Manganese(II)-Oxidizing Bacillus Spores in Guaymas Basin Hydrothermal Sediments and Plumes

Microbial oxidation and precipitation of manganese at deep-sea hydrothermal vents are important oceanic biogeochemical processes, yet nothing is known about the types of microorganisms or mechanisms involved. Here we report isolation of a number of diverse spore-forming Mn(II)-oxidizing Bacillus species from Guaymas Basin, a deep-sea hydrothermal vent environment in the Gulf of California, where rapid microbially mediated Mn(II) oxidation was previously observed. mnxG multicopper oxidase genes involved in Mn(II) oxidation were amplified from all Mn(II)-oxidizing Bacillus spores isolated, suggesting that a copper-mediated mechanism of Mn(II) oxidation could be important at deep-sea hydrothermal vents. Phylogenetic analysis of 16S rRNA and mnxG genes revealed that while many of the deep-sea Mn(II)-oxidizing Bacillus species are very closely related to previously recognized isolates from coastal sediments, other organisms represent novel strains and clusters. The growth and Mn(II) oxidation properties of these Bacillus species suggest that in hydrothermal sediments they are likely present as spores that are active in oxidizing Mn(II) as it emerges from the seafloor.     

Recovery of Benthic Megafauna from Anthropogenic Disturbance at a Hydrocarbon Drilling Well (380 m Depth in the Norwegian Sea)

Recovery from disturbance in deep water is poorly understood, but as anthropogenic impacts increase in deeper water it is important to quantify the process. Exploratory hydrocarbon drilling causes physical disturbance, smothering the seabed near the well. Video transects obtained by remotely operated vehicles were used to assess the change in invertebrate megafaunal density and diversity caused by drilling a well at 380 m depth in the Norwegian Sea in 2006. Transects were carried out one day before drilling commenced and 27 days, 76 days, and three years later. A background survey, further from the well, was also carried out in 2009. Porifera (45% of observations) and Cnidaria (40%) dominated the megafauna. Porifera accounted for 94% of hard-substratum organisms and cnidarians (Pennatulacea) dominated on the soft sediment (78%). Twenty seven and 76 days after drilling commenced, drill cuttings were visible, extending over 100 m from the well. In this area there were low invertebrate megafaunal densities (0.08 and 0.10 individuals m⁻²) in comparison to pre-drill conditions (0.21 individuals m⁻²). Three years later the visible extent of the cuttings had reduced, reaching 60 m from the well. Within this area the megafaunal density (0.05 individuals m⁻²) was lower than pre-drill and reference transects (0.23 individuals m⁻²). There was a significant increase in total megafaunal invertebrate densities with both distance from drilling and time since drilling although no significant interaction. Beyond the visible disturbance there were similar megafaunal densities (0.14 individuals m⁻²) to pre-drilling and background surveys. Species richness, Shannon-Weiner diversity and multivariate techniques showed similar patterns to density. At this site the effects of exploratory drilling on megafaunal invertebrate density and diversity seem confined to the extent of the visible cuttings pile. However, elevated Barium concentration and reduced sediment grain size suggest persistence of disturbance for three years, with unclear consequences for other components of the benthic fauna.     

Phylogenetic Diversity of Nitrogenase (nifH) Genes in Deep-Sea and Hydrothermal Vent Environments of the Juan de Fuca Ridge

The subseafloor microbial habitat associated with typical unsedimented mid-ocean-ridge hydrothermal vent ecosystems may be limited by the availability of fixed nitrogen, inferred by the low ammonium and nitrate concentrations measured in diffuse hydrothermal fluid. Dissolved N₂ gas, the largest reservoir of nitrogen in the ocean, is abundant in deep-sea and hydrothermal vent fluid. In order to test the hypothesis that biological nitrogen fixation plays an important role in nitrogen cycling in the subseafloor associated with unsedimented hydrothermal vents, degenerate PCR primers were designed to amplify the nitrogenase iron protein gene nifH from hydrothermal vent fluid. A total of 120 nifH sequences were obtained from four samples: a nitrogen-poor diffuse vent named marker 33 on Axial Volcano, sampled twice over a period of 1 year as its temperature decreased; a nitrogen-rich diffuse vent near Puffer on Endeavour Segment; and deep seawater with no detectable hydrothermal plume signal. Subseafloor nifH genes from marker 33 and Puffer are related to anaerobic clostridia and sulfate reducers. Other nifH genes unique to the vent samples include proteobacteria and divergent Archaea. All of the nifH genes from the deep-seawater sample are most closely related to the thermophilic, anaerobic archaeon Methanococcus thermolithotrophicus (77 to 83% amino acid similarity). These results provide the first genetic evidence of potential nitrogen fixers in hydrothermal vent environments and indicate that at least two sources contribute to the diverse assemblage of nifH genes detected in hydrothermal vent fluid: nifH genes from an anaerobic, hot subseafloor and nifH genes from cold, oxygenated deep seawater.     

