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Biorremediación De Metales Pesados Por Sulfidogénesis Utilizando Comunidades Y Microorganismos Sulfato-Reductores

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Biorremediación De Metales Pesados Por Sulfidogénesis Utilizando Comunidades Y Microorganismos Sulfato-Reductores FACULTAD DE CIENCIAS EXACTAS DEPARTAMENTO DE QUÍMICA BIORREMEDIACIÓN DE METALES PESADOS POR SULFIDOGÉNESIS UTILIZANDO COMUNIDADES Y MICROORGANISMOS SULFATO-REDUCTORES Trabajo de Tesis Doctoral Graciana Willis Poratti Director: Dr. Edgardo Donati Año 2016 Este Trabajo de Tesis Doctoral es presentado para optar al grado de Doctor de la Facultad de Ciencias Exactas Fue realizado en CINDEFI (CCT La Plata-CONICET, UNLP) A Mamá, Papá y Juan, por acompañarme siempre Reconocimientos A la Facultad de Ciencias Exactas de la Universidad Nacional de La Plata y al Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) por haber hecho posible la realización del presente Trabajo de Tesis Doctoral. Al CINDEFI (Centro de Investigación en Fermentaciones Industriales) y a toda la gente que lo conforma por haberme brindado el lugar físico para la realización del presente trabajo. Al Prof. Dr. David Barrie Johnson (Bangor Acidophile Research Team) de la Universidad de Bangor, Gales por permitirme realizar parte del trabajo de Tesis Doctoral en su laboratorio. A los Dres. Goh Kian Mau de la Universiti Teknologi, Malaysia y Kok-Gan Chan de la University of Malaysa, por la colaboración en la secuenciación del genoma de la cepa LMa1. Al Ente Provincial de Termas de Neuquén (EPROTEN) por permitir la toma de muestra en el Parque Geotermal Copahue. Al grupo de la Dra. Alejandra Giaveno perteneciente al IDEPA (CONICET, UNCo) y a la Facultad de Ingeniería de la Universidad Nacional del Comahue, por la organización de las campañas de muestreo a la zona de Copahue. Agradecimientos A mi director Dr. Edgardo Donati, por haber depositado su confianza en la realización de este trabajo, por dirigirme y haberme dado la oportunidad de viajar y formarme. Muchas Gracias. A Sabrina Hedrich e Iván Ñancucheo por recibirme el primer día que llegué a Gales y ayudarme dentro y fuera del laboratorio siempre que los necesité. A Carmen Falagán, por compartir charlas de laboratorio, almuerzos, viajes, bailes y salidas y hacer de mi estadía en Gales una de las mejores experiencias. Te quiero mucho! A Amira Suriaty Yaakop por ayudarme y darme ánimo en la extracción de ADN para la secuenciación del genoma, pero sobre todo por la gran amistad en el corto tiempo que estuvimos juntas. A mis compañeros de laboratorio, los de hoy, y a los que estuvieron durante todo el camino, por ayudarme siempre que lo necesite. A “Los Mosqueteros”: Cami, Pato y Sara por estar en las buenas y en las malas durante estos cinco años y darme ánimo constante. Muchas Gracias! Los quiero! A Marce, por el apoyo y aliento constante en cada ensayo, en cada charla y por estar siempre en los momentos buenos y malos. Gracias por ser una parte importante de mi vida en tan poco tiempo. A Ceci, por las “charlas de café” y el consejo permanente. A mis amigos y colegas del IBBM: Carla, Piru, Fran, Jose, etc., por responder mis “dudas moleculares” cada vez que los necesite. A Piru especialmente por ayudarme con la edición. A mis colegas biotecnólogos, gracias por las risas de los asados y salidas que han hecho estos años muy divertidos. A mis amigas: Fabri, Tefi, Vicky, Agus, María, Silvi, Estefi y Gime. Gracias por estar en cada momento, en cada logro y en cada fracaso para ayudarme a levantarme y salir adelante. Las quiero! A mis compañeros del CINDEFI. A Andre, Leo y Tere por compartir los almuerzos y a todos los que siempre estuvieron para ayudarme. Gracias! A mis tíos: Icha, Eli, Nenona y Huguito, por estar siempre. A mis primos. A mis abuelos que ya no están. Gracias! A Mami, Papi y Juan, gracias por estar incondicionalmente a mi lado. … Y a todos los que estuvieron al lado mío durante todo el recorrido… Índice CAPÍTULO I: Introducción y Objetivos ............................................................................................... 17 I.1. CONTAMINACIONES CON METALES PESADOS ......................................................... 17 I.1.1. Metales pesados y especiación .............................................................................. 17 I.1.2. Origen de la contaminación con metales pesados ................................................ 17 I.1.3. Contaminaciones antropogénicas de metales pesados en recursos naturales: agua, suelo y aire ........................................................................................................ 19 I.1.3.1. Actividades industriales ...................................................................................... 19 I.1.3.2. Actividades agrícolas y residuos domésticos ...................................................... 22 I.1.4. Efectos de los metales pesados sobre los ecosistemas y seres vivos .................... 