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Project Progress

Our team is interested in examining (1) the diverse mechanisms utilized by life to survive extremes of temperature, pressure, salinity and nutrient limitation, (2) the response of life to fundamental changes in the properties water, and (3) how the unique biochemistry associated with extremes can be used for life detection.

1. Life in Deep-Sea Hydrothermal Vents

The astrobiology research objectives of Co-Investigator John Baross and his group are focused on understanding the microbial ecology of the subsurface at hydrothermal vent environments. His research group is also interested in examining the microbial communities that colonize in crust that is millions of years old, and the role that microbial/mineral associations in these environments have on the growth and survival of microorganisms under the most extreme temperature and pH conditions found in vent environments. During the past year a study in subsurface environments at volcanically active sites on the Juan de Fuca Ridge has been launched that has utilized a range of molecular techniques. These approaches allowed Baross’s group to isolate and characterize bacteria unique to these hot environments. Among the novel groups of microorganisms isolated were organisms that can fix carbon dioxide anaerobically utilizing hydrogen and sulfur as reductants. These organisms also form biofilms on mineral surfaces and produce copious amounts of exo-polysaccharides as part of the biofilm process. These organisms, which use a novel, and yet characterized, metabolic pathway for carbon dioxide reduction, are believed to be key components of the primary producers in these environments. Other organisms isolated from these environments include a new genus of thermophilic, anaerobic iron reducers that couple iron reduction with the oxidation of simple organic acids and four new species of halophilic bacteria that are uniquely adapted to grow at high salinities and high pressures. It was also found that a very high diversity of the subsurface microbial community has the capacity to fix gaseous nitrogen using genes referred to as nifH. Some of the nifH gene sequences from these environments are unique and point to a much higher diversity of nitrogen-fixing archaea than previously reported. These results are important because other forms of nitrogen such as ammonia and nitrate are present in low concentrations or at non-detectable levels in these environments. The subsurface associated with hydrothermal systems harbors a diverse microbial community that includes organisms that have novel metabolic and physiological traits that allow for growth in the absence of nutrients derived from photosynthesis.

Baross’s research group recently 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. Using a new sampling technique that involved piercing stainless steel darts into the crust, they were able to collect crustal fluid that was not contaminated with seawater. Preliminary culture and molecular phylogenetic data show the presence of a small but active microbial population dominated by bacteria including thermophiles. This microbial community is different from subseafloor communities near hydrothermal vents and includes unique groups not found in seawater or sediments.

The Lost City hydrothermal field represents a new type of submarine hydrothermal system driven by exothermic chemical reactions in ultramafic oceanic crust and not as the result of the cooling of magma. Lost City hydrothermal field is hosted on 1.5-Ma ultramafic oceanic crust approximately 15 km from the axis on the Mid Atlantic Ridge. The analytical results from samples gathered during cruises in December of 2000 and May of 2003 show that the physical conditions come together to form a hot, highly reducing, alkaline environment. Furthermore, this environment produces high concentrations of hydrogen, methane, and other low-molecular-weight hydrocarbons. The highly alkaline fluids cause the precipitation of seawater carbonate that results in the formation of massive carbonate chimneys on the seafloor. The microbial communities associated with these carbonate structures were dominated by a single group of methane-metabolizing archaea. The predominant phylotype, related to the Methanosarcinales, formed tens of micrometer-thick biofilms in regions adjacent to hydrothermal flow. These results are in contrast to the extremely high diversity of bacteria and archaea found in active, hot sulfide structures. These findings expand the range of known geological settings that support biological activity to include submarine hydrothermal systems that are not dependent upon magmatic heat sources. The early history of Mars may have included liquid water and exposures to ultramafic rocks that would have favored geochemical processes similar to those at Lost City. It should be noted that the recent discovery of atmospheric methane and the confirmation of hematite and other iron-bearing minerals on Mars indicates that the conditions for such processes to occur on Mars are present.

