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Hydrocarbons such as CH4 are known to be formed through the Fischer-Tropsch or Sabatier type reactions in hydrothermal systems usually at temperatures above 100°C. Weathering of olivine is sometimes suggested to account for abiotic formation of CH4 through its redox lowering and water splitting properties. Knowledge about the CH4 and H2 formation processes at low temperatures is important for the research about the origin and cause of early Earth and Martian CH4 and for CO2 sequestration. We have conducted a series of low temperature, long-term weathering experiments in which we have tested the CH4 and H2 formation potential of forsteritic olivine.

The results show low temperature CH4 production that is probably influenced by chromite and magnetite as catalysts. Extensive analyses of a potential CH4 source trapped in the crystal structure of the olivine showed no signs of incorporated CH4. Also, the available sources of organic carbon were not enough to support the total amount of CH4 detected in our experiments. There was also a linear relationship between silica release into solution and the net CH4 accumulation into the incubation bottle headspaces suggesting that CH4 formation under these conditions could be a qualitative indicator of olivine dissolution.

It is likely that minerals such as magnetite, chromite and other metal-rich minerals found on the olivine surface catalyze the formation of CH4, because of the low temperature of the system. This may expand the range of environments plausible for abiotic CH4 formation both on Earth and on other terrestrial bodies.

Abstract

The boundary between ice and basalt on Earth is an analogue for some near-surface environments of Mars. We investigated neutrophilic iron-oxidizing microorganisms from the basalt-ice interface in a lava tube from the Oregon Cascades with perennial ice. One of the isolates (Pseudomonas sp. HerB) can use ferrous iron Fe(II) from the igneous mineral olivine as an electron donor and O2 as an electron acceptor. The optimum growth temperature is ∼12–14°C, but growth also occurs at 5°C. Bicarbonate is a facultative source of carbon. Growth of Pseudomonas sp. HerB as a chemolithotrophic iron oxidizer with olivine as the source of energy is favored in low O2 conditions (e.g., 1.6% O2). Most likely, microbial oxidation of olivine near pH 7 requires low O2 to offset the abiotic oxidation of iron. The metabolic capabilities of this bacterium would allow it to live in near-surface, icy, volcanic environments of Mars in the present or recent geological past and make this type of physiology a prime candidate in the search for life on Mars. Key Words: Extremophiles—Mars—Olivine—Iron-oxidizing bacteria—Redox. Astrobiology 12, 9–18.

Mineral carbonation of basic silicate minerals regulates atmospheric CO2 on geological time scales by locking up carbon. Mining and spreading onto the earth's surface of fast-weathering silicates, such as olivine, has been proposed to speed up this natural CO2 sequestration (‘enhanced weathering’). While agriculture may offer an existing infrastructure, weathering rate and impacts on soil and plant are largely unknown. Our objectives were to assess weathering of olivine in soil, and its effects on plant growth and nutrient uptake. In a pot experiment with perennial ryegrass (Lolium perenne L.), weathering during 32 weeks was inferred from bioavailability of magnesium (Mg) in soil and plant. Olivine doses were equivalent to 1630 (OLIV1), 8150, 40700 and 204000 (OLIV4) kg ha−1. Alternatively, the soluble Mg salt kieserite was applied for reference. Olivine increased plant growth (+15.6%) and plant K concentration (+16.5%) in OLIV4. At all doses, olivine increased bioavailability of Mg and Ni in soil, as well as uptake of Mg, Si and Ni in plants. Olivine suppressed Ca uptake. Weathering estimated from a Mg balance was equivalent to 240 kg ha−1 (14.8% of dose, OLIV1) to 2240 kg ha−1 (1.1%, OLIV4). This corresponds to gross CO2 sequestration of 290 to 2690 kg ha−1 (29 103 to 269 103 kg km−2.) Alternatively, weathering estimated from similarity with kieserite treatments ranged from 13% to 58% for OLIV1. The Olsen model for olivine carbonation predicted 4.0% to 9.0% weathering for our case, independent of olivine dose. Our % values observed at high doses were smaller than this, suggesting negative feedbacks in soil. Yet, weathering appears fast enough to support the ‘enhanced weathering’ concept. In agriculture, olivine doses must remain within limits to avoid imbalances in plant nutrition, notably at low Ca availability; and to avoid Ni accumulation in soil and crop.

