Dissertations / Theses on the topic 'Petroleum – Microbiology'

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.

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

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.

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

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.

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

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
Degradation of petroleum hydrocarbons by micro-organisms in coastal environments is a very interesting strategy for remediation of these compounds. However, microbial degradation is often limited by nutrients avaiability, particularly nitrogen and phosphorus. In this work, we compared the effect of addition of water-soluble (urea and KH2PO4) or oleophilic (urea-formaldehyde resin and soy lecithin) nitrogen and phosphorus sources on oil degradation by indigenous micro-organisms and bacteria in consortia in microcosms composed by beach sand contaminated with crude oil. The microcosms were submitted to daily water exchanges representing tides activities. We also analysed the retention capability of both nutrient sources in contaminated sediment. For this, we performed UFC.mL-1 counts and quantification of nitrogen and phosphorus concentrations in the water during the experiment. The remaining oil in the sand was extracted with hexane and quantified by spectrophotometry, in which the degradation corresponds to the difference between the initial and final amounts of oil. Water-soluble nutrients have been shown to be gradually washed away during the water exchange and not remained in the sediment for bacterial supply. However, an opposite pattern was verified for oleophilic nutrients, which is a desirable feature for application in environments with intense action of waves and tides. Despite the increased retention of oleophilic nutrients, there were no significant differences in microbial counts between both treatments. In addition, oil degradation occurred mainly in those microcosms supplied with water-soluble nutrients. Although the inoculated micro-organisms are able to use the oleophilic nutrient sources, more detailed studies on the concentration of nutrients to be applied and the availability of nutrients for micro-organisms are necessary in order to maximize the stimulus generated.
A degradação de hidrocarbonetos de petróleo por micro-organismos em ambientes costeiros é uma estratégia extremamente interessante de remediação desses compostos enquanto contaminantes ambientais. No entanto, essa biodegradação é frequentemente limitada pela disponibilidade de nutrientes, principalmente nitrogênio e fósforo. Neste trabalho foi comparado o efeito da adição de fontes de nitrogênio e fósforo solúveis em água (uréia e KH2PO4) e oleofílicas (resina uréia-formaldeído e lecitina de soja) sobre a degradação de petróleo cru, em microcosmos de areia de praia, por micro-organismos nativos ou adicionados em consórcios. As fontes de nutrientes foram ainda comparadas quanto a sua retenção no sedimento contaminado após trocas diárias de água. Para isso, realizou-se contagens de UFC.mL-1 e análises da concentração de nitrogênio e de fósforo na água. O óleo remanescente na areia foi extraído com hexano e quantificado por espectrofotometria, sendo a degradação correspondente a diferença entre as quantidades inicial e final de petróleo. Verificou-se que os nutrientes hidrossolúveis não permaneceram no sedimento, mas foram gradativamente lavados pela remoção diária de água. Um comportamento inverso ocorreu para os nutrientes oleofílicos, sendo essa uma característica desejável para sua aplicação em ambientes com intensa ação de ondas e marés. Apesar da maior retenção de nutrientes quando esses são oleofílicos, não se verificou diferenças consideráveis nas contagens microbianas entre os dois tratamentos. Além disso, a degradação do óleo ocorreu principalmente naqueles microcosmos supridos com os nutrientes solúveis em água. Embora os micro-organismos inoculados sejam capazes de utilizar as fontes oleofílicas como nutrientes, estudos mais detalhados da concentração a ser aplicada e da disponibilização desses nutrientes para os micro-organismos são necessários, a fim de se maximizar o estímulo gerado.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
