Orientador: Valeria Maia de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T21:04:00Z (GMT). No. of bitstreams: 1 Crespim_Elaine_M.pdf: 1613061 bytes, checksum: 0510cf7cad319e1cd26f150b983f83bd (MD5) Previous issue date: 2008 / Resumo: A abordagem tradicional empregada em estudos de Microbiologia Ambiental, baseada em métodos de isolamento seletivo e cultivo de microrganismos em laboratório, embora seja útil para a determinação do potencial fisiológico dos organismos isolados, é inadequada para a realização de uma caracterização abrangente da comunidade microbiana destes ambientes ou para detectar microrganismos de difícil cultivo ou que vivem em consórcios. O emprego de técnicas moleculares em estudos de comunidades microbianas, as quais envolvem o isolamento direto de DNA a partir de amostras ambientais, a amplificação de genes conservados pela técnica de PCR e a análise de seqüências de rDNA 165, tem demonstrado resultados promissores em Ecologia Microbiana, uma vez que permite identificar organismos de ambientes naturais sem a necessidade de cultivo dos mesmos. A detecção de microrganismos com potencial para biodeterioração, biodegradação e biocorrosão encontrados em depósitos petrolíferos é de grande importância na medida em que estes organismos podem estar relacionados com a perda da qualidade do petróleo nos reservatórios e etapas subseqüentes de exploração (extração, . armazenamento e refino). O presente trabalho teve como objetivos desenvolver um método molecular para a detecção rápida de grupos específicos de microrganismos degradadores de hidrocarbonetos e avaliar a sua distribuição e abundância em diferentes amostras de óleo e água de formação provenientes de depósitos petrolíferos da bacia de Campos (RJ). Nossos resultados revelaram a presença dos grupos Bacíllus spp., Streptomyces spp., Achromobacter xy/osoxidans, Bacíllus pumilus, Micrococcus spp. e Dietzia spp. nas amostras de petróleo estudadas. Alguns destes microrganismos apresentaram ampla ocorrência, embora em baixa abundância, nas amostras dos cinco poços avaliados, independentemente do grau de biodegradação dos óleos e condições de pro{undidade e temperatura dos reservatórios. O desenvolvimento de um método molecular para a rápida detecção de grupos de microrganismos potencialmente biodegradadores em amostras ambientais seria extremamente útil como uma ferramenta de apoio para a avaliação da qualidade do óleo em reservatórios de produção / Abstract: The traditional approach used in Environmental Microbiology studies, based on selective isolation and cultivation methods, although very useful for determination of the physiological potential of the isolated organisms, is inadequate for a broad characterization of the microbial community in these environments, since it does not allow the recovery of fastidious microorganisms or the ones that live in consortia. The use of molecular techniques in microbial community studies, which involve the direct DNA isolation from environmental samples, amplification of conserved genes by PCR and sequence analysis, has shown promising results in Microbial Ecology, as it allows the identification of organisms trom natural environments without the need of cultivation. The detection of microorganisms with potential for biodeterioration, biodegradation and biocorrosion in petroleum deposits is of great importance, as these organisms may be related to a decrease in petroleum quality in the reservoirs and subsequent exploration steps (extraction, storage and refining). The present work aimed at developing a molecular method for the rapid detection of specific groups of hydrocarbondegrading microorganisms and evaluating their distribution and abundance in different oil and formation water samples originated trom petroleum reservoirs in the Campos basin (RJ). Our results revealed the presence of the target groups Bacillus spp., Streptomyces spp., Achromobacter xylosoxidans, Bacillus pumilus, Micrococcus spp. and Dietzia spp. in the environmental samples under study. Some of these microorganisms were of broad occurrence, although in low abundance, in ali the five samples trom the petroleum wells analyzed, in spite of the oil biodegradation levei and depth and temperature conditions. The development of a molecular method for the rapid detectión of specific groups of biodegrading microorganisms in environmental samples would be extremely useful as a supporting toei for the evaluation of oil. quality in the production reservoirs / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

Two weathered, petroleum contaminated soils were studied to determine if weathered products are amenable to bioremediation and to determine which TPH (total petroleum hydrocarbon) fractions were degrading during particular time frames under different remediation alternatives. Delineation of fractional degradation patterns results in inferences regarding the efficacy of different treatment methods on various petroleum products. A sandy loam and a clay soil were both studied to determine if soil matrix affects the degradation patterns. The experimental matrix included sacrificial static microcosms, soil columns and aerated slurry reactors. Both soils were evaluated under all bioreactor configurations using both a nutrient amended water and a water lacking nutrients. Controls were also used to evaluate abiotic losses. Biodegradation rates generally followed a biphasic pattern, initially rapid then followed by a slow or stagnant period. Degradation rates increased from static microcosms to soil columns to slurry reactors. The slow phase was controlled by the presence of recalcitrant