Recent Findings

Environmental controls on intragroup diversity of the uncultured benthic archaea of the miscellaneous Crenarchaeotal group lineage naturally enriched in anoxic sediments of the White Oak River estuary (North Carolina, USA)
Lazar, Cassandre Sara;Biddle, Jennifer;Travis Meador, Travis ;Blair, Nic;Teske, Andreas P.;Hinrichs, Kai-Uwe Environmental Microbiology, (2015). DOI:10.1111/1462-2920.12659

Developing Deep-Life Continental Drilling Projects
Onstott, Tullis;Kieft, Thomas Eos, Transactions of the American Geophysical Union, (2015). DOI:10.1029/2015EO024937

Intact preservation of environmental samples by freezing under an alternating magnetic field
Morono, Yuki;Yamamoto, Yuhji;Xiao, Nan;Hirose, Takehiro;Sugeno, Masaya;Ohwada, Norio;Terada, Takeshi;Inagaki, Fumio Environmental Microbiology Reports, (2015). DOI:10.1111/1758-2229.12238

Iron reduction by the deep-sea bacterium Shewanella profunda LT13a under subsurface pressure and temperature conditions
Picard, Aude;Testemale, Denis;Wagenknecht, Laura;Hazael, Rachael;Daniel, Isabelle Frontiers in Microbiology, (2015). DOI:10.3389/fmicb.2014.00796

Low frequency of endospore-specific genes in subseafloor sedimentary metagenomes
Kawai, Mikihiko;Uchiyama, Ikuo;Takami, Hideto;Inagaki, Fumio Environmental Microbiology Reports, (2015). DOI:10.1111/1758-2229.12254

Origins of lithium in submarine mud volcano fluid in the Nankai accretionary wedge
Nishio, Yoshiro;Ijiri, Akira;Toki, Tomohiro;Morono, Yuki;Tanimizu, Masaharu;Nagaishi, Kazuya;Inagaki, Fumio Earth and Planetary Science Letters, (2015). DOI:10.1016/j.epsl.2015.01.018

Presence of oxygen and aerobic communities from seafloor to basement in deep-sea sediment
D'Hondt, Steven;Inagaki, Fumio ;Zarikian, Carlos;Abrams , L J;Dubois , N;Engelhardt , T;Evans, Helen;Ferdelman, Timothy G.;Gribsholt , B;Harris, R N;Hoppie, B W;Hyun, J H;Kallmeyer, Jens;Kim, Jinwook;Lynch , J E;McKinley , C C;Mitsunobu, S;Morono, Yuki;Murray, R W;Pockalny, Robert;Sauvage , J;Shimono, T;Shiraishi, F;Smith, David C;Smith-Duque, C;Spivack, A J;Steinsbu, B. O.;Suzuki, Y.;Szpak, M;Toffin, L;Uramoto, Go-Ichiro;Yamaguchi, Takashi;Zhang, Guoliang;Zhang, Xiao-Hua;Ziebis, W Nature Geoscience, (2015).

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Projects

A Cold-Tolerant, Acetogenic, and Lithoautotrophic Microbial Community in a Subglacial Lake

Project Investigators: Gaidos, Eric; Marteinsson, V T

Start Date: 2011-01-01

End Date: 2013-12-31

Summary:

The east Skaftá lake is one of three perennial lakes maintained by volcanic heat under a 300- to 400-meter-thick ice cap in Iceland. These lakes are permanently in the dark, maintained at 0-4°C by melting ice, and are anoxic and sulfidic (small quantities of oxygen may be introduced by meltwater). Our investigations of the water column of this lake and the neighboring western lake have revealed unusual communities dominated by a few taxa, among them one most closely related to a lithoautotrophic, psychrotolerant homoacetogen. Homoacetogens convert carbon dioxide and hydrogen into acetate (CH3COOH) and compete with hydrogenotrophic methanogens for substrate. Acetogenesis may be a major pathway of carbon into these lakes. The objectives are: (a) measure any spatial structure in the microbial community with depth and location; (b) compare the east and west communities to evaluate the hypothesis that there is exchange between them; and (c) determine whether any lake taxa appear in a sample of water flowing out from underneath the ice cap.

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A Window to the Deep Biosphere: Investigating Succession and Functional Shifts in Marine Deep Subsurface Microbial Communities Exposed to the Seafloor by Mud Volcanism

Project Investigators: Boetius, Antje; Ramette, Alban ; Felden, Janine ; Knittel, Katrin

Start Date: 2011-01-01

End Date: 2013-12-31

Summary:

Mud volcanoes are commonly called “windows to the deep biosphere”, because they represent surface expressions of a rapid upward transport of subsurface sediments of as deep as a few kilometers. On land and in the sea we know of tens of thousands of active mud volcanoes, which expel muds, anoxic fluids, gases, and hydrocarbons. The first microbiological investigations of submarine mud volcanoes have indicated that the subsurface muds are inhabited by typical subsurface microorganisms (e.g., marine benthic crenarchaeal groups, Cytophaga/Flavobacteria groups), but that they are overgrown by anaerobic and aerobic methanotrophs after exposure to electron acceptors such as sulfate and oxygen. However, the time scales at which this succession occurs, the fate of the original deep biosphere communities and the potential for their dispersal remain unknown. In 2009 and 2010, we had the opportunity to sample transects across very recent mud flows at the Håkon Mosby Mud Volcano (HMMV), including a major flow from a recent eruption. We propose to analyze samples of different sediment depths, spatial and temporal distances from the fresh mud flow for both their bacterial and archaeal communities to improve knowledge on the composition, persistence, and dispersal of deep biosphere communities which are exposed to surface environments.

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Alkaline springs of the Voltri Massif, N. Italy

Project Investigators: Lilley, Marvin; Brazelton, William; Lang, Susan; Schrenk, Matthew; Bernasconi, Stefano ; Schwarzenbach, Esther

Start Date: 2014-01-01

End Date: 2014-12-31

Summary:

We are conducting comparative studies that focus on geochemical and microbial processes during serpentinization and the precipitation of carbonate (±brucite) in two high alkaline, ultramafic environments: the active marine Lost City hydrothermal system on the Mid-Atlantic Ridge (MAR, 30°N), and high alkaline, Ca-OH springs associated with present-day serpentinization and carbonate deposits in the Voltri Massif (Liguria, N. Italy). Since 2009 we have sampled fluids and carbonates at twelve alkaline (pH 10-11.6) springs and investigated volatile content, organic and stable isotope geochemistry, and microbiology.

Our field studies are designed to evaluate carbon cycling and microbial metabolisms and thus have focused on co-registered rock and fluid sampling for biological and chemical analyses. We have conducted laboratory analyses to fully characterize the carbon content of the serpentinites, the springs, and the carbonate deposits.   By comparing a modern marine system with a modern meteoric environment, we aim to evaluate the importance of abiotic versus biotic processes in the origin of organic compounds associated with serpentinization and to address the question as to whether Lost City is a good bio-geochemical analogue for present-day serpentinization processes on land and in ancient marine systems.

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Andaman Sea Deep Marine Sediment

Project Investigators: Colwell, Frederick S.; Briggs, Brandon

Start Date: 2010-05-01

End Date: 2015-01-01

Summary: This Census of Deep Life grant examined the relationship of microbial community diversity to environmental parameters in deep methane hydrate-bearing marine sediments. Analysis of the microbial communities using deep DNA sequencing technology provided an assessment of the key microbes in these samples where microbially-produced methane accumulates in hydrates.

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Application of 454 Tag Sequence Technology to Novel Terrestrial Deep Subsurface Habitats of the North American Basin and Range, Black Hills, and Canadian Shield

Project Investigators: Moser, Duane; Sherwood Lollar, Barbara; Bruckner, Jim ; Anderson, Cynthia

Start Date: 2011-01-01

End Date: 2013-12-31

Summary:

This project targets deep microbial ecosystems of three significant, mostly unexplored (for microbiology) North American geological provinces: the Basin and Range, Black Hills, and Canadian Shield. In addition to their value for the exploration of novel deep habitats, these samples also allow us to address the following questions: (1) Is everything truly everywhere? (2) How does one recognize indigenous deep life? and (3) Is it possible via existing excavations to sample below the lower biosphere limit? How these project objectives are responsive to the key research questions outlined in the RFP are detailed below.

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Biogeographic Comparison of Subsurface Microbial Habitats Associated with the Serpentinization of Uplifted Mantle Materials

Project Investigators: Brazelton, William; Schrenk, Matthew

Start Date: 2011-01-01

End Date: 2013-12-31

Summary:

Ultramafic rocks in the Earth’s mantle represent a tremendous reservoir of carbon and reducing power. Upon tectonic uplift and exposure to fluid flow, serpentinization of these materials generates copious energy, sustains abiogenic synthesis of organic molecules, and provides opportunities for subsurface microbial communities to interact with deep carbon pools. To date, however, the “serpentinite microbiome” is poorly constrained -- almost nothing is known about the microbial diversity endemic to rocks actively undergoing serpentinization. We will compare 16S rRNA pyrotag sequences from subsurface fluids collected from three geographically distinct continental sites of active serpentinization. We also request sequencing of serpentinite drill cores from one of the continental sites for comparison with its companion fluids and with archived samples of seafloor serpentinites collected from the Lost City Hydrothermal Field. In the context of an array of microbiological, geological, geochemical, and isotopic data, this work will greatly expand our knowledge of microbial diversity in both continental and marine subsurface environments and be used to develop tools to quantify the functions, activities, and evolution of microorganisms in serpentinite ecosystems.

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Biophysical and Biochemical Characterization of Novel Piezophilic Cultivars from Deep Subsurface Environments

Project Investigators: Hazael, Rachael; Daniel, Isabelle; McMillan, Paul; Bartlett, Douglas; Morgan-Smith, Danielle; Foglia, Fabrizia; Narasingarao, Priya

Start Date: 2012-01-01

End Date: 2014-06-30

Summary:

This sub-project of the RHC grant sought to determine underlying physical-chemical principles relating to the functioning of life under high hydrostatic pressures. The work required adaptation of existing technologies for high-pressure biophysical research to be compatible with relevant deep biosphere conditions. The work attempted to obtain both a fundamental description of biological systems at high pressure as well as integrating new cultivars and insights provided by other areas of the RHC project.

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C-Fe-Mn Interactions Within Suspended Particulates in North Pond Basement Fluids

Project Investigators: Glazer, Brian T

Start Date: 2014-06-01

End Date: 2014-11-30

Summary:

In recent years, there has been growing interest in exploring the interactions between chemically reduced basaltic rock and a hydrologically active (often oxic) subseafloor aquifer, from the perspective of small-scale microbe-mineral interactions to global-scale biogeochemical cycling. This project focuses on understanding the relationship between fluid chemistry, mineral phases, and biomass production in subseafloor fluids from a relatively young (~8 million years) oceanic ridge crust that is hydrologically very active and dominated by aquifer mixing with oxic bottom seawater. Specifically, the speciation of solid-phase C, Fe, Mn, will be measured in samples collected from CORK borehole observatories. This project will perform in situ filtration of basement fluids from multiple isolated horizons at the North Pond CORK observatory suite (western mid-Atlantic Ridge Flank), and examine particle composition and elemental distribution for C, Fe, and Mn using synchrotron techniques. 

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Census of Deep Life Phase II-III

Project Investigators: Boetius, Antje; Colwell, Frederick S.; Sogin, Mitchell; D'Hondt, Steven

Start Date: 2014-01-01

End Date: 2015-12-31

Summary:

The Census of Deep Life (CoDL) seeks to identify the diversity and distribution of microbial life in continental and marine deep subsurface environments. This project directly addresses deep life’s Decadal Goal I to develop a global 3-D census of diversity in continental and marine deep subsurface environments and to explore mechanisms that govern microbial evolution and dispersal in the deep biosphere. It contributes to Decadal Goal II by informing us about the limits of life and metabolic properties necessary to adapt to deep subsurface environments. With the introduction of shotgun metagenomics, the CoDL will address key functional questions about deep life.  We will switch from 454 to more efficient Illuimina sequencing technology. This project is funded under the umbrella of the Deep Life Community proposal Deep Life 2013-10-03. 

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Census of Microbial Diversity in Ultra-deep, Fault-Bordered Sedimentary Rocks of a Foreland Basin in Taiwan

Project Investigators: Cheng, Ting-Wen ; Wang, Pei-ling; Lin, Li-Hung

Start Date: 2011-01-01

End Date: 2013-12-31

Summary:

To enhance the understanding of microbial ecosystems in deep consolidated sedimentary rocks, we propose to analyze community assemblages for samples retrieved from a foreland basin in Taiwan. The target samples at depths of 0.4 to 3 kmbl (kilometers below land surface) are Mio-Pleistocene sedimentary rocks formed during the convergence between the Eurasian and Philippine Sea Plates. The northwestward subduction leads to the oblique collision of the overlying Luzon arc with the Eurasian continent starting about 5 million years ago. The compressional uplift driven by the arc-continent collision propagates from north to south and from east to west through a series of thrusts and folds trending perpendicular to the major stress trajectories. From east to west, metamorphic complexes, Oligo-Miocene shelf-to-slope sedimentary rocks, and Mio-Pleistocene foreland sedimentary rocks were exhumed sequentially to accommodate the stress accumulation. The deformation front currently propagates to offshore southwestern Taiwan. We propose to apply 454-based tag sequencing to a suite of samples collected from the coring to undeformed and displaced sedimentary formations at depths of as great as 3 km below land surface in Taiwan to investigate the community patterns and shifts potentially controlled by the intrinsic rock property and geological framework. We anticipate that the sequencing results combined with other geochemical and cultivation data would provide insights into the distributions, community structures, and metabolic functions of microbial communities across various lithology units disturbed by the ongoing thrusting activities over millions of years in the active tectonic region.

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Characterization of a Novel, Thermophilic, Subsurface Bacterial Genus Related to Candidatus Desulforudis audaxviato

Project Investigators: Orphan, Victoria; Moser, Duane; Hamilton-Brehm, Scott D.

