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Biodegradación De Hidrocarburos Totales De Petróleo Por Hongos Endófitos De La Amazonia Ecuatoria.Pdf PONTIFICIA UNIVERSIDAD CATÓLICA DEL ECUADOR FACULTAD DE CIENCIAS EXACTAS Y NATURALES ESCUELA DE CIENCIAS BIOLÓGICAS Biodegradación de Hidrocarburos Totales de Petróleo por Hongos Endófitos de la Amazonia Ecuatoriana Disertación previa a la obtención del Título de Licenciado en Ciencias Biológicas FERNANDO JAVIER MARÍN MINDA Quito, 2018 Certifico que la Disertación de Licenciatura en Ciencias Biológicas de la Sr. Fernando Javier Marín Minda ha sido concluida de conformidad con las normas establecidas; por lo tanto, puede ser presentada para la calificación correspondiente. M.Sc. Alexandra Narváez Trujillo Directora de la Disertación Quito, 28 de noviembre de 2018 iii “La ciencia no es solo una disciplina de razón, sino también de romance y pasión.” -Stephen Hawking iv AGRADECIMIENTOS A Alexandra Narváez-Trujillo, directora de mi proyecto de tesis por su incondicional apoyo, motivación, tiempo compartido y por creer en mi desde el primer momento en que me incorporé a su grupo de trabajo, al acogerme en su laboratorio y transmitirme su amor por la ciencia y los hongos endófitos. A la Pontificia Universidad Católica del Ecuador por el financiamiento de esta investigación. A Hugo Navarrete Zambrano y al Centro de Servicios Ambientales y Químicos de la PUCE (CESAQ-PUCE) por su valioso aporte en el análisis de los resultados de este estudio. A Carolina Portero, por haberme brindado su confianza y por enseñarme todo lo necesario para realizar mi trabajo de titulación de manera exitosa. Por siempre estar dispuesta a brindarme un poco de su tiempo cada vez que lo necesité. En especial le agradezco por su amistad y por siempre ser esa persona que cuida y vela por el bienestar de sus compañeros de trabajo. A Javier Carvajal, por sus valiosas aportaciones, contribuyendo en la realización de un trabajo de calidad y rigurosidad científica. A Elisa Jara por su gran apoyo durante la realización del análisis de los resultados de este trabajo. A mis padres Fernando y Julia, que a pesar de los tiempos difíciles nunca se han rendido conmigo, espero seguir adelante sin decepcionar a su confianza y verdadero amor. A mis hermanos Pedro y Pablo, compañeros de vida y juegos, quienes me han servido de motivación y a quienes espero haber alentado a seguir adelante y nunca rendirse. v A Elizabeth Veloz por su incondicional apoyo durante todo este tiempo de carrera, haciendo de los momentos duros y difíciles simples tormentas pasajeras, siempre ayudándome a ver esa luz en medio de la oscuridad. A Eliana Veloz Villavicencio por su amistad, su gran apoyo y ayuda durante la realización de este trabajo, por las risas compartidas y nuestras tardes de fútbol y ciencia. A Caro Castro, Stephy Villota y Sofi López por su apoyo y por ser grandes compañeras tanto de trabajo como de risas en nuestro querido Laboratorio de Biotecnología Vegetal. A mis compañeros de carrera por todos los buenos momentos compartidos, tanto en clases como en las salidas al campo. La mejor de las suertes en todos sus emprendimientos. ¡Gracias a todos! vi LISTA DE ABREVIATURAS Abreviatura Significado AA Agar Agua ADN Ácido Desoxirribonucleico ADNr Ácido Desoxirribonucleico ribosómico AEM Agar Extracto de Malta ANOVA Analysis of Variance APD Agar Papa Dextrosa BLAST Basic Local Aligment Search Tool BLASTn Basic Local Aligment Search Tool of nucleotides CEQCA Colección de Endófitos Quito Católica CESAQ Centro de Servicios Ambientales y Químicos CG Cromatografía de Gases cm2 Centímetro cuadrado CV Coeficiente de variación DCFIF Diclorofenol Indofenol DCM Diclorometano FID Flame Ionization Detector gtr General time reversible HAP Hidrocarburos Aromáticos Policíclicos HQCA Herbario Quito-Católica HTP Hidrocarburos Totales de Petróleo ID Identidad IR Espectroscopia Infrarroja ITS Internal Transcribed Spacer Km Kilómetro min Minuto ml Mililitro MSM Medio de Sales al Mínimo Muscle Multiple sequence alignment by log-expectation O Oeste ° Grados vii Abreviatura Significado PCR Reacción en Cadena de la Polimerasa pH Potencial de hidrógeno ppm Partes por millón PUCE Pontificia Universidad Católica del Ecuador RAxML Randomized Axelerated Maximum Likelihood rpm Revoluciones por minuto S Sur Sig Significancia SDS Dodecilsulfato sódico SPSS Statistical Package for the Social Sciences TPH Total Petroleum Hydrocarbon viii TABLA DE CONTENIDO LISTA DE ABREVIATURAS …………………………………..……….…………..….vi 1. RESUMEN …..………………………………………………...…….…………..…….1 2. ABSTRACT ....………………………………………………...…….…………..…….2 3. INTRODUCCIÓN …………………………………………………….………..……..3 3.1 Objetivos ……...……...…………………………………………....…….…6 3.1.1 Objetivo general ...…………...................................……………...…..6 3.1.2 Objetivos específicos .……………...………………..………………..6 4. MATERIALES Y MÉTODOS …………………………………….