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Metal-reducing microorganisms reduce a variety of metals in metabolic processes coupled to the oxidation of organic compounds. These bacteria play an important role in the biogeochemical cycling of metals and organic matter in anaerobic aquatic and sediment ecosystems. It has been proposed recently that metal-reducing microorganisms also are active in deep subsurface environments such as petroleum reservoirs. Only two metal-reducing bacteria have been isolated from petroleum reservoir fluids, Shewanella putrefaciens and Deferribacter thermophilus. This project studied the occurrence and distribution of metal-reducing microorganisms in petroleum reservoirs. The research focused on the isolation, characterisation and identification of anaerobic bacteria from petroleum reservoirs that were capable of reducing metals and the potential roles of these isolates in the microbial ecology and biogeochemical cycling of petroleum reservoirs. Petroleum reservoirs were selected for this study on the basis of physio-chemical conditions such as temperature, salinity, pH and the presence of organic and inorganic compounds, that were likely to provide a suitable environment for anaerobic bacteria capable of reducing metals. Factors such as the stratigraphic features of the sedimentary basin, age of reservoir and past oil field practices also were considered in choosing the reservoir for study. Seven petroleum reservoirs in the USA and Azerbaijan were chosen for extensive investigations. The physico-chemical conditions in these reservoirs varied substantially. A systematic study of the production water from these petroleum reservoirs revealed a consistent presence of iron- and manganese-reducing microorganisms. It was found that salinity and temperature play a significant and defining role in the occurrence and distribution of these metal-reducing microorganisms. Biotic metal reduction was detected from production waters from all but one of the oil wells sampled. It was significant that the water from this well (Neftcala #1074) was the most saline (78 g/l NaCI). Metal-reducing activity was detected at temperatures up to 70°C. Two pure cultures, strains RED1 for Redwash petroleum reservoir (USA) and NEF1 from the Neftcala petroleum reservoir (Azerbaijan) were isolated and characterized. The strains had diverse physiological and metabolic properties including the ability to oxidize a wide range of carbon compounds and reduce a variety of metals. Their temperature, salinity and pH optima varied markedly. Phylogenetic analyses of the 16S rRNA of strain RED1 showed that the strain represented a new species of a new genus in the domain Bacteria. The bacterium most closely related to strain RED1 is the fermentative Fe(III)-reducer, Pelobacter acetylenicus (similarity value, 92.8%). Strain NEF1 possesses a unique combination of phenotypic traits and a low mol % G+C. From preliminary analyses and comparative biochemistry, NEF1 appears to be a novel metal-reducing bacterium of the Flexistipes group. The bacteria isolated in this study were able to grow at temperatures and salinities consistent with the reservoir from which they were isolated. This indicated that petroleum reservoirs are a new source of physiologically diverse, novel, metal-reducing microorganisms. The bacteria isolated also demonstrated a number of characteristics that would enable them to survive and persist in extreme subsurface conditions and develop a selective ecological advantage in petroleum reservoir environments. Significantly, the metal-reducing bacteria isolated were able to utilize an array of metabolic products produced by bacteria indigenous to petroleum reservoirs. This has resulted in a new proposed model for the ecological succession of bacteria in petroleum reservoirs.

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.

Currently standard first order single-point upstream weighting methods are employed in reservoir simulation for integrating the essentially hyperbolic system components. These methods introduce both coordinate-line numerical diffusion (even in 1-D) and cross-wind diffusion into the solution that is grid and geometry dependent. These effects are particularly important when steep fronts and shocks are present and for cases where flow is across grid coordinate lines. In this thesis, families of novel edge-based and cell-based truly multidimensional upwind formulations that upwind in the direction of the wave paths in order to minimise crosswind diffusion are presented for hyperbolic conservation laws on structured and unstructured triangular and quadrilateral grids in two dimensions. Higher resolution as well as higher order multidimensional formulations are also developed for general structured and unstructured grids. The schemes are coupled with existing consistent and efficient continuous CVD (MPFA) Darcy flux approximations. They are formulated using an IMPES (Implicit in Pressure Explicit in Saturation) strategy for solving the coupled elliptic (pressure) and hyperbolic (saturation) system of equations governing the multi-phase multi-component flow in porous media. The new methods are compared with single point upstream weighting for two-phase and three-component two-phase flow problems. The tests arc conducted on both structured and unstructured grids and involve full-tensor coefficient velocity fields in homogeneous and heterogeneous domains. The comparisons demonstrate the benefits of multidimensional and higher order multidimensional schemes in terms of improved front resolution together with significant reduction in cross-wind diffusion.

