Dissertations / Theses on the topic 'Petroleum - Microbial'

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
O petróleo é a principal fonte de energia no Brasil, onde o consumo de óleo continua subindo desde 2013, atingindo atualmente 2.2% do total de energia consumida no mundo. A descoberta recente de petróleo nas baias do Espirito Santo, Campos e Santos, pode representar uma excelente oportunidade para atender as demandas energéticas nacionais. Entretanto, a exploração de petróleo oferece riscos a microbiota e toda a vida marinha. Microrganismos são responsáveis pela ciclagem de nutrientes, podem degradar compostos orgânicos recalcitrantes e muitas espécies são reportadas como sensíveis à contaminação por hidrocarbonetos do petróleo. Esse trabalho teve o objetivo de avaliar as alterações na comunidade microbiana em solos sob a contaminação por petróleo e avaliar os efeitos do Co-produto de biodiesel (BCP) como um agente protetor da microbiota do solo perante a adição de petróleo. Foram utilizados solos da Ilha da Trindade, e da estação de pesquisa Highfield no Rothamsted Research, UK. Foram montados microcosmos com 20 gramas de solo e os tratamentos utilizaram petróleo intemperizado. Os solos foram incubados a 26° C com correção da umidade para cerca de 60% da capacidade de retenção de água dos solos. Foi utilizada a medição de evolução de CO2 para avaliar a atividade do solo, durante o período de incubação, e a extração de DNA genômico do solo, ao final do período de incubação, para avaliar as mudanças nas comunidades microbianas dos tratamentos e controles. O DNA foi submetido para o sequenciamento de amplicons de 16S rDNA para a avaliação de Bacteria e Archaea e de amplicons da região ITS1 para a avaliação de Fungos utilizando a plataforma Illumina HiSeq. Foi feita a comparação das diversidades alpha e beta e análise das alterações taxonômicas. Essa tese está dividida em dois capítulos. O primeiro descreve os efeitos do petróleo nas comunidades microbianas do solo da Ilha da Trindade. No segundo capítulo foi testado o efeito protetor do BCP sobre a microbiota dos solos da Ilha da Trindade, do campo Bare Fallow e do campo Grassland do Rothamsted Research contra a adição de óleo. O petróleo teve um grande efeito negativo sobre a diversidade microbiana da Ilha da Trindade, mas não mudou a diversidade microbiana dos solos agrícolas do Rothamsted. A comparação taxonômica mostrou aumento do filo Actinobacteria, mudanças em várias classes de Proteobacteria e redução da classe Nitrosphaerales do filo Archaea. Esse é o primeiro esforço para aquisição de conhecimento sobre o efeito da contaminação de solos de uma ilha oceânica brasileira com petróleo. Essa informação é importante para guiar qualquer futura estratégia de biorremediação que se faça necessária.
Crude oil is still the dominant energy source in Brazil where oil consumption keeps rising since 2013, reaching nowadays 2.2% of the world‟s energy consumption. A recent discovery of crude oil reservoirs at the Espirito Santo, Campos and Santos basins, can represent an excellent opportunity to meet the country‟s economic and energetic demands. However, offshore exploration offers risks to the microbiota and the whole sea life. Microbes are responsible for nutrient cycling can degrade recalcitrant organic compounds and several species have been reported as sensitive to petroleum hydrocarbons. This work aimed to evaluate microbial community shifts in soils under crude oil contamination and assess the effects of Biodiesel co-product (BCP) as a protecting agent of soil microbiota under crude oil addition. We used soils from the Trindade Island and from the Highfield research station at Rothamsted Research, UK. We assembled microcosms of 20 grams and contaminated the soils using weathered crude oil. Soils were incubated at 26° C with moisture correction to ca. 60% water holding capacity. We used CO2 evolution measurements to evaluate soil activity, during the incubation, and soil genomic DNA extraction, at the end of incubation period, to evaluate microbial community changes from treatments and controls. DNA was submitted to amplicon sequencing of 16S rDNA for Bacteria and Archaea and the ITS1 region for Fungi using Illumina MiSeq platform. We compared alpha and beta-diversity and taxonomic shifts. This thesis is divided in two chapters. The first describes the effects of crude oil on Trindade Island‟s soil microbial communities. In the second chapter we tested the protective effects of BCP on Trindade Island, Rothamsted‟s Bare Fallow and Grassland soils, against the amendment with crude oil. Crude oil had a major negative effect on microbial diversity for Trindade Island, but didn‟t change the diversity of Rothamsted agricultural soils. Taxonomy comparisons showed rise of the Actinobacteria phylum, shifts in several Proteobacteria classes and reduction of the Archaea class Nitrososphaerales. This is the first effort in acquiring knowledge concerning the effect of crude oil contamination in soils of a Brazilian oceanic island. This information is important to guide any future bioremediation strategy that can be required.

Environmental pollution by petroleum hydrocarbons has serious environmental consequences on critical natural resources upon which all living things (including mankind) largely depend. Microbial fuel cells (MFCs) could be employed in the treatment of these environmental pollutants with concomitant bioelectricity generation. Therefore, the overarching objective of this study was to develop an MFC system for the effective and efficient treatment of petroleum hydrocarbons in both liquid and particulate systems. Biodegradation of target hydrocarbons, phenanthrene and benzene, was investigated in dual-chambered microbial fuel cells (MFCs) using different inoculum types - Shewanella oneidensis MR1 14063, Pseudomonas aeruginosa NCTC 10662, mixed cultures and their combinations thereof. All the inocula showed high potentials for phenanthrene and benzene degradation in liquid systems with a minimum degradation efficiency of 97 % and 86 % respectively with concomitant power production (up to 1.25 mWm-2). The performance of MFCs fed with a mixture of phenanthrene and benzene under various operating conditions - temperature, substrate concentration, addition of surfactants and cathodic electron acceptor type – was investigated. The interaction effects of three selected operating parameters - external resistance, salinity and redox mediator were also investigated using response surface methodology. The outcomes of this study demonstrated the robustness of MFCs with good degradation performance (range 80 - 98 %) and maximum power production up to 10 mWm-2 obtained at different treatment conditions. Interactive effects existed among the chosen independent factors with external resistance having a significant impact on MFC performance, with maximum power output of 24 mWm-2 obtained at optimised conditions - external resistance (69.80 kΩ) , redox mediator (29.30μM, Riboflavin) and salinity (1.3 % w/v NaCl). The treatment of a mixture of phenanthrene and benzene using two different tubular MFCs designed for both in situ and ex situ applications in aqueous systems was investigated over long operational periods (up to 155 days). The outcomes of this work demonstrated stable MFC performance at harsh nutrient conditions and ambient temperatures. Simultaneous removal of petroleum hydrocarbons (> 90 %) and bromate, used as catholyte, (up to 79 %) with concomitant biogenic electricity generation (i.e. peak power density up to 6.75 mWm-2) were observed. The performance of a tubular MFC system in phenanthrene-contaminated soil was investigated in the last study. The outcomes of this work has demonstrated the simultaneous removal of phenanthrene (86%) and bromate (95%) coupled with concomitant bioelectricity generation (about 4.69 mWm-2) using MFC systems within a radius of influence (ROI) up to 8 cm. The overall outcomes of this study suggest the possible application of MFC technology in the effective treatment of petroleum hydrocarbons contaminated groundwater or industrial effluents and soil systems (mostly in subsurface environments), with concomitant energy recovery. MFC technology could potentially be utilised as an independent system in lieu of other bioremediation technologies (e.g. pump and treat, electrobioremediation or permeable reactive barriers) or integrated with existing infrastructure such as monitoring wells or piezometers.

There is a vast worldwide consumption of petroleum hydrocarbons and accidental release in to the environment is common. For example petroleum forecourt retail outlets have 'interceptors' to prevent release of hydrocarbons into the environment. The aim of this study was to investigate options for in-situ bioremediation of the hydrocarbon substrates within these 'interceptors' in a laboratory model. The initial studies on bioremediation were undertaken with diesel as the substrate. It was shown that the addition of nitrogen and phosphorus to the system increased hydrocarbon mineralisation by a factor of 16, resulting in increased carbon dioxide evolution. There was strong evidence indicating that nitrogen and phosphorus were the limiting factor for hydrocarbon metabolism in this aqueous system. Trichoderma harzianum and a soil bacterial isolate LFC D1 FI were assessed and shown to degrade hexadecane and pristane. The positive affect of adding a cosubstrate was evident in flask studies; the rates of degradation by LFC D1 FI and T. harzianum were approximately doubled and tripled respectively in the presence of glucose compared to treatments without glucose. Previous attention has focused on the ability of Phanerochaete chrysosporium to degrade polycyclic aromatic hydrocarbons; in this study the degradation of aliphatics was investigated. Spores from P. chrysosporium induced on the hydrocarbon substrate were found to be necessary to degrade hexadecane. Pseudomonas putida was unable to grow in liquid media containing hydrocarbons, however on solid media and in an aqueous environment containing acid-washed sand, degradation of hydrocarbons was evident, hi the presence of sand P. putida degraded both hexadecane and pristane by 70% of the initial concentration added; in the absence of sand no degradation in the aqueous system was seen. This suggests surface attachment plays an important role in hydrocarbon degradation by P. putida. The attachment and use of the sessile P. putida in aliphatic hydrocarbon degradation is discussed.

Orientador: Valéria Maia Merzel
Dissertação (mestrado) - Universidade Estadual de Campinas, Insituto de Biologia
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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

Petroleum-based plastics have replaced many natural materials in their former applications. With their excellent properties, they have found widespread uses in almost every area of human life. However, the high recalcitrance of many synthetic plastics results in their long persistence in the environment, and the growing amount of plastic waste ending up in landfills and in the oceans has become a global concern. In recent years, a number of microbial enzymes capable of modifying or degrading recalcitrant synthetic polymers have been identified. They are emerging as candidates for the development of biocatalytic plastic recycling processes, by which valuable raw materials can be recovered in an environmentally sustainable way. This review is focused on microbial biocatalysts involved in the degradation of the synthetic plastics polyethylene, polystyrene, polyurethane and polyethylene terephthalate (PET). Recent progress in the application of polyester hydrolases for the recovery of PET building blocks and challenges for the application of these enzymes in alternative plastic waste recycling processes will be discussed.

