Subsurface Microbiology

Earth's subsurface is often isolated from phototrophic energy sources and characterized by chemotrophic modes of life. These environments are often oligotrophic and limited in electron donors or electron acceptors, and include continental crust, subseafloor oceanic crust, and marine sediment as well as subglacial lakes and the subsurface of polar desert soils. These low energy subsurface environments are therefore uniquely positioned for examining minimum energetic requirements and adaptations for chemotrophic life. Current targets for astrobiology investigations of extant life are planetary bodies with largely inhospitable surfaces, such as Mars, Europa, and Enceladus. Subsurface environments on Earth thus serve as analogs to explore possibilities of subsurface life on extraterrestrial bodies. The purpose of this review is to provide an overview of subsurface environments as potential analogs, and the features of microbial communities existing in these low energy environments, ...

Sediment-hosted CO 2 -rich aquifers deep below the Colorado Plateau (USA) contain a remarkable diversity of uncultivated microorganisms, including Candidate Phyla Radiation (CPR) bacteria that are putative symbionts unable to synthesize membrane lipids. The origin of organic carbon in these ecosystems is unknown and the source of CPR membrane lipids remains elusive. We collected cells from deep groundwater brought to the surface by eruptions of Crystal Geyser, sequenced the community, and analyzed the whole community lipidome over time. Characteristic stable carbon isotopic compositions of microbial lipids suggest that bacterial and archaeal CO 2 fixation ongoing in the deep subsurface provides organic carbon for the complex communities that reside there. Coupled lipidomic-metagenomic analysis indicates that CPR bacteria lack complete lipid biosynthesis pathways but still possess regular lipid membranes. These lipids may therefore originate from other community members, which also adapt to high in situ pressure by increasing fatty acid unsaturation. An unusually high abundance of lysolipids attributed to CPR bacteria may represent an adaptation to membrane curvature stress induced by their small cell sizes. Our findings provide new insights into the carbon cycle in the deep subsurface and suggest the redistribution of lipids into putative symbionts within this community.

To describe the compartmentalization of the gut and the microbial activity in the digestive tract, the histology of the gut wall, enzymatic activity, pH of the gut content, abundance and composition of the microbial community (direct counts, plate counts on various media, and phospholipid fatty acid analyses-PLFA) were studied in the Penthetria holosericea larvae. The highest secretion activity was observed in the caeca and in the part of the anterior midgut before caeca openings. A strong increase in pH (10.5) was observed in the anterior part of the midgut. Some enzymes produced in the caeca might have their maximum activity in a different part of the gut as a result of their pH optimum; e.g. amylase was most active in the strongly alkaline anterior part of the mesenteron. Maltase, hydrolyzing products of starch degradation, was most active in the posterior part of the gut and in the ectoperithrophic space. The microbial community was reduced in the anterior part of the midgut. In the posterior part of the gut, bacterial numbers increased and peaked in excrements. Caeca had an abundant microbial community.

Observations from NASA's Cassini spacecraft established that Saturn's moon Enceladus has an internal liquid ocean. Analysis of a plume of ocean material ejected into space suggests alkaline hydrothermal vents on Enceladus' seafloor. On Earth, such deep-sea vents harbor microbial ecosystems rich in methanogenic archaea. Here, we use a Bayesian statistical approach to quantify the probability that methanogenesis (biotic methane production) might explain the escape rates of molecular hydrogen and methane in Enceladus' plume, as measured by Cassini instruments. We find that the observed escape rates (i) cannot be explained solely by the abiotic alteration of the rocky core by serpentinization; (ii) are compatible with the hypothesis of habitable conditions for methanogens; (iii) score the highest likelihood under the hypothesis of methanogenesis, assumed the probability of life emerging is high enough. If the probability of life emerging on Enceladus is low, the Cassini measurements are consistent with habitable yet uninhabited hydrothermal vents and point to unknown sources of methane (e.g., primordial methane) awaiting to be discovered by future missions.

The candidate Division MSBL1 (Mediterranean Sea Brine Lakes 1) comprises a monophyletic group of uncultured archaea found in different hypersaline environments. Previous studies propose methanogenesis as the main metabolism. Here, we describe a metabolic reconstruction of MSBL1 based on 32 single-cell amplified genomes from Brine Pools of the Red Sea (Atlantis II, Discovery, Nereus, Erba and Kebrit). Phylogeny based on rRNA genes as well as conserved single copy genes delineates the group as a putative novel lineage of archaea. Our analysis shows that MSBL1 may ferment glucose via the Embden–Meyerhof–Parnas pathway. However, in the absence of organic carbon, carbon dioxide may be fixed via the ribulose bisphosphate carboxylase, Wood-Ljungdahl pathway or reductive TCA cycle. Therefore, based on the occurrence of genes for glycolysis, absence of the core genes found in genomes of all sequenced methanogens and the phylogenetic position, we hypothesize that the MSBL1 are not methanogens...

