Microbial communities in high altitude altiplanic wetlands in northern Chile: phylogeny, diversity and function DISSERTATION Zur Erlangung des Doktorgrades der Mathematisch-Naturwissenchaflichen Fakultät der Christian-Albrechts-Universität zu Kiel vorgelegt von Cristina Inés Dorador Ortiz aus Antofagasta, Chile Max Planck Institut für Evolutionsbiologie, Plön IFM-GEOMAR, Kiel Mai 2007 Referent: Prof. Dr. Johannes F. Imhoff Korreferent: Prof. Dr. Irma Vila Tag der mündlichen Prüfung: 06. Juli 2007 Zum Druck genehmigt: 06. Juli 2007 Der Dekan The work of this thesis was conducted between April 2004 and Mai 2007 in the MPIL (now Max Planck Institute for Evolutionary Biology) in Plön under the supervision of Dr. Karl-Paul Witzel and at Leibniz Institute for Marine Sciences (IFM-GEOMAR) in Kiel under the supervision of Prof. Dr. Johannes F. Imhoff. I received funds from the Deutscher Akademischer Austausch Dienst (DAAD) for the realization of this thesis. TABLE OF CONTENTS Summary ……………………………………………………………………………….1 Zusammenfassung ……………………………………………………………………...2 Resumen ………………………………………………………………………………..3 Thesis outline …………………………………………………………………………..5 1. Introduction ………………………………………………………………………….6 2. Materials and Methods ……………………………………………………………..22 3. Comparative analysis of bacterial and archaeal communities in different high altitude wetlands in Northern Chile ……………………………………………………………35 4. Diversity of Archaea in environmental samples from Salar de Huasco ……………58 5. Diversity and composition of photosynthetic bacterial communities in Salar de Huasco …………………………………………………………………………………74 6. Salt tolerance of enrichment cultures of ammonia oxidizing bacteria from Salar de Huasco ………………………………………………………………………………..102 7. Molecular analysis of halophilic bacteria isolates from Salar de Huasco …………115 8. Discussion …………………………………………………………………………124 Conclusion ……………………………………………………………………………133 Individual scientific contributions to multiple-author publications ………………….134 References ……………………………………………………………………………136 Acknowledgements …………………………………………………………………..163 Curriculum Vitae ……………………………………………………………………..165 Erklärung ……………………………………………………………………………..166 Appendix ……………………………………………………………………………..167 Summary SUMMARY The phylogeny, diversity and function of microbial communities from several altiplanic wetlands was examined using an array of different but complimentary techniques. Results highlighted that microbial diversity exhibited a specific pattern in each wetland. Bacteria were dominant over Archaea in both freshwater and saline systems. Bacterial and archaeal diversity were both higher in sediment than in water samples. Lago Chungará, Laguna de Piacota and Bofedal de Parinacota are freshwater wetlands located at high altitude (>4400 m) in the north of Chile. They support microbial communities closely related to psychrophilic bacteria (e.g. Psychrobacter sp., Pseudomonas congelans, Flavobacterium psychrolimnae) in water and Proteobacteria and Actinobacteria in sediment samples. Salar de Huasco and Salar de Ascotán are located further south at an altitude of 3800 m and exhibit a wide range of salinities (varying between freshwater to 120 gL-1 of total dissolved salts). Microbial communities in these sites were characterized by bacteria tolerant to salt (e.g. halophilic Bacteria: Halomonas sp., halophilic Archaea: Halorubrum sp.). Cytophaga-Flavobacteria- Bacteroidetes was the most frequent group reported at the sites. In-depth studies focussing on the Salar de Huasco revealed a particular diversity of Archaea, characterized by a number of sequences related to uncultured groups and ammonia-oxidizing Crenarchaeota. Cyanobacteria from the Salar de Huasco were closely related to Cyanobacteria previously described from Antarctica. Isolates of halophilic bacteria and phototrophic bacteria displayed an elevated tolerance to different salt concentrations. The particular microbial diversity found in high altitude wetlands provides a new and exciting area of research. 1 Zusammenfassung ZUSAMMENFASSUNG Die Phylogenie, Diversität und Funktion von mikrobiellen Gemeinschaften aus verschiedenen altiplanischen Feuchtgebieten wurde mit mehreren Techniken untersucht. Die Ergebnisse zeigen dass an jedem Standort die mikrobielle Diversität eine spezifische Struktur hat. Bakterien und Archaeen Diversität war höher im Sediment als in den Wasserproben. Lago Chungará, Laguna de Piacota und Bofedal de Parinacota sind auf über 4400 Metern Höhe gelegene Süßwasser-Feuchtgebiete in Nord-Chile. Die mikrobiellen Gemeinschaften waren im Wasser verschiedenen psychrophilen Bakterien (z. B. Psychrobacter sp., Pseudomonas congelans, Flavobacterium psychrolimnae) und im Sediment Proteobakterien und Actinobakterien ähnlich. Salar de Huasco und Salar de Ascotán liegen