nai

Project Progress

5. Life in Extreme Environments

1. Life in Deep-Sea Hydrothermal Vents

The astrobiology research objectives of Baross and his group are focused on understanding the microbial ecology and physiology of Earth environments that share geophysical and geochemical characteristics with other planets and satellites. Baross and colleagues have targeted magma-hosted and peridotite-hosted hydrothermal systems, because they abiotically produce high concentrations of biologically important carbon and energy sources. 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. The group’s research questions address hydrothermal vent ecosystems where hydrogen is the main energy source and the strategies used by microbes to exploit the carbon and energy sources available, extract essential nutrients from rocks, and grow and survive under the extreme conditions of these environments.

Baross’s team also continued its studies of nitrogen fixation in vent environments. 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 these environments. Recent results indicate that subseafloor archaea that are related to the most abundant group of archaea in seawater have genes for nitrogen fixation (nifH). These potential nitrogen-fixing archaea are also found in deep seawater near mid-ocean ridges but not in deep seawater off-axis, suggesting that this group of archaea circulates through the N-poor subseafloor at mid-ocean ridges as part of their life cycle.

Baross and colleagues 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. The results are surprising and show a much higher diversity of microorganisms than expected, including anaerobic hyperthermophiles and hydrogen-utilizing thermophiles. Their results point to the hypothesis that the subsurface associated with hydrothermal systems in off-axis crust harbors a high diversity of microorganisms that includes archaea and bacteria that have novel metabolisms and physiologies that allow for growth in the absence of nutrients derived from photosynthesis.

Much of this work involves the isolation of microorganisms from subsurface fluids and high-temperature sulfide and rock samples. The group demonstrated that isolates of Thermococcales from different subseafloor habitats at Axial Volcano on the Juan de Fuca Ridge show different physiological characteristics linked to the chemistry of the subseafloor fluids. Matt Schrenk, who will be completing his Ph.D. in summer 2005, focused on microbial biofilms formed on minerals at high temperatures. He obtained evidence for microbial biofilm formation at temperatures considerably higher than 121°C, the highest growth temperature so far for isolated microbes.

Research at the Lost City Hydrothermal Field (LCHF) continued. The LCHF is located 15 km off the axis of the Mid-Atlantic Ridge and consists of a network of large carbonate chimney structures. The fluids from these structures reach temperatures greater than 90°C, have pHs from 9 to 11, and are driven by exothermic serpentization reactions that produce high concentrations of hydrogen, methane, and organic compounds. Baross and colleagues found that the most actively venting chimneys are dominated by a single phylotype of Methanosarcinales distinct from the two known groups. These organisms form dense biofilms on the carbonate structures. Recently, they showed the presence of 16S rDNA and methyl coenzyme-M reductase (mcrA) sequences related to the anaerobic methane oxidation group of Methanosarcinales (ANME-1) indicating that LCHF may be a high-temperature analogue to cold methane seep environments. They also completed the phylogenetic characterization of the bacterial and archaeal communities from carbonate and fluid samples and showed evidence for the widespread occurrence of methane and sulfur-oxidizing bacteria. Baross and colleagues will be participating on another cruise to LCHF in August 2005 and will attempt to measure methane production and consumption rates in situ using stable isotopes.

{{ 1 }}

2. Iron-Based Metabolic Strategies for Microbial Life

During the past year Emerson and colleagues made significant progress in their study of bacteria that oxidize iron as a source of energy at neutral pH. Emerson and Postdoctoral Research Scientist Jeremy Rentz participated in a field expedition to the Loihi Seamount in October 2004 and collected a number of new samples for analysis from the extensive microbial Fe mats that exist in association with hydrothermal vents at this site. Highlights from this work include more efficient methods for obtaining and culturing DNA from these organisms, which are very difficult to manipulate in the laboratory. This work has led to the reclassification of isolates obtained from the Loihi system as a deeply branching group of proteobacteria, in contrast to previous data that had indicated they were close relatives of known bacteria. Emerson and his group developed real-time PCR assays that will allow them to track and enumerate their populations in the environment. A simple method was also developed to study the early phase of colonization of microbial mats by these bacteria in situ; study results to date indicate that the Fe-oxidizing bacteria initiate growth rapidly and are probably responsible for catalyzing most of the iron oxidation that occurs in the early phase of Fe mat development.

{{ 2 }}

In collaboration with Steele and Vicenzi, Emerson and colleagues initiated a project aimed at high-resolution imaging of some of the unique morphological structures produced by Fe-oxidizing bacteria using Raman spectroscopy coupled to light microscopy and TOF-SIMs coupled to electron microscopy. Initial results with field samples indicate that Fe-mineralogies can be distinguished and elemental maps resolved at the sub-micrometer scale. Emerson and his group are in the process of designing and conducting experiments using both pure cultures of Fe-oxidizing bacteria and natural samples to understand how diagenetic processes affect these structures. This work will allow them to interpret signatures for similar structures that are commonly found in the rock record dating back billions of years on Earth.

