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In microbiology, cultivation is a central approach for uncovering novel physiology, ecology, and evolution of microorganisms, but conventional methods have left many microorganisms found in nature uncultured. To overcome the limitations of traditional methods and culture indigenous microorganisms, we applied a two-stage approach: enrichment/activation of indigenous organisms by using a continuous-flow down-flow hanging sponge bioreactor and subsequent selective batch cultivation. Here, we provide a protocol for this bioreactor-mediated technique using activation of deep marine sediment microorganisms and downstream isolation of a syntrophic co-culture containing an archaeon closely related to the eukaryote ancestor (Candidatus Promethearchaeum syntrophicum strain MK-D1) as an example. Both stages can easily be tailored to target other environments and organisms by modifying the inoculum, feed solution/gases, attachment material and/or cultivation media. We anaerobically incubate polyurethane sponges inoculated with deep-sea methane seep sediment in a reactor at 10 °C and feed anaerobic artificial seawater medium and methane. Once phylogenetically diverse and metabolically active microorganisms are adapted to synthetic conditions in the reactor, we transition to growing community samples in glass tubes with the above medium, simple substrates and selective compounds (e.g., antibiotics). To accommodate for the slow growth anticipated for target organisms, primary cultures can be incubated for ≥6-12 months and analyzed for community composition even when no cell turbidity is observed. One casamino acid- and antibiotic-amended culture prepared in this way led to the enrichment of uncultured archaea. Through successive transfer in vitro combined with molecular growth monitoring, we successfully obtained the target archaeon with its partner methanogen as a pure syntrophic co-culture., (© 2022. Springer Nature Limited.)

The origin of eukaryotes remains unclear 1-4 . Current data suggest that eukaryotes may have emerged from an archaeal lineage known as 'Asgard' archaea 5,6 . Despite the eukaryote-like genomic features that are found in these archaea, the evolutionary transition from archaea to eukaryotes remains unclear, owing to the lack of cultured representatives and corresponding physiological insights. Here we report the decade-long isolation of an Asgard archaeon related to Lokiarchaeota from deep marine sediment. The archaeon-'Candidatus Prometheoarchaeum syntrophicum' strain MK-D1-is an anaerobic, extremely slow-growing, small coccus (around 550 nm in diameter) that degrades amino acids through syntrophy. Although eukaryote-like intracellular complexes have been proposed for Asgard archaea 6 , the isolate has no visible organelle-like structure. Instead, Ca. P. syntrophicum is morphologically complex and has unique protrusions that are long and often branching. On the basis of the available data obtained from cultivation and genomics, and reasoned interpretations of the existing literature, we propose a hypothetical model for eukaryogenesis, termed the entangle-engulf-endogenize (also known as E 3 ) model.

Various lineages of ammonia-oxidizing archaea (AOA) are present in deep waters, but the mechanisms that determine ecotype formation are obscure. We studied 18 high-quality genomes of the marine group I AOA lineages (alpha, gamma and delta) from the Mariana and Ogasawara trenches. The genomes of alpha AOA resembled each other, while those of gamma and delta lineages were more divergent and had even undergone insertion of some phage genes. The instability of the gamma and delta AOA genomes could be partially due to the loss of DNA polymerase B (polB) and methyladenine DNA glycosylase (tag) genes responsible for the repair of point mutations. The alpha AOA genomes harbour genes encoding a thrombospondin-like outer membrane structure that probably serves as a barrier to gene flow. Moreover, the gamma and alpha AOA lineages rely on vitamin B 12 -independent MetE and B 12 -dependent MetH, respectively, for methionine synthesis. The delta AOA genome contains genes involved in uptake of sugar and peptide perhaps for heterotrophic lifestyle. Our study provides insights into co-occurrence of cladogenesis and anagenesis in the formation of AOA ecotypes that perform differently in nitrogen and carbon cycling in dark oceans., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

A stable d-lactate electrochemical sensing system was developed using a dye-linked d-lactate dehydrogenase (Dye-DLDH) from an uncultivated thermophilic archaeon, Candidatus Caldiarchaeum subterraneum. To develop the system, the putative gene encoding the Dye-DLDH from Ca. Caldiarchaeum subterraneum was overexpressed in Escherichia coli, and the expressed product was purified. The recombinant enzyme was a highly thermostable Dye-DLDH that retained full activity after incubation for 10 min at 70°C. The electrode for detection of d-lactate was prepared by immobilizing the thermostable Dye-DLDH and multi-walled carbon nanotube (MWCNT) within Nafion membrane. The electrocatalytic response of the electrode was clearly observed upon exposure to d-lactate. The electrode response to d-lactate was linear within the concentration range of 0.03-2.5 mM, and it showed little reduction in responsiveness after 50 days. This is the first report describing a d-lactate sensing system using a thermostable Dye-DLDH., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The Challenger Deep is the deepest ocean on Earth. The present study investigated microbial community structures and geochemical cycles associated with the trench bottom sediments of the Challenger Deep, the Mariana Trench. The SSU rRNA gene communities found in trench bottom sediments were dominated by the bacteria Chloroflexi (SAR202 and other lineages), Bacteroidetes, Planctomycetes, "Ca. Marinimicrobia" (SAR406), and Gemmatimonadetes and by the archaeal α subgroup of MGI Thaumarchaeota and "Ca. Woesearchaeota" (Deep-sea Hydrothermal Vent Euryarchaeotic Group 6). The SSU rRNA gene sequencing analysis indicated that the dominant populations of the thaumarchaeal α group in hadal water and sediments were similar to each other at the species or genus level. In addition, the co-occurrence of nitrification and denitrification was revealed by the combination of pore water geochemical analyses and quantitative PCR for nitrifiers.

