A MOLECULAR ANALYSIS OF SUBSURFACE MICROBIAL COMMUNITIES ACROSS A HYDROTHERMAL GRADIENT IN OKINAWA TROUGH SEDIMENTS

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A MOLECULAR ANALYSIS OF SUBSURFACE MICROBIAL COMMUNITIES ACROSS A HYDROTHERMAL GRADIENT IN OKINAWA TROUGH SEDIMENTS

ABSTRACT

Decades ago, life in the deep subseafloor was assumed to be non-existent. At thousands of meters under the surface and decoupled from the photic zone, the deep marine world is a hostile environment. However, the discovery of life thriving around deep-sea hydrothermal vent systems revolutionized our perception of the extent and tenacity of life on Earth and set in motion a movement to understand life at the seafloor and its significant in global biogeochemical and nutrient cycling. Seafloor sediments host an incredible diversity of microbial life, and much interest has sought to understand the spatial and stratigraphic extent of the biosphere, and also taxonomic and functional capacities of such resilient organisms. This dissertation represents a series of studies centered around these concepts – I analyze samples from a sediment profile exposed to a hydrothermal gradient as a scaled-down proxy for how microbial life may exist and/or adapt to conditions as they become buried deeper into the subsurface.

I have focused on the application of culture-independent, molecular methods to understand whether the taxonomic and functional data reflect changes through this temperature gradient in support of a more temperature-adapted microbial community. In chapter 2, I examined the microbial community composition at approximately meter intervals by analyzing the taxonomically specific 16S rRNA marker gene. We presumed the biosphere to be restricted to only the upper 15 m, based on phylotype vetting and decreased sequencing recovery below. However, we observed a significant proportion of archaeal sequences throughout the 15 m, with a particularly high abundance in the deepest 15 m horizon. An in-depth look at the taxa at 15 m indicates an appearance of an uncultured, high-temperature taxon here, and an abundance of a thermophilic, methane-oxidizing archaeon, which suggests thermophilic niche at this particular temperature/depth regime. The exciting results from Chapter 2 were the motivation for a continued metagenomics analysis of select samples in the same sediment profile for Chapter 3.

 

Probing through total genomic DNA from six samples, I found evidence for temperaturedependent trends through the detection of genes of specific proteins involved in thermal processes, and attempted to correlate these genes with a taxonomic identity. I found that the deepest, hottest sample encompassed organisms from both thermophilic and hyperthermophilic temperature regimes. The dichotomy reflected between the existence of two temperature-specific niches implies that, due to the dynamic nature of the hydrothermal vent system, the deepest horizon may be undergoing a transition in temperature, thus, microbial community. Lastly, Chapter 4 was intended to provide a dataset from extractable RNA in order to distinguish representatives of the active microbial population from those represented from extant DNA; however, many challenges encountered in extraction and sequencing yield have restricted the dataset and ability to make reliable interpretations.

Considering the current state of knowledge in the marine subsurface due to its challenges in sampling, low biomass yield, and diversity distantly related to what is known from surface life, this work herein contributes greatly to our understanding of microbial biogeography in terms of temperature constraints in marine subsurface sediments. The challenges in phylotype vetting and need for quality controls speak to the degree of complexities in performing and interpreting molecular analyses from subseafloor sediments. Moreover, I have also produced significant datasets, both 16S rRNA gene and metagenomics, that can continue to be used for future investigations and comparisons of microbial life in the marine seafloor.

 

TABLE OF CONTENTS

LIST OF FIGURES ………………………………………………………………………………………………….. vii

LIST OF TABLES ……………………………………………………………………………………………………. xii

ACKNOWLEDGEMENTS ……………………………………………………………………………………….. xiv

Chapter 1  Introduction ……………………………………………………………………………………………… 1

The Subsurface Beneath the Seafloor …………………………………………………………………… 1

Challenges in Studying the Marine Subsurface ……………………………………………………… 2

Progress in Marine Subsurface Research ………………………………………………………………. 4

Temperature and the Extent of the Deep Biosphere ……………………………………………….. 6

IODP Expedition 331: Okinawa Backarc Basin and the Deep, Hot Biosphere …………… 9         Iheya North Hydrothermal Vent: Site Descriptions …………………………………………. 10

