The Archaean Park Project, which is funded by Special Coordination Fund of the Ministry of Education, Science and Technology (MEXT) is a major program to analyze the interaction between the sub-vent biosphere and the geosphere of the seafloor hydrothermal system. The convection of hydrothermal fluid within the oceanic crust gives rise to various sub-surface environments near hydrothermal vents. Such environmental conditions are analogous to the site of origin and/or early evolution of life during the Archaean age. Since the Domain Bacteria and Domain Archaea near the root of the Universal Tree are hyper-thermophilic, we named our proposal the "Archaean Park Project". There are four sub-themes in the project; 1) Physical processes of hydrothermal circulation systems, 2) Observation of the chemical environment of the sub-vent biosphere, 3) Microbiology and molecular ecology in the sub-vent biosphere; and 4) Interaction between microbial and geological processes. Here, we summarize the results of the research mainly performed in the first three-year period in terms of the deep-sea hydrothermal system at Suiyo Seamount, Izu-Bonin arc (Ogasawara arc), western Pacific.

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Surface geophysical, airgun-OBS (ocean bottom seismograph), and deep-towed surveys by R/V Kairei KR01-15 cruise, and OBS array observations were conducted to characterize geophysical features of a hydrothermal system in Suiyo Seamount as a part of Archaean Park Project. Geomagnetic results from surface and deep towed magnetometers suggest 1) the seamount was formed during Brunhes Epoch (after 0.78 My), and 2) a low magnetization area exists on its summit and is limited only on the eastern side with a diameter of 800 meters. 2-D ray tracing results, using four OBS data with a GI gun across the seamount in NE-SW direction, indicate that a P wave velocity of 2.2∿4.2 km s^-1 (2 km thickness) is located all over the observation line. The OBS array observations reveal the seismic activity and the hypocenter determination indicates that the locations of these earthquakes are focused in a chimney-like shape within its diameter of 3 km just beneath the Suiyo volcanic cone at a depth of 3∿7km. We propose a model to explain these geophysical observations; the former main conduit of Suiyo Seamount is in the middle of cooling after the last eruption, and its heat drives the hydrothermal system in Suiyo Seamount. We perform a numerical simulation of a magma injection in the conduit with its diameter of 1 km and its cooling process has two different mechanisms; 1) a major hydrothermal circulation exists in the uppermost crust with P wave velocity of 2.2∿4.2 km s^-1 (2 km thickness), suggesting a higher porosity region, and 2) thermal conduction is dominated in the deeper part. In the thermal conduction region, the existence of a cooling area inside and a heating area outside near the former main conduit leads to thermal differential stress, which is the cause of earthquakes below a depth of 3 km. This is a reasonable explanation of chimney-like shape hypocenter locations. The magma injection in the conduit with its diameter of 1 km has enough heat to drive hydrothermal circulation at least for a few hundreds years and the hydrothermal circulation is maintained for long times enough to make the low magnetization area with its diameter of 800 meters through the high alteration of the rock. This stable existence of the hydrothermal system in Suiyo Seamount would be an important factor to discuss regarding the origin and evolution of subsurface-vent biospheres, especially when the hydrothermal system has been isolated.

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To understand the surface/subsurface geophysical structure of the Suiyo seamount submarine caldera, which is one of the active Izu-Bonin arc hydrothermal systems, we employed a newly developed acoustic instrument to make detailed imageries of the caldera (sidescan sonar imagery and subbottom reflection profile). To improve the spatial resolution of the seafloor mapping, we chose a deep-tow survey method and developed a new profiling system called the DAIPACK (Deepsea Acoustic Imaging Package). Using this system, we could successfully obtain a high-resolution seafloor imagery (pixel resolution of 1m or better) of the entire hydrothermal area in the Suiyo caldera. We still have difficulty in direct comparison between the imagery obtained and the video/camera images of the seafloor, because of the lack in the navigational accuracy of the ROV system. We have reprocessed the previously interpreted deep-tow seismic reflection data obtained during the KR01-15 cruise in 2001. Applying geometry correction and migration to the data, we could reduce the spatial distortion and the diffraction patterns from the imagery. Both processes made the structural interpretation easier. Interpreting the final cross section, we propose to revise the shallow structure (from the sea bottom to 200 m in depth) of the Suiyo caldera from the previous "layered model" to the "piecewise-continuous block model". We need further discussion on the consistency of our new results to other geophysical observations and models.

