Microbes on the edge of global biosphere]

The search for life on the edge of global biosphere is a frontier to bridge conventional bio/ecology and exo/astrobiology. This communication reviews the foci of microbiological studies on the inhabitants of the selected "edges", i.e., deep-sea, deep subsurface and Antarctic habitats. The deep-sea is characterized as the no-light (non-photosynthetic) habitat, and the primary production is mostly due to the chemosynthetic autotrophy at the hydrothermal vents and methane-rich seeps. Formation of the chemosynthesis-dependent animal communities in the deep leads to the idea that such communities may be found in "ocean" of the Jovian satellite, Europa. The oxygen minimal layer (OML) in mid-water provides another field of deep-sea research. Modern OML is a relatively thin layer, found between the water depth of 200 and 1000 m, but was much thicker during the periods of oceanic anoxia events (OAEs) in the past. The history of oceanic biosphere is regarded as the cycle of OAE and non-OAE periods, and the remnants of the past OAEs may be seen in the modem OML. Anoxic (no-O2) condition is also characteristic of deep subsurface biosphere. Microorganisms in deep subsurface biosphere exploit every available oxidant, or terminal electron acceptor (TEA), for anaerobic respiration. Sulfate, nitrate, iron (III) and CO2 are the representative TEAs in the deep subsurface. Subsurface of hydrothermal vents, or sub-vent biosphere, may house brine (high salt) habitats and halophilic microorganisms. Some sub-vent halophiles were phylogenetically closely similar to the ones found in the Antarctic habitats which are extremely dry by the liophilizing climate. Below the 3000-4000 m-thick glacier on Antarctica, there have been >70 lakes with liquid water located. One of such sub-glacial lakes, Lake Vostok, has been a target of "life in extreme environments" and is about to be drill-penetrated for microbiological studies. These 'microbiological platforms' will provide new knowledge about the diversity and potential of the Earth's life and facilitate the capability of astrobiologial exploration.     

Microbial diversity of deep-sea extremophiles--Piezophiles, Hyperthermophiles, and subsurface microorganisms]

Knowledge of our Planet's biosphere has increased tremendously during the last 10 to 20 years. In the field of Microbiology in particular, scientists have discovered novel "extremophiles", microorganisms capable of living in extreme environments such as highly acidic or alkaline conditions, at high salt concentration, with no oxygen, extreme temperatures (as low as -20 degrees C and as high as 300 degrees C), at high concentrations of heavy metals and in high pressure environments such as the deep-sea. It is apparent that microorganisms can exist in any extreme environment of the Earth, yet already scientists have started to look for life on other planets; the so-called "Exobiology" project. But as yet we have little knowledge of the deep-sea and subsurface biosphere of our own planet. We believe that we should elucidate the Biodiversity of Earth more thoroughly before exploring life on other planets, and these attempts would provide deeper insight into clarifying the existence of extraterrestrial life. We focused on two deep-sea extremophiles in this article; one is "Piezophiles", and another is "Hyperthermophiles". Piezophiles are typical microorganisms adapted to high-pressure and cold temperature environments, and located in deep-sea bottom. Otherwise, hyperthermophiles are living in high temperature environment, and located at around the hydrothermal vent systems in deep-sea. They are not typical deep-sea microorganisms, but they can grow well at high-pressure condition, just like piezophiles. Deming and Baross mentioned that most of the hyperthermophilic archaea isolated from deep-sea hydrothermal vents are able to grow under conditions of high temperature and pressure, and in most cases their optimal pressure for growth was greater than the environmental pressure they were isolated from. It is possible that originally their native environment may have been deeper than the sea floor and that there had to be a deeper biosphere. This implication suggests that the deep-sea hydrothermal vents are the windows to a deep subsurface biosphere. A vast array of chemoautotrophic deep-sea animal communities have been found to exist in cold seep environments, and most of these animals are common with those found in hydrothermal vent environments. Thus, it is possible to consider that the cold seeps are also one of slit windows to a deep subsurface biosphere. We conclude that the deep-sea extremophiles are very closely related into the unseen majority in subsurface biosphere, and the subsurface biosphere probably concerns to consider the "exobiology".     