südlich auf 3800 m Höhe und die Salinität des Wassers schwankt in den verschiedenen Habitaten von Süßwasser bis 120 gL-1 Salz gelöst im Wasser. Die mikrobielle Diversität an diesen Standorten ist charakterisiert durch salztolerante Bakterien (z.B. halophile Bakterien der Gattung Halomonas und halophile Archaeen der Gattung Halorubrum. Vertreter der CFB-Gruppe (Cytophaga-Flavobacterium- Bacteroidetes) waren in den Klonbibliotheken häufig. Im Salar de Huasco war die Diversität von Archaeen besonders hoch, und es wurden zahlreiche Sequenzen gefunden, die nicht kultivierten Archaeen und Ammoniak-oxidierenden Crenarchaeota ähnlich waren. Die Cyanobacterien aus dem Salar de Huasco waren sehr ähnlich mit Cyanobacterien aus der Antarktis. Isolate von halophilen Bakterien und phototrophen Bakterien zeigten eine hohe Toleranz gegenüber verschiedenen Salzkonzentrationen. Die besondere mikrobielle Diversität dieser Feuchtgebiete im Hochland der Anden bietet ein neues und spannendes Forschungsgebiet. 2 Resumen RESUMEN La filogenia, diversidad y función de las comunidades microbianas fueron estudiadas en varios humedales altiplánicos con técnicas distintas y complementarias. Los resultados señalan que la diversidad microbiana exhibe un patrón específico en cada humedal. Bacteria fue dominante sobre Archaea en sistemas de agua dulce y salinos. La diversidad de Bacteria y Archaea fue mayor en las muestras de sedimento que en las de agua. El lago Chungará, la laguna de Piacota y el Bofedal de Parinacota son humedales de agua dulce ubicados a gran altura (>4400 m de altitud) en el norte de Chile. Estos sistemas contienen comunidades microbianas altamente relacionadas con bacterias psicrófilas en agua (por ejemplo, Psychrobacter sp., Pseudomonas congelans, Flavobacterium psychrolimnae) y en sedimentos Proteobacteria y Actinobacteria. El salar de Huasco y el salar de Ascotán están ubicados hacia el sur a una altura de 3800 m de altitud y muestra un amplio rango de salinidad (desde agua dulce hasta 120 gL-1 de sales totales disueltas). Las comunidades microbianas en estos sitios están caracterizadas por ser tolerantes a la sal (por ejemplo, Bacteria halófila: Halomonas sp., Archaea halófila: Halorubrum sp.). Cytophaga-Flavobacteria-Bacteroidetes fue el grupo más frecuente en los sitios estudiados. Los estudios realizados en el Salar de Huasco revelaron una diversidad particular de Archaea caracterizada por un número de secuencias altamente relacionadas con grupos no cultivados y con Crenarchaea oxidadoras de amonio. Las cianobacterias del Salar de Huasco presentaron altas similitudes con las cianobacterias previamente descritas en la Antártida. Los aislados de bacterias halófilas y bacterias fototrófas muestran una alta tolerancia a la sal a distintas concentraciones. La particular 3 Resumen diversidad microbiana encontrada en humedales altoandinos proporciona una nueva e interesante área de investigación. 4 Thesis outline THESIS OUTLINE Here, I use PCR-DGGE as a fingerprinting tool to compare microbial patterns from samples and clone libraries of 16S rRNA gene and amoA gene (Archaea and Bacteria) to infer phylogenetic relationships between the sequences. Bacteria and archaeal communities of Lago Chungará, Laguna de Piacota, Bofedal de Parinacota, Salar de Huasco and Salar de Ascotán are described using clone libraries of 16S rRNA gene (Chapter 3). Chapter 4 focuses on four sites of the Salar de Huasco and describes the composition of archaeal communities and the presence of archaeal amoA. Chapter 5 describes diversity and composition of photosynthetic bacteria including Cyanobacteria and phototrophic bacteria. Chapter 6 reports salt-tolerant enrichment cultures of ammonia oxidizing bacteria using 16S rRNA gene and bacterial amoA gene. Chapter 7 reports the growth of isolates of halophilic Bacteria at moderate and high concentrations of salt. 5 Introduction 1. INTRODUCTION The aim of this thesis was the study of several diverse aspects of the microbial communities of wetlands located in the Chilean Altiplano, including phylogeny, diversity and function. The wetlands are located in the Altiplano, a high altitude plateau of the Andes mountain range. 1.1 Features of the Altiplano 1.1.1 Geomorphology The north of Chile exhibits a particular and extreme relief. There is a 15000 m difference between the marine Atacama Trench in the Pacific Ocean (maximum depth 8065 m) and the summit of the Llullaillaco volcano (6723 m altitude) in the Andes, over a linear distance of less than 300 km. Within this region, several distinct morphostructural units have been identified, running from the west to the east (Chong, 1984; Charrier and Muñoz, 1997; Risacher et al., 2003): i) the Coast Range: a mountain chain (mean altitude 1500 m) located in the west,