Pollution from human and animal faecal waste is a major cause of deteriorating water quality and increased nutrient loads in coastal and inland waterways. Management of this problem depends on knowing which sources of faecal matter are the cause and what is the degree and extent of the pollution. Bacterial indicator organisms have long been the principal method used to test water samples for faecal contamination. However, none of the currently used bacterial indicators on their own are source specific enough to distinguish different sources of faecal matter. The use of faecal sterol biomarkers in conjunction with existing bacterial indicators offers a new way to distinguish sources of faecal contamination. This study investigates the sources of faecal sterols, the relationship of coprostanol to existing bacterial indicators of faecal pollution, the degradation of faecal sterols and the problem of determining the sources of faecal contamination and the distribution of faecal contamination using faecal sterol biomarkers. 5p-Stanols (i.e. faecal sterols) were found to be significant constituents of human, herbivore (i.e. cows, sheep etc.) and pig and cat faeces. Human faeces contained 73 ± 4% coprostanol in relation to the sum of coprostanol and 24-ethylcoprostanol and primary treated effluent contained 86 ± 0.4% coprostanol. Herbivore faeces contained 38 ± 4% coprostanol and 62 ± 4% 24-ethylcoprostanol whereas pig faeces contained 50 � 5% of each compound. Both birds and dogs faeces contained either trace amounts of 5B-stanols or they could not be detected. Notable differences were observed in the abundance of Closthdium perfringens spores between the faeces of birds and domestic pets such as cats and dogs. The above differences were subsequently exploited to distinguish faecal contamination in Lake Tuggerah. An examination of the relationships between coprostanol and bacterial indicator concentrations from several environments revealed that 60 and 400 ng L of coprostanol corresponded to currently defined primary and secondary contact limits for bacteria measured as either thermotolerant coliforms or enterococci in the environment. Four degradation experiments showed faecal sterols and related sterols such as cholesterol decay at similar rates. An induction period was observed in all experiments which meant that simple exponential equations to describe the rate of decay of coprostanol were inadequate; a complimentary log - log transformation of the data was used and the equation: Y = l-Exp(-Exp(time x -0.01 + temp x -0.158 + 3.33)) x 100 was derived where Y equals the predicted percentage of coprostanol remaining over time at a given temperature. In terms of persistence in the environment, Clostridium perfringens spores > coprostanol > enterococci > thermotolerant coliforms. Two field studies were undertaken to highlight the use of faecal sterols. In the Lake Tuggerah study, the results indicated that faecal contamination of receiving waters in the Tuggerah Lakes during rain events was significant, but was not derived from human faecal matter; rather it appears to be principally derived from native birds and, to a lesser extent, domestic pets. In the Derwent Estuary study, based on the distribution of the faecal biomarker coprostanol, the mid estuary and parts of the upper estuary (from Newtown Bay to Taroona), were found to be severely contaminated by sewage. In summary, the use of faecal sterols to trace faecal contamination were found to be an invaluable addition to the tools water managers use to investigate faecal pollution.

No description available.

Faecal indicator bacteria have traditionally been used in the detection of faecal pollution in water, but due to concerns about the lack of reliability of these indicators, alternative methods have been developed. One of which is the detection of sterols present in human and animal excreta via GC-MS analysis of water in this study. The Szűcs method was used to detect six target sterols (coprostanol, cholesterol, dehydrocholesterol, stigmasterol, β-sitosterol, and stigmastanol) in environmental water samples. An initial study was done by analysing raw sewage and effluent (human faecal sterol biomarkers) and water samples were spiked with excreta from cattle, chickens, horses, pigs, and sheep to determine faecal sterol fingerprints. The method was evaluated for quantitation and differences between the water samples from each species. Following liquid-liquid extraction, silylation and derivatization, samples were analysed by GC-MS. Standard curve assays were linear up to 160ng and the limit for quantification was 20ng. The human faecal sterol biomarker was coprostanol, while herbivore profiles were dominated by terrestrial sterol biomarkers (stigmasterol and stigmastanol). Sterol fingerprints and differences in concentrations of sterols between various animals and between animals and humans occurred, providing the opportunity to determine whether faecal pollution was from humans or from animals. The method proved sensitive enough to evaluate faecal contamination in environmental water. Groundwater was collected from bore-holes and surface water samples were collected from the Baberspan Inland Lake. Physico-chemical parameters analysed indicated that pH for surface water samples was above 6.9. The total dissolved solids (TDS) in groundwater indicated that the water was not suitable for human consumption, but could be used for livestock watering. Surface water electrical conductivity (EC) and inorganic nitrates was too high to be used for irrigational purposes. Nitrates in groundwater were too high to be consumed by humans. In groundwater, the total coliform target water quality range (TWQR) was exceeded at 53% of sites analysed and faecal coliform TWQR were exceeded