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Project Progress

Project 5: Geolological-Biological Connections
5.1 Novel culturing, cruises, biofilms: (Schrenk and Baross)

During the past year, Matthew Schrenk’s group Lab at East Carolina University continued the investigation of environments where mineral-catalyzed reactions contribute energy and potentially organic compounds to microbial ecosystems. The focus of their research was on ecosystems dominated by the serpentinization of ultramafic rocks, characteristic of the Earth’s mantle. The team participated in the establishment of a subsurface microbial observatory in the serpentinites of the Coast Range Ophiolite in Northern California with funding from the NAI Director’s Discretionary Fund supporting the recovery of drill cores in this actively serpentinizing environment. Ongoing studies are following the taxonomic and functional diversity of these environments using next generation sequencing approaches. They have also obtained laboratory cultures of alkaliphilic microbes from this system and are focusing upon their genetic and physiological characterization (Fig. 1 and 2). This work is forming the basis of a Ph.D. thesis by Katrina Twing in my laboratory, and contributing to work by William Brazelton, postdoctoral scientist. Additional work is taking place in the Ligurian ophiolites of Italy, which sustains seeps of serpentinizing, low biomass, >pH 12 fluids. Our laboratory has participated in two expeditions to the Ligurian ophiolites in the past year working with collaborators from the ETH in Zurich, Switzerland. They are also engaged in collaborations studying serpentinizing ecosystems in Canada, Portugal, and Oman, in an effort to generate a global perspective on the microbiology and extent of a serpentinite hosted microbial biosphere (Fig 3).

Matt Schrenk and colleagues expanded the study of the sub-surface environments associated with hydrothermal vents, to include viruses so as to assess their possible role in effecting the genetic landscape of the microbial communities. He and coworkers have obtained metagenomes of both a viral assemblage and a bacterial/archaeal assemblage from 170 liters of a >100°C diffuse-flow fluids from a hydrothermal vent on the Juan de Fuca Ridge. They also have “deep-sequencing” ¹⁶S rRNA genes that provide a comprehensive view of the bacterial and archaeal species diversity including the rare groups. They are In the process of annotating these data. Some initial analyses show that microbial communities in subsurface hydrothermal fluids are exposed to a high rate of viral infection, as well as viral metagenomic data suggesting that the vent viral assemblage is particularly enriched in genes that facilitate horizontal gene transfer and host adaptability. Therefore, viruses are likely to play a crucial role in facilitating adaptability to the extreme conditions of these regions of the deep biosphere. Our long-term goals are to identify the genes that are most frequently transferred and to determine the potential for inter-phylum/domain horizontal gene transfer. One of our primary astrobiology goals is to examine the possibility that viruses that infect hyperthermophilic Archaea are model systems for understanding the origin and evolution of viruses and their role in the early diversification of life.

John Baross’ group (Univ. of Washington) continues to test the hypothesis that a tectonically active planet with water is essential for an origin and maintenance of life. Geophysical processes that result of plate tectonics generate the carbon, chemical energy sources and essential trace elements necessary to Earth organisms during the earliest stages of Earth history. For example, there is isotopic and modeling evidence that hydrogen, produced abiotically in hydrothermal systems, may have been the source of energy for the earliest microbial communities on Earth. It would follow that present day microbial communities that thrive on hydrogen, such as found at Lost City and other vent sites, may be useful model systems for studying early life. Likewise, elucidating the carbon-fixation and nitrogen fixation pathways in anaerobic, hyperthermophilic vent microorganisms could provide insights in the evolution of metabolic networks while providing a model of an ancient microbial community for testing hypothesis. The role of viruses in the evolution of these metabolic networks also fits with these goals. These studies could also result in the identification of bio-signatures that could be applicable to our search for evidence of hydrothermal systems on other planets and help identify the source of methane on Mars.

His students include Rika Anderson, a PhD student in Oceanography and Astrobiology; her research project is distribution and molecular characterization of high temperature archaeal viruses from hydrothermal vent and subsurface environments and the role of viruses in the origin and early evolution of life; Aditya Chopra, a PhD student in Astronomy, Australian National University (JAB as co-advisor); Billy Brazelton: NAI post-doctoral fellow; co-advised by Matt Schrenk and JAB. He works on the microbial ecology of serpentinization environments and participates with Rika Anderson on subsurface virus research; Aaron Goldman: NAI post-doctoral fellow at Princeton (JAB as co-advisor); and Stephen Jensen undergraduate student in biochemistry working on bacterial, archaeal and viral ecology of subsurface and hydrothermal vent environments as part of his senior research project.

The Baross group’s astrobiology research objective continues to focus on microbial ecology in the extreme environments associated with deep-sea hydrothermal systems and particularly on sub-surface environments associated with hydrothermal systems. The main components of our research include 1) the molecular and physiological characterization of subseafloor microbial communities with particular emphasis on anaerobic biofilms supported by hydrogen and abiotically synthesized organic compounds and 2) the incidence and characterization of viruses in subseafloor hydrothermal environments and their possible role in the carbon and other nutrient cycles and in the evolution of microbial communities.

Their research includes two types of hydrothermal systems: magma-driven and peridotite-hosted systems. A characteristic of both types of hydrothermal systems, and a focus of our research, is the widespread incidence of vent microbial communities that use mineral-catalyzed hydrogen and sulfur as the primary energy source and exist as biofilms. Autotrophic microbial communities that used hydrogen or sulfur as the primary energy source are believed to be ancient and perhaps even the first metabolic life forms on earth. The dominant microbial community at the Lost City hydrothermal environment consists of hydrogen and methane metabolizing archaeal biofilms. The team completed the analysis of the Lost City samples (MBio, 2011) and awaiting another opportunity to obtain additional samples. They have also completed analyses of the microbial community structure in hydrothermal fluid gradients associated with diffuse-flow vents and vent plumes (FEMS Microbiology Ecology 2012). Surprisingly, a high incidence of a single species of sulfur metabolizing bacteria was found in the plume samples. These sulfur-oxidizing species are cosmopolitan and are frequently seen in oxygen minimum zones. Our report is the first to show their dominance in vent plume environments.

Baross continues cross team collaborations. He is also affiliated with the NAI Icy Moons team at JPL and the VPL team at the University of Washington in the capacity of providing expertise on hydrothermal systems, microbial ecology, geo-microbiology and the evolution of metabolic pathways over the course of Earth history.