Start Date: 2014-04-01

End Date: 2014-12-31

Summary: This project will characterize the first cultivated representative of a novel bacterial genus (denoted DRI-14) isolated from a deep, regional aquifer beneath the Nevada National Security Site (NNSS). This thermophilic anaerobe may have particular relevance to all three of the decadal goals of the Deep Life Community (DLC) with a particular focus on Goal 3: “Determine the interaction between deep life and carbon cycling” due to DRI-14’s apparent physiological linkage to the major source of mobile carbon in the deep biosphere (e.g., it grows in pure culture with methane as the sole C and energy source) and on Goal 1: “Determine the processes that define the diversity and distribution of deep life as it relates to the carbon cycle” as its status as the nearest cultivated neighbor (91% over the full-length 16S rRNA gene) to Candidatus Desulforudis audaxviator.

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Coast Range Ophiolite Microbial Observatory (CROMO)

Project Investigators: Brazelton, William; Kubo, Mike; Schrenk, Matthew; Twing, Katrina; Crespo-Medina, Melitza; Cardace, Dawn; Hoehler, Tori M.

Start Date: 2012-01-01

End Date: 2014-06-30

Summary:

The Coast Range Ophiolite Microbial Observatory (CROMO) sub-project of the RHC grant sought to document and track microbial populations in a series of groundwater wells in actively serpentinizing terrane in northern California. The work capitalizes upon the recovery of microbiologically validated drill cores in 2011 from the site, and from quarterly records of microbial diversity, abundance, and associated geochemistry. The substantial sampling suite and convenient laboratory facilities and infrastructure at CROMO also allows more complicated efforts to be undertaken, including transcriptomics studies, tracer studies, hydrogeological tests, etc.  The work seeks to link the occurrence, function, and consequences of microbial communities in serpentinizing environments and their linkages to both the subsurface and the surface carbon cycle.

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Comparative Genomics of Novel Alkaliphilus Species Isolated from Carbonate Chimneys at a Shallow Marine Serpentinized Site, the Prony Hydrothermal Field, New Caledonia

Project Investigators: Erauso, Gaël ; Bes, Méline ; Quémeneur, Marianne ; Ménez, Bénédicte; Postec, Anne ; Karkouri , Khalid El

Start Date: 2014-07-01

End Date: 2014-12-31

Summary:

Serpentinization and associated geochemical reactions are important sources of energy and organic carbon for subsurface habitats, but very few studies have explored the distribution, ecology, and evolution of serpentinite-hosted microbial communities. The most fully characterized example of a serpentinization-driven microbial ecosystem is that found in the carbonate chimneys of the Lost City hydrothermal field, near the Mid-Atlantic Ridge, which have been the focus of many interdisciplinary studies that aim to understand the relationship between deep carbon, deep energy, and deep life. Furthermore, such systems have been highlighted as potentially providing important clues about the origins and early evolution of life. Until recently, the Lost City carbonate chimneys were thought to be a unique microbial ecosystem, drastically different from any that had been discovered. However recently, similar hydrothermal chimneys in the Bay of Prony, New Caledonia, have been found to be analogous to the Lost City chimneys in many respects, while at the same time providing an interesting point of comparison. In the Bay of Prony, New Caledonia, carbonate chimneys vent 22-43°C, pH 11 fluids rich in hydrogen and methane at shallow depth. The fluids represent mixing of seawater with low-salinity groundwater derived from continental ultramafic rock. In each chimney, bacterial richness was at least an order of magnitude higher than that of archaea, in agreement with previous studies at Lost City. Interestingly, many sequences were affiliated with thermophilic anaerobic organisms related to marine and terrestrial hydrothermal systems, despite the moderate temperatures of chimney fluids. Remarkably, the archaeal community was dominated by uncultured Methanosarcinales represented by three phylotypes, of which one is found in the Lost City chimneys and another is found in a continental serpentinite site (The Cedars, California). The most abundant and diverse bacterial communities were mainly composed of Chlorofexi, Deinococcus-Thermus, Proteobacteria, and Firmicutes, the later two phyla containing phyloptypes also common to either Lost City or the Cedars. Therefore, Prony site represents an interesting transition between continental serpentinite springs. In spite of the considerable interest to the microbiology of serpentinite ecosystems it appears that the large majority of the microorganisms thriving in this extreme environment resisted cultivation attempts because until very recently no isolate has been described. Culture-based approaches of representative isolates, allowing detailed and reproducible physiological and genomic studies, constitute a key step in understanding the ecophysiological properties of these extremophiles, that is how they adapted to such harsh conditions and how their metabolic activities potentially interact with the geochemistries of their environments. The importance of such studies is exemplified by the recent description of a new genus of alkaliphilic betaproteobacteria, Serpentinomonas, isolated from The Cedars springs. This CoDL research aims to sequence the genome of the first alkaliphilic bacteria representative of a marine serpentinization-driven ecosystem. 

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Costa Rica Margin Subseafloor Microbiology

Project Investigators: Martino, Mandi ; House, Chris

Start Date: 2013-10-04

End Date: 2014-10-04

Summary: The subsurface environment off the shore of Costa Rica has been targeted by the broader scientific community for intense research efforts due to the unique history and current conditions of the plate subduction system. The unusualness of the system revolves around hypothesized changes in its behavior from those of an accretionary subduction system to those of an erosive subduction system. 

We are analyzing the microbial community of deeply buried sediments within two drill-site locations on the upper plate of this subduction system, collected during the Integrated Ocean Drilling Program Expedition 334.  454 tag sequencing revealed initial phylogenetic identifications, potential correlations with geochemical properties, and an extensive look at the influence of drilling methods on molecular work.  As an extension, Illumina metagenomic sequencing from three depths at one drill-site location allows for not only an independent view of taxonomy and phylogenetic relationships, but also insight into how functional properties of these communities change with depth, and the potential metabolic capabilities of several largely uncharacterized subseafloor microbial lineages.

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DARCLIFE / DARCSEAS I

Project Investigators: Elvert, Marcus *; Lin, Yu-Shih ; Hinrichs, Kai-Uwe; Heuer, Verena; Lipp, J S; Schröder, Jan ; Schmidt, Frauke; Travis Meador, Travis ; Wendt, Jenny ; Wörmer, Lars ; Arndt, Jessica ; Yoshinaga, Marcos; Zabel, Matthias

Start Date: 2011-02-02

End Date: 2011-02-22

Summary: DARCSEAS I - Deep subseafloor Archaea in the eastern Mediterranean, Marmara and Black seas: carbon cycle, life strategies, and role in sedimentary ecosystems.

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Dark energy in the deep, cold ecosystem of Subglacial Lake Whillans, West Antarctica

Project Investigators: Christner, Brent C.; Achberger, Amanda M.; Michaud, Alexander B.; Priscu, John C.; Vick-Majors, Trista J.

Start Date: 2014-11-01

End Date: 2016-11-01

Summary: Research over the past few decades has substantially increased our appreciation of the deep biosphere as a major reservoir of cellular carbon and biodiversity. Antarctic subglacial aquatic environments are hypothesized to contain a significant pool of biomass and biogeochemically relevant gases such as methane; however, only recently have these environments been accessible for study. Subglacial aqueous habitats harbor microbial ecosystems that are hydraulically linked to the oceans and have the potential to affect marine biological and chemical systems. In spite of their biogeochemical importance, the microbial structure and function in subglacial aquatic environments remains relatively understudied. We are currently characterizing a microbial ecosystem in Subglacial Lake Whillans (SLW), a lake that exists beneath ~800 m of ice in West Antarctic. Our molecular and biogeochemical data indicate that SLW is a chemosynthetically driven ecosystem inhabited by a diverse assemblage of bacteria and archaea. We propose to use metagenomic data to investigate the structure and functional potential of the microbial communities in the upper 40 cm of SLW sediments. In particular, we aim to focus our analysis on lineages and pathways responsible for chemolithoautotrophic carbon fixation and methane oxidation. These data will provide an unprecedented understanding of the ways in which microorganisms harvest “dark energy” and transform carbon in the deep, cold biosphere.

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Deep Life Investigations That Support the ICDP Collisional Orogeny in the Scandinavian Caledonides (COSC) Drilling Project

Project Investigators: Schrenk, Matthew; Onstott, Tullis; Kieft, Thomas

Start Date: 2014-05-01

End Date: 2014-11-01

Summary:

The Collisional Orogeny in the Scandinavian Caledonides (COSC) Project is a drilling effort sponsored by the International Continental Drilling Program (ICDP), the Swedish Deep Drilling Program, and the Swedish Science Research Council (Swedish NSF), which funded the purchase of the drill rig.  The main theme of the project is to understand the role of an ancient (400 Ma) thrust zone in the formation of the mountains of central Sweden and also as a model of other mountain-building processes, such as those that formed the Himalayas.  The DCO Deep Life Community provided funds ($24k + $1K for DNA sequencing) to add a geomicrobiological component to this ICDP project.  Funds were awarded to Kieft, Onstott, and Schrenk; they are collaborating with Karsten Pedersen of Chalmers University in Sweden, who is the lead microbiologist on the COSC project.  They are also collaborating with geologists from Uppsala University and geophysicists from Lund University.  The main emphases of the geomicrobiological efforts are to characterize the biodiversity and metabolic activities of microbes living within fractures, and to determine the importance of rock-water interactions in generating H2 and CH4 to support microbial activities.

 

The plan for the DCO-funded geomicrobiology is to collect samples of drill core at various depth intervals along the planned 2500-meter planned depth of the drilling and to collect a larger number of samples within the main thrust zone.  Three types of tracers will be deployed to quantify contamination from drilling fluids.  These tracers are (1) fluorescein dye, which has been added to the drilling mud, (2) a perfluorinated hydrocarbon, to be added to the drilling mud immediately before it’s pumped down the borehole, and (3) fluorescent microspheres to be added in a plastic bag attached to the bottom of a polycarbonate liner immediately before sampling. The polycarbonate liner serves as the third, or inner, tube, of a triple tube coring tool to minimize contamination of the rock core from drilling mud and handling. Cores to be used for geomicrobiology analyses will be removed from the polycarbonate liner and subcored by removing the outer portions of the core (“parings”), leaving an inner subcore.  Tracers will be quantified in the subcore, parings, and drilling muds. 

 

Cores will also be collected by Pedersen and Onstott for chemical and isotopic analyses of gases and pore fluids within the rock matrix and for 35S autoradiography.  Cores will be sealed in canisters and then incubated for several months, during which time the gases will diffuse from the rock into the atmosphere of the canister. Swabs of fractures for DNA and ATP analyses are also being collected for analyses in Pedersen’s lab.  Whole round cores and drilling mud samples have also been collected and archived in a freezer for later analyses.

 

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Deep Terrestrial Biosphere Observatory: Shield Brines, Microbial Mats, and Banded Iron Formations at the Soudan Iron Mine

Project Investigators: Toner, Brandy M.; Dick, Gregory J; Gralnick, Jeffrey A

Start Date: 2011-01-01

End Date: 2013-12-31

Summary:

The Soudan Iron Mine is located on the Vermillion Iron Range in Northern Minnesota. At 2341 feet underground, the lowest level of the Soudan Iron Mine is home to an extraordinary and extreme environment where the fields of microbiology, geochemistry, and mineralogy converge. The sedimentary iron-rich rock that was mined for 80 years until the mine was closed in the early 1960s is known as a "Banded Iron Formation," or BIF. BIFs can be found across the planet (Brazil, Australia, and South Africa have some of the largest deposits). We propose to use 454 pyrotag sequencing of microbial community DNA retrieved from a BIF at the Soudan Iron Mine. Obtaining a thorough inventory of the microbial communities across a range of geochemical and geological conditions will help develop hypotheses regarding species distribution with depth and along geochemical gradients in a deep terrestrial ecosystem. 

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Deep subsurface evaporite deposit

Project Investigators: Payler, Samuel J; Cockell, Charles

Start Date: 2014-11-01

End Date: 2016-11-01

Summary: Evaporite deposits can be found in many deep subsurface settings around the globe. Despite their ubiquity, little is known about how the identity of these organisms and their role in biogeochemical cycling. This project addresses this knowledge gap by examining brine seeps located 1.1km below the surface in Boulby Mine, UK, using metagenomic techniques. Metagenomics will enable us to study the phylogenetic affiliations of organisms present and the presence of functional genes related to particular metabolisms and elemental cycling capabilities. This work will advance our understanding of the role organisms play in deep subsurface carbon cycling and how ancient evaporite minerals shape deep subsurface communities.

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Deep-Life I: Microbial Carbon Transformation in Rock-Hosted Deep Subsurface Habitats

Project Investigators: van Heerden, Esta; Itavaara, Merja; Onstott, Tullis; Huber, Julie; McMillan, Paul; Stepanauskas, Ramunas; Schrenk, Matthew; Daniel, Isabelle; Bartlett, Douglas; Hoehler, Tori M.

Start Date: 2012-01-01

End Date: 2014-06-30

Summary:

The Rock-Hosted Communities (RHC) grant sought to provide baseline data related to the distribution and function of microbial communities in rocky subsurface habitats underlying the continents and oceans. The grant served to link both field and experimental studies of rock-hosted microbial communities, with a particular focus upon the metabolism of carbon compounds.  Application of molecular biological approaches, utilizing next-generation sequencing technology, provided a foundation to understand the potential, the diversity, and the evolution of metabolic pathways. These surveys were linked to the physiological characterization of novel cultivars, particularly at high pressure, and to activity measurements using stable isotope tracer approaches. These activities were coordinated across participating laboratories through face-to-face meetings, virtual meetings, and research exchanges.

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Deepsea Challenge Expedition

Project Investigators: Bartlett, Douglas; Narasingarao, Priya; Tarn, Jonathan

Start Date: 2013-04-03

End Date: 2014-12-01

Summary: We performed 16S rDNA amplicon V6 region sequencing of Bacteria and Archaea on bottom water samples collected from four locations, and three filter pore size fractions (3. 0.2 and 1 microns).  The locations in question are the west pond and the middle pond of the Challenger Deep (~ 10.9 km water column depth) within the Mariana Trench, the Sirena Deep (~10.7 km water column depth) within the Mariana Trench, and a control location outside the Mariana Trench at a water column depth of about 750 m.  

The Mariana Trench lies at the boundary between the eastern edge of the Philippine Sea plate and the subducting Pacific plate. The Challenger Deep represents the deepest ocean depth on Earth. Its main depression is about 40 km long, with depths greater than 10,400 m occurring throughout this zone. The Sirena Deep is the second deepest site in the Mariana Trench, with a maximum depth of 10,732.  It is located south of the shallow Santa Rosa Bank, about 90 miles south of the island of Guam.