at 77% sites. Surface water samples complied with TWQR with regards to faecal coliforms for full contact recreational activities and livestock watering. The TWQR for E. coli, with regards to full contact recreational activities, was within a safe range for surface water. Faecal streptococci were found in 85% of groundwater sampling sites. And surface water faecal streptococci counts exceeded the TWQR for full contact recreational activities. There is no TWQR for faecal sterols in water, but concentrations of cholesterol and coprostanol was found at three of the groundwater sites analysed. This indicates faecal contamination from possible animal and human origin. Surface water samples analysed showed that the Harts River water is clean and free of faecal sterols, while the water analysed from the inflow, hotel and outflow, cholesterol eluted, which showed faecal contamination, possibly from animals. Faecal sterol markers could be detected in groundwater and surface water, adding an extra dimension to determining the quality of water systems. An optimization and sensitivity study of the method was done on waste water treatment plant (WWTP) raw sewage and effluent. The WWTP sample analysed form Potchefstroom and Carletonville WWTP yielded all six target sterols in the raw sewage water samples, but no sterols eluted in the effluent samples. The raw sewage water sample taken from the Fochville WWTP yielded all six target sterols as well, however, the effluent yielded an unknown compound as well as cholesterol. An alternative study was done where the effluent sample volume was increased. By increasing the volume of water, one can possibly increase the amount (“load”) of sterols extracted and analysed, resulting in a higher abundance of target sterols. By using the target qualifier ions of the six target sterols, and the GC-TOF/MS software, the target sterols could still be qualitatively determined. Optimal volume for raw sewage is 300 ml water sample as this is enough to yield all 6 target sterols. For optimum water quality monitoring via faecal sterol analysis of effluent and other environmental samples, at least 1L sample volume needs to be collected and analysed. The methods described here can be applied to the analysis of environmental water samples. The technical advantages also make it suitable for routine environmental monitoring of faecal pollution. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Faecal indicator bacteria have traditionally been used in the detection of faecal pollution in water, but due to concerns about the lack of reliability of these indicators, alternative methods have been developed. One of which is the detection of sterols present in human and animal excreta via GC-MS analysis of water in this study. The Szűcs method was used to detect six target sterols (coprostanol, cholesterol, dehydrocholesterol, stigmasterol, β-sitosterol, and stigmastanol) in environmental water samples. An initial study was done by analysing raw sewage and effluent (human faecal sterol biomarkers) and water samples were spiked with excreta from cattle, chickens, horses, pigs, and sheep to determine faecal sterol fingerprints. The method was evaluated for quantitation and differences between the water samples from each species. Following liquid-liquid extraction, silylation and derivatization, samples were analysed by GC-MS. Standard curve assays were linear up to 160ng and the limit for quantification was 20ng. The human faecal sterol biomarker was coprostanol, while herbivore profiles were dominated by terrestrial sterol biomarkers (stigmasterol and stigmastanol). Sterol fingerprints and differences in concentrations of sterols between various animals and between animals and humans occurred, providing the opportunity to determine whether faecal pollution was from humans or from animals. The method proved sensitive enough to evaluate faecal contamination in environmental water. Groundwater was collected from bore-holes and surface water samples were collected from the Baberspan Inland Lake. Physico-chemical parameters analysed indicated that pH for surface water samples was above 6.9. The total dissolved solids (TDS) in groundwater indicated that the water was not suitable for human consumption, but could be used for livestock watering. Surface water electrical conductivity (EC) and inorganic nitrates was too high to be used for irrigational purposes. Nitrates in groundwater were too high to be consumed by humans. In groundwater, the total coliform target water quality range (TWQR) was exceeded at 53% of sites analysed and faecal coliform TWQR were exceeded at 77% sites. Surface water samples complied with TWQR with regards to faecal coliforms for full contact recreational activities and livestock watering. The TWQR for E. coli, with regards to full contact recreational activities, was within a safe range for surface water. Faecal streptococci were found in 85% of groundwater sampling sites. And surface water faecal streptococci counts exceeded the TWQR for full contact recreational activities. There is no TWQR for faecal sterols in water, but concentrations of cholesterol and coprostanol was found at three of the groundwater sites analysed. This indicates faecal contamination from possible animal and human origin. Surface water samples analysed showed that the Harts River water is clean and free of faecal sterols, while the water analysed from the inflow, hotel and outflow, cholesterol eluted, which showed faecal contamination, possibly from animals. Faecal sterol markers could be detected in groundwater and surface water, adding an extra dimension to determining the quality of water systems. An optimization and sensitivity study of the method was done on waste water treatment plant (WWTP) raw sewage and