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Te Sis D O C to R a L Microorganismos De Las MICROORGANISMOS DE LAS CUEVAS VOLCÁNICAS DE LA PALMA (ISLAS CANARIAS) DIVERSIDAD Y POTENCIAL USO BIOTECNOLÓGICO TESISDOCTORAL Jose Luis González Pimentel | 2019 Directores: Cesáreo Sáiz Jiménez Leonila Laiz Trobajo MICROORGANISMOS DE LAS CUEVAS VOLCÁNICAS DE LA PALMA (ISLAS CANARIAS): DIVERSIDAD Y POTENCIAL USO BIOTECNOLÓGICO Memoria que presenta José Luis González Pimentel para optar al título de Doctor en Biotecnología 2019 MICROORGANISMOS DE LAS CUEVAS VOLCÁNICAS DE LA PALMA (ISLAS CANARIAS): DIVERSIDAD Y POTENCIAL USO BIOTECNOLÓGICO Visado en Sevilla, a 12 de Abril de 2019 LOS DIRECTORES Prof. Dr. D. Cesáreo Sáiz Jiménez Dra. D. Leonila Laiz Trobajo Profesor de Investigación “Ad Honorem” Científica Titular EL TUTOR Dr. D. Fernando Manuel Govantes Romero Profesor Titular Memoria que presenta D. José Luis González Pimentel para optar al grado de Doctor en Biotecnología DOCTOR D. José Enrique Fernández Luque, DIRECTOR DEL INSTITUTO DE RECURSOS NATURALES Y AGROBIOLOGÍA DE SEVILLA DEL CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS Certifica: Que la presente Memoria de Investigación titulada “Microorganismos de las cuevas volcánicas de La Palma (Islas Canarias): Diversidad Y potencial uso biotecnológico”, presentada por D. Jose Luis González Pimentel para optar al grado de Doctor en Biotecnología, ha sido realizada en el Departamento de Agroquímica, Microbiología Ambiental Y conservación de suelos, bajo la dirección de los Drs. D. Cesáreo Sáiz Jiménez Y Leonila Laiz Trobajo, reuniendo todas las condiciones exigidas a los trabajos de Tesis Doctorales. En Sevilla, a 12 de Abril de 2019 El presente trabajo se ha realizado en el marco del proYecto CGL2013-41674-P del Plan Estatal de Investigación Científica Y Técnica Y de Innovación 2013-2016, dentro del Programa Estatal de Promoción del Talento Y su Empleabilidad en I+D+i. Agradecimientos Me gustaría expresar mi agradecimiento al Prof. Dr. Cesáreo Sáiz Jiménez, a la Dra. Leonila Laiz Trobajo y, en especial, a mis compañeras la Dra. Valme Jurado Lobo, Isabel GalocHa Zapata, la Dra. Irene Dominguez Moñino y el Dr. Miguel Ángel Rogerio Candelera, por la confianza depositada en mi y por toda la ayuda prestada para el desarrollo de esta tesis doctoral. A la Dra. Ana Miller, por dirigir los muestreos. A Octavio Fernández Lorenzo, espeleólogo Que nos ayudó en los accesos y permisos a los tubos de lava. A la dirección del ParQue Nacional de la Caldera del Taburiente (La Palma), por facilitarnos el acceso al tubo de lava “Cueva Honda del Bejenado”. A la ayuda para la formación de doctores del Ministerios de Ciencia, innovación y Universidades y al Consejo Superior de Investigaciones Científicas, por Haberme permitido realizar este proyecto en el Instituto de Recursos Naturales y Agrobiología de Sevilla. A Valme, por estar siempre dispuesta a ayudarme en los distintos ámbitos de la tesis doctoral. Ha sido apoyo y desahogo en incontables ocasiones. A Isabel, Que siempre Que He tenido un momento de agobio me Ha prestado su ayuda. A mis padres, José Luis y Mercedes, por su apoyo incondicional en todo momento. Gracias a sus ánimos y confianza en mi, He conseguido terminar un largo proyecto de 10 años Que culmina con esta tesis. A Mª Carmen, Eduardo, CHari y Manolo, por ser parte activa de mi vida. A mi Hermano, Mario, Que conoce la cantidad de Horas y esfuerzo Que He vertido en el desarrollo de este trabajo. A ti, Maca, a la persona Que Ha aguantado fines de semana en casa desde Que, allá por el 2009, decidiera reencontrarme con los estudios después de tantos años en un trabajo en el Que no disfrutaba. Por ayudarme en tantos y tantos momentos, por Haber estado conmigo, por Hacerme disfrutar la vida a tu lado. Esta tesis es también tuya. Índice ABSTRACT - RESUMEN ........................................................................................................... 3 CAPÍTULO 1: INTRODUCCIÓN ................................................................................................. 7 1.1 TUBOS DE LAVA ............................................................................................................................ 8 1.1.1 Depósitos secundarios de origen biogénico .................................................................. 10 1.2 BIODIVERSIDAD MICROBIANA Y SU PAPEL BIOGEOQUÍMICO EN LOS TUBOS DE LAVA ................................. 16 1.3 WHOLE GENOME SEQUENCING Y METAGENÓMICA FILOGENÉTICA: LA SECUENCIACIÓN MASIVA COMO IMPULSO A LA MICROBIOLOGÍA DE AMBIENTES SUBTERRÁNEOS ................................................................. 24 1.3.1 El origen: Técnicas dependientes de cultivos ................................................................ 25 1.3.2 Primera revolución metodológica: Técnicas independientes de cultivo ....................... 