Feast and famine--microbial life in the deep-sea bed

The seabed is a diverse environment that ranges from the desert-like deep seafloor to the rich oases that are present at seeps, vents, and food falls such as whales, wood or kelp. As well as the sedimentation of organic material from above, geological processes transport chemical energy--hydrogen, methane, hydrogen sulphide and iron--to the seafloor from the subsurface below, which provides a significant proportion of the deep-sea energy. At the sites on the seafloor where chemical energy is delivered, rich and diverse microbial communities thrive. However, most subsurface microorganisms live in conditions of extreme energy limitation, with mean generation times of up to thousands of years. Even in the most remote subsurface habitats, temperature rather than energy seems to set the ultimate limit for life, and in the deep biosphere, where energy is most depleted, life might even be based on the cleavage of water by natural radioisotopes. Here, we review microbial biodiversity and function in these intriguing environments.     

Genomic markers of ancient anaerobic microbial pathways: sulfate reduction,methanogenesis, and methane oxidation

Genomic markers for anaerobic microbial processes in marine sediments-sulfate reduction, methanogenesis, and anaerobic methane oxidation-reveal the structure of sulfate-reducing, methanogenic, and methane-oxidizing microbial communities (including uncultured members); they allow inferences about the evolution of these ancient microbial pathways; and they open genomic windows into extreme microbial habitats, such as deep subsurface sediments and hydrothermal vents, that are analogs for the early Earth and for extraterrestrial microbiota.     

High-temperature deep-subsurface microbial communities as a possible equivalent of ancient ecosystems

Microbiological, molecular biological, and radioisotopic studies suggest that active and complex microbial communities exist in the deep layers of the subsurface biosphere. This review discusses only one group of such communities, i.e., those developing at high (above 60°C temperatures). Oil wells, subsurface water reservoirs (e.g., the Great Artesian Basin in Australia), deep mines (in South Africa), and high-temperature horizons below the seafloor in the areas of underwater volcanic activity contain the best-studied high-temperature subsurface ecosystems. These microbial communities differ considerably from one another in biodiversity, initial energy substrate, and major microbiological processes. However, before they can be considered as equivalents of the Earth’s primordial ecosystems, it is necessary to demonstrate that they are energetically independent of the modern biosphere.     

Characterization of Bacterial Communities in Deep-Sea Hydrothermal Vents from Three Oceanic Regions

Deep-sea hydrothermal vents are considered to be one of the most spectacular ecosystems on Earth. Microorganisms form the basis of the food chain in vents controlling the vent communities. However, the diversity of bacterial communities in deep-sea hydrothermal vents from different oceans remains largely unknown. In this study, the pyrosequencing of 16S rRNA gene was used to characterize the bacterial communities of the venting sulfide, seawater, and tubeworm trophosome from East Pacific Rise, South Atlantic Ridge, and Southwest Indian Ridge, respectively. A total of 23,767 operational taxonomic units (OTUs) were assigned into 42 different phyla. Although Proteobacteria, Actinobacteria, and Bacteroidetes were the predominant phyla in all vents, differences of bacterial diversity were observed among different vents from three oceanic regions. The sulfides of East Pacific Rise possessed the most diverse bacterial communities. The bacterial diversities of venting seawater were much lower than those of vent sulfides. The symbiotic bacteria of tubeworm Ridgeia piscesae were included in the bacterial community of vent sulfides, suggesting their significant ecological functions as the primary producers in the deep-sea hydrothermal vent ecosystems. Therefore, our study presented a comprehensive view of bacterial communities in deep-sea hydrothermal vents from different oceans.     