2. Stress Adaptation on Microorganisms and Expansion of Habitability

The research of Co-I Scott and colleagues is focused on understanding the response of life to extreme-pressures (> 0.3 Gpa) and a variety of temperatures. Their initial work focused on the ability of two piezo-sensitive bacteria, Shewanella oneidensis MR1 and Escherichia coli, to survive at pressures at which water undergoes fundamental changes in its ability interact with organic material, and to stabilize non-covalent interactions such as hydrogen bonding and van der Waals interactions. Van der Waals interactions or forces are temporary fluctuating dipole and dipole-dipole interactions that cause attractions between individual molecules. Because of water’s polar nature, it plays a key role of mediating van der Waals interactions and hydrogen bonding between biological materials. These interactions are profoundly affected by changes in the ordering properties of water due to phase transitions.

Both Shewanella MR1 and E. coli were able to survive in a phase stability field imposed using diamond anvil cells (DAC) designed and manufactured by the high-pressure group at the Carnegie Institution of Washington (CIW). DACs have become a ubiquitous method for imposing pressures on minerals approaching those observed at the center of the planet. Unlike traditional cold-seal pressure systems and pressurized growth vessels, DACs allow real-time observation and interrogation by a range of microscopic and spectrometric techniques. The group demonstrated the utility of using the DAC as a way of examining the effects of GPa-pressures on Shewanella MR1 and E. coli. For example, they showed through Raman spectroscopy that formate oxidation by Shewanella MR1 continued after cells were exposed to pressures above 1 GPa. Furthermore, because of their transparency, DACs provide an ideal optical platform for microscopic examination. The group is developing a range of techniques utilizing epi-fluorescent dyes (Figure 1) to examine the viability and metabolic activity of cells at pressures where water can form dense ices (e.g., ice-VI and ice-VII) at temperature well above 4°C.

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Co-I Hemley and colleagues are developing DACs that can hold larger volumes to allow the recovery of cells and other biological materials after exposure to GPa pressures. Scott is presently developing techniques for measuring the metabolic activity of cells while in the DAC and for the recovery and culturing of cells when grown at pressures where ice-VI and ice-VII are formed. He has been able to recover and culture E. coli cells exposed to pressures between 0.3 and 0.4 GPa in one of several cold-seal pressure systems at CIW (Figure 2). He and his collaborators are at the initial stages of utilizing genomic arrays to examine gene expression in E. coli and Shewanella in response to the extreme conditions in an environment where ice-VI and ice-VII are stable. As part of this goal Scott was a collaborator and co-author on the recently completed sequencing of Shewanella MR1’s genome. During the past year there has been an effort to develop the molecular and other tools to determine if there are genes that are preferentially expressed in response to changes in pressure.

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Despite speculation that ice-VI and -VII may be stable in deep subduction zones it is very likely that there are no ecosystems on the modern Earth where an ice-VI and -VII zone can be found. However, the subsurface of the Jovian satellites, in particular Europa, likely contain zones where ice-VI and -VII are plentiful. This work, therefore, may provide insight into the nature of habitability deep within such a body. It has been suggested that organic veins in ice-VI on a young Solar System body might have facilitated life arising in a cold icy organic soup by concentrating prebiotic solutes and decreasing the kinetic barriers to advantageous reaction pathways. However, most laboratory work in support of this idea was done at the eutectic where ice-Ih is formed at ambient pressures. Under such conditions there is a significant kinetic barrier due to the low temperatures. By doing the experiments at the eutectic where high densities such as ice-VI and -VII are formed, the activation energy for the formation of the biologically useful macromolecules such as amino acids is significantly reduced. Scott is presently designing experiments for examining the kinetics of reaction mechanisms for forming amino acids and organic molecules at such conditions.

3. Iron-based Metabolic Strategies for Microbial Life

Iron isotopes in natural samples exhibit a range of compositions, plausibly reflecting abiotic equilibrium exchange reactions and kinetic effects, as well as mediated chemistry by microbial activity. Having developed a technique to measure precisely the stable isotope variations of Fe using cold plasma ICP-MS, Co-Is Hauri and Emerson have continued to examine samples from specific settings in which Fe isotopic fractionation may occur. Their work presently focuses on Fe precipitated in bacterial mats near hydrothermal vents on the Loihi seamount near Hawaii collected by Emerson during several field expeditions.