In another project that was partially supported by NAI, Emerson, Masters Student Melissa Floyd, and others completed a survey of environmental prokaryotes held at the ATCC. This work quantified anecdotal evidence that there is a wide gulf between the microbes that are typically used in the laboratory and those that have been surveyed in the environment using cultivation-independent methods. This work also pointed out the large discrepancy in discovery and culture of microbes from the developed nations compared with the less-developed nations (which are also home to the bulk of animal and plant diversity).

3. The Effect of Pressure on Carbon Compounds, Water, and Microbial Physiology

The chemistry of carbon and carbonaceous materials over a broad range of pressures and temperatures characteristic of those found within planets defines the limits of life (as we know it) in extreme environments and must guide all thinking about life’s origins. It is well known that terrestrial carbon exists in several forms: native, oxidized, and reduced in a wide variety of hydrocarbons. This complexity is demonstrated by many examples: diamonds in kimberlite formations, graphite in metamorphic rocks, volcanic CO₂ emissions, ubiquitous carbonate minerals in the crust, methane hydrates on the ocean floor, and petroleum reservoirs in sedimentary basins. The stability and formation of reduced species such as methane and higher hydrocarbons are of particular interest from the standpoint of prebiotic chemistry and astrobiology. Indeed, information about these species in other bodies of the solar system has expanded greatly with recent planetary probes.

Hemley, NAI Postdoctoral Research Associate Henry Scott, and colleagues reported the first in situ high-pressure and high-temperature experiments to show that methane readily forms by reduction of carbonate under conditions typical for terrestrial planetary mantles. Starting materials were natural CaCO₃ calcite, FeO wüstite, and distilled H₂O. Experiments were conducted using new diamond anvil cell (DAC) techniques; simultaneous high-pressure and high-temperature conditions were produced by both resistive (<600°C) and laser heating (>1,000°C) methods. Additional calculations of hydrocarbon stability have been pursued with a group at the Lawrence Livermore National Laboratory, and new experiments are underway to examine the formation of higher hydrocarbons and carbon isotope fractionation.

Studies were also carried out in other organic-rich systems under a wide range of conditions with the aim of directly monitoring processes such as structural phase transformations, organic synthesis reactions, fluid-phase immiscibility, reactions kinetics, and pathways. These experiments included investigations of the stability and properties of various clathrate hydrates in a broad range of planetary environments. New data were obtained by Raman spectroscopy and by neutron scattering in a variety of systems (e.g., H₂O-H₂). One focus was the so-called novel clathrates or van der Waals compounds that may be stable in the satellites of the outer planets. One of these, CH₄(H₂)₄, was found to be stable over a remarkably wide range of pressure and temperature. Plans were developed for inelastic neutron scattering measurements on clathrate hydrates to understand further the structure, dynamics, and reactivity of these systems, and preliminary tests were performed.

The behavior of H₂O under pressure underlies Hemley and his team’s work in prebiotic chemistry and high-pressure microbiology. New, and indeed unexpected, transformations in ice were documented by X-ray diffraction and Raman spectroscopy. In this regard, observational data indicate that the water/ice in bodies such as Europa likely contains considerable dissolved salts. Thus determination of the phase relations in aqueous solutions over a broad P-T range is essential for understanding processes within these planetary bodies. Preliminary studies of KCl-H₂O solutions revealed a series of low-temperature transformations in this system under Europan interior conditions.

Scott and Collaborator Anurag Sharma (Rensselaer Polytechnic Institute) made significant progress in understanding the adaptations that allow microbial life to persist at pressures approaching 2 GPa. Using light microscopy and epiflourescent microscopy, the group showed that gram-negative bacteria have much greater resistance to pressure than gram-positive bacteria. This work provided the framework for efforts to identify key proteins and genes that are triggered in response to pressures between 0.1 and 1.5 GPa. The effort to identify pressure-sensitive genetic elements has been a collaborative effort spearheaded by Scott, Steele, and Sharma. mRNA from cells altered by pressure has been isolated by Doctoral Student Verena Starke for DNA microarray analysis to identify genes that are turned off and on in Escherichia coli in response to pressure. Collaborator Grigoriy Pinchuk (Microbial Dynamics Group, Pacific Northwest National Laboratory, or PNNL) has developed continuous culturing approaches to identify pressure-sensitive proteins in E. coli and Shewanella oneidensis MR1.

4. Formate Metabolism in Shewanella oneidensis

In the course of collaborative research with the Microbial Dynamics Group at PNNL, Scott identified a novel carbon assimilation pathway utilized by S. oneidensis MR1 during dissimilatory reduction of ferric iron. Through the utilization of microarray and genomic analysis he confirmed that Shewanella MR1 contains a novel formate assimilation pathway that exploits genes and proteins usually associated with organisms that grow on methane, methylamines, and methanol as their sole sources of carbon. This work should lead to a re-examination of the topic of carbon assimilation by bacteria and archaea and to the identification of other novel pathways.