Nematode succession at deep-sea hydrothermal vents after a recent volcanic eruption with the description of two dominant species

Nematodes are very common in the deep sea and are an important component of deep-sea hydrothermal vent communities. In early 2006, the eruption of the underwater volcano at 9°50’N East Pacific Rise wiped out almost the entire faunal communities of the area. This provided us with the opportunity to study nematode primary succession at vents as well as on adjacent seafloor basalt. Nematode abundance and richness were extremely low at all studied sites in late 2006 and 2007, and increased only slightly in 2009. Interestingly, the most abundant species during early succession were also prominent in this area prior to the eruption. Our results show that nematodes are extremely influenced by volcanic eruptions and need a long period of time to colonize the lava-flooded area in greater numbers and richness. We hypothesize that low food availability on the young bare basalt and harsh environmental conditions at early succession vent sites might hinder a more successful nematode establishment. In addition to the newly established active vent sites we also studied an inactive vent site that was not directly hit by the eruption but whose vent fluid had ceased after the eruption. At this inactive and older vent, diversity was also relatively low but was higher than at the younger, newly established sites. In addition to the ecological analyses, we here describe the two most abundant species found at inactive vents, namely Neochromadora aff. poecilosoma De Mann 1893 and Linhomoeus caudipapillosus sp. n.     

Mineralogy Drives Bacterial Biogeography of Hydrothermally Inactive Seafloor Sulfide Deposits

Mid-ocean ridge hydrothermal venting creates sulfide deposits containing gradients in mineralogy, fluid chemistry, and temperature. Even when hydrothermal circulation ceases, sulfides are known to host microbial communities. The relationship between mineralogy and microbial community composition in low-temperature, rock-hosted systems has not been resolved at any spatial scale, local or global. To examine the hypothesis that geochemistry of seafloor deposits is a dominant parameter driving environmental pressure for bacterial communities at low-temperature, the shared community membership, richness, and structure was measured using 16S rRNA gene sequences. The focus of the study was on hydrothermally inactive seafloor deposits from multiple locations within one deposit (e.g., single extinct chimney), within one vent field (intra-vent field), and among globally distributed vent fields from three ocean basins (inter-vent field). Distinct mineral substrates, such as hydrothermally inactive sulfides versus basalts, host different communities at low temperature in spite of close geographic proximity and contact with the same hydrothermally influenced deep-sea water. Furthermore, bacterial communities inhabiting hydrothermally inactive sulfide deposits from geographically distant locations cluster together in community cladograms to the exclusion of other deep-sea substrates and settings. From this study, we conclude that at low temperature, mineralogy was a more important variable determining microbial community composition than geographic factors. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the supplemental file.     

Bacteria dominate the ammonia-oxidizing community in a hydrothermal vent site at the Mid-Atlantic Ridge of the South Atlantic Ocean