Dissertation Outline …………………………………………………………………………………………… 12

References ………………………………………………………………………………………………………… 13

Chapter 2  Marine subsurface microbial community shifts across a hydrothermal gradient

in Okinawa Trough sediments …………………………………………………………………………….. 19

Abstract ……………………………………………………………………………………………………………. 19

Introduction ………………………………………………………………………………………………………. 20

Experimental Procedures ……………………………………………………………………………………. 22

Sample Collection and Extraction …………………………………………………………………. 22

16S rRNA Gene Amplification and Sequencing of DNA …………………………………. 23

Analysis of 16S rRNA Gene Amplicons ………………………………………………………… 25

Analysis of Geochemical Data ……………………………………………………………………… 26

Correlation Analysis ……………………………………………………………………………………. 27

Results and Discussion ……………………………………………………………………………………….. 28

Domains of Life Represented in the Subsurface ……………………………………………… 28

Subsurface Prokaryotic Diversity ………………………………………………………………….. 31

Shifts in Subsurface Archaeal Relative Abundance …………………………………………. 36

Shifts in Subsurface Archaeal Taxa ………………………………………………………………. 38

Conclusions ………………………………………………………………………………………………………. 43

Acknowledgements ……………………………………………………………………………………………. 45

References ………………………………………………………………………………………………………… 46

Chapter 3  Temperature-dependent taxonomic and functional changes in microbial communities through a hydrothermal gradient in Okinawa Trough sediments …………… 54

Abstract ……………………………………………………………………………………………………………. 54

Introduction ………………………………………………………………………………………………………. 55

Experimental Procedures ……………………………………………………………………………………. 58

Whole Genome Amplification and Sequencing ………………………………………………. 58

Pipelines for Metagenomic Data Analysis ……………………………………………………… 59

Results and Discussion ……………………………………………………………………………………….. 61

General Taxonomic Changes through the Hydrothermal Gradient ……………………. 61

High Temperature Adapted Communities Detected through Functional Genes …… 65

Metagenomic Binning: Metabolic Insight into Individual Microbial Populations .. 77

Conclusions ………………………………………………………………………………………………………. 87

Acknowledgements ……………………………………………………………………………………………. 89

References ………………………………………………………………………………………………………… 90

Chapter 4  Investigating the Active Microbial Populations in Near Hydrothermal Vent

Sediments …………………………………………………………………………………………………………. 98

Abstract ……………………………………………………………………………………………………………. 98

Introduction ………………………………………………………………………………………………………. 98

Experimental Procedures ……………………………………………………………………………………. 100

RNA Extraction ………………………………………………………………………………………….. 100

Post-extraction Treatments of RNA ………………………………………………………………. 102

DNA Sequencing ………………………………………………………………………………………… 103

Analysis of 16S rRNA (Gene) Amplicons ……………………………………………………… 104

Results and Discussion ……………………………………………………………………………………….. 105

General Sequencing Yield ……………………………………………………………………………. 106

Contaminant Vetting: Taxonomic Classification of Sequencing Results ……………. 107

RNA dataset comparison to published DNA amplicons …………………………………… 116

Conclusions ………………………………………………………………………………………………………. 118

Acknowledgements ……………………………………………………………………………………………. 119

References ………………………………………………………………………………………………………… 120

Chapter 5  Conclusions ……………………………………………………………………………………………… 124

Appendix A  Supplemental Materials for Chapter 2 ……………………………………………………… 126

Supplemental Discussion of Methane Data …………………………………………………………… 129

Identification of External or Background DNA ……………………………………………………… 131

Taxonomic Classification Discrepancies ………………………………………………………………. 132

Supplemental Discussion of Amplicon Data …………………………………………………………. 132

Appendix B  Supplemental Information for Chapter 3 …………………………………………………… 138

Chapter 1

 

Introduction

The Subsurface Beneath the Seafloor

The Earth’s deep marine biosphere represents a frontier for investigating the extent, distribution, and perseverance of life. Buried beneath meters to hundreds of meters of sediments, microbial life is far removed from sunlight, strong oxidants, abundant organic matter, and other nutrients circulating in seawater (Orcutt et al., 2011). Yet, there is evidence of microbial life taking advantage of seemingly limiting and unfavorable conditions. Although the sedimentary subsurface microbial abundance is estimated to represent approximately 1-10% of Earth’s total living biomass (Kallmeyer et al., 2012), the importance of this ecosystem bridges the biological and geological element cycles, where microbial life at this interface is directly related to whether elements are sequestered over geologic time, or returned to the ocean as active components of biological, chemical, and climatic cycling (Hinrichs and Inagaki, 2012). The discovery of a vast subsurface community has channeled research efforts regarding energetic requirements and limits for life, the contribution of the subsurface on global biogeochemical cycling, and the potential for life in subsurface environments on other planetary bodies.