This study aimed the prospection of bacteria capable of using petroleum hydrocarbons as their source of carbon and energy, and the analysis of the structure and dynamics of the microbial community that colonizes the oil after its scattering in the sea. We used data from T-RFLP multiplex, and simulate and analyze the efficiency of different petroleum bioremediation strategies by Trindade Island s shoreline. For these analyses, an experiment was settled 100 meters away from Turtles Beach, an aluminum rod, measuring 1.5 meters containing 20 acrylic slabs (4 x 3 cm) with a thin petroleum layer over them, was stuck to a fixed float and rested there for 60 days. During this time, triplicates for a posterior extraction of DNA present in the biofilms formed over the oil layer were made. Passed the 60 days, the last 5 slabs were collected with the purpose of isolation in enrichment media, thus, colonies capable of using hydrocarbons as carbon source could be obtained. The biofilm DNA was used in PCR multiplex for the detection of Bacteria, Archaea and fungi and a posterior diversity analysis. Water from the island s shoreline was used in an experiment that simulates different bioremediation strategies: natural attenuation, bioestimulation, bioaugmentation and the mutual use of the last two strategies. The simulation in this experiment was conducted using nutrients, a bacteria previously isolated from the environment Rhodococcus rhodochrous, biosurfactant and crude oil, previously heated up to 210 oC for the elimination of the more volatile compounds. Fifteen isolates capable of growing in media containing hydrocarbon were isolated. The microbial community structure was studied based on Shannon-Weaver's index (H ), Simpson's dominance (D) and Margalef's richness (R). The Bacteria domain showed to be most diversified during the oil colonization. The composition of the microbial community over the oil showed some variation in the in situ experiment during the first 30 days of sea exposition. The addition of nutrients and a biosurfactant generated significant raise in the oil degradation. The treatment that provided the best solution was the one with nutrients, biosurfactant and the inoculum. In conclusion, the biostimulation with nutrients and biosurfactants, along with bioaugmentation with an autochthonous isolated, are recommended bioremediation strategies in case of accidents with oil spills by Trindade Island's coast.
Este estudo teve como objetivos prospectar bactérias capazes de utilizar hidrocarbonetos do petróleo como fonte de carbono e energia, analisar a estrutura e dinâmica da comunidade microbiana que coloniza o petróleo após sua exposição no mar utilizando dados de T­RFLP multiplex, assim como simular e analisar a eficiência de diferentes estratégias de biorremediação de petróleo no litoral da Ilha da Trindade Brasil. Para tanto, foi montado um experimento à aproximadamente 100 m da praia das Tartatugas na Ilha da Trindade, onde uma haste de alumínio de 1,5 m de comprimento, contendo 20 cupons de acrílico (4 cm x 3cm) com uma delgada camada de petróleo em sua superfície, foi presa a uma bóia fixa e permaneceu no local por 60 dias. Ao longo desse período, foram realizadas coletas em triplicata para posterior extração do DNA dos biofilmes formados sobre a camada de petróleo nas placas. Após os 60 dias, as últimas cinco placas foram coletadas para o isolamento em meios de enriquecimento para a obtenção de colônias capazes de utilizar hidrocarbonetos como fonte de carbono. O DNA extraído dos biofilmes foi utilizado em PCR multiplex para a detecção de Bacteria, Archaea e fungos e posterior análise de índices de diversidade. Água coletada no litoral da Ilha da Trindade foi utilizada em um experimento simulando diferentes estratégias de biorremediação: atenuação natural, bioestimulação, bioaumentação e o conjunto das duas últimas. Nas simulações, foram utilizados nutrientes, uma bactéria isolada do próprio ambiente como inóculo (Rhodococcus rhodochrous), biossurfactante e petróleo previamente aquecido a 210 °C para remoção dos compostos voláteis. Foram obtidos 15 isolados capazes de crescer em meios contendo hidrocarbonetos. A estrutura da comunidade microbiana foi avaliada com base nos índices de Shannon­Weaver (H ), dominância de Simpson (D) e riqueza de Margalef (R). O domínio Bacteria mostrou ser o mais diverso durante a colonização do óleo. Foram observadas flutuações significativas na composição da comunidade microbiana aderida ao petróleo no experimento in situ durante os primeiros 30 dias de exposição do petróleo no mar. A adição de nutrientes e de biossurfactante promoveu aumento significativo da biodegradação do petróleo. O tratamento que forneceu a melhor resposta foi aquele em que foram adicionados nutrientes, inóculo e biossurfactante. Conclui­se que a bioestimulação com nutrientes e biossurfactantes, além da bioaumentação com um isolado autóctone, são estratégias de biorremediação recomendadas em caso de acidentes envolvendo derramamentos de petróleo na região litorânea da Ilha da Trindade.

