ARG levels and its horizontal transfer to various members of the soil community. Addition of waste from livestock is a historical method for increasing nutrients needed in the soil for the cultivation of crops, and in turn causes pronounced shifts in soil microbial communities due to the addition of large amounts of carbon, nutrients, foreign microbes, and other material. This study is unique because it utilizes a novel and relatively large landscape-scale to determine if there are discernable and repeatable patterns of bacterial community structure change in response to amendment regardless of exact soil type or source of chicken litter amendment. In the future, these data can also provide insight into the changes in the relative abundance antibiotic related genes associated with community change.
M.S.
Soil is complicated, both in terms of its physical makeup and the organisms that live inside of it. Predicting changes in soil based on the addition of foreign material such as chemicals or biological waste is not an easy process, and whether or not it is even possible to reliably predict those changes is a matter of some dispute. This study is designed to illustrate that such changes can in fact be reliably and consistently predicted even with regard to the addition of complicated materials to the soil. In this study, specifically, the material in question is chicken litter. A mix of the bedding and waste produced by chickens, litter is commonly handled by composting and is added to soil in farms as a fertilizer rich in organic matter. It is possible to point at specific elements of the soil such as the chemistry and bacteria and see how it is changed with the addition of chicken litter, which allows us to determine the nature and extent of the change that chicken litter has on soil. This study is conducted on a larger scale than similar experiments conducted in the past, making it apparent that these relationships exist on a repeated basis. It is the object of this study to pave the way and make it easier for scientists in the future to determine these relationships in other unique contexts.

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37

Thompson, Andrew Robert. "Heterotrophic Protists as Useful Models for Studying Microbial Food Webs in a Model Soil Ecosystem and the Universality of Complex Unicellular Life". BYU ScholarsArchive, 2019. https://scholarsarchive.byu.edu/etd/8575.

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Heterotrophic protists, consisting largely of the Cercozoa, Amoebozoa, Ciliophora, Discoba and some Stramenopiles, are a poorly characterized component of life on Earth. They play an important ecological role in soil communities and provide key insights into the nature of one of life’s most enigmatic evolutionary transitions: the development of the complex unicell. Soil ecosystems are crucial to the functioning of global biogeochemical cycles (e.g. carbon and nitrogen) but are at risk of drastic change from anthropogenic climate change. Heterotrophic protists are the primary regulators of bacterial diversity in soils and as such play integral roles in biogeochemical cycling, nutrient mobilization, and trophic cascades in food webs under stress. Understanding the nature of these changes requires examining the rates, diversity, and resiliency of interactions that occur between soil organisms. However, soils are the most taxonomically diverse ecosystems on Earth and disentangling the complexities of dynamic and varied biotic interactions in them requires a unique model system. The McMurdo Dry Valleys of Antarctica, one of the harshest terrestrial environments on Earth, serve as a model soil ecosystem owing to their highly reduced biological diversity. Exploring the functioning of heterotrophic protists in these valleys provides a way to test the applicability of this model system to other soil food webs. However, very little is known about their taxonomic diversity, which is a strong predictor of function. Therefore, I reviewed the Antarctic literature to compile a checklist of all known terrestrial heterotrophic protists in Antarctica. I found significant geographical, methodological, and taxonomic biases and outlined how to address these in future research programs. I also conducted a molecular survey of whole soil communities using 18 shotgun metagenomes representing major landscape features of the McMurdo Dry Valleys. The results revealed the dominance of Cercozoa and point to an Antarctic heterotrophic protist soil community that is taxonomically diverse and reflects the structure and composition of communities at lower latitudes. To investigate whether biotic interactions or abiotic factors were a larger driver for Antarctic heterotrophic protists, I conducted variation partitioning using environmental data (e.g. moisture, pH and electrical conductivity). Biotic variables were more significant and accounted for more of the variation than environmental variables. Taken together, it is clear that heterotrophic protists play key ecological roles in this ecosystem. Deeper insights into the ecology of these organisms in the McMurdo Dry Valleys also have implications for the search for complex unicellular life in our universe. I discuss the theoretical underpinnings of searching for these forms of life outside of Earth, conclude that they are likely to occur, and postulate how future missions could practically search for complex unicells.

