Award Type: Graduate Fellowships

Abstract

My research focused on developing quantitative and theoretical frameworks to better understand microbial diversity. I explored topics which include:

1. How microbial community diversity influences genome (metagenome-assembled genome) discovery in short read sequencing studies. I proposed using a coupon collector, a general probability sampling model, to predict the sequencing effort needed to sequence rare genomes.
2. Evaluating the relationship between microbial lineage and genomic trait composition. I evaluated ~120,000 publicly available genomes for patterns in cluster of orthologous groups with respect to taxonomy. Taxonomic ranks spanning domain through genus had significant explanatory power on how traits are distributed across the tree of life. More so, this indicates taxonomy, even at higher taxonomic resolutions, can act as a proxy for certain genomic features.
3. Quantitatively measuring how disperse microbial traits are across microbial communities. Traits are said to be more functionally redundant when possessed by more community members. I applied concepts from traditional diversity theory to measure how evenly microbes in communities contribute to an aggregated ecosystem function.

Abstract

Along the eastern flank of the Juan de Fuca Ridge (JdFR), an array of boreholes drilled through sediments and into subseafloor basalt are fitted with CORKs (Circulation Obviation Retrofit Kits) that hydrologically seal off boreholes from sediment pore water and enable direct sampling of pristine basalt-hosted fluids. In this system, the members of the bacterial phylum Nitrospirae, which are closely related to the thermophilic anaerobic sulfate-reducing Thermodesulfovibrio, are among the most abundant bacteria. However, the metabolic and evolutionary profile of this microbe in this ridge system has yet to be fully elucidated. Here, we analyzed previously extracted and sequenced crustal fluid metagenomes from 2 CORKS (U1362A and U1362B) with total lengths of ~269Mbp and ~140Mbp, respectively, post quality control. The 2 metagenomes were assembled into scaffolds in metaSPades and assessed via read recruitment. Read recruitment results were further assessed, and visualized using a suite of programs in Anvi’o. Binning of scaffolds and subsequent manual refinement of bins were done based on the hierarchical clustering of tetra-nucleotide frequencies and GC content of scaffolds via the Anvi’o interactive and refine interface. Bins taxonomically identified as Nitrospirae using the GTDB-Tk classify workflow were placed in a separate Anvi’o collection, manually refined and assessed for completion and contamination using ‘–anvi-summarize’. Based on the preliminary analyses, we have identified seven bins in U1362A and 2 bins in U1362B belonging to the Nitrospirae phylum. All JdFR Nitrospirae bins have >50% completion and <5% redundancy. Also, the presence of two distinct GC contents (~40% vs 60%) of Nitrospirae may suggest that different populations exist within and between samples.

Abstract

Members of the Archaeal genus Thermococcus are sulfur-dependent hyperthermophiles found in hydrothermal vents throughout the world. Previous analysis of a Thermococcus culture collection containing isolates from the Juan de Fuca Ridge, Gorda Ridge, and South East Pacific Rise using amplified fragment length polymorphism analysis and multilocus sequence typing revealed a distinct clade of Thermococcus isolated from the 1996 megaplume event at Gorda Ridge, indicating that they originated from a deep-subsurface habitat. The aim of this study was to elucidate the functional adaptations that allow for the enhanced survival of the Gorda Ridge clade in the deep-subsurface as compared to representative Thermococcus isolates from shallow subsurface habitats. This was accomplished through a pangenomic analysis of isolates from within this clade along with other representatives from this culture collection. The Gorda Ridge megaplume group was enriched for genes relating to DNA repair and stabilization including a predicted endonuclease distantly related to Archaeal Holliday junction resolvase, DNA mismatch repair ATPase mutS, CRISPR/Cas elements, and dnaK (hsp70). This group was also enriched for the ABC-type branched-chain amino acid (BCAA) transport system, enzymes for the Shikimate pathway for aromatic amino acid synthesis, as well as TupA for tungstate transport. This suggests that Thermococcus populations inhabiting deep-subsurface fluid reservoirs require the ability to prevent and repair damage to their DNA, presumably due to the energy demands of DNA replication. The enrichment in BCAA and tungstate transporters may indicate the use of an amino acid catabolism pathway followed by fermentation catalyzed by the tungstopterin containing enzymes aldehyde ferredoxin oxidoreductase and alcohol dehydrogenase, suggesting a preference for peptides over
carbohydrates as an energy source in the deep-subsurface.

The opportunity to sequence, analyze and compare genomes from within the Gorda Ridge megaplume group along with other Thermococcus isolates has revealed several functional adaptations unique to isolates from the deep-subsurface habitat. These functions largely represent either DNA repair mechanisms, high-temperature protein folding capabilities, and/or strategies for amino acid utilization.