effluent. The WWTP sample analysed form Potchefstroom and Carletonville WWTP yielded all six target sterols in the raw sewage water samples, but no sterols eluted in the effluent samples. The raw sewage water sample taken from the Fochville WWTP yielded all six target sterols as well, however, the effluent yielded an unknown compound as well as cholesterol. An alternative study was done where the effluent sample volume was increased. By increasing the volume of water, one can possibly increase the amount (“load”) of sterols extracted and analysed, resulting in a higher abundance of target sterols. By using the target qualifier ions of the six target sterols, and the GC-TOF/MS software, the target sterols could still be qualitatively determined. Optimal volume for raw sewage is 300 ml water sample as this is enough to yield all 6 target sterols. For optimum water quality monitoring via faecal sterol analysis of effluent and other environmental samples, at least 1L sample volume needs to be collected and analysed. The methods described here can be applied to the analysis of environmental water samples. The technical advantages also make it suitable for routine environmental monitoring of faecal pollution. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-05-10T17:52:51Z No. of bitstreams: 1 michelefabrideresende.pdf: 2146232 bytes, checksum: b545847eac43403b82893dd1f747d002 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-11T13:28:46Z (GMT) No. of bitstreams: 1 michelefabrideresende.pdf: 2146232 bytes, checksum: b545847eac43403b82893dd1f747d002 (MD5) / Made available in DSpace on 2017-05-11T13:28:46Z (GMT). No. of bitstreams: 1 michelefabrideresende.pdf: 2146232 bytes, checksum: b545847eac43403b82893dd1f747d002 (MD5) Previous issue date: 2011-05-06 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os esteróis fecais são considerados importantes indicadores de contaminação fecal em ambientes aquáticos. São compostos orgânicos hidrofóbicos e encontram-se associados ao material particulado e podem ser preservados por um longo tempo com biodegradação pouco significativa. As relações entre o coprostanol e os demais esteróis encontrados no meio ambiente são importantes para confirmar a presença de esgoto doméstico em ambientes aquáticos. O presente trabalho desenvolveu uma metodologia analítica para extração, separação e determinação dos esteróis coprostanol, epicoprostanol, colesterol, colestanol e sitosterol em amostras de sedimento. Em seguida, aplicou-se o método em amostras de sedimentos coletadas em 6 córregos da cidade de Juiz de Fora. O método de análise dos 5 esteróis foi otimizado em Cromatografia Líquida de Alta Eficiência (HPLC) utilizando coluna cromatográfica de fase reversa octadecilsilano (C-18) com detecção na região do ultravioleta. Para otimizar o processo de separação dos esteróis por HPLC, a composição e o fluxo da fase móvel por eluição isocrática foram otimizados, assim como o comprimento de onda de detecção. Como os esteróis não possuem absortividade molar nessa região do espectro, houve a necessidade de realizar o processo de derivatização. Para isso, foram testados dois reagentes derivatizantes para aumentar a sensibilidade do método. Desse modo, pode-se separar e quantificar os 5 esteróis fecais. Ao concluir essa etapa, aplicou-se essa metodologia para os estudos de tratamento de amostra. O resultado desse estudo permitiu percentuais adequados de recuperação dos analitos. Após o tratamento da amostra, realizou-se o procedimento de derivatização otimizado dos extratos para posterior análise por HPLC. Os níveis de concentração de coprostanol nas amostras de sedimento analisadas variaram entre 0,0050 e 14,48 µg.g-1. Com exceção da amostra do córrego Fazenda Floresta, todas as outras foram consideradas contaminadas por fezes utilizando os parâmetros e critérios de avaliação para esse tipo de contaminação. / The fecal sterols are considered important indicators of fecal contamination in aquatic environments. Organic compounds are hydrophobic and are associated with the particulate material and can be preserved for a long time with negligible degradation. The relationships between coprostanol and other sterols found in the environment are important to confirm the presence of domestic sewage into aquatic environments. The present work developed an analytical method for extraction, separation and determination of sterols coprostanol, epicoprostanol, cholesterol, cholestanol and sitosterol in sediment samples. Then applied the method in sediment samples collected from six streams in the city of Juiz de Fora. The method of analysis of the five sterols was optimized (High Performance Liquid Chromatography (HPLC) using reverse phase chromatography column octadecylsilane (C-18) with detection in the ultraviolet region. To optimize the separation of sterols by HPLC, the composition and flow of mobile phase for isocratic elution were optimized, as well as the wavelength of detection. Because sterols are not molar absorptivity in this spectral region, it was necessary to make the process of derivatization. For this, we tested two derivatizing reagents to increase the sensitivity of the method. Thus, it can separate and quantify the five fecal sterols. When you complete this step, we applied this methodology for studies of treatment samples. The result of this study allowed percentage recovery of analytes suitable. After treatment the sample was held optimized the procedure for derivatization of the extracts for analysis by HPLC. The concentrations of coprostanol in the sediment samples analyzed varied between 0.0050 and 14.48 µg.g-1. Except for the sample stream Farm Forest, all others were considered contaminated by feces using the parameters and evaluation criteria for this type of contamination.