Ammonia oxidation is the first and rate-limiting step of nitrification, which is carried out by two groups of microorganisms: ammonia-oxidizing bacteria (AOB) and the recently discovered ammonia-oxidizing archaea (AOA). In this study, diversity and abundance of AOB and AOA were investigated in five rock samples from a deep-sea hydrothermal vent site at the Mid-Atlantic Ridge (MAR) of the South Atlantic Ocean. Both bacterial and archaeal ammonia monooxygenase subunit A (amoA) gene sequences obtained in this study were closely related to the sequences retrieved from deep-sea environments, indicating that AOB and AOA in this hydrothermal vent site showed typical deep ocean features. AOA were more diverse but less abundant than AOB. The ratios of AOA/AOB amoA gene abundance ranged from 1/3893 to 1/242 in all investigate samples, indicating that bacteria may be the major members responding to the aerobic ammonia oxidation in this hydrothermal vent site. Furthermore, diversity and abundance of AOA and AOB were significantly correlated with the contents of total nitrogen and total sulfur in investigated samples, suggesting that these two environmental factors exert strong influences on distribution of ammonia oxidizers in deep-sea hydrothermal vent environment.     

Microbial Community Stratification Controlled by the Subseafloor Fluid Flow and Geothermal Gradient at the Iheya North Hydrothermal Field in the Mid-Okinawa Trough (Integrated Ocean Drilling Program Expedition 331)

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.     

Archaeal Diversity and Distribution along Thermal and Geochemical Gradients in Hydrothermal Sediments at the Yonaguni Knoll IV Hydrothermal Field in the Southern Okinawa Trough

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°C). A moderate temperature gradient extends both horizontally and vertically (5 to 69°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.Deep-sea hydrothermal activity results in diverse physical and chemical environments for the resident microbial communities. Using cultivation techniques and culture-independent molecular analyses, diverse lineages of archaea and bacteria have so far been observed from chimney structures, retrieved in situ colonization systems settled in or on the hydrothermal conduit, microbial mats, sediments, and chemosynthetic macrofaunal bodies (19, 35, 62). Especially in the domain Archaea, most of lineages derived from hydrothermal environments have not yet been cultivated, and little is known about their physiological and metabolic traits.Environmental conditions of the habitat for a particular uncultivated archaeal lineage permit us to speculate about the physiological and metabolic traits of the archaea. For instance, the acidophilic and thermophilic archaeon “Aciduliprofundum boonei,” representing the previously uncultivated deep-sea hydrothermal vent euryarchaeotic group I (DHVEG I) subgroup 2 (DHVE2), has been isolated from a chimney habitat in the Lau Basin (49). In fact, before the cultivation of A. boonei, the DHVE2 was assumed to consist of thermophilic and acidophilic heterotrophs because their habitats had similar characteristics (13, 48, 60, 68). In order to elucidate the distribution patterns of the functionally unknown microbial components in response to the dynamically varying physico-chemical conditions, hydrothermally influenced sediments are considered better study targets than hot vent chimney structures to determine the eco-physiological roles of uncultivated microbes. This is because, unlike vent chimneys, sedimentary habitats affected by subseafloor hydrothermal fluid are expected to have more moderate physico-chemical gradients from mixing of hydrothermal fluid and ambient seawater due to the relatively lower heat convection and hydrothermal fluid penetration. Several studies have already examined the phylogenetic diversity of archaea and bacteria in hydrothermal sediments from the Guaymas Basin (7, 66), the Rainbow vent field in the Mid-Atlantic Ridge (39), and the Iheya Ridge and the Yonaguni Knoll IV in the Okinawa Trough (14, 57). However, only the relationship between the distribution pattern of microbial components and the physico-chemical conditions of these environments has been addressed.The Yonaguni Knoll IV hydrothermal field located at the southern end of the Okinawa Trough is characterized as having thick sediment, several Cl-enriched black smoker sites, and numerous vapor-enriched clear fluid sites (25, 56). The geochemical characterization of these hydrothermal fluids revealed that hydrothermal fluids undergo phase separation under the seafloor (25, 56). Furthermore, the emission of liquid CO₂ droplets has been reported, and occurrence of subseafloor CO₂ hydrate is assumed to have arisen in response to pore water chemistry in the sediments at liquid CO₂ emission sites (14, 25). According to pore water chemistry, it seems likely that these vapor-enriched hydrothermal fluids permeate the sediments around hydrothermal vent sites, and the subseafloor formation-dissociation processes of gas hydrates produce a variety of hydrothermally affected sedimentary habitats (25).In this study, we focused on the “abyss vent” site, which is characterized by 90°C hydrothermal emissions that discharge directly from the seafloor sediments (56). Sediment cores (>25 cm in length) were taken at horizontal distances of 0.5, 2, and 5 m from the hydrothermal emission while the in situ temperature of sediments was measured simultaneously. Vent fluids and interstitial water chemistry of the sediments were characterized along vertical and horizontal gradients of subseafloor mixing zones. Microbial distributions, particularly of archaea, were ascertained by culture-independent molecular analyses targeting the small subunit (SSU) rRNA gene and, mcrA (gene for methyl coenzyme A [CoA] reductase subunit A) and archaeal amoA (gene for ammonia monooxygenase large subunit). Molecular analyses for the functional genes, mcrA and amoA, are expected to indicate diversity and abundance of methanogens, anaerobic methanotrophs, and archaeal ammonium oxidizers that utilize hydrogen, methane, and ammonium, respectively, in hydrothermal fluids as electron donors. In addition, we inferred the phylogenetic diversity and distribution patterns of the bacterial SSU rRNA genes that provide insight into the potential metabolic characteristics and microbial ecosystems in each habitat.     