Subsurface data derived from ~388 ft of drill core from Martin County (TX) were used to understand the depositional setting of the Wolfcamp-D, a petroleum producing interval in the Midland Basin. Elemental geochemistry collected via x-ray fluorescence revealed a highly variable depositional history marked by the deposition of diverse siliciclastic and carbonate lithofacies. Integration of multiple datasets resulted in the interpretation of nine lithofacies, whose deposition appears cyclical. Correlations between molybdenum and total organic carbon indicate slow recharge of bottom waters and anoxic/euxinicconditions within the basin. The presence of phosphatic nodules coinciding with siliceous black mudrocks suggested high levels of primary productivity driven by upwelling. High-frequency sea level variability, driven by far-field glaciation and regional paleoclimate, were key controls on both the chemostratigraphy and lithofacies. Along-strike variability is seen throughout the basin due to paleobathymetry, proximity and connections to paleochannels, and localized structures. Rhenium-osmium (Re/Os) geochronology was conducted on siliceous mudrocks with high total organic carbon. A depositional age of 300 ± 18 Ma was obtained, partially confirming previous correlations to shelf biostratigraphic data. Scatter in the Re/Os data is likely due to mixing in the basin or non-hydrogenous Os incorporated into the analysis due to the method of preparation.

La récupération assistée des hydrocarbures par des microorganismes (MEOR) est une technologie potentiellement utilisable pour améliorer l’efficacité de l’extraction pétrolière. Cette technique utilise les capacités métaboliques de certaines souches bactériennes pour récupérer les huiles des réservoirs. Cependant, le manque de connaissances sur la physiologie et les activités métaboliques des microorganismes des réservoirs est un obstacle majeur pour le développement de cette approche. L’objectif de ce travail était d’étudier la physiologie des microorganismes indigènes de réservoirs pétroliers, en déterminant leurs capacités métaboliques, leurs conditions de croissances et leur positionnement taxonomique. Pour cela, trois activités physiologiques d’intérêt pour la MEOR : (1) la dégradation de l’hexadécane ;(2) la production de biosurfactant et (3) la formation de biofilm ont été évaluées sur 84 souches bactériens anaérobies isolées exclusivement de plusieurs réservoirs pétroliers. Ces isolats appartiennent à deux groupes métaboliques : les bactéries sulfato-réductrices (BSR) et les anaérobies fermentaires. Ainsi, cette prospection à donner une image de la diversité des souches de réservoirs possédant des activités appropriées pour la MEOR. Le séquençage et l’analyse phylogénétique du gène codant pour L’ARNr 16S a permet d’identifier deux nouvelles espèces bactériennes d’anaérobies fermentaires, SRL 4223 et SRL 4209 capables de produire des biosurfactants. Ainsi la caractérisation de ces deux isolats a révélé que la souche SRL 4223 présentait toutes les caractéristiques phénotypiques et génétiques autorisant sa classification un nouveau genre nommé Pleomorphochaeta caudata et que la souche SRL 4209 peut être affilié comme une nouvelle espèce de ce nouveau genre. / The Microbial enhanced oil recovery (MEOR) is a potentially useful technology to improve the efficiency of oil extraction. This technique utilizes microorganisms and/or their metabolites (biosurfactants, polymers, biomass…etc.) to recover oil from reservoirs. However, the lack of basic knowledge about physiology and metabolic capacities of reservoir microorganisms is a major obstacle for the development of this approach. The objective of this work was to study the physiology of indigenous reservoir microorganisms by determining their metabolic capacities, their growth conditions and their taxonomic position. For this, three activities related to MEOR: (1) hexadecane degradation; (2) biofilm formation and (3) biosurfactant production were evaluated on 84 anaerobic bacterial strains isolated exclusively from several petroleum reservoirs. These isolates belong to two metabolic groups: sulfate-reducing bacteria (SRB) and anaerobic fermentative bacteria. This study gives a picture of the diversity of indigenous strains possessing proper activities for MEOR. Sequencing and phylogenetic analysis of 16S rRNA gene identified two new species of fermentative bacteria: SRL 4223 and SRL 4209, capable of producing biosurfactants. Characterization of these isolates revealed that the strain SRL 4223 had all the phenotypic and genetic characteristics allowing its classification as a new genus named Pleomorphochaeta caudata and the strain SRL 4209 was affiliated as a new species of this genus.

O presente trabalho teve como objetivos caracterizar uma coleção de 98 bactérias isoladas de amostras de petróleo e água de formação de reservatórios da Bacia de Campos (RJ) utilizando técnicas de taxonomia molecular e avaliar o potencial de degradação de biomarcadores do petróleo. O sequenciamento e análise filogenética do gene RNAr 16S revelaram Bacillus firmus, megaterium, pumilus, sphaericus, simplex, cereus/B. thuringiensis Marinobacter lutaoensis, Halomonas shengliensis/H. alimentaria/ H.campisalis, Citreicella thiooxidans, Stenotrophomonas maltophilia, Achromobacter xylosoxidans, Micrococcus luteus, Kocuria rosea, Streptomyces alboniger/S. chartreusis /S. moderatus, Staphylococcus hominis e Staphylococcus pasteuri/S. warneri. Os resultados evidenciaram a preferência pela biotransformação do ácido nonadecanóico e esqualano. A caracterização da microbiota presente nos reservatórios e avaliação do potencial de biodegradação pode contribuir para fornecer subsídios para estudos futuros sobre os mecanismos biológicos responsáveis pela biodegradação do petróleo. / This study is aimed to characterize a collection of 98 bacteria isolated from oil and formation water samples derived from reservoirs of the Campos Basin (RJ) using molecular biology-based techniques and to evaluate the degradation potential of petroleum biomarkers. Further sequencing and phylogenetic analysis of 16S rRNA genes revealed species of Bacillus firmus, megaterium, pumilus, sphaericus, simplex, cereus/thuringiensis, Marinobacter lutaoensis, Halomonas shengliensis/H. alimentaria/H. campisalis, Citreicella thiooxidans, Stenotrophomonas maltophilia, Achromobacter xylosoxidans, Micrococcus luteus, Kocuria rosea, Streptomyces alboniger/S. chartreusis/S. moderatus, Staphylococcus hominis and Staphylococcus pasteuri/S. warneri. The results showed the preference of bacteria for the biotransformation of nonadecanoic acid and squalane. The characterization of the microbiota associated to reservoirs and the evaluation of their biodegradation potential may provide subsidies for future studies about the biological mechanisms responsible for petroleum biodegradation.