Bacterial Communities Associated with Subsurface Geochemical Processes in Continental Serpentinite Springs

Reactions associated with the geochemical process of serpentinization can generate copious quantities of hydrogen and low-molecular-weight organic carbon compounds, which may provide energy and nutrients to sustain subsurface microbial communities independently of the photosynthetically supported surface biosphere. Previous microbial ecology studies have tested this hypothesis in deep sea hydrothermal vents, such as the Lost City hydrothermal field. This study applied similar methods, including molecular fingerprinting and tag sequencing of the 16S rRNA gene, to ultrabasic continental springs emanating from serpentinizing ultramafic rocks. These molecular surveys were linked with geochemical measurements of the fluids in an interdisciplinary approach designed to distinguish potential subsurface organisms from those derived from surface habitats. The betaproteobacterial genus Hydrogenophaga was identified as a likely inhabitant of transition zones where hydrogen-enriched subsurface fluids mix with oxygenated surface water. The Firmicutes genus Erysipelothrix was most strongly correlated with geochemical factors indicative of subsurface fluids and was identified as the most likely inhabitant of a serpentinization-powered subsurface biosphere. Both of these taxa have been identified in multiple hydrogen-enriched subsurface habitats worldwide, and the results of this study contribute to an emerging biogeographic pattern in which Betaproteobacteria occur in near-surface mixing zones and Firmicutes are present in deeper, anoxic subsurface habitats.     

Antarctic marine biodiversity and deep-sea hydrothermal vents

The diversity of many marine benthic groups is unlike that of most other taxa. Rather than declining from the tropics to the poles, much of the benthos shows high diversity in the Southern Ocean. Moreover, many species are unique to the Antarctic region. Recent work has shown that this is also true of the communities of Antarctic deep-sea hydrothermal vents. Vent ecosystems have been documented from many sites across the globe, associated with the thermally and chemically variable habitats found around these, typically high temperature, streams that are rich in reduced compounds and polymetallic sulphides. The animal communities of the East Scotia Ridge vent ecosystems are very different to those elsewhere, though the microbiota, which form the basis of vent food webs, show less differentiation. Much of the biological significance of deep-sea hydrothermal vents lies in their biodiversity, the diverse biochemistry of their bacteria, the remarkable symbioses among many of the marine animals and these bacteria, and the prospects that investigations of these systems hold for understanding the conditions that may have led to the first appearance of life. The discovery of diverse and unusual Antarctic hydrothermal vent ecosystems provides opportunities for new understanding in these fields. Moreover, the Antarctic vents south of 60°S benefit from automatic conservation under the Convention on the Conservation of Antarctic Marine Living Resources and the Antarctic Treaty. Other deep-sea hydrothermal vents located in international waters are not protected and may be threatened by growing interests in deep-sea mining.     

How Deep-Sea Wood Falls Sustain Chemosynthetic Life

Large organic food falls to the deep sea – such as whale carcasses and wood logs – are known to serve as stepping stones for the dispersal of highly adapted chemosynthetic organisms inhabiting hot vents and cold seeps. Here we investigated the biogeochemical and microbiological processes leading to the development of sulfidic niches by deploying wood colonization experiments at a depth of 1690 m in the Eastern Mediterranean for one year. Wood-boring bivalves of the genus Xylophaga played a key role in the degradation of the wood logs, facilitating the development of anoxic zones and anaerobic microbial processes such as sulfate reduction. Fauna and bacteria associated with the wood included types reported from other deep-sea habitats including chemosynthetic ecosystems, confirming the potential role of large organic food falls as biodiversity hot spots and stepping stones for vent and seep communities. Specific bacterial communities developed on and around the wood falls within one year and were distinct from freshly submerged wood and background sediments. These included sulfate-reducing and cellulolytic bacterial taxa, which are likely to play an important role in the utilization of wood by chemosynthetic life and other deep-sea animals.     

Thermophilic Microbial Communities of Deep-Sea Hydrothermal Vents

The most recent publications on the phylogenetic and functional diversity of thermophilic prokaryotes inhabiting thermal deep-sea environments are reviewed. Along with a general physicochemical characterization of the biotope studied, certain adaptation mechanisms are discussed that are peculiar to the microorganisms inhabiting it. A separate chapter addresses the phylogenetic analysis of deep-sea hydrothermal microbial communities and uncultivated microorganisms recently discovered therein using molecular biological techniques. Physiological groups of thermophilic microorganisms found in deep-sea hydrothermal vents are considered: methanogens, sulfate-, iron-, and sulfur-reducers, aerobic hydrogen-oxidizing prokaryotes, aerobic and anaerobic organotrophs. In most cases, the isolates represent novel taxons.     