Today, hyperthermophilic (‘superheat-loving’) bacteria and archaea are found within high-temperature environments, representing the upper temperature border of life. They grow optimally above 80°C and exhibit an upper temperature border of growth up to 113°C. Members of the genera, Pyrodictium and Pyrolobus, survive at least 1 h of autoclaving. In their basically anaerobic environments, hyperthermophiles (HT) gain energy by inorganic redox reactions employing compounds like molecular hydrogen, carbon dioxide, sulphur and ferric and ferrous iron. Based on their growth requirements, HT could have existed already on the early Earth about 3.9 Gyr ago. In agreement, within the phylogenetic tree of life, they occupy all the short deep branches closest to the root. The earliest archaeal phylogenetic lineage is represented by the extremely tiny members of the novel kingdom of Nanoarchaeota, which thrive in submarine hot vents. HT are very tough survivors, even in deep-freezing at −140°C. Therefore, during impact ejecta, they could have been successfully transferred to other planets and moons through the coldness of space.

Before the advent of oxygenic photosynthesis, the biosphere was driven by anaerobic metabolisms. We catalogue and quantify the source strengths of the most probable electron donors and electron acceptors that would have been available to fuel early-Earth ecosystems. The most active ecosystems were probably driven by the cycling of H2 and Fe2+ through primary production conducted by anoxygenic phototrophs. Interesting and dynamic ecosystems would have also been driven by the microbial cycling of sulphur and nitrogen species, but their activity levels were probably not so great. Despite the diversity of potential early ecosystems, rates of primary production in the early-Earth anaerobic biosphere were probably well below those rates observed in the marine environment. We shift our attention to the Earth environment at 3.8 Gyr ago, where the earliest marine sediments are preserved. We calculate, consistent with the carbon isotope record and other considerations of the carbon cycle, that marine rates of primary production at this time were probably an order of magnitude (or more) less than today. We conclude that the flux of reduced species to the Earth surface at this time may have been sufficient to drive anaerobic ecosystems of sufficient activity to be consistent with the carbon isotope record. Conversely, an ecosystem based on oxygenic photosynthesis was also possible with complete removal of the oxygen by reaction with reduced species from the mantle.

Viruses are powerful manipulators of microbial diversity, biogeochemistry, and evolution in the marine environment. Viruses can directly influence the genetic capabilities and the fitness of their hosts through the use of fitness factors and through horizontal gene transfer. However, the impact of viruses on microbial ecology and evolution is often overlooked in studies of the deep subsurface biosphere. Subsurface habitats connected to hydrothermal vent systems are characterized by constant fluid flux, dynamic environmental variability, and high microbial diversity. In such conditions, high adaptability would be an evolutionary asset, and the potential for frequent host–virus interactions would be high, increasing the likelihood that cellular hosts could acquire novel functions. Here, we review evidence supporting this hypothesis, including data indicating 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 subsurface biosphere. We also discuss how these results might apply to other regions of the deep subsurface, where the nature of virus–host interactions would be altered, but possibly no less important, compared to more energetic hydrothermal systems.

Sunlight, the ultimate energy source for life on our planet, enters the biosphere as a direct consequence of the evolution of photoautotrophy. Photoautotrophs must balance the light energy absorbed and trapped through extremely fast, temperature-insensitive photochemistry with energy consumed through much slower, temperature-dependent biochemistry and metabolism. The attainment of such a balance in cellular energy flow between chloroplasts, mitochondria and the cytosol is called photostasis. Photoautotrophs sense cellular energy imbalances through modulation of excitation pressure which is a measure of the relative redox state of QA, the first stable quinone electron acceptor of photosystem II reaction centers. High excitation pressure constitutes a potential stress condition that can be caused either by exposure to an irradiance that exceeds the capacity of C, N, and S assimilation to utilize the electrons generated from the absorbed energy or by low temperature or any stress that decreases the capacity of the metabolic pathways downstream of photochemistry to utilize photosynthetically generated reductants. The similarities and differences in the phenotypic responses between cyanobacteria, green algae, crop plants, and variegation mutants of Arabidopsis thaliana as a function of cold acclimation and photoacclimation are reconciled in terms of differential responses to excitation pressure and the predisposition of photoautotrophs to maintain photostasis. The various acclimation strategies associated with green algae and cyanobacteria versus winter cereals and A. thaliana are discussed in terms of retrograde regulation and the “grand design of photosynthesis” originally proposed by Arnon (1982).

