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Departamento De Ciencias De La Vida Y La Agricultura DEPARTAMENTO DE CIENCIAS DE LA VIDA Y LA AGRICULTURA CARRERA DE INGENIERÍA EN BIOTECNOLOGÍA TRABAJO DE TITULACIÓN PREVIO A LA OBTENCIÓN DE TÍTULO DE INGENIERO EN BIOTECNOLOGÍA TEMA: IDENTIFICACIÓN DE BACTERIAS, HONGOS Y ALGAS MESÓFILOS Y TERMÓFILOS DE LAS FUENTES GEOTERMALES: EL RIÑÓN-..PROVINCIA DEL AZUAY Y GUAPÁN- PROVINCIA DEL CAÑAR. AUTOR: NARANJO ROJAS YANARA GABRIELA DIRECTOR: MSc. KOCH KAISER ALMA ROSEL SANGOLQUÍ 2017 ii CERTIFICACIÓN iii AUTORIA DE RESPONSABILIDAD iv AUTORIZACIÓN v DEDICATORIA A mis padres Amanda del Carmen Rojas López y Wilson Raúl Naranjo. Pues a su manera tan distinta han sabido estar en los momentos clave. Los amo con el corazón. Gracias por haberme permitido vivir y desarrollarme, esto es para Uds. vi AGRADECIMIENTOS Ahora me doy cuenta de lo difícil que es ponerle punto final a una etapa de la vida. Es una sensación extraña y hasta cierto punto difícil de afrontar, por momentos quisiera que regresara el tiempo y volver a iniciar, pero luego entiendo que solo es miedo a lo desconocido. En realidad es tanta la felicidad que siento por haber llegado al momento de culminar, y la dicha de saber que aunque fue complicado, es momento de continuar. Muchas han sido las personas que se han visto involucradas de una u otra manera en este tiempo de investigación, principalmente los docentes que han sido una guía y me han brindado su apoyo, no solo en el laboratorio, sino también mediante conversaciones y consejos, que me han permitido crecer como persona y profesional. Quiero presentar mi agradecimiento a la Máster Alma Koch, directora de este trabajo, quien ha sido una de las mejores maestras que he tenido a lo largo de la carrera, sembrando en mí el gusto por la microbiología y todo lo que la materia comprende. Además me permitió ser parte del Laboratorio de Microbiología, en donde aprendí a manejar nuevas técnicas de identificación microbiana y donde pude poner en práctica todos los conocimientos adquiridos. También me gustaría mencionar al Doctor Andrés Izquierdo, quien es el director del proyecto de identificación de microorganismos provenientes de fuentes geotermales del Ecuador y que ha estado desde el inicio con el grupo de “Termas”, permitiendo que nos relacionemos como buenos compañeros de trabajo, valorando la unión y la amistad que se ha creado. Me gustaría mencionar a mis compañeros tesistas: Carito, Mopo y Roque, con quienes iniciamos esta recta final, fuimos a los muestreos y tuvimos la oportunidad de conocernos y compartir muy buenas experiencias. A Beto, que a pesar de entrar después al grupo, también nos ayudó en la organización de algunos eventos. Chicos, sin ustedes el laboratorio no hubiera sido lo mismo, y entre risas y complicidades se llegaron a ganar un lugar en mi corazón. vii Espero que ahora que la vida nos aparta sigamos en contacto y que cada uno cumpla sus sueños. ¡Gracias chicos! Mi agradecimiento a mis amigas de la universidad: Ali, Mishu, Chovis y Aleja, que son las personas con las que compartí los últimos semestres de la carrera, entre bromas y estudio pudimos salir adelante. Las quiero mucho chicas y que su vida sea llena de bendiciones. A mis amigas del colegio Cynthia, Jahaira y Katy, que forman parte de mi vida hasta ahora pues son el regalito de vida que me dio los años y espero que nunca nos apartemos. A Kathe, quien es más que mi mejor amiga, es mi hermana de otra madre, creo que siempre serás parte de mi familia, un pequeño y reducido grupo. Creo que mi vida no sería la misma sin ninguna de ellas. Hubieron momentos a lo largo del tiempo de tesis, en los que el peso de los días pasaban dejando frustración y estrés, muchas veces pensé que era demasiado trabajo. En ese instante Leo fue la persona que me dio ánimos y de una forma divertida, me permitió mirar todo desde un enfoque diferente, haciendo que recobre energía. A ti ¡Muchas gracias! Finalmente quiero agradecer a mi familia, mis padres y hermana. Quiero que sepan que a pesar de todo lo que vivimos día a día, son las únicas personas con las que podré contar de manera certera. Mi pequeña hermanita Vane, quiero agradecerte por estar ahí en todo momento y por ser mi pilar fundamental, todo este esfuerzo y trabajo es por ti. Espero un día poderte ver en la misma etapa en la que me encuentro y créeme que no me sentiré menos orgullosa de lo que estoy ahora de ti. Sigue siendo luchadora. Te amo pequeña. A los que están y a lo que por la premura del asunto no he podido mencionar. Millón de gracias. viii ÍNDICE DE CONTENIDOS CERTIFICACIÓN ................................................................................................................. ii AUTORIA DE RESPONSABILIDAD................................................................................ iii AUTORIZACIÓN ................................................................................................................. iv DEDICATORIA ......................................................................................................................... v AGRADECIMIENTOS ........................................................................................................ vi ÍNDICE DE CONTENIDOS .............................................................................................. viii LISTADO DE FIGURAS .................................................................................................... xi LISTADO DE TABLAS ..................................................................................................... xv LISTADO DE ABREVIATURAS .................................................................................... xvii RESUMEN ....................................................................................................................... xviii CAPÍTULO 1 ......................................................................................................................... 1 1.1. Formulación del problema ................................................................................... 1 1.2. Justificación del problema ................................................................................... 2 1.3. Objetivos de la investigación .............................................................................. 3 1.3.1. Objetivo general .............................................................................................. 3 1.3.2. Objetivos específicos ..................................................................................... 3 1.4. Marco teórico ........................................................................................................... 3 1.4.1. Microorganismos extremófilos .................................................................... 3 1.4.2. Características de los microoganismos extremófilos ............................ 3 1.4.3. Adaptaciones de microorganismos a condiciones ambientales extremas ....................................................................................................................... 4 1.4.4. Identificación de microorganismos mediante técnicas independientes de cultivo ........................................................................................ 9 1.4.5. Descripción de las Fuentes Geotermales ............................................... 18 1.5. Hipótesis de la investigación............................................................................. 20 CAPÍTULO 2 ....................................................................................................................... 21 2.1. Participantes .......................................................................................................... 21 2.2. Localización geográfica ...................................................................................... 21 2.2.1. Localización de la fase de campo ............................................................. 21 2.2.2. Localización de la fase de laboratorio ..................................................... 22 2.3. Periodo de investigación .................................................................................... 23 2.4. Metodología ........................................................................................................... 23 2.4.1. Fase de Campo .............................................................................................. 23 ix 2.4.2. Fase de Laboratorio ...................................................................................... 26 2.4.2.8. Análisis de RFLPs .......................................................................................... 30 2.4.3. Análisis de secuencias ................................................................................ 31 2.4.3.3. Análisis filogenético .................................................................................. 32 2.4.3.3. Análisis de la composición microbiana de las fuentes geotermales mediante secuenciación masiva - Illumina ........................................................ 32 CAPÍTULO 3 ....................................................................................................................... 34 3.1. Caracterización de los sitios de muestreo ..................................................... 34 3.2. Análisis fisicoquímico ......................................................................................... 34 3.3. Obtención de cultivos puros ............................................................................. 35 3.4. Características microscópicas de los cultivos ............................................. 36 3.5. Extracción de ADN ..............................................................................................
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