Abstract

The oceanic crust near mid-ocean ridge spreading centers is a rich source of reducing chemistries and biologically relevant energy sources. It is here that countless microbial metabolic strategies crowd the hydrothermal emissions billowing into the sea, having reacted with the mantle-derived rock deep within. The relative energetic payoff of disparate strategies—a function of electron donor/acceptor availability—changes as a function of host rock composition, temperature, and water-to-rock ratio. This project has constructed chemical maps of these subsurface environments to all realistic extents of these variables, depicting how subtle changes in each generates changes in solute flux and energetic payoffs.

Abstract

Sediment underlying ocean gyres receives minimal input of fresh organic matter yet sustains a small but active heterotrophic microbial community. The concentration and composition of the organic carbon (OC) available to this deep biosphere however is unknown. We analyzed the content and composition of OC in pelagic sediment in order to identify mechanism(s) that dictate the balance between OC preservation and utilization by microorganisms. Sediment cores from the North Atlantic gyre (KN223), South Pacific Gyre (Knox02-RR), and Peru Basin (IODP site 1231) allowed for a global comparison and a test of how sediment lithology and redox state affect OC preservation. OC was present in low concentrations in all samples (0.01—0.61%), at depths up to 112 meters below seafloor and estimated sediment ages of up to 50 million years. Synchrotron-based near edge X-ray absorption fine structure (NEXAFS) spectroscopy was conducted on over 100 samples, one of the first applications of NEXAFS to sedimentary environments. NEXAFS revealed an OC reservoir dominated by amide and carboxylic functionalities in a scaffolding of O-alkyl and aliphatic carbons. Detection of extractable, extracellular proteins supports this composition and suggests that sedimentary OC is protein-derived. This composition was common across all sites and depths, implicating physical rather than chemical mechanisms in OC preservation on long timescales. This study thereby points to physical access rather than energy or metabolic potential as a key constraint on subsurface heterotrophic life.

Abstract

The subsurface biosphere represents one of the final frontiers on Earth and may provide a model for how life can survive on other planets. While focusing on terrestrial subsurface and depths of over 3,000 meters underground, we present and apply a variety of computational tools and techniques for exploring the deep biosphere. Life at this depth is scarce and nutrients are often limited to hydrogen, sulfate, and single carbon compounds such as methane, carbon monoxide and carbon dioxide. Metagenomics and other sequencing techniques shed light onto how subsurface microorganisms transform these limited nutrients into energy and survive underground.

When appropriate, such methods are combined with geochemical measurements and thermodynamic predictions to provide the most accurate picture of life underground. In that direction, we find that the South African subsurface fluids exhibit a spectrum of redox conditions (influenced by the origin and age of the subsurface fluids) that directly affect the microbial community composition and function. Although these large-scale community shifts are believed to occur over long periods underground, we also present evidence for an adaptive methane oxidizing community that responds to changes in geochemistry over relatively short periods. Combined, these results provide a picture of how microbial communities function in the terrestrial subsurface as well as a theoretical framework for understanding the selective pressures these organisms face.

Abstract

Igneous oceanic crust contains the largest aquifer on earth (Johnson & Pruis, 2003). The basaltic layer contains ~2300 m² kg^-1 of surface area that supports an extensive subsurface microbial ecosystem (Heberling et al., 2010; Nielsen & Fisk, 2010; Santelli et al., 2008).  Biological activities in this ecosystem can impact global carbon cycling and increase productivity in the ocean (Edwards et al., 2011; McCarthy et al., 2010). Previous studies of surface layer rocks and mineral deposits have suggested that mineralogy dictates microbial community structure in the upper oceanic crust (Flores et al., 2011; Sylvan et al., 2013; Toner et al., 2013) and subseafloor planktonic communities have been previously investigated (Cowen et al., 2003; Jungbluth et al., 2014, 2013); however, the role of mineralogy and composition in the attached community that comprises the bulk of the habitable zone in the subseafloor has not been previously determined. Planktonic and mineral-attached microbial communities in aquifers are distinct from one another (Lehman, 2007), and investigating the attached community will lead to a more holistic view of this largest crustal aquifer. As a C-DEBI graduate fellow, I analyzed microbial communities attached to igneous minerals and glasses incubated in IODP Hole 1301A of the Juan de Fuca Ridge. I found that Archaeal communities on olivine (a Fe-bearing silicate mineral common in the crust) were distinct from those on other common minerals and glasses, while bacterial communities were influenced by mineral composition. Using olivine bioreactors in the laboratory, I found that isolated subseafloor microbes increased the dissolution of olivine and promoted mineralization of secondary phases, potentially influencing biogeochemical cycles in the ocean. Finally, preliminary metagenomic analysis of attached communities from the JFR indicate that genes for fermentation, one-carbon metabolism, protein synthesis, aromatic compound metabolism, respiration, and fatty acid metabolism are more abundant in the subseafloor than other subsurface or marine environments. These new insights into the attached subseafloor biosphere, through support from C-DEBI, are helping to propel the field of deep biosphere research into a new era and challenging our view of this extensive subseafloor biosphere.