Douglas Bartlett was the Chief Scientist during the James Cameron Deepsea Challenge Expedition, February – April, 2012.  During this expedition an autonomous lander was used to collect bottom seawater from the Mariana Trench and south of the Mariana Trench near the Ulithi Atoll.  This lander was equipped with lights, 3D videography, a high definition camera, Niskin water samplers and baited traps.

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Disko Island as a Test-bed for Carbon Pathways

Project Investigators: Cockell, Charles; Jones, Adrian; Fox-Powell, Mark; Bryce, Casey; Mikhail, Sami; Steele, Andrew; Cousins, Claire

Start Date: 2013-08-05

End Date: 2013-08-13

Summary:

A team of four scientists—P.I. Dr. Claire Cousins, Dr. Sami Mikhail, and PhD students Casey Bryce and Mark Fox-Powell—spent two weeks in August of 2013 sampling basalts and thermal spring materials on Disko Island, Greenland. The primary goal of this work was to collect a robust sample set to support ongoing research of interest to the DCO community.

Disko presents an ideal field site due to its unusual basalts, whose compositions are rare for surface samples, containing FeNi alloys and Fe-carbides. Study of these basalts may lead to increased understanding of their formation. Additionally, Disko is home to many understudied deep thermal springs, including mud volcanoes, salt springs, and cold to warm springs. The investigation of the microbes within these thermal springs may provide insights into subsurface microbiological communities’ carbon pathways and life cycles.

Due in part to its remote location, Disko Island is relatively under-examined. Many of the logistical issues addressed by this team will be invaluable to researchers coming to the island for future study.

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Diversity of Microbes on Igneous Minerals Incubated for Four Years in the Ocean Crust

Project Investigators: Smith, Amy ; Fisk, Martin ; Mason, Olivia ; Popa, Radu

Start Date: 2011-01-01

End Date: 2013-12-31

Summary:

The goals of this study are to characterize the microbial communities that colonized the minerals and glasses incubated in Hole 1301A in the Juan de Fuca Ridge to study the relationship between microbial diversity and mineral type, determine if microbial communities are distributed homogeneously or heterogeneously in ocean crust, and explore the biogeochemical role of organisms colonizing each of the igneous phases. This is the first step to understanding how microbial community structure is influenced by mineral surfaces and what roles they play in the ocean crust. We isolated microbes for identification purposes and to study their physiology in the presence of igneous minerals. However, large-scale sequence analysis of DNA extracted from our incubated minerals would help us obtain key information about the correlation between ocean crust minerals and microbial diversity. The igneous minerals and glasses incubated in the subseafloor at the Juan de Fuca Ridge provide an opportunity to evaluate a number of hypotheses about microbial colonization in the ocean crust.

 

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Dziani Dzaha

Project Investigators: Ader, Magali; Cadeau, Pierre ; Leboulanger, Christophe; Gerard, Emmanuelle; Bouvy, Marc; Jézéquel, Didier ; Sarazin, Gérard

Start Date: 2014-04-01

End Date: 2014-11-01

Summary:

In April 2014, a French research expedition, led by Magali Ader of the Institut de Physique du Globe de Paris, made its first of four planned field expeditions to the Dziani Dzaha lake to begin characterizing the physical chemistry and biology of the lake during different seasons. This crater lake is located in the Comoros Archipelago, a small volcanic hotspot chain in the Indian Ocean between Madagascar and Mozambique. Dziani Dzaha presents a very unusual combination of characteristics making it a likely analog for some hydrocarbon-bearing lacustrine rocks of economic importance and possibly certain Precambrian environments, including expanding rift valleys and basins.

The field team performed on-site measurements of the water chemistry and microorganisms’ metabolic activity. They also sampled gases, waters at various depths, and procured a 1.8-meter-long sediment core for subsurface biodiversity, chemical, mineralogical, and isotopic analyses. DCO support is being provided to the highly leveraged research program to partially cover the cost of 14C analyses of the dissolved inorganic carbon and biomass, and of detrital remains of terrestrial plants found at various levels in the core. These analyses will test the hypothesis that the biomass feeds directly on volcanic CO2 and provide an approximate model age for the core. Information and a short video about the research program and field study is available on the DCO website: https://deepcarbon.net//feature/new-video-field-expedition-indian-ocean#.U-5QEVaF5mo

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Establishing a Platform to Assess Bacterial Endospore Abundance in the Deep Biosphere by Quantification of the Sporulation-Related Molecule Dipicolinic Acid

Project Investigators: Wörmer, Lars

Start Date: 2014-03-01

End Date: 2014-08-30

Summary: This project will develop novel technology for life detection in deep sediments by supplying the Deep Life Community (DLC) with a platform to address one of the stated goals of DLC, the quantification of spores vs. vegetative cells, by accurate chemical detection of the sporulation-related molecule dipicolinic acid (DPA).

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Evaluation of Sample Collection and DNA Extraction Procedures for Characterizing Bacterial Diversity in Ultrabasic Groundwaters ‐ CoDL Option 1

Project Investigators: Schrenk, Matthew; Tiago, Igor

Start Date: 2014-07-01

End Date: 2014-12-31

Summary:

Over the past several years, a number of studies have used culture-independent molecular sequencing surveys to describe the microbial diversity of groundwaters hosted in continental bedrock. Water-rock reactions in many of these systems, especially those associated with the process of serpentinization, lead to H2  and CH4 enriched fluids at ultrabasic pH (10-12). The diversity of microbial communities in these systems is commonly extremely low, consisting of only a few major taxa. Clostridia and related species are predominant inhabitants of serpentinizing subsurface environments. Also, Candidatus ‘Desulforudis audaxviator’ is a persistent member of microbial populations in a multi-year study of a well at Cabeço de Vide (CVA) in Portugal. Furthermore, bacterial taxa related to the order Clostridiales have been detected in numerous subsurface environments, and many of these are related to spore-forming species. Additionally, recent studies have shown that spores originating in warm subsurface environments are easily dispersed, and persist in subsurface environments. This proposal is spurred by our observations of discrepancies between the taxonomic composition of microbial communities at CVA which seem to be dictated by whether cells were collected by filtration on 0.2 μm filters and extracted using gentle (chemical) cell lysis, versus collection on 0.1 μm filters and extracted using physical lysis (bead beating). The 0.1 μm samples had a high percentage (5-24%) of Desulforudis sequences, whereas the 0.2 μm samples did not.  Furthermore, it is known that cell sizes in energy- and nutrient-poor environments can lead to the formation of “dwarf cells” which can pass through 0.2 μm filters. Hydrogenophaga-like organisms, which are common inhabitants of serpentinizing subsurface environments are known to form such miniaturized cells. Our objective is to use samples of ultramafic groundwaters collected in parallel on 0.1 and 0.2 μm filters from the CROMO site in northern California, USA and the CVA site to test the hypotheses that (a) microbial populations <0.2 μm in size contribute to the natural microbial diversity of ultrabasic groundwaters, and (b) a significant portion of the microbial diversity described using physical lysis methods is derived from recalcitrant bacterial endospores. We will compare the taxonomic compositions of community DNA extract by gentle (chemical) and rigorous (physical) lysis approaches. These studies will be conducted in concert with the quantification of spore abundances using differential centrifugation and light microscopy, and through analysis of glass fiber filters and determination of the spore protein dipicolinic acid. We propose to have the purified DNA samples sequenced for bacterial 16S RNA via CoDL Option 1.

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Experimental Calibration of Methane Clumped Isotopologue Systematics for Microbial Production and Consumption

Project Investigators: Könneke, Martin ; Hinrichs, Kai-Uwe; Ono, Shuhei; Gruen, Danielle

Start Date: 2014-05-01

End Date: 2014-10-31

Summary: Determining the origin of organic molecules such as methane in deep Earth is a central objective of the Deep Carbon Observatory program. However, the differentiation between biological and abiological methane-forming processes can be complicated since microbial formation of methane occurs even at temperatures where abiotic processes may generate substrates for microbial methanogenesis. In addition, deep-sourced abiotic methane produced during serpentinization may diffuse into shallower settings. In order to study the interaction between deep life and the C-cycle, we aim to validate methane clumped isotopologue (13CH3D) detection during methanogenesis at different temperatures and using varying substrates. In addition, we want to understand the discrepancy of isotopologue thermometery of methane formed abiotically and methane produced via methanogenesis. Furthermore, we will study the influence of methane consuming processes, such as AOM, on clumped methane isotopologues.

 

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Exploration of Fungal Activities in the Deep Subseafloor

Project Investigators: Pachiadaki, Maria ; Edgcomb, Virginia

Start Date: 2014-10-01

End Date: 2015-04-01

Summary: This project will develop a eukaryote-focused metatranscriptome analysis of deep subsurface Peru Margin sediments where some evidence has been found previously for active fungal populations. This study would provide a more comprehensive understanding of the abilities of fungi to survive on a variety of possible C substrates in the deep buried subsurface and their potentially important impact on biogeochemical cycles, and in particular, on carbon turnover in subseafloor sediments. We will employ Paired-end Illumina MiSeq sequencing of expressed genes in core samples from Peru Margin site 1229A to determine if fungi are actively involved in organic matter degradation (e.g.,  amino acid, lipid and carbohydrate metabolism) in these samples, which activities associated with degradation of specific C sources are the most highly expressed, and what enzymes and metabolic pathways are involved. Samples from Peru Margin site 1229A will be obtained from A. Teske at UNC Chapel Hill. Replicate meta-transcriptome libraries will be generated from 4 g sediment samples from 6 mbsf and 95 mbsf using poly-A selection to enrich for eukaryotic transcripts. Replicate iTAG (18S rRNA) libraries generated from inside and outside each core will aid detection of contaminants.

 

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Genomes of Single-Cell Organisms

Project Investigators: Stepanauskas, Ramunas; Onstott, Tullis; Kieft, Thomas

Start Date: 2012-01-01

End Date: 2014-06-30

Summary:

A large challenge in environmental microbiology is linking taxonomic marks to physiology and metabolic functions. Single-cell genomics provides unequivocal linkage information for multiple genes and enables the reconstruction of entire genomes from uncultured microbial species. This project will undertake genomic sequencing of individual cells from deep subsurface microbes in order to (1) reconstruct metabolic properties of predominant microbial types in studied communities, (2) serve as references when interpreting metagenomic and metatranscriptomic data, and (3) reveal evolutionary processes for deep subsurface microbial populations.

The Deep Fracture (DF) portion of the Rock Hosted Communities Project sought to build upon previous studies on the microbial diversity of Precambrian Shield deep ecosystems and use cell sorting and single-cell genome sequencing approaches to evaluate the adaptations and the population genetics of these deep isolated communities. The work built primarily upon sampling opportunities in South Africa and Finland where populations of the deep biosphere organism Candidatus Desulforudis audaxviator were found to be prevalent.

 

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IODP Leg 347: Baltic Sea Paleoenvironment

Project Investigators: Lloyd, Karen ; Bird, Jordan

Start Date: 2013-09-12

End Date: 2013-11-01

Summary: As part of the onshore science party in IODP Expedition 347: Baltic Sea Paleoenvironment, we are sequencing single cell genomes of uncultured archaea and bacteria in sediments as deep as 85 m. The vast majority of the microbial diversity in deep subsurface environments has eluded efforts to be cultured. Single cell genomics affords us the opportunity to uncover new prospective ecophysiological roles carried out by enigmatic yet abundant organisms. We now have 40 single amplified genomes (SAGs) from 37.5 mbsf and 20 SAGs from 85 mbsf at Baltic Sea Basin hole 60B. The SAGs include members of the deeply-branching archaeal and bacterial groups lacking cultured representatives. To our knowledge, these are the first SAGs ever successfully obtained from deep (> 10 mbsf) marine sediments. We currently have funding to sequence 10 SAGs. Adding 3 more would therefore greatly expand our understanding of the genetic composition of uncultured microorganisms in deep Baltic Sea sediments.

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Iceland Subglacial Lakes

Project Investigators: Gaidos, Eric

Start Date: 2007-01-01

End Date: 2007-12-31

Summary: Subglacial lakes beneath the Vatnajökull ice cap in Iceland host endemic communities of microorganisms adapted to cold, dark and nutrient-poor waters, but the mechanisms by which these microbes disseminate under the ice and colonize these lakes are unknown. We present new data on this subglacial microbiome generated from samples of two subglacial lakes, a subglacial flood and a lake that was formerly subglacial but now partly exposed to the atmosphere. These data include parallel 16S rRNA gene amplicon libraries constructed using novel primers that span the v3–v5 and v4–v6 hypervariable regions. Archaea were not detected in either subglacial lake, and the communities are dominated by only five bacterial taxa. Our paired libraries are highly concordant for the most abundant taxa, but estimates of diversity (abundance-based coverage estimator) in the v4–v6 libraries are 3–8 times higher than in corresponding v3–v5 libraries. The dominant taxa are closely related to cultivated anaerobes and microaerobes, and may occupy unique metabolic niches in a chemoautolithotrophic ecosystem. The populations of the major taxa in the subglacial lakes are indistinguishable (>99% sequence identity), despite separation by 6 km and an ice divide; one taxon is ubiquitous in our Vatnajökull samples. We propose that the glacial bed is connected through an aquifer in the underlying permeable basalt, and these subglacial lakes are colonized from a deeper, subterranean microbiome.