5.2 Life in extreme environments: (Steele, Fogel)

Verena Starke was awarded her PhD this summer from the University of Maryland. One aspect of her work on calcite precipitation at an Arctic geothermal spring on endolith colonization and ecological succession with coauthors Andrew Steele and Marilyn Fogel is summarized below. A critical question in microbial ecology concerns how environmental conditions affect community makeup. Troll Springs, a geothermal spring located at 79°23’N, 13°26’E on Svalbard in the high Arctic, provides an exceptional environment for studying microbial communities and succession along steep environmental gradients that impose strong selective pressures. At Troll, warm water is released into cold, dry climate conditions. Precipitation of travertine (calcite) from the spring’s carbonate-rich waters has built terraces that host a range of microbial communities with microbes inhabiting a wide range of environmental conditions: in warm water as periphyton, in moist granular materials as what?, and in cold, dry rock as endoliths. Basically, Troll Springs has two distinct ecosystems, aquatic and terrestrial, in close proximity, with different underlying environmental factors shaping their microbial communities.

Microscopic and phylogeny-based molecular methods were used to study microbial community makeup at Troll Springs. Periphyton are continuously entrapped during precipitation of calcite from the spring’s waters, becoming precursors for endolithic communities. Much of the pore space originally occupied by periphyton becomes inhabited either by organisms that were already present in minor quantities in the periphyton, or by new organisms that colonized an environment for which they were well suited. This colonization process differs from most endolith colonization, where rock predates the communities that colonize it.

In the aquatic environments, where water is abundant, the strongest dependence of community makeup is on pH and temperature, with gradual changes in community makeup along a pH/temperature gradient among the pools. Illumination (as limited by calcite precipitation) and thermal stability also appear to exert an influence. In contrast, in the granular and endolithic terrestrial environments, lack of soil moisture exerts selective pressure. In those environments, they observed a strong relationship between community makeup and water content of the matrix.

The richness, evenness, and diversity of microbial taxa are all strongly correlated at Troll Springs. These parameters all increase with decreasing temperature across the aquatic samples, and all decrease with decreasing water content across the terrestrial samples. We attribute the trends in evenness to the balance of competition, with evenness limited in the most calcite-free environments by competition with photosynthetic eukaryotes, and in the driest endolith by competition for water and possibly nutrients. We suggest that the trends in richness are a result of the availability of physical niches, with niche availability first increasing as calcite grain surfaces become available for colonization, and later decreasing as pore volume becomes potentially limiting.

The microbial community structure at Troll can be understood as a consequence of ecological succession. It begins at the spring source with a few dominant phylotypes, progressing as conditions change into a more stable and even community. The succession is characterized by gradual changes in environmental parameters that produce a sequence of small, incremental environmental disturbances. Small, cumulative disturbances change resource availability, and alter diversity slowly by affecting growth, reproduction and competition, leading to successional transitions.

Mihaela Glamoclija, Fogel, Liane Benning (Univ. of Leeds), Pamela Conrad (Goddard) and Steele are completing a study on microbial cycling of nitrogen and carbon in an Arctic travertine spring (Jotun Spring, Svalbard Archipelago). Jotun geothermal spring from Spitsbergen Island (79°27’N, 13°17’E) is dominated by travertine gravel, which has formed a terrace around the spring’s source area and down the slope/stream. This spring and associated terraces are dominated by microbial biomass, probably because the shifting environment of the spring offers little opportunity for higher organisms to thrive. We studied two transects to illustrate two different environmental gradients: (1) from the spring source to the end of the stream, which has a continuous source of nutrients, is fully aquatic, and rich in microbial mass; (2) along the gravely travertine plateau, from the spring source to the plateau edge with limited nutrient access, primarily travertine gravel with no visible microbial biomass at the surface. A combination of environmental physicochemical conditions, stable isotopes, and molecular biology were used to determine the characteristics of environmental gradients and communities and to decipher nodes of nitrogen and carbon cycles.

Gasses collected from source water were mainly CO₂ (30%) and N₂ (70%) [see 1]. The NH₄ and NO₃ concentrations, measured in multiple years between 2004 and 2011 years, showed that along the stream transect the concentrations of NH₄ decreased gradually, while those of NO₃ increased (i.e. NH4 22.25 to 8.2 μM; NO3 0 to 3.56 μM). The δ¹⁵N values of biomass from the stream ranged from -3 to +6‰. Both patterns indicate three possible microbial processes: nitrogen fixation, ammonia incorporation, and nitrification. The NH₄ – NO₃ pattern measured at the travertine plateau transect showed a high predominance and increase of NO₃ over NH₄ concentrations (NH₄ 0.35 to 1.9 mg/gm; NO₃ 30±5 mg/gm) suggesting that nitrification is significant process in this setting. δ¹⁵N of the terrace transect were less variable but generally positive values, which could be related to microbial assimilation and nitrification. Total N content of sediments in the stream transect averaged 1.20 wt., whereas the gravel transect had 0.08 wt. indicates that in the stream samples were enriched in biomass. The δ¹³C value of total carbon travertine carbonates varied from -1 to 2.5‰, whereas the δ¹³C of organic carbon ranged from -26.5 to -28‰. In contrast, biofilm δ¹³C measured within the stream was more negative: -30 to -31.5‰,, utilizing the high concentrations of dissolved CO₂ [1].

Nitrogen fixation genes were detected in all of the samples, supported by many of the δ¹⁵N measurements in the stream. A variety of ammonium oxidation genes (β-, γ-proteobacteria, archaea) were detected in all of the samples, which supports the nitrification hypothesis suggested by nitrogen analyses. Denitrification genes were detected in all of the settings, showing that microbial organisms from the analyzed habitats do have abilities to complete the nitrogen metabolic circuit. Further work on reverse transcriptase will reveal the metabolic pathways that were active and in what measure genetic analyses reflect the biogeochemical data.