Many thermophiles thriving in a natural high-temperature environment remain uncultivated, and their ecophysiological functions in the biogeochemical cycle remain unclear. In the present study, we performed long-term continuous cultivation at 65°C and 70°C using a microbial mat sample, collected from a subsurface geothermal stream, as the inoculum, and reconstructed the whole genome of the maintained populations using metagenomics. Some metagenome-assembled genomes (MAGs), affiliated into phylum-level bacterial and archaeal clades without cultivated representatives, contained genes involved in nitrogen metabolism including nitrification and denitrification. Our results show genetic components and their potential interactions for the biogeochemical nitrogen cycle in a subsurface geothermal environment.

Subseafloor microbes beneath active hydrothermal vents are thought to live near the upper temperature limit for life on Earth. We drilled and cored the Iheya North hydrothermal field in the Mid-Okinawa Trough, and examined the phylogenetic compositions and the products of metabolic functions of sub-vent microbial communities. We detected microbial cells, metabolic activities and molecular signatures only in the shallow sediments down to 15.8 m below the seafloor at a moderately distant drilling site from the active hydrothermal vents (450 m). At the drilling site, the profiles of methane and sulfate concentrations and the δ 13 C and δD isotopic compositions of methane suggested the laterally flowing hydrothermal fluids and the in situ microbial anaerobic methane oxidation. In situ measurements during the drilling constrain the current bottom temperature of the microbially habitable zone to ~45 °C. However, in the past, higher temperatures of 106-198 °C were possible at the depth, as estimated from geochemical thermometry on hydrothermally altered clay minerals. The 16S rRNA gene phylotypes found in the deepest habitable zone are related to those of thermophiles, although sequences typical of known hyperthermophilic microbes were absent from the entire core. Overall our results shed new light on the distribution and composition of the boundary microbial community close to the high-temperature limit for habitability in the subseafloor environment of a hydrothermal field.

Unlabelled: Ammonia oxidation regulates the balance of reduced and oxidized nitrogen pools in nature. Although ammonia-oxidizing archaea have been recently recognized to often outnumber ammonia-oxidizing bacteria in various environments, the contribution of ammonia-oxidizing archaea is still uncertain due to difficulties in the in situ quantification of ammonia oxidation activity. Nitrogen and oxygen isotope ratios of nitrite (δ(15)NNO2- and δ(18)ONO2-, respectively) are geochemical tracers for evaluating the sources and the in situ rate of nitrite turnover determined from the activities of nitrification and denitrification; however, the isotope ratios of nitrite from archaeal ammonia oxidation have been characterized only for a few marine species. We first report the isotope effects of ammonia oxidation at 70°C by thermophilic Thaumarchaeota populations composed almost entirely of "Candidatus Nitrosocaldus." The nitrogen isotope effect of ammonia oxidation varied with ambient pH (25‰ to 32‰) and strongly suggests the oxidation of ammonia, not ammonium. The δ(18)O value of nitrite produced from ammonia oxidation varied with the δ(18)O value of water in the medium but was lower than the isotopic equilibrium value in water. Because experiments have shown that the half-life of abiotic oxygen isotope exchange between nitrite and water is longer than 33 h at 70°C and pH ≥6.6, the rate of ammonia oxidation by thermophilic Thaumarchaeota could be estimated using δ(18)ONO2- in geothermal environments, where the biological nitrite turnover is likely faster than 33 h. This study extended the range of application of nitrite isotopes as a geochemical clock of the ammonia oxidation activity to high-temperature environments., Importance: Because ammonia oxidation is generally the rate-limiting step in nitrification that regulates the balance of reduced and oxidized nitrogen pools in nature, it is important to understand the biological and environmental factors underlying the regulation of the rate of ammonia oxidation. The discovery of ammonia-oxidizing archaea (AOA) in marine and terrestrial environments has transformed the concept that ammonia oxidation is operated only by bacterial species, suggesting that AOA play a significant role in the global nitrogen cycle. However, the archaeal contribution to ammonia oxidation in the global biosphere is not yet completely understood. This study successfully identified key factors controlling nitrogen and oxygen isotopic ratios of nitrite produced from thermophilic Thaumarchaeota and elucidated the applicability and its limit of nitrite isotopes as a geochemical clock of ammonia oxidation rate in nature. Oxygen isotope analysis in this study also provided new biochemical information on archaeal ammonia oxidation., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Hadal oceans at water depths below 6,000 m are the least-explored aquatic biosphere. The Challenger Deep, located in the western equatorial Pacific, with a water depth of ∼11 km, is the deepest ocean on Earth. Microbial communities associated with waters from the sea surface to the trench bottom (0∼10,257 m) in the Challenger Deep were analyzed, and unprecedented trench microbial communities were identified in the hadal waters (6,000∼10,257 m) that were distinct from the abyssal microbial communities. The potentially chemolithotrophic populations were less abundant in the hadal water than those in the upper abyssal waters. The emerging members of chemolithotrophic nitrifiers in the hadal water that likely adapt to the higher flux of electron donors were also different from those in the abyssal waters that adapt to the lower flux of electron donors. Species-level niche separation in most of the dominant taxa was also found between the hadal and abyssal microbial communities. Considering the geomorphology and the isolated hydrotopographical nature of the Mariana Trench, we hypothesized that the distinct hadal microbial ecosystem was driven by the endogenous recycling of organic matter in the hadal waters associated with the trench geomorphology.