[pt] O presente trabalho tem como objetivo investigar a contaminação por esgotos domésticos do sistema estuarino formado pelo rio Iguaçu e canal de Sarapuí, localizados na porção noroeste da Baía de Guanabara, através da determinação de esteróis e composição elementar (C e N) da matéria orgânica em amostras de material particulado em suspensão e sedimentos, e da caracterização físico-química e química da água e sedimento. As amostragens foram realizadas em março/2004 e setembro/2004, em 7 estações de água e 10 de sedimento, distribuídas entre 6 km a montante do rio Iguaçu e 4 km dentro da baía. Também foi coletada uma amostra composta de esgoto bruto na Estação de Tratamento da Ilha do Governador (ETIG). Na determinação dos parâmetros foram utilizados métodos analíticos já estabelecidos, tais como cromatografia a gás/espectrometria de massa (esteróis) e oxidação a seco (C e N), após otimização para as condições específicas de campo e de laboratório. As concentrações dos principais esteróis variam em escala temporal e espacial, em função de fatores climáticos e da localização de fontes difusas de esgoto. Há gradientes decrescentes de concentração de esteróis entre rio e a baía, com predomínio de esteróis fecais sobre fitoesteróis. Coprostanona (5b-colestan-3b-ona) atingindo concentrações de até 21 mg gC-1, epicolestanol (5a-colestan-5a-ol) e coprostanol (5b-colestan-3b- ol) foram os mais abundantes. A detecção de epicolestanol (inédita para a baía de Guanabara) em concentrações elevadas no material particulado e sedimento (assim como no esgoto bruto), as diferenças na composição entre esteróis no esgoto bruto em comparação com o material particulado e sedimento, e os resultados das razões entre determinados esteróis, são resultados que refletem a importância dos processos bacterianos em alterar a composição original da matéria orgânica e dos esteróis associados. O conjunto de informações levantadas confirma o elevado nível de degradação ambiental da região estudada. Por outro lado, a dinâmica do sistema e processos pré- e pós- deposicionais influenciam na geoquímica dos esteróis e, portanto, seus efeitos devem ser levados em consideração em estudos sobre marcadores moleculares em estuários com características semelhantes ao do presente trabalho. / [en] The contamination by domestic sewage of the Iguaçu River and Sarapuí channel, located in the north-western sector of Guanabara Bay, was investigated by the determination of sterols and elemental composition (C and N) of organic matter in suspended matter and superficial sediments. Physico-chemical and chemical characterization of water and sediments were considered as well. Conventional analytical methods, like gas chromatography/mass spectrometry (sterols) and dry chemical oxidation (C and N), were employed after optimization for specific field and laboratory conditions. In March and September/2004 water/suspended particles (7 sample stations) and sediment (10 stations) were collected in a transect from 6 km up the river until 4 km off the river, in the bay. An additional sample of bulk sewage was also collected at a sewage treatment plant close to the studied area. The faecal sterols predominate over phyto sterols in suspended matter and sediments. Among the 14 sterols quantified, higher concentrations (up to 21 mg gC-1) were measured for coprostanone (5b-cholestan- 3b-one), epicholestanol (5a-cholestan-3a-ol) and coprostanol (5b-cholestan-3b- ol). In general, there is a river-bay decreasing gradient in sterol concentration, associated with the location of diffuse sources of sewage. Seasonal variation in climatic factor also influenced in the sterols distribution. The presence of epicholestanol (reported here for the first time in Guanabara Bay) in relatively high concentrations in suspended matter and sediments (as well as in raw sewage), the differences in sterol composition between raw sewage and suspended matter/sediments, and the values obtained for selected sterols source-diagnostic ratios, suggested the occurrence of significant microbial alteration of organic matter, with implications on the geochemistry of sterols. The elevated degradation

[pt] A avaliação do grau de contaminação de um ambiente aquático é dada por uma correlação de dados obtidos experimentalmente. A interpretação dos dados visa, além da classificação das condições ambientais locais, encontrar um comportamento previsível dos parâmetros, indicando prováveis consequências dos danos causados pela poluição. Este entendimento permite mapear as principais fontes de contaminação, inter-relacionando os dados obtidos com condições políticas, geográficas e sociais. Um conjunto de indicadores químicos (esteróis, delta elevado a 13 C) e biológicos (bactérias coliformes) foram determinados na matéria orgânica particulada de amostras de água de rios contribuintes a Baía de Guanabara (BG). As análises visaram à investigação das contribuições naturais e do aporte de esgoto doméstico em rios representativos da região hidrográfica da Baía de Guanabara (RHBG). Foram avaliadas 8 bacias da RHBG, sendo 3 no setor leste (Caceribu, Guapimirim e Suruí) e 5 do setor oeste (Iguaçu, Meriti, Irajá, Canal do Cunha e Canal do Mangue), sendo coletadas amostras em três campanhas (set/2014, jan/2015 e abril/2015). Os resultados de coliformes Escherichia Coli encontrados variaram entre 400 e 1,1x10 elevado a 12 nmp/100ml, com um predomínio, em todas as campanhas, de valores mais altos nos rios do setor oeste da RHBG. A concentração de coprostanol apresentou variação semelhante à dos coliformes, com média de 21,9 mais ou menos 43,4 microgramas L elevado a menos 1 e máximo de 205 microgramas L elevado a menos 1 no Canal do Mangue. Índices