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Detailed heat flow surveys were carried out in the hydrothermal area within the caldera of Suiyo Seamount. Heat flow was highest (>10 W m^-2) in the hydrothermally active area. In contrast, very low heat flow, 0.04∿0.4 W m^-2, was observed only several tens of meters away from the venting sites. To the east, heat flow was uniform around 4 W m^-2. Non-linear temperature vs. depth profiles, which are likely to be direct evidence for discharge and recharge activity around the vents, were identified next to the isolated vents. Hydrothermal regime at the Suiyo site reflected a 100 m scale stable circulation, to a local scale of 1 m.

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Many attempts have been made to acquire time-series measurements at Suiyo Seamount, Izu-Bonin Arc, Japan, during August 2001 and August 2002. Among many instruments deployed on the seafloor at hydrothermal sites in the summit caldera of the Suiyo Seamount, we report the results by Medusa hydrothermal monitoring system and SAHF (Stand Alone Heat Flow meter). We deployed Medusa systems at hydrothermal systems in August 2002 for about two weeks. The Medusa system monitors the flow velocities and temperatures of effluent fluid out of cased seafloor boreholes and areas of warm diffusive flow. Spectral analyses of these data provide strong evidence for tidal modulations in case of the diffuse seafloor areas. Effluent flow rate atop a cased borehole drilled into the seafloor indicates peaks at semi-diurnal and diurnal frequencies. It might reflect the tidal response of hydrothermal activity.

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We briefly review modeling studies of subseafloor hydrothermal systems. We discuss critical properties of water, phase separation of seawater, and precipitation of minerals, which are important for the circulation pattern of hydrothermal systems. Physical properties of water drastically change around its critical point. They confine the width of upwelling plumes, and keep the maximum temperature of hydrothermal fluids in the range of 350∿400°C. Seawater separates into two phases with different salinities, when it is heated. This is the major reason for the salinity variation of hydrothermal fluids. High salinity fluids sink to form a stagnant layer beneath the vigorously convecting fluid layer. Since the stable stratification in the brine layer decreases the heat transfer, the heat output from the hydrothermal system with a brine layer is less than that without one. Precipitation and dissolution of minerals occur when the temperature of the fluid changes and when two compositionally different fluids mix. Precipitation decreases pore space the crustal rocks, and obstructs flow paths. Another role of precipitation is to stabilize the circulation by preventing two different fluids from mixing.

