Diversity of Microbial Habitats at Marine Methane Seeps
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Microbial community ecology of marine methane seeps Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften - Dr. rer. nat.- Dem Fachbereich 2 Biologie/Chemie der Universität Bremen vorgelegt von S. Emil Ruff Bremen, November 2013 1. Gutachter: Prof. Dr. Antje Boetius 2. Gutachter: Prof. Dr. Rudolf Amann 1. Prüfer: Prof. Dr. Ulrich Fischer 2. Prüfer: Dr. Katrin Knittel « You know, if I could have my time again, I think I would be a microbial ecologist. » Edward O. Wilson Summary The detailed investigation of microbial communities, e.g. of soil or hydrothermal vent ecosystems, greatly improved our understanding of the diversity, habitat preferences and functions of microorganisms and their impact on global element cycles. The aim of this thesis was a detailed analysis of the diversity, abundance and distribution of micoorganisms at marine methane seeps and the mechanisms that govern community assembly at these sites. The seep ecosystems were investigated using geochemical analyses, gene libraries, pyrosequencing, community fingerprinting and fluorescence in situ hybridization. Cold seep ecosystems hosted distinct microbial communities that differed from those of the surrounding seabed and were unique microbial habitat patches in the deep sea. The communities also greatly differed between seeps, covered broad ranges of richness and evenness and showed high degrees of endemism. However, despite the differences all seeps were inhabited by certain organisms – the cold seep microbiome - including key functional clades of anaerobic methane oxidizing archaea (ANME) and sulfate-reducing bacteria. Additionally, aerobic methanotrophs and thiotrophs were found at all seeps where oxygen was present. These key functional clades seemed to be influenced by environmental parameters, such as temperature, fluid flux, sediment depth and faunal activity. Bioirrigation by ampharetid tubeworms, for instance, created a habitat for aerobic Methylococcales, whereas vesicomyid clams seemed to favor the establishment of the clade ANME-2c. Thus, niche-based processes played an important role for the community assembly at seep ecosystems. However, most of the seeps seemed to be clearly dominated by a few, globally distributed operational taxonomic units at 97% 16S rRNA gene identity (OTU0.03) of each key functional clade. Some of these OTU0.03 were rare at some seep ecosystems and abundant at others. Moreover, some findings suggested that rare organisms became abundant because the environmental conditions at the seep changed supporting the importance of species sorting at seep communities. Finally, the succession of microbial communities and the emergence of ecosystem function at a cold seep were monitored showing that it may take years to develop fully functioning communities that efficiently remove the potential greenhouse gas methane. Overall this work may help to resolve the mysteries of microbial community ecology at cold seep ecosystems. Zusammenfassung Die ausführliche Erforschung mikrobieller Lebensgemeinschaften, z.B. in Böden oder an Hydrothermalquellen, erweiterte unsere Kenntnisse hinsichtlich der Vielfalt und der Stoffwechselleistungen von Mikroorganismen und ihrem Einfluss auf globale Stoffkreisläufe erheblich. Gegenstand dieser Dissertation war die Untersuchung der Vielfalt, Häufigkeit und Verteilung der Mikroorganismen an kalten Methanquellen, sowie der ökologischen Mechanismen, welche diese Gemeinschaften beeinflussten. Die Ökosysteme wurden mittels geochemischer Analysen, Genbanken, Pyrosequenzierung, Community Fingerprinting und Fluoreszenz in situ Hybridisierung beschrieben. Die mikrobiellen Gemeinschaften an Methanquellen unterschieden sich stark von jenen im umliegenden Meeresboden. Zudem unterschieden sich die Gemeinschaften einzelner Methanquellen deutlich bezüglich ihres Artenreichtums und der Artengleichheit und waren stark endemisch. Trotz der Unterschiede fanden sich an allen Standorten bestimmte Organismen – das Methanquellen-Mikrobiom – darunter waren anaerobe methan- oxidierende Archaeen und sulfatreduzierende Bakterien, sowie aerobe Methan- und Schwefeloxidierer, sofern Sauerstoff vorhanden war. Diese funktionellen Gruppen schienen von Umweltfaktoren wie Temperatur, Fluidflüssen, Sedimenttiefe und Makrofauna-Aktivität beeinflusst zu werden. Bioturbation durch bestimmte Röhrenwürmer, beispielsweise, schaffte einen Lebensraum für aerobe Methanotrophe, während die Aktivität von Calyptogena-Muscheln scheinbar das Ansiedeln anaerober Methanoxidierer begünstigte. Ökologische Nischen spielten also eine wichtige Rolle für die Entwicklung der Ökosysteme. Die meisten Methanquellen wurden von wenigen, weltweit vorkommenden taxonomischen Einheiten (OTU0.03 - mit