Marine sediments are the result of accumulating particles that sink to the seafloor from the overlying water column (Orcutt et al., 2011). As in the terrestrial realm, not all sediments are compositionally the same or are affected by the same geological and geochemical parameters. Thickness of sediments varies from relatively thin near newly formed crust at mid-ocean ridges and beneath low productivity zones (e.g. South Pacific Gyre), to kilometers-thick at trenches and highly productive continental margins (Orcutt et al., 2011). The chemical composition of sediments is also variable, depending on the origin of the deposited material, such as “rain” down of biological oozes, terrestrial matter delivered by rivers, or deposits from vent-derived minerals (Orcutt et al., 2011). This spatial geographic and geochemical diversity of the seafloor is responsible for creating a range of habitats and niches.

One of the more enigmatic relationships in marine subsurface research is the stratigraphic relationship of bioavailable organic matter and inorganic compounds with community composition through a sediment profile. In an early study looking at suboxic diagenesis of organic matter in the top 0-80 cm of pelagic sediments, pore water profiles indicated that oxidants were generally consumed in order of decreasing energy production per mole of organic carbon oxidized (Froelich et al., 1978), suggesting that these distinct redox zones were shaped by organisms with specific metabolic traits (Jorgensen et al., 2012). While this hypothesis has been supported by down-core stratification of specific microbial groups, such as anaerobic methane oxidizers (ANME) (Boetius et al., 2000) or anaerobic ammonium oxidizers (anammox) (Strous et al., 1999), predictions about microbial communities based on geochemical conditions have not been consistent across sedimentary marine environments (Jorgensen et al., 2012). Understanding the stratigraphic variability in geochemical and lithological properties with concomitant changes in the total microbial community structure and the relative abundance of individual taxa is an ongoing area of investigation and remains to be considered a complex relationship (Jorgensen et al., 2012).

Challenges in Studying the Marine Subsurface

There are many factors that make the exploration of subseafloor microbial communities particularly challenging. First, the opportunities to obtain samples from the ocean seafloor are limited by equipped drilling vessels or submersibles. Both sampling options are very expensive and require an experienced crew to operate and engineer the ship and equipment. For these reasons, there are many regions of the seafloor that remain completely unexplored, and our concept of the global marine subsurface relies on predicting or modeling those regions that are not sampled (Colwell and D’Hondt, 2013, Martino, 2014). In order to penetrate compacted sediments and rock layers within the ocean crust, scientists have relied on the drilling technology, which was not designed for sterile microbiological sampling. Thus, scientists have had to devise ways to ensure that the subsampling of cores has not been subject to external seawater or drilling mud contamination.

Aside from sampling logistics and cost, extremely low rates of cellular activity make laboratory cultivation very difficult (D’Hondt et al., 2002; 2009). Scientists have, therefore, been reliant on culture-independent analysis of nucleic acids to better understand community composition and functional potential within marine sediments. Often, the preliminary extractions and amplifications of environmental DNA and RNA from marine sediments are especially challenging in sediments where biomass densities are extremely low or in sediments with complex or altered clay compositions. Downstream taxonomic and phylogenetic datasets, in particular, rely on amplification of DNA and RNA marker genes via oligonucleotide primersets, which are designed from databases of known sequenced organisms. This can introduce biases in amplification by excluding “novel” lineages or intensifying signals of sequences that are compatible with the primer design (Colwell and D’Hondt, 2013). Within sequence data produced from extractable and amplifiable DNA, many taxa appear to be only distantly related to known representatives from pure cultures or surface environments (Sørensen et al., 2004; Inagaki et al., 2006; Lipp et al., 2008; Fry et al., 2008; Teske and Sørensen, 2008). Hence, when sequences are obtained, there are uncertainties and limitations in classifying and inferring metabolic capabilities within the subsurface communities.

