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Estudio De La Composición De La Comunidad Bacteriana De Manantiales Salinos Ubicados En Los Departamentos De Risaralda Y Boyaca

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Estudio De La Composición De La Comunidad Bacteriana De Manantiales Salinos Ubicados En Los Departamentos De Risaralda Y Boyaca PONTIFICIA UNIVERSIDAD JAVERIANA FACULTAD DE CIENCIAS DOCTORADO EN CIENCIAS BIOLÓGICAS ESTUDIO DE LA COMPOSICIÓN DE LA COMUNIDAD BACTERIANA DE MANANTIALES SALINOS UBICADOS EN LOS DEPARTAMENTOS DE RISARALDA Y BOYACA CAROLINA DIAZ CARDENAS TESIS Presentada como requisito parcial Para optar al título de DOCTOR EN CIENCIAS BIOLÓGICAS Bogotá D.C. 20 de Junio de 2011 NOTA DE ADVERTENCIA "La Universidad no se hace responsable por los conceptos emitidos por sus alumnos en sus trabajos de tesis. Solo velará por que no se publique nada contrario al dogma y a la moral católica y por que las tesis no contengan ataques personales contra persona alguna, antes bien se vea en ellas el anhelo de buscar la verdad y la justicia". Artículo 23 de la Resolución No13 de Julio de 1946. AGRADECIMIENTOS A la Unidad de Saneamiento y Biotecnología Ambiental (USBA) de la Pontifica Universidad Javeriana por facilitarme sus instalaciones e infraestructura para llevar a cabo este trabajo y a todos sus integrantes por compartir estos años, siempre con una calidad sonrisa y generando un buen ambiente de trabajo. Agradezco a mi directora Sandra Baena por sus enseñanzas, paciencia, amistad, confianza, entusiasmo y buenas ideas. A mis compañeros y amigos Luisa Bernal, Carolina Rubiano, Gina López, Habib Yanine y Javier Gómez, por sus aportes en el desarrollo de este trabajo y por su ayuda incondicional. A Colciencias por su apoyo financiero mediante el programa de créditos condonables para el desarrollo de doctorados nacionales. Al Dr. Bharat Patel, por recibirme en su laboratorio y por facilitarme los recursos económicos y logísticos para llevar a cabo parte de este trabajo. Al grupo de Genómica y Bioinformática de Ambientes Extremos – GEBIX, por su aporte en el proceso de pirosecuenciación y análisis de secuencias. Al geólogo Michael Tistl y a Claudia Alfaro, por proporcionarme información relacionada con las características fisicoquímicas y geológicas de los manantiales salinos. A mis padres, Marleny Cárdenas y Aberlado Díaz, por haberme dado la oportunidad de tener la educación que deseaba, por su ejemplo de perseverancia y compromiso y por su apoyo incondicional en todos los aspectos de mi vida. A mi hermana Ximena Díaz y a Alejandro Reyes por su paciencia, ánimo y colaboración. A todos mis amigos, quienes de una u otra forma me acompañaron durante este proceso, siempre con palabras de aliento y buena energía. RESUMEN En este estudio se determinó la composición de las comunidades bacterianas de dos manantiales salinos ubicados en el departamento de Risaralda, el Salado de Consotá y La Cristalina y uno ubicado en el departamento de Boyacá, denominado Salpa. Los tres manantiales, importantes desde una perspectiva histórica y económica, fluyen a partir de aguas subterráneas con más de 50 años antigüedad y pueden diferenciarse a partir de sus características fisicoquímicas. Aunque no se detectó diferencias significativas en la abundancia y composición de organismos del dominio Bacteria entre los tres manantiales a lo largo del tiempo, la composición de la comunidad bacteriana sí presentó variaciones espaciales, indicando que cada manantial alberga una comunidad bacteriana diferente, esta variación fue explicada en parte por los parámetros fisicoquímicos estudiados y principalmente por la concentración del ion calcio y de la temperatura. A pesar de las diferencias al interior de la comunidad bacteriana de cada manantial, se observó predominio de OTUs relacionadas con organismos de las clases y α-Proteobacteria, ampliamente reportadas en hábitats salinos incluyendo ambientes marinos, aunque en el manantial Salpa también se detectó predominio de OTUs relacionadas con organismos de la clase β-Proteobacteria. Además, se observó que estos manantiales son hábitats de nueva diversidad taxonómica como se deduce del alto número de secuencias del 16S ARNr con menos del 97% de similitud con secuencias de organismos cultivados y por las nuevas especies aisladas e identificadas en el curso de este estudio, las cuales además presentaron capacidades hidrolíticas que pueden ser exploradas más adelante. ABSTRACT Our study determined the composition of bacterial communities in two salt springs located in the department of Risaralda, the Salado de Consotá and La Cristalina and one located in the department of Boyacá, called Salpa. The three springs, which are important from a historical and economic perspective, flow from groundwaters that are more than 50 years old and can be recognized by their physicochemical characteristics. Although no significant differences were detected among the abundance and composition