Claire Cousins (University College London) worked with Fogel and Roxane Bowden on a study of stable isotope fractionation at a glacial hydrothermal field: implications for biogeochemistry and biosignatures on Mars along with collaborators Charles Cockell (Astrobiology Center, Edinburgh), Ian Crawford (UCL), Matt Gunn (Aberystwyth University), John Ward (UCL), Magnus Karlsson, and Thorsteinn Thorsteinnson (Icelandic Glaciological Society). Hydrothermal environments that arise through the interaction between volcanogenic heat and glacial ice are ideal sites for understanding microbial biogeochemical processes on Earth, and also potentially on Mars where similar volcano-cryosphere interactions are thought to have occurred in the past. The Kverkfjöll subglacial basaltic volcano in central Iceland is geographically isolated, with little influence from flora, fauna, and human activity. Major environmental inputs include geothermal heat, meltwater from ice and snow, and outgassing of CO₂, H₂S, and SO₂. Large physiochemical gradients exist, from steaming fumaroles and boiling hydrothermal pools, to frozen geothermal ground and glacial ice. Stable isotope measurements of total organic carbon, total sulphur, and total nitrogen were coupled with metagenomic analysis of the residing microbial communities, with the aim to identify biogeochemical relationships and processes operating within the Kverkfjöll geothermal environment, and also to identify any isotopic biosignatures that could be preserved within geothermal sediments. This study focused on a variety of samples taken along a hot spring stream that fed into a large ice-confined geothermal lake. Samples analysed range from unconsolidated hot spring sediments, well-developed microbial mats, and dissolved sulphate from hot spring fluids. From the anoxic spring source, the stream water increases in dissolved oxygen, decreases in temperature, yet maintains a pH of ~4. The spring environment is dominated by dissolved sulphate (~2.3 mM), with lower levels of nitrate (~50 μM), phosphorous (~5μM), and ammonium (~1.5 μM). Stable S isotope analysis reveals a fractionation of ~3.2 ‰ between sediment sulphide (as pyrite; δ³⁴S ~0‰), and dissolved water sulphate (δ³⁴S ~3.2 ‰) consistently along the hot spring stream, indicating the presence of an active sulphur cycle, although not one dominated by sulphate reduction (e.g. very negative sulphide δ³⁴S). This fractionation trend was absent within lake sediments, possibly due to a number of mixed sources feeding into the lake, in addition to the spring stream. δ¹³C in sediments becomes increasingly more negative going downstream, and is associated with increasing removal of TOC. Microbial mats were similar with very positive C isotope ratios (δ¹³C -9.4 to -12.6 ‰) typical of sulfur oxidizing microbes. Bulk genomic DNA was extracted from sediments and mats in order to identify firstly the community composition via 454-pyrosequencing, and secondly the functional diversity within these physiochemically varied environments. This metagenomic data will be combined with stable isotope patterns to elucidate the metabolic potential of hydrothermal environments at Kverkfjöll, which can be used to infer biogeochemical pathways on Mars in similar, past environments.

For two weeks, Derek Smith and Marilyn Fogel studied the microbial mats in mangroves on cayes along the barrier reef of Belize. The water column of these mangroves is depleted in nitrogen and phosphorus, but the microbial communities are diverse and thriving. The microbial communities are able to metabolize and recycle the small concentrations of available nitrogen and phosphorous which enables dense mat communities. This serves as an analog to early earth conditions when nutrients were in very low concentrations but microbes were able to colonize and diversify. They performed incubation experiments examining the carbon, nitrogen, and sulfur cycling of different microbial populations. Enriched HCO₃- and acetate with ¹³C were used to examine the role of autotrophy and heterotrophy in two photosynthetic mats. The potential importance of nitrogen fixation and anaerobic ammonia oxidation (ANAMOX) was explored by the addition and monitoring of ammonium concentrations. Enrichments for phototrophic sulfur bacteria and sulfate reducing bacteria were performed, and revealed that sulfur oxidation was the predominant process in the sulfur cycle of these communities. Stable isotope measurements for δ¹³C, δ¹⁵N, and δ³⁴S were made on bulk biomass as well as precipitated substrates, i.e. SO₄^-2 and HCO₃^-, to better understand the measurements observed in the field and elucidate key metabolic processes, i.e. sulfur oxidation and autotrophy. They collected three one-half meter long cores through one of the larger photosynthetic mats, analyzed pigments, measured ammonium concentrations, and measured δ¹³C, δ¹⁵N, and δ³⁴S values. They were able to discern changes in microbial populations via pigment variations, and alterations in the nitrogen cycle of the microbial mat. It is precisely these comprehensive studies of diverse microbial systems that enable better understanding of early earth conditions, and can guide astrobiological explorations.

Research Scientist Karyn L. Rogers along with Brian Hynek and Thomas McCollom (Both from Univ. of Colorado) are studying microbes from Cerro Negro Volcano, Nicaragua, to gain insight into the potential for Photosynthesis within Mars’ volcanic craters. Discrete locales of sulfate-rich bedrocks exist on Mars and in many cases represent the products of acid-sulfate alteration of martian basalt. In some places, the products have been attributed to hydrothermal processes from local volcanism. In order to evaluate the habitability of such an environment, we are investigating the geochemical and biological composition of active fumaroles at Cerro Negro Volcano, Nicaragua, where fresh basaltic cinders similar in composition to martian basalts are altered by acidic, sulfur-bearing gases. Temperatures at active fumaroles can reach as high as 400°C and the pH of the steam ranges from <0 to 5. Adjacent to some fumaroles, silica is being precipitated from condensing steam on the crater walls and endolithic photosynthetic mats are found at 1-2 cm depth within these silica deposits. We have analyzed one of these mats, Monkey Cheek (T=65°C, pH ~4.5), for both Archaeal and Bacterial diversity. Cloning of PCR-amplified ¹⁶S rRNA genes reveals a diverse community of Bacteria, with eight phyla represented. The most common bacterial sequences belonged to the Cyanobacteria and Ktedonobacteria, however Actinobacteria, alpha-Proteobacteria and Acidobacteria were also identified. Many of the cyanobacterial sequences were similar to those of the eukaryotic Cyanidiales, red algae that inhabit acidic, geothermal environments. Many of sequences related to Ktedonobacteria and Actinobacteria have also been found in acid mine drainage environments. The Archaeal community was far less diverse, with sequences matching those of unclassified Desulfurococcales and unclassified Thermoprotei. These sequences were more distant from isolated species than the bacterial sequences. Similar bacterial and archaeal communities have been found in hot spring environments in Yellowstone National Park, Greenland, Iceland, New Zealand and Costa Rica. Some of Mars’ volcanoes were active for billions of years and by analogy to Cerro Negro, may have hosted photosynthetic organisms that could have been preserved in alteration mineral assemblages. Even on a generally cold and dry Mars, volcanic craters likely provided long-lived warm and wet conditions and should be a key target for future exploration assessing habitability.