Progress in Marine Subsurface Research

Advances in drilling recovery, molecular extraction methodologies, and sequencing technology over the past several decades have yielded high through-put datasets and more streamline analyses to better understand the micro- to macroscale relationships of the marine subsurface ecosystem. Investigative approaches in studying microbial diversity have advanced since the 1980s from laboratory isolations of organisms to analyses of macromolecules directly from environmental samples, as previously mentioned (Olsen et al., 1986 and Rappé and Giovannoni, 2003). This progression towards a cultivation-independent methodology has dramatically increased our knowledge of the unculturable community, estimated to represent approximately 99% of all microorganisms, due to the expansion of standard reference libraries (Rappé and Giovannoni, 2003). The transition of DNA sequencing chemistries and technologies from Sanger-based capillary sequencing to massively parallel, high-throughput “next-generation” sequencing has revolutionized ecological science. Large amounts of data, on the order of millions of sequence reads, at a relatively low cost per sequence yield have lead to a better representation of sample diversity (Shokralla et al., 2012). With current piqued interests in deep sea drilling, scientists have been able to apply these molecular tools directly to a suite of recovered, deep-sea sediment cores to answer questions regarding microbial populations and processes within the extent of the deep biosphere.

The Ocean Drilling Program (ODP) expedition to the Peru Margin (ODP Leg 201) was a pivotal breakthrough for marine subsurface research that involved concurrent molecular and geochemical analyses along meter-scale vertical sediment profiles (Jørgensen et al., 2006). The comprehensive dataset produced from ODP Leg 201 represents the first synthesis of correlating taxonomic and functional data from DNA and RNA with geochemical interpretations from approximately 87 m of recovered core. Sediment cores recovered from the Peru Margin were from underlying, highly productive surface waters off the Peru shelf and slope. The profiled microbial ecosystems appeared to be stratified with respect to geochemistry and stimulated at interfaces between seawater sulfate and methane (Jørgensen et al., 2006, Schrenk et al., 2010). Based on porewater chemistry interpretations, several horizons were predicted to be dominant biological methanogenic or methanotrophic zones, yet, known lineages of Archaea involved in these processes were sparse in the taxonomic analyses (Jørgensen et al., 2006). Instead, extractable 16S ribosomal RNA (rRNA) was classified within uncultured archaeal lineages that have cosmopolitan distributions across other marine environments (Biddle et al., 2006). Furthermore, the carbon isotopic composition of both archaeal cells and lipids from these sulfatemethane transition zones (SMTZs) suggested that bulk assimilation of carbon was derived from fossil organic matter, rather than methane. Overall, the results from multiple analytical approaches did not accurately reflect what had been initially hypothesized from initial porewater geochemistry profile.

The results from ODP Leg 201 demonstrate that the relationships between phylogenetic diversity, functional diversity, and geochemical interpretations in subsurface sediments are still poorly constrained. However, this study has brought to light the significance of Archaea in the marine subsurface as well as the potential eco-physiological flexibility of certain widespread, uncultured archaeal lineage. Since ODP Leg 201, deep biosphere research has continued to gain momentum across a range of marine environments, from the ultraoligotrophic sediments in the South Pacific Gyre (IODP 329), to a deep coal bed formation off the Shimokita Peninsula (IODP 337). Many ongoing studies are even focusing on biogeographical trends in specific uncultured archaeal lineages (e.g. representatives from the Miscellaneous Crenarchaeotic Group in Kubo et al., 2012) and their potential functional roles in the subsurface (e.g. Lloyd et al., 2013). In most cases, the utilization of next-generation DNA sequencing platforms has been a valuable and integral part of advancing our knowledge in marine subsurface ecology.

Temperature and the Extent of the Deep Biosphere

Within sediment profiles, cell concentrations generally decrease with depth, which is attributed to the decreasing porosity of sediments and bioavailability of organic carbon needed to fuel metabolic reactions and cell growth (Parkes et al., 2000). But, the extent and density of the biosphere vary across marine environments (Figure 1-1). One of the major factors limiting microbial distributions to great depths within the subsurface is the increasing temperature during burial (Parkes et al., 2000). The upper temperature limits of life for organisms (up to 122°C) have been studied in great detail from energy-rich hydrothermal vent fluids. However, temperature estimates associated with the more energy-limited sedimentary subsurface are derived from studies that indicate biodegradation of petroleum is inactivated at temperatures above 80°C (Wilhelms et al., 2001, Head et al., 2003). It has been suggested that this lower temperature fringe, relative to hyperthermophilic isolates, is linked to inadequate requirements to support rapid re-synthesis of essential biomolecules (Head et al., 2003). The study of hyperthermophilic communities in sediments, though, has been limited to the very surface and has not involved extensive taxonomic and metagenomic analyses.