O presente trabalho teve como objetivos caracterizar uma coleção de 98 bactérias isoladas de amostras de petróleo e água de formação de reservatórios da Bacia de Campos (RJ) utilizando técnicas de taxonomia molecular e avaliar o potencial de degradação de biomarcadores do petróleo. O sequenciamento e análise filogenética do gene RNAr 16S revelaram Bacillus firmus, megaterium, pumilus, sphaericus, simplex, cereus/B. thuringiensis Marinobacter lutaoensis, Halomonas shengliensis/H. alimentaria/ H.campisalis, Citreicella thiooxidans, Stenotrophomonas maltophilia, Achromobacter xylosoxidans, Micrococcus luteus, Kocuria rosea, Streptomyces alboniger/S. chartreusis /S. moderatus, Staphylococcus hominis e Staphylococcus pasteuri/S. warneri. Os resultados evidenciaram a preferência pela biotransformação do ácido nonadecanóico e esqualano. A caracterização da microbiota presente nos reservatórios e avaliação do potencial de biodegradação pode contribuir para fornecer subsídios para estudos futuros sobre os mecanismos biológicos responsáveis pela biodegradação do petróleo. / This study is aimed to characterize a collection of 98 bacteria isolated from oil and formation water samples derived from reservoirs of the Campos Basin (RJ) using molecular biology-based techniques and to evaluate the degradation potential of petroleum biomarkers. Further sequencing and phylogenetic analysis of 16S rRNA genes revealed species of Bacillus firmus, megaterium, pumilus, sphaericus, simplex, cereus/thuringiensis, Marinobacter lutaoensis, Halomonas shengliensis/H. alimentaria/H. campisalis, Citreicella thiooxidans, Stenotrophomonas maltophilia, Achromobacter xylosoxidans, Micrococcus luteus, Kocuria rosea, Streptomyces alboniger/S. chartreusis/S. moderatus, Staphylococcus hominis and Staphylococcus pasteuri/S. warneri. The results showed the preference of bacteria for the biotransformation of nonadecanoic acid and squalane. The characterization of the microbiota associated to reservoirs and the evaluation of their biodegradation potential may provide subsidies for future studies about the biological mechanisms responsible for petroleum biodegradation.

Neste trabalho apresentamos um esquema numérico para a simulação computacional de escoamentos bifásicos, água-óleo, em reservatórios de petróleo. O modelo matemático consiste em um sistema de equações diferenciais parciais não-linear nas incógnitas velocidade, pressão e saturação. Uma quebra de operadores a dois níveis possibilita uma maior eficiência ao método permitindo que a velocidade, fornecida pelo problema de velocidade-pressão, seja atualizada somente para determinados intervalos de tempo associados ao problema de transporte advectivo-difusivo em termos da saturação. O método dos volumes finitos é empregado na resolução numérica do problema de velocidade-pressão e do transporte de massa por advecção e difusão. Na solução do problema de transporte de massa utilizamos limitadores de fluxo na aproximação dos termos advectivos e diferenças centradas para os termos difusivos. O nosso simulador foi validado a partir de confrontações dos seus resultados com as soluções teóricas conhecidas para os problemas unidimensionais, equações de Burgers e de Buckley-Leverett, e com outros resultados numéricos em se tratando do escoamento bifásico água-óleo bidimensional em meios porosos heterogêneos. / A new numerical method is proposed for the solution of two-phase flow problem in petroleum reservoirs. The two-phase (water and oil) flow problem is governed by a pressure-velocity equation coupled to a saturation equation. For computational eficiency an operator spliting technique is used; distinct time steps can be used for the computation of transport and pressure-velocity problems. The finite volume method is used in the numerical solution of the velocity-pressure and mass transport problems. A flux limiter is used for the numerical discretization of the advective terms while centered schemes are employed for the diffusion terms in the mass transport problem. In the validation of our numerical method we compared numerical and theoretical solutions for one dimensional problems, Burgers and Buckley-Leverett equations, and compared our numerical results to others, in the case of oil-water flows in two dimensions for an heterogeneous porous media.