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.

Les fossiles moléculaires sont de puissants outils pour étudier l'origine, la transformation et le transfert de la matière organique dans les écosystèmes modernes et anciens. Marqueurs d'origine, ils livrent des informations sur le milieu de dépôt, en précisant notamment les sources de la matière organique et la place de leur précurseurs biologiques dans les chaînes alimentaires. De manière surprenante, la découverte de certains fossiles "orphelins" peut conduire à l'identification de nouvelles substances biologiques. L'analyse isotopique de marqueurs végétaux des sols permet d'étudier la dynamique de l'humus au niveau moléculaire. Marqueurs de processus, leur structure et leur composition isotopique révèlent la nature des réactions géochimiques opérant dans les milieux très complexes tels les sols et les sédiments. Marqueurs de maturité, les fossiles moléculaires mesurent l'intensité du stress thermique subi par la matière organique au cours de l'enfouissement dans les sédiments. À cet égard, ils s'avèrent utiles à la fois pour la recherche pétrolière et pour authentifier la présence de contamination pétrolière dans les écosystèmes modernes. Ainsi, le concept de marqueur moléculaire développé en géochimie pétrolière est à la base d'un domaine scientifique en émergence, la géochimie organique environnementale.

Bioremediation is the process whereby the degradation of organic polluting compounds occurs as a result of biochemical activity of macro- and microorganisms. Bioremediation of hydrocarbon contaminated soils can be practised in situ or ex situ by either stimulating the indigenous microorganisms (biostimulation) or introducing adapted microorganisms which specifically degrade a contaminant (bioaugmentation). This investigation focused on ex situ remediation processes with special attention to the processes and microbiology of landfarming and thermal bioventing. Landfarming was investigated at pilot-scale and full-scale, and thermal bioventing at laboratory and pilot-scale. This study indicated that pilot-scale bioremediation by landfarming was capable of effecting a total petroleum hydrocarbon concentration (TPHC) reduction of 94% (m1m) from an initial concentration of 320 gkg-I soil to 18 gkg-I soil over a period of 10 weeks. Reactors receiving biosupplements showed greater rates of bioremediation than those receiving nutrients. Promotion of TPHC catabolism by addition of a commercial or a site-specific microbial biosupplement was similar. Seedling experiments proved that bioremediation did not necessarily leave the soil in an optimal condition for plant growth. The full-scale landfarming operation reduced the TPHC concentrations from 5 260 - 23 000 mgkg- I to 820 - 2335 mgkg- I soil over a period of 169 days. At full-scale, the larger fraction of more recalcitrant and weathered petroleums. and the less intensive treatment resulted in a slower rate of TPHC reduction than was found in the pilot-scale study. Three distinct decreases in the TPHC were observed during the full-scale treatment. These presented an ideal opportunity to investigate the microbiology of the soil undergoing treatment. The dominant culturable microorganisms were isolated and identified. The bioremediation process was dominated by Bacillus and Pseudomonas species. The method used to study the population was, however, biased to culturable, fast growing microorganisms which represent a small portion of the total microbial population. For this reason, a method to study the total eubacterial population in situ with rRNA targeted oligonucleotide probes was adapted and found to be a valuable technique. Soil microorganisms respiratory activity was investigated at different times in the full-scale treatment. A clear correlation between activity and degradation was recorded. The effect of a supplement. anaerobically digested sludge, was also assessed by this method. Thermal bioventing was investigated as an ex situ in-vessel treatment technology for small volumes of highly contaminated soils. This proved to be a viable technique for the bioremediation of petroleum hydrocarbons at laboratory-scale. Volatilisation contributed to at least 40% of the reduction. Of the two supplements evaluated. dried sludge promoted degradation to a greater extent than chicken manure. The pilot-scale study proved that a chemical contaminant reduction of at least 50% could be achieved in 13 weeks by thermal bioventing. Of the supplemented reactors. the presence of dried sludge and commercial biosupplement etfected the largest contaminant decrease. As a possible supplement to increase the rate of bioremediation. dried anaerobically digested sludge was more effective than chicken manure. A parallel laboratory-scale experiment gave similar results. Gravimetric analyses were found to be conservative indications of the remediation process. The results of this study shed some light on our. still. limited understanding of bioremediation. The gap between the technology in the laboratory and field was narrowed and a better understanding of the soil microbiology was achieved. Due to the limited control of environmental parameters in the case of landfarming. thermal bioventing was investigated and proved to be an effective alternative. The latter technology is novel in Southern Africa.
Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1996.