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Integrated Metagenomic and Metatranscriptomic Analyses of Active Microbial Communities Involved in Carbon Metabolism at a Tropical Serpentinizing Environment (Santa Elena Ophiolite, Costa Rica)

Project Investigators: Sanchez-Murillo, Ricardo ; Crespo-Medina, Melitza

Start Date: 2014-05-01

End Date: 2014-12-31

Summary:

The Santa Elena Ophiolite comprises 250 km2 of ultramafic rocks and mafic associations along the northwestern Pacific coast of Costa Rica. Uplifted ultramafic rocks originating from the Earth’s mantle represent a reservoir of carbon and reducing power, which during the process of serpentinization, are liberated into the surface environment, potentially supporting microbial growth. The extremely variable weather conditions that occur on the Santa Elena Peninsula between the wet (May – October) and dry (November – April) seasons result in a unique hydrogeological environment. Contrary to other continental serpentinization environments (e.g., Tablelands, Canada; Western Coastal Range, USA; Gruppo di Voltri, Italy), in this tropical scenario, precipitation quantities and intensities are usually greater and occur during extended periods of time, which facilitate infiltration, accelerating water-rock interactions and liberating carbon and energy to the surface environment. These extreme seasonal events provide a unique opportunity to investigate the response of microbial metabolisms (at the interface of deep carbon and energy fluxes) under a dynamic subsurface hydrology setting. During an expedition in March 2013, a series of alkaline springs along the peninsula were sampled. Preliminary V4-V5 sequence data suggests that these fluids had low diversity and that indicated the presence of microorganisms involved in hydrogen, methane, and methanol metabolism, including bacteria from the genera Hydrogenophaga and Methylibium and from the family Methylophilaceae, as well as archaea from the orders Methanobacteriales, Methanocellales, and Methanomicrobiales. In this project we intend to demonstrate the links between geochemistry, hydrology, and microbial activities related to subsurface processes at Santa Elena Ophiolite. We propose to apply metagenomic and metatranscriptomic analysis to one representative alkaline spring (Spring 9) and its corresponding background sample (Murcielago Upstream). 

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Investigating Microbial Community Transitions at the Sediment-Basement Interface

Project Investigators: Orcutt, Beth ; Edwards, Katrina J.; Sylvan, Jason ; Lever, Mark

Start Date: 2011-01-01

End Date: 2013-12-31

Summary:

Previous studies of microbial abundance and geochemistry in deep marine sediments indicate a stimulation of microbial activity near the sediment-basement interface. Furthermore, the few studies thus far conducted in deep oceanic crust reveal an active microbial community in this habitat. Yet, the extent to which microbial communities in bottom sediments and underlying crustal habitats interact is unclear. Are microbial communities in sediments seeded from basement communities, or vice versa? If there is influence from one habitat type on the other, how far does this influence extend, both with vertical distance away from the interface and laterally along the flow path of fluids within basement? We propose to conduct tag pyrosequencing on samples from a spectrum of sediment-basement habitats to identify patterns in microbial community shifts across sediment-basement interfaces. Such comparisons will allow us to evaluate whether microbial groups from one habitat are found in another, and to examine whether members of the ‘rare biosphere’ in one habitat come to dominate in another. Analysis of the pyrosequencing data will help us better understand how subsurface microbial communities are established and evolve, in addition to expanding our knowledge of microbial biogeography across sediment-basement interfaces in different oceanic basins.

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Isolation and Characterization of Novel Piezophiles from Deep Subsurface Environments

Project Investigators: Itavaara, Merja; Kieft, Thomas; Narasingarao, Priya; Bartlett, Douglas; Huber, Julie; Schrenk, Matthew

Start Date: 2012-01-01

End Date: 2014-06-30

Summary:

This aspect of the RHC grant sought to enrich for and characterize novel piezophilic cultivars from deep, rock-hosted microbial habitats with a central focus upon the metabolism of carbon compounds at high hydrostatic pressure. Research efforts used samples from the Mid Cayman Rise and Deep Fracture projects in particular as a source of unique samples to enrich for new microorganisms, which may serve as type strains for future deep biosphere studies.

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Juan de Fuca Ridge Flank Deep Biosphere

Project Investigators: Edwards, Katrina J.; Lever, Mark; Orcutt, Beth

Start Date: 2010-07-05

End Date: 2010-09-05

Summary: This project included the sequencing of sediment-basement interface samples from the eastern flank of the Juan de Fuca (JdF) Ridge and from the western flank of the Mid-Atlantic Ridge (North Pond, NP) in an effort to document the microbial community connection among deep sediments, oceanic crust, and circulating seawater.  The sequenced samples were collected during IODP Exp. 327 (JdF; Fisher et al. 2011) and via gravity coring during cruise MSM11-1 (NP, Ziebis et al. 2012).  Microsphere contamination controls were used during IODP Exp 327, and background control samples were included in this sequencing project.

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Ligurian Ophiolites and Ophicalcites, N. Italy

Project Investigators: Boschi, Chiara; Schwarzenbach, Esther; Bernasconi, Stefano ; Vogel, Monica

Start Date: 2007-04-01

End Date: 2015-03-31

Summary:

A number of the Tethyan ophiolites in the Alps and northern Apennines are considered relicts of oceanic lithosphere and contain lower crustal and upper mantle sequences that are believed to have been exposed by detachment faults onto the seafloor. These ophiolite complexes often contain sections of carbonate-veined serpentinites and carbonate-serpentine breccias, so-called ophicalcites, which show similarities to the serpentinite-hosted carbonate deposits recovered at Lost City.

Our long-standing research targets the Jurassic Bracco-Levanto and Val Graveglia ophiolite complexes in Liguria (Italy), which provide spatial information (e.g., three-dimensional distributions and structures) and constraints on the preservation of geochemical fingerprints over time – information that is commonly unavailable in the modern marine system. An overall goal of our project is to obtain a better understanding of the sources of carbon and sulfur in these systems, how they are cycled from the basement to the fluids, the deposits and the biosphere, and how the carbon and sulfur budgets change with mineral-fluid and or microbe-fluid interactions over time (PhD thesis E. Schwarzenbach). Another aim is to study the links among fluid-rock interaction, hydrothermal deposits and deformation processes and to compare these to modern marine systems along the Mid-Atlantic Ridge (PhD thesis M. Vogel). We have been conducting petrological, major element, trace element and isotopic (O, C, S, Sr, B) analyses of basement rocks and the hydrothermal deposits to quantify fluid flow paths, mass transfer, and carbon cycles during progressive hydrothermal activity, with emphasis on processes leading to the formation of the ophicalcites. This comparative study will provide a better understanding of evolving, subsurface processes in serpentinite-hosted hydrothermal systems and will contribute to a comprehensive, integrated model of end-member hydrothermal systems in oceanic sequences formed at slow spreading ridge environments. Our work has been supported by the Swiss National Science Foundation.

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Linking Microbial Populations, Ocean Basins, and Paleoenvironmental Conditions in the Eastern Mediterranean Sea and Black Sea

Project Investigators: Teske, Andreas; Hinrichs, Kai-Uwe

Start Date: 2011-01-01

End Date: 2013-12-31

Summary:

This project examined the relationship between subseafloor microbial diversity and paleoceanographic conditions in samples from three sediment cores from the Eastern Mediterranean Sea and the Black Sea. All three cores record dramatic changes in oceanographic/limnic conditions resulting from oscillation between oligotrophic and euxinic conditions, yet they also differ remarkably between sites. Our motivation for this study is based on the premise that paleoenvironmental conditions in the overlying water column are intimately linked to the composition of aquatic microbial communities and thus the inoculum for subseafloor communities. Our study will address the long‐standing question to what degree the composition of subseafloor microbial communities is linked to paleoenvironmental conditions at the time of deposition. The sample set at both Mediterranean sites is composed of carbonate‐rich, organic‐lean sediments and interspersed so‐called sapropels, that is, organic‐rich black layers that were deposited during climates that were probably warmer and more humid than today, when the Mediterranean was euxinic. The samples from the discovery basin are influenced by a highly saline brine and thus may select for halophilic sedimentary microbes. The Black Sea sample set is composed of organic‐lean coccolith ooze deposited during the recent few thousand years, an organic‐rich sapropel deposited during the early Holocene and of sediment deposited during the late glacial/early Holocene when the Black Sea was still a freshwater lake. On a set of eight selected sediment samples we propose to examine both archaeal and bacterial diversity with a 454‐based tag sequencing survey. We can address questions relating to the paleoenvironmental history at each site as well as to the relationship of microbial communities in sediments deposited during euxinic conditions at three sites along a salinity gradient remote to each other, in two different ocean basins. The sample set will be accompanied by an in‐depth biogeochemical characterization of its sedimentary habitat. Sediment sampling and characterization are performed and mutually integrated on the same cruise (RV Meteor, M84‐1, February 9‐22, 2011).

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Loihi Seamount Subsurface and Seafloor Connections

Project Investigators: Moyer, Craig L.; Sylvan, Jason

Start Date: 2014-06-01

End Date: 2014-12-31

Summary: Loihi Seamount is a model system for mid-plate hotspot magmatism. The newest volcano in the Hawaiian Seamount Chain, it is located ~15 km off the southeast corner of the island of Hawaii.  Low-temperature vents emitting fluids up to ~70˚C dominate hydrothermal activity at Loihi, and contain elevated levels of Fe, CO2, CH4 and NH4+.  Loihi Seamount is an ideal site for studying subsurface crustal microbes because its chemistry is so well known, with plenty of background information from multiple samplings available from each site. 

We are performing Option 1 sequencing (24 samples of V4V5 amplicon sequencing) of subsurface fluid samples and microbial mats sampled directly above the venting fluids to further our understanding of subsurface microbial populations in a low sulfide, high iron hydrothermal environment. These experiments will also allow us to determine the extent of interaction between microbial communities in subsurface diffuse fluids and seafloor microbial mats.

We will test the following hypotheses: 1. The subsurface microbial communities in the Hiolo area and Spillway area have different compositions that are related to local fluid chemistry 2. The dominant species in microbial mats at Loihi Seamount are seeded by subsurface populations present in diffuse flow fluids.  It is likely that the dominant mat species are present but not dominant in the subsurface, and bloom in the transition zone at the seafloor where warm, reduced anoxic fluids mingle with cool, oxic seawater.

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Louisville Seamounts Subsurface Microbiology

Project Investigators: Edwards, Katrina J.; Sylvan, Jason ; Tully, Ben

Start Date: 2011-06-01

End Date: 2013-12-31

Summary: The Louisville Seamounts are a hotspot seamount trail in the southwest Pacific Ocean, formed in an analogous process to the Hawaiian Islands.  Seamounts are known conduits of fluid flow into and out of subseafloor basaltic crust. 

In an effort to understand microbial life in 64-70 million year old subsurface marine basement, we collected samples from two guyots in the Louisville Seamount Chain. We extracted DNA from fifteen samples, for amplification of the bacterial 16S rRNA gene and analysis of the bacteria present. Additionally, metagenomes were sequenced from three of these sites.  While some new recent studies have focused on microbiology of relatively young (<10 million years old) subseafloor crust, this is the first detailed study of microbial diversity in older basement samples.


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Marcellus Shale Gas Wells

Project Investigators: Mouser, Paula ; Trexler, Ryan ; Hartsock, Angela ; Wrighton, Kelly ; Wilkins, Michael ; MacRae, Jean

Start Date: 2012-06-08

End Date: 2013-04-26

Summary: Natural gas energy extraction using horizontal drilling and hydraulic fracturing technologies in deep hydrocarbon-bearing shale significantly alters biogeochemical conditions and microbial ecological function at depth. We tracked changes in microbial community dynamics and functional potential via genome reconstruction over an 11-month period in fluids produced from three wells drilled and fractured in the Marcellus shale. A marked shift in 16S rRNA sequences occurred over time; communities shifted from those closely associated to low-salt tolerance, mesophilic aerobic bacteria to dominance by halophilic, thermotolerant anaerobic associated bacteria and archaea. Genomes from halotolerant microorganisms, including Halolactibacillus, Vibrio, Marinobacter, Halanaerobium and Halomonas encoded the potential for fermentation, hydrocarbon oxidation, and sulfur-cycling metabolisms, while Arcobacter contained potential for chemoautotrophic sulfide oxidation metabolism. Relative to earlier samples, the 11-month samples were enriched in genes for the acquisition and degradation of aromatic compounds, sulfur, iron, and nitrogen, supporting the importance of these processes in later produced fluids when anaerobic conditions prevail. Later time points also show the enrichment of microorganisms closely related to Methanohalophilus and Methanolobus. These methanogens are known to disproportionate methylamines, and the recovery of genes associated with the fermentation of trimethylamine to dimethylamine suggests a pathway for methane production exists within detected genomes. These data provide insight into the microbial changes that occur from hydraulic fracturing in deep shale ecosystems.

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Metabolic Reconstruction of Desulforudis audaxviator, a Subsurface Sentinel Species

Project Investigators: Onstott, Tullis; Moser, Duane; Rowland, Sara ; Robb, Frank

Start Date: 2014-06-01

End Date: 2014-12-31

Summary:

The subsurface sentinel species Candidatus Desulforudis audaxviator is a bacterium in the Firmicutes phylum and forms a unique one-species ecosystem in deep-fracture hot aquifers. So far, Desulforudis and its relatives have resisted persistent efforts at cultivation and thus their growth physiology and capacity for carbon transformation remain speculative. This project aims to initiate reconstruction of the C utilization pathway of this strain in a CO-utilizing surface hot spring model organism in order to obtain specific data on C uptake and processing systems. The key subsurface strains are known only by their genomes, which inform us that they are strict anaerobes and that the Wood-Ljungdahl Pathway generating Acetyl CoA from carbon monoxide is likely to be an important route for primary carbon acquisition. Our approach could yield information critical to isolation of Cand. D. audaxviator, leading to subsequent projects in collaboration with Drs. Onstott and Moser. The long-term goal of this consortium will be to isolate and characterize deep-dwelling strains of Firmicutes and other extremophiles.

 

Our objectives include:

1. Obtain insights into the temperature range of D. audaxviator by studying the thermoactivity of two recombinant enzymes expressed from genes deduced from its genome.

2. Complement the deficient C1 utilization pathway in the model organism with a carbon monoxide dehydrogenase from D. audaxviator.

 

We will achieve these objectives by synthesizing genes encoding the Carbon Monoxide Dehydrogenase and the Group III Chaperonin, cloning genes into shuttle vectors for expression in E. coli and in a model organism, the CO-utilizing thermophile Carboxydothermus hydrogenoformans and by purifying and characterizing recombinant Cpn60 and CODH enzymes. The work applies directly to Decadal Goal II, "Determine the principal pathways of carbon transformations in the subsurface and quantify the rates of these reactions." Further study of the metagenomes or metatranscriptomes from well effluents will have diminishing returns of relevant data on the rates and mechanisms of carbon biotransformation unless key species can be successfully isolated and their primary carbon fixation pathways studied.