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The benthic Multi-coring System (BMS) is a remotely operated seafloor coring system installed on R/V "Hakurei Maru No. 2". The only connection between BMS and the research vessel "Hakurei Maru No.2" is via an armored umbilical cable. BMS has 5 TV camera systems for finding the drilling site and a monitoring borehole during drilling operation. BMS has remotely operated 3 telescoping legs for leveling, each capable of extending 1m for adjusting BMS position on rough seafloor. To obtain a coring depth of 20 m, BMS uses rotating storage magazine to store up to 33 drilling tool (e.g. 15 sections of 2.2m core barrels, 9 sections of 3m rods and 6 sections of 1.5m casings). Temperature logging of boreholes after BMS drilling was performed using Kuster tools set in the core barrel. Ten holes were drilled at the Suiyo Seamount in 2001 and 2002. The total penetration was 51.86m with individual penetration ranging from 2.88m to 8.99m and core recoveries of 6% to 90%. These cores preserve original textures of volcanic rocks and sulfide-sulfate veins. These cores are suitable for geological and mineralogical studies using optical microscope, X-ray fluorescence (XRF) microscope, Electron probe micro analyzer (EPMA), X-ray diffractometer (XRD) and analytical transmission microscope (TEM). A sulfide-rich rock sample contains high concentrations of Au (42mg kg^-1), Ag (112mg kg^-1), As (1, 440mg kg^-1), Hg (55mg kg^-1), Cu (>2wt%), and Zn (>10wt%), and Pb (5, 890mg kg^-1). The relatively low Pb concentration, with respect to Cu and Zn, shows that magma is the only metal source for mineralization. Hydrothermally altered rock samples are characterized by montmorillonite, chlorite/montmorillonite mixed-layer minerals (Chl/M), mica, and chlorite. These hydrothermal clay minerals change with depth from montmorillonite to chlorite and mica via Chl/M, suggesting temperature increased from 150 to 300°C with depths. TEM observation on Chl/M shows the presence of some 1.6nm phase (chlorite) among 2.8nm phase (Chl/M). Transformation of montmorillonite to chlorite is not simply due to temperature and/or chemical environmental change during their formation. Chlorite can precipitate instead of Chl/M, if the temperature of hydrothermal alteration increased due to rapid change of mixing ratios of hydrothermal fluid and seawater. Heterogeneous strontium concentration of anhydrite crystals, determined by X-ray fluorescence microscopic analysis, may suggest chemistry and temperature of the hydrothermal fluid was variable and a high degree of supersaturation existed for anhydrite precipitation.

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The active submarine hydrothermal field of Suiyo Seamount was directly drilled in order to understand the geological structure and hydrothermal processes at the subsurface region. A contrasting geological structure was found between the central and eastern areas. A thick accumulation of volcanoclastic sediments on the seafloor is a notable feature of the central area. These sediments were extensively altered by hydrothermal fluids and heavily cemented by anhydrite. Networks of hydrothermal veins and vesicles are developed in this cemented zone. This cementing process isolates the reducing subsurface environment from the oxic seawater environment. In contrast, less porous dacite lavas and breccia are main constituents of the eastern area. Hydrothermal fluids migrate along fractures developed in dacite lavas and breccia at the eastern hydrothermal area. Analyses of sulfur isotope compositions were performed on sulfide minerals separated from drilled core samples. Sulfur isotope compositions of the eastern samples (APSK01) are rather homogeneous, ranging from +1.0 to +1.3‰. These homogenized values suggest simple precipitation of sulfides using isotopically homogeneous H₂S. Sulfur isotope compositions of sulfides from the central area are heterogeneous, ranging from +0.9 to +5.3‰. This indicates that isotope exchange occurred directly or indirectly between seawater sulfate and hydrothermal sulfides. Such regional difference in sulfur isotope compositions is tightly related to the difference in subsurface geological structure between the eastern and central regions of the Suiyo Seamount.

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Deep-sea hydrothermal systems are natural laboratories for the study of organic geochemistry and microbial habitats in extreme environments. A high-temperature deep-sea hydrothermal system related to dacitic arc-volcanism was drilled using a tethered, submarine rock-drill system as part of the Archaean Park Project. The benthic multi-coring system (BMS) employed allowed for direct sampling of microorganisms, rocks and fluids beneath hydrothermal vents. Based on the vertical distribution of organic compounds derived from this vigorous sub-vent environment, a description of deep-sea subterranean chemistry and biology was determined detailing optimal microbial activities. Deep-sea hydrothermal sub-vent core samples of dacitic arc-volcanism obtained at the Suiyo Seamount, Izu-Bonin Arc, in the Western Pacific Ocean were analyzed for total organic carbon (TOC), total organic nitrogen (TON), hydrolyzed amino acids including their D/L ratios, and enzymatic activities such as alkaline and acid phosphatases. Amino acid distributions and their D/L ratios indicated that sub-vent organic matter was influenced by biogenic activity rather than by abiotic chemical synthesis. Useful biomarkers of acid phosphatase (ACP) and alkaline phosphatase (ALP) enzymatic activities were positively correlated with each other and were greatest at the partial middle core sequences; ACP and ALP activities were determined to be as high as 5.1 nmol min^-1 g-rock^-1 and 6.8 nmol min^-1 g-rock^-1, respectively. Biochemical indicators of ACP and ALP were consistent with the origin of total hydrolyzed amino acids (THAA) and the chiral ratio of D- and L-amino acid forms. The significant enzymatic activities demonstrated in this study provide crucial evidence that sub-vent regions represent an extreme-environment biosphere, extending the known subterranean habitable spaces of, for example, extremophilic microbes.

