Anaerobic Oxidation of Methane in Cold Seeps and Gas Hydrates
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ANAEROBIC OXIDATION OF METHANE IN COLD SEEPS AND GAS HYDRATES: RESPONSIBLE MICROORGANISMS, RATES OF ACTIVITY, AND INTERACTIONS WITH OTHER PROCESSES by Beth! N. Orcutt (under the supervision of Samantha B. Joye) ABSTRACT This work utilized a multidisciplinary approach to explore the microbial biogeochemistry of methane cycling in the marine environment with the aim of identifying which processes were occurring and which microorganisms were involved, focusing on two methane-rich systems: oil- laden, gas hydrate-bearing and/or brine-charged cold seeps in the Gulf of Mexico and the Hydrate Ridge deep biosphere. The research presented here expands our knowledge of microbially-mediated anaerobic oxidation of methane (AOM) and associated process such as sulfate reduction (SR) and methanogenesis in surficial and deep methane-rich environments. This work confirmed that the ANME-1 and -2 clades of methanotrophic archaea are responsible for AOM in surficial sediments in the Gulf of Mexico and was the first to demonstrate that the ANMEs are also involved in the production of methane, although at a fraction of the rate of AOM. Our work was the first to directly document microbial activity within gas hydrate material collected from the Gulf of Mexico, which suggests that microbial activity may impact the biogeochemical cycling of methane within the unique gas hydrate niche. Active populations of both Bacteria and Archaea were observed in a methane-rich deep biosphere environment, in contrast to previous studies which suggest that one or the other domain is dominant. In methane- rich areas of the deep subsurface, ANME were detected for the first time in an area where sulfate levels were elevated, although AOM may be limited in the deep biosphere by the availability of sulfate coupled with slow growth rates. Unlike at other cold seeps which have been studied to date, SR in Gulf of Mexico surficial sediments is often uncoupled from AOM and is also fueled by other endogenous hydrocarbons and petroleum derivatives; the identity of the microorganisms which mediate sulfate-dependent hydrocarbon oxidation in situ is unclear but likely includes members of the seep-endemic Deltaproteobacteria sulfate reducing bacterial (SRB) clades. Modeling approaches to determine the potential intermediate compound exchanged within consortia of ANME and SRB to sustain AOM and SR revealed that hydrogen, acetate and formate cannot sustain rates of activity that match measured values due to diffusion-limited removal of the intermediate compound. INDEX WORDS: Anaerobic oxidation of methane, Sulfate Reduction, Methanogenesis, Biogeochemistry, Molecular Ecology, Lipid biomarkers, Gas hydrate, Cold seeps, Gulf of Mexico, Oil, Reaction transport model, Deep biosphere, Hydrate Ridge, Thermodynamics, Archaea, Ocean Drilling Program ANAEROBIC OXIDATION OF METHANE IN COLD SEEPS AND GAS HYDRATES: RESPONSIBLE MICROORGANISMS, RATES OF ACTIVITY, AND INTERACTIONS WITH OTHER PROCESSES by BETH! NOELLE ORCUTT B.S. Interdisciplinary Studies, University of Georgia, 2002 A Dissertation Submitted to the Graduate Faculty of the University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2007 © 2007 Beth! N. Orcutt All Rights Reserved ANAEROBIC OXIDATION OF METHANE IN COLD SEEPS AND GAS HYDRATES: RESPONSIBLE MICROORGANISMS, RATES OF ACTIVITY, AND INTERACTIONS WITH OTHER PROCESSES by BETH! NOELLE ORCUTT Major Professor: Samantha B. Joye Committee: Antje Boetius Christof Meile Mary Ann Moran Stuart Wakeham William Whitman Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2007 ACKNOWLEDGEMENTS There are many people I would like to thank for helping make this dissertation possible, either for helping me with my work or for offering support, encouragement and good jokes, or both. I am so thankful for my advisor, Mandy Joye, for giving me the opportunity as an undergraduate to explore the fascinating world of environmental microbial biochemistry, for providing incredible research opportunities, for inspiring me to pursue a career in science, and for being a friend. None of this would have been possible without the initial support of Steve Carini, who brought me into the lab and taught me much of what I know. Many thanks to my other UGA labmates – Steve Bell, Meaghan Bernier, Marshall Bowles, Jon Clark, Matty Erickson, Kim Hunter, Rosalynn Lee, Bill Porubsky, Kate Segarra, and Nat Weston – for keeping the energy level high and the machines working. There are others at UGA who have helped me along the way that I would like to thank – Jen Fisher, Tim Hollibaugh, Christof Meile, Mary Ann Moran, Rachel Poretsky, Stuart Wakeham and Barny Whitman. I owe a huge debt of gratitude to Antje