This thesis explores bio-mediated processes in geotechnical and petroleum engineering. Worldwide energy consumption is rapidly increasing as the world population and per-capita consumption rises. The US Energy Information Agency (EIA) predicts that hydrocarbons will remain the primary energy source to satisfy the surging energy demands in the near future. The three topics described in detail in this document aim to link microbiology with geotechnical engineering and the petroleum industry. Microorganisms have the potential to exploit residual hydrocarbons in depleted reservoirs in a technique known as microbial enhanced oil recovery, MEOR. The potential of biosurfactants was analyzed in detail with a literature review. Biosurfactant production is the most accepted MEOR technique, and has been successfully implemented in over 700 field cases. Temperature is the main limiting factor for these techniques. The dissolution of carbonates by microorganisms was investigated experimentally. We designed a simple, economical, and robust procedure to monitor diffusion through porous media. This technique determined the diffusion coefficient of H+ in 1.5% agar, 1.122 x 10-5 cm2 sec-1, by using bromothymol blue as a pH indicator and image processing. This robust technique allows for manipulation of the composition of the agar to identify the effect of specific compounds on diffusion. The Red Sea consists of multiple seeps; the nearby sediments are telltales of deeper hydrocarbon systems. Microbial communities associated with the sediments function as in-situ sensors that provide information about the presence of carbon sources, metabolites, and the remediation potential. Sediments seeps in the Red Sea revealed different levels of bioactivity. The more active seeps, from the southern site in the Red Sea, indicated larger pore sizes, higher levels of carbon, and bioactivity with both bacteria and archaeal species present.

The potential for in situ anaerobic benzene degradation in petroleum-contaminated aquifers was investigated. Sediments collected from a contaminated aquifer near Bemidji, MN in which Fe(III)-reduction was the dominant microbial process readily mineralized benzene when incubated with [U-14C]benzene while sediments from other aquifers did not. Benzene mineralization was localized within a narrow zone at the downgradient edge of the Fe(III) reduction zone. Analysis of MPN cultures and sediment by a variety of 16S rRNA-based techniques indicated a selective enrichment of Geobacteraceae in benzene degrading, Fe(III)-reducing sediments. Members of the Geobacteraceae are known to couple the oxidation of aromatic compounds to the reduction of Fe(III) and could be responsible for the observed benzene degradation at this site. ^ Bemidji sediments also mineralized other aromatic compounds commonly found in hydrocarbon-contaminated groundwaters. Benzene-degrading sediments readily mineralized toluene and naphthalene indicating that these compounds were also being oxidized in situ. The unusual aromatic degradation activity at the Bemidji site could not be attributed to the presence of Fe(III)-chelators and/or electron shuttling compounds in the groundwater. Uncontaminated sediments could be adapted to benzene suggesting that Bemidji sediments naturally contain a microbial population capable of anaerobic benzene degradation. ^ Results obtained from the Bemidji aquifer encouraged the investigation of anaerobic treatment alternatives for contaminated aquifers. Anaerobic bioremediation of benzene was evaluated at a petroleum-contaminated aquifer in Oklahoma. The injection of sulfate into the subsurface stimulated benzene degradation within a treatment zone located downgradient from an injection gallery. Benzene concentrations in the groundwater decreased by an average of 90% (100% in one well) during the study period. Sulfate concentrations, relative to a bromide tracer, decreased with distance from the injection gallery suggesting that benzene removal was coupled to the removal of sulfate from the groundwater. [U-14C]Benzene mineralization and [2-14C]acetate analysis of sediments confirmed that benzene degradation was indeed coupled to sulfate reduction within treatment zone sediments. Mass balance calculations suggested that as much as 42% of the removed sulfate within the treatment zone could be attributed to the anaerobic oxidation of benzene. The results demonstrate that stimulation of anaerobic processes can be an effective treatment alternative for heavily contaminated aquifers. ^