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Metagenomic Profiling of Microbial Dynamics in Methane Shale after Hydraulic Fracturing

Project Investigators: Wilkins, Mike ; MacRae, Jean ; Mouser, Paula ; Hartsock, Angela

Start Date: 2011-01-01

End Date: 2013-12-31

Summary:

Recent advances in horizontal drilling coupled with hydraulic fracturing techniques have made it possible to extract large volumes of oil and natural gas from methane shale formations located more than a mile below ground surface. The Marcellus and Utica-Point Pleasant shales in the Ohio-Pennsylvania region represent two formations projected to produce large energy resources over the next 25 years. Although Marcellus and Utica core chemistry has been studied, the challenges associated with drilling and sampling at these depths have limited the collection of pristine samples from which to benchmark indigenous microbial populations in these rocks. Our current conceptual model based on wellhead fluids and sparse core observations is that diagenesis temperatures effectively sterilized these formations, and current physicochemical conditions (inc. pressures greater than 50 MPa, temperatures above 60ºC, 20% salt content, and pore sizes less than 1 μm) have limited microbial growth to discrete fracture zones that are connected to regional fluid circulation patterns. The injection of millions of gallons of fluid introduces labile polymeric carbon sources and surface microorganisms to these depths that may colonize new fractures, thereby altering the microbial ecology and biogeochemistry in unknown ways. The fracturing and perturbation of these formations at the current scale has resulted in a significant uncharacterized subsurface environment that may be indefinitely altered through these activities. Understanding microbial survivability and the metabolic potential of organisms that persist is key to elucidating the long-term impacts these technologies will have on deep subsurface microbiota and shale biogeochemistry. Our objective is to profile the metagenomes of dominant microbiota at three time points after fracking. Three samples spanning a short (3 to 14 days) and long (6 or 10 weeks) time frame will be chosen to specifically probe the dominant microbes.

 

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Metagenomic analysis of a continental serpentinizing spring in the Philippines

Project Investigators: Cardace, Dawn; Meyer-Dombard, D'Arcy ; Woycheese, Kristin

Start Date: 2014-11-01

End Date: 2016-11-01

Summary: The Zambales Ophiolite range, located in the Luzon province of the Philippines, hosts several continental serpentinizing fluid seeps in a tropical, densely vegetated environment. Fluid and sediment samples were collected for metagenomic analysis at Manleluag National Park to characterize the deep subsurface microbial community originating in these seeps. This study aims to compare the planktonic microbial deep subsurface community, collected via peristaltic pumping approximately 20 liters of fluid onto Sterivex filters, with sediment-bound microbial communities collected at the source of the seep. Additionally, samples isolated from adjacent springs will be analyzed to provide an indication of the genetic relatedness between two serpentinizing fluid seeps in Manleluag National Park. The results of this analysis will have relevance and be complimentary to studies by the broader deep life community, and will provide baseline data regarding the function and diversity of microbial communities in deeply-sourced tropical serpentinizing fluid seeps.

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Metagenomic sequencing of sediments from the Tonga Trench: A Subsurface island?

Project Investigators: Biddle, Jennifer; Leon-Zayas, Rosa

Start Date: 2014-11-01

End Date: 2016-11-01

Summary: In this project we will investigate the metagenome of the Tonga Trench, at 9.2km water depth and up to 2 m sediment depth. Species of hadal fauna are restricted to single or adjacent trenches and as a result trenches have been referred to as “zoographic provinces”. Put another way, trenches are biogeographically constrained “inverted islands of biodiversity”. At this time it is not clear how different trench microbial communities are distinct from one another or from those present in bathypelagic and abyssal regions. As trenches are an extremely unique ecosystem from the pelagic environment, we anticipate that sediments from within trenches will be equally unique and organisms from the Tonga Trench will show unique adaptations to their environment, which can be determined from comparative sequence analysis.

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Metagenomics in oxic/anoxic transition zones of oligotrophic sediments from the North Atlantic Gyre (North Pond, IODP Expedition 336)

Project Investigators: Jørgensen, Steffen Leth; Zhao, Rui

Start Date: 2014-11-01

End Date: 2016-11-01

Summary:

Microbial metabolism in the oxic/anoxic transition zones (OATZ) of sediments can serve as a barrier preventing the emission of nutrients and greenhouse gases diffusing upwards to the overlying water column. However, microbial diversity and activities in this transition zone are not well understood. Here we propose to employ metagenomic shotgun DNA sequencing on three representative samples from oligotrophic sediment columns retrieved from North Pond during IODP Expedition 336. These sediments offer a unique opportunity to explore the OATZ in oligotrophic sediments, and are characterized by both an upper a lower OATZ. In short we aim at: elucidating the identity to the owners of functional genes that are not “accounted” for in the 16S rRNA libraries. Investigate the genomic inventory of uncharacterized sedimentary lineages of putative archaeal ammonium oxidizers. Expand the current knowledge of the microbial community and its potential activity in the OATZ.

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Metagenomics of the Soudan Iron Mine

Project Investigators: Badalamenti, Jonathan P; Bond, Daniel R; Gralnick, Jeffrey A; Toner, Brandy M.; Sheik, Cody S; Dick, Gregory J

Start Date: 2014-08-01

End Date: 2015-06-30

Summary: Embedded deep within northern Minnesota’s expansive Iron Range, the Soudan Iron Mine transects massive veins of hematite and Archaean (2.7 Gy) banded iron formations, reaching a depth of 713 m (2,341 ft) below the surface. Now an actively maintained State Park serving to preserve Minnesota’s oldest and deepest underground mine, Soudan provides year-round access to the deep terrestrial subsurface and the unique microbial communities it hosts. 

Before mining ceased in 1962, vertical and horizontal exploratory cores were taken at the mine’s lowest level (Level 27, 713 m depth).  Today, these boreholes act as conduits for anoxic, low flow, saline groundwater in an otherwise dry mine.  Calcium chloride brines emanate from seeps with ionic strengths up to three times saltier than seawater, low oxidation-reduction potentials, circumneutral pH, and low concentrations of organic electron donors.  Despite carbon limitation, anoxic brines contain reduced metals and continuously evolve methane gas, raising fundamental questions about the microbes responsible for primary production and carbon flux in this subsurface ecosystem. 

Our project takes a shotgun metagenomics approach to address fundamental questions about the biogeochemical roles of Soudan’s microbial life and how these processes structure subsurface food webs. Having taken samples from several different borehole seeps on Level 27, we specifically focused on understanding how redox potential constrains the metabolism of microbial communities, identifying the microorganisms responsible by leveraging cutting edge, post-assembly genome reconstruction methods, and building conceptual metabolic food webs of subsurface communities based on previous ribosomal sequencing efforts and physiological insights gained from genome annotation.

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Microbial Census of Deep-Sea Sediments

Project Investigators: Walsh, Emily ; D'Hondt, Steven

Start Date: 2011-01-01

End Date: 2014-08-31

Summary: We proposed applying 454-based tag pyrosequencing to investigate how microbial communities change with sediment depth, age, lithology, and porewater geochemistry in the sediment that underlies one of the highest productivity regions in the world ocean. To accomplish this goal, we analyzed extracted DNA from 16 samples taken in high resolution in conjunction with detailed porewater geochemistry. This dataset provides an unprecedented insight into the microbial ecology of a highly productive open ocean site (25 cm/k.y) to a sediment depth of 404 m and an age of 1.6 Ma.

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Microbial Census of the Organic-Rich Deep-Sea Sediments of the Highly Productive Bering Sea

Project Investigators: Walsh, Emily

Start Date: 2011-01-01

End Date: 2014-12-31

Summary:

We propose to apply 454-based tag pyrosequencing to investigate how microbial communities change with depth, lithology, and porewater geochemistry in the sediment that underlies the Bering Sea, one of the highest productivity regions in the world ocean. To accomplish this goal, we propose to analyze extracted DNA from 16 samples taken in high resolution in conjunction with detailed porewater geochemistry. This dataset will provide an unprecedented insight into the microbial ecology of a highly productive open ocean site (35 cm/k.y) to a sediment depth of 750 m and an age of 2.1 Ma. We anticipate that through this use of next-generation sequencing technology in combination with biogeochemical and cell count datasets collected during IODP expedition 323, we will obtain significant insight into this previously uncharted ecosystem.

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Microbial Communities in Deep Sedimentary Rocks

Project Investigators: Lin, Li-Hung

Start Date: 2012-09-12

End Date: 2013-11-13

Summary: The deep biosphere is the largest habitat on Earth. Previous studies suggest that microorganisms inhabiting the deep subsurface environments metabolize at extremely slow rates and constitute up to 30% of the total biomass, exerting a profound impact on global elemental cycling among different reservoirs over geological time. Most previous efforts are, however, diverted to examine microbial ecosystems associated with unconsolidated marine sediments at relatively shallower depths (<1 km below seafloor). In contrast, terrestrial settings are composed of primarily crystalline or consolidated sedimentary rocks with hydrological circulation and substrate availability distinct from those in marine sediments. While most groundwater in consolidated rock settings is confined in the fracture network, the distribution and assemblage of microbial communities in pore space with limited nutrient exchange and fluid transport are largely unexplored. Furthermore, the retrieval of terrestrial samples dedicated to geomicrobiology research remains a rare opportunity. In this study, we collected a suite of rock samples distributed from 1500 to 3000 meters below land surface in a foreland basin of Taiwan through a coring operation designed to retrieve the formation property for CO2 sequestration. We will perform contamination assessment, cell abundances, and community assemblages.

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Microbial Communities in Finnish Deep Groundwater

Project Investigators: Itavaara, Merja; Pyrhönen, Mirva; Bomberg, Malin ; Miettinin, Hanna; Vikman, Minna ; Purkamo, Lotta; Salavirta, Heikki; Eriksson, Tarja

Start Date: 2012-01-01

End Date: 2014-12-31

Summary:

Our research concentrates on the Fennoscandian Shield terrestrial deep biosphere. We have characterized the microbial communities (bacteria, archaea and fungi) at several Finnish sites and are presently investigating the possible functions of the uncultured microbial communities in deep groundwater using next generation sequencing techniques, metagenomics, enrichment techniques, such as SIP, cell sorting and genome sequencing. So far we have participated in the DCO Deep Life with a project where we have tried to gain more insight into the use of simple carbon compounds by the deep subsurface microorganisms and their preferences to different common electron acceptors.

This project involves screening of metabolically activated cells by FACS and single cell sorting. We are involved in the 'Enigmatic life underneath us: Genomic analysis of deep subsurface microorganisms' project (Dr. Stephanauskas, Lead) where genomes of single bacterial cells from deep terrestrial groundwater will be sequenced. In addition, we have participated in the high throughput community sequencing opportunity by the CoDL and are presently working on the data obtained from that effort.

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Microbial Diversity and Carbon Cycling in Deep Subsurface Basalts Targeted for Geologic Carbon Sequestration

Project Investigators: Lavalleur, Heather ; Colwell, Frederick S.

Start Date: 2011-01-01

End Date: 2013-12-31

Summary:

Large igneous provinces (LIPs) are being examined as potential geological storage sites for carbon dioxide in an attempt to sequester CO2 away from the atmosphere to alleviate temperature increases due to climate change. The Wallula pilot well, the location of a geologic carbon sequestration project in eastern Washington State and located in the Columbia River Basalt Group (CRBG), provides a window to the subsurface where the microbial diversity of these geologically important regions can be explored. In addition, the well provides insight into the microbial communities present in the basalts that could play a role in carbon cycling in the deep subsurface where supercritical CO2 (scCO2) is injected. Analyzing samples from the CRBG using deep DNA sequencing technology will further the understanding of the unique microbial diversity of the subsurface, especially with respect to community composition of LIPs and different microbes present in different formations. Pyrosequencing will also establish an important baseline for understanding the microbial communities in the aquifers of the Wallula pilot well prior to the injection of scCO2. These communities will certainly change following the injection and pyrosequencing could play a critical role in the analysis of the samples obtained after the injection of scCO2 into the system. The high resolution associated with deep sequencing technology would allow the detection of shifts in the diversity of the microbial communities.

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Microbial Diversity in Deep Subsurface Basalts

Project Investigators: Colwell, Frederick S.; Lavalleur, Heather

Start Date: 2011-06-07

End Date: 2013-03-18

Summary: Geological carbon sequestration in basalts is a promising solution to mitigate carbon emissions into Earth’s atmosphere. The Wallula pilot well in Eastern Washington State, USA, provides an opportunity to investigate how native microbial communities in basalts are affected by the injection of supercritical carbon dioxide into deep, alkaline formation waters of the Columbia River Basalt Group. Our objective was to characterize the microbial communities at five depth intervals in the Wallula pilot well prior to CO2 injection to establish a baseline community for comparison after the CO2 is injected. We examined microbial communities using quantitative polymerase chain reaction to enumerate bacterial cells and 454 pyrosequencing to compare and contrast the diversity of the native microbial communities. The deepest depth sampled contained the greatest amount of bacterial biomass, as well as the highest bacterial diversity. The shallowest depth sampled harbored the greatest archaeal diversity. Pyrosequencing revealed the well to be dominated by the Proteobacteria, Firmicutes, and Actinobacteria, with microorganisms related to hydrogen oxidizers (Hydrogenophaga), methylotrophs (Methylotenera), methanotrophs (Methylomonas), iron reducers (Geoalkalibacter), sulfur oxidizers (Thiovirga), and methanogens (Methermicocccus). Thus, the Wallula pilot well is composed of a unique microbial community in which hydrogen and single-carbon compounds may play a significant role in sustaining the deep biosphere.