The microbial ecology of 45 high temperature (> 50 ° C) petroleum reservoirs was investigated by isolating and characterizing bacteria that were present in their produced fluids. Initial work was aimed at selecting a suitable high temperature petroleum reservoir for the study of natural microbial populations. Experimental work then focussed on establishing the physico-chemical conditions that prevail in the selected reservoir and on developing media and enrichment conditions for the isolation of microorganisms indigenous to the reservoir. The ability of reservoir bacteria to grow and survive under the physical and chemical conditions found in the selected reservoir was used to assess the likelihood of an indigenous origin for these bacteria. The petroleum reservoir selected for study was the Alton petroleum reservoir (SW Queensland, Australia). It was established that most of the physico-chemical conditions in the Alton reservoir had remained unchanged since oil recovery began. The stability of redox conditions (90 mV) in the reservoir over its operating life was identified as an important factor in the coexistence of strict aerobic and strict anaerobic bacterial populations within the reservoir. An important change that has occurred in the Alton reservoir over its operating life because of oil recovery was an increase in water pH from 6.41 to 8.42 as a result of carbon dioxide loss (1.36 atm to 0.0134 atm) from the reservoir. Development of novel enrichment procedures that simulated Alton reservoir conditions led to the isolation of previously unreported aerobic and anaerobic populations of thermophilic bacteria. The aerobic bacteria isolated were identified as either endosporeforming heterotrophic bacteria from the genus Bacillus or nonspore-forming heterotrophic bacteria resembling members of the genus Thermoleophilum. All aerobes grew on carbon sources such as acetate and n-heptadecane that are normal constituents of the reservoir. The anaerobic bacteria isolated were characterized as sheathed fermentative bacteria from the order Thermotogales or non-sheathed fermentative bacteria. In parallel studies, the natural microbial populations in other reservoirs were investigated and I concluded that fermentative microorganisms were common inhabitants of high temperature petroleum reservoirs. The isolation of fermentative bacteria from these high temperature petroleum reservoirs established that fermentative bacteria are a fourth major microbial group, together with hydrocarbon-oxidizers, sulphate-reducers and methanogens, to be reported in petroleum reservoirs. The fermentative bacteria use organic nutrients and carbohydrates, but not contemporary crude oil as the principal nutrient source within reservoir waters. The thermophilic bacteria isolated from Alton petroleum reservoir demonstrated growth characteristics such as temperature (optima 50-70 ° C and range 37-85 ° C), pH (optima 6.0-9.0 and range 5.0-9.0 and salinity (optima 0-15 g per litre and range 0-30 g per litre), that were consistent with conditions encountered in the Alton reservoir (temperature 75 � C, pH 8.5 and TDS 2.7 g per litre). The isolated bacteria also demonstrated a number of characteristics that might enable them to survive adverse conditions that could be encountered in a petroleum reservoir environment. The characteristics that contribute to aerobic bacteria surviving in and overcoming periods of oxygen limitation include well-documented processes such as sporulation, by Bacillus spp., and microaerophily. The characteristics that contribute to fermentative bacteria surviving were: (1) a natural tolerance to reservoir physico-chemical fluctuations, (2) an ability to remain viable when metabolic activity was suppressed to very low rates by the growth-limiting conditions imposed, and (3) possible formation of viable ultramicrobacteria (UMB). Formation of UMB (bacteria smaller than 0.3 |im) by thermophilic bacteria has not been reported previously. The recovery of thermophilic UMB by filtration from the Alton reservoir water indicates that these bacteria occur in natural habitats. This study found the formation of thermophilic UMB and their survival characteristics differed considerably from that reported for the mesophilic, marine bacterium Vibrio sp. DWI. Unlike mesophilic marine bacteria, thermophilic bacteria did not always respond to nutrient deprivation by forming UMB and that these UMB did not show any increased ability to survive in the face of adverse conditions. Although the formation of UMB as part of routine cell growth and division was not demonstrated directly in this study, circumstantial evidence suggests that they form part of a natural life cycle. The exact conditions that result in UMB formation and their role in survival remain unresolved. The capacity of nonspore-forming indigenous populations from Alton to survive sudden shifts in environmental conditions that might result from common oilfield operations was poor. Such operations were demonstrated to be inhibitory or lethal to Alton reservoir bacteria. It also was concluded that such oilfield operations suppress indigenous microbiota. However, the impacts of most oilfield operations within a reservoir are likely to be confined to the immediate area surrounding injection and producing wells. Minimizing the localized effects of oilfield practices on indigenous reservoir populations will lead to the better management of undesirable microbial activity in reservoirs such as H2S formation (souring) and facilitate development of better microbially mediated oil recovery process. This study showed that selected reservoir isolates possess characteristics which are suitable for in situ biotechnological applications such as microbially enhanced oil recovery (MEOR). Characteristics favourable for enhanced oil recovery include a capability for UMB formation, which would enable better dispersion, and resistance to high concentrations of reservoir components such as calcium, magnesium, strontium, heavy metals and hydrocarbons.

A field survey was conducted following the Deepwater Horizon blowout and it was noted that resulting coastal petroleum deposits possessed distinct geometries, ranging from small tar balls to expansive horizontal oil sheets. A laboratory study evaluated the effect of oil deposit geometry on localized gradients of electron acceptors and microbial community composition, factors that are critical to accurately estimating biodegradation rates. One-dimensional top-flow sand columns with 12-hour simulated tidal cycles compared two contrasting geometries (isolated tar â ballsâ versus horizontal â sheetsâ ) relative to an oil-free control. Significant differences in the effluent dissolved oxygen and sulfate concentrations were noted among the columns, indicating presence of anaerobic zones in the oiled columns, particularly in the sheet condition. Furthermore, quantification of genetic markers of electron acceptor and catabolic conditions via quantitative polymerase chain reaction of dsrA (sulfate-reduction), mcrA (methanogenesis), and cat23 (oxygenation of aromatics) genes in column cores suggested more extensive anaerobic conditions induced by the sheet relative to the ball geometry. Denaturing gradient gel electrophoresis similarly revealed that distinct gradients of bacterial communities established in response to the different geometries. Thus, petroleum deposit geometry impacts local redox and microbial characteristics and may be a key factor for advancing attenuation models and prioritizing cleanup.
Master of Science

Orientador: Valéria Maia Merzel
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O processo de biodegradação do petróleo em reservatórios pode resultar em mudanças na composição e propriedades físico-químicas de óleos brutos e gases naturais, as quais levam à diminuição do teor de hidrocarbonetos saturados, produzindo óleos mais pesados e com baixo valor econômico. O uso combinado de técnicas dependentes e independentes de cultivo pode nos permitir um melhor entendimento acerca da comunidade de micro-organismos que habita os reservatórios de petróleo, incluindo aqueles responsáveis por esta biodegradação. O conhecimento sobre a composição microbiana, suas funções e interações com outros micro-organismos e com o ambiente pode levar à definição de estratégias de monitoramento e/ou controle da biodegradação em reservatórios. Este estudo teve como finalidade avaliar a diversidade de micro-organismos e genes envolvidos na degradação de hidrocarbonetos presentes em amostras de petróleo provenientes de dois poços terrestres da Bacia Potiguar (RN), identificados como GMR75 (poço biodegradado) e PTS1 (poço não-biodegradado), através da construção de bibliotecas de genes catabólicos (alcano monooxigenases - alk, dioxigenases que hidroxilam anéis aromáticos ¿ ARHDs e 6-oxocyclohex-1-ene-1-carbonyl-CoA hidroxilase - bamA) e sequenciamento em larga escala de metagenoma e metatranscriptoma de enriquecimentos microbianos aeróbios. Os resultados obervados mostraram uma distribuição diferencial dos genes catabólicos entre os reservatórios, sendo o óleo biodegradado mais diverso para os genes alk e bamA. As sequências foram semelhantes aos genes alkB dos gêneros Geobacillus, Acinetobacter e Streptomyces, aos genes ARHD dos gêneros Pseudomonas e Burkholderia, e aos genes bamA do gênero Syntrophus. A análise quantitativa dos genes catabólicos de degradação de hidrocarbonetos presentes e expressos nos enriquecimentos microbianos em diferentes etapas da biodegradação do óleo, através de PCR Tempo Real, demonstrou maior atividade do gene que codifica a enzima dioxigenase nas comunidades microbianas enriquecidas, e os resultados obtidos pela técnica de microarray sugeriram a existência de novas sequências dos genes alk e ARHD provindas do reservatório de petróleo. As análises das sequências obtidas a partir do metagenoma e metatranscriptoma mostraram que a comunidade bacteriana recuperada no enriquecimento aeróbio é bastante diversa, com predominância do Filo Actinobacteria, seguido de Proteobacteria. As sequências com maior abundância e níveis de expressão foram relacionadas aos genes que codificam as proteínas ligase CoA de ácido graxo de cadeia longa, envolvida na degradação de compostos aromáticos; descarboxilase, envolvida com o ciclo do glioxilato, e o fator sigma da RNA polimerase, envolvida com a regulação da resposta ao estresse oxidativo, sugerindo uma adaptação da comunidade microbiana às condições do enriquecimento e um processo inicial de biodegradação dos hidrocarbonetos. Os resultados obtidos neste trabalho fornecem dados inéditos sobre a diversidade de genes catabólicos e de membros da comunidade microbiana potencialmente envolvidos com a degradação do óleo em reservatórios de petróleo
Abstract: The process of oil biodegradation in reservoirs may result in changes in the composition and physico-chemical properties of crude oils and natural gases, which lead to the decrease of the content of saturated hydrocarbons, producing heavy oils and with low economic value. The combined use of both dependent and independet cultivation techniques may allow us to better understand the microbial community inhabiting oil reservoirs, including those microorganisms responsible for oil degradation. The knowledge about the microorganisms, ther functions and interactions with other microorganisms and the environment may lead to the definition of monitoring and/or control strategies of biodegradation in oil reservoirs. This study aimed at evaluating the diversity of microorganisms and genes involved in the degradation of hydrocarbons present in oil samples from two onshore reservoirs at Potiguar Basin (RN), identified as GMR75 (biodegraded) and PTS1 (non- biodegraded), through the construction of catabolic gene libraries (alkane monooxygenases - alk, aromatic ring hydroxylating dioxygenases ¿ ARHD and 6-oxocyclohex-1-ene-1-carbonyl-CoA hydroxylase - bamA) and highthroughput sequencing of metagenome and metatranscriptome from aerobic microbial enrichments. Results observed showed a differential distribution of catabolic genes between the reservoirs, being the biodegraded oil more diverse for the alk and bamA genes. The sequences were similar to alkB genes from Geobacillus, Acinetobacter and Streptomyces genera, to the ARHD genes from Pseudomonas and Burkholderia genera, and to the bamA genes from Syntrophus genus. Quantitative analysis of the hydrocarbon degradation genes present and expressed in the microbial enrichments during the different phases of oil biodegradation by Real-Time PCR showed that there was a higher activity of dioxygenase enzymes in the enriched microbial communities and results from microarray assays suggested the existence of new alk and ARHD gene sequences originated from the oil reservoir. Metagenomic and metatranscriptomic analyses showed a highly diverse bacterial community, dominated by the Phylum Actinobacteria, followed by Proteobacteria. The most abundant and active sequences were affiliated to the Long-chain-fatty-acid-CoA ligase protein, involved in the degradation of aromatic compounds; decarboxylase, which is involved with the glyoxylate cycle, and RNA polymerase sigma factor, which is involved in regulating the oxidative stress response, suggesting an adaptation of the microbial community to the enrichment conditions and an initial process of biodegradation of hydrocarbon compounds. The results obtained in this work bring innovative data on the diversity of catabolic genes and microbial community members potentially involved with oil degradation in petroleum reservoirs
Doutorado
Genetica de Microorganismos
Doutor em Genetica e Biologia Molecular