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The geochemical environment within active hydrothermal fields varies greatly, reflecting mixing of hydrothermal fluid and ambient cold seawater. Because chemosynthetic microorganisms utilize both reductive species in hydrothermal fluid and oxidants in seawater, description of the geochemical environment should provide important clues to activities of the microbiosphere within the hydrothermal fields. Specific devices have been developed to collect samples in order to study geochemical gradients in detail. Moreover, in situ chemical analyzers and chemical sensors for oceanographic use have made significant progress in monitoring time-series variations in the geochemical environment. During the Archaean Park Project (2000-2005), several instruments were developed and deployed in the Suiyo Seamount hydrothermal field. The results demonstrate that the geochemical environment above the seafloor in the active hydrothermal sites is strongly affected by bottom currents.

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The extent of biosphere in its ecological association with the seafloor hydrothermal system was studied based on the distribution and stable isotopic composition of organic matter in sediments, cores, and biological samples. The origins of organic matter were estimated by the following geochemical approach; i.e., primarily by the analysis of lipid compositions and their molecular-level isotopic ratios, as well as by bulk analysis of organic carbon content and isotopic ratios of each sample. We also developed two new sampling devices for the organic matter analyses. One is a sediment and rock scooper, which has a tight cap for preventing exposure to surface seawater. The other is a stand-alone in situ filtering system for concentrating dissolved organic matter in the hydrothermal fluid. We found large variations in isotopic signatures of organic carbon in bulk samples (δ¹³C=-12∿-31‰) and at the molecular-level (-22∿-31‰) in the core and seafloor sediment samples. Although the organic carbon content of core samples was very low, a large variation in bulk stable carbon isotopic ratios suggests that a high diversity of microbes inhabits the subvent environment.

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This paper summerizes the geochemical studies on a Suiyo Seamount hydrothermal plume during the Archaean Park project. Effluent hydrothermal water samples in and around the water column of the Suiyo Seamount caldera (ca. 2km diameter), Izu-Bonin arc, were taken to determine concentrations and stable carbon isotopic compositions (δ¹³C) of methane (CH₄) and carbon monoxide (CO) in the plume. As well, concentrations of Mn and Fe were determined in the hydrothermal plume using an in situ analyzer, together with the magnitude of light attenuation anomaly. Venting fluids on the caldera floor (ca. 1, 370m depth) were also examined, using the manned submersible "Shinkai 2000", to compare the chemical and isotopic composition with those in the hydrothermal plume. Strong CH₄, Mn, and Fe enrichments and light attenuation anomaly were detected not only in the water columns within the caldera, but also in those outside the caldera at a depth of ca. 1, 100m, the sill depth of the caldera wall. The hydrothermal plume that accmulates in the caldera may leak horizontally and episodically from the inside to the outside of the caldera through the three deepest saddle points on the caldera wall. From the δ¹³C tracer study, at least more than 70% of hydrothermal CH₄ escapes to the outside of the caldera without microbial oxidation, while ca. 50% of hydrothermal Mn is removed from the plume within the caldera, through microbial and/or inorganic oxidation. Within the plume, significant CO enrichment was also detected. The δ¹³C of CO in the plume, however, exhibited highly ¹³C-depleted values (-110 to -60‰_VPDB) compared with those in seafloor venting hydrothermal fluids (around -31‰_VPDB). The plume samples also exhibited higher CO/CH₄ ratios than those in seafloor venting hydrothermal fluids. Some unknown in situ microbial activity in the plume must be responsible for the CO enrichment in the hydrothermal plume.