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Microbial Diversity in Groundwater from Bedrock Sites in Sweden and Finland

Project Investigators: Pedersen, Karsten

Start Date: 2011-01-01

End Date: 2013-12-31

Summary:

Underground research laboratories (URL) have been constructed in geographical locations chosen for high-level radioactive waste repositories (HLRW) in Sweden and Finland. The Swedish URL is named Äspö Hard Rock Laboratory and is situated on the South East coast of Sweden about 400 km south of Stockholm. The Finnish URL is named ONKALO and is located in the Olkiluoto area on the west coast of Finland, about 300 km northwest of Helsinki. Both URLs reach about 450 m underground and they are excavated in crystalline bedrock. Both URLs reach about 450 m underground and are excavated in crystalline bed-rock. The sites have been investigated thoroughly for microbiology over a period of about 20 years. The main difference between the sites is that in Olkiluoto, there are large amounts of methane below about 300 m depth that can reach up to 1000 mL methane or more per liter of groundwater at 800 – 1000 m, while the Äspö groundwater typically have amounts of methane in the 0.1 - 5 mL L-1 range. Olkiluoto with its large amounts of methane consequently is of great interest as a deep carbon observatory. Äspö groundwater is rich in sulphate and sulphate reducing bacteria (SRB) down to 500 m and deeper, while ONKALO groundwater is almost free from sulphate and SRB at depths deeper than 350 m. General questions that we will address include: 1) Are there depth dependent distributions of diversity in Fennoscandian hard rock aquifers (both sites)? and 2) Are there differences in diversity between Äspö and the Olkiluoto/ONKALO sites (both sites)?

 

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Microbial Diversity in Subseafloor Sediments Hosting Gas Hydrates

Project Investigators: Colwell, Frederick S.; Briggs, Brandon

Start Date: 2011-01-01

End Date: 2013-12-31

Summary:

Gas hydrate systems are often composed of biologically produced methane, yet we are still trying to understand fundamental aspects about the microbial processes that cycle the carbon in hydrate-rich sediments. Microbes responsible for the creation of the methane that exists in hydrates and the processes that they carry out need to be characterized in order to fully understand the role of the largest methane reservoir on the planet. Furthermore, a more complete accounting of the types of microbes present and the diversity of Bacteria and Archaea that occur in and near sediments where hydrates exist is needed in order to develop conceptual models of the biological representatives of these globally significant deep strata. Our goal is to examine the relationship of microbial community diversity to environmental parameters in deep methane hydrate-bearing marine sediments. Analysis of the microbial communities using deep DNA sequencing technology will provide an assessment of the key microbes in these samples where microbially produced methane accumulates in hydrates and where other microbes may play a role in consuming the methane.

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Microbial Diversity in the Deep Hot Biosphere

Project Investigators: House, Chris ; Brandt, Leah D

Start Date: 2013-04-03

End Date: 2015-07-01

Summary: Site C0014 from IODP Exp. 331 has a unique geochemistry down core because of increasing hydrothermal conditions. This project is intended to explore how microbial communities change with depth.

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Microbial Ecology of IODP Site U1387 - Population Response to Dynamic Depositional Environments

Project Investigators: Biddle, Jennifer; Russell, Joseph A.

Start Date: 2014-11-01

End Date: 2016-11-01

Summary: IODP Site U1387 is a unique location to study microbial assemblage in relation to varying depositional environment. Its distal location to the Mediterranean Outflow has exposed it to a dynamic history of changing currents, water masses, and turbidite events. We will investigate potential effects of these events on the down-core microbial community via 16S rRNA sequencing of bacteria and archaea.

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Microbial Genomic Reconstructions from Hydraulically Fractured Shale Fluids Reveal Community-wide Biogeochemical Cycling and Phage Signatures

Project Investigators: Mouser, Paula ; Wrighton, Kelly

Start Date: 2014-05-01

End Date: 2014-12-31

Summary:

The shale gas basins in the Ohio-Pennsylvania-New York region contain the largest natural gas reserves in the U.S. In 2012, over 7,000 Marcellus shale gas wells were active in Pennsylvania, and Ohio is expecting a similar level of development in its deeper, Utica-Point Pleasant shale over the next 5 years. These shales have insufficient permeability to produce natural gas at economical rates, thus their development requires horizontal drilling coupled to hydraulic fracturing, or “fracking”. The hydraulic fracturing process involves wellbore detonation and high-pressure injection of large volumes (up to 20 million L) of freshwater and sand mixed with chemical additives to propagate fissures in the shale matrix, maximizing the surface area for natural gas release to the wellbore. As a result of fracturing, larger flow paths and newly exposed shale surfaces offer greater biogeochemical gradients for microbial colonization with greater opportunities for nutrient and genetic exchange. We know little about the indigenous microbial membership of Marcellus shale, but given current physicochemical conditions of this formation (depths > 1000 m, pressures > than 50 MPa, temperatures > 60ºC, 20% salt content, and pore sizes < 1 μm) we anticipate that organisms enriched during energy development will encode adaptations to these physical and geochemical conditions. Our motivation is to understand the biotic and engineered factors responsible for altering microbial community structure and biogeochemical cycling in these deep subsurface ecosystems. To better understand the metabolic capabilities and adaptation of these organisms, the DCO Census of Deep Life program supported our 2013 proposal to conduct community genomic sequencing for three time points after fracturing. Our goal for this new CoDL project is to identify viral roles in controlling microbial abundance, manipulating genetic content, and impacting biogeochemical cycling in deep shale environments.

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Microbial Life in an Underground Ancient Ocean: Metagenomics of the Soudan Iron Mine

Project Investigators: Gralnick, Jeffrey A; Bond, Daniel R; Sheik, Cody S; Toner, Brandy M.; Badalamenti, Jonathan P; Dick, Gregory J

Start Date: 2014-07-01

End Date: 2014-12-31

Summary:

Embedded deep within northern Minnesota’s expansive Iron Range, the Soudan Iron Mine transects massive veins of hematite and Archaean (2.7 Gy) banded iron formations, reaching a depth of 713 m (2,341 ft) below the surface. Before mining ceased in 1962, vertical and horizontal exploratory cores were taken at its lowest level (Level 27, 713 m depth) to track the iron formation. Today these boreholes act as conduits for anoxic, low flow, saline groundwater in an otherwise dry mine. Despite carbon limitation, anoxic brines contain reduced metals and continuously evolve methane gas, raising questions about the microbes responsible for primary production and carbon flux in this subsurface ecosystem. Amplicon sequencing surveys and isolate genomes suggest novelty within these low-diversity communities, and establish an opportunity to study physiological adaptations of microorganisms that thrive in this fascinating subsurface environment. Our objective is to use shotgun metagenomic sequencing to address fundamental questions regarding the biogeochemical roles of Soudan’s microbial life and how these processes structure subsurface food webs. 

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Microbiology of the Costa Rica Margin Subsurface

Project Investigators: House, Chris ; Martino, Mandi

Start Date: 2011-01-01

End Date: 2013-12-31

Summary:

To date, efforts to study the microbial communities in the marine subsurface biosphere have established the widespread occurrence of microbes in deeply buried sediments, revealed initial phylogenetic identities of microbial groups present, and provided some subsurface microbial cultures. We used 454-based tag sequencing of the 16S rRNA gene to characterize the microbial communities along depth profiles from two Costa Rica Margin subseafloor sites, located on the upper plate of a unique subduction zone system. This research will allow a more thorough image of the diversity within these subsurface samples than possible in previous subseafloor diversity studies. In particular, this deep diversity data will provide a greater perspective into previously proposed relationships of lithology and chemistry to the microbial diversity seen in subsurface sediments.

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Mid-Cayman Rise Hydrothermal Vents

Project Investigators: Huber, Julie; Seewald, Jeff ; Reveillaud, Julie

Start Date: 2012-01-01

End Date: 2014-06-30

Summary:

The goal of this subproject of the RHC grant was to characterize the diversity of microbial communities in hydrothermal vent habitats from the Mid-Cayman Rise, the world’s deepest hydrothermal system discovered to date. The vent samples encompassed a range of subseafloor environments from those that were high-temperature, deep, and basalt-hosted to those that were shallower, ultramafic-hosted, and potentially influenced by serpentinization. The work also intended to use metagenomics and metatranscriptomics to characterize the potential function of microbial communities associated with these sites and stable isotope tracer approaches to characterize activities in the vent samples.

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Mid-Cayman Rise, Piccard and Von Damm Vent Fields (Field Study)

Project Investigators: German, Chris

Start Date: 2013-05-31

End Date: 2014-07-02

Summary: The exploration-based science in this project will focus upon three types of field sites that represent widespread rock-hosted subsurface environments: deep sea hydrothermal vents, serpentinizing ophiolites, and deep fracture systems. The overarching hypothesis will test whether H2 and sunlight-independent organic carbon fuel microbial activities in deep subsurface ecosystems. Our interdisciplinary studies of field sites will use comparative taxonomic, metagenomic, and transcriptomic technologies linked to thorough characterization of all sites, single-cell genomics analysis, high-pressure enrichments, and experimental microbiology of novel cultivars, and measurements of microbial utilization of H2 and various carbon compounds. These analyses will (1) create a catalogue of microbial activities relative to their environmental context; (2) resolve the extent and diversity of microbial communities in subsurface environments; (3) identify the relationships between deep subsurface microbial processes and carbon flux; and (4) stimulate applied research aimed at alternative energy approaches, biotechnology, and carbon sequestration. The overall questions include; “How do organisms in the rock-hosted subsurface transform and assimilate carbon compounds and how quickly do they do this?” and “How do these processes relate to ambient pressure, their geologic context (fluid flux, carbon sources and sinks), to microbial physiology, and to evolution in the subsurface environment?”

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North Pole Dome, Australia

Project Investigators: Hazen, Robert

Start Date: 2014-06-17

End Date: 2014-06-27

Summary:

The North Pole Dome in Western Australia is a small feature within the ancient Pilbara Complex, which represents a remarkable raft of virtually unaltered crust from Earth’s Archean Eon. It is a roughly circular ring of hills, approximately 12 kilometers in diameter, surrounding a relatively flat depression that represents a “caldera”—the collapsed center of a 3.5 billion-year-old volcano. Following the collapse, the caldera gradually filled with layers of sediments, some of which contain pristine microbial fossil mounds called “stromatolites,” as well as other features that point to a dynamic shallow water environment.  

The  North Pole Dome holds Earth’s oldest unambiguous fossils, as well as extensive carbon-bearing rocks and hints regarding Earth’s early geochemical environment. This study will include detailed mapping of North Pole Dome hydrothermal systems and will investigate distributions of organic carbon, the geochemistry of carbonate minerals, and the nature and extent of diagnostic detrital minerals in these ancient rocks.

Geological Context: Life may have arisen in a hydrothermal environment, in an alkaline, low-temperature (<100°C) system. Indeed, the oldest convincing evidence for life occurs in just such a system, within the exceptionally well-preserved volcanic caldera and associated hydrothermal vein system of the ~3.5 Ga North Pole Dome, Pilbara Craton, Western Australia. Previous work documented a variety of early life signatures in this area, but it remains unclear whether early life was exclusively linked to hydrothermal systems, or if it occupied a variety of niches that reflect diverse microbial environments.

This field study will entail detailed mapping of carbon-bearing zones of the North Pole Dome hydrothermal system. We will also leverage ongoing detailed geological mapping and laboratory analysis of the North Pole Dome to explore three topics tied to DCO Decadal Goals related to Deep Life, Reservoirs and Fluxes, and Deep Energy. First, we will characterize the composition and distribution of carbonaceous materials within the North Pole hydrothermal system, to search for co-variation with changes in fluid temperature, system chemistry, and depth. These studies will assist with discriminating between a biogenic vs. abiogenic origin for the stromatolites, microfossils, and carbonaceous materials preserved in hydrothermal veins, in footwall basalts, in bedded sedimentary rocks, and perhaps in sulfide minerals. Second, we will characterize trace elements in carbonate minerals, including stromatolites. We will test hypotheses on redox-sensitive element distributions and will include these geochemical data as part of the DCO-sponsored Mineral Evolution Database project to develop an open access data infrastructure to probe Earth’s changing C cycle through deep time. Third, we will collect and analyze heavy detrital grains. The study of ancient detrital zircon (and to a lesser extent monazite) grains has opened a new window on early Earth. However, other potentially revealing ancient detrital heavy minerals, such as ilmenite, rutile, cassiterite, and uraninite, have not received comparable attention. We propose to acquire and analyze suites of these heavy mineral separates from the most ancient sedimentary terrains of Western Australia. These studies will complement and amplify decades of field and analytical research, representing millions of dollars in grants for investigations of North Pole Dome geology.

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Ocean Crust Mineral Metagenomes

Project Investigators: Popa, Radu ; Mason, Olivia ; Colwell, Frederick S.; Fisk, Martin ; Smith, Amy

Start Date: 2015-07-15

End Date: 2015-07-18

Summary: Our findings from 454 pyrotag sequencing revealed that the mineral olivine was dominated by the uncultured, and thus uncharacterized, archaeum Archaeoglobaceae OTU 5. Given that olivine is one of the most abundant minerals in oceanic crust, understanding the functional role of the uncultured Archaeoglobaceae in biogeochemical cycling in the marine subsurface is imperative and has the potential to provide transformative knowledge regarding mineral weathering on a global scale.

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Oman Drilling Project

Project Investigators: Nicolas, Adolphe; Sherwood Lollar, Barbara; Blackman, Donna; Shock, Everett; Pézard, Phillipe ; Al Rajhi, Ali; Langmuir, Charles H.; Klein, Frieder; Michibayashi, Katsuyoshi; Bani, Philipson ; Hirth, Greg; Miller, Jay; Coogan, Laurence; Matter, Jürg; MacLeod, Chris; Sonnenthal, Eric ; Jamveit, Bjorn; Manning, Craig; Umino, Susumu; Ceuleneer, Georges; Godard, Marguerite; Goldstein, Steven; Miyashita, Sumio ; Koepke, Jurgen; Nasir, Sobhi; Singh, Satish; Gouze, Philippe; Ildefonse, Benoit; Hofmann, Albrecht; Kelemen, Peter; Arai, Shoji; Teagle, Damon; Warren, Jessica; Takazawa, Eiichi

Start Date: 2014-04-01

End Date: 2017-03-31

Summary: The Samail ophiolite in Oman and the United Arab Emirates (UAE) is the world’s largest, best-exposed, and most-studied subaerial block of oceanic crust and upper mantle. In an ongoing dialogue between geological studies of the ophiolite and seagoing investigations along modern oceanic ridges, observations from Oman and the UAE are central to scientific understanding of oceanic plates formed at spreading centers. Observations of mantle peridotites overlying the subduction zone thrust, which carried the ophiolite onto the Arabian continental margin, reveal an unexpected reservoir of carbon, derived from subducted sediments and precipitated as carbonate minerals in the mantle wedge. This could form an important, hitherto unrecognized part of the global carbon cycle. And, following on ground-breaking work in the 1980’s, there has been a recent surge of interest in the Samail ophiolite as the ideal site for studies of weathering in mantle peridotite, together with the subsurface biosphere fueled by microbial catalysis of low temperature alteration reactions. Such studies will contribute to understanding of microbial ecosystems in extreme environments and the origin of life.  