23 I.2. TECNOLOGÍAS DE REMEDIACIÓN ............................................................................. 26 I.2.1. Métodos fisicoquímicos de remediación de metales pesados............................... 26 I.2.2. Alternativas biológicas de remediación ................................................................. 28 I.2.3. Mecanismos biológicos de remoción de metales pesados de sitios contaminados .................................................................................................................................... 31 I.3. BIOPRECIPITACIÓN DE METALES PESADOS .............................................................. 33 I.4. OBJETIVOS ................................................................................................................ 45 CAPÍTULO II: Búsqueda de consorcios de microorganismos sulfato-reductores. Caracterización molecular .................................................................................................................... 51 II.1 INTRODUCCIÓN ....................................................................................................... 51 II.2. DESCRIPCIÓN DEL SITIO DE MUESTREO .................................................................. 53 II.2.1. Ubicación del área ............................................................................................... 53 II.2.2. El volcán Copahue, Río Agrio y Lago Caviahue ..................................................... 55 II.2.3. Las Máquinas ........................................................................................................ 58 II.2.4. Las Maquinitas...................................................................................................... 59 II.2.5. Termas de Copahue .............................................................................................. 60 II.3. RECOLECCIÓN DE LAS MUESTRAS Y ANÁLISIS FISICOQUÍMICO ............................. 62 II.3.1. Sitios de recolección de muestras ........................................................................ 62 II.3.2. Parámetros fisicoquímicos .................................................................................... 64 II.3.3. Resultados y Discusión ......................................................................................... 65 II.4. PROCEDIMIENTOS DE ENRIQUECIMIENTO ............................................................. 69 II.4.1. Consorcios de MSR a pH neutro ........................................................................... 69 II.4.1.1. Discusión de los resultados ................................................................................ 70 II.4.1.2. Cinética de crecimiento de los consorcios de MSR a pH neutro ........................ 72 II.4.1.2.1. Discusión de los resultados ............................................................................. 74 II.4.2. Consorcios acidófilos de MSR ............................................................................... 77 II.4.2.1. Discusión de los resultados ................................................................................ 79 II.5. CARACTERIZACIÓN MOLECULAR ............................................................................ 83 II.5.1. Sedimentos anaerobios del volcán Copahue ....................................................... 83 II.5.1.1. Metodología ...................................................................................................... 83 II.5.1.2. T-RFLP arqueas y bacterias - Resultados y Discusión ....................................... 85 II.5.1.3. Bibliotecas del gen 16S rARN de bacterias - Resultados y Discusión................ 92 II.5.1.3.1. Muestra AL1 ................................................................................................... 92 II.5.1.3.2. Muestra LMi ................................................................................................... 98 II.5.2. Consorcios de MSR ............................................................................................. 105 II.5.2.1. Metodología .................................................................................................... 105 II.5.2.2. Consorcios acidófilas de MSR. Resultados y discusión .................................... 106 II.5.2.3. Consorcios a pH neutro de MSR. Resultados y discusión ................................ 112 II.6. POSIBLES MODELOS GEOMICROBIOLOGICOS ...................................................... 116 II.7. CONCLUSIONES FINALES DEL CAPITULO .............................................................. 120 CAPÍTULO III: Aislamiento y caracterización de microorganismos sulfato-reductores ................... 125 III.1. INTRODUCCIÓN ................................................................................................... 125 III.2. AISLAMIENTO DE MICROORGANISMOS SULFATO-REDUCTORES ......................
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