Hidden Historical Habitat-Linked Population Divergence and Contemporary Gene Flow of a Deep-Sea Patellogastropod Limpet

Hydrothermal vents and hydrocarbon seeps in the deep ocean are rare oases fueled by chemosynthesis. Biological communities inhabiting these ecosystems are often distributed in widely separated habitats, raising intriguing questions on how these organisms achieve connectivity and whether habitat types facilitate intraspecific divergence. The deep-sea patellogastropod limpet Bathyacmaea nipponica that colonizes both vents and seeps across ∼2,400 km in the Northwest Pacific is a feasible model to answer these questions. We analyzed 123 individuals from four vents and three seeps using a comprehensive method incorporating population genomics and physical ocean modeling. Genome survey sequencing and genotyping-by-sequencing resulted in 9,838 single-nucleotide polymorphisms for population genomic analyses. Genetic divergence and demographic analyses revealed four habitat-linked (i.e., three seep and one vent) genetic groups, with the vent genetic group established via the opportunistic invasion of a few limpet larvae from a nearby seep genetic group. TreeMix analysis uncovered three historical seep-to-vent migration events. ADMIXTURE and divMigrate analyses elucidated weak contemporary gene flow from a seep genetic group to the vent genetic group. Physical ocean modeling underlined the potential roles of seafloor topography and ocean currents in shaping the genetic connectivity, contemporary migration, and local hybridization of these deep-sea limpets. Our study highlighted the power of integrating genomic and oceanographic approaches in deciphering the demography and diversification of deep-sea organisms. Given the increasing anthropogenic activities (e.g., mining and gas hydrate extraction) affecting the deep ocean, our results have implications for the conservation of deep-sea biodiversity and establishment of marine protected areas.     

Deep-sea hydrothermal vent Epsilonproteobacteria encode a conserved and widespread nitrate reduction pathway (Nap)

Despite the frequent isolation of nitrate-respiring Epsilonproteobacteria from deep-sea hydrothermal vents, the genes coding for the nitrate reduction pathway in these organisms have not been investigated in depth. In this study we have shown that the gene cluster coding for the periplasmic nitrate reductase complex (nap) is highly conserved in chemolithoautotrophic, nitrate-reducing Epsilonproteobacteria from deep-sea hydrothermal vents. Furthermore, we have shown that the napA gene is expressed in pure cultures of vent Epsilonproteobacteria and it is highly conserved in microbial communities collected from deep-sea vents characterized by different temperature and redox regimes. The diversity of nitrate-reducing Epsilonproteobacteria was found to be higher in moderate temperature, diffuse flow vents than in high temperature black smokers or in low temperatures, substrate-associated communities. As NapA has a high affinity for nitrate compared with the membrane-bound enzyme, its occurrence in vent Epsilonproteobacteria may represent an adaptation of these organisms to the low nitrate concentrations typically found in vent fluids. Taken together, our findings indicate that nitrate reduction is widespread in vent Epsilonproteobacteria and provide insight on alternative energy metabolism in vent microorganisms. The occurrence of the nap cluster in vent, commensal and pathogenic Epsilonproteobacteria suggests that the ability of these bacteria to respire nitrate is important in habitats as different as the deep-sea vents and the human body.     