…….…..……….7 4.1 Sitio de muestreo y colección de muestras vegetales …..……...…..…….…7 4.2 Aislamiento de hongos endófitos de tejido vegetal……...……………...…..7 4.3 Ensayos cualitativos de degradación de petróleo por hongos endófitos..…..8 4.4 Ensayos cuantitativos y evaluación de Hidrocarburos Totales de Petróleo (HTP) y cuantificación mediante Espectroscopia Infrarroja (IR) y Cromatografía de Gases (CG) ………………………………………….………8 4.5 Identificación taxonómica de hongos endófitos ………………....…………9 4.6 Análisis estadístico ………..………………………………………..……..10 5. RESULTADOS …………………………………………………………..…………..11 5.1 Identificación de muestras vegetales …………..………………….………11 5.2 Aislamiento de hongos endófitos de tejido vegetal ……..………...............11 5.3 Ensayos cualitativos de degradación de petróleo por hongos endófitos..…11 5.4 Ensayos cuantitativos y evaluación de Hidrocarburos Totales de Petróleo (HTP) y cuantificación mediante Espectroscopia Infrarroja (IR) y Cromatografía de Gases (CG) ………………………………………….……..12 5.5 Identificación taxonómica de hongos endófitos ……………….………….12 5.6 Análisis estadístico …………………………………………….………….13 6. DISCUSIÓN …………………………………………………………………….……14 6.1 Colección de muestras vegetales ………………………………….………14 6.2 Aislamiento de hongos endófitos y ensayos cualitativos ………...……….15 6.3 Cuantificación de Hidrocarburos Totales de Petróleo (HTP) …...………..16 6.4 identificación taxonómica de hongos endófitos ……………………..……19 7. CONCLUSIONES …………………………………………………………………...23 8. REFERENCIAS …………………..…………………………………………………24 9. FIGURAS ………………………………………………………………….…………32 ix 10. TABLAS …………………………………………………………..………………….37 11. ANEXOS ……………………………………………………………...……….……..45 x LISTA DE FIGURAS Figura 1. Sitio de colección de muestras vegetales. A. Vista de la localidad “Las Minas” desde el nivel de la carretera. B. Suelo contaminado con petróleo crudo. C. Pequeño riachuelo donde se observa el nivel de contaminación del agua de la zona. …………………….………………………………..………………....…...32 Figura 2. Figura 2. Ensayo cualitativo de biodegradación de hidrocarburos. A. Control negativo: donde se observa la apariencia inicial del medio de cultivo producto del petróleo crudo B. Resultado positivo: apariencia del medio de cultivo al producirse la biodegradación de hidrocarburos por un hongo endófito, en este caso se observa la degradación resultante con Xylaria sp. 8, observándose una aclaración total del medio de cultivo después de 10 días de ensayo.………..33 Figura 3. Tasas de remoción de hidrocarburos. Resultados obtenidos tras 30 días de incubación en el medio de cultivo MSM suplementado con petróleo crudo al 1%. Se puede observar los resultados cuantificados por espectroscopia infrarroja en azul (IR) y por cromatografía de gases en rojo (CG) con los 10 y 5 mejores biodegradadores de hidrocarburos respectivamente…....……………..…………34 Figura 4. Árbol filogenético de máxima verosimilitud de las secuencias ITS de hongos endófitos biodegradadores de hidrocarburos. Los aislados se muestran con el código CEQCA (verde), agrupados en sus respectivo ordenes taxonómicos (amarillo). Spizellomyces punctatus fue usado como grupo externo.…………………………………………...………………………...…….35 Figura 5. Ordenes taxonómicos de hongos endófitos biodegradadores de hidrocarburos. Porcentajes de hongos clasificados en cada orden (N=53). El 4% de los endófitos identificados no pudieron asignárseles la categoría taxonómica de orden. Los ordenes con mayor número de aislados Diaporthales (26%) y Xylariales (22%).……………………………………………………………...…36 xi LISTA DE TABLAS Tabla 1. Identificación taxonómica de las plantas colectadas en el campo y número de endófitos aislados por planta………………………………………………………37 Tabla 2. Resultados de bioactividad positiva de hongos endófitos para ensayos cualitativos de biodegradación de hidrocarburos.………………………………………..……..39 Tabla 3. Tasas de biodegradación de hidrocarburos cuantificados por espectroscopia infrarroja (IR) y cromatografía de gases (CG)…………………………………….41 Tabla 4. Cuantificación de hidrocarburos de petróleo persistentes en el medio de cultivo por cromatografía de gases (CG) después de 30 días de incubación con hongos endófitos…………………………………………………………………………...42 Tabla 5. Identificación taxonómica de los 53 aislados fúngicos endófitos biodegradadores de hidrocarburos…………………………………………………………………...43 xii LISTA DE ANEXOS Anexo 1. Cromatogramas resultado de la cuantificación de HTP por cromatografía de gases (CG) de los ensayos cuantitativos. A. Resultados de la muestra control (verde), junto con la muestra blanco de DCM (azul), en rojo se observan los picos con los compuestos identificados. B. Resultados obtenidos con el hongo endófito Clonostachys sp. 2.………………………………………………………………...45 Anexo 2. Resultados ANOVA de un factor con los resultados de la cuantificación de HTP por espectroscopia infrarroja………………………………………………………46 Anexo 3.
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