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

In 1990, an oil recycling plant situated in Hammarsdale, South Africa, was decommissioned and a decision was taken by management to rehabilitate the site in preparation for resale. The heavily impacted area covered over two hecatares and oil contamination penetrated soil to depths in excess of three metres, making excavation and removal of the soil very expensive. The options for remediation of the site were limited. No facility for incineration of contaminated soil exists in South Africa, and landfilling was not permitted. The emphasis in developing a remediation strategy, therefore, focussed upon the possibility of in situ remediation with minimal excavation of soil. This study, the first of its kind in South Africa, was subsequently initiated to assess the feasibility of this approach, the results of which would underpin a full-scale cleanup programme. The development of such a strategy involved four key stages of work : (1) a comprehensive site investigation to evaluate and fully understand the particular problems at the site; (2) treatability studies to determine the potential for biological treatment of the contaminated soil and the optimisation of such treatments, particularly in terms of time and cost; (3) the testing of some of the more effective treatments on a pilot-scale; and (4) recommendations for full-scale bioremediation of the contaminated site. various conditions unique to South Africa had to be considered at each stage viz. the lack of funds and remediation experience, which created numerous problems and emphasised the requirement for a simple, "low-tech" approach. Site investigations revealed that in situ remediation may be possible due to the high permeability of the sandy soils and low concentrations of heavy metals. Laboratory experiments also showed that a mixed association of indigenous microorganisms was present which, once stimulated by nutrient supplementation at C:N:P, ratios of between 10:1:1 and 20:1:1, was capable of degrading total petroleum hydrocarbons at an average rate of 11% week -1. Further experimentation, aimed at reducing the cost of remediation and improving the soil quality, focussed on the efficacy of oil solubilisers, a soil ameliorant (composted pine-bark), indigenous fungi and higher plants in the remedial process. Three commercial surfactants (Arkopal N-050, N-060 and E2491) and one natural solubiliser (soybean lecithin) were tested for their biotoxicity, solubilisation and biodegradability at various concentrations (0.01 - 1.0%). Formulation E2491 was able to support a microbial population and was selected as the preferred commercial surfactant if soil washing was to be recommended; however, lecithin was considered to be more useful in situ because of its localised solubilising effect, biological origin and nutritional contribution. The use of fungi was of particular interest in addressing the persistent organic compounds, such as the heavy fractions of oil, for which bacterial remediation methods have been slow or ineffective. While it was not possible, however, to demonstrate in the laboratory that the indigenous fungi contributed significantly towards the degradation of the contaminating oil, the basic trends revealed that the fungal component of the indigenous microbial population was readily stimulated by the addition of nutrient supplements. The bulking-up process was also a success and additional exploratory work was proposed in the form of a larger scale composting design. Finally, the potential for using higher plants and 20% (v / v) composted pinebark (in addition to nutrients) to increase the microbial degradation of the contamination was investigated in both greenhouse and field plot studies. Greenhouse investigations employed soybeans which were postulated to have soil quality and cost benefits. However, although the soybeans were found to significantly enhance the remedial process, the complex soil-contaminant- plant interactions gave rise to strange nutritional effects and, in some cases, severe stunting. In contrast, the field studies employed grasses that had previously established on the site and which ultimately demonstrated a better tolerance for the contaminated conditions. Scanning electron microscopy revealed that there were considerable differences between the root tips of soybean plants which had been grown in contaminated soil and those which had been grown in uncontaminated soil. It was concluded that toxicity symptoms, which are readily observed in the root, could be used as an early indicator for determining the suitability of vegetation for remediation purposes. In both instances, despite the differences, the addition of composted pine-bark and nutrients (nitrogen and phosphorus) resulted in total petroleum hydrocarbon reductions of >85%, illustrating the benefits of plant establishment and oxygen availability. The need to link results from laboratory or pilot-scale experiments to achieve reliable predictions of field-scale behaviour was an essential component of this research. The results of the field study provided evidence, similar to that found in the pot trial, of the accelerated disappearance of organic compounds in the rhizosphere. All experiments incorporated parallel measurements of hydrocarbon residues, microbial activity and pH changes in the contaminated soil, the results of which strongly supported the argument that biodegradation was the dominant component of the remediation process. Thus, after consideration of the significant interactions which dominated the study (time-contaminant-nutrient; time-contaminant-pine-bark; and time-contaminant- pine-bark-plant), it was clear that, aside from these limiting factors, little should preclude the in situ bioremediation of the impacted soil.
Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1996.