Mihaela Glamoclija, Karyn Rogers, Roxane Bowden, Fogel, and Steele are starting a new exciting project studying carbon cycling related to volcanic processes at the Campi Felgrei Deep Drilling Site, Southern Italy. Glamoclija, Rogers, and Bowden begin this project in late September. Deep drilling at Campi Flegrei area near Naples in Italy provides a rare opportunity to study deep carbon cycling related to volcanic processes at depths spanning from the surface to the magma chamber. The Campi Flegrei Deep Drilling Project (CFDDP) is approved, underway, and supported by the International Continental Drilling Project (ICDP) (see references).

The drilling project will be carried out in two steps. The first step is drilling of a pilot hole, which has started on July 15^th. The aim of this step was to drill to 200 m deep and the next step is to drill to 500m depth in order to test mechanical properties and temperatures of the host rocks, to plan for successful drilling and the deviation detail. The pilot hole was also used for testing of novel borehole sensors for monitoring of volcanic activity and risk mitigation. The later step includes the drilling of a 3.8 km deep borehole that is deviated 25° at about 500m depth (see figure 1). This type of drilling will reach subsurface temperatures around 500°C and will likely encounter magma pockets on its way towards the magma chamber. We have got a permission to participate in the drilling activities and have been invited to acquire our biologically relevant samples in October during the acquisition of the second portion of the pilot hole.

The subterranean geology of the region includes carbonate basement that forms an open-system in which mixing of magma and assimilation of carbonate rock played role in volcanic evolution. The extensional tectonic activity controls rise of magma from deep to shallow reservoirs and eventually to the surface. Magmas are situated partially beneath the sea and possibly equilibrate at depths above the carbonate basement (2 to 3 km beneath the surface). The past interaction of water and magmas caused numerous explosive eruptions of the Campi Flegrei. Today, the volcanic gasses are reaching the surface and allow for the volcanic system to partially degas through the continuous fumarolic activities of Solfatara Crater at Pozzuoli.

Their intention is to piggyback on this drilling/scientific effort and to expand the scientific return by the addition of our investigations of carbon cycling in a volcanic/hydrothermal system. We will characterize different pools of carbon in this system (biologic, organic, magmatic, sedimentary from carbonate basement and sea water) and their interactions. Surface sources of organic carbon have been studied through a number of studies, including extremophilic microbial communities from the Solfatara Crater; however, the insight into subterranean distribution of the ecosystem is completely unknown. The carbon compounds from minerals, fluid and gasses will provide a unique opportunity to characterize behavior, solubility and mixing of carbon at few km depths.

Glamoclija and Steele are leading a new ASTEP effort that will dovetail with the NAI team at Carnegie and elsewhere with a study of terrestrial analogues to define important technical and scientific parameters for space flight missions. Studies of terrestrial analogues offer natural systems that in some way resemble particular planetary or solar system settings. Many of terrestrial analogue field sites have been used to mimic geological or environmental settings of the moon, Mars, or Europa. However, most of the planetary bodies in our solar system (i.e. Venus, Titan, Io, Enceladus) are very different from Earth, and in these cases meteorites, laboratory experiments and simulations provide alternative samples to expand the palette of analogues. At this time however, no central database or standard collection, or protocol for collection storage and curation of analogue samples has been agreed by or made available to the community. This makes standardization of observation across developing flight instruments and science goals difficult to achieve, resulting in often confusing detection limits, sensitivities, sample preparation protocols and science definition for new missions. They will address these issues by using their experience of sample collection and protocol development to engage the community and curators in workshops to define sample collection and curation issues, agree a suitable standard set of analogue settings and samples that are highly characterized and recommended (by the community) for use in instrument and science testing.

They will also begin the activity of preparing and maintaining a database of current analogue sites and curated samples and analysis techniques for use in solar system exploration with the intent of allowing planetary scientists and engineers access to a single resource during science and mission development. They will undertake testing of the developed curation protocols using samples from the evaporitic White Sands deposit and continue to develop instrumentation and testing protocols through existing work on Martian meteorites. Furthermore, allowing universal access to the locations, samples and developed protocols to wide community will streamline the archiving and curatorial issues, which will ultimately, we hope, improve the scientific return of terrestrial research and future missions to other solar system bodies.

Our research on the geochemistry and microbiology of hydrothermally influenced environments, has also extended to shallow-water vents systems, where biological primary production occurs simultaneously from both chemosynthetic and photosynthetic microbial processes. During May of 2012 Dionysis Foustoukos and Ileana Pérez-Rodríguez participated in a multi-institutional / international / interdisciplinary project in collaboration with Stefan Sievert (WHOI), Costantino Vetriani (Rutgers), and Nadine Le Bris (CNRS) to study these systems in Palaeochori Bay off Milos (Greece). This site represents an excellent natural laboratory to perform these types of studies because it is easily accessible and its gradients are well- characterized. Currently, they are working on 1) characterizing the chemical and isotopic signatures associated to the microbial community composition at these sites as well as 2) performing culture-dependent studies to constrain chemolithoautotrophic dissimilatory Fe(III) reduction metabolism from microorganisms belonging to these communities.

5.3 Isotope fractionation with metabolic growth: (Farquhar, Fogel, Cody, Steele)

Research by the group at the University of Maryland focused on a variety of questions related to understanding sulfur isotope fractionations produced by the microbial sulfur metabolisms and developing ways to apply information about these fractionations to natural systems. Research by six members of James Farquhar’s group was supported by NAI funds.

Research by graduate student Brian Harms, undergraduate student Jonathan Banker, and laboratory manager Joost Hoek focused on analysis of experiments with A. fulgidus and D. autotrophicum. Brian Harms presented the results of his research with A. fulgidus at the 2012 V.M. Golsdchmidt conference of the geochemical society in Montréal, Canada (Harms et al, 2012). Brian’s work focused on using the rare sulfur isotopes to understand the way that this organism’s metabolism responds at different temperatures. His experiments used isotopes to examine how differences in the proportions of sulfur transported into and out of the cell as sulfate is related to differences in the proportions of sulfur reduced from intermediate oxidation states to the terminal product sulfide. Work by Jonathan Banker was undertaken as part of a senior thesis project entitled “Sulfur isotope effects associated with microbial metabolism”. Jonathan’s thesis and poster can be accessed at http://www.geol.umd.edu/undergraduates/paper/paper_banker.pdf and is located at http://www.geol.umd.edu/undergraduates/poster/poster_banker.pdf. Joost Hoek’s role in these research projects was is a secondary advisor. Joost also initiated a research study into a third organism, Thermodesulfatator Indicus which was grown under nonlimiting hydrogen concentrations to evaluate its response to changes in temperature. Plans for the coming year include: (1) preparing a publication, most likely for Geochimica et Cosmochimica Acta describing the results of Brian Harms work; (2) conducting additional analyses of the samples had studied by Jonathan Banker with the goal of ultimately preparing a manuscript for peer review; (3) conducting research to analyze the results of experiments with Thermodesulfatator Indicus.