Enceladus has a subsurface ocean in the South Pole that has been inferred due to the presence of water vapor and other molecules like molecular hydrogen and ammonia detected by the Cassini mission from the ejection of material through the plumes in that region. The chemical composition of this ocean could give some information about the evolutionary stage of the icy moon if its components are found to be similar with the aqueous chemistry of the primitive oceans on Earth during glacial periods. Here we present a comparative geochemical analysis between the ocean of Enceladus and the aqueous composition of the oceans on Earth during the Snowball Event, in order to figure out if there are similar species, how the interaction of the metabolic processes between them works and if, in the future, those molecules could evolve making possible the emergence of life.

Rocks that react with liquid water are widespread but spatiotemporally limited throughout the solar system, except for Earth. Rock-forming minerals with high iron content and accessory minerals with high amounts of radioactive elements are essential to support rock-hosted microbial life by supplying organics, molecular hydrogen, and/or oxidants. Recent technological advances have broadened our understanding of the rocky biosphere, where microbial inhabitation appears to be difficult without nutrient and energy inputs from minerals. In particular, microbial proliferation in igneous rock basements has been revealed using innovative geomicrobiological techniques. These recent findings have dramatically changed our perspective on the nature and the extent of microbial life in the rocky biosphere, microbial interactions with minerals, and the influence of external factors on habitability. This study aimed to gather information from scientific and/or technological innovations, such as omi...

Observations from NASA's Cassini spacecraft established that Saturn's moon Enceladus has an internal liquid ocean. Analysis of a plume of ocean material ejected into space suggests alkaline hydrothermal vents on Enceladus' seafloor. On Earth, such deep-sea vents harbor microbial ecosystems rich in methanogenic archaea. Here, we use a Bayesian statistical approach to quantify the probability that methanogenesis (biotic methane production) might explain the escape rates of molecular hydrogen and methane in Enceladus' plume, as measured by Cassini instruments. We find that the observed escape rates (i) cannot be explained solely by the abiotic alteration of the rocky core by serpentinization; (ii) are compatible with the hypothesis of habitable conditions for methanogens; (iii) score the highest likelihood under the hypothesis of methanogenesis, assumed the probability of life emerging is high enough. If the probability of life emerging on Enceladus is low, the Cassini measurements are consistent with habitable yet uninhabited hydrothermal vents and point to unknown sources of methane (e.g., primordial methane) awaiting to be discovered by future missions.

Recent studies postulated the viability of a suite of metabolic pathways in Enceladus’ ocean motivated by the detection of H2 and CO2 in the plumes – evidence for available free energy for methanogenesis driven by hydrothermal activity at the moon's seafloor. However, these have not yet been explored in detail. Here, a range of experiments were performed to investigate whether microbial iron reduction could be a viable metabolic pathway in the ocean by iron-reducing bacteria such as Geobacter sulfurreducens. This study has three main outcomes: (i) the successful reduction of a number of crystalline Fe(III)-bearing minerals predicted to be present at Enceladus was shown to take place to differing extents using acetate as an electron donor; (ii) substantial bacterial growth in a simulated Enceladus ocean medium was demonstrated using acetate and H2(g) separately as electron donors; (iii) microbial iron reduction of ferrihydrite was shown to partially occur at pH 9, the currently a...

The detection of silica-rich dust particles, as an indication for ongoing hydrothermal activity, and the presence of water and organic molecules in the plume of Enceladus, have made Saturn’s icy moon a hot spot in the search for potential extraterrestrial life. Methanogenic archaea are among the organisms that could potentially thrive under the predicted conditions on Enceladus, considering that both molecular hydrogen (H2) and methane (CH4) have been detected in the plume. Here we show that a methanogenic archaeon, Methanothermococcus okinawensis, can produce CH4 under physicochemical conditions extrapolated for Enceladus. Up to 72% carbon dioxide to CH4 conversion is reached at 50 bar in the presence of potential inhibitors. Furthermore, kinetic and thermodynamic computations of low-temperature serpentinization indicate that there may be sufficient H2 gas production to serve as a substrate for CH4 production on Enceladus. We conclude that some of the CH4 detected in the plume of E...

The tree of terrestrial life probably roots in non-photosynthetic microbes. Chemoautotrophs were the first primary producers, and the globally dominant niches in terms of primary productivity were determined by availability of carbon dioxide and hydrogen for methanogenesis and sulfite reduction. Methanogen niches were most abundant where CO 2-rich ocean water flowed through serpentinite. Black smoker vents from basalt supplied comparable amount of H 2. Hydrogen from arc volcanoes supported a significant methanogenic niche at the Earth's surface. SO 2 from arc volcanoes reacted with organic matter and hydrogen, providing a significant surface niche. Methane ascended to the upper atmosphere where photolysis produced C-rich haze and CO, and H escaped into space. The CO and C-rich haze supported secondary surface niches. None of these ecologies were bountiful; less than 1% of the CO 2 vented by ridge axes, arcs, and metamorphism became organic matter before it was buried in carbonate. In contrast, a photosynthetic biosphere leaves copious amounts of organic carbon, locally concentrated in sediments. Black shales are a classic geologic biosignature for photosynthesis that can survive subduction and high-grade metamorphism.