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.

The central aim of this study was to determine if indigenous fungi of an oil-contaminated soil could be effectively used in a low-cost bioremediation of the soil. Since some of the contaminant had been present at the site for over two decades, the indigenous microbial species had been subjected to specific selection pressures for a protracted period, thus facilitating key enzymatic capabilities for hydrocarbon degradation. Analysis of the pertinent influential parameters of soil bioremediation indicated that an ex situ technique, utilising the catabolic activities of the indigenous soil fungi, was a feasible low-cost option. Fungi were isolated from the contaminated soil through a variety of techniques. The abilities of these isolates to degrade the contaminant oil and a range of representative hydrocarbon molecules was evaluated by a systematic screening programme. Sixty-two isolates were initially examined for their growth potential on hydrocarbon-supplemented agar. A bioassay, utilising hydrocarbon-impregnated filter paper discs, was then used to examine the abilities of 17 selected isolates to catabolise three representative hydrocarbon molecules (hexadecane, phenanthrene and pristane) in different concentrations. In the same bioassay, the influence of a co-metabolite (glucose) on growth potential was also examined. Eight fungal species: Trichophyton sp.; Mucor sp.; Penicillium sp.; Graphium sp.; Acremoniwn sp.; Chaetomium sp.; Chrysosporium sp.; and an unidentified basidiomycete were then selected. Liquid batch cultures with a hydrocarbon mixture of hexadecane, phenanthrene, pristane and naphthalene facilitated quantitative analysis (HPLC) of the hydrocarbon catabolic abilities of the selected isolates. Ex situ bioremediation was evaluated at laboratory-scale by both bioaugmentation and biostimulation in soil microcosm trials. During the course of the study, total petroleum hydrocarbon (TPH) concentration (U.S. EPA Method 418.1) was used as a simple and inexpensive parameter to monitor hydrocarbon disappearance in response to soil treatments. Soil microbial activities were estimated by use of a fluorescein diacetate hydrolysis bioassay. This was found to be a reliable and sensitive method to measure the activity of respiring heterotrophs as compared with the unreliable data provided by plate counts. In the bioaugmentation trial, the eight selected isolates were individually used to inoculate (30% v/v) the contaminated soil. The highest rate of biodegradation (50.5% > than the non-sterile control) was effected by an Acremonium species after 50 days incubation (25°C). The second highest rate of biodegradation (47% > than the non-sterile control) was achieved with a soil treatment of sterile barley/beer waste only. Comparable rates of hydrocarbon degradation were achieved in simple biostimulation trials. Thus, due to its lower cost, biostimulation was the preferred remediation strategy and was selected for further laboratory investigation. Common agricultural or industrial lignocellulosic wastes such as: wood chips; straw; manure; beer brewery waste; mushroom compost; and spent mushroom substrate were used as soil treatments, either alone or in combination. The effect of the addition of a standard agricultural fertiliser was also examined. The highest level of biodegradation (54.4% > the non-sterile control) was recorded in a microcosm supplemented (40% v/v) with chicken manure. Finally, an ex situ bioremediation technique was examined in a pilot-scale field trial. Wood chips and chicken manure were co-composted with the contaminated soil in a low-cost, low-maintenance bioremediation system know as passive thermal bio venting. Extensive monitoring of the thermal environment within the biopile was made as an indirect measure of microbial activity. These data were then used to optimise the composting process. Three-dimensional graphical representations of the internal temperatures, in time and space, were constructed. From these graphs, it was determined that an inner core region of approximately 500 cm3 provided a realistic simulation of conditions within a full-scale biopile. During this trial a TPH reduction of 68% was achieved in 130 days. The findings of this research suggested that the utilisation of fungal catabolism is applicable to soils contaminated with a wide range of hydrocarbon contaminants. Passive thermal bioventing offers a bioremediation strategy which is highly suitable for South African conditions in terms of its low level of technological sophistication, low maintenance design and, most importantly, its relatively low cost.
Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1997.