The impacts of lithologic structure and geothermal gradient on subseafloor microbial communities were investigated at a marginal site of the Iheya North hydrothermal field in the Mid-Okinawa Trough. Subsurface marine sediments composed of hemipelagic muds and volcaniclastic deposits were recovered through a depth of 151 m below the seafloor at site C0017 during Integrated Ocean Drilling Program Expedition 331. Microbial communities inferred from 16S rRNA gene clone sequencing in low-temperature hemipelagic sediments were mainly composed of members of the Chloroflexi and deep-sea archaeal group. In contrast, 16S rRNA gene sequences of marine group I Thaumarchaeota dominated the microbial phylotype communities in the coarse-grained pumiceous gravels interbedded between the hemipelagic sediments. Based on the physical properties of sediments such as temperature and permeability, the porewater chemistry, and the microbial phylotype compositions, the shift in the physical properties of the sediments is suggested to induce a potential subseafloor recharging flow of oxygenated seawater in the permeable zone, leading to the generation of variable chemical environments and microbial communities in the subseafloor habitats. In addition, the deepest section of sediments under high-temperature conditions (∼90°C) harbored the sequences of an uncultivated archaeal lineage of hot water crenarchaeotic group IV that may be associated with the high-temperature hydrothermal fluid flow. These results indicate that the subseafloor microbial community compositions and functions at the marginal site of the hydrothermal field are highly affected by the complex fluid flow structure, such as recharging seawater and underlying hydrothermal fluids, coupled with the lithologic transition of sediments., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Anaerobic oxidation of methane (AOM) in marine sediments is an important global methane sink, but the physiological characteristics of AOM-associated microorganisms remain poorly understood. Here we report the cultivation of an AOM microbial community from deep-sea methane-seep sediment using a continuous-flow bioreactor with polyurethane sponges, called the down-flow hanging sponge (DHS) bioreactor. We anaerobically incubated deep-sea methane-seep sediment collected from the Nankai Trough, Japan, for 2,013 days in the bioreactor at 10°C. Following incubation, an active AOM activity was confirmed by a tracer experiment using 13C-labeled methane. Phylogenetic analyses demonstrated that phylogenetically diverse Archaea and Bacteria grew in the bioreactor. After 2,013 days of incubation, the predominant archaeal components were anaerobic methanotroph (ANME)-2a, Deep-Sea Archaeal Group, and Marine Benthic Group-D, and Gammaproteobacteria was the dominant bacterial lineage. Fluorescence in situ hybridization analysis showed that ANME-1 and -2a, and most ANME-2c cells occurred without close physical interaction with potential bacterial partners. Our data demonstrate that the DHS bioreactor system is a useful system for cultivating fastidious methane-seep-associated sedimentary microorganisms.

There has been much progress in understanding the nitrogen cycle in oceanic waters including the recent identification of ammonia-oxidizing archaea and anaerobic ammonia oxidizing (anammox) bacteria, and in the comprehensive estimation in abundance and activity of these microbial populations. However, compared with the nitrogen cycle in oceanic waters, there are fewer studies concerning the oceanic benthic nitrogen cycle. To further elucidate the dynamic nitrogen cycle in deep-sea sediments, a sediment core obtained from the Ogasawara Trench at a water depth of 9760 m was analysed in this study. The profiles obtained for the pore-water chemistry, and nitrogen and oxygen stable isotopic compositions of pore-water nitrate in the hadopelagic sediments could not be explained by the depth segregation of nitrifiers and nitrate reducers, suggesting the co-occurrence of nitrification and nitrate reduction in the shallowest nitrate reduction zone. The abundance of SSU rRNA and functional genes related to nitrification and denitrification are consistent with the co-occurrence of nitrification and nitrate reduction observed in the geochemical analyses. This study presents the first example of cooperation between aerobic and anaerobic nitrogen metabolism in the deep-sea sedimentary environments., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Viruses play important roles in marine surface ecosystems, but little is known about viral ecology and virus-mediated processes in deep-sea hydrothermal microbial communities. In this study, we examined virus-like particle (VLP) abundances in planktonic and attached microbial communities, which occur in physical and chemical gradients in both deep and shallow submarine hydrothermal environments (mixing waters between hydrothermal fluids and ambient seawater and dense microbial communities attached to chimney surface areas or macrofaunal bodies and colonies). We found that viruses were widely distributed in a variety of hydrothermal microbial habitats, with the exception of the interior parts of hydrothermal chimney structures. The VLP abundance and VLP-to-prokaryote ratio (VPR) in the planktonic habitats increased as the ratio of hydrothermal fluid to mixing water increased. On the other hand, the VLP abundance in attached microbial communities was significantly and positively correlated with the whole prokaryotic abundance; however, the VPRs were always much lower than those for the surrounding hydrothermal waters. This is the first report to show VLP abundance in the attached microbial communities of submarine hydrothermal environments, which presented VPR values significantly lower than those in planktonic microbial communities reported before. These results suggested that viral lifestyles (e.g., lysogenic prevalence) and virus interactions with prokaryotes are significantly different among the planktonic and attached microbial communities that are developing in the submarine hydrothermal environments.