Past environments on Mars contained abundant water, suggesting certain regions may have been conducive to life as we know it and implying the potential for microbial inhabitants. Gale and Jezero craters, home of the Perseverance and Curiosity rovers, hosted ancient lakes that experienced periods of active hydrologic cycling and prolonged drying intervals. Exploration of these basins (and future operations on Mars) will benefit from detailed characterizations of analogous environments on Earth, where life detection strategies at various spatial scales (i.e., rover to orbiter) can be tested and validated. Investigations of terrestrial analogs are critical for understanding (1) how microorganisms generate chemical biosignatures in environments characterized by multiple extreme conditions; (2) the impact of environmental conditions and mineralogy on biosignature preservation; and (3) what technologies and techniques are needed to detect biosignatures remotely or in situ. Here, we survey...

Subglacial Lake Whillans (SLW) is located beneath ∼800 m of ice on the Whillans Ice Stream in West Antarctica and was sampled in January of 2013, providing the first opportunity to directly examine water and sediments from an Antarctic subglacial lake. To minimize the introduction of surface contaminants to SLW during its exploration, an access borehole was created using a microbiologically clean hot water drill designed to reduce the number and viability of microorganisms in the drilling water. Analysis of 16S rRNA genes (rDNA) amplified from samples of the drilling and borehole water allowed an evaluation of the efficacy of this approach and enabled a confident assessment of the SLW ecosystem inhabitants. Based on an analysis of 16S rDNA and rRNA (i.e., reverse-transcribed rRNA molecules) data, the SLW community was found to be bacterially dominated and compositionally distinct from the assemblages identified in the drill system. The abundance of bacteria (e.g., Candidatus Nitrotoga, Sideroxydans, Thiobacillus, and Albidiferax) and archaea (Candidatus Nitrosoarchaeum) related to chemolithoautotrophs was consistent with the oxidation of reduced iron, sulfur, and nitrogen compounds having important roles as pathways for primary production in this permanently dark ecosystem. Further, the prevalence of Methylobacter in surficial lake sediments combined with the detection of methanogenic taxa in the deepest sediment horizons analyzed (34-36 cm) supported the hypothesis that methane cycling occurs beneath the West Antarctic Ice Sheet. Large ratios of rRNA to rDNA were observed for several operational taxonomic units abundant in the water column and sediments (e.g., Albidiferax, Methylobacter, Candidatus Nitrotoga, Sideroxydans, and Smithella), suggesting a potentially active role for these taxa in the SLW ecosystem. Our findings are consistent with chemosynthetic microorganisms serving as the ecological foundation in this dark subsurface environment, providing new organic matter that sustains a microbial ecosystem beneath the West Antarctic Ice Sheet.

The genetic diversity of streptomycetes in colliery spoil heaps (Sokolov, Czech Republic) was investigated by restriction pattern analysis of 16S-internal transcribed spacer rDNA and 16S sequences. We sampled freshly excavated Miocene sediment (17-19-million-year-old) and four sites of primary succession (initial, early, middle, and late stages; aged 1-44 years) on the same sediment. Active bacteria were present even in fresh Miocene sediment, and the relative proportion of actinomycetes among total bacterial and their genetic diversity increased significantly with the age of the sampling site. The replacement of pioneer species by late succession species during succession was observed. Plate assays of Streptomyces strains revealed 27% antibiotic-producing strains. Screening for nonribosomal peptide synthases and type I polyketide synthases systems suggested that 90% and 55% streptomycetes, respectively, are putative producers of biologically active compounds. The frequencies of tetracycline-, amoxicillin-, and chloramphenicol-resistant streptomycetes were 6%, 9%, and 15%, respectively. These findings document the occurrence of genetic elements encoding antibiotic resistance genes and the production of antibiotics by streptomycetes located in pristine environments. Our results indicate key roles for ancient streptomycetes related to S. microflavus, S. spororaveus, and S. flavofuscus in pioneering community development in freshly excavated substrates.

While the importance of anaerobic methane oxidation has been reported for marine ecosystems, the role of this process in soils is still questionable. Grasslands used as pastures for cattle overwintering show an increase in anaerobic soil micro-sites caused by animal treading and excrement deposition. Therefore, anaerobic potential methane oxidation activity of severely impacted soil from a cattle winter pasture was investigated in an incubation experiment under anaerobic conditions using 13 C-labelled methane. We were able to detect a high microbial activity utilizing CH 4 as nutrient source shown by the respiration of 13 CO 2. Measurements of possible terminal electron acceptors for anaerobic oxidation of methane were carried out. Soil sulfate concentrations were too low to explain the oxidation of the amount of methane added, but enough nitrate and iron(III) were detected. However, only nitrate was consumed during the experiment. 13 C-PLFA analyses clearly showed the utilization of CH 4 as nutrient source mainly by organisms harbouring 16 : 1ω7 PLFAs. These lipids were also found as most 13 Cenriched fatty acids by Raghoebarsing et al. (2006) after addition of 13 CH 4 to an enrichment culture coupling denitrification of nitrate to anaerobic oxidation of methane. This might be an indication for anaerobic oxidation of methane by relatives of "Candidatus Methylomirabilis oxyfera" in the investigated grassland soil under the conditions of the incubation experiment.

