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Universidad Politécnica De Valencia UNIVERSIDAD POLITÉCNICA DE VALENCIA ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS DIVERSIDAD DE ACTINOMICETOS NOCARDIOFORMES PRODUCTORES DE ESPUMAS BIOLÓGICAS AISLADOS DE PLANTAS DEPURADORAS DE AGUAS RESIDUALES DE LA COMUNIDAD VALENCIANA TRABAJO FINAL DE CARRERA INGENIERO AGRÓNOMO PRESENTADO POR: ALBERT SOLER HERNÁNDEZ DIRIGIDO POR: GONZALO CUESTA AMAT JOSE LUIS ALONSO MOLINA VALENCIA, JULIO DE 2008 ESCUELA TÉCNICA Anexo 4 SUPERIOR DE INGENIEROS Autorización del director/a, AGRÓNOMOS codirector/a o tutor/a Datos del trabajo de fin de carrera Autor: Albert Soler Hernández DNI: 22584259-F Título: DIVERSIDAD DE ACTINOMICETOS NOCARDIOFORMES PRODUCTORES DE ESPUMAS BIOLÓGICAS AISLADOS DE PLANTAS DEPURADORAS DE AGUAS RESIDUALES DE LA COMUNIDAD VALENCIANA Área o áreas de conocimiento a las que corresponde el trabajo: Microbiología y Medio Ambiente Titulación: Ingeniero Agrónomo A cumplimentar por el director/a, codirector/a o tutor/a del trabajo Nombre y apellidos: Gonzalo Cuesta Amat Departamento: Biotecnología En calidad de: X director/a codirector/a tutor/a Autorizo la presentación del trabajo de fin de carrera cuyos datos figuran en el apartado anterior y certifico que se adecua plenamente a los requisitos formales, metodológicos y de contenido exigidos a un trabajo de fin de carrera, de acuerdo con la normativa aplicable en la ETSIA. (Firma)* Gonzalo Cuesta Amat Jose Luis Alonso Molina Valencia, 21 de Julio de 2008 En el caso de codirección, han de firmar necesariamente los que sean profesores de esta Escuela; si existe tutor o tutora, tiene que ser éste quien firme esta autorización. DIRECCIÓN DE LA ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS ESCUELA TÉCNICA Anexo 5 SUPERIOR DE INGENIEROS Ficha resumen del Trabajo Fin de AGRÓNOMOS Carrera Datos personales Nombre y apellidos: Albert Soler Hernández Datos del trabajo de fin de carrera Título del TFC: DIVERSIDAD DE ACTINOMICETOS NOCARDIOFORMES PRODUCTORES DE ESPUMAS BIOLÓGICAS AISLADOS DE PLANTAS DEPURADORAS DE AGUAS RESIDUALES DE LA COMUNIDAD VALENCIANA Lugar de realización: ETSIA, Departamento de Biotecnología Fecha de lectura: Septiembre 2008 Titulación: Ingeniero Agrónomo Especialidad: Recursos Naturales y Medio Ambiente Director/a: Gonzalo Cuesta Amat Codirector/a: Jose Luis Alonso Molina Resumen La formación de espumas de origen biológico en la superficie de los reactores y de los clarificadores secundarios en las Estaciones Depuradoras de Aguas Residuales (EDARs) es uno de los problemas de separación de sólidos más importantes. Entre las bacterias responsables de estas espumas se encuentran los actinomicetos nocardioformes que contienen ácidos micólicos (mycolata) que pertenecen al phylum Actinobacteria. Este grupo de microorganismos contiene especies filamentosas que causan problemas de formación de espumas biológicas (“foaming”) en plantas de tratamiento de aguas residuales con sistemas de fangos activos. Estas espumas interfieren en el proceso de depuración de depuración de aguas residuales, ya que retienen hasta el 40% de los sólidos en suspensión. La presencia de especies patógenas en este grupo de microorganismos implica un riesgo para la salud pública y, por lo tanto, la necesidad de detectar estas especies. En el presente trabajo se han investigado varias EDARs de la Comunidad Valenciana para identificar a nivel de especie las bacterias causantes de estas espumas biológicas. Para ello se han utilizado cuatro técnicas: caracterización morfológica, caracterización quimiotaxonómica, caracterización genotípica y caracterización fenotípica. En la caracterización morfológica se realizó un estudio micro y macroscópico de los asilados. Para caracterizar quimiotaxonómicamente los asilados se llevaron a cabo tres marcadores quimiotaxonómicos: extracción de ácidos micólicos, extracción de los isómeros del ácido diaminopimélico y extracción de los azúcares predominantes de la pared celular. En la caracterización genotípica se llevó a cabo la extracción del DNA de los aislados seleccionados y la amplificación por PCR del 16S rDNA. Los productos de PCR se purificaron y se enviaron a secuenciar. Las secuencias se analizaron mediante los programas BLAST, CHROMAS y PHYDIT. Posteriormente se realizaron árboles filogenéticos y matrices de similaridad. En la caracterización fenotípica se realizaron una serie de pruebas para completar los resultados obtenidos. Las pruebas se basaron en tests de tolerancia a diferentes temperaturas, tests de degradación y tests en donde se hacía crecer a los aislados con diferentes fuentes de carbono y/o nitrógeno. De los 25 aislados seleccionados el 48% pertenece al género Gordonia, el 36% al género Tsukamurella, el 8% al género Dietzia, el 4% al género Rhodococcus y el último 4% al género Mycobacterium. Todas