physical, metabolic, and energetic investigations of methane-metabolizing microbial communities

300 Pages·2015·28.2 MB·English

PHYSICAL, METABOLIC, AND ENERGETIC
INVESTIGATIONS OF METHANE-METABOLIZING
MICROBIAL COMMUNITIES
Thesis by
Jeffrey Marlow
In Partial Fulfillment of the Requirements for the degree of
Doctor of Philosophy
CALIFORNIA INSTITUTE OF TECHNOLOGY
Pasadena, California
2016
(Defended June 5th, 2015)
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© 2015
Jeffrey Marlow
All Rights Reserved
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ACKNOWLEDGEMENTS
Whatever information of consequence that may be encompassed by this compilation is a testament
to the generous network of support I have enjoyed during its completion. Most fundamentally, my
family and dear friends, scattered across time zones, have provided critical grounding that has
bolstered my faith in both the importance and limits of my scientific pursuits. Administration, staff
members, and other researchers across the Caltech campus have helped in ways both seen and
unseen to enable a frictionless scientific experience. I greatly appreciate the personal
encouragement and scientific insight of all members of the Orphan Lab past and present –
especially Jan Haskell, Stephanie Connon, Shana Goffredi, Joshua Steele, Greg Wanger, Connor
Skennerton, Anne Dekas, Jennifer Glass, David Case, Derek Smith, Shawn McGlynn, Alexis
Pasulka, and Roland Hatzenpichler – as well as other colleagues, most notably Joel Scheingross,
Megan Newcombe, James Hemp, Sebastian Kopf, Ryan Hunter, Ajay Limaye, Doug LaRowe,
Bethany Ehlmann, and Andrew Thurber. I am very grateful for the logistical, financial, and
scientific support of John Grotzinger, Wiebke Ziebis, Lisa Levin, Peter Girguis, and Michael
Roukes. Jan Amend has witnessed and shepherded my development as a scientist, from a
dishwashing freshman at Washington University to a continuing collaborator in the study of
microbial ecosystems. Countless scientific conversations with all of the remarkable colleagues
above have shaped my scientific identity and set the groundwork for my future investigations.
Financial support from the National Science Foundation’s Graduate Student Fellowship Program,
Caltech’s Center for Environmental Microbial Interactions, and the National Research Council have
made this work and its dissemination possible. My thesis advisory committee – Doug Rees,
Woodward Fischer, Tori Hoehler, Dianne Newman, and Victoria Orphan – has provided generous
guidance, overcoming busy schedules to discuss results and prioritize ballooning investigatory
ambitions. In particular, Dianne Newman kindled my scientific drive through insightful
interrogation and encouraged me to follow empirically testable questions with a focused tenacity.
Victoria Orphan led and taught by the most effective means possible, through the quiet stimulation
of wonder and awe rather than a fear of insufficiency or unworthiness. Her appreciation that, in its
purest form, the curiosity that constitutes the most essential seed of a scientist’s soul knows no
bounds, allowed me to cultivate a wide range of interests that has – inevitably – enabled unforeseen
scientific opportunities and broadened their ultimate impact. This list is a poor approximation of my
indebtedness, but the deep support and prodding intellectual stimulation I have received from the
vivacious scientific community in which I find myself inspires a brimless excitement for future
questions we will engage and answers we will chase, together.
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ABSTRACT
Understanding the roles of microorganisms in environmental settings by linking phylogenetic
identity to metabolic function is a key challenge in delineating their broad-scale impact and
functional diversity throughout the biosphere. This work addresses and extends such questions in
the context of marine methane seeps, which represent globally relevant conduits for an important
greenhouse gas. Through the application and development of a range of culture-independent tools,
novel habitats for methanotrophic microbial communities were identified, established settings were
characterized in new ways, and potential past conditions amenable to methane-based metabolism
were proposed. Biomass abundance and metabolic activity measures – both catabolic and anabolic
–demonstrated that authigenic carbonates associated with seep environments retain methanotrophic
activity, not only within high-flow seep settings but also in adjacent locations exhibiting no visual
evidence of chemosynthetic communities. Across this newly extended habitat, microbial diversity
surveys revealed archaeal assemblages that were shaped primarily by seepage activity level and
bacterial assemblages influenced more substantially by physical substrate type. In order to reliably
measure methane consumption rates in these and other methanotrophic settings, a novel method
was developed that traces deuterium atoms from the methane substrate into aqueous medium and
uses empirically established scaling factors linked to radiotracer rate techniques to arrive at absolute
methane consumption values. Stable isotope probing metaproteomic investigations exposed an
array of functional diversity both within and beyond methane oxidation- and sulfate reduction-
linked metabolisms, identifying components of each proposed enzyme in both pathways. A core set
of commonly occurring unannotated protein products was identified as promising targets for future
biochemical investigation. Physicochemical and energetic principles governing anaerobic methane
oxidation were incorporated into a reaction transport model that was applied to putative settings on
ancient Mars. Many conditions enabled exergonic model reactions, marking the metabolism and its
attendant biomarkers as potentially promising targets for future astrobiological investigations. This
set of inter-related investigations targeting methane metabolism extends the known and potential
habitat of methanotrophic microbial communities and provides a more detailed understanding of
their activity and functional diversity.