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38

Karolski, Bruno. "Metagenômica comparativa e perfil metabólico in silico de solos no município de Cubatão, SP". Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-05112013-111556/.

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Cubatão, o maior pólo industrial da américa latina também já foi uma das cidades mais poluídas do mundo. Os 30 anos de intensa atividade industrial vêm pressionando o meio ambiente com substâncias tóxicas e afetando gravemente a saúde da população. Dentre as substâncias contaminantes mais importantes da região estão os derivados de petróleo como o benzeno, tolueno, etilbenzeno e xilenos. Conhecidos como BTEX, eles são produzidos e utilizados em larga escala e a contaminação ocorre frequentemente através de vazamentos. Nos solos, devido à sua solubilidade em água, essas substâncias podem se espalhar por longas distâncias a partir do ponto afetado contaminando locais distantes. Já foi comprovada a capacidade de micro-organismos de sobreviver e até utilizar BTEX como fonte de carbono. Os micro-organismos adaptados catabolizam os contaminantes transformando-os em substâncias menos tóxicas e até mesmo eliminando-os do ambiente, capacidade de grande interesse econômico e ambiental. Nessa linha, nossa proposta visa o estudo das comunidades microbianas de solos afetados e não afetados por BTEX. Para isso foi utilizada a metagenômica como abordagem de estudo identificando-se diferenças qualitativas e quantitativas nas estruturas microbianas de três diferentes locais do município de Cubatão, sendo um deles afetado diretamente por BTEX. Pelo método utilizado e aqui desenvolvido, foi possível identificar um panorama metabólico geral identificando-se genes relevantes e o potencial de degradação de hidrocarbonetos aromáticos de micro-organismos conhecidos e desconhecidos, revelando melhor o potencial metabólico dos solos identificados. Os resultados apresentados podem contribuir para um melhor entendimento da dinâmica in situ de uma comunidade microbiana afetada por BTEX assim como melhorar o conhecimento sobre a comunidade microbiana de um local altamente impactado como Cubatão.
Cubatão is the largest industrial site in Latin America and was in the past one of the most polluted cities in the world. 30 years of intense industrial activity has pushed environmental limits with toxic substances and has severely affected the inhabitants\' health. Among the contaminants found in the region, the petroleum derivatives benzene, toluene, ethylbenzene and xylenes are the most important. Known collectively as BTEX, they are produced and used at a large scale and contamination frequently occurs. Because it is highly soluble in water, when in soil BTEX can spread long distances from the original contamination site, thus affecting large areas. Some microorganisms are known to live in contaminated environments and use contaminants such as BTEX as a unique carbon source for energy production. They catabolize contaminants into less dangerous products or even eliminate them from environment, a feature which has great commercial and environmental interest. We therefore compared the microbial communities in soils which were affected and un-affected by BTEX contamination. To this end, we used a metagenomics approach and developed a comparison method to identify microorganisms and degradation potential of soils studied. We found qualitative and quantitative differences in microbial structures from three different sites in Cubatão County, one of which is contaminated with BTEX. We constructed a metabolic overview identifying important genes, degradation potential and microorganisms related to BTEX degradation. The results presented here could contribute to understanding the in situ dynamics of a BTEX affected microbial community as well as improving our knowledge of the microbial community of Cubatão, a highly environmentally impacted place.

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39

Souza, Dennis Góss de. "Comparative analyses of microbial phylogenetic and functional processes following long-term land-use change". Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/91/91131/tde-05012016-152905/.

