This thesis investigates four microbial systems, with a particular focus for how spatial considerations shape the behavior and evolution of microorganisms. After a general introduction in Chapter 1, Chapter 2 presents the results of experiments demonstrating how cellular activity varies through space within an anode-reducing biofilm. Chapter 3 presents a comprehensive comparative genomic analysis of all known marine anaerobic methanotrophic archaea, supporting the notion that these organisms share many energetic similarities with the anode reducing organisms in Chapter 2. These are interpreted as specific adaptations to life in highly structured microbial communities. Chapter 4 describes the enrichment and characterization of a new member of the purple sulfur bacteria, and the adaptations that may improve substrate acquisition beyond the normal limitations of diffusion. Chapter 5 describes the convergent evolution of novel Complex I gene clusters that have incorporated new proton pumping subunits, and the modifications made to the protein structure to facilitate the incorporation of these new subunits into the quaternary structure of the complex.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Orphan, Victoria J.}, } @phdthesis{10.7907/f3k8-ck13, author = {Mullin, Sean William Alexander}, title = {Spatial and Temporal Dynamics of Microorganisms Living Along Steep Energy Gradients and Implications for Ecology and Geologic Preservation in the Deep Biosphere}, school = {California Institute of Technology}, year = {2020}, doi = {10.7907/f3k8-ck13}, url = {https://resolver.caltech.edu/CaltechTHESIS:09232020-172452048}, abstract = {The deep biosphere represents a massive repository of life with unknown effects on global biogeochemical cycles. Even the fundamental life strategies of the endemic microorganisms that inhabit this biome remain enigmatic; some studies have indicated that subsurface organisms subsist in energetic regimes below the theoretical lower limit for life. A boom-bust life cycle, mediated by tectonic disturbances and subsurface fractures, may help explain these phenomena. This work addresses and expands on this question, first by exploring the response of continental deep biosphere microorganisms to an in situ organic matter amendment, then by analyzing the microbial community dynamics of the sediments and carbonate along a naturally-occurring energy gradient at a methane seep. Our experiments in the continental deep biosphere confirmed that mineralogical heterogeneity can drive differential colonization of the native microorganisms, implying that selection and adaptation to in situ conditions occurs, differentiating individual microbial niches. We also observed the formation of secondary framboidal iron sulfide minerals, a well-known phenomenon in marine sediments but not extensively observed in the deep subsurface, that were correlated to the presence of abundant sulfur-metabolizing microorganisms. Chapters 2 and 3 are instead focused on the microbial ecology of a methane seep on the Pacific margin of Costa Rica. Cold methane seeps themselves represent sharp boundaries between the generally low-energy background seafloor and abundant chemical energy in the form of methane. Chapter 2 describes that the microorganisms living at these seeps occupy a significantly narrower spatial scale than the endemic megafauna. In addition, by correlating community dissimilarity and geographic distance, the functional center of the seep was identified, allowing for insight into the ecological differentiation between clades of anaerobic methanotrophic archaea (ANME). Chapter 3 examines in greater detail the endolithic microbial community, principally composed of ANME-1. By conducting transplantation experiments of carbonates on the seafloor, we tested the response of the in situ endolithic communities and found that carbonates moved distinctly outside the active zone changed less than communities moved to regions of less activity.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Orphan, Victoria J.}, } @phdthesis{10.7907/ve4r-k526, author = {Metcalfe, Kyle Shuhert}, title = {Symbiotic Diversity and Mineral-Associated Microbial Ecology in Marine Microbiomes}, school = {California Institute of Technology}, year = {2020}, doi = {10.7907/ve4r-k526}, url = {https://resolver.caltech.edu/CaltechTHESIS:08122020-110818529}, abstract = {This thesis investigates ecological interactions in the seafloor between microbial taxa (Chapters 1 and 2) and between these microorganisms and their mineral hosts (Chapters 2 through 4). In seafloor sediments, electron acceptors are often limited, forcing microorganisms inhabiting these sediments to acquire symbiotic partners and/or perform extracellular electron transfer to insoluble electron acceptors. Seafloor methane seeps present an endmember case wherein extremely reducing fluids charged with methane advect through sediment. In these benthic ecosystems, anaerobic methanotrophic archaea (ANME) form symbiotic partnerships with sulfate-reducing bacteria (SRB), but it remained unclear if certain ANME exhibit a preference for certain SRB partners. In Chapter 1, I present results documenting such a pattern of partnership specificity in ANME-SRB consortia. In Chapter 2, I further examine these patterns in rare ANME taxa through development and application of a density-separation protocol refined from published work. This protocol exploits the co-association of microbial taxa on mineral surfaces to aid in the detection of novel symbioses, and further is useful to detect microbial interactions with certain minerals. In Chapter 3, I focus on the interaction between ANME-SRB consortia and authigenic silicates that have been observed on consortium exteriors, finding evidence to support that the precipitation of these silicates is actively mediated by ANME-SRB. In Chapter 4, I perform geochemical modeling benchmarked by synchrotron X-ray analysis to examine the imprint of extracellular electron transport by metal-reducing microorganisms on Precambrian manganese-rich sedimentary rocks.