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.     

Phylogenetic diversity of nitrogenase (nifH) genes in deep-sea and hydrothermal vent environments of the Juan de Fuca Ridge

The subseafloor microbial habitat associated with typical unsedimented mid-ocean-ridge hydrothermal vent ecosystems may be limited by the availability of fixed nitrogen, inferred by the low ammonium and nitrate concentrations measured in diffuse hydrothermal fluid. Dissolved N2 gas, the largest reservoir of nitrogen in the ocean, is abundant in deep-sea and hydrothermal vent fluid. In order to test the hypothesis that biological nitrogen fixation plays an important role in nitrogen cycling in the subseafloor associated with unsedimented hydrothermal vents, degenerate PCR primers were designed to amplify the nitrogenase iron protein gene nifH from hydrothermal vent fluid. A total of 120 nifH sequences were obtained from four samples: a nitrogen-poor diffuse vent named marker 33 on Axial Volcano, sampled twice over a period of 1 year as its temperature decreased; a nitrogen-rich diffuse vent near Puffer on Endeavour Segment; and deep seawater with no detectable hydrothermal plume signal. Subseafloor nifH genes from marker 33 and Puffer are related to anaerobic clostridia and sulfate reducers. Other nifH genes unique to the vent samples include proteobacteria and divergent ARCHAEA: All of the nifH genes from the deep-seawater sample are most closely related to the thermophilic, anaerobic archaeon Methanococcus thermolithotrophicus (77 to 83% amino acid similarity). These results provide the first genetic evidence of potential nitrogen fixers in hydrothermal vent environments and indicate that at least two sources contribute to the diverse assemblage of nifH genes detected in hydrothermal vent fluid: nifH genes from an anaerobic, hot subseafloor and nifH genes from cold, oxygenated deep seawater.     

Geochemical and Visual Indicators of Hydrothermal Fluid Flow through a Sediment-Hosted Volcanic Ridge in the Central Bransfield Basin (Antarctica)

In the austral summer of 2011 we undertook an investigation of three volcanic highs in the Central Bransfield Basin, Antarctica, in search of hydrothermal activity and associated fauna to assess changes since previous surveys and to evaluate the extent of hydrothermalism in this basin. At Hook Ridge, a submarine volcanic edifice at the eastern end of the basin, anomalies in water column redox potential (E_h) were detected close to the seafloor, unaccompanied by temperature or turbidity anomalies, indicating low-temperature hydrothermal discharge. Seepage was manifested as shimmering water emanating from the sediment and from mineralised structures on the seafloor; recognisable vent endemic fauna were not observed. Pore fluids extracted from Hook Ridge sediment were depleted in chloride, sulfate and magnesium by up to 8% relative to seawater, enriched in lithium, boron and calcium, and had a distinct strontium isotope composition (⁸⁷Sr/⁸⁶Sr  = 0.708776 at core base) compared with modern seawater (⁸⁷Sr/⁸⁶Sr ≈0.70918), indicating advection of hydrothermal fluid through sediment at this site. Biogeochemical zonation of redox active species implies significant moderation of the hydrothermal fluid with in situ diagenetic processes. At Middle Sister, the central ridge of the Three Sisters complex located about 100 km southwest of Hook Ridge, small water column E_h anomalies were detected but visual observations of the seafloor and pore fluid profiles provided no evidence of active hydrothermal circulation. At The Axe, located about 50 km southwest of Three Sisters, no water column anomalies in E_h, temperature or turbidity were detected. These observations demonstrate that the temperature anomalies observed in previous surveys are episodic features, and suggest that hydrothermal circulation in the Bransfield Strait is ephemeral in nature and therefore may not support vent biota.     