Following a successful workshop in September 2012, our international team of 38 investigators proposes a comprehensive drilling program in the Samail ophiolite in the Sultanate of Oman. Via observations on core, geophysical logging, fluid sampling, hydrological measurements, and microbiological sampling in a series of diamond- and rotary-drilled boreholes, we will address long-standing, unresolved questions regarding melt and solid transport in the mantle beneath oceanic spreading ridges, mass transfer between the oceans and the crust via hydrothermal alteration, and recycling of volatile components in subduction zones. We will undertake frontier exploration of subsurface weathering processes in mantle peridotite, natural mechanisms of carbon dioxide uptake from surface waters and the atmosphere via alteration and weathering, the process of reaction-driven cracking, and the nature of the subsurface biosphere in peridotite undergoing alteration and weathering. This aspect of the Oman Drilling Project is the one that is co-funded by the Sloan Foundation as part of the Integrative Field Studies for the Deep Carbon Observatory, Sloan Foundation Grant No: G-2014-3-01  Societally relevant aspects of our project include the involvement and training of university students in earth science research, including numerous students from Sultan Qaboos University in Oman. Studies of the natural system of mineral carbonation in peridotite will contribute to design of engineered systems for geological carbon dioxide capture and solid storage. More generally, our studies of alteration will contribute to fundamental understanding of the mechanisms of reaction-driven cracking: chemical reactions that cause subsurface cracking, enhancing permeability and reactive surface area, in a positive feedback mechanism. The results of these studies studies could enhance geothermal power generation and extraction of unconventional hydrocarbon resources.

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Produced Water from Hydraulically Fractured Shale

Project Investigators: Wrighton, Kelly ; Mouser, Paula ; Daly, Rebecca

Start Date: 2012-06-12

End Date: 2013-05-01

Summary: The shale gas basins in the Ohio-Pennsylvania-New York region contain the largest natural gas reserves in the U.S. In 2012, over 7,000 Marcellus shale gas wells were active in Pennsylvania, and Ohio is expecting a similar level of development in its deeper, Utica-Point Pleasant shale over the next 5 years. As a result of fracturing, larger flow paths and newly exposed shale surfaces offer greater biogeochemical gradients for microbial colonization with greater opportunities for nutrient and genetic exchange. We know little about the indigenous microbial membership of Marcellus shale, but given current physicochemical conditions of this formation (depths > 1000 m, pressures > than 50 MPa, temperatures > 60C, 20% salt content, and pore sizes < 1 μm) we anticipate that organisms enriched during energy development will encode adaptations to these physical and geochemical conditions. We previously reconstructed genomes from three samples from post-fracturing produced water in Marcellus shale gas wells and identified a large fraction of prophage and lytic phage contigs. This glimpse into the subsurface virome suggests phage may pay an important role in structuring microbial community function in deep shale environments. Sequencing of additional samples in Marcellus shale gas well produced fluids provides a rare opportunity to investigate phage diversity, phylogeny, dynamics, and auxiliary metabolic gene pools in deep terrestrial environments. The goal of our project is to identify viral roles in controlling microbial abundance, manipulating genetic content, and impacting biogeochemical cycling in deep shale environments.

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Proposal to Apply Pyro-Tag 16S rRNA Gene Sequence Analyses to the Deep Microbial Communities in the Witwatersrand Basin, South Africa

Project Investigators: Kieft, Thomas; Onstott, Tullis; Litthauer, Derek ; Sherwood Lollar, Barbara; van Heerden, Esta

Start Date: 2011-01-01

End Date: 2013-12-31

Summary:

The Witwatersrand Basin is ~300 km long x 150 km wide and comprised  of 2.9 Ga terrigenous, siliclastic detrital sediments of the Witwatersrand Supergroup, overlain uncomformably by the 2.7 Ga Ventersdorp Supergroup continental volcanic units, which in turn are overlain by the 2.5 Ga Transvaal Supergroup marine dolomite and banded iron formation. The entire sequence was metamorphosed to 250-300°C during circulation of hydrothermal fluids, first from the intrusion of outliers of the 2.06 Ga Bushveld Igneous Complex (BIC) and then from the 2.02 Ga Vredefort Impact  at the center of the basin. The Au and Pt mines offer access to fracture fluid over a range of depths (approaching 4 km), temperatures (geothermal gradients vary from 9-15°C/km), geochemical conditions, and subsurface residence times. The fracture water represents a mixture of low salinity, hypoxic, paleometeoric water, some as young as 5 to 40 kyr old, and anoxic, saline water, ~2.0 byr old, with bulk ages of ~1 to 100 million years old. The younger fresh and typically shallower fracture water originates from the Karroo and Transvaal Supergroups’ aquifers and contains a diverse bacterial and archaeal planktonic community with potential eukaryotic components. The typically deeper, older, saline fracture water contains abiogenic hydrocarbons and radiolytically produced H2 and sulfate that fuels a planktonic microbial community comprised of sulfate-reducers and autotrophic methanogens. Our objectives for this CoDL project include exploring the “rare” biosphere in the fracture systems, differentiating the sessile from planktonic communities, and identify the distribution of the Desulforudis audaxviator in the mine subsurface.

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Proposal to Apply for 454-Based Tag Sequencing: Exploring Microbial Diversity in the Deep, Hot Biosphere

Project Investigators: Brandt, Leah D; House, Chris

Start Date: 2011-01-01

End Date: 2013-12-31

Summary:

Hydrothermal systems are one of the deep ocean’s extreme ecosystems. Though studied to some extent at the surface, hydrothermal systems have not been extensively studied beneath the seafloor. The subseafloor contains 10-30% of Earth’s total living biomass, yet, we know little about the contribution or microbial populations of subseafloor hydrothermal systems. On IODP Expedition 331 to the Okinawa backarc basin, several tens of meters of sediment cores were collected in close proximity of a hydrothermal vent chain. Our objective is to apply 454-based tag sequencing to explore the microbial diversity around a subvent biosphere as temperature becomes a limiting factor with depth. This sequencing of the V4 through V6 hypervariable regions of rDNA gene will supplement current work aimed at characterizing the microbial community composition in this understudied, subsurface environment.

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Proposal to Sequence Genomes of Uncultivated Microorganisms from 30 m and 80 m Deep in Baltic Sea Sediments Using Single-Cell Genomics

Project Investigators: Lloyd, K G; Bird, Jordan

Start Date: 2014-05-01

End Date: 2014-12-31

Summary:

As part of the onshore science party in IODP Expedition 347: Baltic Sea Paleoenvironment, we are sequencing single-cell genomes of uncultured archaea and bacteria in sediments as deep as 85 m. The vast majority of the microbial diversity in deep subsurface environments has eluded efforts to be cultured. Single-cell genomics affords us the opportunity to uncover new prospective ecophysiological roles carried out by enigmatic yet abundant organisms. We now have 40 single amplified genomes (SAGs) from 37.5 mbsf and 20 SAGs from 85 mbsf at Baltic Sea Basin hole 60B. The SAGs include members of the deeply branching archaeal and bacterial groups lacking cultured representatives. To our knowledge, these are the first SAGs ever successfully obtained from deep (>10 mbsf) marine sediments. We currently have funding to sequence 10 SAGs. Adding three more would therefore greatly expand our understanding of the genetic composition of uncultured microorganisms in deep Baltic Sea sediments.

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Proposition for Sequencing of Amplicon Libraries from Fennoscandian Shield Groundwater

Project Investigators: Bomberg, Malin ; Itavaara, Merja

Start Date: 2011-01-01

End Date: 2013-12-31

Summary:

The microbial communities of the Fennoscandian shield have been characterized by both cultur-dependent and independent methods since the 1980s. These approaches have revealed a diversity of microorganisms of wide metabolic variety. Only a small part of the microbial communities appear to be truly autotrophic. Methane is an important carbon source in these deep groundwaters and is very abundant. Methane composes the clear majority of the gasses in these groundwater samples. It has still not been shown, however, that any specific group of microorganisms would utilize this methane in the Finnish deep terrestrial groundwater. Typical methane oxidizing bacterial or archaeal linages have not yet been detected. Nevertheless, an enrichment of sulphides at methane-sulphate transition zones where other carbon sources than methane are scarce, suggest that methane is oxidized in combination with sulphate reduction. The number of methanogens and sulphate reducers is low.  Our objective is to study the community composition of archaea and bacteria at different depth of Olkiluoto groundwater. We want to clarify the co-existence of specific microbial groups in relation to hydrogeochemistry and the presence of methane and sulphides.

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Proteome Profiling to Delineate Subsurface Microbial CH4-Cycling Pathways

Project Investigators: Perlman, David ; Onstott, Tullis; Lau, Maggie C.Y.

Start Date: 2014-04-01

End Date: 2014-09-30

Summary: This project will provide resources to characterize the metaproteome expressed at deep fracture water sites in South Africa and it will supplement existing metagenome, metatranscriptome, and single-cell genome data. The project will develop protocols for extraction and purification of proteins from the same samples used for the metagenome and metatranscriptome studies. It will identify proteins and enzymes involved in active C and N utilization pathways associated with CH4 cycling and will establish links to aqueous, gaseous, and isotopic geochemistry studies.

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Santa Elena Ophiolite, Costa Rica

Project Investigators: Sanchez-Murillo, Ricardo ; Crespo-Medina, Melitza

Start Date: 2014-06-03

End Date: 2014-07-03

Summary: The Santa Elena Ophiolite comprises 250 km2 of ultramafic rocks and mafic associations along the northwestern Pacific coast of Costa Rica. Uplifted ultramafic rocks originating from Earth’s mantle represent a reservoir of carbon and reducing power, which, during the process of serpentinization, are liberated into the surface environment, potentially supporting microbial growth.

The extremely variable weather conditions that occur on the Santa Elena Peninsula between the wet (May – October) and dry (November – April) seasons result in a unique hydrogeological environment. Contrary to other continental serpentinization environments (e.g., Tablelands, Canada; Western Coastal Range, USA; Gruppo di Voltri, Italy), in this tropical scenario, precipitation quantities and intensities are usually greater and occur during extended periods of time. This facilitates infiltration, accelerating water-rock interactions and liberating carbon and energy to the surface environment.

Such extreme seasonal events provide a unique opportunity to investigate the response of microbial metabolisms (at the interface of deep carbon and energy fluxes) under a dynamic subsurface hydrology setting. During an expedition in March 2013, we sampled a series of alkaline springs along the peninsula. Preliminary V4-V5 sequence data suggests these fluids have low diversity and indicates the presence of microorganisms involved in hydrogen, methane, and methanol metabolism, including bacteria from the genera Hydrogenophaga and Methylibium and from the family Methylophilaceae, as well as archaea from the orders Methanobacteriales, Methanocellales, and Methanomicrobiales. In this project we intend to demonstrate links between geochemistry, hydrology, and microbial activities related to subsurface processes at Santa Elena Ophiolite.

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Short‐Chain Hydrocarbon Oxidation by Archaea

Project Investigators: Harding, Katie ; Wegener, Gunter

Start Date: 2014-05-01

End Date: 2014-10-31

Summary:

The anaerobic oxidation of shortchain hydrocarbons (methane, ethane, propane, butane, pentane) with sulfate as terminal electron acceptor is an important process in the marine subsurface, for example in deep oil and gas reservoirs. Besides the anaerobic oxidation of methane which is mediated by archaea, anaerobic oxidation of C3C5 alkanes is only known for bacteria. Recently we succeeded with the enrichment of butane oxidizing archaea which form aggregates with partner bacteria that they appear to share with anaerobic methanotrophs (ANME). The 16S rRNA gene sequences of these archaea abundantly occur in subsurface seep environments, suggesting an important role of these clades in the environment. A first physiological description has been performed during a six-month Master's project. The aim of the present six-month project is to elucidate the identity, functioning, and physiological limitation of these novel archaea. An in silico analysis of public 16S gene libraries will help to estimate the role of this and closely related archaea (Thermoplasmatales) in shortchain hydrocarbons.

The project will perform physiological and stable isotope probing experiments on the butane oxidizing communities and will perform genome analyses of the involved organisms. The archaeal bacterial interaction will be investigated by stable isotope probing combined with nanoscale secondary ion mass spectrometry (NanoSIMS). To elucidate the pathways of hydrocarbon oxidation, a metagenomic analysis of this culture will be performed.

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South African Terrestrial Deep Subsurface

Project Investigators: Lau, Maggie C.Y.; Schrenk, Matthew; Linage-Alvarez, Borja; Kuloyo, Olukayode

Start Date: 2012-01-01

End Date: 2013-12-31

Summary: Recent studies of the continental subsurface microbial ecosystem present in the Witwatersrand Basin, South Africa have shown that with increasing depth and fracture water age and salinity, biogenic methane diminishes and abiogenic hydrocarbons and H2 increase and the concentration of planktonic cells slowly declines. Similarly, studies of the 16S rRNA gene in the planktonic community revealed a decrease in the relative abundance of methanogens and an increase in the relative abundance of low G+C Firmicutes with increasing depth.   Metagenome analyses of one member of this Firmicutes community, D. Audaxviator, indicated that it was capable of both heterotrophic and chemoautotrophic activity. An in situ incubation experiment suggests that some Firmicutes are acetogens that may support the aceticlastic methanogens.

Our geochemical and isotopic analyses indicate that abiogenic hydrocarbons are not utilized by the microbial community. Instead, a chemoautotrophic ecosystem is sustained by radiolytic generation of H2 and oxidants. Abiogenic hydrocarbon gases and elevated H2 have also been reported from the Canadian and Fennoscandian Precambrian shields and from ocean floor vents. The subsurface ecosystem present in the Witwatersrand Basin, therefore, is potentially wide spread in both the continental and marine crust, yet quite distinct from continental sedimentary basins or coastal margin sea floor sediments where degradation of photosynthetically produced organic matter deposited within the strata is believed to limit the extent of the subsurface microbial ecosystem. Alternatively, organic acids, thermogenically produced ~2 by ago from the thin shale layers present in the Witwatersrand Basin, could be sustaining the microbial community observed today.