Microbial-based strategies were investigated for eventual bioremediation of petroleum hydrocarbon-contaminated, acidic soils from Resolution Island (RI), Nunavut. A biotreatability assessment phase one study determined that supplementation of soil with commercial fertilizer and lime enhanced hydrocarbon mineralization. Phase two applied these conditions to large scale mesocosm trials, containing ~150 kg soil, incubated in a temperature cycle that represented the ambient summer conditions on RI (10 d of 1°C - 10°C for 60 d). Culture-dependent and –independent analyses of RI soil microbial communities showed the mesocosm treatment enhanced hexadecane mineralization, increased the enumerations of total microbes and viable, cold-adapted hydrocarbon-degrading microorganisms. DGGE analyses indicated emergence of a hydrocarbon-degrading community and 16S rRNA gene clone libraries showed bacterial population shift in mesocosm soils. Potentially novel isolated strains included those able to grow on hydrocarbons alone while under acidic or sub-zero conditions. This microbiological study addressed RI site conditions and presents a potential bioremediation.
Des techniques s'appuyant sur la microbiologie ont été utilisée pour évaluer la biorestauration future de sols acides, contaminés par des hydocarbures pétroliers, à Resolution Island (RI), Nunavut. Premièrement, une étude de biotraitabilité a permis de determiner que l'amendement du sol avec des fertilisants de type commercial et de la chaux améliore la dégradation des hydrocarbures. La phase deux a consisté en l'application de ces conditions à des essais de mesocosmes à grande échelle incubés à des températures représentant les conditions estivales de RI, i.e. cycle de 10 jrs (1°C-10°C) pendant 60 jrs. Des analyses de microbiologie classique et de biologie moléculaire des communatés microbiennes du sol de RI ont démontré que l'amendement des mésocosmes a permis une augmentaion de la minéralisation de l'hexadécane et un accroîssement du dénombrement de total de microorganismes ainsi que des microorganismes viables, adaptés au froid et dégradant les hydrocarbures. Des analyses par DGGE ont démontré l'apparition d'un communauté microbienne dégradant les hydrocarbures et une librairie de clones d'ARNr 16S a souligné un réarrangement des populations microbiennes présentes dans les sols de mesocosmes. Des nouvelles souches ont été isolées, incluant certaines pouvant croître sur une source unique d'hydrocarbures sous des conditions acides ou sous-zéro. Cet étude microbiologique a été faite sous des conditions respectant celles présente à RI et présente des procédés pouvant être utilisées pour la bioremediation du site.

Regulatory acceptance of monitored natural attenuation (MNA) requires demonstration that natural processes, such as sorption and biodegradation, attenuate specific contaminants of concern on a time scale that is comparable to other remediation options while concurrently preventing contaminant migration to site-specific points of contact. Two of the tools used to demonstrate the efficacy of MNA, microcosm experiments and numerical fate and transport modeling, were examined in this study. In the first phase of this work, laboratory microcosm studies were initiated as part of an overall MNA site assessment to determine whether a native microbial consortia collected with a soil sample from a petroleum-hydrocarbon contaminated site was capable of biodegrading specific polynuclear aromatic hydrocarbon (PAH) compounds. Results indicated that selected PAH compounds were biodegraded under simulated natural conditions using oxygen and sulfate as electron acceptors. In the second phase of this study, a numerical experiment was conducted using the three-dimensional, multiple substrate, multiple electron acceptor fate and transport model SEAM3D (Waddill and Widdowson, 1997) to evaluate the impact of including iron(III)-reducing conditions during numerical simulations of natural attenuation. Results for this phase of the study indicated that the mass of hydrocarbon simulated as biodegraded by the iron(III)-reducing population was significantly larger than hydrocarbon biodegradation under aerobic conditions. The final component of research used the SEAM3D model to interpret field observations recorded during a natural attenuation experiment where the fate and transport of selected hydrocarbon contaminants (BTEX and naphthalene) was tracked through an extremely heterogeneous, but well-instrumented test aquifer. Results from the calibrated model for the NATS experiment indicated that the majority of the contaminant remained in the non-aqueous phase during the first year of the experiment, and that aerobic biodegradation was the dominant natural attenuation process. Model results were particularly sensitive to the rate of contaminant release and the starting mass of electron acceptor.
Ph. D.

This research has investigated the biodegradation of hydrocarbons particularly n-alkanes using open mixed culture which is relevant for both microbial enhanced oil recovery (MEOR) and the bioremediation of hydrocarbon contaminated soils. Biodegradation of n-C12, C14, C16, C18, C20 and some readily biodegradable substrates (glucose, acetic acid and ethanol) was studied using a respirometric method developed to assess the biodegradability of these compounds. Laboratory batch and semi-continuous experiments were performed in small-scale bioreactors at room temperature and 40oC under various conditions i.e. aerobic, anoxic with nitrate, sulfate reducing and completely anaerobic conditions using two different sources of open mixed microbial cultures obtained from an agricultural site and anaerobic digestion plant. Glucose, acetic acid, ethanol, C12, C14 and C16 were degraded microbially under aerobic batch conditions to nondetectable levels at room temperature and 40oC using the two sources of inoculum whereas C18 and C20 were degraded partially under room temperature and to nondetectable levels at 40oC with the two inocula sources. Under aerobic semi-continuous, glucose and the n-alkane substrate were biodegraded even at low hydraulic retention time (HRT). Under anaerobic conditions, the n-alkanes were utilized by the soil inoculum at room temperature and at 40oC with nitrate as the electron acceptor but no microbial activity was observed under sulfate reducing and completely anaerobic conditions. The open mixed cultures require an initial acclimation period before utilizing the substrates. The acclimation period was significantly shorter under aerobic conditions than anaerobic conditions for the n-alkanes. Acclimation periods of approximately 1-2 days under aerobic conditions was observed for the readily biodegradable substrates and 2 days for glucose under anoxic conditions. The acclimation periods for the nalkanes was between 3-5 days under aerobic conditions and approximately 2 weeks under anoxic conditions. The acclimation period was not affected by the substrate concentration and inoculum type however, for the n-alkanes, the acclimation period was reduced by 1-2days under aerobic conditions at 40oC. The biodegradation of the liquid hydrocarbons was more significant than the solids at room temperature but in general higher temperature increased the degree of biodegradation. The electron acceptor consumption data i.e. dissolved oxygen and nitrate consumption data obtained was mathematically modelled using Monod kinetics to obtain biokinetic parameters. Good fittings between the model solution and the experimental data was obtained. The biokinetic parameters obtained were within the range of values reported in literature. The use of respirometric data for the estimation of biodegradation kinetic parameters can be very reliable. The consistency of the data obtained show that the approach is very reproducible and quality information can be obtained. The results of this study showed that the open mixed microbial cultures from soil and AD inocula contained diverse microorganisms capable of utilizing both liquid and solid n-alkanes at room temperature and 40oC under aerobic and anoxic conditions.

Civil Engineering
M.S.Env.E.
The Deepwater Horizon (DWH) blowout, in the Gulf of Mexico, heavily contaminated miles of sandy beaches. Previous experience of petroleum contamination has shown that oil residues can persist in the sediments for decades. Biodegradation is the major mechanism of remediation regarding petroleum hydrocarbons. There is an urgent need to evaluate the competent indigenous microbial biomass in contaminated sediments if the risks posed by toxic oil residues, for the coastal ecosystem, are to be minimized. We report a field investigation during December 2010 and January 2011 regarding measurement of microbial activity in a sandy beach at the Bon Secour National Wildlife Refuge in Alabama. One transect of wells for sampling was installed in the beach; starting with multiport one, being most landward and thought to be least exposed to oil residue and ending with multiport four being the most seaward and exposed to the open waters of the Gulf of Mexico. Sediment samples were collected from different depths purposely chosen from above, inside, and below the oil layers for microbial analysis. Dissolved oxygen (DO) measurements were obtained and temperature was recorded while collecting the oxygen measurements. Pore water samples were collected for nutrient content and were monitored using the multiport sampling wells. Moisture content was analyzed from the sediments extracted at various depths at each well. pH and salinity were also analyzed for their contributing affect on the microbial community. Grain size distribution analyses were conducted on samples collected at all wells and at multiple depths to characterize the field study location. Results show that the bacterial biomass, as measured by Adenosine-5-triphosphate (ATP) and numbers of alkane and polycyclic aromatic hydrocarbon (PAH) degraders determined by Most Probable Number (MPN), are consistently higher in the sediment layers where oil had been detected. A very good correlation was observed among the relative abundance of bacteria in the different samples using MPN and ATP measurements. As expected, ATP based estimates of the microbial populations were two orders of magnitude higher than the alkane and PAH numbers determined by MPN, which reflect the non-cultivability of most environmental bacteria. The lower concentrations of PAH degraders than alkane degraders that were observed in this study are consistent with other studies, even though both populations are lower than in studies involving fresh oil trapped in beach or wetland sediments. PAHs (aromatics) are notoriously more resistant to biodegradation than alkanes, therefore allowing a lower number of biomass to grow using them. The overall smaller size of the bacterial numbers could be explained by the naturally occurring low-organic content of beach sand. On the other hand, this may be due to the highly weathered nature of the oil or it could reflect some other limitation.
Temple University--Theses

Current prices and demand for petroleum hydrocabons have generated an increase of oil spills around the country and the world. Health and environmental impacts associated to these organic pollutants represent a huge concern for the general public, leading the public and private sector to develop new technologies and methods to minimize or eliminate those risks. Ex-Situ bioremediation through biopiles, as a main remediation technique to treat a wide range of hydrocarbons, has been a topic of considerable research interest over the last years. It provides an economical and environmental solution to restore the environment to background levels. Nevertheless, successful bioremediation under cold climate conditions is of considerable concern in countries like Canada, as low temperatures can delay the rate of bioremediation of oil hydrocarbons, thus limiting the operation of soil treatment facilities to certain times of the year. Recent research has found out that bioremediation could be conducted even at low or cold temperatures with larger periods of times. And even more, the addition of petroleum degrading microorganisms (bioaugmentation) and nutrients or biosurfactants (biostimulation) could enhance the process in some cases. In the present study, a comprehensive assessment of bioaugmentation and biostimulation strategies for ex-situ bioremediation of petroleum contaminated soil under cold climate conditions is proposed. Field scale biopiles were constructed and subjected to different concentrations of commercial microbial consortia and mature compost, as bioaugmentation and biostimulation strategies, in a soil treatment facility at Moose Creek, Ontario over a period of 94 days (November 2012 to February 2013). Assessment and comparison of the biodegradation rates of total petroleum hydrocarbons (TPH) and their fractions were investigated. Furthermore, a response surface methodology (RSM) based on a factorial design to investigate and optimize the effects of the microbial consortia application rate and amount of compost on the TPH removal was also assessed. Results showed that biopiles inoculated with microbial consortia and amended with 10:1 soil to compost ratio under aerobic conditions performed the best, degrading 82% of total petroleum hydrocarbons (TPHs) with a first-order kinetic degradation rate of 0.016 d_1, under cold temperature conditions. The average removal efficiencies for TPHs after 94 days for control biopiles, with no amendments or with microbial consortia or compost only treatments were 48%, 55%, and 52%, respectively. Statistical analyses indicated a significant difference (p < 0.05) within and between the final measurements for TPHs and a significant difference between the treatment with combined effect, and the control biopiles. On the other hand, the modeling and optimization statistical analysis of the results showed that the microbial consortia application rate, compost amendment and their interactions have a significant effect on TPHs removal with a coefficient of determination (R2) of 0.88, indicating a high correlation between the observed and the predicted values for the model obtained. The optimum concentrations predicted via RSM were 4.1 ml m-3 for microbial consortia application rate, and 7% for compost amendment to obtain a maximum TPH removal of 90.7%. This research contributes to provide valuable knowledge to practitioners about cost-effective and existing strategies for ex-situ bioremediation under cold weather conditions.