The rates of metabolism in animals vary tremendously throughout the biosphere. The origins of this variation are a matter of active debate with some scientists highlighting the importance of anatomical or environmental constraints, while others emphasize the diversity of ecological roles that organisms play and the associated energy demands. Here, we analyse metabolic rates in diverse marine taxa, with special emphasis on patterns of metabolic rate across a depth gradient, in an effort to understand the extent and underlying causes of variation. The conclusion from this analysis is that low rates of metabolism, in the deep sea and elsewhere, do not result from resource (e.g. food or oxygen) limitation or from temperature or pressure constraint. While metabolic rates do decline strongly with depth in several important animal groups, for others metabolism in abyssal species proceeds as fast as in ecologically similar shallow-water species at equivalent temperatures. Rather, high metabolic demand follows strong selection for locomotory capacity among visual predators inhabiting well-lit oceanic waters. Relaxation of this selection where visual predation is limited provides an opportunity for reduced energy expenditure. Large-scale metabolic variation in the ocean results from interspecific differences in ecological energy demand.

Next-generation sequencing technologies have led to recognition of a so-called ‘rare biosphere'. These microbial operational taxonomic units (OTUs) are defined by low relative abundance and may be specifically adapted to maintaining low population sizes. We hypothesized that mining of low-abundance next-generation 16S ribosomal RNA (rRNA) gene data would lead to the discovery of novel phylogenetic diversity, reflecting microorganisms not yet discovered by previous sampling efforts. Here, we test this hypothesis by combining molecular and bioinformatic approaches for targeted retrieval of phylogenetic novelty within rare biosphere OTUs. We combined BLASTN network analysis, phylogenetics and targeted primer design to amplify 16S rRNA gene sequences from unique potential bacterial lineages, comprising part of the rare biosphere from a multi-million sequence data set from an Arctic tundra soil sample. Demonstrating the feasibility of the protocol developed here, three of seven recovered phylogenetic lineages represented extremely divergent taxonomic entities. These divergent target sequences correspond to (a) a previously unknown lineage within the BRC1 candidate phylum, (b) a sister group to the early diverging and currently recognized monospecific Cyanobacteria Gloeobacter, a genus containing multiple plesiomorphic traits and (c) a highly divergent lineage phylogenetically resolved within mitochondria. A comparison to twelve next-generation data sets from additional soils suggested persistent low-abundance distributions of these novel 16S rRNA genes. The results demonstrate this sequence analysis and retrieval pipeline as applicable for exploring underrepresented phylogenetic novelty and recovering taxa that may represent significant steps in bacterial evolution.

By coupling an atmospheric general circulation model asynchronously with an equilibrium vegetation model, manifold equilibrium solutions of the atmosphere-biosphere system have been explored. It is found that under present-day conditions of the Earth's orbital parameters and sea-surface temperatures, two stable equilibria of vegetation patterns are possible: one corresponding to present-day sparse vegetation in the Sahel, the second solution yielding savannah which extends far into the south-western part of the Sahara. A similar picture is obtained for conditions during the last glacial maximum (21 000 years before present (BP)). For the mid-Holocene (6000 years BP), however, the model finds only one solution: the green Sahara. We suggest that this intransitive behaviour of the atmosphere-biosphere is related to a westward shift of the Hadley-Walker circulation. A conceptual model of atmosphere-vegetation dynamics is used to interpret the bifurcation as well as its change in terms of stability theory.

International drilling projects for the study of microbial communities in the deep-subsurface hot biosphere have been expanded. Core samples obtained by deep drilling are commonly contaminated with mesophilic microorganisms in the drilling fluid, making it difficult to examine the microbial community by 16S rRNA gene clone library analysis. To eliminate mesophilic organism contamination, we previously developed a new method (selective phylogenetic analysis [SePA]) based on the strong correlation between the guanine-plus-cytosine (G+C) contents of the 16S rRNA genes and the optimal growth temperatures of prokaryotes, and we verified the method's effectiveness (H. Kimura, M. Sugihara, K. Kato, and S. Hanada, Appl. Environ. Microbiol. 72:21-27, 2006). In the present study we ascertained SePA's ability to eliminate contamination by archaeal rRNA genes, using deep-sea hydrothermal fluid (117°C) and surface seawater (29.9°C) as substitutes for deep-subsurface geothermal samples and drilling fluid, respectively. Archaeal 16S rRNA gene fragments, PCR amplified from the surface seawater, were denatured at 82°C and completely digested with exonuclease I (Exo I), while gene fragments from the deep-sea hydrothermal fluid remained intact after denaturation at 84°C because of their high G+C contents. An examination using mixtures of DNAs from the two environmental samples showed that denaturation at 84°C and digestion with Exo I completely eliminated archaeal 16S rRNA genes from the surface seawater. Our method was quite useful for culture-independent community analysis of hyperthermophilic archaea in core samples recovered from deep-subsurface geothermal environments.