In spite of the acknowledged importance of growth-promoting bacteria, only a reduced number of studies were conducted with these microorganisms on Theobroma cacao. The objectives of this work were to study the population densities and genetic diversity of actinomycetes associated with the rhizosphere of cacao as a first step in their application in plant growth promotion and biological control. The populations densities of actinomycetes in soil and cacao roots were similar, with mean values of 1,0 x 10 6 CFU/g and 9,6 x 10 5 CFU/g, respectively. All isolates selected and used in this study were identified through sequencing analyses of a fragment of the rpoB gene that encodes the β-subunit of the RNA polymerase as species of the genus Streptomyces. In vitro cellulolytic, xilanolytic and chitinolytic activity, indolacetic acid production and phosphate solubilization activities were observed in most of the isolates tested. The data obtained in this study demonstrate that actinomycetes account for a higher percentage of the total population of culturable bacteria in soil than on cacao roots. Additionally, actinomycetes from the cacao rhizosphere are genetically diverse and have potential applications as agents of growth promotion.

The icy satellites of Jupiter and Saturn are perhaps the most promising places in the Solar System regarding habitability. However, the potential habitable environments are hidden underneath km-thick ice shells. The discovery of Enceladus’ plume by the Cassini mission has provided vital clues in our understanding of the processes occurring within the interior of exooceans. To interpret these data and to help configure instruments for future missions, controlled laboratory experiments and simulations are needed. This review aims to bring together studies and experimental designs from various scientific fields currently investigating the icy moons, including planetary sciences, chemistry, (micro-)biology, geology, glaciology, etc. This chapter provides an overview of successful in situ, in silico, and in vitro experiments, which explore different regions of interest on icy moons, i.e. a potential plume, surface, icy shell, water and brines, hydrothermal vents, and the rocky core.

Actinomycete bacteria have previously been reported from reproductive structures (infructescences) of Protea (sugarbush/suikerbos) species, a niche dominated by fungi in the genera Knoxdaviesia and Sporothrix. It is probable that these taxa have symbiotic interactions, but a lack of knowledge regarding their diversity and general ecology precludes their study. We determined the diversity of actinomycetes within Protea repens inflorescence buds, open inflorescences, young and mature infructescences, and leaf litter surrounding these trees. Since the P. repens habitat is fire-prone, we also considered the potential of these bacteria to recolonise infructescences after fire. Actinomycetes were largely absent from flower buds and inflorescences but were consistently present in young and mature infructescences. Two Streptomyces spp. were the most consistent taxa recovered, one of which was also routinely isolated from leaf litter. Lower colonisation rates were evident in samples from a recently burnt site. One of the most consistent taxa isolated from older trees in the unburnt site was absent from this site. Our findings show that P. repens has a distinct community of actinomycetes dominated by a few species. These communities change over time and infructescence developmental stage, season and the age of the host population. Mature infructescences appear to be important sources of inoculum for some of the actinomycetes, seemingly disrupted by fire. Increased fire frequency limiting maturation of P. repens infructescences could thus impact future actinomycete colonisation in the landscape. Streptomyces spp. are likely to share this niche with the ophiostomatoid fungi, which merits further study regarding their interactions and mode of transfer.

We investigated soil streptomycete communities associated with four host plant species (two warm season C4 grasses: Andropogon gerardii, Schizachyrium scoparium and two legumes: Lespedeza capitata, Lupinus perennis), grown in plant communities varying in species richness. We used actinobacteria-selective PCR coupled with pyrosequencing to characterize streptomycete community composition and structure. The greatest pairwise distances between communities were observed in contrasts between monocultures of different plant species, indicating that plant species exert distinct selective effects on soil streptomycete populations. Increasing plant richness altered the composition and structure of streptomycete communities associated with each host plant species. Significant relationships between plant community characteristics, soil edaphic characteristics, and streptomycete community structure suggest that host plant effects on soil microbial communities may be mediated through changes to the soil environment. Co-occurring streptomycete taxa also shared consistent relationships with soil edaphic properties, providing further indication of the importance of habitat preference for taxon occurrence. Physical distance between sampling points had a significant influence on streptomycete community similarity. This work provides a detailed characterization of soil streptomycete populations across a field scale and in relation to plant host identity and plant community richness.

Terrestrial analogue studies underpin almost all planetary missions and their use is essential in the exploration of our Solar system and in assessing the habitability of other worlds. Their value relies on the similarity of the analogue to its target, either in terms of their mineralogical or geochemical context, or current physical or chemical environmental conditions. Such analogue sites offer critical ground-truthing for astrobiological studies on the habitability of different environmental parameter sets, the biological mechanisms for survival in extreme environments and the preservation potential and detectability of biosignatures. The 33 analogue sites discussed in this review have been selected on the basis of their congruence to particular extraterrestrial locations. Terrestrial field sites that have been used most often in the literature, as well as some lesser known ones which require greater study, are incorporated to inform on the astrobiological potential of Venus, Mars, Europa, Enceladus and Titan. For example, the possibility of an aerial habitable zone on Venus has been hypothesized based on studies of life at high-altitudes in the terrestrial atmosphere. We also demonstrate why many different terrestrial analogue sites are required to satisfactorily assess the habitability of the changing environmental conditions throughout Martian history, and recommend particular sites for different epochs or potential niches. Finally, habitable zones within the aqueous environments of the icy moons of Europa and Enceladus and potentially in the hydrocarbon lakes of Titan are discussed and suitable analogue sites proposed. It is clear from this review that a number of terrestrial analogue sites can be applied to multiple planetary bodies, thereby increasing their value for astrobiological exploration. For each analogue site considered here, we summarize the pertinent physiochemical environmental features they offer and critically assess the fidelity with which they emulate their intended target locale. We also outline key issues associated with the existing documentation of analogue research and the constraints this has on the efficiency of discoveries in this field. This review thus highlights the need for a global open access database for planetary analogues.