Understanding the mechanistic basis of the disruption of tRNA genes, as manifested in the intron-containing and split tRNAs found in Archaea, will provide considerable insight into the evolution of the tRNA molecule. However, the evolutionary processes underlying these disruptions have not yet been identified. Previously, a composite genome of the deep-branching archaeon Caldiarchaeum subterraneum was reconstructed from a community genomic library prepared from a C. subterraneum-dominated microbial mat. Here, exploration of tRNA genes from the library reveals that there are at least three types of heterogeneity at the tRNA(Thr)(GGU) gene locus in the Caldiarchaeum population. All three involve intronic gain and splitting of the tRNA gene. Of two fosmid clones found that encode tRNA(Thr)(GGU), one (tRNA(Thr-I)) contains a single intron, whereas another (tRNA(Thr-II)) contains two introns. Notably, in the clone possessing tRNA(Thr-II), a 5' fragment of the tRNA(Thr-I) (tRNA(Thr-F)) gene was observed 1.8-kb upstream of tRNA(Thr-II). The composite genome contains both tRNA(Thr-II) and tRNA(Thr-F), although the loci are >500 kb apart. Given that the 1.8-kb sequence flanked by tRNA(Thr-F) and tRNA(Thr-II) is predicted to encode a DNA recombinase and occurs in six regions of the composite genome, it may be a transposable element. Furthermore, its dinucleotide composition is most similar to that of the pNOB8-type plasmid, which is known to integrate into archaeal tRNA genes. Based on these results, we propose that the gain of the tRNA intron and the scattering of the tRNA fragment occurred within a short time frame via the integration and recombination of a mobile genetic element.

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A variety of archaeal lineages have been identified using culture-independent molecular phylogenetic surveys of microbial habitats occurring in deep-sea hydrothermal environments such as chimney structures, sediments, vent emissions, and chemosynthetic macrofauna. With the exception of a few taxa, most of these archaea have not yet been cultivated, and their physiological and metabolic traits remain unclear. In this study, phylogenetic diversity and distribution profiles of the archaeal genes encoding small subunit (SSU) rRNA, methyl coenzyme A (CoA) reductase subunit A, and the ammonia monooxygenase large subunit were characterized in hydrothermally influenced sediments at the Yonaguni Knoll IV hydrothermal field in the Southern Okinawa Trough. Sediment cores were collected at distances of 0.5, 2, or 5 m from a vent emission (90 degrees C). A moderate temperature gradient extends both horizontally and vertically (5 to 69 degrees C), indicating the existence of moderate mixing between the hydrothermal fluid and the ambient sediment pore water. The mixing of reductive hot hydrothermal fluid and cold ambient sediment pore water establishes a wide spectrum of physical and chemical conditions in the microbial habitats that were investigated. Under these different physico-chemical conditions, variability in archaeal phylotype composition was observed. The relationship between the physical and chemical parameters and the archaeal phylotype composition provides important insight into the ecophysiological requirements of uncultivated archaeal lineages in deep-sea hydrothermal vent environments, giving clues for approximating culture conditions to be used in future culturing efforts.

The potential for microbial nitrogen fixation in the anoxic methane seep sediments in a mud volcano, the number 8 Kumano Knoll, was characterized by molecular phylogenetic analyses. A total of 111 of the nifH (a gene coding a nitrogen fixation enzyme, Fe protein) clones were obtained from different depths of the core sediments, and the phylogenetic analysis of the clones indicated the genetic diversity of nifH genes. The predominant group detected (methane seep group 2), representing 74% of clonal abundance, was phylogenetically related to the nifH sequences obtained from the Methanosarcina species but was most closely related to the nifH sequences potentially derived from the anoxic methanotrophic archaea (ANME-2 archaea). The recovery of the nif gene clusters including the nifH sequences of the methane seep group 2 and the subsequent reverse transcription-PCR detection of the nifD and nifH genes strongly suggested that the genetic components of the gene clusters would be operative for the in situ assimilation of molecular nitrogen (N(2)) by the host microorganisms. DNA-based quantitative PCR of the archaeal 16S rRNA gene, the group-specific mcrA (a gene encoding the methyl-coenzyme M reductase alpha subunit) gene, and the nifD and nifH genes demonstrated the similar distribution patterns of the archaeal 16S rRNA gene, the mcrA groups c-d and e, and the nifD and nifH genes through the core sediments. These results supported the idea that the anoxic methanotrophic archaea ANME-2c could be the microorganisms hosting the nif gene clusters and could play an important role in not only the in situ carbon (methane) cycle but also the nitrogen cycle in subseafloor sediments.