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Subsurface Microbial CO2 Fixation

Project Investigators: Menez, Benedicte; Mansilla, Rosalia Trias; Gerard, Emmanuelle

Start Date: 2012-01-10

End Date: 2013-06-09

Summary: Our research group has been monitoring the microbial diversity in the different available deep wells in the basalt hosted aquifers of Hellisheidi (SW Iceland) since 2008, using bacterial and archaeal 16s rRNA gene cloning, 454-pyrosequencing (hypervariable regions V1-V3) and qPCR. The Hellisheidi site has different strategically located wells, used by Reykjavik Energy, the operator of the associated geothermal plant that has been performing CO2 and CO2-H2-H2S injections at a depth of 400–800 m and temperatures up to ~80°C as mitigation strategy for its main waste products. It provides the unique opportunity to benefit from a large-scale injection experiment mimicking natural processes. The basaltic injection site can accordingly be seen as an observatory of the oceanic crust that encounters similar CO2/H2S-rich fluid percolation, over larger periods of time. Preliminary results suggest that there is a diverse and abundant community that changes drastically after the gas injections. The increase of autotrophic microbial groups after the first injection suggests an enhancement of C fixation, and therefore, consequences for the nutrients cycling in this system. With this project we aim to better understand the microbial communities function and response to pure CO2/CO2-H2-H2S injections in relation to C, S, and N cycles through metagenomics analysis.

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Subsurface Microbial Communities in Volcanic Basement Along an Ancient Hotspot Seamount Trail

Project Investigators: Edwards, Katrina J.; Sylvan, Jason

Start Date: 2011-01-01

End Date: 2013-12-31

Summary:

One of the objectives of Integrated Ocean Drilling Program (IODP) Expedition 330, Louisville Seamount Trail, was to sample and learn about the subsurface biosphere in the Louisville Seamount Chain (LSC). This seamount chain consists of submerged, inactive volcanoes formed at the Louisville hotspot, one of three primary hotspots in the Pacific Ocean. The drilled seamounts range in age from 65 to 77 Ma and basement composition at all three consists of alternating layers of massive lava flows and basaltic breccia. Diverse rock lithologies were recovered from the subsurface seamount environment along the LSC, all of which are potential habitats for microbial colonization. We focus here on analysis of samples collected from Sites U1372, U1374 and U1376 that were recovered deeper than the interface between sedimentary rock and volcanic basement rock. The purpose of this selection is to examine life specifically in deeply buried volcanic basement rocks. The goal of the work is to answer the following questions: (1) What is the diversity of the resident microbes in the subsurface of the LSC? (2) Do the different seamounts host different communities, and are there systematic changes with age? (3) Does the microbial community composition change with depth and/or lithology?

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Subsurface Microbial Contribution to Dark CO2 Fixation in Geological Storage Sites

Project Investigators: Mansilla, Rosalia Trias

Start Date: 2014-07-01

End Date: 2014-12-31

Summary:

The relatively recently discovered microbial communities inhabiting the Earth subsurface are still poorly studied but have to be considered as important contributors to the global C, N, and S cycles, notably in basalts where high diversity was revealed. With the aim of understanding the diversity and dynamics of microbial communities in the basalt hosted aquifers of Hellisheidi (SW Iceland), our research group has been monitoring the microbial diversity in the different available deep wells since 2008, using bacterial and archaeal 16s rRNA gene cloning, 454-pyrosequencing (hypervariable regions V1-V3) and qPCR. The Hellisheidi site has different strategically located wells, used by Reykjavik Energy, the operator of the associated geothermal plant that has been performing CO2 and CO2-H2-H2S injections at a depth of 400800 m and temperatures up to ~80°C as mitigation strategy for its main waste products. It provides the unique opportunity to benefit from a large-scale injection experiment mimicking natural processes. The basaltic injection site can accordingly be seen as an observatory of the oceanic crust that encounters similar CO2/H2S-rich fluid percolation, over larger periods of time. Our objective is to understand the microbial communities function and response to pure CO2/CO2-H2-H2S injections in relation to C, S, and N cycles through metagenomics analysis.

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Sulfidic Deep Terrestrial Subsurface-FS

Project Investigators: McCauley, Rebecca ; Jones, Daniel ; Macalady, Jenn

Start Date: 2009-07-01

End Date: 2015-12-31

Summary:

Sulfur is one of the 10 most abundant elements in the solar system. Its complex redox chemistry and tendency to polymerize have led to a comparably complex array of microbial metabolisms linking sulfur with carbon, nitrogen, and iron in biogeochemical cycles. The Frasassi Cave aquifer has emerged as a model ecosystem for studying sulfur-based chemosynthesis outside the oceans. Our previous work indicated that the chemosynthetic microbial community is directly dependent on reducing power from ancient organic matter in stratigraphically adjacent shales, as well as oxidant anions (sulfate, nitrate) in evaporites immediately underlying the shales. This juxtaposition of rock types (evaporite, shale, carbonate) is common in sedimentary rock sequences globally, and thus the biogeochemistry of the Frasassi deep aquifer may reveal subsurface processes, metabolisms, and microbial taxa that have global significance. Continuing exploration of the Frasassi aquifer by cave divers has recently revealed that there are conspicuous and widespread microbial biofilms throughout the system. Our preliminary study of one of these biofilms using genetic approaches showed that the microbial community contained a large number of novel and uncultivated taxa. The CoDL project is motivated by our new awareness of the large biomass and widespread extent of these enigmatic biofilms throughout the aquifer, which encompasses more than 30 kilometers of passages navigable by humans.

Deep caves in central Italy offer a unique opportunity to investigate the diversity of microoxic and anoxic microbial communities in sulfidic sedimentary aquifers 100-600 m below ground surface. We expect the proposed pyrotag-enabled study to reveal the spatial distributions and redox/temperature niches of enigmatic Epsilonproteobacteria previously detected in extremely microoxic aquifer zones. We also expect to extend our understanding of a diverse anoxic aquifer community dominated by Deltaproteobacteria, Chloroflexi, and a large number of taxa in unnamed and novel clades.

 

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Tag 16S Sequencing of Deeply Buried Sediments Within the Baltic Sea, IODP Exp. 347

Project Investigators: Lloyd, K G; Reese, Brandi; Marshall, Ian

Start Date: 2014-05-01

End Date: 2014-12-31

Summary:

The microbiological objectives of IODP Expedition 347 Baltic Sea Paleoenvironment focused on (1) how the phylogenetic diversity of the deep biosphere in this intracontinental sea differs from that of deep-ocean communities, and (2) are microorganisms that presently live in the deep sediments remnants of limnic and marine populations, or does the modern sedimentary environment select for the community. Our objective for this CoDL project is to conduct tag 16S sequencing for both bacteria and archaea from the Baltic Sea paleoenvironment.

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The Deep Drilling Project in Songliao Basin, China

Project Investigators: Dong, Hailiang

Start Date: 2014-06-01

End Date: 2015-05-31

Summary: This sub-project will leverage major investments by the International Continental Deep Drilling Project (ICDP) and China, who have initiated studies of scientific drilling, pilot CO2 sequestration and oil displacement, geothermal resource development, and seismic and volcanic monitoring in the Songliao Basin, one of the largest hydrocarbon-rich basins in the world. ICDP has funded a project in this basin to drill a 7000-meter borehole into the Cretaceous/Jurassic boundary, to commence in 2014. Chinese scientists have also proposed building a deep underground laboratory in the Songliao Basin, so-called deep multi-well (1000 to 6000 m) underground laboratory (MW-DUL) using a large number of existing boreholes in the Songliao Basin.  These are unique opportunities to investigate deep life and deep energy. Specifically, we propose 3 complementary approaches to the study of the deep biosphere. (1) We will employ CORK in-situ observation systems and a multi-level U-tube fluid sampling system uniquely suited to monitor and recover samples at these depths. (2) We will investigate microbial abundance, diversity, activity, and metabolic pathways in fluids and rocks and correlate these with temperature, pressure, lithological properties (porosity, hydraulic conductivity, and fracture distribution), deep fluid/gas compositions, and radioactivity to determine key environmental conditions that control microbial distribution and activity. (3) We use molecular microbiology approaches to compare microbial community structure and functions before and after CO2 injection to assess the impact of this oil recovery technology on subsurface deep biosphere.  We also propose studies related to deep energy. The sources of hydrocarbons found in the lower parts of the basin stratigraphy have been a controversial topic. We will secure samples for isotopologue and noble gas isotope measurements, lipid biogeochemistry, and organic geochemistry studies. These comprehensive efforts will further constrain the origin of hydrocarbons in the Songliao Basin and their relations with serpentinization.

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The Microbial Biogeography of the Sulfate-Methane Transition Zone

Project Investigators: Walsh, Emily ; D'Hondt, Steven

Start Date: 2014-08-01

End Date: 2014-12-31

Summary: This project will sample SMTZ’s from multiple sites within four globally separated geographic regions of the global ocean including the Bering Sea (IODP Expedition 323 sites U1343-U1345),  Indian Ocean  (NGHP Expedition 1 Sites 3B, 7A, 10A, 14A, 15A, 17A), Peru Margin (ODP Leg 201 Sites U1227-1230) and the Oregon Margin (ODP Leg 204 Sites 1244 and 1251). Samples from the Bering Sea,  Indian Ocean and Peru Margin are currently in hand while Oregon Margin samples will be requested from the IODP sample repository. By examining the SMTZ community composition of these globally dispersed sites, the project will elucidate: (a) variation in microbial communities within and between geographic locations;  (b)  examine differences in “active” community members (RNA vs DNA) and (c) determine the environmental factors responsible [total organic carbon (TOC), sediment age, sediment depth, water depth, dissolved chemistry (e.g., [H2S] concentrations and fluxes of [SO4-], [CH4]), lithology, etc.] for observed differences in SMTZ community composition and diversity.

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Tracing Conversion of Specific Carbon Compounds using Stable Isotopes

Project Investigators: Kieft, Thomas; Seewald, Jeffrey; Onstott, Tullis; Hoehler, Tori M.

Start Date: 2012-01-01

End Date: 2014-06-30

Summary: Stable isotope tracers can measure metabolism of specific compounds in the absence of pure cultures. This project will measure carbon fixation and metabolism of small organic molecules using stable isotope analyses in order to link the occurrence of specific compounds to the organisms that metabolize them under different pressure, temperature, and energy conditions.

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Understanding the Lost City Hydrothermal System

Project Investigators: Lilley, Marvin; Schrenk, Matthew; Brazelton, William; Bernasconi, Stefano ; Kelley, Deborah S.; Lang, Susan

Start Date: 2001-01-01

End Date: 2014-12-31

Summary:

This project is a comparative geochemical and stable isotope study of modern serpentinite-carbonate systems, focused on understanding geochemical and microbial processes associated with the formation of high alkaline fluids during serpentinization. Our study builds on an immense set of data from the peridotite-hosted Lost City hydrothermal system (MAR, 30°N), produced in collaboration with an international team of scientists over more than ten years. Lost City is unlike all other hydrothermal known to date and is characterized by metal- and CO2-poor, high pH fluids (9-11) with elevated hydrogen and methane contents resulting from serpentinization processes at depth.

We specifically address open questions about the links between the inorganic reactions in the ultramafic basement rocks (i.e. serpentinizing reactions), cycling of carbon and sulfur, and microbial activity in these high pH systems. The overall goal of our project is to quantify C and S pools in active serpentinite-carbonate systems and to constrain their changes over time. To achieve this goal our project involves a comparative organic geochemical and C-and S-isotope study of Lost City with modern high alkaline Ca-OH springs and carbonate deposits associated with present-day serpentinization processes in Liguria (Italy). Field and laboratory studies are specifically aimed to (1) characterize the organic matter at a molecular and isotopic level; (2) constrain the origin and cycling of C and S in modern marine and meteoric systems; and (3) make quantitative volume estimates of the amount of abiotic and biotic organic carbon and CO2 that is sequestered in these environments. Our work has been supported by the Swiss National Science Foundation and has involved developing a number of novel analytical techniques to measure stable and radiogenic carbon species in natural samples. Multidiscipline studies of Lost City and the Atlantis Massif provide the basis for IODP drilling during Expedition 357 in 2015.

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Unveiling Carbon Fixation in Three Deep Serpentinization-Driven Hyper-Alkaline Springs

Project Investigators: Tiago, Igor

Start Date: 2014-01-01

End Date: 2014-12-31

Summary:

This project will perform a shotgun metagenomic anlaysis of samples from three continental serpentinization-driven deep aquifers. The samples will be processed using different pore size filters, namely 0.1 μm and 0.2 μm for generation of metagenomes and transcriptomes. The objectives include (1) determining if the microbial populations inhabiting these environments are identical to populations from similar ecosystems, (2) identifying potential presence of carbon fixation pathways and if they are expressed (active), (3) determining if they are the same as those identified in similar ecosystems, (4) determining the nature of the energy network and carbon fixation in the different springs are perpetrated by the same populations and/or by the same functionalities, (5) comparing these environments to other similar deep ecosystems, (6) establishing whether there is a correlation between "deep chemistry" and "carbon fixation/energy flux," (7) evaluating whether commonly used sampling methodology in these environments reveals the community composition and activity? and (8) exploring whether there is a major difference between the "0.2 μm microbial diversity" and "0.1 μm microbial diversity."

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Workshop: Carbon Cycling in Deep Ocean Crust Revealed Through Single-Cell Genomics

Project Investigators: Orcutt, Beth

Start Date: 2014-06-15

End Date: 2014-11-30

Summary:

This project hosted a three-day workshop that focused on synthesizing recently available genomic and metagenomic data from fluids collected from the Juan de Fuca Ridge (JFR) flank subsurface "CORK" borehole observatories with multi-year time-series geomicrobiological data, with a focus on (1) developing a proposal for continued work at the observatories to explicitly test functional hypotheses developed from this synthesis, (2) writing manuscripts, and (3) experiment and sample planning for an August 2014 cruise to these observatories. The workshop primarily supported HI-based scientists and covered the cost of travel for the PI and several invitees.  

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