Microdistribution of Faunal Assemblages at Deep-Sea Hydrothermal Vents in the Southern Ocean

Chemosynthetic primary production by microbes supports abundant faunal assemblages at deep-sea hydrothermal vents, with zonation of invertebrate species typically occurring along physico-chemical gradients. Recently discovered vent fields on the East Scotia Ridge (ESR) in the Southern Ocean represent a new province of vent biogeography, but the spatial dynamics of their distinct fauna have yet to be elucidated. This study determines patterns of faunal zonation, species associations, and relationships between faunal microdistribution and hydrothermal activity in a vent field at a depth of 2,400 m on the ESR. Remotely operated vehicle (ROV) dives obtained high-definition imagery of three chimney structures with varying levels of hydrothermal activity, and a mosaic image of >250 m² of seafloor co-registered with temperature measurements. Analysis of faunal microdistribution within the mosaiced seafloor reveals a consistent pattern of faunal zonation with increasing distance from vent sources and peak temperatures. Assemblages closest to vent sources are visibly dominated by a new species of anomuran crab, Kiwa n. sp. (abundance >700 individuals m⁻²), followed by a peltospiroid gastropod (>1,500 individuals m⁻²), eolepadid barnacle (>1,500 individuals m⁻²), and carnivorous actinostolid anemone (>30 individuals m⁻²). Peripheral fauna are not dominated by a single taxon, but include predatory and scavenger taxa such as stichasterid seastars, pycnogonids and octopus. Variation in faunal microdistribution on chimneys with differing levels of activity suggests a possible successional sequence for vent fauna in this new biogeographic province. An increase in δ³⁴S values of primary consumers with distance from vent sources, and variation in their δ¹³C values also indicate possible zonation of nutritional modes of the vent fauna. By using ROV videography to obtain a high-resolution representation of a vent environment over a greater extent than previous studies, these results provide a baseline for determining temporal change and investigations of processes structuring faunal assemblages at Southern Ocean vents.     

Manganese(II)-oxidizing Bacillus spores in Guaymas Basin hydrothermal sediments and plumes

Microbial oxidation and precipitation of manganese at deep-sea hydrothermal vents are important oceanic biogeochemical processes, yet nothing is known about the types of microorganisms or mechanisms involved. Here we report isolation of a number of diverse spore-forming Mn(II)-oxidizing Bacillus species from Guaymas Basin, a deep-sea hydrothermal vent environment in the Gulf of California, where rapid microbially mediated Mn(II) oxidation was previously observed. mnxG multicopper oxidase genes involved in Mn(II) oxidation were amplified from all Mn(II)-oxidizing Bacillus spores isolated, suggesting that a copper-mediated mechanism of Mn(II) oxidation could be important at deep-sea hydrothermal vents. Phylogenetic analysis of 16S rRNA and mnxG genes revealed that while many of the deep-sea Mn(II)-oxidizing Bacillus species are very closely related to previously recognized isolates from coastal sediments, other organisms represent novel strains and clusters. The growth and Mn(II) oxidation properties of these Bacillus species suggest that in hydrothermal sediments they are likely present as spores that are active in oxidizing Mn(II) as it emerges from the seafloor.     

Growth and Phylogenetic Properties of Novel Bacteria Belonging to the Epsilon Subdivision of the Proteobacteria Enriched from Alvinella pompejana and Deep-Sea Hydrothermal Vents

Recent molecular characterizations of microbial communities from deep-sea hydrothermal sites indicate the predominance of bacteria belonging to the epsilon subdivision of Proteobacteria (epsilon Proteobacteria). Here, we report the first enrichments and characterizations of four epsilon Proteobacteria that are directly associated with Alvinella pompejana, a deep sea hydrothermal vent polychete, or with hydrothermal vent chimney samples. These novel bacteria were moderately thermophilic sulfur-reducing heterotrophs growing on formate as the energy and carbon source. In addition, two of them (Am-H and Ex-18.2) could grow on sulfur lithoautrotrophically using hydrogen as the electron donor. Optimal growth temperatures of the bacteria ranged from 41 to 45°C. Phylogenetic analysis of the small-subunit ribosomal gene of the two heterotrophic bacteria demonstrated 95% similarity to Sulfurospirillum arcachonense, an epsilon Proteobacteria isolated from an oxidized marine surface sediment. The autotrophic bacteria grouped within a deeply branching clade of the epsilon Proteobacteria, to date composed only of uncultured bacteria detected in a sample from a hydrothermal vent along the mid-Atlantic ridge. A molecular survey of various hydrothermal vent environments demonstrated the presence of two of these bacteria (Am-N and Am-H) in more than one geographic location and habitat. These results suggest that certain epsilon Proteobacteria likely fill important niches in the environmental habitats of deep-sea hydrothermal vents, where they contribute to overall carbon and sulfur cycling at moderate thermophilic temperatures.     

