Graduate Fellowships

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This study assessed deep subsurface-to-surface transitional microbial communities in two serpentinizing ecosystems: a flaming gas/fluid seep in Yanartaş, Turkey and a bubbling fluid seep in Manleluag Spring National Park, the Philippines. These systems exhibited some similar geochemical and taxonomic attributes, but different physical properties that help demonstrate the effect surface conditions have on the subsurface. 16S rRNA gene sequencing, coupled with metagenomic and geochemical analysis, reveal dynamic microbial ecosystems supported partly by the products of active serpentinization. At Yanartaş, the fluid seeps may be ephemeral. Large travertine deposits are visible on the mountain slope as relicts of former fluid seeps. At Manleluag, the tropical climate causes monsoon and “dry” seasons, which influence dissolved inorganic and organic carbon input in the system. The 16S rRNA gene sequencing and metagenomic shotgun sequencing data suggest that, despite differences in regional climate and vegetation cover, Manleluag and Yanartaş exhibit taxonomic and functional similarities. Metabolisms involving methane, nitrogen, iron, and sulfur cycling, hydrogen oxidation, and respiration were detected in both 16S rRNA amplicon and metagenomic sequence datasets. Metagenomic analysis detected genes involved with osmotic and oxidative stress at both sites, and sporulation and dormancy genes at Manleluag. Several transposable elements were also reported at both Manleluag and Yanartaş. These mechanisms may allow subsurface microbial communities to adapt to surface conditions. In general, the surface and subsurface environment appear to be inherently connected at Yanartaş and Manleluag; the surface and subsurface both shape the microbial community in these ecosystems, and the microbial community alters the biogeochemistry.

The microbially mediated anaerobic oxidation of methane (AOM) is critical for regulating the flux of methane from the ocean. AOM is coupled to sulfate reduction (SR) in many anoxic marine environments, which has been extensively studied at cold seeps, hydrothermal vents, and the sulfate-methane transition zone at the seafloor. Sulfate-dependent AOM is performed by specialized groups of anaerobic methane-oxidizing (ANME) archaea, which are thought to form consortial relationships with sulfate-reducing bacteria (SRB). Certain ANME and SRB groups have been shown to occupy different ecological niches in both hydrocarbon seep and hydrothermal vent sediments. However, the environmental parameters that select for certain phylogenetic variants across these hydrocarbon-rich marine ecosystems are still unknown. In this study, we generated the largest dataset to date of 16S rRNA gene sequences for these uncultivable deep sea microorganisms using Illumina sequencing. Sediment strata were collected from the cold seeps of Hydrate Ridge, metalliferous sediments of Juan de Fuca Ridge, and organic-rich hydrothermal sediments of Guaymas Basin. We then used the Illumina MiSeq platform to assess archaeal and bacterial richness, diversity, and taxonomic composition followed by phylogenetic analyses of ANME and SRB phylotypes across environmental gradients and geographic ranges. Environmental metadata were used to establish the relationships between ANME and SRB phylotype distribution and environmental gradients as well as the extent of these functional groups in different hydrocarbon-rich ecosystems. Our results indicate that physicochemical constraints, particularly temperature and substrate availability, drive the distribution of different ANME and SRB ecotypes and the associated communities in spatially separated sites.

Light hydrocarbon gas mixtures are commonly found in organic-rich marine sediments. Methane (C1) is typically the dominant constituent in these mixtures, but ethane (C2) and propane (C3) are nearly always present in trace amounts. C1 dynamics are typically associated with either thermal cracking of deeply buried organic matter or the metabolic end-product of organic matter degradation. Ethane and propane production had typically been associated with thermocatalytic processes in deeply buried sediments, but limited studies suggested C2/C3 production in biogenic C1 gas mixtures was likely attributable to the activity of methanogenic archaea. However, very few of these studies looked at C1/C2 production in deep-sea sediments, and quantification of rates had either not been attempted, or were absent from the literature. We attempted to use organic-rich, cold seep sediments from the Green Canyon area of the Gulf of Mexico (GC600) to determine C2/C3 dynamics in the first ten meters of sediment (i.e. 0 – 10 m). We found C2/C3 production in near surface cold-seep sediments to be indistinguishable from the background degassing signatures of clay minerals. Surface sediments (i.e. < 4 m) are hypothesized to be dominated by communities of organisms that oxidize C2/C3 compounds, rather than communities that produce them. Experiments determining the controls and magnitude of C2/C3 oxidation in surface sediments in cold-seep environments are ongoing. We hypothesized that C2/C3 production likely occurs deeper in the sediment column (i.e. > 4 m), based primarily on ethane and propane profiles of similar environments. Such material proved difficult to acquire; efforts are ongoing to obtain deep piston cores (i.e. >10 m) for environmental profiling and experimental manipulation in the deeper sediment layers where C2/C3 production likely occurs.

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The project focused on the efficacy by which microorganisms can obtain nutrient Fe from silicate minerals. Silicate minerals are a particularly abundant mineral phase in the oceanic crust and thus the bio-availability of silicate-bound nutrients has important implications for microbial activity in the deep subseafloor (C-DEBI theme 1) and the limits to microbial life (C-DEBI theme 3). The specific goal of this project was to quantitatively determine how metal-binding organic compounds (siderophores) produced by microorganisms under Fe-limited conditions affect the rate of Fe-silicate mineral dissolution using laboratory experiments. The exact effect of microbial activity on Fe-silicate mineral dissolution has previously been hard to discern due to the complicating effects of feedbacks associated with microbial growth, siderophore production, and mineral dissolution rates. To limit the effects of these feedbacks, my experimental design used purified microbial siderophores and a silicate mineral (olivine) that dissolves at a rate that is relatively insensitive to the accumulation of its constituent ions in solution. My results showed that sub-millimolar siderophore concentrations lead to an order of magnitude increase in olivine dissolution rates. The accelerating effect of siderophores was linked to the removal of an inhibiting surface Fe-oxide coating that forms during the reaction of olivine at circum-neutral pH in the presence of O₂. By combining the experimental results with a numerical model of the relevant biological feedbacks, this work further constrained the maximum extent to which microbial activity may affect silicate mineral dissolution rates under conditions of Fe-limitation. The results of this study are presently under review for publication in Geobiology.