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Orphan, Victoria J.}, } @phdthesis{10.7907/Z93776RJ, author = {Magyar, Paul Macdonald}, title = {Insights into Pathways of Nitrous Oxide Generation from Novel Isotopologue Measurements}, school = {California Institute of Technology}, year = {2017}, doi = {10.7907/Z93776RJ}, url = {https://resolver.caltech.edu/CaltechTHESIS:05262017-143517395}, abstract = {The accumulation of nitrous oxide (N2O) in the atmosphere is a significant manifestation of human perturbations of the nitrogen cycle. This thesis reports the development and first applications of a novel isotopic technique for characterizing nitrous oxide sources. Chapter 1 describes the development of methods to use the newly available technology of high- resolution dual-inlet multi-collector mass spectrometry to measure six isotopic parameters in nitrous oxide. It reports the standardization and initial biological application of these methods. Chapter 2 presents a model for the generation of isotope effects in an important N2O generating enzyme, the bacterial nitric oxide reductase; this model and published isotopic constraints are used to provide insights into the mechanism of that enzyme. Chapter 3 describes the six-dimensional isotopic characterization of nitrous oxide from bacterial denitrifiers, while Chapter 4 describes nitrous oxide generated by ammonia oxidizing bacteria.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, } @phdthesis{10.7907/Z9XD0ZQQ, author = {Yu, Hang Hank}, title = {Understanding the Symbiosis in Anaerobic Oxidation of Methane Through Metabolic, Biosynthetic and Transcriptomic Activities}, school = {California Institute of Technology}, year = {2017}, doi = {10.7907/Z9XD0ZQQ}, url = {https://resolver.caltech.edu/CaltechThesis:06082017-134458390}, abstract = {Microorganisms provide essential ecological services to our planet. Their combined activities control and shape our environment as we know today. In the deep sea, a microbial mediated process known as anaerobic oxidation of methane (AOM) consumes large amounts of methane, a potent greenhouse gas and a valuable energy resource. How this symbiosis works is poorly understood.
In this thesis, I tested current hypotheses on the symbiotic mechanisms in AOM microbial consortia, consisting of a partnership between anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Sediments collected from methane seeps offshore Oregon and California and dominated by AOM consortia were used in these investigations. A range of compounds were amended to sediment microcosms, and their effects on the metabolic activities of ANME or SRB were monitored by tracking the rates of methane oxidation or sulfate reduction on timescales varying from hours to months. A lack of stimulation or inhibition on the AOM consortia, combined with long-term community profiles, suggest that diffusible compounds are unlikely to be involved in the symbiosis in AOM. I further examine ANME genomes, focusing the role of sulfur in methane seep ecosystems. Phylogenetic analyses revealed multiple poorly characterized genes in the sulfur pathway, and comparisons with methanogenic archaea related to ANME provided a better understanding of their roles in the cell. Transcriptional responses combined with protein modeling were used to predict the potential substrate of a sulfite reductase related enzyme. These predictions were validated using genetics, and together point to an assimilatory rather than dissimilatory sulfur pathway in methane-utilizing archaea in general. Then, the AOM symbiosis was decoupled for the first time using soluble electron acceptors. ANME remained metabolically and biosynthetically active without their SRB partner, suggesting that the electrons are transferred directly in this partnership. This observation was investigated to a greater depth with transcriptomics. Membrane proteins and multiheme cytochromes critical in extracellular electron transfer in ANME and SRB were expressed. These results together illuminate the path electrons may take to exit or enter the AOM consortia. Overall, multiple activity analyses used here piece together a clearer view on how the symbiosis in AOM works, with potential applications in future energy generation from methane.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Orphan, Victoria J.}, } @phdthesis{10.7907/Z9TD9V84, author = {Case, David Hamilton}, title = {Carbonate-Associated Microbial Ecology at Methane Seeps: Assemblage Composition, Response to Changing Environmental Conditions, and Implications for Biomarker Longevity}, school = {California Institute of Technology}, year = {2016}, doi = {10.7907/Z9TD9V84}, url = {https://resolver.caltech.edu/CaltechTHESIS:05262016-170051580}, abstract = {Methane seeps are globally distributed geologic features in which reduced fluid from below the seafloor is advected upward and meets the oxidized bottom waters of Earth’s oceans. This redox gradient fuels chemosynthetic communities anchored by the microbially-mediated anaerobic oxidation of methane (AOM). Both today and in Earth’s past, methane seeps have supported diverse biological communities extending from microorgansisms to macrofauna and adding to the diversity of life on Earth. Simultaneously, the carbon cycling associated with methane seeps may have played a significant role in modulating ancient Earth’s climate, particularly by acting as a control on methane emissions.