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.

Les champignons mycorhiziens à arbuscules (CMA) forment un groupe de champignons qui appartient à l'embranchement des Gloméromycètes (Glomeromycota). Les CMA forment des associations symbiotiques, connus sous le nom des mycorhizes à arbuscules avec plus de 80 % des plantes vasculaires terrestres. Une fois que les CMA colonisent les racines de plantes, ils améliorent leurs apports nutritionnels, notamment le phosphore et l'azote, et protègent les plantes contre les différents pathogènes du sol. En contrepartie, les plantes offrent un habitat et les ressources de carbone nécessaires pour le développement et la reproduction des CMA. Des études plus récentes ont démontré que les CMA peuvent aussi jouer des rôles clés dans la phytoremédiation des sols contaminés par les hydrocarbures pétroliers (HP) et les éléments traces métaliques. Toutefois, dans les écosystèmes naturels, les CMA établissent des associations tripartites avec les plantes hôtes et les microorganismes (bactéries et champignons) qui vivent dans la rhizosphère, l'endosphère (à l'intérieur des racines) et la mycosphère (sur la surface des mycéliums des CMA), dont certains d'entre eux jouent un rôle dans la translocation, l’immobilisation et/ou la dégradation des polluants organiques et inorganiques présents dans le sol. Par conséquent, la diversité des CMA et celle des microorganismes qui leur sont associés sont influencées par la concentration et la composition des polluants présents dans le sol, et aussi par les différents exsudats sécrétés par les trois partenaires (CMA, bactéries et les racines de plantes). Cependant, la diversité des CMA et celle des microorganismes qui leur sont associés demeure très peu connue dans les sols contaminés. Les interactions entre les CMA et ces microorganismes sont aussi méconnus aussi bien dans les aires naturelles que contaminées. Dans ce contexte, les objectifs de ma thèse sont: i) étudier la diversité des CMA et les microorganismes qui leur sont associés dans des sols contaminés par les HP, ii) étudier la variation de la diversité des CMA ainsi que celle des microorganismes qui leur sont associés par rapport au niveau de concentration en HP et aux espèces de plantes hôtes, iii) étudier les correlations (covariations) entre les CMA et les microorganismes qui leur sont associés et iv) comparer les communautés microbiennes trouvées dans les racines et sols contaminés par les HP avec celles trouvées en association avec les CMA. Pour ce faire, des spores et/ou des propagules de CMA ont été extraites à partir des racines et des sols de l'environnement racinaire de trois espèces de plantes qui poussaient spontanément dans trois bassins de décantation d'une ancienne raffinerie de pétrole située dans la Rive-Sud du fleuve St-Laurent, près de Montréal. Les spores et les propagules collectées, ainsi que des échantillons du sol et des racines ont été soumis à des techniques de PCR (nous avons ciblés les genes 16S de l'ARNr pour bactéries, les genes 18S de l'ARNr pour CMA et les régions ITS pour les autres champignons), de clonage, de séquençage de Sanger ou de séquençage à haut débit. Ensuite, des analyses bio-informatiques et statistiques ont été réalisées afin d'évaluer les effets des paramètres biotiques et abiotiques sur les communautés des CMA et les microorganismes qui leur sont associés. Mes résultats ont montré une diversité importante de bactéries et de champignons en association avec les spores et les propagules des CMA. De plus, la communauté microbienne associée aux spores des CMA a été significativement affectée par l'affiliation taxonomique des plantes hôtes et les niveaux de concentration en HP. D'autre part, les corrélations positives ou négatives qui ont été observées entre certaines espèces de CMA et microorganismes suggérèrent qu’en plus des effets de la concentration en HP et l'identité des plantes hôtes, les CMA peuvent aussi affecter la structure des communautés microbiennes qui vivent sur leurs spores et mycéliums. La comparaison entre les communautés microbiennes identifiées en association avec les spores et celles identifiées dans les racines montre que les communautés microbiennes recrutées par les CMA sont différentes de celles retrouvées dans les sols et les racines. En conclusion, mon projet de doctorat apporte de nouvelles connaissances importantes sur la diversité des CMA dans un environnement extrêmement pollué par les HP, et démontre que les interactions entre les CMA et les microorganismes qui leur sont associés sont plus compliquées que ce qu’on croyait précédemment. Par conséquent, d'autres travaux de recherche sont recommandés, dans le futur, afin de comprendre les processus de recrutement des microorganismes par les CMA dans les différents environnements.