Research by graduate students Daniel Eldridge and Nanping Wu as well as undergraduate Zahra Mansaray focused on developing approaches to study the expression of sulfur metabolism in nature. Daniel Eldridge conducted a series of experiments examining abiotic oxidation of reduced and intermediate sulfur compounds as part of this study that will examine both biological and abiological pathways for sulfur oxidation. Daniel presented the first results of his graduate research at the 2012 V.M. Golsdchmidt conference of the geochemical society in Montréal, Canada (Eldridge et al. 2012). Nanping Wu is presently in the process of completing his PhD. NanPing’s research focuses on developing techniques to understand the factors that control the fractionation between sulfate and pyrite that is buried in oceanic sediments. Nanping also presented an abstract at the V.M. Golsdchmidt conference (Wu et al., 2012). Undergraduate Zahra Mansaray conducted research on the temperature response of natural populations of sulfate reducers in the Severn River, near Annapolis, Maryland. It has only recently been recognized that the Severn River becomes sulfidic during the spring summer and fall. Zarha’s research involved collecting sediment cores and water from the Severn River and using incubations to study the rates of sulfide production by sulfate reducers in Severn River sediments at different temperature conditions. She chose temperature conditions that would be representative of summer bottom water temperatures and winter bottom water temperatures. Her undergraduate research thesis and poster entitled “Sulfide flux as a function of temperature in the Severn River” can be accessed at http://www.geol.umd.edu/undergraduates/paper/paper_mansaray.pdf and http://www.geol.umd.edu/undergraduates/poster/poster_mansaray.pdf.

Student Derek Smith (Dartmouth College and Geophysical Lab) is in the final year of his dissertation studies. He studies the effects of metabolism and physiology on the production of okenone and bacteriochlorophyll a in Purple Sulfur Bacteria. The anaerobic cycling of carbon and sulfur in the photic zone is linked by phototrophic green and purple sulfur bacteria, and molecules that constitute the pigments synthesized by these bacteria, as well as diagenetically reduced fragments of these carotenoids are well-documented biomarkers for past and present euxinic conditions. Okenane is the only recognized molecular fossil unique to purple sulfur bacteria (PSB), in the geologic record. Okenane is widely understood to be of biologic origin, derived from the complete hydrogenation of the carotenoid pigment okenone, which has only been documented in eleven species of Chromatiaceae. The potential functions of carotenoids in PSB are known, however, the specific function of okenone is still not clear. Bacteriochlorophyll a (Bchl a), has also been used as a paleo-proxy as it is a well-studied porphyrin-like pigment that is required for photosynthetic function. We undertook a comprehensive study examining the effects of metabolism and physiology on the production of pigments in PSB. Specific growth rates were determined in PSB grown autotrophically and photoheterotrophically. The four strains studied produce the carotenoid okenone: Marichromatium purpuratum DSMZ 1591, Marichromatium purpuratum DSMZ 1711, Thiocapsa marina DSMZ 5653, and FGL21, a strain isolated from Fayetteville Green Lake, New York. Bchl a and okenone concentrations were quantified using Ultra Performance-Liquid Chromatography-Mass Spectrometry (UP-LC-MS) and spectrophotometry. The ratio of okenone:Bchl a differs among species and strains of PSB. Photoheterotrophically grown PSB have statistically significant lowered okenone:Bchl a ratios, than when under autotrophic metabolism, which is interpreted to indicate a decreased requirement for okenone when PSB are provided with a complex (> C1) carbon source. Stable isotopic analysis, using Isotope Ratio Mass Spectrometry (IR-MS), for d¹³C, d¹⁵N, dD and d³⁴S of bulk cell biomass showed variations in substrate fractionations across different strains and treatments. The d¹³C values reveal the switch from autotrophic to photoheterotrophic metabolism, and disparities in carbon incorporation. The compound specific d¹³C values of okenone and Bchl a were representative of the substrate on which the organism was grown, and may provide a means of ‘fingerprinting’ the compounds. Collectively, these results indicate that pigment production is heterogeneous across species and strains of PSB and that photoheterotrophic growth leads to a depression in the production of okenone, which suggests that okenone is connected with a light-harvesting role in PSB.

Currently, Smith and Fogel are working on experiments to measure complex sulfur isotope fractionation by these microbes which use sulfate, sulfide, and thiosulfate for their metabolism. This work utilizes the DDF instrument that was purchased to experiment with a rapid method for organic and inorganic sulfur isotope measurements of d³⁴S and D³³S.

Ileana Pérez-Rodríguez along with Stefan Sievert (Woods Hole), Marilyn Fogel and Dionysis Foustoukos are studying nitrogen metabolisms of deep-sea vent chemosynthetic microbes in an experimental study at in situ seafloor pressures. The biological cycling of nitrogen in the world’s oceans involves to a great extent ammonia and nitrate. Several of the key reduction-oxidation (redox) reactions involving nitrate and ammonia cycling are carried out in nature almost exclusively by microorganisms; thus, microbial involvement in the ocean’s nitrogen cycle is of great importance. The increasing concentration of nitrate with depth in the open ocean suggests that nitrate plays an important role for deep-sea microorganisms, and that nitrate reduction may be relevant in global transformation of nitrate in the ocean. Moreover, the physico-chemical conditions at near-seafloor hydrothermal sites, reflect their potential importance on sustaining nitrate-based activities in deep-sea microorganisms.