diagnósticos com esteróis selecionados confirmaram o quadro geral de contaminação fecal no setor oeste da RHBG. Os valores mais baixos de ambos os marcadores prevaleceram nos rios do setor Leste da BG, particularmente o Caceribu e o Guapimirim, que atravessam a Área de Proteção Ambiental de Guapimirim. Fundamentado em dados da literatura de caracterização da MO de amostras de esgoto bruto (EB), foram calculadas o percentual, para cada amostra do estudo, correspondente à EB. Isto é, a fração da água do rio que equivale à puramente esgoto não tratado. Desta forma, a partir da vazão dos rios foram estimadas as cargas de esgoto exportadas à Baía. Os resultados obtidos foram preocupantes. A menor carga calculada ultrapassou 2 toneladas de esgoto bruto exportado a cada mês, dado referente ao rio Caceribu. Em contrapartida, o rio São João de Meriti indicou um despejo mensal de mais de 140 toneladas. Somando os oito rios do estudo, estipulou-se, mensalmente, o lançamento de mais de 400 toneladas de esgoto bruto na BG. Foi possível também identificar o aporte de matéria orgânica de origem continental nos rios com baixa contaminação fecal. Foi estimado que níveis de coprostanol entre 0,060 microgramas L elevado a menos 1 e 0,240 microgramas L elevado a menos 1 correspondem ao limite de coliformes estipulado para balneabilidade pelo CONAMA 274/2000. Portanto, o coprostanol pode vir a ser um indicador adicional de avaliação da contaminação fecal para fins de balneabilidade das águas da baía. A carga fluvial oriunda do Canal do Mangue, Canal do Cunha e Rio Meriti são as maiores contribuintes para o aporte de esgotos para a Baía de Guanabara. Esse quadro é reflexo direto da densidade populacional e da falta de saneamento básico nas bacias desses rios. / [en] The evaluation of the degree of contamination of an aquatic environment is obtained by a correlation of data obtained experimentally. The data interpretation, besides the classification of local environmental conditions, is to find a predictable parameter behavior, indicating probable consequences of pollution damage. This understanding allows you to map the main sources of contamination, interrelating the data obtained with political, geographical and social conditions. A set of chemical (sterols, delta to the power of 13 C) and biological (thermotolerant coliforms) indicators were determined in particulate organic matter in order to investigate the natural contributions and domestic sewage contribution in representative rivers of the catchment region of Guanabara Bay (CRGB). Eight rivers in the CRBG were evaluated, three in the eastern sector (Caceribu , Guapimirim and Surui) and five in the western sector (Iguaçu , Meriti , Irajá, Cunha Channel and Mangue Channel). Samples were collected in three campaigns (Sep/2014, Jan/2015 and April/2015). The results of thermotolerant coliforms ranged between 400 and 1,1x10 to the power of 12 NMP/100ml, with a predominance, in all campaigns, of higher values in rivers of the west sector of the CRGB. The concentration of coprostanol showed similar variation as that of the coliforms, averaging 21,9 plus/minus 43,4 Microgram L to the power of minus 1 with a maximum value of 205 Microgram L to the power of minus 1 in the Mangue Channel. Diagnostic indexes based on selected sterols confirmed the general scenario of fecal contamination in the western sector of the CRGB. . The lowest values of both markers prevailed in the rivers of eastern Guanabara bay, particularly in the Environmental Protection Area of Guapimirim crossing rivers Caceribu and Guapimirim. Based on data from the literature of characterization of the organic matter from raw sewage (RS) samples, the percentage were calculated, for each study sample, that corresponds to RS. That is the fraction of river water equivalent to the untreated sewage. Thus, from the flow of the rivers were estimated sewage loads exported to the Bay. The results were worrisome. The lowest calculated load exceeded 2 tons of raw sewage exported every month, as referring to Caceribu river. In contrast, the São João de Meriti river indicated a monthly dump of more than 140 tonnes. Adding the eight rivers of the study, was stipulated monthly, the release of more than 400 tons of raw sewage in Guanabara Bay. It was also possible to identify the contribution of organic matter of continental origin in rivers with low fecal contamination. It was estimated that coprostanol levels between 0,060 Microgram L to the power of minus 1 and 0,240 g L tothe power of minus 1 correspond to the coliforms limited by CONAMA 274/2000 for primary contact. Therefore, the coprostanol may prove to be an additional indicator for assessing fecal contamination for the purpose of bathing in the bay waters. The river flow coming from the Mangue Channel, Cunha Channel and Meriti River are the largest contributors to the sewage supply to the Guanabara Bay. That situation is a direct reflection of population density and poor sanitation in the basins of these rivers.