The remediation of chromium-contaminated sites requires knowledge of the processes that control the migration and transformation of chromium. Advection, dispersion, and diffusion are physical processes affecting the rate at which contaminants can migrate in the subsurface. Heterogeneity is an important factor that affects the contribution of each of these mechanisms to the migration of chromium-laden waters. Redox reactions, chemical speciation, adsorption/desorption phenomena, and precipitation/dissolution reactions control the transformation and mobility of chromium. The reduction of CrVI to CrIII can occur in the presence of ferrous iron in solution or in mineral phases, reduced sulfur compounds, or soil organic matter. At neutral to alkaline pH, the CrIII precipitates as amorphous hydroxides or forms complexes with organic matter. CrIII is oxidized by manganese dioxide, a common mineral found in many soils. Solid-phase precipitates of hexavalent chromium such as barium chromate can serve either as sources or sinks for CrVI. Adsorption of CrVI in soils increases with decreasing chromium concentration, making it more difficult to remove the chromium as the concentration decreases during pump-and-treat remediation. Knowledge of these chemical and physical processes is important in developing and selecting effective, cost-efficient remediation designs for chromium-contaminated sites.

Highly concentrated NaCl brines are important geothermal fluids; chloride complexation of metals in such brines increases the solubility of minerals and plays a fundamental role in the genesis of hydrothermal ore deposits. There is experimental evidence that the molecular nature of the NaCl–water system changes over the pressure–temperature range of the Earth's crust. A transition of concentrated NaCl–H2O brines to a "hydrous molten salt" at high P and T has been argued to stabilize an aqueous fluid phase in the deep crust.

In this work, we have done molecular dynamic simulations using classical potentials to determine the nature of concentrated (0.5–16 m) NaCl–water mixtures under ambient (25°C, 1 bar), hydrothermal (325°C, 1 kbar) and deep crustal (625°C, 15 kbar) conditions. We used the well-established SPCE model for water together with the Smith and Dang Lennard-Jones potentials for the ions (J. Chem. Phys., 1994, 100, 3757). With increasing temperature at 1 kbar, the dielectric constant of water decreases to give extensive ion-association and the formation of polyatomic (NanClm)n-m clusters in addition to simple NaCl ion pairs. Large polyatomic (NanClm)n-m clusters resemble what would be expected in a hydrous NaCl melt in which water and NaCl were completely miscible. Although ion association decreases with pressure, temperatures of 625°C are not enough to overcome pressures of 15 kbar; consequently, there is still enhanced Na–Cl association in brines under deep crustal conditions.

Microorganisms adapted to piezopsychrophilic growth dominate the majority of the biosphere that is at relatively constant low temperatures and high pressures, but the genetic bases for the adaptations are largely unknown. Here we report the use of transposon mutagenesis with the deep-sea bacterium Photobacterium profundum strain SS9 to isolate dozens of mutant strains whose growth is impaired at low temperature and/or whose growth is altered as a function of hydrostatic pressure. In many cases the gene mutation-growth phenotype relationship was verified by complementation analysis. The largest fraction of loci associated with temperature sensitivity were involved in the biosynthesis of the cell envelope, in particular the biosynthesis of extracellular polysaccharide. The largest fraction of loci associated with pressure sensitivity were involved in chromosomal structure and function. Genes for ribosome assembly and function were found to be important for both low-temperature and high-pressure growth. Likewise, both adaptation to temperature and adaptation to pressure were affected by mutations in a number of sensory and regulatory loci, suggesting the importance of signal transduction mechanisms in adaptation to either physical parameter. These analyses were the first global analyses of genes conditionally required for low-temperature or high-pressure growth in a deep-sea microorganism.