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In the last years, microbial ecologists have dramatically increased their efforts to elucidate the \"black box\" of patterns and processes that modulate the diversity and functionality of soil microorganisms, examining their genetic diversity (e.g. through metagenomic) and measuring their functional characteristics. The aim of this thesis was to evaluate the interaction of the ecological processes of dispersion, diversification, selection and genetic drift on (1) the soil microbial communities, after conversion of forest to grassland or no-till cropping in long-term and (2) on the microbial communities in the rhizosphere of soybean in long-term no-till system. The cultivation of grassland in long-term led to a homogenizing selection of microbial communities, reducing beta-diversity, with consequent changes in the soil functions related to stress. No-till long-term led to minor changes of diversity, maintaining the functions found in the forest. The soybean plant has shown homogenizing power selection, and this increased with time. However, the functions selected in the rhizosphere were maintained, indicating functional resilience.
Nos últimos anos, ecologistas microbianos aumentaram drasticamente seus esforços para elucidar a \"caixa preta\" dos padrões e processos que modulam a diversidade e funcionalidade dos microrganismos do solo, examinando sua diversidade genética (e.g. através de metagenômica) e medindo suas características funcionais. O objetivo dessa tese foi avaliar a interação dos processos ecológicos de dispersão, diversificação, seleção e deriva gênica, sobre (1) as comunidades microbianas do solo, após a conversão da floresta em pastagem ou plantio direto, em longo período e (2) sobre as comunidades microbianas da rizosfera de soja, em sistema de plantio direto, em longo período. O cultivo de pastagens em longo período levou a uma seleção homogeneizante das comunidades microbianas, reduzindo a beta-diversidade, com conseguinte alteração em funções no solo relacionadas ao estresse. O plantio direto em longo período levou a uma menor alteração da diversidade, com manutenção das funções encontradas na floresta. A planta de soja demonstrou poder de seleção homogeneizante, e este aumentou com o tempo. Contudo, as funções selecionadas na rizosfera foram mantidas, indicando resiliência funcional.

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40

Moreno, Lilliana I. "The Effect of Sample and Sample Matrix on DNA Processing: Mechanisms for the Detection and Management of Inhibition in Forensic Samples". FIU Digital Commons, 2015. http://digitalcommons.fiu.edu/etd/1764.

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The presence of inhibitory substances in biological forensic samples has, and continues to affect the quality of the data generated following DNA typing processes. Although the chemistries used during the procedures have been enhanced to mitigate the effects of these deleterious compounds, some challenges remain. Inhibitors can be components of the samples, the substrate where samples were deposited or chemical(s) associated to the DNA purification step. Therefore, a thorough understanding of the extraction processes and their ability to handle the various types of inhibitory substances can help define the best analytical processing for any given sample. A series of experiments were conducted to establish the inhibition tolerance of quantification and amplification kits using common inhibitory substances in order to determine if current laboratory practices are optimal for identifying potential problems associated with inhibition. DART mass spectrometry was used to determine the amount of inhibitor carryover after sample purification, its correlation to the initial inhibitor input in the sample and the overall effect in the results. Finally, a novel alternative at gathering investigative leads from samples that would otherwise be ineffective for DNA typing due to the large amounts of inhibitory substances and/or environmental degradation was tested. This included generating data associated with microbial peak signatures to identify locations of clandestine human graves. Results demonstrate that the current methods for assessing inhibition are not necessarily accurate, as samples that appear inhibited in the quantification process can yield full DNA profiles, while those that do not indicate inhibition may suffer from lowered amplification efficiency or PCR artifacts. The extraction methods tested were able to remove >90% of the inhibitors from all samples with the exception of phenol, which was present in variable amounts whenever the organic extraction approach was utilized. Although the results attained suggested that most inhibitors produce minimal effect on downstream applications, analysts should practice caution when selecting the best extraction method for particular samples, as casework DNA samples are often present in small quantities and can contain an overwhelming amount of inhibitory substances.

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41

Zayed, Ahmed Abdelfattah. "Microbe-Environment Interactions in Arctic and Subarctic Systems". The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1562494472055278.

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42

HUANG, YI-SHIANG y 黃翊翔. "Using Metagenomic Method to Analyze Soil Microbial Communities in Taitung". Thesis, 2017. http://ndltd.ncl.edu.tw/handle/kjsae4.