Since evidence indicates that the Last Universal Common Ancestor (LUCA) was phylogenetically closest to Methanopyrus kandleri among living organisms with elucidated genomes, this study has been directed to a search for the most primitive Methanopyrus lineage. For this purpose, the divergence of valyl-tRNA synthetase (ValRS) and isoleucyl-tRNA synthetase (IleRS) was employed as a measure of primitivity. Comparison of Methanopyrus kandleri and the Methanopyrus isolates GC34 and GC37 from the Pacific Ocean and KOL6, TAG1, TAG11, and SNP6 from the Atlantic Ocean established that the Pacific lineages are more primitive than the Atlantic lineages. Both the groups, however, are younger than environmental genomes from the Kairei Field of Central Indian Ridge in the Indian Ocean. These results showed that different Methanopyrus isolates differ significantly with respect to ValRS-IleRS divergence. On this basis, genomes giving rise to the ValRS and IleRS gene fragments from the Central Indian Ridge represent the most primitive Methanopyrus, phylogenetically the oldest living lineage closest to LUCA.

The subseafloor microbial communities in the turbidite depositional basins Brazos-Trinity Basin IV (BT Basin) and the Mars-Ursa Basin (Ursa Basin) on the Gulf of Mexico continental slope (IODP holes U1319A, U1320A, U1322B and U1324B) were investigated by PCR-dependent molecular analyses targeted to the small subunit (SSU) rRNA genes, dsrA and mcrA, and hydrogenase activity measurements. Biomass at both basins was very low, with the maximum cell or the SSU rRNA gene copy number <1 x 10(7) cells mL(-1) or copies g(-1) sediments, respectively. Hydrogenase activity correlated with biomass estimated by SSU rRNA gene copy number when all data sets were combined. We detected differences in the SSU rRNA gene community structures and SSU rRNA gene copy numbers between the basin-fill and basement sediments in the BT Basin. Examination of microbial communities and hydrogenase activity in the context of geochemical and geophysical parameters and sediment depositional environments revealed that differences in microbial community composition between the basin-fill and basement sediments in the BT Basin were associated with sedimentation regimes tied to the sea-level change. This may also explain the distributions of relatively similar archaeal communities in the Ursa Basin sediments and basement sediments in the BT Basin.

Uncultured archaeal anaerobic methanotrophs (ANMEs) are known to operate the anaerobic oxidation of methane process, an important sink for the greenhouse gas methane in natural environments. In this study, we designed 16S rRNA gene-specific primers for each of the phylogenetic groups of ANMEs (ANME-1, Guaymas Basin hydrothermal sediment clones group within the ANME-1, ANME-2a, ANME-2b, ANME-2c and ANME-3) based on previously reported sequences. The newly designed primers were used for the detection of the various groups of ANMEs in the sulphate-limited anaerobic environmental samples, i.e. methanogenic sludges, rice field soils, lotus field sediments and natural gas fields. The ANME 16S rRNA gene sequences were detected only in a natural gas field sample among the environments examined in this study and were of the ANME-1 and -2c groups. In addition, the quantitative real-time PCR analysis using the designed primers showed that abundances of ANME-1 and -2c were estimated to be <0.02% of the total prokaryotic 16S rRNA gene community. The newly designed ANME group-specific primers in this study may be useful to survey the distribution and quantitative determination of ANMEs.

Microbial communities associated with a variety of hydrothermal emissions at the Yonaguni Knoll IV hydrothermal field, the southernmost Okinawa Trough, were analyzed by culture-dependent and -independent techniques. In this hydrothermal field, dozens of vent sites hosting physically and chemically distinct hydrothermal fluids were observed. Variability in the gas content and formation in the hydrothermal fluids was observed and could be controlled by the potential subseafloor phase-separation and -partition processes. The hydrogen concentration in the hydrothermal fluids was also variable (0.8-3.6 mmol kg(-1)) among the chimney sites, but was unusually high as compared with those in other Okinawa Trough hydrothermal fields. Despite the physical and chemical variabilities of the hydrothermal fluids, the microbial communities were relatively similar among the habitats. Based on both culture-dependent and -independent analyses of the microbial community structures, members of Thermococcales, Methanococcales and Desulfurococcales likely represent the predominant archaeal components, while members of Nautiliaceae and Thioreductoraceae are considered to dominate the bacterial population. Most of the abundant microbial components appear to be chemolithotrophs sustained by hydrogen oxidation. The relatively consistent microbial communities found in this study could have been because of the sufficient input of hydrogen from the hydrothermal fluids rather than other chemical properties.