Archaea are abundant and drive critical microbial processes in the Earth's cold biosphere. Despite this, not enough is known about the molecular mechanisms of cold adaptation and no biochemical studies have been performed on stenopsychrophilic archaea (e.g., Methanogenium frigidum). This study examined the structural and functional properties of cold shock proteins (Csps) from archaea, including biochemical analysis of the Csp from M. frigidum. csp genes are present in most bacteria and some eucarya but absent from most archaeal genome sequences, most notably, those of all archaeal thermophiles and hyperthermophiles. In bacteria, Csps are small, nucleic acid binding proteins involved in a variety of cellular processes, such as transcription. In this study, archaeal Csp function was assessed by examining the ability of csp genes from psychrophilic and mesophilic Euryarchaeota and Crenarchaeota to complement a cold-sensitive growth defect in Escherichia coli. In addition, an archaeal gene with a cold shock domain (CSD) fold but little sequence identity to Csps was also examined. Genes encoding Csps or a CSD structural analog from three psychrophilic archaea rescued the E. coli growth defect. The three proteins were predicted to have a higher content of solvent-exposed basic residues than the noncomplementing proteins, and the basic residues were located on the nucleic acid binding surface, similar to their arrangement in E. coli CspA. The M. frigidum Csp was purified and found to be a single-domain protein that folds by a reversible two-state mechanism and to exhibit a low conformational stability typical of cold-adapted proteins. Moreover, M. frigidum Csp was characterized as binding E. coli single-stranded RNA, consistent with its ability to complement function in E. coli. The studies show that some Csp and CSD fold proteins have retained sufficient similarity throughout evolution in the Archaea to be able to function effectively in the Bacteria and that the function of the archaeal proteins relates to cold adaptation. The initial biochemical analysis of M. frigidum Csp has developed a platform for further characterization and demonstrates the potential for expanding molecular studies of proteins from this important archaeal stenopsychrophile.

The kinetics of pyrite oxidation by Metallosphaera sedula were investigated with mineral pyrite and two coals with moderate (Pittsburgh no. 8) and high (New Brunswick, Canada) pyritic sulfur content. M. sedula oxidized mineral pyrite at a greater rate than did another thermophile, Acidianus brierleyi, or a mesophile, Thiobacillus ferrooxidans. Maximum rates of coal depyritization were also greater with M. sedula, although the magnitude of biological stimulation above abiotic rates was notably less than with mineral pyrite. Coal depyritization appears to be limited by the oxidation of pyrite with ferric ions and not by the rate of biotic oxidation of ferrous iron, as evidenced by the maintenance of a high ratio of ferric to ferrous iron in solution by M. sedula. Significant precipitation of hydronium jarosite at elevated temperature occurred only with New Brunswick coal.

Background

Oceans cover approximately 70% of the Earth's surface with an average depth of 3800 m and a pressure of 38 MPa, thus a large part of the biosphere is occupied by high pressure environments. Piezophilic (pressure-loving) organisms are adapted to deep-sea life and grow optimally at pressures higher than 0.1 MPa. To better understand high pressure adaptation from a genomic point of view three different Photobacterium profundum strains were compared. Using the sequenced piezophile P. profundum strain SS9 as a reference, microarray technology was used to identify the genomic regions missing in two other strains: a pressure adapted strain (named DSJ4) and a pressure-sensitive strain (named 3TCK). Finally, the transcriptome of SS9 grown under different pressure (28 MPa; 45 MPa) and temperature (4°C; 16°C) conditions was analyzed taking into consideration the differentially expressed genes belonging to the flexible gene pool.

Results

These studies indicated the presence of a large flexible gene pool in SS9 characterized by various horizontally acquired elements. This was verified by extensive analysis of GC content, codon usage and genomic signature of the SS9 genome. 171 open reading frames (ORFs) were found to be specifically absent or highly divergent in the piezosensitive strain, but present in the two piezophilic strains. Among these genes, six were found to also be up-regulated by high pressure.

Conclusion

These data provide information on horizontal gene flow in the deep sea, provide additional details of P. profundum genome expression patterns and suggest genes which could perform critical functions for abyssal survival, including perhaps high pressure growth.

