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Project Progress

1. Water at Gigapascal Pressures

The behavior of H₂O under pressure underlies the work of Co-Investigator Hemley and his colleagues in prebiotic chemistry and high-pressure microbiology. Collaborator Yukihiro Yoshimura documented new transformations in ice by X-ray diffraction and Raman spectroscopy. Studies of metastable transformations in H₂O continued, as they could be important for characterizing cold, water-rich environments. In addition, Collaborator Ho-kwang Mao and colleagues have found evidence for the facile dissociation of H₂O to form H₂ and O₂ under pressure. These observations may limit the stability range of H₂O in planetary interiors.

Related experiments involved investigations of the stability and properties of various clathrate hydrates in a broad range of planetary environments. Hemley, Mao, and colleages report that novel compounds that may be stable in the satellites of the outer planets such as H₂O-H₂ and CH₄(H₂)₄ were found to be stable over wide ranges of pressure and temperature. Neutron scattering measurements on clathrate hydrates under pressure were begun in order to better understand the structure, dynamics, and reactivity of these systems. The stability and formation of reduced species such as methane and higher hydrocarbons are of particular interest to astrobiology. Studies in this area have continued, including new experiments that address the question of high-pressure catalysis and multiple reaction mechanisms.

2. Microbial Adaptation at Gigapascal Pressures

Co-Investigator Scott and Collaborator Anurag Sharma examined the ability of microorganisms to adapt and survive at higher pressures and temperatures than in experiments reported previously. For the past year Scott and colleagues have been developing techniques for measuring the ability of Shewanella oneidensis MR-1 to reduce ferric oxides over the pressure range 2 to 3 GPa.

The alterations that occur to water at gigapascal pressures lead to a novel environment for microorganisms. Scott and Collaborator Grigoriy Pinchuck are in the initial stages of identification of putative proteins involved in the adaptation of Shewanella oneidensis MR-1 and Escherichia coli K-12. The goal is to develop a set of expression arrays that will allow them to identify novel proteins involved in the adaptation of microorganisms to novel environments.

Studies of the viability of sustained biological activity at very high pressures also continued during this past year. One of the key components of this study is to understand the role of temperature in concert with pressure. Scott and Sharma performed new experiments to determine whether pressure can raise the temperature limits for Gram-positive bacteria and Shewanella oneidensis MR-1 and Escherichia coli K-12. A student project, supervised by Co-I Scott, was initiated to explore several effects of temperature on the viability of Shewanella oneidensis MR-1 at 2 GPa pressure. In addition, a series of new, specially adapted high-pressure microbiology diamond cells were designed and are being built for these experiments.

3. Iron-based Metabolic Strategies for Microbial Life

Co-Investigator Emerson and colleagues continued to make good progress on bacteria that oxidize Fe at neutral pH. On the basis of results achieved from a field expedition to the Loihi seamount in 2004, and coupled with the results of collaborators outside the NAI, his group has evidence that one of their isolates of a marine Fe-oxidizing bacterium, PV-1, is a dominant organism at a variety of hydrothermal vents sites in the Pacific Ocean. Furthermore, this organism is a very deep-branching member of the most common bacterial phylum, the Proteobacteria. Emerson and his team are proposing that it composes a new class, the zeta-proteobacteria. This past spring a draft genome sequence was completed by the Venter Institute, and Emerson and colleagues are in the process of annotating and closing the genome. The genome confirms that this organism is adapted for a lithoautotrophic lifestyle of living on Fe(II) and has a very limited metabolic reportoire aside from growing in Fe(II). Furthermore, this organism forms a unique filamentous Fe-oxide. Figure 1 shows high-resolution transmission electron microscope images of this oxide structure, which is composed of fibrils of Fe-oxides. In the left-hand photomicrograph there are 8 fibrils with individual diameters of approximately 40 nm. The number of fibrils varies, and in some cases they are twisted. In the right-hand image a secondary mineral, akaganeite, has been deposited on the fibrils. Emerson and his team are not aware of any abiological processes that can produce oxides with these characteristics and are continuing to study their properties as possible biomarkers

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Emerson’s lab also focused on freshwater Fe-oxidizing bacteria. Emerson and colleagues named two new genera and three new species of lithotrophic Fe-oxidizing bacteria from circumneutral freshwater habitats. They developed real-time PCR assays%, FISH probes, and molecular fingerprinting techniques to determine if these organisms are present in a given environment. In addition, Postdoctoral Fellow Matthew Schrenk, working with Co-I Fogel, has helped carry out elemental analysis of Fe-mat samples. In work unrelated to iron oxidation, Emerson and colleagues have provided advice and cultures to Co-I Steele and Doctoral Student Verena Starke for their development of a bacterial identification chip with Affymetrix.