Culture-Dependent and -Independent Characterization of Microbial Communities Associated with a Shallow Submarine Hydrothermal System Occurring within a Coral Reef off Taketomi Island, Japan

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, ~23 m) on the coral reef seafloor. The vent fluid (maximum temperature, >52°C) contained 175 μM 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°C, and another novel type I methanotroph exhibited optimum growth at 45°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.     

Caging experiment in the deep sea: Efficiency and artefacts from a case study at the Arctic long-term observatory HAUSGARTEN

Present theories of deep-sea community organization recognize the importance of small-scale biological disturbances, originated partly from the activities of epibenthic megafaunal organisms, in maintaining high benthic biodiversity in the deep sea. However, due to technical difficulties, in situ experimental studies to test hypotheses in the deep sea are lacking. The objective of the present study was to evaluate the potential of cages as tools for studying the importance of epibenthic megafauna for deep-sea benthic communities. Using the deep-diving Remotely Operated Vehicle (ROV) “VICTOR 6000”, six experimental cages were deployed at the sea floor at 2500 m water depth and sampled after 2 years (2y) and 4 years (4y) for a variety of sediment parameters in order to test for caging artefacts. Photo and video footage from both experiments showed that the cages were efficient at excluding the targeted fauna. The cage also proved to be appropriate to deep-sea studies considering the fact that there was no fouling on the cages and no evidence of any organism establishing residence on or adjacent to it. Environmental changes inside the cages were dependent on the experimental period analysed. In the 4y experiment, chlorophyll a concentrations were higher in the uppermost centimeter of sediment inside cages whereas in the 2y experiment, it did not differ between inside and outside. Although the cages caused some changes to the sedimentary regime, they are relatively minor compared to similar studies in shallow water. The only parameter that was significantly higher under cages at both experiments was the concentration of phaeopigments. Since the epibenthic megafauna at our study site can potentially affect phytodetritus distribution and availability at the seafloor (e.g. via consumption, disaggregation and burial), we suggest that their exclusion was, at least in part, responsible for the increases in pigment concentrations. Cages might be suitable tools to study the long-term effects of disturbances caused by megafaunal organisms on the diversity and community structure of smaller-sized organisms in the deep sea, although further work employing partial cage controls, greater replication, and evaluating faunal components will be essential to unequivocally establish their utility.     

Detection of Putatively Thermophilic Anaerobic Methanotrophs in Diffuse Hydrothermal Vent Fluids

The anaerobic oxidation of methane (AOM) is carried out by a globally distributed group of uncultivated Euryarchaeota, the anaerobic methanotrophic arachaea (ANME). In this work, we used G+C analysis of 16S rRNA genes to identify a putatively thermophilic ANME group and applied newly designed primers to study its distribution in low-temperature diffuse vent fluids from deep-sea hydrothermal vents. We found that the G+C content of the 16S rRNA genes (P_GC) is significantly higher in the ANME-1GBa group than in other ANME groups. Based on the positive correlation between the P_GC and optimal growth temperatures (T_opt) of archaea, we hypothesize that the ANME-1GBa group is adapted to thrive at high temperatures. We designed specific 16S rRNA gene-targeted primers for the ANME-1 cluster to detect all phylogenetic groups within this cluster, including the deeply branching ANME-1GBa group. The primers were successfully tested both in silico and in experiments with sediment samples where ANME-1 phylotypes had previously been detected. The primers were further used to screen for the ANME-1 microorganisms in diffuse vent fluid samples from deep-sea hydrothermal vents in the Pacific Ocean, and sequences belonging to the ANME-1 cluster were detected in four individual vents. Phylotypes belonging to the ANME-1GBa group dominated in clone libraries from three of these vents. Our findings provide evidence of existence of a putatively extremely thermophilic group of methanotrophic archaea that occur in geographically and geologically distinct marine hydrothermal habitats.