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Biodegradación De Hidrocarburos Alifáticos En Sedimentos Marinos Subantárticos: Estudios Poblacionales Y Metagenómicos

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Biodegradación De Hidrocarburos Alifáticos En Sedimentos Marinos Subantárticos: Estudios Poblacionales Y Metagenómicos Facultad de Ciencias Bioquímicas y Farmacéuticas Universidad Nacional de Rosario Tesis de Doctorado Biodegradación de Hidrocarburos Alifáticos en Sedimentos Marinos Subantárticos: Estudios Poblacionales y Metagenómicos Presentada por Lilian Marcela Guibert Rosario, Argentina 2014 Biodegradación de Hidrocarburos Alifáticos en Sedimentos Marinos Subantárticos: Estudios Poblacionales y Metagenómicos Lilian Marcela Guibert Licenciada en Biotecnología Universidad Nacional de Rosario Esta Tesis es presentada como parte de los requisitos para optar al grado académico de Doctor en Ciencias Biológicas, de la Universidad de Rosario y no ha sido presentada previamente para la obtención de otro título en esta u otra Universidad. La misma contiene los resultados obtenidos en investigaciones llevadas a cabo en Laboratorio de Microbiología Ambiental, Centro Nacional Patagónico, dependiente del CONICET, durante el período comprendido entre el 17 de diciembre de 2009 y el 12 de diciembre de 2014, bajo la dirección de las Dras. Mariana Lozada y Hebe Dionisi. A mi familia y a Ezequiel Agradecimientos No se confundan, esta es la parte más importante de la tesis, porque sin la ayuda y el apoyo de estas personas nada de esto hubiera sido posible. Quiero agradecer a mi familia, por su apoyo incondicional, por la confianza, por escuchar, por los consejos, por las visitas y los llamados que ayudaron a acortar las distancias. A Eze, por todo, por estar a mi lado en los buenos y en los malos momentos, por hacerme reir, por la paciencia y la contención, por bajarme a la realidad, y por la asistencia informática. A la familia de Eze, por integrarme a su familia y hacerme sentir cómoda. A los padres de Eze por su ayuda y su confianza. A Pili por su magia y su hermosura. Quiero agradecer a mis directoras Mariana y Hebe, por su trabajo de dirección y acompañamiento a lo largo de esta tesis, y todo lo que eso implica. Por las correcciones a último momento (hasta altas horas de la noche, Mariana sabe de lo que estoy hablando…), los consejos, y las levantadas de ánimo para seguir adelante. Quiero agradecer a mis grandes compañeras de aventuras laboratoriles y demás: Magui y Clau, por estar desde el principio, haciendo el aguante, haciendo mucho mejor el trabajo de cada día, por los mates revividores y los tererés de gustos exóticos. Por integrarme a la vida de Madryn, y ayudarme a que no me sintiera sola. Al resto de mis amigos de Madryn, Sole, Agus, Nati Muffin, Juli, Jime, Lau, Poly, Ana, Gabi, Noe, Marian, Nati Pessaq, Jose, Gastón, Juanpi, Lucas, Lucio, también por su amistad, por las risas y los momentos compartidos, y (ya se los dije) por enseñarme que los amigos también pueden ser familia. Sole, roommate compañera de aventuras de la casa cero!. Agus, Clau, Juli, Mari, compañeras de aventuras pileteras, confidentes, y a las “lechuzas” gracias por el fútbol!. Muffin, gracias por la compañía, las llamadas de larga duración, y las visitas. Magui, Clau, Agus, Sole y Muffin, gracias por el apoyo desde que volví a Rosario para darme fuerzas para terminar esta tesis. A mis amigos de toda la vida: Ana, Maru, Vir, y Fede, por tantos años de amistad y momentos compartidos, por hacerme sentir que nada había cambiado a pesar de la distancia. A todos mis compañeros del CENPAT, en especial a los integrantes del Laboratorio de Oceanografía y Contaminación de Aguas del CENPAT, por permitirme utilizar sus instalaciones, por los análisis, y por recolectar las muestras de Bahía Ushuaia. Al trabajo y la predisposición de mis tutores Christian Magni y Alejandra Mussi. Por último, quisiera agradecer al CONICET por otorgarme las becas doctorales que permitieron financiar mis estudios. Parte de los resultados que se describen en el presente trabajo de Tesis fueron divulgados en las revistas y congresos que se detallan a continuación: Trabajos publicados: Guibert LM, Loviso CL, Marcos MS, Commendatore MG, Dionisi HM, Lozada M. Alkane Biodegradation Genes from Chronically Polluted Subantarctic Coastal Sediments and Their Shifts in Response to Oil Exposure. Microb Ecol (2012) 64:605–616. DOI 10.1007/s00248-012- 0051-9. (Capítulos 1 y 2). Trabajos en preparación: Guibert LM, Loviso CL, Jansson J, Dionisi HM, Lozada M. Contribution of Bacteroidetes and Planctomycetes to alkane biodegradation potential in the metagenome of Subantarctic marine sediments. (Manuscrito en preparación, fecha probable de envío a la revista Microbial Ecology: fines del 2014 o principios del 2015). (Capítulo 3). Loviso CL, Guibert LM, Sarango-Cárdenas SV, Lozada M, Dionisi HM. Metagenomic fragments containing multiple ring-hydroxylating oxygenase