Abundance of reverse tricarboxylic acid cycle genes in free-living microorganisms at deep-sea hydrothermal vents

Since the discovery of hydrothermal vents more than 25 years ago, the Calvin-Bassham-Benson (Calvin) cycle has been considered the principal carbon fixation pathway in this microbe-based ecosystem. However, on the basis of recent molecular data of cultured free-living and noncultured episymbiotic members of the epsilon subdivision of Proteobacteria and earlier carbon isotope data of primary consumers, an alternative autotrophic pathway may predominate. Here, genetic and culture-based approaches demonstrated the abundance of reverse tricarboxylic acid cycle genes compared to the abundance of Calvin cycle genes in microbial communities from two geographically distinct deep-sea hydrothermal vents. PCR with degenerate primers for three key genes in the reverse tricarboxylic acid cycle and form I and form II of ribulose 1,5-bisphosphate carboxylase/oxygenase (Calvin cycle marker gene) were utilized to demonstrate the abundance of the reverse tricarboxylic acid cycle genes in diverse vent samples. These genes were also expressed in at least one chimney sample. Diversity, similarity matrix, and phylogenetic analyses of cloned samples and amplified gene products from autotrophic enrichment cultures suggest that the majority of autotrophs that utilize the reverse tricarboxylic acid cycle are members of the epsilon subdivision of Proteobacteria. These results parallel the results of previously published molecular surveys of 16S rRNA genes, demonstrating the dominance of members of the epsilon subdivision of Proteobacteria in free-living hydrothermal vent communities. Members of the epsilon subdivision of Proteobacteria are also ubiquitous in many other microaerophilic to anaerobic sulfidic environments, such as the deep subsurface. Therefore, the reverse tricarboxylic acid cycle may be a major autotrophic pathway in these environments and significantly contribute to global autotrophic processes.     

Sub-seafloor biogeochemical processes and microbial life in the Baltic Sea

The post-glacial Baltic Sea has experienced extreme changes that are archived today in the deep sediments. IODP Expedition 347 retrieved cores down to 100 m depth and studied the climate history and the deep biosphere. We here review the biogeochemical and microbiological highlights and integrate these with other studies from the Baltic seabed. Cell numbers, endospore abundance and organic matter mineralization rates are extremely high. A 100-fold drop in cell numbers with depth results from a small difference between growth and mortality in the ageing sediment. Evidence for growth derives from a D:L amino acid racemization model, while evidence for mortality derives from the abundance and potential activity of lytic viruses. The deep communities assemble at the bottom of the bioturbated zone from the founding surface community by selection of organisms suited for life under deep sediment conditions. The mean catabolic per-cell rate of microorganisms drops steeply with depth to a life in slow-motion, typical for the deep biosphere. The subsurface life under extreme energy limitation is facilitated by exploitation of recalcitrant substrates, by biochemical protection of nucleic acids and proteins and by repair mechanisms for random mismatches in DNA or damaged amino acids in proteins.     

Advances in Taxonomy,Ecology, and Biogeography of Dirivultidae (Copepoda) Associated with Chemosynthetic Environments in the Deep Sea

Background

Copepoda is one of the most prominent higher taxa with almost 80 described species at deep-sea hydrothermal vents. The unique copepod family Dirivultidae with currently 50 described species is the most species rich invertebrate family at hydrothermal vents.

Methodology/Principal Findings

We reviewed the literature of Dirivultidae and provide a complete key to species, and map geographical and habitat specific distribution. In addition we discuss the ecology and origin of this family.

Conclusions/Significance

Dirivultidae are only present at deep-sea hydrothermal vents and along the axial summit trough of midocean ridges, with the exception of Dirivultus dentaneus found associated with Lamellibrachia species at 1125 m depth off southern California. To our current knowledge Dirivultidae are unknown from shallow-water vents, seeps, whale falls, and wood falls. They are a prominent part of all communities at vents and in certain habitat types (like sulfide chimneys colonized by pompei worms) they are the most abundant animals. They are free-living on hard substrate, mostly found in aggregations of various foundation species (e.g. alvinellids, vestimentiferans, and bivalves). Most dirivultid species colonize more than one habitat type. Dirivultids have a world-wide distribution, but most genera and species are endemic to a single biogeographic region. Their origin is unclear yet, but immigration from other deep-sea chemosynthetic habitats (stepping stone hypothesis) or from the deep-sea sediments seems unlikely, since Dirivultidae are unknown from these environments. Dirivultidae is the most species rich family and thus can be considered the most successful taxon at deep-sea vents.     