However, the role of deep seawater NO₃- has not been fully explored, despite the highly energetic nature of the microbially-mediated nitrate respiration reactions. Because chemosynthetic Epsilonproteobacteria are increasingly recognized as an ecologically relevant group at deep-sea vents, we are using Caminibacter mediatlanticus, a nitrate-reducing epsilonproteobacterium, as a model system. To this end, we are assessing dissimilatory nitrate reduction to ammonium (DNRA) pathway at pressure conditions similar to those encountered in deep-sea vent environments. Through a constrained investigation of the nitrogen stable isotope exchange between the dissolved nitrogen species (NO₃^-; NH₄⁺) and the nitrogen assimilated into biomass, we are researching the chemical signatures of nitrogen-based metabolic processes during C. mediatlanticus growth.

In detail, measurements of ¹⁵N/¹⁴N fractionation between NO₃^- and NH₄⁺ obtained during C. mediatlanticus growth under different hydrostatic pressure regimes have been obtained together with growth rates and rates of dissimilatory nitrate reduction to ammonium (DNRA). Our data includes the successful establishment and sampling of batch cultures growing under hydrostatic pressures of 50 and 200 bar, utilizing a flexible gold/titanium reaction cell. This batch reactor allows for samples to be retrieved during the course of experiments, and thus, to monitor growth at in-situ seafloor pressures. Results indicate that higher hydrostatic pressure conditions (50 bars and 200 bar) significantly decrease the microorganism’s growth rates, cell biomass and rates of catalyzed nitrate reduction reactions. These data help to improve the understanding of nitrogen metabolism in anaerobic chemosynthetic nitrate reducing microorganisms at deep-sea hydrothermal vents, and provide new proxies for estimating the importance of this process for biomass production in the subseafloor at deep-sea vents.

One of the main challenges to high-pressure, high-temperature microbiology is replicating environmental conditions in the laboratory. Of particular importance are (i) realizing in situ volatile compositions, (ii) emulating dynamic flow environments, and (iii) sub-sampling experiments without disrupting the experimental conditions (e.g. via decompression or cooling). Many of the high-pressure apparatus currently in use are batch-style systems, and sampling during experiments requires decompression (sometimes cooling) and volume loss of the entire experiment1. A few continuous flow designs have been used, but many of these do not have a pressurized media reservoir or adequate pressure ranges20. A pressurized media reservoir is essential for supplying a growth medium that contains representative quantities of volatiles to mimic conditions found in the deep biosphere.

Recently, a system that overcomes many of these obstacles and can reach 60 MPa and 120°C has been developed in the laboratory of Jens Kallmeyer (University of Potsdam). Rogers is currently developing the laboratory capabilities to investigate thermophile growth under high-pressure conditions to explore the following hypotheses: (1) Pressure is a key parameter in evaluating habitability on Earth (and throughout the solar system), where life is likely confined to the subsurface; (2) Physiological parameters (i.e. growth rate, temperature range, metabolic activity and fractionation of light stable isotopes) are functions of growth pressure. Central to this objective is the construction of a high-temperature, high-pressure chemostat at the Carnegie Institution of Washington. A recent proposal submitted to the NASA Exobiology Program would support the construction of a system that would be dedicated to high- temperature, high-pressure microbial growth and that builds on the designs of Dr. Kallmeyer21. Currently, she is working with Dr. Kallmeyer to adapt this system to high-temperature, high- pressure deep-sea microbes, and extend the pressure limit to shallow crustal pressure regimes (~100 MPa). Essential to the success of a continued collaboration is for me to gain experience operating this system and learn how it can be applied to microbial systems. This summer, Rogers visited the laboratory of Dr. Kallmeyer at the University of Potsdam and to work with him and his graduate student, Patrick Sauer.

Post Doc Wang and CoI’s Cody, Fogel and Steele have developed a novel approach towards understanding hydrogen isotope fractionation in bacteria during exponential growth. Experiments were performed where E. coli was grown on 10 % deuterated glucose, in 10 % D₂O, and a control. The distribution of deuterium and hydrogen in the cells was determined using D and H solid state NMR. Assessment of levels of deuteration of specific molecules was determined with structural mass spectrometry. Interesting we find that that fatty acids are enriched in deuterium over protein by 600 permil. This work was submitted and is under review in Organic Geochemistry.

5.4 Carbonaceous Bio- and Abiosignatures: (Steele, Cody, Fogel, and Papineau)
Steele and colleagues published a paper in Science documenting the presence of organic carbon on Mars. The work is the culmination of many years of work and engaged an international team of scientists. The source and nature of carbon on Mars has been a subject of intense speculation. Steele’s team reports the results of confocal Raman imaging spectroscopy on eleven Martian meteorites, spanning ~4.2 Ga of Martian history. Ten of the meteorites contain macromolecular carbon (MMC) phases included within high temperature mineral phases. Along with the MMC phases are polycyclic aromatic hydrocarbons, which were detected in association with small oxide grains. The association of this organic carbon within magmatic minerals indicates Martian magmas favored precipitation of reduced carbon species during their crystallization. The ubiquitous distribution of this abiotic organic carbon in Martian igneous rocks is important for understanding the Martian carbon cycle and has implications for future missions to detect possible past Martian life.

Steele and Fogel also contributed to a study that is in review at Science. Carl Agee and Francis McCubbin (Univ. of New Mexico) and coauthors report the first data on a new type of martian meteorite, Northwest Africa (NWA) 7034, which shares some petrologic and geochemical characteristics with known martian (SNC, i.e., Shergottite, Nakhlite, and Chassignite) meteorites, but also possesses some unique characteristics that would exclude it from the current SNC grouping. The NWA 7034 meteorite is a geochemically enriched crustal rock bearing a striking compositional resemblance to basalts and average martian crust measured by recent NASA rover and orbiter missions. The formation age of NWA 7034 as determined by Rb-Sr is 2.089±0.081 Ga, making it the first sample from the early Amazonian epoch in Mars’ geologic history. NWA 7034 has an order of magnitude more indigenous water than most SNC meteorites, with at least 3330 ppm extraterrestrial H₂O released during stepped heating. It also has bulk oxygen isotope values of Δ¹⁷O=0.56±0.06‰ and a heat-released water oxygen isotope average value of Δ¹⁷O=0.330±0.011‰ suggesting the existence of multiple oxygen reservoirs on Mars. When considered in the context of other martian meteorites, NWA 7034 could indicate multiple oxygen isotopic reservoirs within its lithosphere, which is difficult to envision in the context of a well-mixed global magma ocean. Alternatively, atmospheric components could have interacted with minerals in NWA 7034 and its bulk represents a mixture of distinct lithospheric and atmospheric oxygen isotopic reservoirs on Mars.