Sulfate-reducing prokaryotes (SRP) are ubiquitous and quantitatively important members in many ecosystems, especially in marine sediments. However their abundance and diversity in subsurface marine sediments is poorly understood. In this study, the abundance and diversity of the functional genes for the enzymes adenosine 5′-phosphosulfate reductase (aprA) and dissimilatory sulfite reductase (dsrA) of SRP in marine sediments of the Peru continental margin and the Black Sea were analyzed, including samples from the deep biosphere (ODP site 1227). For aprA quantification a Q-PCR assay was designed and evaluated. Depth profiles of the aprA and dsrA copy numbers were almost equal for all sites. Gene copy numbers decreased concomitantly with depth from around 108/g sediment close to the sediment surface to less than 105/g sediment at 5 mbsf. The 16S rRNA gene copy numbers of total bacteria were much higher than those of the functional genes at all sediment depths and used to calculate the proportion of SRP to the total Bacteria. The aprA and dsrA copy numbers comprised in average 0.5–1% of the 16S rRNA gene copy numbers of total bacteria in the sediments up to a depth of ca. 40 mbsf. In the zone without detectable sulfate in the pore water from about 40–121 mbsf (Peru margin ODP site 1227), only dsrA (but not aprA) was detected with copy numbers of less than 104/g sediment, comprising ca. 14% of the 16S rRNA gene copy numbers of total bacteria. In this zone, sulfate might be provided for SRP by anaerobic sulfide oxidation. Clone libraries of aprA showed that all isolated sequences originate from SRP showing a close relationship to aprA of characterized species or form a new cluster with only distant relation to aprA of isolated SRP. For dsrA a high diversity was detected, even up to 121 m sediment depth in the deep biosphere.

The coupled biosphere–atmosphere system entails a vast range of processes at different scales, from ecosystem exchange fluxes of energy, water and carbon to the processes that drive global biogeochemical cycles, atmospheric composition and, ultimately, the planetary energy balance. These processes are generally complex with numerous interactions and feedbacks, and they are irreversible in their nature, thereby producing entropy. The proposed principle of maximum entropy production (MEP), based on statistical mechanics and information theory, states that thermodynamic processes far from thermodynamic equilibrium will adapt to steady states at which they dissipate energy and produce entropy at the maximum possible rate. This issue focuses on the latest development of applications of MEP to the biosphere–atmosphere system including aspects of the atmospheric circulation, the role of clouds, hydrology, vegetation effects, ecosystem exchange of energy and mass, biogeochemical interactions and the Gaia hypothesis. The examples shown in this special issue demonstrate the potential of MEP to contribute to improved understanding and modelling of the biosphere and the wider Earth system, and also explore limitations and constraints to the application of the MEP principle.

Oceanic crust comprises the largest hydrogeologic reservoir on Earth, containing fluids in thermodynamic disequilibrium with the basaltic crust. Little is known about microbial ecosystems that inhabit this vast realm and exploit chemically favorable conditions for metabolic activities. Crustal samples recovered from ocean drilling operations are often compromised for microbiological assays, hampering efforts to resolve the extent and functioning of a subsurface biosphere. We report results from the first in situ experimental observatory systems that have been used to study subseafloor life. Experiments deployed for 4 years in young (3.5 Ma) basaltic crust on the eastern flank of the Juan de Fuca Ridge record a dynamic, post-drilling response of crustal microbial ecosystems to changing physical and chemical conditions. Twisted stalks exhibiting a biogenic iron oxyhydroxide signature coated the surface of mineral substrates in the observatories; these are biosignatures indicating colonization by iron oxidizing bacteria during an initial phase of cool, oxic, iron-rich conditions following observatory installation. Following thermal and chemical recovery to warmer, reducing conditions, the in situ microbial structure in the observatory shifted, becoming representative of natural conditions in regional crustal fluids. Firmicutes, metabolic potential of which is unknown but may involve N or S cycling, dominated the post-rebound bacterial community. The archaeal community exhibited an extremely low diversity. Our experiment documented in situ conditions within a natural hydrological system that can pervade over millennia, exemplifying the power of observatory experiments for exploring the subsurface basaltic biosphere, the largest but most poorly understood biotope on Earth.

Since the days of Darwin, scientists have used the framework of the theory of evolution to explore the interconnectedness of life on Earth and adaptation of organisms to the ever-changing environment. The advent of molecular biology has advanced and accelerated the study of evolution by allowing direct examination of the genetic material that ultimately determines the phenotypes upon which selection acts. The study of evolution has been furthered through examination of microbial evolution, with large population numbers, short generation times, and easily extractable DNA. Such work has spawned the study of microbial biogeography, with the realization that concepts developed in population genetics may be applicable to microbial genomes (Martiny et al., 2006; Manhes and Velicer, 2011). Microbial biogeography and adaptation has been examined in many different environments. Here we argue that the deep biosphere is a unique environment for the study of evolution and list specific factors that can be considered and where the studies may be performed. This publication is the result of the NSF-funded Center for Dark Energy Biosphere Investigations (C-DEBI) theme team on Evolution (www.darkenergybiosphere.org).