Abstract

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.

Abstract

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.

Abstract

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.

Abstract

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.

Abstract

Heterotrophic organisms are central to subsurface microbial communities and play an important role in carbon cycling. Most approaches to measuring enzymatic activities rely on the addition of a fluorescently labeled substrate to a sediment incubation. However, quantifying rates of extracellular enzymatic hydrolysis of organic matter is often problematic due to the tendency for a fluorescently labeled organic substrate to sorb to the sediment matrix. This results in lower fluorescence intensities and distorted, inaccurate hydrolysis rate calculations. In this project, a desorption treatment was developed to counteract the adverse effects of sorption on enzymatic activity measurements. Upon subsampling a sediment incubation amended with a fluorescently labeled substrate, the subsample is treated with a concentrated solution of unlabeled substrate, along with 0.2% sodium dodecyl sulfate (SDS), in order to competitively desorb the adsorbed, fluorescent substrate target. This treatment improves measured fluorescence intensities by a median of 62.5%, and is particularly effective at desorbing high molecular weight substrate products, resulting in debiased hydrolysis rates that are 14.75 nM/hr lower on average. Competitive desorption treatment was demonstrated to be effective for multiple substrates and in a broad range of sediments from diverse geological and geochemical contexts. Future applications of this method will result in more quantitative and comparable hydrolysis rates in subsurface sediments, will enable enzymatic activity measurements in problematic sediments that were previously infeasible, and will facilitate physiological characterization of microbial communities and model organisms in order to better understand heterotrophic carbon cycling in the subsurface environment.

Abstract

The Iheya North Hydrothermal Field in the Okinawa Backarc Basin represents an ideal environment in which to investigate the biotic temperature fringe of microbial life at depth because of its subsurface hydrothermal activity within its continental margin-type sediment profile. Geographically, the Okinawa Backarc Basin is situated along a continental margin, which is a sediment profile type commonly sampled and studied across the seafloor (e.g. Peru Margin, Costa Rica Margin, Cascadia Margin). The hydrothermal network within the subsurface here supplies an additional temperature obstacle to microbial life existing in the sediments. In particular, the sediment profile at Site C0014 exhibits a transition from hemipelagic ooze with pumiceous volcaniclastic sediments and low temperature (4°C) to a hydrothermally altered sequence of clays within the top ~10 mbsf of sediment. Temperature measurements indicate a gradient of approximately 3°C/m, which is roughly an order of magnitude greater than continental margin sites (e.g. Cascadia Margin, IODP 311 and Costa Rica Margin, IODP 344), but is more gradual than intense, centimeter-scale gradients from other hot, surface sediments. We have focused on the application of culture-independent, molecular methods to understand taxonomic and functional characteristics through this hydrothermal gradient. Confidence in DNA recovery suggests a microbial biosphere extent of approximately 15 mbsf (55°C). Results from both 16S rRNA gene surveys and metagenomics analyses suggest a temperature-dependent stratigraphy of taxonomic and functional adaptations between the shallowest and deepest sample horizons. Cosmopolitan marine subsurface bacterial and archaeal taxa are present throughout the top 10 mbsf, whereas, hyperthermophilic heterotrophic as well as thermophilic anaerobic methanotrophic archaea appear in varying local abundances in deeper, hydrothermal clay horizons.