Dominic Papineau studied ultramafic rocks from Mars to examine non-biological organic matter associated with apatite in the Chassigny meteorite. The confirmation that organic matter was associated with apatite in this rock was done in his lab at Boston College by Raman micro-spectroscopic imaging, which was combined with adopted correlated micro-analytical approach. Two FIB foils were micro-fabricated and analyzed in the Chassigny meteorite. The first phase of this work is now completed.

Papineau also successfully lead the micro-fabrication of 10 FIB foils subsequently analyzed by Raman spectroscopy. For example, he investigated the microfossils collected from the Doushantuo phosphorite deposits. Micro-analyses were performed on a thin section from the Mesoproterozoic Vindhyan Basin that had more spectacularly preserved and diverse microfossils than the best thin section of the Gunflint Fm. available in PBEL. This thin section belongs to new collaborator Dr. Purnima Srivastava and had been studied by her and reported to preserve 28 species and 18 genera (Kumar and Srivastava, 1995). Systematic petrographic mapping of the different microfossil colonies has begun and a first double- extracted FIB foil were successfully prepared on a target of tetraphycus mixed with myxococcoides. Results of the correlated micro-analyses are shown in Figure 1, described briefly in the caption, and given preliminary interpretations below. Some striking new observations of morphological and geochemical biosignature were produced by these correlated micro-analyses. In the first FIB foil extracted from this specimen, the two phases detected by Raman (Fig. 1b-c) represent areas of two different electronic densities in TEM bright field images, with chert being bright electron-lucentand organic matter being electron dense and black (Fig. 1g). This is another example of how “finely disseminated organic matter” appears in a crypto- or micro-crystalline chert matrix. High resolution TEM images of a portion of the foil revealed previously unidentified nanoporous honey- combed organic-chert structure that is clearly biological in origin. These never-before seen structures represent Mesoproterozoic small shelly fossils or the internal structure of the cyanobacteria tetraphycus or myxococcoides. The significance of this discovery is still not fully appreciated and more FIB foils from this thin section are now required to pursue the meaning of this remarkable morphological biosignature. In the second foil (~1 μm thick) of this doubly-extracted micro-fabricated target, NanoSIMS C isotope secondary ion maps revealed the presence of organic-walled myxococcoides (Fig. 1m). This confirms the site-specificity of our targeting approach (estimated to be +/- 1.5 μm in this case) and the double extraction of these foils allowed for the unexpected discovery of the nanoporous honey-comb pattern. More work will be need to either reproduce these observations or find complementary observations that could help interpreting this morphological biosignature.

Papineau continued his work on finely disseminated organic matter in the Jhamarkotra stromatolitic phosphorite. Fine apatite layers are composed of microscopic elongated apatite granules whereas interlayers of carbonate are dominated by calcite and cemented by late diagenetic dolomite. Nanoscopic globular particles of organic matter occur as fine disseminations dominantly in the apatite that precipitated during early diagenesis, along with microcrystalline calcite, which contains only rare organic nano- particles. Nanoscopic dendritic to chaotic filaments of organic matter occur in occasional calcite crystals. These occurrences are distinct from what was observed and may be a metamorphic feature. Some fringes could be seen in an organic nano-particle from an apatite grain, consistent with Raman spectra with a D-band/G-band intensity ratio of about 0.5 (Papineau et al., 2009) and greenschist facies metamorphism.

Numerous collaborators and students worked with Papineau on these and other projects: Paul Strother (Weston Observatory, Boston College); Christian Hallmann (U. of Bremen), Jianhua Wang (CIW), Larry Nittler; Timothy Rose (Smithsonian Institution); Purnima Srivastava (Lucknow University, India); Shuhai Xiao (Virginia Tech); Wouter Bleeker (Geological Survey of Canada); Leigh Maniscalco (B.A. 2012); Katie Interlichia (M.Sc. 2013); Thomas Kelly (B.A. 2013); Rhianna James (B.A. 2014)

Robert Hazen, Cody, Fogel and Steele worked with Univ. of Maryland undergraduate student, John Nance, studying 15-20 Million year old organic matter with exceptional preservation. The genus Ecphora of Muricid gastropods from the mid-Miocene Calvert Cliffs, Maryland is characterized by distinct coloration that ranges from tan to dark brown to reddish-brown hues. This unusual, and as yet uncharacterized, pigmentation presents an opportunity to study biomineralization and the possible preservation of protein within 8-18 Ma shells. Ecphora in its various species and subspecies spans nearly 10 million years of time along Calvert Cliffs, MD within the Calvert, Choptank, and St. Marys Formations. Mollusc shells predominantly form by a process of biomineralization wherein calcium carbonate precipitates in association with an organic matrix made of proteins and polysaccharides. The coloring of these molluscs is the result of shell-binding proteins associated with pigments within the outer calcite portion of the shell. Micro-Raman spectroscopy indicates the presence of a carotenoid like pigment. To extract the organic material, we dissolved shells in dilute HCL. A sheet-like residue of the same color as the initial shell is concentrated in the solution (Hazen-Area5-Fig.5). C:N elemental and isotopic analysis confirmed that the sheet-like material released from shells is organic. Total organic carbon in the residue from acid treatment ranges from 4 to 40%, with 11 < C/N < 14. Isotope values for carbon (-17 < δC¹³ < -15‰) indicate a marine environment, while values for nitrogen (3 < δN¹⁵ < 5‰) confirm Ecphora’s role as a predator. The remarkable preservation of this pigmentation and shell-binding organic material presents a unique opportunity to study the ecology of the Chesapeake Bay region 8-18 million years ago.