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碩士
大葉大學
生物產業科技學系
105
Microbes are small biological lives distribute all over the earth, including soil, rivers, ocean and inside the body of animals. Microbes in the environment would form communities that interact with the environment and influence the environmental conditions. Traditional studies usually relied on pure culture of the bacterium, however, only very few portion of the environment bacteria can be cultured using conventional methods. Therefore, analysis of specific universal DNA such as the gene of ribosomal subunit, and use the sequences as markers for taxonomic analysis. This thesis analyzed the bacterial communities in the soil sampled from paddy field in Taitung. A total of 18 soil samples were taken and marked by their global positions. DNA was extracted and the 16S rDNA from each sample was amplified by polymerase chain reaction using V3 region primers. The amplified DNA was then purified and sequenced by next generation sequencer. The data was blasted by NCBI BLASTN program on a local server, and the results were analyzed by MEGAN software.

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43

BACCI, GIOVANNI. "Mining Microbiomes. Computational Biology approaches to uncover the complexity of bacterial communities". Doctoral thesis, 2015. http://hdl.handle.net/2158/986409.

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44

Yu, Wen-Han y 郁文涵. "Construction of the soil metagenomic library from the agricultural soil and discovery of the novel biocatalysts". Thesis, 2004. http://ndltd.ncl.edu.tw/handle/38921573501607245423.

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碩士
國立臺灣大學
農業化學研究所
92
Environmental microorganisms hold the greatest abundant resource applied to medicine, food and industry. But the source of natural-products in microbiota is constrained because the majority of the microbial species in the biosphere cannot be cultured in the laboratory; these sources must be discovered by direct-cloning method and applied further by construction of metagenomic library. A large number of different methods have been published for extraction of total microbial soil DNA. But DNA fragments were sheared seriously due to physical disruption. We have developed newer method to extract DNA from the agricultural soils and obtain high molecular weight and pure DNA (100~1000kb). To access these uncultured genetic materials, we have used bacterial artificial chromosome (BAC) vector to construct 12,000-clones libraries of microbiota genomes. The average insert size of BAC library we prepared is about 60 kb. The phylogenetic analysis of 16S rRNA and 18S rRNA gene sequences from the soil metagenomic library approximately belonged to the undescribed microbes, and revealed the divergent microbial phyla such as Proteobacteria, Nitrospina, High G+C Gram positive, Sporomusa, Ascomycota, and Basidiomycota. N-acylamino acid racemase (NAAAR) is an enzyme that specifically catalyzes the racemization of N-acetyl-D, L-amino acids but not general amino acids and was applied to the production of optically active amino acids. The BAC library was screened by colony PCR and seminested PCR for sequences similar to the high conserved region of NAAAR and related homologous proteins. Four positive recombinant clones (pBAB1 to pBAB4) of 12,000 clones were obtained by double PCR screening. Analysis the cell extracts of four positive clones with different substrates and cofactors, the NAAAR activity in pBAB3 and pBAB4 were two folds higher than the negative control E.coli/BAC. All of related naaar genes were identified during subcloning and sequencing of the inserts of pBAB1 to pBAB4. After analysis of BLAST, ORF finder, and CD-search programs, the deduced gene functions showed similarities to homocysteine methyltransferase (pBAB1), Hypothetical protein (pBAB2), Uncharacterization protein (pBAB3), sensor kinase (pBAB4). But the sequences homologous to naaar gene cannot be found. Therefore, it is possible that the pBAB3 and pBAB4 utilize the substrates (N-acetyl-D-methionine, N-acetyl-D-phenylalanine) in a way with the unknown functions. Based on these data, we provide a strong platform for searching the novel-type enzymes.

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45

Kai-BoChang y 張凱博. "Metagenomic analyses on the bioremediation process of soil contaminated with petroleum and heavy metal". Thesis, 2019. http://ndltd.ncl.edu.tw/handle/f9xn76.

