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Caracterización Y Tratamiento Fotocatalítico De Hongos Y Bacterias De Aire Interior

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Caracterización Y Tratamiento Fotocatalítico De Hongos Y Bacterias De Aire Interior UNIVERSIDAD AUTÓNOMA DE MADRID FACULTAD DE CIENCIAS DEPARTAMENTO DE BIOLOGÍA MOLECULAR CARACTERIZACIÓN Y TRATAMIENTO FOTOCATALÍTICO DE HONGOS Y BACTERIAS DE AIRE INTERIOR TESIS DOCTORAL MARTA SÁNCHEZ MUÑOZ MADRID, 2013 FACULTAD DE CIENCIAS DEPARTAMENTO DE BIOLOGÍA MOLECULAR CARACTERIZACIÓN Y TRATAMIENTO FOTOCATALÍTICO DE HONGOS Y BACTERIAS DE AIRE INTERIOR Memoria presentada por Marta Sánchez Muñoz para optar al grado de Doctor en Ciencias por la Universidad Autónoma de Madrid. Junio de 2013 Director de Tesis: Benigno Sánchez Cabrero Tutor: Ricardo Amils Pibernat CIEMAT (Centro de Investigaciones Energéticas Medioambientales y Tecnológicas) Centro de Biología Molecular Severo Ochoa A mi madre, Mamá, Chelo o Consuelito para sus grandes amigos, Esta tesis finaliza hoy gracias a tu insistencia y apoyo Gracias por ese último aliento que me llenó de fuerza para seguir adelante AGRADECIMIENTOS A mi padre y mi hermano Javi, por estar siempre a mi lado ofreciéndome calor familiar y apoyo en mi carrera profesional. A mi yayo, y a toda mi gran familia de tíos y primos. Al doctor Aldo Gónzalez, por abrirme las puertas de su laboratorio y haberme enseñado tanto sobre hongos, gracias por compartir conmigo todas aquellas “horas y horas” de microscopio. Al doctor Ricardo Amils por haber confiado en mí y haberme dado su apoyo en todo momento. Muchas gracias a los dos por supervisar mi trabajo y proporcionarme los medios que necesitaba para llevarlo a cabo. Al grupo de Aplicaciones Medioambientales de la Radiación Solar en Aire en el CIEMAT, liderado por Benigno Sánchez, por haberme acogido en su seno todos estos años y haber abierto mis ojos al mundo del aire interior. Gracias a todos los que comparten o compartieron su día a día conmigo y no han dudado en ayudarme a resolver dudas e incidencias. Gracias Silvia, Loli, Juan Coronado, Raquel Portela e Ingrid. Gracias a Rafa, Raúl y Gloria por ayudarme en mi trabajo diario. Quiero agradecer también al doctor Guillermo Cobas, que empezó conmigo, todas las lecciones que me dio sobre la vida y el seguir preocupándose por mí después de tanto tiempo y a Emiliano E. Díaz, porque me ayudó a sentar las bases en aquellos primeros días. Por supuesto, no me olvido de María Muñoz, que trabajó codo a codo conmigo todos estos años, por querer aprender de mi y enseñarme tanto al mismo tiempo. A todos los “becarios” del CIEMAT, que han sido mi familia todo este tiempo, gracias por esas comidas aliñadas con carcajadas en el comedor y los “findes rurales”. Entre ellos, los chicos del despacho de enfrente: Jose y Sofía, gracias por hacerme reír y por escucharme tantas veces, y por supuesto también a Fran y Víctor. A los chicos de la planta baja del 42, a David, Alberto, por vuestra compañía y afecto, y en especial a Pepín por estar siempre el primero para ofrecerme su mano y levantarme con cada tropiezo, gracias por toda tu ayuda. A Bea, y a las chicas del otro lado de la carretera: Cecilia, Ana, Vane, Natalia y Mary, por vuestra alegría contagiosa. A los que ya se fueron: Juan, Nairoby, Cristóbal, Irene, Henar, Roberto, porque seguís formando parte de mi vida a pesar de la distancia. A toda la gente que conocí en el CBM que no dudó en hacerme allí un hueco, aconsejarme y ayudarme como Carlotta Vizioli, Nuria 2, Irma Marín y José Luis Sanz. Gracias a Mónica Oggerin y a Nuria Rodríguez por su colaboración en la identificación de mis muestras bacterianas y en especial quiero agradecer a Catalina del Moral y Diego Sánchez su gran trabajo en el análisis de las regiones ITS de mis muestras de hongos. A Juan Carlos Villar, y a su grupo de Celulosas y papel del departamento de Productos forestales del INIA, por permitirme entrar libremente en sus instalaciones y utilizar sus microscopios. A mis amigos de Villalba, Dani y Sonsoles y a las biólogas, Aurora, Silvia, Sandra, Helena, Ana, que se formaron conmigo y estarán siempre a mi lado haciendo piña. A Francesca y Luna, por la compañía y cariño que me dan en casa y a Rubén, por animarme a convertirme en doctora. Finalmente, quiero agradecer a Fernando Feldman y la empresa AIRE LIMPIO por habernos permitido desarrollar y ensayar un primer prototipo de fotorreactor a escala de demostración con el que poner en práctica todo lo aprendido en este trabajo. INDICE ABSTRACT 1.INTRODUCCIÓN 1.1. CALIDAD DE AIRE INTERIOR ............................................................... - 1 - 1.1.1. Contaminantes de aire interior ...................................................................................................- 2 - 1.1.2. Normativa........................................................................................................................................