 

Figure 1-1:  Source: Parkes et al., 2014. Depth (mbsf) distribution of prokaryotic cells in subsurface sediments at 106 locations, including 17 ODP/IODP Legs (black dots). Orange circles are mud volcano breccia samples, green circles are hydrothermal samples, purples circles are South Pacific Gyre sediments (Kallmeyer et al., 2012).

 

One of the first studies to investigate a possible hyperthermophilic community in marine sediments comes from the Parkes et al, 2000 compilation of ODP Juan de Fuca Ridge sediment cores from 1990s expeditions. Access to sediments that reach temperatures above 100°C would require up to 4 km of recovered sediment core, based on a geothermal gradient of 25°C/km. Juan de Fuca Ridge sediments were appealing in such an investigation because of their large thermal gradients, thus, high temperatures at relatively shallow sediment depth. Parkes et al., 2000 found that cell enumerations declined with depth, but persisted, or even increased, in certain high temperature intervals, which suggested that certain microbial life might occupy a unique hightemperature subsurface habitat resulting from mixing of recharging seawater and hydrothermal fluid. In an even broader sense, it is quite possible that certain groups of microbes are capable of persisting much deeper into the surface that what has been sampled. Thus, modeling exercises that incorporate microbial extent into the ocean crust should consider the possibility of these extreme communities. While the Parkes et al., 2000 study has extended the concept of the subseafloor biosphere to new depth, the techniques used would, today, be considered rudimentary and lacking comprehensive support of a hyperthermophilic subsurface biosphere. An expansion of the Parkes et al., 2000 using present-day molecular tools would be a pivotal step in understanding the relationship between 1) the putative temperature limit of life and life in subsurface sediments and 2) taxonomic, functional, and activity changes as a function of subsurface temperature limits.

Much like the Middle Valley sediments of Juan de Fuca Ridge, hydrothermal sediments represent few places in the crust where a similar physical environment (i.e. sediments) is in contact with multiple geothermal regimes (Biddle et al., 2012). At the sediment-water interface of most hydrothermal vents, microbial communities change rapidly across intense gradients over a much shorter spatial scale. But, the ability to analyze taxonomically similar microbial populations across a gentle temperature gradient has not been thoroughly studied in microbial ecology. Hydrothermal vent systems are formed at tectonic boundaries and mid-plate hot spots of volcanic activity. Fluids circulating through the crust experience extreme heating and water-rock interactions, resulting in a net gain of thermally energy and gaseous compounds (Orcutt et al., 2011). These evolved hydrothermal fluids, which are chemically reduced in comparison to source seawater, are transported to the surface, where they interact with seawater to often produce precipitated mineral deposits, or “chimneys” (Orcutt et al., 2011). Generally, most hydrothermal venting occurs in areas with little sediment cover on young oceanic crust, so sedimented systems, such as Guaymas Basin, Juan de Fuca Ridge, and the Okinawa Backarc basin are more of an anomaly than the norm. The Okinawa Backarc Basin hydrothermal system, in particular, is one of the few environments sampled that formed as a result of plate subduction, rather than a spreading center. In this PhD dissertation, I communicate the results of a comprehensive study that implements a suite of molecular analyses to answer profound questions about the sedimentary subsurface in the first survey of a hydrothermal vent system in a continental margin setting.

IODP Expedition 331: Okinawa Backarc Basin and the Deep, Hot Biosphere

The Integrated Ocean Drilling Program (IODP) Expedition 331 to the Okinawa backarc basin provided an opportunity to study the biosphere within the sedimentary subsurface of a deep, hot “subvent” system. The Okinawa Backarc Basin was formed as a result of the subduction of the Philippine Sea plate beneath the Eurasian plate. Generally, backarc basins are characteristic features of oceanic convergent plate boundaries. Unsurprisingly, hydrothermal vent networks have been documented here, and several studies have investigated surface microbiology and geochemistry immediately surrounding a few specific vents. In contrast to other backarc basins, the Okinawa Backarc Basin is the only example of a young backarc basin that has developed along a continental margin (Letouzey and Kimura, 1985).