ellas presentaban ácidos micólicos, forma meso-DAP y arabinosa y galactosa como azúcares predominantes. Con ello demostramos que en las EDARs de la Comunidad Valenciana existe una amplia diversidad de especies formadoras de espumas biológicas. Palabras clave Fangos activos, foaming, mycolata, BLAST, PCR, Gordonia, Rhodococcus, Dietzia, Tsukamurella, Mycobacterium Resum La formació de bromeres d'origen biològic en la superfície dels reactors i dels clarificadors secundaris en les estacions Depuradores d'Aigües Residuals (EDARs) és un dels problemes de separació de sòlids més importants. Entre els bacteris responsables d'estes bromeres es troben els actinomicets nocardioformes que contenen àcids micòlics (mycolata) que pertanyen al phylum Actinobacteria. Este grup de microorganismes conté espècies filamentoses que causen problemes de formació de bromeres biològiques (“foaming”) en plantes de tractament d'aigües residuals amb sistemes de fangs actius. Estes bromeres interferixen en el procés de depuració de depuració d'aigües residuals, ja que retenen fins al 40% dels sòlids en suspensió. La presència d'espècies patògenes en este grup de microorganismes implica un risc per a la salut pública i, per tant, la necessitat de detectar estes espècies. En el present treball s'han investigat diverses EDARs de la Comunitat Valenciana per a identificar a nivell d'espècie els bacteris causants d'estes bromeres biològiques. Per a això s'han utilitzat quatre tècniques: caracterització morfològica, caracterització quimiotaxonòmica, caracterització genotípica i caracterització fenotípica. En la caracterització morfològica es va realitzar un estudi micro i macroscòpic dels aïllats. Per a caracteritzar quimiotaxonòmicament els aïllats es van dur a terme tres marcadors quimiotaxonòmics: extracció d'àcids micòlics, extracció dels isòmers de l'àcid diaminopimèlic i extracció dels sucres predominants de la paret cel·lular. En la caracterització genotípica es va dur a terme l'extracció del ADN dels aïllats seleccionats i l'amplificació per PCR del 16S rDNA. Els productes de PCR es van purificar i es van enviar a seqüenciar. Les seqüències es van analitzar per mitjà dels programes BLAST, CHROMAS i PHYDIT. Posteriorment es van realitzar arbres filogenètics i matrius de similaritat. En la caracterització fenotípica es van realitzar una sèrie de proves per a completar els resultats obtinguts. Les proves es van basar en tests de tolerància a diferents temperatures, tests de degradació i tests on es feia créixer als aïllats amb diferents fonts de carboni i/o nitrogen. Dels 25 aïllats seleccionats el 48% pertany al gènere Gordonia, el 36% al gènere Tsukamurella, el 8% al gènere Dietzia, el 4% al gènere Rhodococcus i l'últim 4% al gènere Mycobacterium. Tots ells presentaven àcids micòlics, forma meso-DAP i arabinosa i galactosa com a sucres predominants. Amb això demostrem que en les EDARs de la Comunitat Valenciana hi ha una àmplia diversitat d'espècies formadores de bromeres biològiques. Paraules clau Fangs actius, foaming, mycolata, BLAST, PCR, Gordonia, Rhodococcus, Dietzia, Tsukamurella, Mycobacterium Abstract The formation of thick viscous scums layers in activated sludge tanks and secondary clarifiers of wastewater treatment plants is one of the most important problems of solid separation. The most significant filamentous bacteria of foaming are nocardioform actinomycetes (mycolata), which belong to phylum Actinobacteria. That group of microorganisms has filamentous species that causes foaming in wastewater treatment plants. These foams interfere in that process because retain until 40% of suspension solids. The presence of pathogen species in that group is a danger to public health. In this study had been study a lot of wastewater treatment plants of the Comunidad Valenciana to identificate the species that causes foaming. We have been use four procedures: morphologic, chemotaxonomy, genotypic and phenotypic characterization. In the morphologic characterization we did a micro and macro study of the isolates. In the chemotaxonomy characterization we did micolic acids extraction, DAP extraction and Whole-cell sugar. In the genotypic characterization we did the DNA extraction and PCR amplification of the 16s rDNA. The sequences were analysed trough BLAST, CHROMAS and PHYDIT programs. Later we did the philogenetic trees and the similarity values. In the phenotypic characterization we did a lot of tests to complete the results (tolerance tests and degradation tests). 48% of isolates belongs to the Gordona genus, 36% belongs to the Tsukamurella genus, 8% belongs to the Dietzia genus, 4% to the Rhodococcus genus and the last 4% belongs to the Mycobacterium genus. All of these had micolic acids, meso-Dap form and galactose and arabinose. In conclusion, the results
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