The AOM metabolism generates alkalinity and dissolved inorganic carbon (DIC) and at a 2:1 ratio, promoting the abiogenic, or authigenic, precipitation of carbonate minerals. Over time, these precipitates can grow into pavements covering hundreds of square meters on the seafloor and dominating the volumetric habitat space available in seep ecosystems. Importantly, carbonates are incorporated into the geologic record and therefore preserve an inorganic (i.e., d13C) and organic (i.e., lipid biomarker) history of methane seepage. However, the extent to which preserved biomarkers represent a snapshot of microorganisms present at the time of primary precipitation, a time-integrated history of microbial assemblages across the life cycle of a methane seep, or a view of the final microorganisms inhabiting a carbonate prior to incorporation in the sedimentary record is unresolved.
This thesis addresses the ecology of carbonate-associated seep microorganisms. Chapters One and Two contextualize the extant microbial diversity on seep carbonates versus within seep sediments, as determined through 16S rRNA gene biomarkers. Small, protolithic carbonate “nodules” recovered from within seep sediments are observed to be capable of capturing surrounding sediment-hosted microbial diversity, but in some cases also diverge from sediments. Meanwhile, lithified carbonate blocks recovered from the seafloor host microbial assemblages demonstrably distinct from seep sediments (and seep nodules). Microbial 16S rRNA gene diversity within carbonate samples is well-differentiated by the extent of contemporary seepage. In situ seafloor transplantation experiments further demonstrated the microbial assemblages associated with seep carbonates to be sensitive to seep quiescence and activation on short (13-month) timescales. This was particularly true for organisms whose 16S rRNA genes imply physiologies dependent on methane or sulfur oxidation. With an improved understanding of the modern ecology of carbonate-associated microorganisms, Chapter Three applies intact polar lipid (IPL) and core lipid analyses to begin describing whether, and to what extent, geologically relevant biomarkers mimic short-term dynamics observed in 16S rRNA gene profiles versus archive a record of historic microbial diversity. Biomarker longevity is determined to increase from 16S rRNA genes to IPLs to core lipids, with IPLs preserving microbial diversity history on timescales more similar to 16S rRNA genes than core lipids. Ultimately, individual IPL biomarkers are identified which may be robust proxies for determining whether the biomarker profile recorded in a seep carbonate represents vestiges of active seepage processes, or the profile of a microbial community persisting after seep quiescence.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Orphan, Victoria J.}, } @phdthesis{10.7907/Z96Q1V6Q, author = {Trembath-Reichert, Elizabeth}, title = {Molecular and Geochemical Insights into Microbial Life Centimeters to Kilometers Below the Seafloor}, school = {California Institute of Technology}, year = {2016}, doi = {10.7907/Z96Q1V6Q}, url = {https://resolver.caltech.edu/CaltechTHESIS:05262016-132232540}, abstract = {At the broadest scale, this thesis is an investigation of how life modulates the movement of essential elements (carbon, sulfur, nitrogen, and silicon) on modern and geologic timescales. Chapters 1 and 2 explore carbon and sulfur cycling microbial communities found centimeters below the seafloor in hydrocarbon-rich methane seep ecosystems. At the Hydrate Ridge methane seep, we investigated how microbial partnerships direct the flow of methane and sulfide in these benthic oases by using identity-based physical separation methods developed in our lab (Magneto-FISH) in conjunction with community profiling and metagenomic sequencing. This method explores the middle ground between single cell and bulk sediment analysis by separating target microbes and their physically associated community for downstream sequencing applications. Magneto-FISH captures were done at a range of microbial taxonomic group specificities and sequenced with both clone library and next-gen iTag 16S rRNA gene methods. Chapter 1 provides a demonstration of how FISH probe taxonomic specificity correlates to resultant Archaeal taxonomic diversity in Magneto-FISHed seep sediments, with specific attention to preparation of Archaea-enriched samples for downstream metagenomic sequencing. In Chapter 2, a Bacteria-focused parallel environmental isolation and sequencing effort was subjected to co-occurrence analyses which suggested there may be far more microbial associations in methane seep systems than are currently appreciated, including partnerships that do