Microbial biodegradation is the primary mechanism by which petrogenic hydrocarbons (PHCs) are removed from the environment. Though hydrocarbon biodegradation is widely studied in marine systems, knowledge of how it occurs in freshwater systems is still lacking. The Peace-Athabasca Delta (PAD), located in northeastern Alberta, is an ideal location to study microbial hydrocarbon degradation since it has a long history of exposure to PHCs. What’s more, these PHCs are predominately sourced from bituminous deposits and are therefore relevant to the Canadian Oil Sands Industry. This thesis investigated the genetic potential for hydrocarbon degradation of PHCs via metagenomic reconstruction of microbial communities in lakes of the Peace and Athabasca Deltas, as well as reference lakes in the nearby boreal uplands. In order to properly evaluate the microbial community and its potential for hydrocarbon degradation, a comprehensive analysis of PHCs (including n-alkanes, polycyclic aromatic compounds (PACs), and petroleum biomarkers of terpanes, hopanes, and steranes) was performed. PHC analysis showed that n-alkanes in lake sediments from all three regions were highly similar and predominately biogenic, while PAC composition was significantly different in each region. Restricted-drainage lakes of the Athabasca Delta had the highest concentrations of PACs from petrogenic sources. Closed-drainage lakes in the Peace Delta had lower concentrations of PACs that likely originated from a mixture of pyrogenic and petrogenic sources. Closed-drainage lakes in the boreal upland region had the lowest concentrations of PACs likely sourced from pyrogenic wood combustion with traces of petrogenic PACs, possibly from atmospheric deposition of dust. Petroleum biomarkers of terpanes, hopanes, and steranes were successfully used to identify the long-range fluvial, and possibly atmospheric, transport of bituminous compounds more than one hundred kilometers from their potential source. This validates the future use of these biomarkers in environmental forensics. Microbial communities in all three regions under study were highly diverse, and their composition was significantly different in both sediment and water. Targeted gene analysis identified a total of 3885 genes involved in the degradation of n-alkanes and PACs in sediment and water. The results show that organic carbon, nitrogen, and sulfur content, as well as PAC and short-chain alkane concentrations were important chemical predictors of change in degradation gene composition. Furthermore, genes for anaerobic degradation of PHCs were identified in syntrophic bacteria, methanogens, nitrate and sulfate reducers, demonstrating the potential for syntrophic hydrocarbon degradation in PAD lakes. Though this thesis confirms the genetic potential for hydrocarbon degradation in PAD and boreal upland lakes, further research is necessary to determine whether these microbial communities can actively degrade the PHCs present in these lakes.

Currently available physical and chemical remediation technologies are not effective in the deep sea, where ultimate fate of oil is strongly dependent on degradation by microorganisms. In order to setup an efficient bioremediation strategy, the effect of the environmental constraints on oil degrading communities’ metabolism needs to be assessed. In this work, natural surface seawater communities were incubated under different T, P and in different dilutions of the medium ONR7a. Then, after 1 day, 1 week and 3 weeks, cell number, SO4, NH4 and DIC concentration were assessed. When only one environmental stress (low T or high HP) is applied, a positive effect of nutrients’ concentration is highlighted, with increased growth rates and DIC production; at 23 °C and 20 MPa, a lower cell growth is observed with respect to atmospheric pressure samples, but a relatively high value of oil degradation is mostly sustained by respiration to DIC: this result evidence the possibility to setup an effective biostimulation strategy in deep seas characterized by milder temperatures (Sulu, Mediterranean, Red Sea). High Ammonium concentrations are needed to sustain large production of proteins for cell maintenance process required for adaptation to HP. Moreover, at 23 °C and 20 MPa, significant Sulphate uptake occurs, whose role in HP adaptation needs to be clarified. Also at low T and atmospheric pressure oil biodegradation mostly relies on respiration to DIC, however biodegradation rates seems to be more affected by T reduction than HP increase. When both low T and high HP are applied, cellular metabolism is still active but any growth is observed, drastically reducing the oil biodegradation, that relies only on respiration to DIC. Surface communities subject to combination of 2 stresses may need more than 3 weeks to adapt to the deep sea environment and start a growth phase.

Laboratory batch and long-term column experiments were conducted to investigate the effects of wood-chip biochar and coal-derived activated carbon amendment on the microbiology of a volatile petroleum hydrocarbon (VPH) - contaminated gravelly sand. First, a stable isotope-labelled mono aromatic compound – toluene was used as a model VPH to gain insights into the mineralization of VPHs by soil microorganisms in the presence and absence of biochar or activated carbon. The biodegradation of a mixture of 12 VPHs was subsequently monitored in batch microcosms over a duration of 6-19 days by measuring headspace CO2 concentration. Further analysis was carried out by characterizing changes in the soil microbial community composition using next generation sequencing techniques – 454 pyrosequencing and Ion torrent sequencing. Increases in the levels of headspace CO2 in contaminated soil batches as compared to live and abiotic controls to which no VPHs were added indicated a stimulation of microbial activity in the batches through VPH addition. By fitting a maximum specific growth rate of 0.6 h-1 (in line with published rates), it was possible to match model predictions of 45CO2 and 44CO2 concentrations with the experimentally determined data. Half-saturation constants of 4.06 x 103 mgL-1, 7.76 x 102 mgL-1 and 1.83 x 102 mgL-1 were predicted for soil, soil & BC and soil & AC respectively, much higher than values reported in the literature. Differences in the half-saturation constant suggests that sorbent amendment affects the microbial ecology, by making microorganisms which can utilize substrates at lower concentrations more competitive. Yield coefficients (g biomass-C relative to g (biomass-C + CO2-C)) compared more closely in the nutrient (N & P) amended soils ranging from 4.83±0.46 in soil and biochar to 7.86±0.72 in unamended soil, than in the batches without nutrients, 4.1±3.1 in soil & BC, 17.7±5.2 in soil and 13.7±4.6 in soil & AC. Sorbent amendment thus reduced yield coefficients, thereby slowing the growth of VPH degrading biomass. Microbial community structure analysis revealed an increase in the relative abundance ranking of members of the genera Pseudomonas, Pseudoxanthomonas, and Arenimonas by up to 32 folds and in the families Nocardioidaceae and Pseudomonadaceae by at least 32 folds in sorbent amended and unamended soil batches and columns compared to their initial soil conditions. Consequently, amending soils with 2% BC or AC changed the biokinetics of VPH degradation by rendering VPHs less bioavailable, but did not appear to have any detrimental effects on the VPH degrading bacteria both in the short- and long-term, and may serve as a sustainable, cost-effective approach for enhancing the natural attenuation of VPHs in soil, thus addressing the challenge of petroleum hydrocarbon contamination.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Os métodos tradicionais de estimular a produção de petróleo, envolvendo a injeção de água, vapor, gás ou outros produtos, estabeleceram a base conceitual para novos métodos de extração de óleo, utilizando micro-organismos e processos biológicos. As tecnologias que empregam os processos de bioestimulação e bioaumentação já são amplamente utilizadas em inúmeras aplicações industriais, farmacêuticas e agroindustriais, e mais recentemente, na indústria do petróleo. Dada a enorme dimensão econômica da indústria do petróleo, qualquer tecnologia que possa aumentar a produção ou o fator de recuperação de um campo petrolífero gera a expectativa de grandes benefícios técnicos, econômicos e estratégicos. Buscando avaliar o possível impacto de MEOR (microbial enhanced oil recovery) no fator de recuperação das reservas de óleo e gás no Brasil, e quais técnicas poderiam ser mais indicadas, foi feito um amplo estudo dessas técnicas e de diversos aspectos da geologia no Brasil. Também foram realizados estudos preliminares de uma técnica de MEOR (bioacidificação) com possível aplicabilidade em reservatórios brasileiros. Os resultados demonstram que as técnicas de MEOR podem ser eficazes na produção, solubilização, emulsificação ou transformação de diversos compostos, e que podem promover outros efeitos físicos no óleo ou na matriz da rocha reservatório. Também foram identificadas bacias petrolíferas brasileiras e recursos não convencionais com maior potencial para utilização de determinadas técnicas de MEOR. Finalmente, foram identificadas algumas técnicas de MEOR que merecem maiores estudos, entre as técnicas mais consolidadas (como a produção de biossurfatantes e biopolímeros, e o controle da biocorrosão), e as que ainda não foram completamente viabilizadas (como a gaseificação de carvão, óleo e matéria orgânica; a dissociação microbiana de hidratos de gás; a bioconversão de CO2 em metano; e a bioacidificação). Apesar de seu potencial ainda não ser amplamente reconhecido, as técnicas de MEOR representam o limiar de uma nova era na estimulação da produção de recursos petrolíferos existentes, e até mesmo para os planos de desenvolvimento de novas áreas petrolíferas e recursos energéticos. Este trabalho fornece o embasamento técnico para sugerir novas iniciativas, reconhecer o potencial estratégico de MEOR, e para ajudar a realizar seu pleno potencial e seus benefícios.
The traditional methods of stimulating production, involving the injection of water, steam, gas or other products, have established the conceptual basis for new methods of oil extraction, utilizing microorganisms and biological processes. Technologies that employ biostimulation and bioaugmentation processes are widely utilized in numerous industrial, pharmaceutical and agroindustrial applications, and, more recently, in the oil industry. Given the enormous economic dimension of the oil industry, any technology that can increase production or recovery of an oil field creates the expectation of large technical, economic and strategic benefits. In order to assess the possible impact of MEOR (Microbial Enhanced Oil Recovery) on the recovery factor of oil and gas reserves in Brazil, and which techniques might be most indicated, a wide ranging study of those techniques and of various aspects of the geology of Brazil was carried out. Preliminary studies of a MEOR technique (bioacidification) with possible application in Brazilian reservoirs were also carried out. The results demonstrate that MEOR techniques can be effective in the production, solubilization, emulsification or transformation of several compounds, and that they can promote other physical effects in the oil or the reservoir rock matrix. Brazilian oil basins and unconventional resources with potential for utilization of certain MEOR techniques were also identified. Finally, certain MEOR techniques that deserve further studies were identified, involving both more consolidated techniques (such as biosurfactant and biopolymer production, and the control of microbially induced corrosion), as well as those that have not yet fully proven their viability (such as coal, oil and organic matter gasification; microbial dissociation of gas hydrates; bioconversion of CO2 into methane; and bioacidification). Despite the fact that their potential is not yet fully recognized, MEOR techniques represent the dawn of a new era in the stimulation of production of existing oil resources, and even in the production development plans of new oil and other energy resources. This work furnishes the technical basis for suggesting new initiatives, for recognizing the strategic potential of MEOR, and for helping to realize the full potential of MEOR and its benefits.