Thermovibrio ammonificans type strain HB-1T is a thermophilic (Topt: 75°C), strictly anaerobic, chemolithoautotrophic bacterium that was isolated from an active, high temperature deep-sea hydrothermal vent on the East Pacific Rise. This organism grows on mineral salts medium in the presence of CO2/H2, using NO3- or S0 as electron acceptors, which are reduced to ammonium or hydrogen sulfide, respectively. T. ammonificans is one of only three species within the genus Thermovibrio, a member of the family Desulfurobacteriaceae, and it forms a deep branch within the phylum Aquificae. Here we report the main features of the genome of T. ammonificans strain HB-1T (DSM 15698T).

Dihydrofolate reductase (DHFR) maintains the intracellular pool of tetrahydrofolate through catalysis of hydrogen transfer from reduced nicotinamide adenine dinucleotide to 7,8-dihydrofolate. We report results for pre-steady-state kinetic studies of the temperature dependence of the rates and the hydrogen/deuterium-kinetic isotope effects for the reactions catalysed by the enzymes from the mesophilic Escherichia coli and the hyperthermophilic Thermatoga maritima. We propose an evolutionary pattern in which catalysis progressed from a relatively rigid active site structure in the ancient thermophilic DHFR to a more flexible and kinetically more efficient structure in E. coli that actively promotes hydrogen transfer at physiological pH by modulating the tunnelling distance. The E. coli enzyme appeared relatively robust, in that kinetically severely compromised mutants still actively propagated the reaction. The reduced hydrogen transfer rates of the extensively studied Gly121Val mutant of DHFR from E. coli were most likely due to sterically unfavourable long-range effects from the introduction of the bulky isopropyl group.

The oxidation of a pure pyrite by Thiobacillus ferrooxidans is not really a constant phenomenon; it must be considered to be more like a succession of different steps which need characterization. Electrochemical studies using a combination of a platinum electrode and a specific pyrite electrode (packed-ground-pyrite electrode) revealed four steps in the bioleaching process. Each step can be identified by the electrochemical behavior (redox potentials) of pyrite, which in turn can be related to chemical (leachate content), bacterial (growth), and physical (corrosion patterns) parameters of the leaching process. A comparison of the oxidation rates of iron and sulfur indicated the nonstoichiometric bacterial oxidation of a pure pyrite in which superficial phenomena, aqueous oxidation, and deep crystal dissolution are successively involved.

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We report the isolation and physiological characterization of novel, psychrophilic, iron-oxidizing bacteria (FeOB) from low-temperature weathering habitats in the vicinity of the Juan de Fuca deep-sea hydrothermal area. The FeOB were cultured from the surfaces of weathered rock and metalliferous sediments. They are capable of growth on a variety of natural and synthetic solid rock and mineral substrates, such as pyrite (FeS2), basalt glass (∼10 wt% FeO), and siderite (FeCO3), as their sole energy source, as well as numerous aqueous Fe substrates. Growth temperature characteristics correspond to the in situ environmental conditions of sample origin; the FeOB grow optimally at 3 to 10°C and at generation times ranging from 57 to 74 h. They are obligate chemolithoautotrophs and grow optimally under microaerobic conditions in the presence of an oxygen gradient or anaerobically in the presence of nitrate. None of the strains are capable of using any organic or alternate inorganic substrates tested. The bacteria are phylogenetically diverse and have no close Fe-oxidizing or autotrophic relatives represented in pure culture. One group of isolates are γ-Proteobacteria most closely related to the heterotrophic bacterium Marinobacter aquaeolei (87 to 94% sequence similarity). A second group of isolates are α-Proteobacteria most closely related to the deep-sea heterotrophic bacterium Hyphomonas jannaschiana (81 to 89% sequence similarity). This study provides further evidence for the evolutionarily widespread capacity for Fe oxidation among bacteria and suggests that FeOB may play an unrecognized geomicrobiological role in rock weathering in the deep sea.