nai

Project Progress

Task 1. Field Studies and Laboratory Characterization of Hydrothermal Vent Microbes (Baross)

The molecular-phylogenetic analyses of the subsurface bacterial and archaeal communities from the 1998 deep-sea eruption at Axial Volcano, Juan de Fuca Ridge, has been completed for samples collected in 1998,1999, 2000 and 2001. Another cruise to Axial Volcano is planned for August 2002. The results from this study show that the subseafloor archaeal community at diffuse-flow vents is a complex mixture of seawater-entrained and indigenous species. Hyperthermophilic and mesophilic methanogens and Thermococcales are clearly indicator-organisms for an anaerobic subseafloor biosphere. The Thermococcus species, while phylogenetically closely related, have markedly different phenotypic characteristics, including different protein patterns and enzyme activities. There are also a large number of archaeal sequences that are not found in seawater that appear to be unique to the subseafloor. The dominant subseafloor bacteria belong to the ɥ-proteobacteria group, and species diversity increases significantly with time and correlates with increasing levels of seawater electron acceptors in subseafloor fluids. Our results so far have established that there is high diversity of both bacteria and archaea in the subseafloor that is unique to this environment. Included in this group are anaerobic hyperthermophilic methanogens, heterotrophs and new genera of CO₂-fixing mixotrophs, moderately thermophilic and mesophilic, anaerobic methanogens and sulfate-reducing bacteria, and a high diversity of organisms capable of oxidizing sulfur, Fe (II), methane, and hydrogen. We also have evidence that normal life-style for many subseafloor organisms includes the ability to attach to mineral surfaces and form biofilms. We plan to use bacterial and archaeal isolates from the subseafloor as models for understanding how microbes exploit nutrients from minerals and the significance of biofilms in this activity.

A degenerate primer has been designed to detect the nifH gene (one of the nitrogen fixation genes) in archaea and bacteria. Data from the 2000 and 2001 cruises to Axial volcano indicate an extremely high diversity of both bacteria and archaea that have the nifH gene. Most of the archaeal nifH gene sequences appear to be closely related to methanogens and uncultured marine Crenarchaeota. During the 2002 cruises we plan to extract ribonucleic acid (RNA) from subseafloor communities in an effort to determine the diversity of microbes that are expressing the nifH gene in situ. We also plan to isolate nitrogen-fixing microbes so as to better understand the environmental factors controlling gene expression.

A preliminary description of the microbial ecology of active sulfide chimneys has been completed using a combination of molecular and microscopic analyses. Intact microbes have been observed throughout these sulfide structures, including in mineral zones thought to be at temperatures greater than 150°C. This work has also been expanded to include the newly discovered (December 2000) "Lost City" vent field on the Mid-Atlantic Ridge. This is a unique vent system, since the fluids are devoid of sulfide and enriched in carbonate, hydrogen, and methane. The "smoker" structures consist of carbonates and related minerals. Molecular analyses and culture studies show a high abundance of methanogens in these environments along with novel groups of bacteria. A cruise to “Lost City” is scheduled for 2003; we plan to do more extensive microbial analyses, including in situ colonization experiments.

Task 2. Microbial Activity at Gigapascal Pressures (Sharma, Scott, Cody, Fogel, Hazen, Hemley, Huntress)

A group led by Sharma and Scott observed microbial activity at pressures of 68 to 1680 megapascals (MPa). Experiments with two microbial species, Shewenella oneidensis and E. coli, were carried out in diamond anvil cells in situ. The group observed bacteria living inside of ice-VI crystals. Viability of the microbes was determined by measuring rates of formate oxidation using Raman spectrometry. Cells were also stained with dyes that proved that cellular membranes were intact and that cellular machinery was functioning. These cells continued to be viable upon subsequent release of the high pressure.

Sharma and Scott are continuing this work to determine whether cells undergo division and can grow at extreme pressures. Some cells undergoing division have been photographed. The group is also perfecting methods for counting cells, and thereby estimating growth, in situ in the diamond cell. Plans are under development for a new microscope facility for better imaging, and a variety of biochemical dyes and tracers are being tested for their utility in this work.

One of the key directions for this work will be to determine the effect of pressure on the temperature limits for life.

Task 3. Possible Origin of Chirality (Hazen, Filley)

Hazen and collaborators continued studies of the selective adsorption of L- and D-amino acids on calcite – work that has implications for the origin of biochemical homochirality. One of life’s most distinctive biochemical signatures is its strong selectivity for chiral molecular species, notably L-amino acids and D-sugars. Prebiotic synthesis reactions, with the possible exception of some interstellar processes, yield essentially equal amounts of L- and D-enantiomers. A significant challenge in origin-of-life research, therefore, is to identify natural mechanisms for the homochiral selection, concentration and polymerization of molecules from an initially racemic mixture. Symmetry breaking on a chirally selective mineral surface in an aqueous environment offers a viable scenario for the origin of life.

Hazen’s group demonstrated that crystals of the common rock-forming mineral calcite (CaCO₃), when immersed in a racemic aspartic acid solution, display significant adsorption and chiral selectivity of D- and L-enantiomers on pairs of mirror-related crystal growth surfaces. Subsequent studies (now in progress) on glutamic acid, alanine, valine, lysine, tyrosine and glycine reveal both chiral selection and selective adsorption of different amino acids. Studies are also commencing on molecular adsorption on crystal growth surfaces of quartz. We will employ microarray and fluorescent tag technologies developed for the molecular biology field in these studies.

In the coming year, the group plans to study the selective adsorption of other amino acids onto calcite and two other minerals (quartz and gypsum). They will also study the molecular-scale mechanism of amino acid adsorption using fluorescent tags with specific binding characteristics.

Task 4. Emergence (Hazen)

Hazen continues to investigate the role of emergence in the origin of life. Natural systems with many interacting components, such as atoms, molecules, cells or stars, often display complex, “emergent” behavior not associated with their individual components. The geochemical origin of life may be modeled as a sequence of “emergent” events, each of which adds to molecular complexity and order. Each of these steps, if properly formulated, should be amenable to experimental study. Each emergent step, furthermore, may result in characteristic isotopic, molecular, and structural “fossils” that might be measured in extraterrestrial environments that have not been subjected to reworking by biological activity. Studies of the reduction of nitrate to ammonia in the presence of minerals will be expanded to investigate the synthesis and stability of amino acids under hydrothermal conditions, at various levels of pH, both with and without minerals.

Task 5. Iron-Oxiding Lithotrophs (Emerson)

Research in Emerson’s lab has focused on a novel group of lithotrophic Fe-oxidizing bacteria (FeOB). The group showed that these bacteria are abundant and play a major role in deposition of Fe-oxides at Loihi Seamount hydrothermal vent sites. Furthermore, they isolated several strains of FeOB from this site and are in the process of studying them in more detail. One of these strains forms a filamentous Fe-oxide that is common to a number of hydrothermal vent sites around the world and has been seen in ancient micro-fossils as well. The group found that these marine strains and freshwater strains collected from different parts of the United States are all very closely related phenotypically and genotypically, making this particular lineage of FeOB quite easy to trace.

The group has also used a set of bioreactors to determine the contribution of Fe-oxidation. Microbial Fe-oxidation at neutral pH is a complex interplay between biotic and abiotic processes. Their results indicate that the bacteria increase the rate of Fe-oxidation by about 50% and decrease the rate of abiotic oxidation by 25 or 30%. These are the first direct measurements of Fe-oxidation rates and cell yields for neutrophilic, lithotrophic Fe-oxidizers. In total, these results suggest that microbial Fe oxidation will have to be considered as another potential microbial process in astrobiology.