Os impactos da poluição por hidrocarboneto de petróleo sobre a diversidade e funcionalidade das comunidades microbianas em manguezais não são totalmente conhecidos, principalmente devido às limitações metodológicas para acessar os microrganismos nãocultiváveis. No entanto, vários métodos moleculares independentes de cultivo têm sido utilizados para investigar a diversidade e a estrutura das comunidades microbianas em ecossistemas naturais. O objetivo deste trabalho foi avaliar as variações da estrutura das comunidades de Bacteria e Archaea e a diversidade de Bacteria em uma transeção de solo de mangue do rio Iriri (Bertioga, SP) com um gradiente de contaminação por hidrocarbonetos de petróleo. As análises por eletroforese em gel com gradiente desnaturante (DGGE) mostraram que as comunidades de Bacteria e Archaea nas diferentes posições geográficas foram mais similares entre si do que entre diferentes profundidades ao longo do perfil em uma mesma posição geográfica. A análise das seqüências de clones de rDNA 16S de Bacteria dos diferentes pontos amostrados em abril de 2000, mostrou que a diversidade genética, avaliada pelo índice de Shannon, das comunidades microbianas diferem estatisticamente somente entre ponto o P1 (ponto menos contaminado) e P3 (ponto mais contaminado). As estimativas não-paramétricas da riqueza de espécies mostraram que P1, P2 e P3 possuem mais de 3539, 2524 e 1421 espécies bacterianas, respectivamente. Já, para as amostras do ponto P2 coletadas nos anos 2000 e 2004, muito embora os valores dos índices de Shannon tenham sido semelhantes, houve uma provável dominância de grupos específicos nas amostras coletadas em 2004, verificada pelos altos valores da recíproca do índice de Simpson. Os dados mostraram também que o número estimado de espécies bacterianas no ponto P2 diminuiu com o tempo, sendo menor em amostras de 2004, se comparado com amostras de 2000. No geral, a afiliação filogenética dos clones de rDNA 16S mostrou a grande diversidade de espécies, a maioria não conhecidas. Os dados sugerem que a contaminação do solo de mangue do rio Iriri está selecionando microrganismos mais adaptados às fontes de carbono introduzidas no solo.
The impacts of petroleum hydrocarbon pollution on the diversity and functionality of the microbial communities in mangrove soils are not totally understood, mainly due to the methodological limitations to access unculturable microorganisms. However, several cultureindependent molecular methods have been used to investigate the diversity and structure of microbial communities in natural ecosystems. The aim of this work was to evaluate shifts in Bacteria and Archaea community structures and the diversity of Bacteria in a soil transection of the Iriri river mangrove (Bertioga, SP) showing a petroleum hydrocarbon contamination gradient. The analyses by denaturing gradient gel electrophoresis (DGGE) showed that the communities of Bacteria and Archaea in different geographical positions were more similar among them than the communities in different depths along the soil profile at the same geographical position. Sequence analyses of bacterial 16S rDNA clones from different points sampled in April 2000 showed that the genetic diversity of the bacterial communities, based on the Shannon index, differ statistically only between P1 (less polluted) and P3 (more polluted) locations. Nonparametric estimates of species richness showed that P1, P2 and P3 may have more than 3539, 2524 and 1421 bacterial species, respectively. For P2 sampled in years 2000 and 2004, even though the Shannon indices were similar, there was a probable dominance of specific bacterial groups in year 2004, based on the high values of the reciprocal of Simpson\'s index. The data also showed that the estimated number of bacterial species in P2 decreased with the time, being lower in samples collected in 2004, as compared to samples collected in 2000. In the general, the phylogenetic affiliation of the 16S rDNA clones showed high bacterial species diversity, and most of the bacteria were of unknown species. The data suggest that the contamination of Iriri river mangrove soil with petroleum hydrocarbon is selecting microorganisms more adapted to the introduced carbon sources into the soil.

Dans les environnements côtiers, soumis à l’impact des marées noires, les microorganismes jouent un rôle crucial dans le devenir des hydrocarbures pétroliers. Toutefois, de nombreux facteurs influencent les activités microbiennes, notamment par les organismes bioturbateurs qui modifient la pénétration de l’oxygène dans les sédiments. Le travail de la thèse vise à mieux comprendre l’impact d’une pollution pétrolière sur les communautés microbiennes dans des sédiments bio turbés. Il s’agissait de comparer les remaniements structuraux de la communauté microbienne liée à la contamination pétrolière dans des sédiments présentant une faible et une forte activité de bioturbation. Des sédiments marins ont été maintenus en microcosmes durant 9 mois et soumis à quatre conditions : (i) pas de traitement (contrôle), (ii) pétrole, (iii) bioturbation et (iv) pétrole et bioturbation. Les efficacités de dégradation des hydrocarbures pétroliers se sont révélées similaires dans les deux types de sédiments. Par des approches moléculaires, la diversité taxonomique et fonctionnelle des communautés microbiennes totales et métaboliquement actives a été évaluée au cours du temps. Les communautés microbiennes ont subi d’importants remaniements structuraux spécifiques à chaque traitement. Nous suggérons que le fonctionnement global de la communauté est modifié par l’activité bioturbatrice sans pour autant modifier l’activité de dégradation. Ces travaux ont mis en évidence une redondance fonctionnelle de l’activité de biodégradation des hydrocarbures pétroliers des communautés microbiennes. L’isolement de communautés hydrocarbonoclastes a permis de confirmer cette redondance fonctionnelle
Coastal areas such as mudflats are affected by oil spills. In these environments, microorganisms play a crucial role in the fate of petroleum hydrocarbons. However, many factors influence microbial activities, especially the bioturbating organisms, which altered the oxygen penetration in sediments. The present work attempts to better understand the impact of petroleum contamination on microbial community associated with petroleum contamination in sediments with low and high bioturbation activity. This study is based on microcosm experiments with a device simulating tidal cycles. Marine sediments were maintained for 9 months in microcosms and subjected to four conditions: (i) no treatment (control), (ii) oil, (iii) bioturbation and (iv) oil and bioturbation. Chemical, microbiological and biological analyses were conducted throughout the experiment. The efficiencies of degradation of petroleum hydrocarbons were similar in both sediments. By molecular approaches, we assessed the dynamic of the functional and taxonomic diversity of the total and metabolically active communities during the oil contamination. Microbial communities showed significant structural rearrangements specific for each treatment that resulted in distinct microbial communities in both sediments. Hence, the overall microbial community structure was changed by bioturbating activity without changing the degradation capacity revealing a functional redundancy of the biodegradation capacity of hydrocarbons. This result was further supported by the isolation and characterization of hydro carbonoclastic communities

Orientador: Lucia Regina Durrant
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Neste trabalho foi formulado um consórcio microbiano para avaliação de seu potencial em degradar petróleo e seus derivados, em microcosmos e meio mínimo de sais. As 43 bactérias testadas na primeira fase do projeto demonstraram alguma propriedade que as classificam como potencialmente degradadoras de hidrocarbonetos. Todavia, as cepas 1, 9, 22, 23 e 36 demonstraram os melhores e mais homogêneos resultados em praticamente todos os parâmetros analisados, sendo estas, portanto, as cinco cepas selecionadas para a formulação do consórcio. As cepas do consórcio propiciaram uma degradação média para todos os hidrocarbonetos testados de 62,65% para cepa 1, 61,12% para a cepa 9, 53,77% para a cepa 22, 57,87% para a cepa 23 e de 44,03% para a cepa 36. Com exceção de alguns HPA¿s degradados por determinadas cepas, a grande maioria não foi totalmente biodegradado, fato que pode ser comprovado pelos baixos valores de EC50, e, com isso, altos valores de toxicidade aguda. No experimento realizado em microcosmo, avaliando-se a evolução de C-CO2 por modelos estatísticos, pode-se concluir que a melhor condição para a maximização desse parâmetro foi na condição do ponto central para a fonte de nitrogênio (0,10 g/100g de solo), ferro (0,0015 g/100g de solo) e inóculo (0,6ml/100 g de solo) e, na condição de +2 para a fonte de fósforo (0,08 g/100g de solo). Com este experimento foram fixadas as condições ótimas para uma melhor evolução de CCO2 em solos contaminados. No mesmo experimento, analisando a degradação dos hidrocarbonetos por cromatografia gasosa, foi obtida uma melhor degradação para o experimento 3 (uréia 0,07 g/100g de solo, fosfato de potássio 0,065 g/100 g de solo, Sulfato de ferro 0,001 g/100 g de solo, e inoculo 0,4 ml/100 g de solo), com uma degradação de 70,74% dos hidrocarbonetos. Nos microcosmos utilizando solo da REPLAN (Refinaria de Paulínia/PETROBRAS), com histórico de contaminação, foi obtida uma melhor degradação para os hidrocarbonetos analisados de 75% quando se utilizou a bioestimulação e de 51,7% quando se utilizou a bioaumentação e bioestimulação juntas. Para os microcosmos utilizando o consórcio liofilizado, foi obtida uma degradação de 57,85% para o liofilizado sem nutriente e, de 67% para o liofilizado com nutriente. As linhagens foram identificadas através da técnica do RNA 16S, como sendo pertencentes a gênero Bacillus, exceto o microrganismo 23, cuja identificação não foi possível através dos resultados obtidos, o consórcio microbiano tem potencial para ser aplicado em áreas contaminadas com petróleo e seus derivados
Abstract: In this work, a microbial consortium was evaluated regarding to its potential to degrade oil and derivatives, using microcosms in salt minimal medium. Forty three bacteria were tested in the first phase of this project to demonstrate some properties that could identify them as potentially degraders of hydrocarbons. The strains 1, 9, 22, 23 and 36 showed the best and homogeneous results in practically all the analyzed parameters, and five strains were selected to be part of the bacterial consortium. These strains had an average degradation of 62.65% for the strain 1; 61.12% for the strain 9; 53.77% for the strain 22; 57.87% for the strain 23; and 44.03% for the strain 36. Some PAHs were not completelly degraded by some strains, and this fact could be proven by the low values of EC50, with production of high values of acute toxicidade. In the microcosm experiments, the C-CO2 evolution was performed using statistical models, which could be concluded that the best condition for the maximization of this parameter was in the central point condition for the nitrogen source (0.10g/100g of soil), iron (0.0015g/100g of soil) and inoculum (0,6ml/100g of ground), using the condition of +2 for the phosphorus source (0.08g/100g of soil). In this experiments could be set an excellent condition for a better evolution of C-CO2 in contaminated soil. In the same experiment, analyzing the degradation of the hydrocarbons in GC-FID, a better degradation was obtained for the experiment 3 (urea 0,07g/100g of soil, potassium fosfate 0.065g/100g of soil, iron sulphate 0.001g/100g of ground, and inoculum of 0.4 ml/100g of soil), reaching hydrocarbons degradation of 70.74%. In the microcosms using the REPLAN (PAULINIA REFINARY/PETROBRAS) soil with previous contamination history, the best degradation of 75% was promoted when bioestimulating, and 51.7% when this soil was bioaugmented with the bacterial consortium and also bioestimulated. For the microcosms using the lyofilizated consortium, a degradation of 57.85% was verifyed without nutrient, and 67% with nutrient. The strains were identified through the RNA 16S sequencing as Bacillus genus, excepting to the strain 23, whose results of sequencing identification was not possible in this time. These results showed that the microbial consortium has a potential application in contaminated areas with oil and derivatives
Doutorado
Doutor em Ciência de Alimentos