not involve the canonical anaerobic methane oxidizing archaea and sulfate reducing bacteria. With samples from IODP Expedition 337 Shimokita coalbed biosphere, Chapter 3 provides evidence for an active microbial assemblage kilometers below the sea floor in the deepest samples ever collected by marine scientific ocean drilling. Using in situ temperature Stable Isotope Probing (SIP) incubations and NanoSIMS, we investigated whole community activity (with the passive tracer D2O) and substrate specific activity with C1-carbon compounds methylamine and methanol. We found deuterium-based turnover times to be faster (years) than previous deep biosphere estimates (hundreds to thousands of years), but methylotrophy rates to be slower than previous carbon metabolic rates.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Orphan, Victoria J.}, } @phdthesis{10.7907/Z9W66HPS, author = {Marlow, Jeffrey James}, title = {Physical, Metabolic, and Energetic Investigations of Methane-Metabolizing Microbial Communities}, school = {California Institute of Technology}, year = {2016}, doi = {10.7907/Z9W66HPS}, url = {https://resolver.caltech.edu/CaltechTHESIS:07212015-103405937}, 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.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Orphan, Victoria J.}, } @phdthesis{10.7907/H9F5-T161, author = {Dekas, Anne Elizabeth}, title = {Diazotrophy in the Deep: An Analysis of the Distribution, Magnitude, Geochemical Controls, and Biological Mediators of Deep-Sea Benthic Nitrogen Fixation}, school = {California Institute of Technology}, year = {2013}, doi = {10.7907/H9F5-T161}, url = {https://resolver.caltech.edu/CaltechTHESIS:12192012-141624638}, abstract = {Biological nitrogen fixation (the conversion of N2 to NH3) is a critical process in the oceans, counteracting the production of N2 gas by dissimilatory bacterial metabolisms and providing a source of bioavailable nitrogen to many nitrogen-limited ecosystems. One currently poorly studied and potentially underappreciated habitat for diazotrophic organisms is the sediments of the deep-sea. Although nitrogen fixation was once thought to be negligible in non-photosynthetically driven benthic ecosystems, the present study demonstrates the occurrence and expression of a diversity of nifH genes (those necessary for nitrogen fixation), as well as a widespread ability to fix nitrogen at high rates in these locations. The following research explores the distribution, magnitude, geochemical controls, and biological mediators of nitrogen fixation at several deep-sea sediment habitats, including active methane seeps (Mound 12, Costa Rica; Eel River Basin, CA, USA; Hydrate Ridge, OR, USA; and Monterey Canyon, CA, USA), whale-fall sites (Monterey Canyon, CA), and background deep-sea sediment (off-site Mound 12 Costa Rica, off-site Hydrate Ridge, OR, USA; and Monterey Canyon, CA, USA). The first of the five chapters describes the FISH-NanoSIMS method, which we optimized for the analysis of closely associated microbial symbionts in marine sediments. The second describes an investigation of methane seep sediment from the Eel River Basin, where we recovered nifH sequences from extracted DNA, and used FISH-NanoSIMS to identify methanotrophic archaea (ANME-2) as diazotrophs, when associated with functional sulfate-reducing bacterial symbionts. The third and fourth chapters focus on the distribution and diversity of active diazotrophs (respectively) in methane seep sediment from Mound 12, Costa Rica, using a combination of 15N-labeling experiments, FISH-NanoSIMS, and RNA and DNA analysis. The fifth chapter expands the scope of the investigation by targeting diverse samples from methane seep, whale-fall, and background sediment collected along the Eastern Pacific Margin, and comparing the rates of nitrogen fixation observed to geochemical measurements collected in parallel. Together, these analyses represent the most extensive investigation of deep-sea nitrogen fixation to date, and work towards understanding the contribution of benthic nitrogen fixation to global marine nitrogen cycling.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Orphan, Victoria J.