This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. Microbiology and the Limits to Life in Deep Salts Samuel J. Payler Doctor of Philosophy The University of Edinburgh 2017 1 Lay summary Despite some salt deposits being over 250 million years old, they host a range of salt-adapted microbes, some of which are hypothesised to be thousands, even millions of years old. How exactly these communities survive in these environments is still unknown. Interest has spiked recently in these environments due to the discovery of similar salt deposits on Mars. Whilst surface conditions on Mars are very hostile to life, the subsurface may provide a safe haven for any extant life on the planet. On Earth, subsurface salt deposits are remarkably common and can be found underlying a significant portion of the planet’s landmass. The importance of deep subsurface salt deposits to both terrestrial and extraterrestrial science means that our current understanding of life in these environments needs to be improved. This thesis addresses this by significantly advancing our understanding of how the deep biosphere is shaped by salts. It achieves this by sampling waters and salt rocks from an active mine to examine the impact brine and rock salt geochemistry has had on the native microbial communities. The work shows how the remains of a 250-million-year-old sea can still control the habitability of modern deep subsurface waters, sometimes even rendering them completely uninhabitable to all known life. Cutting edge sequencing technologies are used to gain insights into the composition and behaviour of the communities living in these environments. Their remarkable similarity to highly salty environments on the Earth’s surface demonstrates that salt can almost exclusively shape microbial communities, regardless of environmental setting. How microbial communities survive in these environments was also investigated. Ancient sources of carbon were examined to see if the organisms from the salt deposit could use them for sustenance. As well as showing this to be possible, it was found that certain salt rocks greatly increased the growth of the deep subsurface organisms by changing the water chemistry around them. Gases that could provide energy and carbon sources also were found trapped in salt rocks. Potential positive detections of Mars relevant metabolisms were also made, adding to the relevance of the brines as Mars analogue environments. Overall this work greatly enhances the established knowledge base of these fascinating environments. i Abstract Deep subsurface evaporites are common terrestrial deep subsurface environments found globally. These deposits are known to host communities of halophilic organisms, some of which have been suggested to be millions of years old. The discovery of evaporite minerals on Mars has led to these environments becoming of interest to astrobiology, particularly because the subsurface of Mars represents the best chance of finding more clement conditions conducive to life. Despite this interest, deep subsurface evaporites remain poorly understood and we have little insight into how different salts shape the Earth’s biosphere, much of which is underground. This thesis addresses several knowledge gaps present in the literature by sampling a selection of brine seeps and rock salt samples taken from Boulby Potash Mine, UK. The origin and evolution of the brines is determined with geochemical techniques, showing the majority to have been sourced from an aquifer above where they were intersected in the mine. These brines appear to have taken a variety of pathways through the subsurface leading to the presence of a range of different ions dissolved within them. The majority are Na/Cl dominated, whilst one is K/Cl dominated. One brine appears to have a different origin and probably interacted with dolomite becoming very concentrated in Mg. This variety in brine origins and migration pathways has impacted the habitability of the brines. Physicochemical measurements for chaotropicity, water activity and ionic strength, combined with culturing experiments suggest brines from the Sherwood Sandstone were habitable, but the brine from a distinct unknown source was uninhabitable. DNA was successfully extracted from three of the habitable brines and their metagenomes sequenced. These revealed communities largely functionally and phylogenetically similar to surface near saturation brines, indicating that the structure of the communities present in saturated Na/Cl brines are controlled almost exclusively by these ions rather than any other environmental difference between the surface and subsurface. Organisms were also taken from these brines and culturing experiments carried out to determine if any carbon sources were present in ancient salt that might promote growth in the absence of other carbon sources. Controls showed that the geochemical changes to the growth media induced by solving the salts, particularly sylvinite, were responsible for the increases in growth observed, indicating certain salt minerals effectively fertilise the growth of halophiles. Culturing on hydrocarbon seeps collected in the mine suggested they may provide a carbon source periodically to some organisms within the deposit. Work was done to show the presence of dissimilatory sulphate and iron reducing halophiles. Overall this significantly advances our understanding of how ii salts shape the Earth’s biosphere, particularly its deep subsurface component, and what functional capabilities life has to persist in these environments. This work provides a new window on the potential habitability of deep subsurface extraterrestrial environments and how we might go about investigating these environments for habitable conditions. iii Declaration This thesis has been solely composed by the candidate. Unless clearly stated, this work is the candidate’s own. Where results have been obtained through collaborations with other researchers, their contributions are made clear in the text as appropriate. This work has not been submitted for any other degree or professional qualification. Candidate’s signature Date……12/01/2017…………. iv Acknowledgements This thesis would not have been possible without the input of a whole range of different people. Initially, I would like to thank the Engineering and Physical Sciences Research Council (EPSRC), for funding the research. Israel Chemicals Ltd (ICL) also deserve tremendous credit for allowing me access to their mine and providing their superb professionals, particularly Jac Genis and Thomas Edwards, to help advise on the project and accompany me on field work. Thanks too to the brilliant team at STFC’s Boulby Underground Science Facility, namely Dr Sean Paling, Emma Meehan, Louise Yeoman and Christopher Toth, who have been nothing but exceptional in helping organise logistics and facilitating science. I am extremely grateful to my supervisor Professor Charles Cockell for taking me on as a student and helping me along both in this project and a range of other endeavours. Working with him has afforded me some remarkable opportunities, allowing me to experience things I might have once thought I would never have the opportunity to do. I would also like thank Bryne Ngwenya for his input and kind advice. I spent time working with various academics at a range of institutions, without whom the project would not have been possible. These include Professor Jennifer Biddle, who’s help with the metagenomics was invaluable and who’s warmth and generosity whilst I was visiting in the US will not be quickly forgotten. Dr Nicholas Odling, who helped perform XRD on the rock salt samples. Professor Barbara Sherwood Lollar, who analysed the hydrogen and oxygen isotopes in the brine samples. And finally, Dr John Telling, who’s expertise was used to achieve the rock salt gas analysis and who’s long term support for the work was very much appreciated. I would also like to thank all the past and present members of the UKCA in Edinburgh who I have had the pleasure of working with during my time there, including Mark, Sophie, Casey, Tasha, Hanna, Jesse, Derek, Toby, Jenn, Delma, Claire, Sarah, Susanna, Petra and Adam. As ever, huge gratitude is reserved for Mum, Dad, Matt and Harriet, who have supported me throughout my whole educational career, and no doubt will beyond. v Contents Lay Summary ........................................................................................................................... i Abstract ................................................................................................................................... ii Declaration.............................................................................................................................