Most archaea divide by binary fission using an FtsZ-based system similar to that of bacteria, but they lack many of the divisome components described in model bacterial organisms. Notably, among the multiple factors that tether FtsZ to the membrane during bacterial cell constriction, archaea only possess SepF-like homologs. Here, we combine structural, cellular, and evolutionary analyses to demonstrate that SepF is the FtsZ anchor in the human-associated archaeon Methanobrevibacter smithii. 3D super-resolution microscopy and quantitative analysis of immunolabeled cells show that SepF transiently co-localizes with FtsZ at the septum and possibly primes the future division plane. M. smithii SepF binds to membranes and to FtsZ, inducing filament bundling. High-resolution crystal structures of archaeal SepF alone and in complex with the FtsZ C-terminal domain (FtsZCTD) reveal that SepF forms a dimer with a homodimerization interface driving a binding mode that is different from that pre...

An enigmatic uncultured member of Firmicutes, Candidatus Desulforudis audaxviator (CDA), is known by its genome retrieved from the deep gold mine in South Africa, where it formed a single-species ecosystem fuelled by hydrogen from water radiolysis. It was believed that in situ conditions CDA relied on scarce energy supply and did not divide for hundreds to thousand years. We have isolated CDA strain BYF from a 2-km-deep aquifer in Western Siberia and obtained a laboratory culture growing with a doubling time of 28.5 h. BYF uses not only H 2 but also various organic electron donors for sulfate respiration. Growth required elemental iron, and ferrous iron did not substitute for it. A complex intracellular organization included gas vesicles, internal membranes, and electron-dense structures enriched in phosphorus, iron, and calcium. Genome comparison of BYF with the South African CDA revealed minimal differences mostly related to mobile elements and prophage insertions. Two genomes harbored <800 single-nucleotide polymorphisms and had nearly identical CRISPR loci. We suggest that spores with the gas vesicles may facilitate global distribution of CDA followed by colonization of suitable subsurface environments. Alternatively, a slow evolution rate in the deep subsurface could result in high genetic similarity of CDA populations at two sites spatially separated for hundreds of millions of years.

Thawing permafrost promotes microbial degradation of cryo-sequestered and new carbon leading to the biogenic production of methane, creating a positive feedback to climate change. Here we determine microbial community composition along a permafrost thaw gradient in northern Sweden. Partially thawed sites were frequently dominated by a single archaeal phylotype, Candidatus 'Methanoflorens stordalenmirensis' gen. nov. sp. nov., belonging to the uncultivated lineage 'Rice Cluster II' (Candidatus 'Methanoflorentaceae' fam. nov.). Metagenomic sequencing led to the recovery of its near-complete genome, revealing the genes necessary for hydrogenotrophic methanogenesis. These genes are highly expressed and methane carbon isotope data are consistent with hydrogenotrophic production of methane in the partially thawed site. In addition to permafrost wetlands, 'Methanoflorentaceae' are widespread in high methane-flux habitats suggesting that this lineage is both prevalent and a major contributor to global methane production. In thawing permafrost, Candidatus 'M. stordalenmirensis' appears to be a key mediator of methane-based positive feedback to climate warming.

Life can persist under severe osmotic stress and low water activity in hypersaline environments. On Mars, evidence for the past presence of saline bodies of water is prevalent and resulted in the widespread deposition of sulfate and chloride salts. Here we investigate Spotted Lake (British Columbia, Canada), a hypersaline lake with extreme (>3 M) levels of sulfate salts as an exemplar of the conditions thought to be associated with ancient Mars. We provide the first characterization of microbial structure in Spotted Lake sediments through metagenomic sequencing, and report a bacteria-dominated community with abundant Proteobacteria, Firmicutes, and Bacteroidetes, as well as diverse extremophiles. Microbial abundance and functional comparisons reveal similarities to Ace Lake, a meromictic Antarctic lake with anoxic and sulfidic bottom waters. Our analysis suggests that hypersaline-associated species occupy niches characterized foremost by differential abundance of Archaea, uncharacterized Bacteria, and Cyanobacteria. Potential biosignatures in this environment are discussed, specifically the likelihood of a strong sulfur isotopic fractionation record within the sediments due to the presence of sulfate reducing bacteria. With its high sulfate levels and seasonal freeze-thaw cycles, Spotted Lake is an analog for ancient paleolakes on Mars in which sulfate salt deposits may have offered periodically habitable environments, and could have concentrated and preserved organic materials or their biomarkers over geologic time.