}, } @phdthesis{10.7907/Z9125QKD, author = {Saxena, Abigail Green}, title = {Sulfur-Cycling in Methane-Rich Ecosystems: Uncovering Microbial Processes and Novel Niches}, school = {California Institute of Technology}, year = {2013}, doi = {10.7907/Z9125QKD}, url = {https://resolver.caltech.edu/CaltechTHESIS:05282013-062935856}, abstract = {Microbial sulfur cycling communities were investigated in two methane-rich ecosystems, terrestrial mud volcanoes (TMVs) and marine methane seeps, in order to investigate niches and processes that would likely be central to the functioning of these crucial ecosystems. Terrestrial mud volcanoes represent geochemically diverse habitats with varying sulfur sources and yet sulfur-cycling in these environments remains largely unexplored. Here we characterized the sulfur-metabolizing microorganisms and activity in 4 TMVs in Azerbaijan, supporting the presence of active sulfur-oxidizing and sulfate-reducing guilds in all 4 TMVs across a range of physiochemical conditions, with diversity of these guilds being unique to each TMV. We also found evidence for the anaerobic oxidation of methane coupled to sulfate reduction, a process which we explored further in the more tractable marine methane seeps. Diverse associations between methanotrophic archaea (ANME) and sulfate-reducing bacterial groups (SRB) often co-occur in marine methane seeps, however the ecophysiology of these different symbiotic associations has not been examined. Using a combination of molecular, geochemical and fluorescence in situ hybridization coupled to nano-scale secondary ion mass spectrometry (FISH-NanoSIMS) analyses of in situ seep sediments and methane-amended sediment incubations from diverse locations, we show that the unexplained diversity in SRB associated with ANME cells can be at least partially explained by preferential nitrate utilization by one particular partner, the seepDBB. This discovery reveals that nitrate is likely an important factor in community structuring and diversity in marine methane seep ecosystems. The thesis concludes with a study of the dynamics between ANME and their associated SRB partners. We inhibited sulfate reduction and followed the metabolic processes of the community as well as the effect of ANME/SRB aggregate composition and growth on a cellular level by tracking 15N substrate incorporation into biomass using FISH-NanoSIMS. We revealed that while sulfate-reducing bacteria gradually disappeared over time in incubations with an SRB inhibitor, the ANME archaea persisted in the form of ANME-only aggregates, which are capable of little to no growth when sulfate reduction is inhibited. These data suggest ANME are not able to synthesize new proteins when sulfate reduction is inhibited.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Orphan, Victoria J.}, } @phdthesis{10.7907/3891-QQ56, author = {Harrison, Benjamin Kimball}, title = {Microbial Colonization of Minerals in Marine Sediments – Method Development and Ecological Significance}, school = {California Institute of Technology}, year = {2011}, doi = {10.7907/3891-QQ56}, url = {https://resolver.caltech.edu/CaltechTHESIS:05262011-085126961}, abstract = {Interactions between microorganisms and minerals significantly impact microbial diversity and geochemical cycles in diverse settings. However, methodological difficulty has inhibited past study of microbe–mineral interactions in fine-grained subsurface environments. Conventional sampling poorly resolves microbial diversity at the fine scale necessary to perceive overall community differences between mineral substrates that are thoroughly mixed. In particular, the importance of microbial attachment to minerals in unconsolidated marine sediments remains poorly constrained despite extensive geobiological research in these settings. This study presents an approach for characterizing microbial colonization patterns using mineral separation techniques. Differences in density and magnetic susceptibility are used to enrich target minerals from bulk environmental samples, selecting for those minerals which may have importance as substrates for metabolic activity.
The application of this methodology to methane seep sediments of the Eel River Basin (ERB) on the California margin demonstrates that variations in microbial diversity between minerals are comparable to community differences across broad spatial scales and a range of porewater geochemistry. ERB colonization patterns determined by separation are shown to be reproducible and reflect in situ differences in the microbial community. Affinity of putative sulfide-oxidizing bacteria (primarily identified as Gammaproteobacteria) for mineral partitions enriched in authigenic sulfides suggests microbial attachment may reflect a metabolic role in sulfur cycling under reducing conditions. Mineral attachment is also shown to select between key archaeal phylotypes involved in the anaerobic oxidation of methane (AOM), providing insight into physiological differences between these uncultured groups. Preliminary results demonstrate that mineral attachment may be a significant factor in the microbial diversity of the marine subsurface, and that such community differences will be ecologically relevant.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Orphan, Victoria J.}, }