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The seafloor and subsurface microbial world represents a significant portion of life on our planet. The influence on its proximate ambience and global processes, such as element cycles, has potentially been largely underestimated and not always been precisely evaluated. I am interested in the nature of deep biosphere microorganisms in rocks from the Loihi seamount, Hawai’i, the East Pacific Rise, and the Juan de Fuca Ridge, as well as in sediments from North Pond (Mid-Atlantic). In order to assess microbial diversity, metabolic activity, adaptation strategies and biogeographical signatures in the deep subseafloor biosphere, metagenomics by pyrosequencing will be used to complement previous research efforts with the most in-depth and precise data that is available to date.

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The importance of microbial mediation in the biogeochemical cycles of the ocean is well documented. A major source of marine metallic minerals exists as ferromanganese (polymetallic) nodules in the deep ocean (4,000-5,000 m deep). Composed predominantly of iron, manganese, copper, nickel, and zinc, these nodules play a key role in governing the biogeochemical availability of many of these metals in the global ocean. While it is assumed that microorganisms mediate some of the processes that form nodules, it is poorly constrained as to which organisms mediate these processes or how these processes in turn may support microbial metabolisms. We propose using fingerprinting and sequencing methods to examine the microbial community diversity of organism associated with ferromanganese nodule collected from the South Pacific Gyre. Further, because many of the microbial organisms present in the deep-sea are novel and uncultivated, we plan to perform metagenomic analysis to link phylogenetic identity with physiology, with the goal of generating (near-)complete environmental genomes. The proposed research will be the first attempt to determine how the microbiology of deep oceanic nodules shape and are shaped by the environment.

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Most microbiology work in marine subsurface sediments has been focused in the upper 100-200 meters of sediment, partially because the switchover from Advanced Piston Coring (APC) to Extended Core Barrel (XCB) coring generally occurs around this depth, which leads to large increases in drilling-induced contamination. Molecular studies in deeper samples may be greatly hindered by the potential interferences from these contaminating microbes. This project provides an extensive next-generation sequencing based study coupling the analysis of microbial community composition to great depth in the Costa Rica Margin subseafloor to the analysis of the drilling fluid used in the process of obtaining those samples. In nearly all samples examined, the influence of drilling-fluid in molecular analysis of the sediment samples was very low, even in several samples taken with XCB coring. One sample from 496 mbsf was shown to contain a high proportion of sequences potentially originated from drilling fluid, however, which suggests that it is still important to routinely include comparison to drilling fluid as a control in molecular analyses. This study also provides a first and extensive look at the microbial community composition of the Costa Rica Margin subseafloor from 2 sites on the upper plate of the erosive subduction zone between the Cocos and Caribbean plates. These 2 sites, while in close proximity and sharing many physical and chemical properties, showed distinction in terms of the relative abundances of microbial groups, particularly in the proportion of archaea to bacteria. Additionally, correlations assessed between microbial taxa and geochemistry variables suggest directions for future research into the metabolic capabilities of some uncharacterized subseafloor microbial lineages.

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Over the course of two years of C-DEBI support, I have investigated subseafloor microbial ecology in three separate environments; the basaltic crust aquifer underneath the sediments of North Pond, the sediments of North Pond, and the sediments of the Iberian Margin at IODP site U1385. At North Pond, my research was primarily cultivation-based, with enrichments for multiple metabolisms across basalt and sediment samples. Shallow and deep heterotrophic isolates from the sediment column at site U1382B offer an opportunity to ask unique research questions regarding the breakdown of fresher, more labile organic carbon vs. older, more refractory organic carbon. At the Iberian margin, my research was primarily molecular-based, with several enrichment and cultivation efforts initiated after compelling evidence for particular metabolisms associated with individual groups of microbes. Diversity studies using high-throughput sequencing of 16S/18S rRNA amplicons examined the distribution and abundance of bacteria, archaea, and microbial eukaryotes. To further investigate ecological trends and the biology of particular community members, metagenomes were generated from the same DNA pools as the amplicon data.

During my tenure as a C-DEBI graduate fellow, my work fell into Research Theme I: Activity in the Deep Subseafloor Biosphere: function & rates of global biogeochemical processes. Few studies have directly measured sulfate reduction at hydrothermal vents, and relatively little is known about how environmental or ecological factors influence rates of sulfate reduction (SR) in vent environments. This work features laboratory studies using radioisotopic 35S-tracer techniques to elucidate rates of microbially mediated sulfate reduction from hydrothermal vents in the Middle Valley and Main Endeavor vent field along the Juan de Fuca Ridge, as well as assessments of bacterial and archaeal diversity, estimates of total biomass and the abundance of functional genes related to sulfate reduction, and in situ geochemistry. The rates of sulfate reduction measured suggest that-within anaerobic niches of hydrothermal deposits-heterotrophic sulfate reduction may be quite common and can contribute substantially to secondary productivity, underscoring the potential role of this process in both sulfur and carbon cycling at vents and the deep subsurface. Direct metabolic rate measurements of sulfate reduction help to facilitate defining key environmental and energetic parameters for microbial community colonization in hydrothermal vents. Better understanding of the metabolic and taxonomic relationship of these endolithic communities in the geochemical context of hydrothermal vents will help to constrain the microbial impact on global biogeochemical cycling.