29 1.3.3 Segunda revolución metodológica: Técnicas de secuenciación masiva y el desarrollo de las ciencias ómicas ................................................................................................................. 31 1.3.4 Ómicas y estrategias de estudio: Secuenciación de genomas completos y análisis de amplicones.............................................................................................................................. 35 1.4 OBJETIVOS ................................................................................................................................ 39 CAPÍTULO 2: MATERIALES Y MÉTODOS..................................................................................41 2.1 MUESTRAS ESTUDIADAS............................................................................................................... 42 2.2 AISLAMIENTO Y CARACTERIZACIÓN DE CEPAS BACTERIANAS ................................................................. 46 2.2.1 Aislamientos campaña 2015 ......................................................................................... 46 2.2.2 Aislamientos campaña 2016 ......................................................................................... 48 2.2.3 Identificación de las cepas aisladas............................................................................... 49 2.2.4 Estudios de determinación de nueva especie bacteriana ............................................. 57 2.3 ENSAYOS DE INHIBICIÓN DE CRECIMIENTO DE BACTERIAS Y HONGOS ..................................................... 64 2.3.1 Ensayo con bacterias ..................................................................................................... 64 2.3.2 Ensayo con hongos ........................................................................................................ 67 2.4 SECUENCIACIÓN DE GENOMAS COMPLETOS ..................................................................................... 68 2.4.1 Extracción del ADN genómico ....................................................................................... 69 2.4.2 Procesado bioinformático de las secuencias de los genomas ....................................... 71 2.5 CARACTERIZACIÓN DE LAS COMUNIDADES MICROBIANAS PRESENTES EN MUESTRAS NATURALES................. 76 2.5.1 Extracción de ácidos nucleicos de muestras naturales ................................................. 76 2.5.2 Preparación de genotecas de ADN y ARN ..................................................................... 79 2.6 MEDIOS DE CULTIVO ................................................................................................................... 96 2.7 REACTIVOS .............................................................................................................................. 103 2.8 SOLUCIONES Y TAMPONES .......................................................................................................... 104 CAPÍTULO 3: CARACTERIZACIÓN DE LAS COMUNIDADES MICROBIANAS NO CULTIVABLES DE LOS TUBOS DE LAVA ........................................................................................................... 109 3.1 CONSTRUCCIÓN DE GENOTECAS DE ADN Y ARN MEDIANTE LA TÉCNICA DE CLONACIÓN ......................... 110 3.1.1 Genotecas de la muestra EP01-6F: Coraloides ............................................................ 111 3.1.2 Genotecas de MZ03-6F y MZ03-7G: Comparativa ...................................................... 132 3.1.3 Comparación de las genotecas de bacterias de las tres muestras analizadas ........... 158 3.2 CONSTRUCCIÓN DE GENOTECAS MEDIANTE SECUENCIACIÓN MASIVA O HT-NGS .................................. 170 3.2.1 Análisis taxonómico de las genotecas HT-NGS ........................................................... 174 3.2.2 Cálculo de los índices de diversidad ............................................................................ 189 CAPÍTULO 4: CARACTERIZACIÓN DE LAS COMUNIDADES MICROBIANAS CULTIVABLES DE LOS TUBOS DE LAVA ................................................................................................................. 193 4.1 AISLAMIENTO, IDENTIFICACIÓN Y CARACTERIZACIÓN DE BACTERIAS .................................................... 194 4.1.1 Cepas aisladas del tubo de lava “Cueva del Llano de los Caños”: MZ04 .................... 195 4.1.2 Cepas aisladas del tubo de lava “Fuente de La Canaria”: MZ03 ................................. 201 4.1.3 Cepas aisladas del tubo de lava “Búcara II de Montes Luna”: MZ26-11K .................. 213 4.1.4 Cepas aisladas del tubo de lava “Cueva Honda del Bejenado”: EP01-6F .................... 215 4.1.5 Descripción de nuevas especies bacterianas del género Streptomyces ...................... 218 4.2 AISLAMIENTO E IDENTIFICACIÓN
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