5.5 Raman studies of microfossils: (Steele)
The unambiguous identification of biosignatures and detection of extra planetary life is one of the primary goals in astrobiology. Steele, Dina Bower and Marc Fries have been using Raman spectroscopy to study organic carbon as a biosignature. Carbon, however, is also a ubiquitous element in many abiogenic compounds. Because of the complex geologic histories and thermal processing of older rocks, the distinction between biogenic or abiogenic carbon sources can be difficult, making the establishment of an “unambiguous” biosignature nearly impossible. Micro Raman spectroscopy is a non-destructive method that allows for in situ analysis of samples and unambiguous identification of a wide range of minerals and compounds. In addition, the unique mapping capabilities of micro Raman spectroscopy provide a greater view of the spatial relationships between carbonaceous materials and other features in ancient rocks. To tease out a biogenic signal, we analyzed and compared the Raman spectral signatures of carbonaceous material in a suite of microfossils and meteorites. Our approach differs from previous studies, in that we avoid the bias of pre-assigning biogenicity to proposed microfossil samples before analysis. Here, the data from all samples are compared first, and then interpretations are made based on known geologic context and history. The preliminary results of this study show a correlation between trends of the G-and D-band parameters and the complexity and thermal history of precursor carbonaceous material. To achieve our goal to unambiguously identify biosignatures using micro Raman spectroscopy, we are continuing to collect and catalogue Raman spectra for a wide range of carbonaceous materials in natural geologic samples. Only by fully exploring the spatial relationships and spectral characteristics of carbonaceous material and associated features, will the establishment of definite biosignatures for life detection in ancient rocks on Earth and those on other planets be made.

5.6 Mineralogical Co-Evolution of the Geosphere and Biosphere: (Hazen)
Mineral evolution, the study of Earth’s changing near-surface mineralogy through 4.567 billion years of Earth history, is based on the premise that the geosphere and biosphere have co-evolved through a sequence of deterministic and stochastic events. Temporal trends in Earth’s near-surface mineralogy correlate with major events in Earth’s geochemical and tectonic history. Consequently, the near-surface mineralogy of a planet or moon reflects a range of geochemical, tectonic and biological processes. In the past year our research has focused on the minerals of the redox sensitive Hg and Mo.

Analyses of the temporal and geographic distribution of earliest recorded appearances of the 88 IMA approved mercury minerals plus 2 potentially valid species exemplify principals of mineral evolution. Metacinnabar (HgS) and native Hg are the only two species reported from meteorites, specifically, the primitive H₃ Tieschitz chondrite with an age of 4550 Ma. Since the first terrestrial appearance of cinnabar more than 3 billion years ago, mercury minerals have been present continuously at or near Earth’s surface.

Mercury mineral evolution is characterized by episodic deposition and diversification, perhaps associated with the supercontinent cycle. We observe statistically significant increases in the number of reported Hg mineral localities and new Hg species at ~2.8-2.6, ~1.9-1.8, and ~0.43-0.25 Ga—intervals that correlate with episodes of presumed supercontinent assembly and associated orogenies of Kenorland (Superia), Columbia (Nuna), and Pangea, respectively. In constrast, few Hg deposits or new species of mercury minerals are reported from the intervals of supercontinent stability and breakup at ~2.5-1.9, ~1.8-1.2, and 1.1-0.8 Ga. The interval of Pangean supercontinent stability and breakup (~250-65 Ma) is also marked by a significant decline in reported mercury mineralization; however, rocks of the last 65 million years, during which Pangea has continued to diverge, is characterized by numerous ephemeral near-surface Hg deposits.

The period ~1.2-1.0 Ga, during the assembly of the Rodinian supercontinent, is an exception because of the absence of new Hg minerals or deposits from this period. Episodes of Hg mineralization reflect metamorphism of Hg-enriched marine black shales at zones of continental convergence. We suggest that Hg was effectively sequestered as insoluble nanoparticles of cinnabar (HgS) or tiemannite (HgSe) during the period of the sulfidic “intermediate ocean” (~1.85-0.85 Ga); consequently, few Hg deposits formed during the aggregation of Rodinia, whereas several deposits date from 800-600 Ma, a period that overlaps with the rifting and breakup of Rodinia.

Nearly all Hg mineral species (87 of 90 known), as well as all major economic Hg deposits, are known to occur in formations ≤400 million years old. This relatively recent diversification arises, in part, from the ephemeral nature of many Hg minerals. In addition, mercury mineralization is strongly enhanced by interactions with organic matter, so the relatively recent pulse of new Hg minerals may reflect the rise of a terrestrial biosphere at ~400 Ma (Hazen-Area5-Fig.2).

New and published analyses of 422 molybdenite (MoS2) specimens from 135 localities with known ages from 2.91 billion years (Ga) to 6.3 million years (Ma) reveal two statistically significant trends. First, systematic increases in average and maximum trace concentrations of Re in molybdenite since 3.0 Ga point to enhanced oxidative weathering by subsurface fluids (Hazen-Area5-Fig.3). These trace element results, coupled with the delayed appearance of minerals of other redox sensitive elements, suggest that significant terrestrial subsurface oxidation may have postdated the Great Oxidation Event (~2.4 to 2.2 Ga) by hundreds of millions of years. In addition, episodic molybdenum mineralization, as with Hg minerals, correlates with five intervals of supercontinent assembly from ~2.7 Ga (Kenorland) to 300 Ma (Pangaea).

One of the biggest issues faced by geoscientists studying early life is absolutely proving the biogenicity of microstructures in early Archean sedimentary rocks. Bower and colleagues studied the “fossil” microstructures in these rocks and found that they often resembled modern microbial structures, but rarely contained original cellular material. Instead, many fossils are composed of interesting mineral assemblages such as clays and Fe-,Ti- oxides. Unfortunately, the mineral composition of such features we see today in these ancient rocks differs from what was originally formed in the sediments billions of years ago as a result of long, complicated histories and atmospheric influences. The contribution of microbes is not typically considered, and the complex microbe-mineral relationships under different chemical and geologic conditions are still not well constrained. Here the goal is to understand the co-evolutionary path of microbes and minerals in sandy non-hydrothermal environments with laboratory experiments that simulate diagenesis. The precipitation of minerals on microbial cells and extra polymeric substances and the phase changes in natural ilmenites (FeTiO3) were documented to determine if microbes passively influence mineral phase pathways. The precipitates, ilmenite grains, and fossilized cells were analyzed using high-resolution imaging and geochemical techniques: scanning electron microscopy (SEM), X-ray diffraction (XRD), and micro Raman spectroscopy. Preliminary results of this study indicate that microbial fossilization and mineral phase changes occur under early diagenetic conditions in non-hydrothermal sandy environments. The results also show that minerals precipitated in the presence of microbes differ from those without microbes. This provides the geobiological community with much needed information to aid in the understanding of the intricate relationship between microbes and minerals and helps establish geochemical biosignatures in ancient sedimentary rocks both on Earth and on other planets.