La réduction du taux de cholestérol (CH) sanguin est un point clé dans la lutte contre les maladies cardiovasculaires. L’efficacité contrastée des médicaments disponibles actuellement ainsi que l’intérêt porté autour du microbiote intestinal dans la régulation de la physiologie de l’hôte nous amènent à envisager cette voie comme alternative thérapeutique. La production de coprostanol (CO), dérivé très peu absorbé du CH, par des bactéries de ce microbiote a été corrélée positivement à une faible cholestérolémie. Les objectifs de cette thèse sont i) d’isoler et d’identifier de nouvelles souches bactériennes ayant cette activité, ii) d’identifier les gènes bactériens responsables de cette transformation et iii) de détereminer l’impact de ce métabolisme sur la physiologie de l’hôte. Nous avons isolé 22 nouvelles souches productrices de CO à partir des selles d’un individu en produisant beaucoup. Nous avons choisi les souches Bacteroides sp. D8 et Bacteroides sp. BV pour la construction de deux banques génomiques et huit autres pour des essais d’implantation in vivo dans le tractus gastro-intestinal (TGI) de souris axéniques. Nous avons identifié 55 clones potentiellement positifs par le criblage fonctionnel des banques génomiques. Leurs analyses supplémentaires devraient nous apporter des informations sur les gènes impliqués dans cette activité. Toutes les bactéries sélectionnées sont capables de coloniser le TGI de la souris axénique. La souche Parabacteroides distasonis est la meilleure souche productrice de CO in vivo. Nous avons testé son effet sur la cholestéolémie chez des souris axéniques soumises à un régime riche en CH sur 11 semaines en comparaison avec une souche non productrice in vitro, B. dorei, et avec des souris conventionnalisées comme contrôle. La souche B. dorei produit du CO in vivo, soulignant l’importance de l’environnement dans l’activité de production de CO déjà supposée d’après la littérature et nos résultats in vitro. Des gènes impliqués dans l’excrétion du CH de l’organisme vers les selles sont surexprimés chez ces souris et celles colonisées avec P. distasonis. Cependant seules ces dernières présentent une cholestérolémie plus faible que les souris conventionnalisées. Le mécanisme impliqué semble indépendant de la production de CO et de l’excrétion de CH car les mêmes quantités de ces composés sont retrouvées dans les selles indépendamment du statut bactérien. Les concentrations en acides biliaires totaux dans la bile et dans les selles sont supérieures pour les souris monocolonisées comparées au conventionnalisées. Les selles des souris colonisées avec P. distasonis présentent plus d’acides urso- et chénodésoxycholiques que les souris conventionnalisées et plus d’acide cholique que les souris colonisées avec B. dorei. En conclusion, nous avons isolé de nouvelles souches et identifier des clones potentiellement positifs. Les études in vivo tendent à montrer que l’activité de production de coprostanol n’a pas d’effet sur la cholestérolémie. En revanche, la souche P. distasonis semble diminuer la cholestérolémie par un mécanisme encore inconnu. / Cholesterol (CH) level management is a keystone to limit cardiovascular diseases. The contrasted efficiency of the drugs currently available as well as the interest around the intestinal microbiota in regulating the host physiology lead us to consider this pathway as a therapeutic alternative. The production of coprostanol (CO), a very poorly absorbed CH derivative, by bacteria of this microbiota has been positively correlated with low CH plasma level. The aims of this thesis are (i) isolate and identify new bacterial strains possessing this activity, (ii) identify the bacterial genes responsible for this transformation and (iii) determine the impact of this metabolism on host physiology. We isolated 22 new strains producing CO from the stools of a high-coprostanol producing individual. We chose Bacteroides sp. D8 and Bacteroides sp. BV for the construction of two genomic libraries and eight others for in vivo implantation tests in the gastrointestinal tract (GIT) of germ-free mice. We identified 55 potentially positive clones by functional screening of these genomic libraries. Their additional analyzes should provide us with information about the genes involved in this activity. All selected bacteria are capable of colonizing the GIT of germ-free mice. Parabacteroides distasonis is the best strain producing CO in vivo. We tested its effect on blood cholesterol level in germ-free mice subjected to an 11-week CH-rich diet compared to an in vitro non-producing strain, B. dorei, and with conventionalized mice as control. The B. dorei strain produces CO in vivo, emphasizing the importance of the environment in the CO production activity already assumed from the literature and our results in vitro. Genes involved in the excretion of CH from body to feces are overexpressed in these mice and those colonized with P. distasonis. However, only the latter have lower cholesterolemia than conventional mice. The mechanism involved appears to be independent of CO production and CH excretion because the same amounts of these compounds are found in feces independently of bacterial status. Total biliary acids concentrations in bile and feces are higher for monocolonized mice compared to conventionalized mice. The feces of mice colonized with P. distasonis exhibited more urso- and chenodeoxycholic acids than conventionalized mice and more cholic acid than mice colonized with B. dorei. In conclusion, we have isolated new strains and identified potentially positive clones. In vivo studies tend to show that coprostanol production activity has no effect on plasma cholesterol. In contrast, P. distasonis seems to decrease plasma cholesterol by a still unknown mechanism.

