Heat resistant thermophilic endospores in cold estuarine sediments Emma Bell Thesis submitted for the degree of Doctor of Philosophy School of Civil Engineering and Geosciences Faculty of Science, Agriculture and Engineering February 2016 Abstract Microbial biogeography explores the spatial and temporal distribution of microorganisms at multiple scales and is influenced by environmental selection and passive dispersal. Understanding the relative contribution of these factors can be challenging as their effects can be difficult to differentiate. Dormant thermophilic endospores in cold sediments offer a natural model for studies focusing on passive dispersal. Understanding distributions of these endospores is not confounded by the influence of environmental selection; rather their occurrence is due exclusively to passive transport. Sediment heating experiments were designed to investigate the dispersal histories of various thermophilic spore-forming Firmicutes in the River Tyne, a tidal estuary in North East England linking inland tributaries with the North Sea. Microcosm incubations at 50-80°C were monitored for sulfate reduction and enriched bacterial populations were characterised using denaturing gradient gel electrophoresis, functional gene clone libraries and high-throughput sequencing. The distribution of thermophilic endospores among different locations along the estuary was spatially variable, indicating that dispersal vectors originating in both warm terrestrial and marine habitats contribute to microbial diversity in estuarine and marine environments. In addition to their persistence in cold sediments, some endospores displayed a remarkable heat-resistance surviving multiple rounds of autoclaving. These extremely heat-resistant endospores are genetically similar to those detected in deep subsurface environments, including geothermal groundwater investigated from a nearby terrestrial borehole drilled to >1800 m depth with bottom temperatures in excess of 70°C. The ability of these endospores to survive extreme temperatures whilst in a dormant state may enable them to withstand adverse conditions for long periods of time and then germinate in response to changing surroundings. This was investigated further in the context of seawater injection during secondary oil recovery, where cold seawater is injected into hot oil reservoirs, resulting in a cooler reservoir temperature near the injection well bore. Microcosm experiments designed to simulate this showed that cooling triggered the germination of endospores of sulfate-reducing Desulfotomaculum leading to the onset of souring in this model system. The results presented here, indicate that bacterial endospores are transported between terrestrial and marine, surface and subsurface environments. Their survival and distribution therefore has i relevance to understanding deep biosphere processes, and factors shaping microbial diversity in the marine environment. ii Acknowledgements First and foremost, I would like to thank my supervisor Dr. Casey Hubert, for all of his help and guidance throughout my Ph.D. Thank you for all of the inspiring scientific discussions, the constant encouragement and your invaluable support. I would like to thank my co-supervisors, Prof. Ian Head and Dr. Neil Gray for all of their valued advice and contributions. Special thanks to Dr. Angela Sherry for sharing her wisdom, teaching me many techniques in the lab, and guiding me along the way. Thanks to the whole Geomicrobiology group at Newcastle, thanks for making it a great place to learn and work, thank you all for teaching me so much and for your friendship. Thanks to Dave Race for help with my many many sulfate samples and to David Manning for the opportunity to work on the geothermal water, which has made a significant contribution to this thesis. iii iv Table of Contents Abstract ............................................................................................... i Acknowledgements .......................................................................... iii Table of Contents .............................................................................. v List of Figures ................................................................................... ix List of Tables .................................................................................. xvi Chapter 1. ........................................................................................... 1 Introduction ........................................................................................ 1 1.1 Thermophilic Endospores in Cold Sediments ......................................... 1 1.2 Microbial biogeography ........................................................................... 2 1.2.1 Passive dispersal ............................................................................. 3 1.2.2 Dispersal limitation ........................................................................... 4 1.3 Dormancy and the Microbial Seed Bank ................................................. 5 1.3.1 Bacterial endospores ....................................................................... 6 1.3.2 Endospores in the subseafloor sediments ....................................... 7 1.4 Thermophilic spore-forming Desulfotomaculum ..................................... 9 1.4.1 Habitats of thermophilic endospore-forming bacteria .................... 10 1.4.1.1 Petroleum reservoirs ............................................................... 11 1.4.1.2 Mid-ocean ridges .................................................................... 12 1.4.1.3 Terrestrial thermal environments ............................................ 14 1.5 Estuaries ............................................................................................... 15 1.5.1 Microbial distribution in estuarine sediments ................................. 16 1.6 Objectives and Outline of this Thesis .................................................... 17 Chapter 2. ......................................................................................... 18 Materials and Methods .................................................................... 18 2.1 The Tyne estuary .................................................................................. 18 2.1.1 Sampling locations ......................................................................... 18 2.2 Sediment slurry incubations .................................................................. 19 2.2.1 Preparation of microcosms ............................................................ 19 2.2.2 Analysis of sulfate .......................................................................... 20 2.2.3 Analysis of volatile fatty acids (VFA) .............................................. 21 v 2.2.4 Analysis of sulfide .......................................................................... 21 2.3 Microbial community analysis ............................................................... 21 2.3.1 DNA extraction ............................................................................... 21 2.3.2 Polymerase chain reaction (PCR) amplification ............................ 22 2.3.3 Amplification of 16S rRNA genes .................................................. 22 2.3.3.1 Denaturing gradient gel electrophoresis (DGGE) targeting Desulfotomaculum spp. ............................................................................ 23 2.3.3.2 16S rRNA gene clone libraries ............................................... 24 2.3.3.3 Ion Torrent sequencing ........................................................... 24 2.3.4 Amplification of dsrAB genes ......................................................... 26 Chapter 3. ......................................................................................... 27 The distribution of thermophilic Firmicutes along an estuarine gradient reveals multiple dispersal histories for endospores in estuarine and marine sediments ................................................... 27 3.1 Abstract ................................................................................................. 27 3.2 Introduction ........................................................................................... 28 3.3 Methods ................................................................................................ 29 3.3.1 Study sites ..................................................................................... 29 3.3.2 Sediment slurry incubations at elevated temperature .................... 29 3.3.3 Monitoring of time course incubations ........................................... 30 3.3.4 Analysis of 16S rRNA amplicon libraries ....................................... 30 3.3.5 MPN quantification of endospores of thermophilic sulfate-reducing bacteria ……………………………………………………………………………30 3.3.6 Total organic carbon (TOC) ........................................................... 31 3.4 Results .................................................................................................. 32 3.4.1 Endospores of thermophilic SRB in temperate estuarine sediments ……………………………………………………………………………32 3.4.2 Microbial community composition in heated sediments under sulfate- reducing conditions ...................................................................................... 33 3.4.3 Diversity of thermophilic Desulfotomaculum spp. within the Tyne estuary and North Sea sediments ................................................................ 36 3.5 Discussion 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