4. Life in Deep-Sea Hydrothermal Vents

The research objectives of Co-Investigator Baross and his colleagues continue to focus on understanding the microbial ecology and physiology of Earth environments that share geophysical and geochemical characteristics with other planets and moons. Emphasis is on magma-hosted and peridotite-hosted hydrothermal systems. Both systems produce biologically important carbon and energy sources that Baross and his team propose can support microbial ecosystems in the absence of photosynthesis. These environments are also primordial and may be the sites for key chemical reactions that led to the origin of life on Earth and for the earliest microbial ecosystems. In the past year the research of Baross and his colleagues focused on microorganisms and microbial communities that are involved in hydrogen and methane metabolism, carbon dioxide reduction, and the reduction of iron coupled with the oxidation of simple organic compounds such as formate and acetate. These metabolic pathways are believed to be ancient and may have developed before the evolution of catalytic proteins using minerals as the source of catalysis.

Work continued at the Lost City Hydrothermal Field (LCHF) located on the Mid-Atlantic Ridge. The LCHF is the first deep-sea environment discovered where hydrothermal fluid flow is driven by exothermic water-rock reactions in the subseafloor and not by magma sources of heat. These reactions result in a combination of extreme conditions never before seen in the marine environment: the venting of high pH (from 9 to 11), warm (40-91°C) hydrothermal fluids with high concentrations of hydrogen, methane, and other low-molecular-weight hydrocarbons. LCHF may thus represent a new type of life-supporting system in the deep sea and has profound implications for life elsewhere since olivine, the key mineral in these “serpentinization” reactions, has been detected on Mars and is assumed to be present on Europa. Mixing of the warm, high-pH fluids with seawater results in carbonate precipitation and growth of chimneys, which tower up to 60 m above the seafloor. According to work by Doctoral Students Matthew Shrenk, Billy Brazelton, and others, these carbonate towers house extensive microbial biofilms that are dominated by a single group of Archaea, the Methanosarcinales, which include separate methanogenic and anaerobic methane-oxidizing species. One of the extraordinary results from preliminary work by Brazelton and others at LCHF is that one species of Methanosarcinales appears both to produce and to oxidize methane as part of a single biofilm. If correct, this hypothesis would profoundly affect paradigms in microbial ecology and microbial evolution: metabolically diverse cells existing within a community of a single species are novel. Moreover, the association between cells that make methane and oxidize it appears to be obligatory, with neither process occurring without the other. Such a symbiotic association would be the first between two metabolically distinct strains of the same species. Ongoing research is focused on testing this hypotheses using molecular and culture approaches and activity measurements based on stable isotope incorporation into lipids and DNA.

Studies by Baross and his team continue on nitrogen fixation in vent environments. During this past year Doctoral Student Mausmi Mehta isolated a deep subseafloor methanogen that is capable of fixing nitrogen at 92°C (the high temperature for growth of the isolate). This is almost 30°C higher than has ever been observed for nitrogen fixation. Baross, Mehta, and others have been able to express the nitrogen fixation gene (nifH) at 92°C, genetically map most of the nitrogen fixation operon, and demonstrate nitrogen assimilation into proteins using ¹⁵N₂ over the temperature growth range of the isolate. This work is important because other forms of nitrogen such as ammonia and nitrate are present in low concentrations or at non-detectable levels in most subseafloor environments, and the work expands the temperature of nitrogen fixation into to hyperthermophilic range. Given that nitrogen fixation is believed to be ancient and to have evolved before the separation of the three domains of life, these results build on the evidence that hyperthermophily was a characteristic of ancient Earth microbes.

Baross, former Doctoral Student Julie Huber, and others also completed a study of the microbial community in a subseafloor environment at Baby Bare Seamount, a well-described basaltic outcrop located on 3.5-Ma oceanic crust in the northeast Pacific. Results point to a subsurface hydrothermal system in old crust that harbors a high diversity of archaea and bacteria, including hyperthermophiles and organisms that use carbon dioxide as their carbon source and hydrogen as their energy source. It is apparent from these results that there can be active microbial ecosystems that can exist without input of essential electron acceptors from photosynthesis.