Archaeal diversity and community development in deep-sea hydrothermal vents

Over the past 35 years, researchers have explored deep-sea hydrothermal vent environments around the globe and studied a number of archaea, their unique metabolic and physiological properties, and their vast phylogenetic diversity. Although the pace of discovery of new archaeal taxa, phylotypes and phenotypes in deep-sea hydrothermal vents has slowed recently, bioinformatics and interdisciplinary geochemistry-microbiology approaches are providing new information on the diversity and community composition of archaea living in deep-sea vents. Recent investigations have revealed that archaea could have originated and dispersed from ancestral communities endemic to hydrothermal vents into other biomes on Earth, and the community structure and productivity of chemolithotrophic archaea are controlled primarily by variations in the geochemical composition of hydrothermal fluids.     

Metagenomic investigation of the geologically unique Hellenic Volcanic Arc reveals a distinctive ecosystem with unexpected physiology

Hydrothermal vents represent a deep, hot, aphotic biosphere where chemosynthetic primary producers, fuelled by chemicals from Earth's subsurface, form the basis of life. In this study, we examined microbial mats from two distinct volcanic sites within the Hellenic Volcanic Arc (HVA). The HVA is geologically and ecologically unique, with reported emissions of CO₂‐saturated fluids at temperatures up to 220°C and a notable absence of macrofauna. Metagenomic data reveals highly complex prokaryotic communities composed of chemolithoautotrophs, some methanotrophs, and to our surprise, heterotrophs capable of anaerobic degradation of aromatic hydrocarbons. Our data suggest that aromatic hydrocarbons may indeed be a significant source of carbon in these sites, and instigate additional research into the nature and origin of these compounds in the HVA. Novel physiology was assigned to several uncultured prokaryotic lineages; most notably, a SAR406 representative is attributed with a role in anaerobic hydrocarbon degradation. This dataset, the largest to date from submarine volcanic ecosystems, constitutes a significant resource of novel genes and pathways with potential biotechnological applications.     

Abundance of Reverse Tricarboxylic Acid Cycle Genes in Free-Living Microorganisms at Deep-Sea Hydrothermal Vents

Since the discovery of hydrothermal vents more than 25 years ago, the Calvin-Bassham-Benson (Calvin) cycle has been considered the principal carbon fixation pathway in this microbe-based ecosystem. However, on the basis of recent molecular data of cultured free-living and noncultured episymbiotic members of the epsilon subdivision of Proteobacteria and earlier carbon isotope data of primary consumers, an alternative autotrophic pathway may predominate. Here, genetic and culture-based approaches demonstrated the abundance of reverse tricarboxylic acid cycle genes compared to the abundance of Calvin cycle genes in microbial communities from two geographically distinct deep-sea hydrothermal vents. PCR with degenerate primers for three key genes in the reverse tricarboxylic acid cycle and form I and form II of ribulose 1,5-bisphosphate carboxylase/oxygenase (Calvin cycle marker gene) were utilized to demonstrate the abundance of the reverse tricarboxylic acid cycle genes in diverse vent samples. These genes were also expressed in at least one chimney sample. Diversity, similarity matrix, and phylogenetic analyses of cloned samples and amplified gene products from autotrophic enrichment cultures suggest that the majority of autotrophs that utilize the reverse tricarboxylic acid cycle are members of the epsilon subdivision of Proteobacteria. These results parallel the results of previously published molecular surveys of 16S rRNA genes, demonstrating the dominance of members of the epsilon subdivision of Proteobacteria in free-living hydrothermal vent communities. Members of the epsilon subdivision of Proteobacteria are also ubiquitous in many other microaerophilic to anaerobic sulfidic environments, such as the deep subsurface. Therefore, the reverse tricarboxylic acid cycle may be a major autotrophic pathway in these environments and significantly contribute to global autotrophic processes.     