Orientadores: Valéria Maia Merzel, Anete Pereira de Souza
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A ocorrência de óleos biodegradados nos reservatórios de petróleo, juntamente com o isolamento das primeiras bactérias a partir destes ambientes, forneceu evidências de micro-organismos ativos no subsolo profundo. Os micro-organismos, habitantes comuns de reservatórios de petróleo, desenvolveram estratégias eficazes de biodegradação, baseados em sistemas enzimáticos e vias metabólicas especializadas, de maneira a acessar os hidrocarbonetos como fonte de carbono e energia. No entanto, o conhecimento atual da diversidade microbiana e dos processos metabólicos envolvidos na biodegradação em reservatórios de petróleo ainda é limitado, principalmente devido à dificuldade em recuperar a complexa comunidade microbiana presente em tais ambientes extremos. Essa limitação imposta pelo uso de técnicas de cultivo convencionais pode ser superada através do uso de abordagens independentes de cultivo, como a metagenômica, a qual permite o acesso ao potencial metabólico dos micro-organismos não cultivados. Em trabalho prévio, uma biblioteca metagenômica fosmidial derivada de enriquecimentos aeróbios e anaeróbios de petróleo foi construída e avaliada para a capacidade de crescimento microbiano em hexadecano como fonte de única de carbono. No presente estudo, foi analisada a capacidade de biodegradação de hexadecano e fenantreno pelos clones selecionados dessa biblioteca através de cromatografia gasosa acoplada a espectrometria de massas (CG-EM). As análises que se sucederam tiveram como objetivos a identificação e caracterização das sequências metagenômicas responsáveis pela biodegradação de hidrocarbonetos. A estratégia de clonagem aleatória "shotgun" seguida de sequenciamento dos clones foi utilizada para determinar a sequência inteira dos insertos fosmidias que mostraram relevantes capacidades de biodegradação. Cerca de 30 kb de sequência foram montados para o fosmideo 1A (91% de degradação do hexadecano e 5% do fenantreno) e 37 kb para o fosmideo 2B (98% de degradação do hexadecano e 44% do fenantreno), e em cada um foram reconhecidos 23 ORFs e 40 ORFs, respectivamente. As proteínas putativas foram identificadas por comparação das sequências de aminoácidos deduzidas. Várias proteínas envolvidas na degradação aeróbia e anaeróbia de diferentes compostos de hidrocarbonetos foram encontradas. As sequências que codificam estas enzimas não mostraram-se agrupadas em clusters completos de degradação, semelhantes aos encontrados nas bactérias degradadoras de hidrocarbonetos conhecidas. Os fragmentos metagenômicos continham apenas subconjuntos de genes pertencentes a várias vias, mostrando novos arranjos gênicos. Esses resultados reforçam o potencial da abordagem metagenômica para a prospecção e elucidação de novos genes e vias metabólicas, contribuindo para uma visão mais abrangente dos processos de biodegradação que ocorrem nos reservatórios de petróleo
Abstract: The occurrence of biodegraded oil in petroleum reservoirs, along with the isolation of the first bacteria from such environments, provided evidence for active microorganisms in the deep subsurface. Microorganisms are common inhabitants of petroleum reservoirs and they may have effective biodegrading strategies, based on specialized enzyme systems and metabolic pathways, as a form to access hydrocarbons as carbon and energy sources. However, the current knowledge of the microbial diversity and metabolic pathways involved in hydrocarbon biodegradation in petroleum reservoirs is still limited, mostly due to the difficulty in recovering the complex community present in such extreme environments. This limitation imposed by conventional culturing techniques can be circumvented by the metagenomic approach, which is a culture-independent molecular method that allows the access to the metabolic potential of previously uncultured microorganisms. In a previous work, a metagenomic fosmid library derived from aerobic and anaerobic petroleum enrichments were constructed and screened for the detection of microbial growth on hexadecane as sole carbon source. In this study, the biodegradation abilities on hexadecane and phenanthrene of selected clones were analyzed using Gas Chromatography - Mass Spectroscopy (GC-MS) and subsequent analyses aimed at the identification and characterization of metagenomic sequences responsible for the hydrocarbon biodegradation. The shotgun sequencing approach was used to determine the whole insert sequence of two fosmid clones showing different and relevant biodegradation capacities, FOS1A (91% hexadecane and 5% phenanthrene degradation) and FOS2B (98% hexadecane and 44% phenanthrene degradation). About 30 kb in sequence were assembled for the fosmid 1A and 37 kb for fosmid 2B, and 23 ORFs and 40 ORFs were recognized in each one, respectively. Putative proteins were identified by comparison of deduced aminoacid sequences. Several proteins involved in the degradation of different hydrocarbon compounds by aerobic and anaerobic pathways were found. The sequences coding these enzymes were not grouped together into complete clusters of degradation pathways similar to those already described in known hydrocarbon degrading bacteria. The metagenomic fragments contained only subsets of gene belonging to different pathways, showing novel gene arrangements. The results obtained in this study reinforce the potential of the metagenomic approach for the prospection and elucidation of new genes and pathways, contributing to a broader perspective of the biodegradation processes that take place in petroleum reservoirs
Mestrado
Genetica de Microorganismos
Mestre em Genética e Biologia Molecular

Orientadores: Jose Luiz Antunes de Oliveira e Sousa, Paulo Dore Fernandes
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecânica e Instituto de Geociências
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Resumo: O sucesso da estimulação por fraturamento hidráulico ou ácido é dependente da geração de condutividade. No fraturamento hidráulico sustentado, a condutividade é gerada pela introdução de uma quantidade significativa de um agente de sustentação granular e praticamente esférico. No fraturamento ácido, a condutividade é gerada por irregularidades reveladas devido à reação desigual do ácido em cada uma das faces da fratura naturalmente rugosa e da resistência destas mesmas irregularidades para suportar as tensões que atuam após o fechamento da fratura. A baixa resistência das irregularidades leva ao colapso e cicatrização da fratura ácida com a perda total da condutividade. Esta dissertação investiga a viabilidade da estimulação por fraturamento ácido em carbonatos microbiais rasos e profundos. Ensaios de laboratório em escala reduzida foram projetados para verificar se a condutividade de uma fratura induzida por reação química da rocha reservatório com um ácido forte pode ser mantida frente às elevadas tensões normais efetivas que agirão na fase de explotação do poço. A dissertação ainda investiga se o emprego de uma pequena quantidade de agente de sustentação é capaz de dividir os esforços aplicados sobre as irregularidades de uma fratura ácida e manter a condutividade por maiores períodos de tempo. As superfícies que imitam fraturas ácidas são criadas pelo ataque ácido em corpos de prova em condições de laboratório que reproduzem em escala aquelas observadas em um fraturamento ácido real. Imagens digitais são utilizadas para medir a rugosidade das fraturas ácidas e o consumo de rocha carbonática pelo ácido na face do corpo de prova. A resistência das irregularidades é medida indiretamente pelo decréscimo da condutividade da fratura ácida frente a tensões de confinamento crescentes. O efeito ou não da adição de agente de sustentação é medido pela comparação dos parâmetros da correlação empírica desenvolvida por Nierode e Kruk
Abstract: A well succeeded stimulation by hydraulic or acid fracturing is related to conductivity creation. In a hydraulic fracturing, the conductivity is created by means of introducing a significant amount of a propping agent. In an acid fracturing, the conductivity is related to the creation of asperities due to uneven acid reaction on each naturally rougher fracture side and the asperities strength to withstand high stresses that start to act just after fracture closure. Low asperities strength leads to collapse of acid fracture and fracture healing with total conductivity loss. The dissertation investigates the feasibility of acid fracturing stimulation in shallow and deep microbial carbonates. Laboratory tests were designed to verify if the acid fracture conductivity induced by chemical reaction of reservoir rock with a strong acid could be kept when facing the high effective normal stress that is expected in exploitation phase. The dissertation still investigates whether the use of a small amount of proppant is able to share the tension that acts on asperities and maintain the acid facture conductivity for longer periods. The surfaces that mimic acid fractures are created by acid etching specimens in laboratory conditions reduced in scale from those observed in an actual acid fracturing job. Digital images are used to measure fracture roughness and rock consumption by acid on the specimen face. The asperities strength is indirectly measured from acid conductivity decrease under increased confinement tension. The effect of adding or not adding propping agent is accomplished comparing empirical parameters from Nierode and Kruk correlation
Mestrado
Explotação
Mestre em Ciências e Engenharia de Petróleo