zu 97% identischen 16S rRNA Genen) aus jeder funktionellen Gruppe dominiert. Einige dieser OTU0.03 traten an manchen Standorten häufig und an anderen selten auf und schienen durch bestimmten Umweltveränderungen beeinflusst zu werden, was die Bedeutung des Species-sorting Prinzips für die Entstehung der Lebensgemeinschaften bestätigte. Überdies wurde die Sukzession mikrobieller Gemeinschaften beobachtet und gezeigt, dass es möglicherweise Jahre dauert bis voll funktionsfähige Biozönosen entstehen, die das Klimagas Methan effizient entfernen. Insgesamt trug diese Arbeit dazu bei, die ökologischen Prozesse, welche für die Entstehung der Lebensgemeinschaften verantwortlich sind, besser zu verstehen. Table of contents Table of contents…………………………………………………………………… 1 Chapter 1……………………………………………………………………………… ..3 General Introduction………………………………………………………… 4 Thesis Objectives…………………………………………………………….. 21 Chapter 2……………………………………………………………………………… 22 Abstract……………………………………………………………………… 24 Introduction………………………………………………………………… .25 Material and Methods……………………………………………………… 27 Results and Discussion……………………………………………………… 29 Conclusion…………………………………………………………………… 36.. Acknowledgements………………………………………………………… 36 Figures and Tables………………………………………………………… 37 References…………………………………………………………………… 43. Chapter 3……………………………………………………………………………… 48 Abstract……………………………………………………………………… 50 Introduction…………………………………………………………………… 50. Material and Methods………………………………………………………. 51.. Results………………………………………………………………………… 54 Discussion…………………………………………………………………….. 61 Conclusion…………………………………………………………………… 63. Acknowledgements………………………………………………………….. 63 References…………………………………………………………………… 64. Chapter 4………………………………………………………………………… 6…6 Abstract……………………………………………………………………… 68 Introduction…………………………………………………………………… 69. Material and Methods……………………………………………………… 71 Results………………………………………………………………………… 75 Discussion……………………………………………………………………... 79 Conclusion………………………………………………………………… …83.. Acknowledgements………………………………………………………… 83 Figures………………………………………………………………………… 84 References…………………………………………………………………… 89. 1 Chapter 5……………………………………………………………………………… 93 Abstract……………………………………………………………………… 95 Introduction……………………………………………………………………. .96 Material and Methods………………………………………………………… 99 Results…………………………………………………………………………. 103 Discussion…………………………………………………………………….. 107 Conclusion…………………………………………………………………….. 114 Acknowledgements………………………………………………………… 114 Figures………………………………………………………………………… 115 References…………………………………………………………………… .123 Chapter 6……………………………………………………………………………… .130 General Discussion…………………………………………………………… 131 Microbial communities at methane seep ecosystems…………………… 131 Environmental heterogeneity at methane seep ecosystems…………… 138 Microbial community assembly at methane sepp ecosystems………… 140 Conclusion…………………………………………………………………… 145 Outlook………………………………………………………………………… 146 References…………………………………………………………………… 147 Acknowledgements…………………………………………………………… 162 Appendix……………………………………………………………………………… .163 Supporting Information Chapter 2………………………………………… 164 Supporting Information Chapter 3………………………………………… 183 Supporting Information Chapter 4………………………………………… 204 Miscellaneous………………………………………………………………… .212 Statement……………………………………………………………………… 216 2 Chapter 1 3 General Introduction The description and classification of all living organisms may be one of mankinds oldest yet unaccomplished ambitions. It is the tremendous opulence of life that has fascinated generations of naturalists and researchers alike, yet we do not fully comprehend its complexity and meaning to this day. It was estimated that there are around 8.7 million eukaryotic species on Earth of which we classified around 20% (Mora et al., 2011). To complicate the matter this large number is dwarfed by the estimated number of prokaryotic species, as one ton of soil could already harbor as many as 4 million different prokaryotic species (Curtis et al., 2002). Hence, given the roughly 9000 prokaryotic species that have been described so far (Sutcliffe et al., 2012), at least 99% of prokaryotic diversity remains unknown. The need to determine the species diversity of ecosystems and how it is generated and maintained, however, has increased since ecosystems all over the world are being destroyed (Hoekstra et al., 2005). Some ecosystems were threatened by human activities before they were even discovered, for instance in the deep sea of Hikurangi continental margin off the coast of New Zealand (Baco et al., 2010). As diversity is often positively correlated with the productivity and resilience of an ecosystem, both for macroscopic (Walker et al., 1999; Folke et al., 2004; Worm et al., 2006) as well as microscopic organisms (Horner-Devine et al., 2003; Allison and Martiny, 2008; Bienhold et al., 2011) we need to foster surveys of biodiversity in general and surveys of