compounds which decreased in number and concentrations from static microcosms to columns to slurry reactors, and generally with nutrient addition. Nutrient addition enhanced degradation for all sandy soil treatments, but only slurry reactor treatment for the clay soil. The entire TPH spectrum was broken down into five minute parcels based on GC elution time. The compounds that eluted quickest generally were the easiest to degrade. The fraction that effectively covered the TPH components in gasoline was well removed under all treatment modes. All nutrient amended studies resulted in rapid essentially complete removal of the light fractions within two weeks. The fraction encompassing the middle distillates such as diesel fuel and jet fuel was degraded under all treatment methods, however only the slurry reactors resulted in final TPH levels that would have met regulatory limits. Fractions that eluted after 15 minutes were not effectively degraded by the static microcosms or the soil columns for either soil, eliminating in situ bioremediation as a viable treatment alternative for crude oil, fuel oil and gas oil contamination, at concentrations present in this study. Persistence of recalcitrant compounds was the major factor leading to the poor biodegradation observed in the static microcosms and soil columns. Fractional degradation was highly dependent on the initial concentration of the fraction. Generally, fractions present in the largest concentrations degraded fastest. / Master of Science

Orientador: Valéria Maia Merzel / Dissertação (mestrado) - Universidade Estadual de Campinas, Insituto de Biologia / Made available in DSpace on 2018-08-16T20:58:52Z (GMT). No. of bitstreams: 1 Silva_TiagoRodriguese_M.pdf: 5632148 bytes, checksum: 82526f541aaf1c9b32cf5fbca6bc03aa (MD5) Previous issue date: 2010 / Resumo: Estudos realizados em reservatórios de petróleo têm evidenciado que parte da microbiota associada a este tipo de ambiente é representada por bactérias e arqueias de distribuição geográfica bastante ampla e que diversos destes organismos têm potencial para transformar compostos orgânicos e inorgânicos, atuando na interface óleo-água dos reservatórios. A investigação de micro-organismos com potencial para biodeterioração, biodegradação e biocorrosão encontrados em depósitos petrolíferos é de grande importância, uma vez que estes organismos podem estar relacionados com a perda da qualidade do petróleo nos reservatórios e etapas subseqüentes de exploração. Este estudo teve como finalidade comparar a microbiota presente em amostras de óleo de dois poços de petróleo terrestres da Bacia Potiguar (RN), identificados como GMR75 (poço biodegradado) e PTS1 (poço não-biodegradado). As comunidades microbianas foram estudadas usando técnicas de cultivo (enriquecimentos microbianos e isolamento) e independentes de cultivo (construção de bibliotecas de genes RNAr 16S). Os micro-organismos cultivados de ambos os poços mostraram-se afiliados aos filos Actinobacteria, Firmicutes e Proteobacteria. As bibliotecas de gene RNAr 16S foram construídas a partir de DNA total extraído do petróleo bruto. Ambas as bibliotecas de bactérias revelaram uma grande diversidade, com 8 filos diferentes para o poço GMR75, Actinobacteria, Bacteroidetes, Deferribacteres, Spirochaetes, Firmicutes, Proteobacteria, Thermotoga e Synergistetes, e 5 filos para o poço PTS1, Actinobacteria, Chloroflexi, Firmicutes, Proteobacteria e Thermotogae. A biblioteca de genes RNAr 16S de arqueias só foi obtida para o poço GMR75 e todos os clones encontrados mostraram-se relacionados a membros da ordem Methanobacteriales. Os resultados de diversidade sugerem que a metanogênese é o processo terminal dominante no poço, o que indica uma biodegradação anaeróbia. A comparação dos estudos dependente e independente de cultivo mostrou que alguns gêneros, como Janibacter, Georgenia, Saccharopolyspora, Tessaracoccus, Brevundimonas e Brachymonas não foram encontradas na abordagem independente de cultivo, sugerindo que mais clones devam ser seqüenciados para cobrir toda a diversidade presente na amostra. Nossa hipótese de que poderia haver algum agente antimicrobiano inibindo o crescimento de bactérias degradadoras de hidrocarbonetos no poço não-biodegradado não foi confirmada. No entanto, durante os testes realizados, uma bactéria, Bacillus pumilus, isolada em estudos anteriores de reservatórios da Bacia de Campos, apresentou resultados positivos de inibição para todas as linhagens testadas como indicadoras, e os testes de caracterização do composto revelaram ser este um diterpeno da classe das Ciatinas. / Abstract: Recent studies from oil fields have shown that microbial diversity is represented by bacteria and archaea of wide distribution, and that many of these organisms have potential to metabolize organic and inorganic compounds. The potential of biodeterioration, biodegradation and biocorrosion by microorganisms in oil industry is of great relevance, since these organisms may be related with the loss of petroleum quality and further exploration steps. The aim of the present study was to compare the microbial communities present in two samples from terrestrial oil fields from Potiguar basin (RN - Brazil), identified as GMR75 (biodegraded oil) and PTS1 (non-biodegraded oil). Microbial communities were investigated using cultivation (microbial enrichments and isolation) and molecular approaches (16S rRNA gene clone libraries). The cultivated microorganisms recovered from both oil-fields were affiliated with the phyla Actinobacteria, Firmicutes and Proteobacteria. The 16S rRNA gene clone libraries were constructed from