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A strong water current flowing in a southwesterly direction over a 25 hour period was found near the bottom of the caldera. In addition, a downward current and water temperature decrease were observed during the period when the strong current was present. These results suggest that the low-temperature ocean water around the Suiyo Seamount flows toward the bottom of the caldera periodically. Vertical profiles of density in the caldera show that the vertical mixing layer is shifted and that the vertical mixing is attenuated on slack water. The turbidity profiles indicate the transport of hydrothermal particles and microorganisms from the bottom to mid-depths of the caldera by the vertical mixing. During long-term monitoring of pH at the hydrothermal active area in the caldera, decreases of 0.3-0.4 pH in about 25 hours were consistent with the observed water currents. According to the pH mapping of the hydrothermal area in the caldera, patchy low pH areas could be formed by the amalgamation of several hydrothermal plumes.

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Microbial ecosystems associated with hydrothermally active layers (e.g., hydrothermal plume, venting seafloor and sub-vent layers) inside the caldera of Suiyo Seamount (ca. 1, 000∿1, 400 m in the depth), Izu-Bonin Arc, were investigated in the framework of Archaean Park Project using a portable marine driller, manned and unmanned submersibles, water samplers, in situ filtration systems, and in situ growth chambers. Through quantitative population analysis of microbes in the hydrothermal plume, we found that this caldera has functioned as a natural continuous incubator for only two phylotypes of Bacteria in the deep sea. We also discovered several new phylotypes of microbes belonging to the epsilon-Proteobacteria, photosynthetic alpha-Proteobacteria, and chemoautotrophic thermophilic Archaea. These were found in samples collected from natural vents and boreholes using a newly developed catheter-type in situ growth chamber. From one of the growth chamber samples, a new dissimilatory sulfite reductase gene sequence was obtained. We also succeeded in identifying a gene region which regulates optimal growth temperatures of microbes, and we tried to apply this gene information to analysis of natural microbial communities.

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Every ecosystem on the Earth depends mainly on the free energy continuously supplied by sunlight. However, there is a chemoautotrophically supported ecosystem around the marine hydrothermal vent systems, which is dependent on the energy source of reducing compounds in the hydrothermal water. Microbial analysis was carried out on hydrothermal water collected from the casing pipes inserted into the bored holes in the caldera of the Suiyo Seamount, which were drilled as part of the Archaean Park Project. The results suggested the presence of a microbial ecosystem based on hydrogen-dependent hyperthermophilic Archaea and sulfur-dependent chemoautotrophic bacteria as primary producers, and the possible presence of a chemoautotrophically supported ecosystem under the seafloor. Based on the hypothesis that the last common ancestor, Commonote, is a hyperthermophile, the sub-seafloor around the hydrothermal area may retain an ecosystem that developed early in the evolutionary history of life.

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Quite a few microorganisms have been discovered in extreme environments such as hydrothermal vents, hot springs and deep subsurface environments, but a large number of extremophiles in these environments have still remained uncultured. We are searching for those unknown organisms hidden in extreme environments. In our attempts, we have successfully isolated several new extremophiles. One of the isolates, Thermanaeromonas toyohensis, is a spore-forming strictly anaerobic bacterium growing at 70°C in subsurface environments. Its most unique property is that it can use formate, the simplest organic acid, as a sole energy and carbon source in the presence of a suitable electron sink. Formate can be synthesized abiotically in thermal subsurface environments. The ability to use formate is advantageous for living in those subsurface environments that lack more complex organic compounds. Another isolate, Thermodesulfobium narugense, is a thermophilic sulfate-reducing bacterium isolated from a Japanese hot spring (Narugo hot spring in Miyagi). The bacterium is not phylogenetically related to any known sulfate-reducers, and we have proposed a new family, Thermodesulfobiaceae, for the isolate. This isolate occupies an interesting and important phylogenetic position in considering an evolutionary pathway of sulfate-reduction that has remained mysterious. The other isolate, Oceanithermus desulfurans, was found in a deep-sea hydrothermal vent. The new isolate is a thermophilic short rod which grows well under micro-aerobic conditions (less than 5% oxygen tension). It can also grow by reducing sulfur under anaerobic conditions. Generally, Thermus relatives do not use sulfur as an electron acceptor. This is the first report on sulfur-reduction by a Thermus-related species. We also succeeded in isolating a new thiosulfate-reducing archaeon from a hydrothermal vent. The phylogenetic analysis indicated that the isolate belongs to the genus Archaeoglobus, and should be classified as a new species in the genus. The new archaeon may play a significant role in the deep-sea sulfur cycle with other thermophilic sulfur reducers and oxidizers.