The motivation for sampling the Okinawa Backarc Basin hydrothermal systems is twofold. Firstly, backarc basins inherently produce a significantly greater abundance of volatile species that are derived from devolatilization of the subducting slab than spreading center hydrothermal systems. Thus, backarc basins have a different composition of hydrothermal fluid and porewater in sediments. For example, the chemistry of hydrothermal fluids collected from active sulfide chimneys in the Okinawa Trough is characterized by higher concentrations of CO2, CH4, NH4, I, and K, and higher alkalinity than those in sediment-free, mid-ocean ridge hydrothermal fluids (Sakai et al., 1990a, 1990b; Gamo et al., 1991; Konno et al., 2006; Takai and Nakamura, 2001; Kawagucci et al., 2001; Takai et al., 2011). Secondly, IODP Expedition 331 is the first to core an active hydrothermal system within a sediment-filled backarc basin in a continental margin type setting. Most other sedimented hydrothermal vent systems (e.g. Guaymas Basin and Juan de Fuca Ridge) are associated with spreading centers. The sediment profiles sampled from the Okinawa Backarc Basin represent an environment that can be compared to sediments from both other margins and hydrothermal vent systems. Furthermore, this is an opportunity to better understand the changes in microbial life associated with sediments along a temperature gradient and also the extent or limits of the deep biosphere. Collectively, these factors suggest a new environment, in terms of sediment matrix, geochemistry, and potentially microbial community composition, which has not yet been considered in deep biosphere or global biogeochemical research.

Iheya North Hydrothermal Vent: Site Descriptions

The IODP Expedition 331 drilling efforts were focused around the active hydrothermal vent site within the Iheya North hydrothermal system (Figure 1-2). Three sites ~100, 450, and 1500 m east and one site ~600 m northwest of the active vent all showed significant differences of hydrothermal inputs from one another. No temperature data were collected from the closest site to the active vent, Site C0013. But, based on the recovered melted acrylide core liner that cored down to 12 mbsf, the temperature, likely, was well over 82°C (onboard testing later found that these core liners began to deform above 82°C) within that core section. Site C0014, approximately 450 m away from the active vent, had an interpolated temperature gradient of

3°C/m  (Figure 1-3). The furthest site, C0017, had a temperature gradient of approximately 0.5 °C/m (Figure 1-3). Both temperature profiles from Sites C0014 and C0017 display irregularities suggestive of lateral flow. The concave upward profile at Site C0017 is consistent with recharge of cold seawater into the system at this site. On the contrary, the temperature and geochemistry profiles indicate that Site C0014 represents a moderate hydrothermal mixing zone in the subsurface. Site C0015 was upslope 600 m from the active vent on a mound, where many colonies of dead deep-sea mussels and pavements of carbonate and/or sulfate crusts were found. The crest of this hill has been hypothesized to represent an enormous methane seepage field in the past.

Figure 1-2:  Source: Takai et al., 2011. Area map of Iheya North Knoll showing Sites C0013C0017 (stars) drilling during Expedition 331. Inserts show the Iheya North Knoll in relation to Okinawa and Okinawa in relation to major tectonic components. EUR = Eurasian plate, PHS = Philippine Sea plate.

 

Figure 1-3:  Source: Yanagawa et al., 2013. Depth profile of temperature at Sites C0014 and C0017, as measured by the APCT-3 (yellow) temperature shoe and thermoseal strip taped to the outer surface of the core liner. The stars represent the error associated with the thermoseal strips. Site C0017 is representative of a non-hydrothermal continental margin sediment profile.

 

Dissertation Outline

In the following sections I discuss a taxonomic and functional profiling of the continental margin-type sediments impacted by the Iheya North Field subsurface hydrothermal system in the Okinawa backarc basin. I first use a commonly practiced approach of comparing the 16S rRNA taxonomic marker gene among Site C0014 with one from Site C0015 to determine general trends in microbial community composition with depth. I also incorporate porewater geochemical data and published IODP Expedition 331 Proceedings results to make interpretations whether the community composition reflects changes along the temperature profile, or the temperature gradient is too strong or quickly progressing for an adapted biosphere to be established. The subsequent chapter builds off of these results in an analysis of metagenomic data from several depth horizons with the intent to capture relevant metabolic and functional changes through this hydrothermal gradient. I use several data mining approaches to better understand how microbial processes and adaptations are different among several horizons along this transect. The last section was initially intended to use taxonomic information from extractable RNA as a proxy for active microbial populations through the Sites C0014 and C0017 sediment profiles; however, laboratory and sequencing challenges have restricted the dataset and interpretations comparing the indigenous, active microbial community from RNA to that from extant DNA.

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A MOLECULAR ANALYSIS OF SUBSURFACE MICROBIAL COMMUNITIES ACROSS A HYDROTHERMAL GRADIENT IN OKINAWA TROUGH SEDIMENTS

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