Abstract

Subglacial lakes were discovered beneath the Antarctic Ice Sheet in the 1970’s and, given the presence of liquid water and saturated sediments, it has been debated whether or not these deep, cold biosphere habitats harbor active microbial communities. Subglacial Lake Whillans (SLW) was cleanly sampled in January 2013 with the goal of establishing the habitability and presence of life beneath the Antarctic Ice Sheet. The aim of this graduate fellowship project was to further characterize the SLW carbon cycle, in particular, chemolithoautotrophic microbial processes through geochemical and microbiological methods. Geochemical analyses showed that sulfide oxidizing bacteria were active and contribute to mineral weathering in the surficial sediments of SLW. Long water residence times beneath the West Antarctic Ice Sheet (WAIS) create a mineral weathering regime in SLW that is distinctly different from subglacial habitats of mountain glaciers. Concentration and stable isotope measurements of methane confirm a reservoir of methane formed by methanogenic archaea beneath the WAIS. The modeling results show that this biological methane provides a source of energy to an active methane oxidizing population at the sediment-water interface. The methane also is modeled to be an important source of carbon for biomass synthesis in the methane oxidizing population, with rates of biomass incorporation similar to that of ammonia oxidizing archaea in the SLW water column. These results provide evidence that the sub ice sheet environment provides favorable conditions and substrates to support an active microbial ecosystem, thus expanding the extent of the biosphere to include the area beneath the WAIS and, possibly, the entire Antarctic Ice Sheet.

Abstract

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.

Abstract

The aim of this proposal is to gain a better understanding of what subsets of proteins are actually being expressed during neutrophilic, microbial iron (Fe)-oxidation. The recently isolated Mariprofundus ferrooxydans, strain PV-1, will be used as a marine model organism to investigate proteomic differences under different Fe substrates: aqueous Fe²⁺ and solid Fe⁰. Two-dimensional gel electrophoresis (2D-GE) and shotgun proteomic methods (LC-MS/MS) will be employed to obtain results from the cultures grown under different conditions. The research being proposed would constitute the foundation for the development of diagnostic tools for the accordance, distribution, and activity level of Fe-oxidation, a globally important biogeochemical process at and below the ocean floor.

Abstract

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.

Abstract

Shallow subsurface temperatures can reach extreme levels in just 40 cm depth in Guaymas Basin sediments, limiting microbial colonization to thermally tolerable surface sediments. At temperatures beyond approximately 80°C and 100°C, respectively, the ¹³C-isotopic signatures of microbial anaerobic oxidation of methane and organic matter remineralization appear to be thermally restricted. Putative methane consuming archaea dominate the archaeal clone library while sulfur cycling bacteria and Chloroflexi-related sequences dominate the bacterial clone library. Archaeal clone library data suggest that the ANME-1 Guaymas archaea tolerate high in situ temperatures up to approximately 80°C, thereby gaining an advantage in access to the geothermal methane pool in hot Guaymas Basin sediments. Lastly, the results indicate that in situ thermal and/or geochemical gradients structure archaeal community composition and biogeography more than bacterial biogeography. While the average upper thermal temperature for detectable microbial life by RNA recovery in Guaymas Basin sediments appears to be around 80°C, temperatures may fluctuate by 25°C in as little as a day. Isotopic evidence for microbially mediated methane oxidation is only slight, yet putative methanotrophic archaea are commonly recovered in nearly all samples suggesting they may perform other physiological modes or isotopic signatures are not detectable because of high methane concentrations. High temperature associated archaea appear to be represented by OTUs related to uncultured MCG and ANME-1 Guaymas groups. For bacteria the dominant high temperature associated OTU was phylogenetically associated with the Thermodesulfobacteriaceae. Two of the four main themes of C-DEBI research are “Extent of Life” and “Limits of Life”. Using sediment samples acquired from Guaymas Basin, my C-DEBI research links these two themes by examining how the biogeographical distribution of sedimentary microorganisms is shaped by severe, life-limiting conditions. Although these samples are not from deep sediments, they exemplify deep biogeochemical processes that have been compressed to shallower depths by elevated hydrothermal activity. My research demonstrates how thermal and geochemical regimes interact to control the spatial extent of life by focusing on microbial zonation in an energetically diverse hydrothermal environment. My intention with this research was to accurately describe microbial biogeography and the physicochemical factors controlling it in these unique, compressed sediments, which can be a useful asset in preparation for future IODP sampling procedures and analyses as well as investigations in deep subsurface microbiology around the world.

Abstract

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.

Abstract

Phosphorus (P) is an essential nutrient that can limit biological activity in some environments. Yet, many components of its cycle remain unclear, including P uptake and cycling in deep-sea sediments. These are critical, since a significant portion of Earth’s prokaryotes thrives in deep marine sediments, which are thought to mainly contain low bioavailable P in mineral phases. This suggests that microorganisms possess mechanisms to utilize recalcitrant P pools. The work performed focuses on identifying possible P sources to the deep biosphere, as well as microbial uptake mechanisms using stable oxygen isotope ratios in phosphate and ³¹P nuclear magnetic resonance spectroscopy on sediment samples collected during IODP 336 Expedition (North Pond).

Abstract

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.

Abstract

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.