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Euryhaline halophiles are microorganisms that grow in a wide range of saline conditions, from low salinities (about 3%NaCl) to high salinities which prohibit the growth of many marine microorganisms. For that reason, this communication characterizes euryhaline halophiles as growing over a salinity range of 15% (NaCl equivalent) or greater. Most of the known euryhaline halophiles use organic matter as osmotic-compatible solutes; hence extremely halophilic archaea use contrastive inorganic ions. Certain organic osmotic-compatible solutes used by euryhaline halophiles have additional functions of increasing the stress tolerance of cells or enzymes against temperature, hydrostatic pressure, pH, dryness, etc. These special attributes allow euryhaline halophiles to inhabit various extreme environments. Particularly, hydrothermal vents form sub-seafloor "salinity mosaics" of low-and high-salinity waters via two-phase separation of super-heated seawater. Sub-vent "salinity mosaics" provide isolated habitats for euryhaline halophiles to undergo unique evolution within each vent area. This communication describes the phylogeny of representative euryhaline halophiles from the Suiyo Seamount, Izu-Bonin Arc, in comparison with halophiles from other "salinity mosaics", from the viewpoint of phylogeography.

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Many people are interested in marine extreme environments, such as deep-sea hydrothermal vents and sub-vent regions, in order to discover new biological phenomena and economically invaluable bio/gene resources. However, the difficulty in approaching and collecting samples from such extreme environments has prevented further progress in bio-science and bio-technology. Taking advantage of an opportunity of drilling in the vicinity of deep-sea hydrothermal vents in the Archaean Park Project, we started to develop some new instruments and methods such as large-scale in situ filtration systems for aseptic drilling and advanced genomic research, and catheter-and column-type in situ growth chambers for finding new sub-vent microbes and genes. As a result, we managed to concentrate microbes from more than 200 liters of hydrothermal fluid and found several new gene sequences in DNA samples purified from the concentrates. Invaluable sub-vent microbial samples were also successfully collected with the in situ growth chambers from the inside of warm to extremely hot boreholes. To date, we have found several new phylotypes of microbes and very unique distribution patterns of those in the sub-vent. Further innovation in sampling and analysis may also provide a means of elucidating mutual relationships between geological and microbiological processes, as well as leading to further findings of novel microbes and genes.

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The purpose of this study was to develop an automated in situ gene analysis device for use in extreme environmental microbiology. Bacteria or Archaea inhabiting extreme environments such as the deep-sea or the deep subsurface are major targets of gene analysis using this device. By developing a small, fully automated and integrated gene analysis device, a small ROV (Remotely Operated Vehicle) or AUV (Autonomous Underwater Vehicle) can bring the device into underwater extreme environments to analyze microbial communities without risk of contamination and time lag problems. Since a long-term analysis operation will also be available, dynamic properties of microflora can be revealed. In this study, "microfabrication techniques" originated in the field of semiconductor or micromachine engineering are applied to develop an in situ gene analysis device that is capable of flow-through PCR (polymerase chain reaction) analyses. The device is composed of a glass-based "temperature control chip", a silicone rubber-based "microchannel chip" and a pumping system. Microfabrication techniques enable the device to be miniaturized and functionally integrated. Results of evaluation experiments indicate that the developed device can amplify targeted specific DNA fragments from E. coli cells. Furthermore, flow-through PCR operations have been tried under high static pressure conditions up to 30 MPa to simulate analysis in deep-sea environments. PCR is thus possible under high pressure, and the possibility of gene analysis in deep-sea or deep subsurface environments is demonstrated.

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