- 3 - 1.2. AEROBIOLOGÍA. HONGOS Y BACTERIAS DE AIRE INTERIOR .......................... - 4 - 1.2.1. Hongos de aire interior ................................................................................................................- 5 - 1.2.2 Bacterias de aire interior ............................................................................................................- 7 - 1.3. MÉTODOS DE MUESTREO DE MICROORGANISMOS EN AIRE ........................... - 8 - 1.3.1 Muestreo por sedimentación pasiva ...........................................................................................- 9 - 1.3.2 Muestreadores inerciales ............................................................................................................- 9 - 1.3.3. Muestreadores no inerciales .................................................................................................... - 10 - 1.4. TRATAMIENTO DE AIRE. MÉTODOS EXISTENTES. ..................................... - 11 - 1.4.1 Filtración ........................................................................................................................................- 11 - 1.4.2. Radiación ultravioleta (UV)........................................................................................................- 11 - 1.4.3. Purificadores de aire electrónicos...........................................................................................- 11 - 1.4.4 Ozonización ................................................................................................................................... - 12 - 1.4.5. Control de la presurización....................................................................................................... - 12 - 1.5. PROCESOS DE OXIDACIÓN AVANZADA. FUNDAMENTOS DE FOTOCATÁLISIS . ..... - 12 - 1.5.1. Fotocatálisis como sistema de desinfección.......................................................................... - 15 - 1.5.2. Dióxido de titanio ....................................................................................................................... - 17 - 1.5.3. Catalizadores soportados.......................................................................................................... - 17 - 1.5.4. Reactores fotocatalíticos para el tratamiento de bioaerosoles ...................................... - 19 - 2.OBJETIVOS 3.METODOLOGÍA 3.1. MEDIOS DE CULTIVO ....................................................................... - 23 - 3.1.1. Medios para el crecimiento de bacterias...............................................................................- 23 - 3.1.2. Medios para el crecimiento de hongos ...................................................................................- 24 - 3.2. SOLUCIONES, TAMPONES, ADITIVOS .................................................... - 26 - 3.3. MÉTODOS DE TOMA DE MUESTRA DE AIRE ............................................. - 26 - 3.3.1. Métodos ensayados para la puesta a punto de una metodología de toma de muestra de aire............................................................................................................................................................- 26 - 3.3.2. Muestreadores de aire..............................................................................................................- 26 - 3.3.3. Conteo de unidades formadoras de colonia y correcciones realizadas tras el uso de muestreadores por impacto en placa.................................................................................................- 30 - 3.3.4. Aplicación de la norma UNE 171330-2:2009 para el cálculo del número de puntos de muestreos................................................................................................................................................- 30 - 3.4. CARACTERIZACIÓN DE HONGOS Y BACTERIAS DE AIRE INTERIOR .................. - 31 - 3.4.1. Cultivo y aislamiento de hongos ............................................................................................... - 31 - 3.4.2. Identificación de hongos filamentosos y levaduras por métodos no moleculares ....... - 31 - 3.4.3. Caracterización de bacterias ..................................................................................................- 32 - 3.4.4. Muestras bacterianas recogidas mediante medio de cultivo............................................- 33 - 3.4.5. Muestras bacterianas recogidas mediante filtros .............................................................- 33 - 3.4.6. Muestras bacterianas recogidas mediante solución isotónica o PBS..............................- 34 - 3.4.7. Extracción de ADN de hongos filamentosos y levaduras. Caracterización de hongos por métodos moleculares.............................................................................................................................- 34 - 3.4.8. Reacción
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