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia Química, Florianópolis, 2015.
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Um dos maiores problemas nos poços maduros de petróleo é a presença de acidez biogênica (souring), gerada principalmente pela produção biológica de sulfeto na recuperação secundária do petróleo em plataformas offshore. Uma das estratégias para o controle do souring em reservatórios consiste na injeção de nitrato, o qual é utilizado preferencialmente como aceptor de elétron, limitando desta forma a produção de sulfeto. O objetivo do presente estudo foi investigar a relação entre o carbono e o nitrogênio (Relação C:N) necessária para inibição da produção de sulfeto num consórcio microbiano enriquecido a partir de água de produção de um reservatório de óleo offshore. Foi realizado primeiramente um ensaio em batelada com acetato e propionato (ácidos graxos de fácil degradação) para avaliar o melhor doador de elétrons utilizado pelas bactérias redutoras de sulfato (BRS) para produção de sulfeto. A partir do ensaio concluiu-se que o consórcio de bactérias utiliza propionato para produzir acetato e CO2, Com isso, para subseqüentes ensaios, foi utilizado propionato como aceptor de elétrons. Dois ensaios mais foram feitos, a fim, de avaliar a relação C:N inibitória da produção de sulfeto: o primeiro, mudando a concentração de carbono e a concentração de nitrogênio; o segundo, mantendo a concentração de carbono constante e variando a concentração de nitrogênio. No primeiro, evidenciou-se que, nas condições onde a concentração de nitrogênio, expressada em nitrato (N-NO3?), foi superior à concentração de carbono orgânico, expressada em propionato ( ). A produção de sulfeto ficou inibida, possivelmente, pela indisponibilidade de carbono, ou então pelo favorecimento de processos de desnitrificação autotrófica, que resultariam na remoção de algum sulfeto formado. Já, nas condições onde a concentração de nitrato foi inferior à de carbono orgânico houve formação de sulfeto, a partir do ponto de término do nitrito, não havendo uma influência da concentração residual de matéria orgânica no sistema. Os resultados da segunda série de ensaios foi concordante com a primeira, evidenciando que a relação C:N propriamente dita não é um fator que resulte em inibição ou estimulação das BRS num ponto especifico de injeção, mas sim, a presença ou ausência de nitrato ou nitrito, de forma independente da concentração de matéria orgânica. Um ensaio adicional foi realizado para determinação da presença ativa de bactérias autotróficas redutoras de nitrato e oxidadoras de sulfeto (BRN-OS); os resultados mostram presença ativa de BRN-OS.

Abstract : A major problem in oil wells is the presence of biogenic acid (Souring) generated mainly by the production of sulfide in secondary recovery of oil at offshore platforms. The control of souring in reservoirs can be done through nitrate injection, which is preferably used as the electron acceptor, limiting the production of sulphide. The objective of this study was to investigate the carbon-nitrogen ratio required (C:N) for inhibition of sulfide production in an enriched microbial consortia coming from a production water from an offshore oil reservoir. It was first performed a batch assay with acetate and propionate (easily degradable fatty acids) to evaluate the best electron donor used by sulphate reducing bacteria (SBR) to produce sulfide. It was concluded from this assay that the bacteria consortia has a greater affinity to propionate. Two more assays were made with the objective to evaluate the C:N ratio that inhibit sulfide production: the first, it was maintained constant carbon concentration and varying nitrogen concentration; the second, it was maintained nitrogen concentration constant and varying carbon concentration. The first assay showed that, under the conditions where the concentration of nitrate expressed as nitrogen (N- NO3?) was greater than the concentration of organic carbon ( ), sulfide production was possibly inhibited by the lack of organic matter, or it was favored by autotrophic denitrification processes, that contributes to remove of some sulfide formed. When nitrate concentration was lower than organic carbon, sulfide was formed immediately after nitrogen species were totally consumed. There was no influence of residual concentration of organic matter in the system. The second assay results corroborate with the previous one, indicating that the C:N ratio is not a factor that results in SBR inhibition or stimulation, but, what matter is the nitrate, or nitrite, presence or absence, independently of the organic matter concentration. An additional test was performed to determine the presence active of sulfide-oxidizing, nitrate reducing bacteria (NBR - OS).

碩士
雲林科技大學
環境與安全工程系碩士班
98
Benzene, one of the petroleum hydrocarbons has been classified as a first-degree carcinogen by International Agency for Research on Cancer and listed in the control of toxic compound by Environmental Protection Administration, therefore it is imperative to use and dispose benzene. Among the remediation technologies for a petroleum hydrocarbon contaminated site, bioremediation is a common used strategy. The feature of bioremediation is that the microorganisms play a role of degrader in degrading pollutants and transforming to form low toxic or non toxic compounds. During the process of microbial decomposition of pollutants, the generated electrons (e-) and protons (H+) could be used to generate electricity through the use of microbial fuel cell (MFC)(including electrodes, copper wires, external resistors, the proton exchange membrane (PEM) and the electrolytes). This highlights the significance of MFC. In this study, vertical and H-type configurations of the MFC system were used to evaluate the power generation potential as the process of biodegradation of benzene. The vertical-type MFC was assembled by acrylic material of cylinder column and the H-type MFC was assembled by glass material of serum bottles. Both MFC systems were used a PEM to separate the anode chamber from cathode chamber. Carbon cloths were served as anode and cathode materials, and the anodes were connected with cathodes by copper wires and external resistors (1 KΩ). In the experiments, the phosphate buffer was used to maintain pH within 6.5–7 in both chambers. The anode chamber was inoculated with mixed cultures which were acclimated to benzene for a long-term. However, potassium ferricyanide (0-200 mM) as the terminal electron acceptor was added in the cathode chamber. The results showed that: (1) the mixed cultures can use benzene or glucose as the sole carbon source to generate electricity in the H-MFC, showing these two compounds can be used as fuels for MFC; (2) the power generation for H-MFC is higher than vertical-type MFC. This is believed to be due to the shorter distance between anode and cathode in H-MFC compared to in vertical-type MFC. The short distance may reduce the transported distance for e- and H+; (3) the output voltage increases from 4.2 mV to 116 mV with the addition of potassium ferricyanide from zero mM to 200 mM in cathode chamber in H-MFC. The conductivity in the cathode solution changes from 6.71 ms/cm (0 mM potassium ferricyanide) to 46.5 ms/cm (200 mM potassium ferricyanide); (4) the power generation efficiency of MFCs can be improved further with adding the potassium ferricyanide in the cathode solution leading to reduce the internal resistance of MFCs; and (5) the electron transferring materials (mediators) have been detected, according to the results of redox reaction in H-MFC examined by cyclic voltammetry (CV) analysis. The results of this study can be used as a basis for future assessment of using MFC to treat the benzene-type wastewater and the power generation capacity.

The contamination of environmental media by total petroleum hydrocarbons (TPH) is a concern in many parts of the world; particularly as most petroleum components like polycyclic aromatic hydrocarbons (PAHs) are either toxic or carcinogens. In South Africa, the sale of major petroleum products by the South African Petroleum Industry Association (SAPIA) reveals that about 21 billion litres of petroleum products are sold per year. These products include bitumen, diesel, fuel oil, illum paraffin, jet fuel and petrol. In addition, 19.5 million tonnes of crude oil are brought into South Africa annually to feed the country’s four refineries. The production of oily sludges at refineries, transportation, storage, and handling of petroleum products by end users, results in environmental contamination. The soil environment is particularly vulnerable to hydrocarbon contamination as most of the accidental spillages by trucks, rail locomotives and pipelines have a direct impact on the soil medium. As most of the petroleum compounds are either toxic or carcinogenic, their removal from the soil is necessary. The literature reveals that biological treatment of hydrocarbons is cost effective compared to other treatment options. However, in order to improve the efficiency of biological treatments, there is a need to understand the microbial diversity of TPH stressed environments and how simple biomonitoring ‘instruments’ can be used to evaluate the removal of hydrocarbons from the soil. The message from the literature indicates some potential solutions to the existing problems associated with soil microbial diversity and biotreatment of hydrocarbon contaminated soil, which must be investigated. The main aim of this work was to evaluate the microbial diversity of the different soil environments disturbed by Total Petroleum Hydrocarbons (TPHs) and the potential use of plants and microorganisms in monitoring and removing hydrocarbons from the soil. In addition, the potential of the culture-independent methods in complementing, the culture-dependent methods when evaluating soil microbial diversity were also evaluated. The polyphasic approach was successfully used in evaluating microbial diversity in both hydrocarbon-contaminated and uncontaminated soils. The approach involved the use of community level physiological profiles (CLPP) and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to evaluate the effects of hydrocarbons on the soil microbial communities of both the contaminated and non-contaminated soil layers at a diesel contaminated site. Because of the ability of the molecular methods (PCR-DGGE) to complement the CLPP, the polyphasic approach is recommended when evaluating soil microbial diversity and the effect of pollutants on microbial community structure as the approach appears to compensate for the limitations of each of the methods of evaluating microbial diversity. However, further work is needed to improve the recovery of bacteria from the soil, particularly where the interest is to evaluate the availability of the indigenous microbial populations for bioremediation. The substrate utilisation pattern and 16S DNA fragments of the soil microbial communites in different soil layers at a diesel contaminated site were different. The substrate utilisation pattern of the topsoil was different from the substrate utilisation pattern of the soil layers below 1m. In addition, the substrate utilisation pattern of the contaminated and uncontaminated soil layers were different. 16S DNA fragments of the different soil layers were also different. While the metabolic activities of different samples as reflected by CLPP does not necessarily imply the difference in community structure of the samples, PCR-DGGE revealed differences in 16S DNA fragments and this complemented the results of the culture based methods. The results suggest that the use of functional and genetic approaches (in combination) have a better chance of revealing a ‘clearer’ picture of soil microbial diversity. The distribution of hydrocarbon-utilising bacteria and the efficiency of biodegradation of hydrocarbons vary with soil depth. The biodegradation rate of hydrocarbon was highest in the topsoil compared to other soil layers and this was supported by the high number of hydrocarbon-degrading bacteria in the topsoil compared to soil layers at and below 1m. The results suggest that the biological removal of hydrocarbons varies in different soil layers and that microbial diversity as measured by CLPP and PCR-DGGE varies with depth in hydrocarbon-contaminated soil. The information about metabolic activities of different soil layers is important when assessing the footprints of degradation processes during monitored natural attenuation (MNA). However, further studies are required to understand the effect of (not only) other pollutants, but the influence of soil components (pore volume, level of adsorbents and other environmental factors) on the microbial diversity of different soil layers in both ‘shallow’ and deep aquifers. The microbial diversity of different environments contaminated by hydrocarbons has different community level physiological profiles. At diesel depots where similar hydrocarbons are used for maintenance of locomotives, the number of bacteria (both total culturable heterotrophic bacteria and hydrocarbon-degrading bacteria) was proportional to the level of hydrocarbon contamination. However, there was no significant difference in the level of total culturable heterotrophs (TCHs) and the hydrocarbon degrading bacteria. In addition, the biological activities as evaluated by CO2 production were higher in nutrient amended treatments in which high numbers of TCHs were present. Microbial diversity of polluted surfaces needs to be studied further to investigate the concentration or the thickness of the hydrocarbons layer on the rock surfaces that encourages the attachment or colonization of the TCHs and the hydrocarbon-degrading bacteria. The hydrocarbons rather than the geographical origin of the soil sample appear to be more important in determining functional or species diversity within the bacterial communities. The samples from different locations were as different as samples from the same location but from contaminated versus uncontaminated soil. The results of the soils from different locations artificially contaminated by different hydrocarbons also reached the same conclusion. However, further work is required to investigate the importance of soil heterogeneity in community studies of soil environments contaminated by similar hydrocarbons. The removal of Polycyclic Aromatic Hydrocarbons (PAHs) in multi-planted soil microcosm was higher compared to PAHs removal in monoculture soil microcosms. In addition, the PAH removal was higher in the vegetated soil microcosms compared to the non-vegetated microcosms. There was however, no significant difference in the PAH removal in the soil microcosms planted with Branchiaria serrata and the microcosm with Eulisine corocana. The Principle Component Analysis (PCA) and Cluster analysis used to analyse the functional diversity of the different treatments revealed differences in the metabolic fingerprints of the PAH contaminated and non-contaminated soils. However the differences in metabolic diversity between the multi-planted and mono-planted treatments were not clearly revealed. The results suggest that multi-plant rhizoremediation using tolerant plant species rather than monoculture rhizoremediation have the potential to enhance pollutant removal in moderately contaminated soils. Lepidium sativum, a plant with short germination period, was successfully used to monitor, the removal of Polycyclic Aromatic Hydrocarbons (PAHs) from the soil. The sensitivity of L. sativum eased with increasing concentration of the polycyclic aromatic hydrocarbons in the artificially contaminated soil while no germination occurred in the historically polluted soil. When used during phytoremediation of PAH, the germination level of L. sativum was inhibited during the first weeks, after which germination increased, possibly due to PAH dissipation from the soil. The methodology based on the sensitivity of L. sativum to PAH can be used as a monitoring tool in bioremediation of soil contaminated with PAH. However, the methodology should be developed further to gain more knowledge on aspects of bioavailability of PAH in both the aged as well as the freshly spiked soil. Also critical is the sensitivity of the seeds to other pollutants (e.g. heavy metals), which are most likely to occur in the presence of the PAHs. Although the biological activities have the potential to monitor the removal of hydrocarbons from the soil, the methodologies have not been developed sufficiently to cater for the heterogeneity of the soil and to differentiate toxicity by the parent compound and the metabolites. At present, it is best that they be used to complement existing conventional monitoring instruments. Finally, the biological removal of hydrocarbons is cost-effective compared to other treatments. However, inherent physical, chemical and biological limitation hampers the efficient utilisation of the bioremediation technologies. Biostimulation approaches involving the stimulation of indigenous pollutant-degrading bacteria should be preferred ahead of bioaugumentation. The latter approach should be considered when the contaminated site does not have the indigenous pollutant-degrading bacteria. Even in this case, the aim should be to ‘seed’ the biodegradation knowledge to the indigenous microbial populations due to poor survival of the added strains.
Thesis (PhD (Biotechnology))--University of Pretoria, 2007.
Microbiology and Plant Pathology
unrestricted