genes belong to ecologically- relevant bacteria inhabiting polluted Subantarctic sediments. (Enviado para publicación, revista International Microbiology). (Capítulo 3) Trabajos presentados en congresos: Guibert, L., Loviso, C., Jansson, J., Dionisi, H., Lozada, M. Identification of gene clusters involved in alkane biodegradation from polluted subantarctic sediments by two complementary metagenomic approaches. IX Congreso de Microbiología General Sociedad Argentina de Microbiología General (SAMIGE). Rosario, 5-7 agosto 2013. Loviso, C., Guibert, L., Sarango, S., Lozada, M., Dionisi, H. Screening of a Metagenomic Library from Polluted Subantarctic Sediments for the Identification of Gene Clusters Involved in Hydrocarbon Biodegradation. American Society for Microbiology 113th General Meeting. Denver, EEUU, 18–21 mayo 2013. Guibert, L., Loviso, C., Sarango, S., Marcos, M., Lozada, M., Dionisi, H. Culture-independent and metagenomic analyses of hydrocarbon-degrading bacteria from polluted subantarctic marine sediments. VI Simpósio de Microbiología Aplicada. Porto Alegre, Brasil, 5-8 noviembre 2012. Loviso, C., Guibert, L., Marcos, M., Di Marzio, W., Lozada M., Dionisi, H. Characterization and metagenomic analysis of bacterial genes encoding key enzymes for hydrocarbon degradation in Subantarctic marine sediments. New Horizons in Biotechnology. Trivandrum, India, 21 -24 noviembre 2011. Guibert, L., Loviso, C., Marcos, M., Commendatore, M., Dionisi, H., Lozada, M. Comunidades bacterianas de sedimentos costeros subantárticos y sus cambios como consecuencia de la exposición a petróleo crudo. II Jornada de Becarios del CENPAT-CONICET. Puerto Madryn, 11 noviembre 2011. ISBN 978-987-26951-0-1 Guibert, L., Loviso, C., Marcos, M., Dionisi, H., Lozada, M. Alkane monooxygenase gene diversity in subantarctic marine sediments. XLVI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB). Puerto Madryn, 30 noviembre - 3 diciembre 2010. Loviso, C., Guibert, L., Marcos, M., Di Marzio, W., Lozada, M., Dionisi, H. Metagenomic analysis of aromatic ring-hydroxylating dioxygenases from cold marine environments. XLVI Reunión Anual SAIB. Puerto Madryn, 30 noviembre - 3 diciembre 2010. Marcos, M., Loviso, C., Guibert, L., Lozada, M., Dionisi, H. Novel molecular approaches for studying microbiol communities. XLV Reunión Anual SAIB. Tucumán, noviembre 2009. Índice General ÍNDICE GENERAL Abreviaturas y símbolos ............................................................................................. i Lenguaje especial ........................................................................................................ iii 1 Resumen ................................................................................................................... 1 2 Introducción ............................................................................................................ 4 2.1 Los hidrocarburos ......................................................................................................... 4 2.1.1 Fuentes de hidrocarburos en el ambiente marino ........................................................ 4 2.1.2 Fuentes de hidrocarburos en la costa Patagónica ......................................................... 5 2.2 Biodegradación de alcanos ........................................................................................ 6 2.2.1 Mecanismos de acceso.............................................................................................................. 7 2.2.2 Enzimas involucradas en la activación aeróbica de alcanos ..................................... 8 2.2.2.1 Metano monooxigenasas (MMO) ..................................................................................................... 9 2.2.2.2 Enzimas relacionadas a la familia MMO .................................................................................... 10 2.2.2.3 Alcano hidroxilasas de la familia AlkB........................................................................................ 10 2.2.2.4 Monooxigenasas citocromo P450 ................................................................................................. 12 2.2.2.5 Alcano hidroxilasas para alcanos de cadena larga ................................................................ 12 2.2.3 Organización génica y mecanismos de regulación ...................................................... 13 2.2.3.1 Pseudomonas putida ......................................................................................................................... 14 2.2.3.2 Alcanivorax borkumensis ................................................................................................................ 17 2.2.4 Evolución molecular y transferencia horizontal .......................................................... 18 2.2.5 Biodegradación en el ambiente marino ..........................................................................
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