Boetius, my home-away-from-home advisor in Bremen, Germany. Thank you so much for supporting my many visits to your labs, for encouraging me to push the boundaries of what I can do, and for being a fabulous role model. To everyone at the MPI who helped me out over the years – Viola Beier, Imke Busse, Marcus Elvert, Niko Finke, Katrin Knittel, Tina Lösekann, Jochen Neuster, Helge Niemann, and Tina Treude – thank you, especially to my office mate and chaos girl Nina Knab. To the RCOM Organic Geochemistry Group in Bremen, thanks for offering one of the friendliest and most inspiring working environments. Much of my lipid biomarker work would not have been possible without Kai-Uwe Hinrichs allowing me to work in his lab – thank you. Thanks again to Marcus Elvert for always being there with advice and for never relenting in iv telling me to learn German. Julius Lipp has been a great working partner for the deep biosphere project – thanks for putting up with my overseas requests! Daniel Birgel, Solveig Bühring, Verena Heuer, Xavi Prieto, Pamela Rossel, Flo Schübotz, and Julio Sepúlveda – I can’t thank you enough for your encouragement and your enduring friendship. I consider myself blessed to have friends far and wide that share my enthusiasm for understanding the world and trying to make it a better place. Jelte Harnmeijer, I will never forget our adventures. Emily Fleming, you are welcome to stay on a couch I sell out from under you anytime. John Spear, your compassionate instruction in the field, in the lab, in the classroom, and in general render you an invaluable role model in my book. Thanks to the unknown reviewers of my NSF graduate research fellowship application – I couldn’t have done half of what I did without the support their decision led to. And finally, thanks to my high school chemistry teacher – Mr. Lanham – for sparking my imagination and setting me on the path of the pursuit of truth. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS…………………………………………………………………….vi LIST OF TABLES……………………………………………………………………………...viii LIST OF FIGURES…………………………………………………………………………......x CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW…………………………….1 2 MICROBIAL BIOGEOCHEMISTRY OF SULFATE REDUCTION, METHANOGENESIS AND THE ANAEROBIC OXIDATION OF METHANE AT GULF OF MEXICO COLD SEEPS…………………..………37 3 IMPACT OF OIL AND HIGHER HYDROCARBONS ON MICROBIAL DIVERSITY, DISTRIBUTION AND ACTIVITY IN GULF OF MEXICO SEDIMENTS…………………………………………………………………….88 4 ON THE RELATIONSHIP BETWEEN METHANE PRODUCTION AND OXIDATION BY ANAEROBIC METHANOTROPHIC COMMUNITIES FROM COLD SEEPS OF THE GULF OF MEXICO…………………………131 5 LIFE AT THE EDGE OF METHANE ICE: MICROBIAL CYCLING OF CARBON AND SULFUR IN GULF OF MEXICO GAS HYDRATES………162 6 BACTERIA AND ARCHAEA AND THEIR ACTIVITY IN HYDRATE- BEARING DEEP SUBSURFACE SEDIMENTS OF HYDRATE RIDGE (CASCADIA MARGIN, ODP LEG 204)….......................................…………194 vi 7 CONSTRAINTS ON MECHANISMS AND RATES OF ANAEROBIC OXIDATION OF METHANE BY ANME-2 CONSORTIA…………………..249 8 CONCLUSIONS…………………………………………………………….…284 vii LIST OF TABLES Page Table 2.1: Concentrations of sulfide and chloride, total cell abundance, and percentage of cells that were Bacteria, Archaea, of the ANME-1 clade of Euryarchaeota, of the ANME-2 clade of Euryarchaeota, or of the Desulfosarcina/Desulfococcus spp. clades of δ-proteobacteria in sediments from the Gulf of Mexico………………………67 Table 2.2: Isotopic composition of various carbon compounds from gas hydrate and brine pool sites in the Gulf of Mexico…………………………………………………………68 Table 2.3A: Abundance and isotopic composition of select lipid biomarkers extracted from sediments at a brine influence site in the Gulf of Mexico………………………….69 Table 2.3B: Abundance and isotopic composition of select lipid biomarkers extracted from sediments at a gas hydrate site in the Gulf of Mexico…………………………….70 Table 3.1: Gulf of Mexico site and sample descriptions……………………………………….116 Table 3.2: Integrates rates of sulfate reduction, the anaerobic oxidation of methane, methanogenesis from carbon dioxide or from acetate, and acetate oxidation and estimated turnover times of the methane, sulfate, and acetate pools for hydrocarbon-rich sediments from the Gulf of Mexico…………………………………117 Table 3.3: Overview of 16S gene sequences obtained from Gulf of Mexico sediments……….118 Table 4.1: Site descriptions for the Gulf of Mexico sediments used in these experiments…….152 Table 4.2: Abundance of ANME groups in sediments…………………………………………153 Table 5.1: Average hydrocarbon gases of hydrate material and vent gas……………………...183 Table 5.2: Rates of AOM and SR in gas hydrate material collected from two sites in the Gulf of Mexico……………………………………………………………..…………...184 viii Page Table 6.1: Southern Hydrate Ridge (ODP Leg 204) site characteristics……………………….226