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碩士
國立成功大學
生命科學系
107
Since the 20th century, the production of petroleum created an unprecedented prosperity of human civilization. However, due to factors such as transportation and disposal, oil pollution has become a major issue in environmental protection. On the other hand, heavy metal pollution has a long history of human mining. Heavy metals include gold, silver and copper. As the mining area expands, the pollution area also increases. In Taiwan, many heavy metal pollution events in history, such as the RCA pollution. For escaping environmental pollution, animals can leave and find new lands, but plants and microorganisms cannot freely move. In the face of adversity, plants and microorganisms use different bio-metabolism mechanisms to absorb or degrade these toxic substances and protect cells or plants from toxicity. With the rapid development of genomic technology, we are able to study the microbial composition in environment which has pollutants. In this study, I focus on two kinds of contaminated soil, petroleum and heavy metal, and investigate the bacterial community with targeted Metagenomics by 16S rRNA gene. I found that Proteobacteria and Bacteroidetes are the main components of bacterial community in petroleum-polluted soil. Proteobacteria and Actinobacteria are the main components of bacterial community at the site with heavy metal pollution. According to the analysis of specific microbiome, the proportion of Firmicutes and Tenericutes in petroleum-polluted soil is higher than heavy metal contaminated soil. In Macaranga tanarius and Miscanthus floridulus rhizosphere, Proteobacteria and Actinomycetes are the main components of the core microbiome of the two plants. The above results shows that in different polluted area has their own specific microbial community, and different kinds of plants grow in contaminated soil share with similar core microbiome. This thesis uses metagenomics to explore more deeply, hoping to find the best way to degrade pollutants and protect the earth.

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46

Abbasian, Fioruz. "Investigation of the total petroleum hydrocarbon degrading microorganisms in soil and water: a metagenomic approach". Thesis, 2017. http://hdl.handle.net/1959.13/1335807.

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Research Doctorate - Doctor of Philosophy (PhD)
Hydrocarbons are relatively recalcitrant compounds and are classified as high priority pollutants. However, these compounds are slowly degraded by a large variety of aerobic and anaerobic microorganisms. Although the corresponding genes in many phylogenetic groups of microbial species show different levels of diversity in terms of the gene sequence, the organisation of the genes in the genome or on plasmids and the activation mode of several microorganisms show identical hydrocarbon degrading genes. Since the majority of microorganisms in natural environments cannot be cultured in laboratory media, culture-based systems are unable to estimate the full microbial diversity of an environment. Metagenomic methods, however, employ sequencing procedures for the determination of the microbial diversity of a community and for examining a particular functional ability of microorganisms in the environment using genomic DNA obtained directly from environmental samples. Application of metagenomic methods provides a huge amount of data that can be analysed only by using powerful computational bioinformatics tools. In this study, we used next generation technology and metagenomic analysis to investigate the microbial diversity in crude oil and crude oil contaminated soils and to find the functional genes involved in the degradation of hydrocarbons in crude oil. The findings from this study can be used for bioremediation of crude oil spills and also for improvement of the quality of crude oil derivatives in terms of removal of sulfur and nitrogen. As a part of this study, we report a list of microorganisms that are abundant in the crude oil and the crude oil contaminated soil. Furthermore, we found a new operon responsible for removal of sulfur from dibenzothiophenes. The three genes in this operon were cloned and their activities measured in cell free condition.

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47

Mtimka, Sibongile. "Metagenomic discovery and characterisation of restriction endonuclease from Kogelberg Biosphere Reserve". Diss., 2018. http://hdl.handle.net/10500/25147.