metagenomic DNA obtained from crudeoil. Both bacterial libraries revealed a great diversity, encompassing representatives of 8 different phyla for GMR75, Actinobacteria, Bacteroidetes, Deferribacteres, Spirochaetes, Firmicutes, Proteobacteria, Thermotogae and Synergistetes, and of 5 different phyla, Actinobacteria, Chloroflexi, Firmicutes, Proteobacteria and Thermotoga, for PTS1. The archaeal 16S rRNA clone library was obtained only for GMR75 oil and all phylotypes were affiliated with order Methanobacteriales. Diversity resuts suggest that methanogenesis is the dominant terminal process in GMR75 reservoir, driven by anaerobic biodegradation. The cross-evaluation of culture-dependent and independent techniques indicates that some bacterial genera, such as Janibacter, Georgenia, Saccharopolyspora, Tessaracoccus, Brevundimonas and Brachymonas, were not found using the the 16S rRNA clone library approach, suggesting that additional clones should be sequenced in order to cover diversity present in the sample. Our hypothesis that biodegrading bacterial populations could be inhibited by antimicrobialproducing microorganisms in the non biodegraded oil field (PTS1) was not confirmed. However, one Bacillus pumilus strain, previously isolated from Campos Basin reservoirs, showed positive results in inhibitory tests for all indicator strains. Chemical analyses allowed us to identify the compound as a diterpen from the Cyathin class. / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

Soil contamination by gasoline underground storage tanks is a critical environmental problem. The results herein show that in situ bioremediation using indigenous soil microorganisms is the method of choice. Five sites were selected for bioremediation based on the levels of benzene, toluene, ethylbenzene and xylene and the amount of total petroleum hydrocarbons in the soil. Bacteria capable of degrading these contaminants were selected from the contaminated sites and grown in 1,200 I mass cultures. These were added to the soil together with nutrients, water and air via PVC pipes.

The development of analytical methods for emerging contaminants creates many unique challenges for analytical chemists. By their nature, emerging contaminants have inherent data gaps related to their environmental occurrence, fate, and impact. This dissertation is a compilation of three studies related to method development for the structural identification of emerging contaminants, the detection and quantification of chemicals used in unprecedented quantities and applications, and the extraction of compounds from complex matrices where the solvent-solute-matrix interactions are not completely understood. The three studies present analytical methods developed for emerging contaminants in complex matrices, including: fluorochemical surfactants in aqueous film-forming foams, oil dispersant surfactants in seawater, and fullerene nanomaterials in carbonaceous solids. Aqueous film-forming foams, used in military and commercial firefighting, represent environmentally-relevant commercial mixtures that contain a variety of fluorochemical surfactants. Combining the surfactant-selective ionization of fast atom bombardment mass spectrometry with high resolution mass spectrometry, chemical formulas for 11 different fluorochemical classes were identified. Then AFFF-related patents were used to determine the structures. Of the eleven classes of fluorochemicals, ten have little, if any, data on their environmental occurrence, fate, and potential impacts in the peer-reviewed literature. In addition, nine of the identified classes had either cationic or zwitterionic functionalities and are likely to have different transport properties compared to the well-studied anionic fluorochemicals, such as perfluorooctanoate. After the Deepwater Horizon oil spill in the summer of 2010, one of the emergency response methods for the mitigation of the oil's environmental impact was the use of unprecedented amounts of oil dispersant to break down the oil slick and encourage biodegradation. This event illustrated the need for rapid analytical method development in order to respond to the potential environmental disaster in a timely manner. Using large volume injection liquid chromatography with tandem mass spectrometry, an analytical method was developed for the trace analysis of the multiple dispersant surfactant classes and the potential degradation products of the primary surfactant. Limits of detection ranged from 49 ��� 3,000 ng/L. The method provided excellent recovery (86 ��� 119%) and precision (10 ��� 23% RSD), while also accommodating for the high salinity of seawater samples and analyte contamination. Despite the fact that fullerene nanomaterials have been studied for almost three decades, research is still being conducted to fully understand the environmental properties of these materials. Previous studies to extract fullerenes from environmental matrices have resulted in low efficiency, high variability, or the extraction efficiencies have gone unreported. Extraction by ultrasonication with toluene and 1-methylnaphthalene increased the recovery 5-fold of a spiked, isotopically-labeled C������ surrogate from carbon lampblack as compared to that of the conventional approach of extracting with 100% toluene. The study revealed the importance of evaluating experimental variables such as extraction solvent composition and volume, and sample mass, as they have a significant impact on the quantitative extraction of fullerenes from environmental matrices. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from Aug. 15, 2012 - Aug. 15, 2013