Deep-sea vents support productive ecosystems driven primarily by chemoautotrophs. Chemoautotrophs are organisms that are able to fix inorganic carbon using a chemical energy obtained through the oxidation of reduced compounds. Following the discovery of deep-sea vent ecosystems in 1977, there has been an increasing knowledge that deep-sea vent chemoautotrophs display remarkable physiological and phylogenetic diversity. Cultivation-dependent and -independent studies have led to an emerging view that the majority of deep-sea vent chemoautotrophs have the ability to derive energy from a variety of redox couples other than the conventional sulfur-oxygen couple, and fix inorganic carbon via the reductive tricarboxylic acid cycle. In addition, recent genomic, metagenomic and postgenomic studies have considerably accelerated the comprehensive understanding of molecular mechanisms of deep-sea vent chemoautotrophy, even in yet uncultivable endosymbionts of vent fauna. Genomic analysis also suggested that there are previously unrecognized evolutionary links between deep-sea vent chemoautotrophs and important human/animal pathogens. This review summarizes chemoautotrophy in deep-sea vents, highlighting recent biochemical and genomic discoveries.

A quantitative fluorogenic PCR method for detecting methanogenic and methanotrophic orders was established using a refined primer set for the methyl coenzyme M reductase subunit A gene (mcrA). The method developed was applied to several microbial communities in which diversity and abundance of methanogens or anaerobic methanotrophs (ANMEs) was identified by 16S rRNA gene clone analysis, and strong correlations between the copy numbers of mcrA with those of archaeal 16S rRNA genes in the communities were observed. The assay can be applied to detecting and assessing the abundance of methanogens and/or ANMEs in anoxic environments that could not be detected by 16S rRNA gene sequence analyses.

Microbial communities in a shallow submarine hydrothermal system near Taketomi Island, Japan, were investigated using cultivation-based and molecular techniques. The main hydrothermal activity occurred in a craterlike basin (depth, approximately 23 m) on the coral reef seafloor. The vent fluid (maximum temperature, >52 degrees C) contained 175 microM H2S and gas bubbles mainly composed of CH4 (69%) and N2 (29%). A liquid serial dilution cultivation technique targeting a variety of metabolism types quantified each population in the vent fluid and in a white microbial mat located near the vent. The most abundant microorganisms cultivated from both the fluid and the mat were autotrophic sulfur oxidizers, including mesophilic Thiomicrospira spp. and thermophilic Sulfurivirga caldicuralii. Methane oxidizers were the second most abundant organisms in the fluid; one novel type I methanotroph exhibited optimum growth at 37 degrees C, and another novel type I methanotroph exhibited optimum growth at 45 degrees C. The number of hydrogen oxidizers cultivated only from the mat was less than the number of sulfur and methane oxidizers, although a novel mesophilic hydrogen-oxidizing member of the Epsilonproteobacteria was isolated. Various mesophilic to hyperthermophilic heterotrophs, including sulfate-reducing Desulfovibrio spp., iron-reducing Deferribacter sp., and sulfur-reducing Thermococcus spp., were also cultivated. Culture-independent 16S rRNA gene clone analysis of the vent fluid and mat revealed highly diverse archaeal communities. In the bacterial community, S. caldicuralii was identified as the predominant phylotype in the fluid (clonal frequency, 25%). Both bacterial clone libraries indicated that there were bacterial communities involved in sulfur, hydrogen, and methane oxidation and sulfate reduction. Our results indicate that there are unique microbial communities that are sustained by active chemosynthetic primary production rather than by photosynthetic production in a shallow hydrothermal system where sunlight is abundant.

During Integrated Ocean Drilling Program Expedition 301, we obtained a sample of black rust from a circulation obviation retrofit kit (CORK) observatory at a borehole on the eastern flank of Juan de Fuca Ridge. Due to overpressure, the CORK had failed to seal the borehole. Hot fluids from oceanic crust had discharged to the overlying bottom seawater and resulted in the formation of black rust analogous to a hydrothermal chimney deposit. Both culture-dependent and culture-independent analyses indicated that the black-rust-associated community differed from communities reported from other microbial habitats, including hydrothermal vents at seafloor spreading centers, while it shared phylotypes with communities previously detected in crustal fluids from the same borehole. The most frequently retrieved sequences of bacterial and archaeal 16S rRNA genes were related to the genera Ammonifex and Methanothermococcus, respectively. Most phylotypes, including phylotypes previously detected in crustal fluids, were isolated in pure culture, and their metabolic traits were determined. Quantification of the dissimilatory sulfite reductase (dsrAB) genes, together with stable sulfur isotopic and electron microscopic analyses, strongly suggested the prevalence of sulfate reduction, potentially by the Ammonifex group of bacteria. Stable carbon isotopic analyses suggested that the bulk of the microbial community was trophically reliant upon photosynthesis-derived organic matter. This report provides important insights into the phylogenetic, physiological, and trophic characteristics of subseafloor microbial ecosystems in warm ridge flank crusts.