of organisms of the domain Bacteria from the three springs along time, the bacterial community composition did present spatial variations, indicating that each spring hosts a different microbial community; this variation was partly explained by physicochemical parameters and mainly by the calcium ion concentration and temperature. Despite the differences within the bacterial community of each spring, there was a predominance of OTUs related with organisms of the and α-Proteobacteria, widely reported in saline habitats including marine environments; in addition in Salpa spring β-Proteobacteria class was also detected. We also found that these springs are habitats of a new undescribed taxonomic diversity as reveled by the high number of 16S rRNA sequences with less than 97% similarity to sequences of cultured organisms, and by new species isolated and identified in the course of this study, which also showed hydrolytic properties that can be explored in further studies. TABLA DE CONTENIDO Página 1. INTRODUCCIÓN 1 2. MARCO TEÓRICO 4 2.1 Diversidad biológica 4 2.2 Estudio de comunidades microbianas en ambientes naturales 4 2.2.1 Métodos independientes de cultivo 5 2.2.2 Métodos dependientes de cultivo 9 2.2.2.1 Identificación taxonómica de los microorganismos cultivados 10 2.2.2.1.1 Caracterización fenotípica 10 2.2.2.1.2 Características genotípicas 11 2.3 Concepto de especie procariota 13 2.4 Ambientes acuáticos salinos 15 2.5 Comunidades microbianas en sistemas acuáticos salinos 17 2.6 Factores que afectan la composición de las comunidades microbianas en ambientes salinos 21 2.7 Importancia del estudio de la composición de las comunidades microbianas en ambientes salinos 22 3 OBJETIVOS DE INVESTIGACIÓN 25 4 MATERIALES Y MÉTODOS 26 4.1 Descripción de los sitios de estudio 26 4.1.1 Descripción de los manantiales del departamento de Risaralda 26 4.1.1.1 Manantial Salado de Consotá (SC) 26 4.1.1.2 La Cristalina (LC) 30 4.1.1.3 Generalidades hidrológicas y geológicas de la zona aledaña a los Manantiales SC y LC 33 4.1.2 Descripción del manantial Salpa (SP) ubicado en el departamento de Boyacá 34 4.1.2.1 Generalidades hidrológicas y geológicas de la zona aledaña al manantial SP 35 4.2 Muestreos y mediciones 37 4.3 Caracterización de la composición química del agua de los manantiales 39 4.3.1 Análisis de las variables fisicoquímicas 39 4.4 Composición isotópica del agua 40 4.5 Caracterización de la composición de las comunidades microbianas de los manantiales salinos 41 4.5.1 Características morfológicas de las comunidades microbianas presentes en los manantiales salinos SC y SP 41 4.5.2 Determinación del número total de células microbianas y detección de grupos microbianos particulares 43 4.5.2.1 Recuento total de células 43 4.5.2.2 Detección selectiva de grupos microbianos particulares usando la técnica de FISH 44 4.5.2.3 Recuento de células 46 4.5.3 Generación de inventarios biológicos a partir del aislamiento de microorganismos halotolerantes y/o halófilos aerobios y anaerobios heterotróficos 47 4.5.3.1 Inoculación de las series de cultivo 49 4.5.3.2 Aislamiento de cepas microbianas 49 4.5.3.3 Conservación de organismos aislados 50 4.5.3.4 Estudio polifásico de las cepas aisladas 50 4.5.3.4.1 Caracterización fenotípica 51 4.5.3.4.2 Caracterización genotípica 60 4.5.3.4.3 Detección de producción de enzimas hidrolíticas y producción de polihidroxialcanoatos (PHA) 65 4.5.4 Caracterización de la composición microbiana usando métodos independientes de cultivo 69 4.5.4.1 Extracción de ADN comunitario 69 4.5.4.1.1 Cuantificación de ácidos nucleicos 70 4.5.4.2 Clonación y secuenciación del gen que codifica para la sub-unidad pequeña del 16S ARNr de muestras del manantial SC y SP 70 4.5.4.3 Pirosecuenciación 454 de la región V5-V6 del gen codifica para el 16S ARNr del manantial LC 72 4.5.4.4 Análisis computacional de las secuencias del 16S ARNr 73 4.5.4.4.1 Alineamiento de las secuencias del 16S ARNr 74 4.5.4.4.2 Análisis filogenético de las secuencias del 16S ARNr 74 4.5.4.4.3 Agrupación de las secuencias en Unidades Taxonómicas Operacionales (OTUs) 75 4.5.4.4.4 Comparación filogenética de los taxones obtenidos por clonación 75 4.5.4.4.5 Análisis de las secuencias obtenidas por pirosecuenciación 75 4.5.5 Determinación de variaciones espaciales y temporales de las comunidades bacterianas dominantes de los manantiales salinos 76 4.5.5.1 Electroforesis en gen con gradiente desnaturalizante (DGGE) 76 4.5.5.1.1 Identificación de los grupos taxonómicos más abundantes de la comunidad bacteriana a partir de DGGE 77 4.5.5.1.2 Análisis estadístico del patrón de bandas de DGGE 78 5. RESULTADOS Y DISCUSIÓN 80 5.1 Caracterización de la composición fisicoquímica del agua de los manantiales salinos 80 5.1.1 Determinación de las diferencias en los parámetros fisicoquímicos entre los diferentes sitios de muestreo 82 5.1.2 Análisis de Componentes Principales (ACP) 83 5.2 Composición isotópica
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