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Restriction endonucleases are a group of enzymes that cleave DNA at or around specific sequences, which are typically palindromic. A fosmid library was constructed from a metagenome isolated from soil from the Kogelberg Nature Reserve, Western Cape and was functionally screened for restriction endonucleases. Next-generation (NGS) Illumina sequencing technology was used to identify putative endonucleases. The sequence data generated was assembled and analysed using CLC Bio Genomics Workbench and bioinformatics tools (NCBI BLAST, REBASE and MG-RAST). Using these tools, genes encoding restriction-modification systems and endonuclease homologues were discovered. Three genes were identified and were recombinantly produced in Rosetta™ (DE3) pLysS and purified with IMAC using Ni-TED resin and subsequently characterised. These three genes were selected based on the identity percentage when compared to sequences on the NCBI database. Production of Endo8 was scaled up using 2 l fermenter and the purification done using ÄKTA Avant 150 FPLC using a HiScale 50 column packed with Ni-TED resin and the total amount of protein achieved was 58.82 mg.g-1. The productivity achieved at 17 hours (8 h harvest) was 2-fold greater than at 12 hours. Endonuclease activity of endo8 and endo52 was tested, both exhibited strong non-specific activity at 37 °C with an incubation period of 30 min. This work demonstrates that environmental soil samples are a valuable source for discovery of novel enzymes and also the utility of functional metagenomics to discover and purify these enzymes. These endonucleases may contribute to the next generation of reagent enzymes for molecular biology research.
Chemistry
M. Sc. (Life Sciences)

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48

Will, Christiane. "Assessment of the functional diversity of soil microbial communities in the German Biodiversity Exploratories by metagenomics". Thesis, 2011. http://hdl.handle.net/11858/00-1735-0000-0006-AE01-2.

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49

Liao, Yi-Ting y 廖怡婷. "Using root soil & pore water microcosm incubations coupled with metagenomic and qPCR techniques to probe primary Hg-methylating guilds in the paddy rhizosphere". Thesis, 2017. http://ndltd.ncl.edu.tw/handle/gscb7n.

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碩士
國立中央大學
環境工程研究所
105
Recent studies have shown that in addition to intake of piscivorous fish, rice consumption is another critical route of human expose to methylmercury (MeHg), the most toxic form of mercury (Hg) in the environment. Nonetheless, there is still a paucity of data on the biogeochemical mechanisms that control the formation (in the rhizosphere), uptake (by root), and eventual accumulation of MeHg (in rice grain) in the paddy ecosystem. To gain an in-depth understanding of this undesirable environmental process, in 2014 we began our investigation and initiated fieldwork at rice paddies that were proximal to the coal-fired power station in Taichung. Preliminary results of microcosm incubations (of root soil samples) suggested that sulfate-reducing bacteria (SRB) might be the primary Hg methylators at our study sites. However, because the incubation tests were conducted with synthetic media instead of pore water, there was a potential fraud in our methodology that might have resulted in a bias in our observations. Further, a detail look at the microbial community structure has not yet carried out. In light of this, here we aimed at rectifying our former protocols of microcosm incubations to confirm the role of SRB as the principal Hg methylating guild. More importantly, this study incorporated certain advanced molecular biology techniques including real-time polymerase chain reactions (qPCR), metagenomics, as well as the next-generation sequencing (NGS) into this inquiry, hoping to obtain a complementary interpretation of methylation results at the cellular level. Results from root soil/pore water incubations assayed with Hg methylation & demethylation confirmed that SRB indeed were the major Hg-methylating guild in the rhizosphere of our study sites. Relative quantification of the hgcA level by qPCR also indicated that Deltaproteobacteria was the principal Hg-methylators at the class level, consistent with the aforementioned role of SRB. In addition, our data suggested that iron-reducers and methylotrophic- and hydrogentrophic-methanogens, while not prominent, might as well play a certain role in MeHg production in paddies. However, metagenomic analysis of 16S rRNA genes showed that Geobacter was the most abundant genus in all samples, suggesting that there are a significant amount of unknown Hg-methylating microbes inhabiting in paddies that await to be identified. On the basis of all these results, time-course experiments focusing on RT-PCR and RT-qPCR of mRNA transcribed from the hgcAB gene cluster are warranted for the future study to pinpoint Hg-methylators at the species level. Ultimately, information gain from this type of investigations may entail us to devise more efficient and sounder remediation strategies to deal with Hg contamination issues in farmland.

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Tran, Huyen-Trang y 陳玄莊. "Metagenomic and whole-genome analyses revealed a long-term impact of dioxin contamination from the US-Vietnam Waron the soil microbiomes in Central Vietnam". Thesis, 2019. http://ndltd.ncl.edu.tw/handle/csy3ym.

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Tesis sobre el tema "Soil metagenomic"

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