Emerson and colleagues previously identified lithotrophic Fe-oxidizing bacteria in Fe-oxide-rich colonies around hydrothermal vent sites on the Loihi summit. Although some of the Fe oxides present may result from abiotic oxidation of ferrous Fe in the vent water, the morphologies of the precipitated Fe oxides strongly suggest at least partial microbe-induced Fe oxide deposition. Ongoing Fe isotope measurements of bulk precipitates show —0.2 ‰ < δ⁵⁶Fe < +0.5 ‰ (Figure 3). These values are well within the range of fractionations measured in both natural abiotic precipitation of ferrihydrite from aqueous ferrous iron and equilibrium fractionation between ferrous-iron and ferric-iron-bearing compounds in the laboratory. The four marginally isotopically light samples (which are essentially indistinguishable in composition from the terrestrial basalt standard, within uncertainties) possess the highest fraction of amorphous Fe oxide precipitate compared with non-amorphous morphotypes. In contrast, the isotopically heavy mats tend to possess larger fractions of sheath and filament morphologies, whose structures are presumed to result from precipitation reactions mediated by microbial activity. The meaning of this tentative association is unclear, but it could represent a connection between isotopic composition and the degree of microbial Fe processing in the mats. In addition to further characterizing the range of Fe isotope compositions in the Loihi samples, Co-I Scott and colleagues continue to explore connections between isotopic composition and markers of biological activity. Since any extant or past life would have interacted with the iron cycle of the planet, iron isotope fractionation is of interest as a potential biomarker in the search for evidence of past and present life on Mars.

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A parallel project by Co-I Emerson involves the source of carbon for neutrophilic Fe-oxidizing bacteria that grow under microaerobic conditions. While this should be a relatively trivial exercise, for these Fe-oxidizers it has not been so. Circumstantial evidence, growth in very oligotrophic environments, has always suggested that they are autotrophs; furthermore, acidophilic Fe-oxidizers are known autotrophs. Standard ¹⁴C-uptake assays suggest that Emerson’s strains are autotrophs; however, cell yields are also low, and signals are weak. To confirm autotrophy, he has carried out stable isotope ¹³C-uptake studies with Co-I Fogel. Results for the Fe-oxidizers using this assay have consistently been negative for autotrophy. A number of different controls have worked appropriately. One finding from this work is the Fe-oxidizers appear to require certain amino acids for growth, suggesting that these could be their source of carbon. However, feeding them with labeled amino acids yielded negative results. Further investigation suggests that amino acids may have been exerting a small buffering effect, which may have enhanced growth. The question remains unresolved, but another round of experiments is underway to solve this riddle.

In collaboration with George Luther’s group at the University of Delaware, Emerson used nonselective microelectrodes that employ scanning voltammetry to measure iron species. This work has shown that ferrous iron is required for growth and has allowed the team to measure Fe oxidation rates more precisely than has been done before. The group has also made field measurements on in situ ferrous iron concentrations and iron ligands. An interesting finding from this work was a novel form of FeS that appears to exist in the natural habitat. Greg Druschel, formerly a postdoctoral researcher in Luther’s lab, was able to synthesize and further characterize this compound.

An ancillary project in Emerson’s laboratory, not supported by NAI but of general interest, is the development of two rapid, universal methods for aiding in the identification of archaea. One of these is a phenotypic method that uses whole cell maxtrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to compare mass spectral patterns of cell wall components. This method requires a minimum of cell preparation beyond growing the cells, is rapid and accurate, and yields strain-level differentiation. The other method is the use of repetitive primer PCR (repPCR) to genotype archaea. This procedure, available as a kit from Bacterial Barcodes (which Emerson helped to optimize), works well for strain-level differentiation.