碩士
國立成功大學
生物學系碩博士班
92
Petroleum hydrocarbons are the most widespread contaminants in the environment. Several of petroleum aromatic hydrocarbons (PAHs) are known mutagens or carcinogens for human and other organisms and they are difficult to be degraded in the natural environment. Bioremediation is considered an useful method to degrade the PAHs using the PAH-degrading bacteria. In this study, polyphasic approach combined cultivation-dependent with cultivation-independent methods was used to study the microbial communities in petroleum-contaminated soil. A total of 7 strains were isolated and 71% (5 strains) of which were oil-degrading bacteria that belong to Pseudomonas (2 strain)、Bacillus (1 strain)、Mycobacterium (1 strain)、Acinetobacter (1 strain). Phylogenetic analysis of a bacterial 16S rDNA clone library from the petroleum-contaminated soil treated with BTEX as carbon source showed that 9 of 24 OTUs (operational taxonomic units) were the oil-degrading bacteria which belong to Pseudomonas (30.5%)、Burkholderia (24.5%)、Sphingomononas (6.0%)、Bacillus (2.5%) and Nocardioides (0.5%). The results of combined cultivation-dependent with cultivation-independent methods revealed the majority of the oil-degrading bacteria in soil were Pseudomonas. Functional gene (xylE gene；catechol 2,3-dioxygenase) primer was designed in this study could get better and more PCR product from pure bacterium strain and environmental samples than the DEG primers used by Mesarch et al.(2000). The change of the microbial community in petroleum-contaminated soil was investigated by denaturing gel electrophoresis (DGGE) approach. The results indicated that different microbial community structure depended on the soil source and depth. After adding BTEX as carbon source into the petroleum-contaminated soil through one to three weeks, the bacterial number became less and community structure changed. It indicated that BTEX have adverse effects to microbial community structure.

碩士
國立中山大學
生物科學系研究所
91
In this study, we had established a 16S rDNA-DGGE analys is system to detect the microbial community in petroleum polluted soil and assess the feasibility of using this system to monitor the bioremediation process. Three genomic DNA extracted methods, the KIT, the Bead-beating system, and the Freeze-thaw method were used to evaluate the DNA extraction efficiency and purity. These DNA samples were further tested by DGGE to analysis the microbial community in soil samples. The results showed that KIT method performed advantageous not only in the DNA extraction efficiency and purity, but also expressed the richest bacterial community in its PCR products. From the DGGE analysis, our data indicated that composition of bacterial community were different in the soil samples that were taken from the same site but at different time. This indicated that the species and number of microorganisms in a polluted soil were under a dynamic transition. The combination of DGGE and 16S rDNA gene sequence analysis system were also proven useful in identifying the predominant microbes in a soil sample and monitoring its bacterial community.

碩士
國立中山大學
生物科學系研究所
92
Abstract This research used molecular biological techniques such as polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) to analyze the dynamic microbial community and biodiversity in the groundwater contaminated with petroleum-hydrocarbons. The 16S rDNA sequences from all water samples were compared with the sequences of relative bacteria in the Ribosomal Database Project Bank to construct a phylogenetic tree. The results allowed us to understand the composition of the microbial communities in the petroleum-hydrocarbon contaminated groundwater. In this study, groundwater samples taken from the Chinese Petroleum Corporation Kaohsiung Refinery (CPCKR), Chinese Petroleum Corporation at Ciaotou fuel Tank Farm (CPCCTF) and China Petrochemical Development Corporation at Kaohsiung Factory (CPDCKF) were analyzed. The contaminated sites at CPDCKR and CPCCTF are remeated by natural attenuation. While the CPDCKF site is remeated by an enhanced air sparging bioremediation. In CPDCKR, we found that the low polluted area contained the richest microbial community, followed by the non-polluted area, and the high polluted area. At the CPCCTF site, the microbial community in the non-polluted area was richer than the high-polluted area. Increased microbial populations and variation in microbial community have beenobserved in non-polluted, less polluted, and highly polluted areas. The microbial community showed a dynamic succession of complexity during the bioremediation process at the CPDCKF site. From the 16S rDNA sequence analysis, it is possible that all samples contained petroleum-hydrocarbon degrading bacteria. These petroleum-hydrocarbon degrading bacteria include Methylobacterium, Xanthobacter, Xanthomonas and Pseudomonas at CPCKR site, Flavobacterium at CPCCTF site, Nocardia, Pseudomonas, Rubrivivax, Methylobacterium, and Candida at CPDCKF site. This study also demonstrates that it is more economic and reliable of using molecular techuiques to analyze the groundwater. Thus, groundwater samples can be used to replace soil samples for future work.

A standardized method to examine soil microbial health incorporating biomass, activity, and diversity measurements is currently lacking, limiting the use of this ecologically relevant endpoint in ecological risk assessments. The soil microbial health of a petroleum hydrocarbon-contaminated boreal forest soil, relative to a reference soil, was examined using a suite of tests. Microbial health impacts in the contaminated soil were observed using nitrification, organic matter decomposition, bait lamina, denaturing gradient gel electrophoresis, community level physiological profiling, and most enzyme assays. Results of heterotrophic plate count and respiration tests indicated higher culturable numbers and activity in the contaminated soil. A data integration technique was developed to incorporate the results from individual tests into an overall conclusion, indicating that soil contamination at the site moderately to severely impacted microbial health. The research presented lays the foundation for the development of a soil microbial health standardized method.

Sulfate- and Fe(III)-reducing, and methanogenic prokaryotes (SRP, FRP, MGP) are key players in metabolic pathways involved in anaerobic biodegradation processes. Understanding the metabolic activity of these microbes in environments can enhance microbe-mediated processes such as oil spill bioremediation and methane biogas production. In this study, anaerobic microbial activities in Deepwater Horizon oil spill-impacted salt marsh sediments, and in methanogenic coal bed production water enrichment cultures amended with trace elements (TE), were elucidated by employing an approach combining methods in molecular biology and geochemistry. In situ metabolic activity of SRP, FRP and MGP were monitored seasonally and metabolically-active communities were identified in oil-impacted sediments using quantitative real time Reverse Transcription -PCR and clone library analysis of key functional genes: Dissimilatory (bi)sulfite reductase (dsrAB), Geobactereceae-specific citrate synthase (gltA), methyl coenzyme M reductase (mcrA), and benzyl succinate synthase (bssA). In situ application of montmorillonite clay was assessed for its potential at accelerating PHC degradation by stimulating microbial activities. Levels of dsrA, gltA and bssA transcripts suggested that PHC-oxidizing SRP are more active in summer while FRP are more active in winter, indicating their activities linked to the seasonal changes of redox potential and vegetation. BssA gene expression peaked in winter, and was highest at more highly oil-impacted sites. Expression of all genes was higher in clay-amended sites. bssA transcript level and Fe(II) production were highest in clay-amended microcosm. Total petroleum hydrocarbon (TPH) levels were lower in oil and clay-amended microcosm incubation than one with oil only amendment, suggesting enhanced TPH degradation by clay amendment. Pyrosequencing analysis 16S rRNA gene in clay-amended microcosms demonstrated the highest percentage abundance of groups closely related to known anaerobic aromatic degraders. Levels of mcrA transcripts correlated with methane production rates in TE-amended coal bed production water enrichments. The findings of the present study clearly support the advantage of gene expression analyses for estimating microbial activity. To the best of our knowledge, this is the first in situ study which employs key functional gene markers as molecular proxies for metabolic activity and diversity assessments in anaerobic oil-contaminated salt marsh sediment and also elucidates clay-enhanced in situ TPH degradation.