Arbuscular mycorrhizal fungi (AMF) are an important soil fungal group that belongs to the phylum Glomeromycota. AMF form symbiosic associations known as arbuscular mycorrhiza with more than 80% of vascular plants on earth. Once AMF colonize plant roots, they promote nutrient uptake, in particular phosphorus and nitrogen, and protect plants against soil-borne pathogens. In turn, plants provide AMF with carbon resources and habitat. Furthermore, more recent studies demonstrated that AMF may also play key roles in phytoremediation of soils contaminated with petroleum hydrocarbon pollutants (PHP) and trace elements. Though, in natural ecosystems, AMF undergo tripartite associations with host plants and micoorganisms (Bacteria and Fungi) living in rhizosphere (the narrow region of soil surounding the plant roots), endosphere (inside roots) and mycosphere (on the surface AMF mycelia), which some of them play a key role on translocation, immobilization and/or degradation of organic and inorganic pollutants. Consequently, the diversity and community structures of AMF and their associated microorganisms are influenced by the composition and concentration of pollutants and exudates released by the three partners (AMF, bacteria and plant roots). However, little is known about the diversity of AMF and their associated microorganisms in polluted soils and the interaction between AMF and these microorganisms remains poorly understood both in natural and contaminated areas. In this context, the objectives of my thesis were to: i) study the diversity of AMF and their associated microorganisms in PHP contaminated soils, ii) study the variation in diversity and community structures of AMF and their associated microorganisms across plant species identity and PHP concentrations, iii) study the correlations (covariations) between AMF species and their associated microorganisms and iv) compare microbial community structures of PHP contaminated soils and roots with those associated with AMF spores in order to determine if the microbial communities shaped on the surface of AMF spores and mycelia are different from those identified in soil and roots. To do so, AMF spores and/or their intraradical propagules were harvested from rhizospheric soil and roots of three plant species growing spontaneously in three distinct waste decantation basins of a former petrochemical plant located on the south shore of the St-Lawrence River, near Montreal. The harvested spores and propagules, as well as samples of soils and roots were subjected to PCR (we target 16S rRNA genes for bacteria, 18S rRNA genes for AMF and ITS regions for the other fungi), cloning, Sanger sequencing or 454 high throughput sequencing. Then, bioinformatics and statistics were performed to evaluate the effects of biotic and abiotic driving forces on AMF and their associated microbial communities. My results showed high fungal and bacterial diversity associated with AMF spores and propagules in PHP contaminated soils. I also observed that the microbial community structures associated with AMF spores were significantly affected by plant species identity and PHP concentrations. Furthermore, I observed positive and negative correlations between some AMF species and some AMF-associated microorganisms, suggesting that in addition to PHP concentrations and plant species identity, AMF species may also play a key role in shaping the microbial community surrounding their spores. Comparisons between the AMF spore-associated microbiome and the whole microbiome found in rhizospheric soil and roots showed that AMF spores recruit a microbiome differing from those found in the surrounding soil and roots. Overall, my PhD project brings a new level of knowledge on AMF diversity on extremely polluted environment and demonstrates that interaction of AMF and their associated microbes is much complex that we though previously. Further investigations are needed to better understand how AMF select and reward their associated microbes in different environments.