Genetic diversity of archaea in deep-sea hydrothermal vent environments.

Molecular phylogenetic analysis of naturally occurring archaeal communities in deep-sea hydrothermal vent environments was carried out by PCR-mediated small subunit rRNA gene (SSU rDNA) sequencing. As determined through partial sequencing of rDNA clones amplified with archaea-specific primers, the archaeal populations in deep-sea hydrothermal vent environments showed a great genetic diversity, and most members of these populations appeared to be uncultivated and unidentified organisms. In the phylogenetic analysis, a number of rDNA sequences obtained from deep-sea hydrothermal vents were placed in deep lineages of the crenarchaeotic phylum prior to the divergence of cultivated thermophilic members of the crenarchaeota or between thermophilic members of the euryarchaeota and members of the methanogen-halophile clade. Whole cell in situ hybridization analysis suggested that some microorganisms of novel phylotypes predicted by molecular phylogenetic analysis were likely present in deep-sea hydrothermal vent environments. These findings expand our view of the genetic diversity of archaea in deep-sea hydrothermal vent environments and of the phylogenetic organization of archaea.     

Microbial Communities of Deep Marine Subsurface Sediments: Molecular and Cultivation Surveys

Recent molecular analyses show that microbial communities of deep marine sediments harbor members of distinct, uncultured bacterial and archaeal lineages, in addition to Gram-positive bacteria and Proteobacteria that are detected by cultivation surveys. Several of these subsurface lineages show cosmopolitan occurrence patterns; they can be found in cold marine sediments and also in hydrothermal habitats, suggesting a continuous deep subsurface and hydrothermal biosphere with shared microbiota. The physiologies and activities of these uncultured subsurface lineages remain to be explored by innovative combinations of genomic and biogeochemical approaches.     

Geochemical and microbiological evidence for a hydrogen-based,hyperthermophilic subsurface lithoautotrophic microbial ecosystem (HyperSLiME) beneath an active deep-sea hydrothermal field

Subsurface microbial communities supported by geologically and abiologically derived hydrogen and carbon dioxide from the Earths interior are of great interest, not only with regard to the nature of primitive life on Earth, but as potential analogs for extraterrestrial life. Here, for the first time, we present geochemical and microbiological evidence pointing to the existence of hyperthermophilic subsurface lithoautotrophic microbial ecosystem (HyperSLiME) dominated by hyperthermophilic methanogens beneath an active deep-sea hydrothermal field in the Central Indian Ridge. Geochemical and isotopic analyses of gaseous components in the hydrothermal fluids revealed heterogeneity of both concentration and carbon isotopic compositions of methane between the main hydrothermal vent (0.08 mM and –13.8 PDB, respectively) and the adjacent divergent vent site (0.2 mM and –18.5 PDB, respectively), representing potential subsurface microbial methanogenesis, at least in the divergent vent emitting more ¹³C-depleted methane. Extremely high abundance of magmatic energy sources such as hydrogen (2.5 mM) in the fluids also encourages a hydrogen-based, lithoautotrophic microbial activity. Both cultivation and cultivation-independent molecular analyses suggested the predominance of Methanococcales members in the superheated hydrothermal emissions and chimney interiors along with the other major microbial components of Thermococcales members. These results imply that a HyperSLiME, consisting of methanogens and fermenters, occurs in this tectonically active subsurface zone, strongly supporting the existence of hydrogen-driven subsurface microbial communities.     

Geomicrobiology in oceanography: microbe-mineral interactions at and below the seafloor

Oceanography is inherently interdisciplinary and, since its inception, has included the study of microbe-mineral interactions. From early studies of manganese nodules, to the discovery of hydrothermal vents, it has been recognized that microorganisms are involved at various levels in the transformation of rocks and minerals at and below the seafloor. Recent studies include mineral weathering at low temperatures and microbe-mineral interactions in the subseafloor "deep biosphere". A common characteristic of seafloor and subseafloor geomicrobiological processes that distinguishes them from terrestrial or near-surface processes is that they occur in the dark, one or more steps removed from the sunlight that fuels the near-surface biosphere on Earth. This review focuses on geomicrobiological studies and energy flow in dark, deep-ocean and subseafloor rock habitats.