Cryoturbation, the burial of topsoil material into deeper soil horizons by repeated freeze-thaw events, is an important storage mechanism for soil organic matter (SOM) in permafrost-affected soils. Besides abiotic conditions, microbial community structure and the accessibility of SOM to the decomposer community are hypothesized to control SOM decomposition and thus have a crucial role in SOM accumulation in buried soils. We surveyed the microbial community structure in cryoturbated soils from nine soil profiles in the northeastern Siberian tundra using high-throughput sequencing and quantification of bacterial, archaeal and fungal marker genes. We found that bacterial abundances in buried topsoils were as high as in unburied topsoils. In contrast, fungal abundances decreased with depth and were significantly lower in buried than in unburied topsoils resulting in remarkably low fungal to bacterial ratios in buried topsoils. Fungal community profiling revealed an associated decrease in presumably ectomycorrhizal (ECM) fungi. The abiotic conditions (low to subzero temperatures, anoxia) and the reduced abundance of fungi likely provide a niche for bacterial, facultative anaerobic decomposers of SOM such as members of the Actinobacteria, which were found in significantly higher relative abundances in buried than in unburied topsoils. Our study expands the knowledge on the microbial community structure in soils of Northern latitude permafrost regions, and attributes the delayed decomposition of SOM in buried soils to specific microbial taxa, and particularly to a decrease in abundance and activity of ECM fungi, and to the extent to which bacterial decomposers are able to act as their functional substitutes.

When exposed to air and adequate moisture, soils containing sulphides (sulphidic soils with pH > 4) become oxidized and generate sulphuric acid to form 'sulphuric soils' (pH < 4). Treatment of this acidity is undertaken by addition of lime. In this study, we investigated the effectiveness of adding plant organic matter, and simple carbon and nitrogen compounds, as alternatives to lime to sulphuric and sulphidic soils. In sulphuric soils under aerobic conditions, organic matter increased pH, the extent depending on the nitrogen content. Lucerne hay, which had the largest nitrogen content, increased the pH from 3.7 to 8.0, while pea straw and wheat straw effected smaller changes, in proportion to their respective nitrogen contents. Lucerne hay also caused the greatest reductions in soil redox potential and sulphate content, consistent with the action of sulphate-reducing bacteria. Similarly, incorporation of organic matter under aerobic conditions effectively prevented sulphidic soil acidification and reduced the redox potential and sulphate content. The individual effects of carbon and nitrogen compounds were then examined and compared to plant organic material. Glucose was ineffective at both small and large concentrations, while molasses increased the pH slightly to 4.6 and acetate to 5.9. None of these carbon compounds was as effective as complex organic matter. Nitrogen added alone as nitrate or ammonia had little or no effect on pH, whereas organic nitrogen in the form of urea caused the pH to rise to 6.3 and reduced the redox to less than 0 mV but had no significant effect on sulphate content.

Earth's subsurface is often isolated from phototrophic energy sources and characterized by chemotrophic modes of life. These environments are often oligotrophic and limited in electron donors or electron acceptors, and include continental crust, subseafloor oceanic crust, and marine sediment as well as subglacial lakes and the subsurface of polar desert soils. These low energy subsurface environments are therefore uniquely positioned for examining minimum energetic requirements and adaptations for chemotrophic life. Current targets for astrobiology investigations of extant life are planetary bodies with largely inhospitable surfaces, such as Mars, Europa, and Enceladus. Subsurface environments on Earth thus serve as analogs to explore possibilities of subsurface life on extraterrestrial bodies. The purpose of this review is to provide an overview of subsurface environments as potential analogs, and the features of microbial communities existing in these low energy environments, with particular emphasis on how they inform the study of energetic limits required for life. The thermodynamic energetic calculations presented here suggest that free energy yields of reactions and energy density of some metabolic redox reactions on Mars, Europa, Enceladus, and Titan could be comparable to analog environments in Earth's low energy subsurface habitats.

Viable microorganisms were found in Miocene lacustrine clays of the cypris formation excavated from 200-m below the surface as spoil during open-cast brown coal mining (Sokolov Brown Coal Basin, North-Western Bohemia, Czech Republic). Both saprotrophic microfungi of the genera Penicillium, Verticillium, Cladosporium and Aspergillus as well as heterotrophic bacteria were isolated from an intact sediment cores. Heterotrophic bacteria were classified by the MIS Sherlock System as representatives of genera Nocardiopsis, Arthrobacter, Micrococcus, Kocuria, Rothia, Clavibacter, Bacillus, Paenibacillus, Brevibacillus, Microbacterium, Acinetobacter and Pseudomonas. A bacterium found among the strains had an atypical fatty acids profile enriched by branched fatty acids and polyunsaturated fatty acid (18:3x6) and gave no MIS Sherlock match. Phospholipid fatty acids analysis indicates a relatively high (535 pmol g )1 ) but inhomogeneously distributed viable microbial biomass. Fatty acids analyses of non-fractioned lipids (representing viable, storage and dead biomass; 8390 pmol g )1 ) detected rich and homogenous profiles with fungal, bacterial and actinomycetal markers but no protozoan and algal fatty acids markers.