Os rios Iguaçu e Barigüi, nos trechos afetados pelo vazamento da REPAR (2000), foram amostrados em 18 pontos, em 3 campanhas (2007-2008). TPH, BTEX e HPA ocorrem na água com concentrações muito baixas ou nem foram detectados. Não há aporte de hidrocarbonetos proveniente da REPAR. No sedimento, não foram detectados BTEX, HPA têm concentrações baixas ou não foram detectados; TPH ocorre em concentrações 10 vezes menores que o padrão ambiental, mas foi detectado em todas as amostras. As maiores concentrações ocorrem no rio Iguaçu, a jusante da foz do Barigüi. Alguns HPA presentes no óleo derramado só ocorrem neste trecho, sugerindo origem a partir do rio Barigüi. As razões diagnósticas de HPA no sedimento não estabelecem ligação direta com o vazamento da REPAR, por predominarem indicações de fonte pirolítica ou mista. Entretanto, estudo do sedimento do Banhado 4 mostrou que muitas das razões diagnósticas de HPA que indicavam fonte petrogênica no óleo original passaram a indicar fonte pirolítica 7 anos após o acidente. O índice pirogênico é a única razão que manteve indicação de fonte petrogênica. Na estação seca, as concentrações médias de esteróis no sedimento do rio Barigüi são coprostanol (25,97 μg/g), coprostanona (4,67 μg/g), colesterol (9,02 μg/g), colestanol (4,15 μg/g) e colestanona (0,42 μg/g). Na estação chuvosa, as concentrações reduzem-se pela metade (colestanona não foi detectada). O rio Iguaçu apresenta resultados similares a montante de Fazenda Rio Grande. Mas, nesta localidade, as concentrações aumentam apenas na estação chuvosa para coprostanol (249,63 μg/g), coprostanona (11,6 μg/g), colesterol (81,85 μg/g) e colestanol (49,99 μg/g), o que é atribuído à lavagem do solo pela água pluvial. A influência de Araucária é marcada por altas concentrações de coprostanol (329,8 μg/g na estação seca e 229,25 μg/g na estação chuvosa). Daí até General Lúcio as maiores concentrações ocorrem no canal principal, na campanha seca (média de 246,08 μg/g); na campanha chuvosa, a média cai para 101,51 μg/g. Nos canais secundários, o comportamento é contrário, relacionado ao acúmulo de sedimentos contaminados ocorrer aí apenas na cheia. No rio Iguaçu, os valores médios de COT na estação chuvosa e na seca são semelhantes (~28 mg/g) e bem maiores do que no rio Barigüi (estação chuvosa 5,2 μg/g; seca 8,9 mg/g). As relações entre coprostanol e COT indicam que o aporte de esgoto é o principal fator de definição do teor de carbono orgânico sedimentar apenas para o canal principal do rio Iguaçu e na estação chuvosa. Nos canais secundários deste rio e no rio Barigüi, em qualquer estação, ocorre contribuição importante de outras fontes (vegetação e/ou produção primária in situ). Coprostanol e coprostanona apresentam forte correlação nas duas estações. A ausência de correlação entre coprostanol e colesterol na estação seca, nos dois rios, pode ser explicada pela degradação do colesterol e/ou por parte deste provir de fonte diferente do coprostanol. As razões [5β/(5α+5β)estanona] e [5β/(5α+5β)estanol] são superiores a 7 (altamente contaminado) em 16 pontos na estação chuvosa e em todos os 18 pontos na estação seca. / The Iguaçu and Barigui rivers in parts affected by leakage REPAR (2000), were sampled in 18 points in three campaigns (2007-2008). TPH, BTEX and PAH occurring in water with very low concentrations or not detected. There is no input of oil from the REPAR. In the sediment, were not detected BTEX, PAH concentrations are low or not detected; TPH occurs in concentrations 10 times lower than the environmental standard, but was detected in all samples. The highest concentrations occur in the Iguaçu river, downstream from the mouth of Barigui. Some PAHs present in the oil spill occur only in this stretch, suggesting origin from the river Barigüi. The reasons diagnostic of PAH in the sediment do not establish a direct connection with the leaking of REPAR, predominate source of indication by pyrolytic or mixed. However, a study of sediment from the Wetland 4 showed that many of the reasons diagnostic for PAH that indicated petrogenic source in the original oil passed indicate source pyrolytic seven years after the accident. Pyrogenic index is the only reason that kept indication of petrogenic source. In the dry season, the average concentrations of sterols in the sediment of Barigüi river are coprostanol (25.97 μg/g), coprostanona (4.67 μg/g), cholesterol (9.02 μg/g), cholestanol (4.15 μg/g) and Cholestanones (0.42 μg/g). In the rainy season, the concentrations are reduced by half (Cholestanones was not detected). The Iguaçu river shows similar results upstream of Fazenda Rio Grande. But in this location the concentrations increase only during the rainy season to coprostanol (249.63 μg/g), coprostanona (11.6 μg/g), cholesterol (81.85 μg/g) and cholestanol (49.99 μg/g), which is assigned to wash the soil by rainwater. The influence of Araucaria is marked by high concentrations of coprostanol (329.8 μg/g dry season and 229.25 μg/g in the rainy season). Thence to General Lúcio the highest concentrations occur in the main channel in the dry campaign (average of 246.08 μg/g) in the rainy campaign, the average falls to 101.51 μg/g. In the secondary channels, the behavior is opposite, related to the accumulation of contaminated sediments occur there only in full. In the Iguaçu River, the average values of TOC in the rainy and dry seasons are similar (~ 28 mg/g) and much larger than the Barigüi river (wet season 5.2 μg/g dry 8.9 mg/g). The relationships between coprostanol and TOC indicate that the sewage discharges are the main factor defining the sedimentary organic carbon content just for the main channel of the Iguaçu river and the rainy season. In secondary channels this river and in the river Barigüi, in any season, there is significant contribution from other sources (vegetation and/or primary production in situ). Coprostanol and coprostanona have strong correlation in both seasons. The lack of correlation between cholesterol and coprostanol in the dry season in the two rivers can be explained by the degradation of cholesterol and/or part of this come from different source of coprostanol. The reasons [5β / (5α +5 β) estanona] and [5β / (5α +5 β) stanol] are superior to 7 (highly polluted) on 16 points in the rainy season and all 18 points in the dry season.