Increasing levels of CO2 in the atmosphere are expected to cause climatic change with negative effects on the earth's ecosystems and human society. Consequently, a variety of CO2 disposal options are discussed, including injection into the deep ocean. Because the dissolution of CO2 in seawater will decrease ambient pH considerably, negative consequences for deep-water ecosystems have been predicted. Hence, ecosystems associated with natural CO2 reservoirs in the deep sea, and the dynamics of gaseous, liquid, and solid CO2 in such environments, are of great interest to science and society. We report here a biogeochemical and microbiological characterization of a microbial community inhabiting deep-sea sediments overlying a natural CO2 lake at the Yonaguni Knoll IV hydrothermal field, southern Okinawa Trough. We found high abundances (>10(9) cm(-3)) of microbial cells in sediment pavements above the CO2 lake, decreasing to strikingly low cell numbers (10(7) cm(-3)) at the liquid CO2/CO2-hydrate interface. The key groups in these sediments were as follows: (i) the anaerobic methanotrophic archaea ANME-2c and the Eel-2 group of Deltaproteobacteria and (ii) sulfur-metabolizing chemolithotrophs within the Gamma- and Epsilonproteobacteria. The detection of functional genes related to one-carbon assimilation and the presence of highly 13C-depleted archaeal and bacterial lipid biomarkers suggest that microorganisms assimilating CO2 and/or CH4 dominate the liquid CO2 and CO2-hydrate-bearing sediments. Clearly, the Yonaguni Knoll is an exceptional natural laboratory for the study of consequences of CO2 disposal as well as of natural CO2 reservoirs as potential microbial habitats on early Earth and other celestial bodies.

The deep subseafloor biosphere is among the least-understood habitats on Earth, even though the huge microbial biomass therein plays an important role for potential long-term controls on global biogeochemical cycles. We report here the vertical and geographical distribution of microbes and their phylogenetic diversities in deeply buried marine sediments of the Pacific Ocean Margins. During the Ocean Drilling Program Legs 201 and 204, we obtained sediment cores from the Peru and Cascadia Margins that varied with respect to the presence of dissolved methane and methane hydrate. To examine differences in prokaryotic distribution patterns in sediments with or without methane hydrates, we studied >2,800 clones possessing partial sequences (400-500 bp) of the 16S rRNA gene and 348 representative clone sequences (approximately 1 kbp) from the two geographically separated subseafloor environments. Archaea of the uncultivated Deep-Sea Archaeal Group were consistently the dominant phylotype in sediments associated with methane hydrate. Sediment cores lacking methane hydrates displayed few or no Deep-Sea Archaeal Group phylotypes. Bacterial communities in the methane hydrate-bearing sediments were dominated by members of the JS1 group, Planctomycetes, and Chloroflexi. Results from cluster and principal component analyses, which include previously reported data from the West and East Pacific Margins, suggest that, for these locations in the Pacific Ocean, prokaryotic communities from methane hydrate-bearing sediment cores are distinct from those in hydrate-free cores. The recognition of which microbial groups prevail under distinctive subseafloor environments is a significant step toward determining the role these communities play in Earth's essential biogeochemical processes.

Novel hydrothermal activities accompanying effluent white smokers and elemental sulfur chimney structures at the north-east lava dome of the TOTO caldera depression in the Mariana Volcanic Arc have been explored and characterized by geochemical and microbiological surveys. White smoker hydrothermal fluids were observed in the potential hydrothermal activity centre of the field and represented the maximal temperature of 170 degrees C and the lowest pH of 1.6. The chimney structures, all consisting of elemental sulfur (sulfur chimney), were also unique to the TOTO caldera hydrothermal field. Microbial community structures in a sulfur chimney and its formation hydrothermal fluid with a high concentration of hydrogen sulfide (15 mM) have been investigated by culture-dependent and -independent analyses. 16S rRNA gene clone analysis and fluorescence in situ hybridization (FISH) analysis revealed that epsilon-Proteobacteria dominated the microbial communities in the sulfur chimney structure and formed a dense microbial mat covering the sulfur chimney surface. Archaeal phylotypes were consistently minor components in the communities and related to the genera Thermococcus, Pyrodictium, Aeropyrum, and the uncultivated archaeal group of 'deep-sea hydrothermal vent euryarchaeotal group'. Cultivation analysis suggested that the chemolithoautotrophs might play a significant ecological role as primary producers utilizing gas and sulfur compounds provided from hydrothermal fluids.

Phase-separation and -segregation (boiling/distillation of subseafloor hydrothermal fluids) represent the primary mechanisms causing intra-field variations in vent fluid compositions. To determine whether this geochemical process affects the formation of microbial communities, we examined the microbial communities at three different vent sites located within a few tens meters of one another. In addition to chimney structures, colonization devices capturing subseafloor communities entrained by the vent fluids were studied, using culture-dependent and -independent methods. Microbiological analyses demonstrated the occurrence of distinctive microbial communities in each of the hydrothermal niches. Within a chimney structure, there was a transition from a mixed community of mesophiles and thermophiles in the exterior parts to thermophiles in the interior. Beside the transition within a chimney structure, intra-field variations in microbial communities in vent fluids were apparent. Geochemical analysis demonstrated that different vent fluids have distinctive end-member compositions as a consequence of subseafloor phase-separation and -segregation, which were designated gas-depleted, normal and gas-enriched fluids. In comparison to gas-depleted and normal fluids, gas-enriched fluids harbored more abundant chemolithoautotrophs with gaseous component-dependent energy metabolism, such as hydrogenotrophic methanogenesis. Subseafloor phase-separation and -segregation may play a key role in supplying energy and carbon sources to vent-associated chemolithoautotrophs and subvent microbial communities.