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TABLE OF CONTENTS
Acknowledgements ....................................................................................................... iii
Abstract ............................................................................................................................ v
Table of Contents ........................................................................................................... vi
Introduction ..................................................................................................................... 1
Chapter 1: Carbonate Hosted Methanotrophy Represents an
Unrecognized Methane Sink in the Deep Sea Case Study ............................................. 6
Introduction ............................................................................................................... 7
Results ..................................................................................................................... 12
Discussion ............................................................................................................... 21
Methods .................................................................................................................. 31
References ............................................................................................................... 42
Chapter 2: Deuterated Methane: A Novel Approach for Measuring
Rates of Biological Methane Oxidation ........................................................................ 47
Introduction ............................................................................................................. 48
Methods .................................................................................................................. 50
Results and Discussion ........................................................................................... 59
Conclusion .............................................................................................................. 74
References ............................................................................................................... 75
Chapter 3: Microbial Abundance and Diversity Patterns
Associated with Sediments and Carbonates from the Methane
Seep Environments of Hydrate Ridge, OR ................................................................... 78
Introduction ............................................................................................................. 80
Methods .................................................................................................................. 86
Results and Discussion ........................................................................................... 92
Conclusion ............................................................................................................ 112
References ............................................................................................................. 114
Chapter 4: Stable Isotope Probing Metaproteomics Reveals
Dynamic Metabolism at Marine Methane Seeps ........................................................ 121
Introduction ........................................................................................................... 122
Methods ................................................................................................................ 125
Results and Discussion ......................................................................................... 128
Conclusion ............................................................................................................ 150
References ............................................................................................................. 152
Chapter 5: The Potential for Biologically Catalyzed Anaerobic
Methane Oxidation on Ancient Mars .......................................................................... 160
Introduction ........................................................................................................... 161
Data Selection and Methods ................................................................................. 165
Results ................................................................................................................... 177
Discussion ............................................................................................................. 180
Conclusion ............................................................................................................ 191
References ............................................................................................................. 192
Conclusion ................................................................................................................... 201
Appendix 1: Supplementary Information for Chapter 1 ............................................. 206
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Appendix 2: Autoendoliths: A Distinct Type of Rock-Hosted
Microbial Life .............................................................................................................. 221
Appendix 3: Supplementary Information for Chapter 3 ............................................. 233
Appendix 4: Supplementary Information for Chapter 4 ............................................. 237
Appendix 5: Supplementary Metaproteomic Study of Alternative
Electron Acceptors in the Anaerobic Oxidation of Methane ..................................... 273
Appendix 6: Supplementary Information for Chapter 5 ............................................. 286
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1
I n t r o d u c t i o n
Over the last several decades, as exploratory range has broadened and analytical
tools have sharpened, our conception of the biosphere’s extent has expanded dramatically.
Much of the newly characterized habitat is the province of microorganisms: from the deep
subsurface (Whitman et al. 1998) to the upper atmosphere (Wainwright et al. 2003),
microbes are increasingly viewed as a pervasive force permeating Earth’s habitable
volume. These microscopic denizens are not merely passive footnotes in the saga of
planetary evolution and elemental cycling; rather, microbial communities frequently drive
such transformations, marshaling their metabolic versatility to produce oxygen (Farquhar et
al. 2011), fix nitrogen (Burris & Roberts 1993), degrade organic matter (Middelburg et al.
1993), and access rock-bound metals (Haferburg & Kothe 2007).
Researchers have developed a healthy appreciation for microbes’ range, but a wide
gap between presence and functional relevance persists, and developing a more detailed
understanding of the activity-based roles and relationships of these communities remains an
important frontier. The most reliable methods of linking microbial identity to function,
which require pure cultures of isolated species, are largely impotent when applied to
environmental systems, where the vast majority of organisms are recalcitrant to culturing
attempts (e.g., Rappé & Giovannoni 2003; Pace et al. 1986). What’s more, the seemingly
critical role of inter-species interactions (Pham & Kim 2012; West et al. 2007) renders pure
culture studies imprecise at best and dangerously misleading at worst.
In response to the “culturability bottleneck,” two paths have emerged in the quest to
understand microbial roles in environmental contexts: characterizing evidence of