Subsurface microbial life contributes significantly to biogeochemical cycling, yet it remains largely uncharacterized, especially its archaeal members. This ’microbial dark matter’ has been explored by recent studies that were, however, mostly based on DNA sequence information only. Here, we use diverse techniques including ultrastuctural analyses to link genomics to biology for the SM1 Euryarchaeon lineage, an uncultivated group of subsurface archaea. Phylogenomic analyses reveal this lineage to belong to a widespread group of archaea that we propose to classify as a new euryarchaeal order (‘Candidatus Altiarchaeales’). The representative, double-membraned species ‘Candidatus Altiarchaeum hamiconexum’ has an autotrophic metabolism that uses a not-yet-reported Factor420-free reductive acetyl-CoA pathway, confirmed by stable carbon isotopic measurements of archaeal lipids. Our results indicate that this lineage has evolved specific metabolic and structural features like nano- grappling hooks empowering this widely distributed archaeon to predominate anaerobic groundwater, where it may represent an important carbon dioxide sink.

The karst aquifer systems in southern Kentucky can be dynamic and quick to change. Microorganisms that live in these unpredictable aquifers are constantly faced with environmental changes. Their survival depends upon adaptations to changes in
water chemistry, taking advantage of positive stimuli and avoiding negative environmental conditions. The U.S. Geological Survey conducted a study in 2001 to determine the capability of bacteria to adapt in two distinct regions of water quality in a karst aquifer, an area of clean, oxygenated groundwater and an area where the
groundwater was oxygen depleted and contaminated by jet fuel. Water samples containing bacteria were collected from one clean well and two jet fuel contaminated wells in a conduit-dominated karst aquifer. Bacterial concentrations, enumerated through direct count, ranged from 500,000 to 2.7 million bacteria per mL in the clean portion of the aquifer, and 200,000 to 3.2 million bacteria per mL in the contaminated portion of the aquifer over a twelve month period. Bacteria from the clean well ranged in size from 0.2 to 2.5 mm, whereas bacteria from one fuel-contaminated well were generally larger, ranging in size from 0.2 to 3.9 mm. Also, bacteria collected from the clean well had a higher density and, consequently, were more inclined to sink than bacteria collected
from contaminated wells. Bacteria collected from the clean portion of the karst aquifer were predominantly (,95%) Gram-negative and more likely to have flagella present than bacteria collected from the contaminated wells, which included a substantial fraction
(,30%) of Gram-positive varieties. The ability of the bacteria from the clean portion of the karst aquifer to biodegrade benzene and toluene was studied under aerobic and anaerobic conditions in laboratory microcosms. The rate of fuel biodegradation in
laboratory studies was approximately 50 times faster under aerobic conditions as compared to anaerobic, sulfur-reducing conditions. The optimum pH for fuel biodegradation ranged from 6 to 7. These findings suggest that bacteria have adapted
to water-saturated karst systems with a variety of active and passive transport mechanisms.

Acid sulphate (AS) soils along the Baltic coasts contain significantamounts of organic carbon and nitrogen in their
subsoils. The abundance, composition, and activity ofmicrobial communities throughout the AS soil profilewere
analysed. The data from a drained AS soil were compared with those from a drained non-AS soil and a pristine
wetland soil from the same region. Moreover, the potential production of methane, carbon dioxide, and nitrous
oxide from the soils was determined under laboratory conditions. Direct microscopic counting, glucose-induced
respiration (GIR), whole cell hybridisation, and extended phospholipid fatty acid (PLFA) analysis confirmed the
presence of abundant microbial communities in the topsoil and also in the deepest Cg2 horizon of the AS soil. The
patterns of microbial counts, biomass and activity in the profile of the AS soil and partly also in the non-AS soil
therefore differed fromthe general tendency of gradual decreases in soil profiles. High respiration in the deepest
Cg2 horizon of the AS soil (5.66 μg C g−1 h−1, as comparedto 2.71 μg C g−1 h−1 in a top Ap horizon) is unusual
but reasonable given the large amount of organic carbon in this horizon. Nitrous oxide production peaked in the
BCgc horizon of the AS and in the BC horizon of the non-AS soil, but the peak value was ten-fold higher in the AS
soil than in the non-AS soil (82.3 vs. 8.6 ng N g−1d−1). The data suggest that boreal AS soils on the Baltic coast
contain high microbial abundance and activity. This, together with the abundant carbon and total and mineral
nitrogen in the deep layers of AS soils, may result in substantial gas production. Consequently, high GHG emissions
could occur, for example, when the generally high water table is lowered because of arable farming.

The geneticdiversityofstreptomycetesincollieryspoilheaps(Sokolov,Czech
Republic)wasinvestigatedbyrestrictionpatternanalysisof16S-internaltranscribed
spacer rDNAand16Ssequences.WesampledfreshlyexcavatedMiocenesediment
(17–19-million-year-old) andfoursitesofprimarysuccession(initial,early,middle,
and latestages;aged1–44years)onthesamesediment.Activebacteriawere
present eveninfreshMiocenesediment,andtherelativeproportionof
actinomycetes amongtotalbacterialandtheirgeneticdiversityincreased
significantly withtheageofthesamplingsite.Thereplacementofpioneerspecies
by latesuccessionspeciesduringsuccessionwasobserved.Plateassaysof
Streptomyces strains revealed27%antibiotic-producingstrains.Screeningfor
nonribosomal peptidesynthasesandtypeIpolyketidesynthasessystemssuggested
that 90%and55%streptomycetes,respectively,areputativeproducersof
biologically activecompounds.Thefrequenciesoftetracycline-,amoxicillin-,and
chloramphenicol-resistant streptomyceteswere6%,9%,and15%,respectively.These
findings documenttheoccurrenceofgeneticelementsencodingantibioticresistance
genes andtheproductionofantibioticsbystreptomyceteslocatedinpristine
environments. Ourresultsindicatekeyrolesforancientstreptomycetesrelatedto
S. microflavus, S. spororaveus, and S. flavofuscus in pioneeringcommunity
development infreshlyexcavatedsubstrates.