One of the keys to success of many anaerobic ecosystems is the process of syntrophic intercellular hydrogen transfer. This process facilitates the overall reaction by end-product removal, taking advantage of a wide variety of organisms that are able to use hydrogen directly as an energy source by uptake hydrogenases. Thus, the issue is not whether there are hydrogen-driven processes or communities but whether there are hydrogen-driven communities that exist and persist independently of the products of photosynthesis (so-called subsurface lithoautotrophic microbial ecosystems, or SLiMEs). It is the proof of long-term independence from photosynthesis and its products that is the most difficult issue to establish, and perhaps the most important one with regard to searching for SLiMEs both on and off our planet. Although the evidence is not yet unequivocal, a growing body of evidence supports the existence of SLiME-like communities: if they exist, the implications are immense with regard to understanding subsurface environments on Earth, looking for present day analogs of early Earth and the search for life in other worlds.

Samples from three submerged sites (MC, a core obtained in the methane seep area; MR, a reference core obtained at a distance from the methane seep; and HC, a gas-bubbling carbonate sample) at the Kuroshima Knoll in the southern Ryuku arc were analyzed to gain insight into the organisms present and the processes involved in this oxic-anoxic methane seep environment. 16S rRNA gene analyses by quantitative real-time PCR and clone library sequencing revealed that the MC core sediments contained abundant archaea (approximately 34% of the total prokaryotes), including both mesophilic methanogens related to the genus Methanolobus and ANME-2 members of the Methanosarcinales, as well as members of the delta-Proteobacteria, suggesting that both anaerobic methane oxidation and methanogenesis occurred at this site. In addition, several functional genes connected with methane metabolism were analyzed by quantitative competitive-PCR, including the genes encoding particulate methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), methanol dehydrogenese (mxaF), and methyl coenzyme M reductase (mcrA). In the MC core sediments, the most abundant gene was mcrA (2.5 x 10(6) copies/g [wet weight]), while the pmoA gene of the type I methanotrophs (5.9 x 10(6) copies/g [wet weight]) was most abundant at the surface of the MC core. These results indicate that there is a very complex environment in which methane production, anaerobic methane oxidation, and aerobic methane oxidation all occur in close proximity. The HC carbonate site was rich in gamma-Proteobacteria and had a high copy number of mxaF (7.1 x 10(6) copies/g [wet weight]) and a much lower copy number of the pmoA gene (3.2 x 10(2) copies/g [wet weight]). The mmoX gene was never detected. In contrast, the reference core contained familiar sequences of marine sedimentary archaeal and bacterial groups but not groups specific to C1 metabolism. Geochemical characterization of the amounts and isotopic composition of pore water methane and sulfate strongly supported the notion that in this zone both aerobic methane oxidation and anaerobic methane oxidation, as well as methanogenesis, occur.

Subsurface microbial community structure in relation to geochemical gradients and lithology was investigated using a combination of molecular phylogenetic and geochemical analyses. Discreet groundwater and substratum samples were obtained from depths ranging from 182 to 190 m beneath the surface at approximately 10-cm intervals using a multilevel sampler (MLS) that straddled Cretaceous shale and sandstone formations at a site in the southern San Juan Basin in New Mexico. DNA and RNA were extracted directly from quartzite sand substratum loaded into individual cells of the MLS and colonized in situ. Polymerase chain reaction (PCR)-mediated T-RFLP analysis of archaeal rRNA genes (rDNA) in conjunction with partial sequencing analysis of archaeal rDNA libraries and quantitative RNA hybridization with oligonucleotide probes were used to probe community structure and function. Although total microbial populations remained relatively constant over the entire depth interval sampled, significant shifts in archaeal populations, predominantly methanogens, were observed. These shifts coincided with the geochemical transition from relatively high methane (26 mM), low sulphate (< 3 mg l(-1)) conditions in the region adjacent to the organic matter-rich shale to relatively low-methane (< 0.5 mM), high-sulphate (48 mg l(-1)) conditions in the organic-poor sandstone beneath the shale. These results indicated that active, phylogenetically diverse archaeal communities were present in the subsurface Cretaceous rock environment at this site and that major archaeal clades shifted dramatically over scales of tens of centimetres, corresponding to changes in the lithology and geochemical gradients.

A record of the history of the Earth is hidden in the Earth's crust, like the annual rings of an old tree. From very limited records retrieved from deep underground, one can infer the geographical, geological, and biological events that occurred throughout Earth's history. Here we report the discovery of vertically shifted community structures of Archaea in a typical oceanic subseafloor core sample (1410 cm long) recovered from the West Philippine Basin at a depth of 5719 m. Beneath a surface community of ubiquitous deep-sea archaea (marine crenarchaeotic group I; MGI), an unusual archaeal community consisting of extremophilic archaea, such as extreme halophiles and hyperthermophiles, was present. These organisms could not be cultivated, and may be microbial relicts more than 2 million years old. Our discovery of archaeal rDNA in this core sample, probably associated with the past terrestrial volcanic and submarine hydrothermal activities surrounding the West Philippine Basin, serves as potential geomicrobiological evidence reflecting novel records of geologic thermal events in the Pleistocene period concealed in the deep-sea subseafloor. [ABSTRACT FROM AUTHOR]