Os rios Iguaçu e Barigüi, nos trechos afetados pelo vazamento da REPAR (2000), foram amostrados em 18 pontos, em 3 campanhas (2007-2008). TPH, BTEX e HPA ocorrem na água com concentrações muito baixas ou nem foram detectados. Não há aporte de hidrocarbonetos proveniente da REPAR. No sedimento, não foram detectados BTEX, HPA têm concentrações baixas ou não foram detectados; TPH ocorre em concentrações 10 vezes menores que o padrão ambiental, mas foi detectado em todas as amostras. As maiores concentrações ocorrem no rio Iguaçu, a jusante da foz do Barigüi. Alguns HPA presentes no óleo derramado só ocorrem neste trecho, sugerindo origem a partir do rio Barigüi. As razões diagnósticas de HPA no sedimento não estabelecem ligação direta com o vazamento da REPAR, por predominarem indicações de fonte pirolítica ou mista. Entretanto, estudo do sedimento do Banhado 4 mostrou que muitas das razões diagnósticas de HPA que indicavam fonte petrogênica no óleo original passaram a indicar fonte pirolítica 7 anos após o acidente. O índice pirogênico é a única razão que manteve indicação de fonte petrogênica. Na estação seca, as concentrações médias de esteróis no sedimento do rio Barigüi são coprostanol (25,97 μg/g), coprostanona (4,67 μg/g), colesterol (9,02 μg/g), colestanol (4,15 μg/g) e colestanona (0,42 μg/g). Na estação chuvosa, as concentrações reduzem-se pela metade (colestanona não foi detectada). O rio Iguaçu apresenta resultados similares a montante de Fazenda Rio Grande. Mas, nesta localidade, as concentrações aumentam apenas na estação chuvosa para coprostanol (249,63 μg/g), coprostanona (11,6 μg/g), colesterol (81,85 μg/g) e colestanol (49,99 μg/g), o que é atribuído à lavagem do solo pela água pluvial. A influência de Araucária é marcada por altas concentrações de coprostanol (329,8 μg/g na estação seca e 229,25 μg/g na estação chuvosa). Daí até General Lúcio as maiores concentrações ocorrem no canal principal, na campanha seca (média de 246,08 μg/g); na campanha chuvosa, a média cai para 101,51 μg/g. Nos canais secundários, o comportamento é contrário, relacionado ao acúmulo de sedimentos contaminados ocorrer aí apenas na cheia. No rio Iguaçu, os valores médios de COT na estação chuvosa e na seca são semelhantes (~28 mg/g) e bem maiores do que no rio Barigüi (estação chuvosa 5,2 μg/g; seca 8,9 mg/g). As relações entre coprostanol e COT indicam que o aporte de esgoto é o principal fator de definição do teor de carbono orgânico sedimentar apenas para o canal principal do rio Iguaçu e na estação chuvosa. Nos canais secundários deste rio e no rio Barigüi, em qualquer estação, ocorre contribuição importante de outras fontes (vegetação e/ou produção primária in situ). Coprostanol e coprostanona apresentam forte correlação nas duas estações. A ausência de correlação entre coprostanol e colesterol na estação seca, nos dois rios, pode ser explicada pela degradação do colesterol e/ou por parte deste provir de fonte diferente do coprostanol. As razões [5β/(5α+5β)estanona] e [5β/(5α+5β)estanol] são superiores a 7 (altamente contaminado) em 16 pontos na estação chuvosa e em todos os 18 pontos na estação seca. / The Iguaçu and Barigui rivers in parts affected by leakage REPAR (2000), were sampled in 18 points in three campaigns (2007-2008). TPH, BTEX and PAH occurring in water with very low concentrations or not detected. There is no input of oil from the REPAR. In the sediment, were not detected BTEX, PAH concentrations are low or not detected; TPH occurs in concentrations 10 times lower than the environmental standard, but was detected in all samples. The highest concentrations occur in the Iguaçu river, downstream from the mouth of Barigui. Some PAHs present in the oil spill occur only in this stretch, suggesting origin from the river Barigüi. The reasons diagnostic of PAH in the sediment do not establish a direct connection with the leaking of REPAR, predominate source of indication by pyrolytic or mixed. However, a study of sediment from the Wetland 4 showed that many of the reasons diagnostic for PAH that indicated petrogenic source in the original oil passed indicate source pyrolytic seven years after the accident. Pyrogenic index is the only reason that kept indication of petrogenic source. In the dry season, the average concentrations of sterols in the sediment of Barigüi river are coprostanol (25.97 μg/g), coprostanona (4.67 μg/g), cholesterol (9.02 μg/g), cholestanol (4.15 μg/g) and Cholestanones (0.42 μg/g). In the rainy season, the concentrations are reduced by half (Cholestanones was not detected). The Iguaçu river shows similar results upstream of Fazenda Rio Grande. But in this location the concentrations increase only during the rainy season to coprostanol (249.63 μg/g), coprostanona (11.6 μg/g), cholesterol (81.85 μg/g) and cholestanol (49.99 μg/g), which is assigned to wash the soil by rainwater. The influence of Araucaria is marked by high concentrations of coprostanol (329.8 μg/g dry season and 229.25 μg/g in the rainy season). Thence to General Lúcio the highest concentrations occur in the main channel in the dry campaign (average of 246.08 μg/g) in the rainy campaign, the average falls to 101.51 μg/g. In the secondary channels, the behavior is opposite, related to the accumulation of contaminated sediments occur there only in full. In the Iguaçu River, the average values of TOC in the rainy and dry seasons are similar (~ 28 mg/g) and much larger than the Barigüi river (wet season 5.2 μg/g dry 8.9 mg/g). The relationships between coprostanol and TOC indicate that the sewage discharges are the main factor defining the sedimentary organic carbon content just for the main channel of the Iguaçu river and the rainy season. In secondary channels this river and in the river Barigüi, in any season, there is significant contribution from other sources (vegetation and/or primary production in situ). Coprostanol and coprostanona have strong correlation in both seasons. The lack of correlation between cholesterol and coprostanol in the dry season in the two rivers can be explained by the degradation of cholesterol and/or part of this come from different source of coprostanol. The reasons [5β / (5α +5 β) estanona] and [5β / (5α +5 β) stanol] are superior to 7 (highly polluted) on 16 points in the rainy season and all 18 points in the dry season.