Viable microorganisms were found in Miocene lacustrine clays of the cypris formation excavated from
200-m below the surface as spoil during open-cast brown coal mining (Sokolov Brown Coal Basin, North-
Western Bohemia, Czech Republic). Both saprotrophic microfungi of the genera Penicillium, Verticillium,
Cladosporium and Aspergillus as well as heterotrophic bacteria were isolated from an intact sediment cores.
Heterotrophic bacteria were classified by the MIS Sherlock System as representatives of genera Nocardiopsis,
Arthrobacter, Micrococcus, Kocuria, Rothia, Clavibacter, Bacillus, Paenibacillus, Brevibacillus,
Microbacterium, Acinetobacter and Pseudomonas. A bacterium found among the strains had an atypical
fatty acids profile enriched by branched fatty acids and polyunsaturated fatty acid (18:3x6) and gave no
MIS Sherlock match. Phospholipid fatty acids analysis indicates a relatively high (535 pmol g)1) but
inhomogeneously distributed viable microbial biomass. Fatty acids analyses of non-fractioned lipids
(representing viable, storage and dead biomass; 8390 pmol g)1) detected rich and homogenous profiles with
fungal, bacterial and actinomycetal markers but no protozoan and algal fatty acids markers.

Using our example of Earth life as a guide, can we constrain Mars life? Life on Earth has chemotrophic origins, and phototrophy developed on a chemotrophic template. Subsurface ecosystems thrive without photosynthetic input. Plausibly, life emerges within the geochemistry of planetary interiors. Water, heat, chemical disequilibria, energy supplies, and nutrient fluxes are all present below planetary surfaces, and may still be present on Mars. Igneous processes release volatiles as melts drive volcanism. Quenching of volcanic gases establishes disequilibria during cooling. Hydrothermal circulation is stoked from below by volcanic input and from above by atmospheric exchange. Volcanically-derived energy sources permit reductive metabolisms: methanogenesis from CO 2 or CO, NH 3 from N 2 , H 2 S from SO 2 , and O 2 reduction to H 2 O. Hydrothermal extensions allow the addition of ferric iron reduction, sulfide oxidation, ferrous iron oxidation, methane oxidation, and sulfate reduction depending on temperature changes and mixing dynamics. Meanwhile, thermodynamic drives favor simultaneous organic synthesis from carbon oxides and H 2 . Ranking potential energy supplies for autotrophy or heterotrophy is accomplished through theoretical models using petrologic constraints on volcanic gases, martian meteorite compositions, extents of hydrothermal reaction progress, and efficiency of atmospheric entrainment into groundwater. Results for subsurface habitats are recognizably Earth-like, with subtleties unique to the atmosphere and mantle of Mars. This framework needs tuning and tightening against firmer constraints, together with tests of emergent hypotheses. Ultimately, as the present is the key to the past, unlocking the possible biogeochemistries of Mars demands exploring the subsurface biosphere on Earth.

Using our example of Earth life as a guide, can we constrain Mars life? Life on Earth has chemotrophic origins, and phototrophy developed on a chemotrophic template. Subsurface ecosystems thrive without photosynthetic input. Plausibly, life emerges within the geochemistry of planetary interiors. Water, heat, chemical disequilibria, energy supplies, and nutrient fluxes are all present below planetary surfaces, and may still be present on Mars. Igneous processes release volatiles as melts drive volcanism. Quenching of volcanic gases establishes disequilibria during cooling. Hydrothermal circulation is stoked from below by volcanic input and from above by atmospheric exchange. Volcanically-derived energy sources permit reductive metabolisms: methanogenesis from CO 2 or CO, NH 3 from N 2 , H 2 S from SO 2 , and O 2 reduction to H 2 O. Hydrothermal extensions allow the addition of ferric iron reduction, sulfide oxidation, ferrous iron oxidation, methane oxidation, and sulfate reduction depending on temperature changes and mixing dynamics. Meanwhile, thermodynamic drives favor simultaneous organic synthesis from carbon oxides and H 2 . Ranking potential energy supplies for autotrophy or heterotrophy is accomplished through theoretical models using petrologic constraints on volcanic gases, martian meteorite compositions, extents of hydrothermal reaction progress, and efficiency of atmospheric entrainment into groundwater. Results for subsurface habitats are recognizably Earth-like, with subtleties unique to the atmosphere and mantle of Mars. This framework needs tuning and tightening against firmer constraints, together with tests of emergent hypotheses. Ultimately, as the present is the key to the past, unlocking the possible biogeochemistries of Mars demands exploring the subsurface biosphere on Earth.