Michinari Sunamura

Two Bacteria Phylotypes Are Predominant in the Suiyo Seamount Hydrothermal Plume

ABSTRACT Microbial diversity and populations in a hydrothermal plume that was present inside the caldera of the Suiyo Seamount, a submarine volcano on the Izu-Bonin Arc, were investigated by performing a phylogenetic analysis of the 16S rRNA gene and by using fluorescence in situ hybridization (FISH). Corresponding to transmissivity, an indicator of turbidity, the vertical total cell count as determined by 4′,6′-diamidino-2-phenylindole (DAPI) staining varied from 5.6 × 104 to 1.1 × 105 cells ml−1, and the apparent plume layer was assessed to be at a depth of 1,050 to 1,200 m inside the caldera and to contain 1.0 × 105 to 1.1 × 105 cells ml−1. From microbial samples collected in the plume by an in situ filtration system, the following two major phylogenetic groups, which were closely related to sulfur-oxidizing microbes, were obtained: the SUP05 group belonging to the gamma subclass of the Proteobacteria (13 of 20 clones) and the SUP01 group belonging to the epsilon subclass of the Proteobacteria (5 of 20 clones). Specific oligonucleotide probes for these groups (SUP05-187 and SUP01-63) were designed and were used with various water samples obtained from the Suiyo Seamount. In the apparent plume layer, up to 66% of the total counts of microbial cells were estimated to be Bacteria cells that hybridized to EUB338, and few cells were identified by the archaeal probe ARCH915. Almost all Bacteria cells were hard to identify with the known group-specific probes, such as ALF19, GAM42a, and CF319, while 88 to 90% of the Bacteria cells hybridized with SUP05-187 and >98% of them were considered members of the SUP05 and SUP01 populations. In a low-temperature vent fluid emitted from a bivalve-colonized mound, the SUP05 cells accounted for >99% of the Bacteria cells, suggesting that a portion of the plume cells originated on the surface of the seafloor at a depth of about 1,380 m. From further analysis of cell morphology (i.e., cell size and cell elongation index) we inferred that the SUP05 cells were active in the plume layer at a depth of 1,050 to 1,200 m compared to the activity in a near-bottom layer, while many elongated cells were found between these layers. These findings suggest that the morphology and distribution of SUP05 cells have complex relationships with hydrothermal activities and water circulation. Although growth and production rates remain to be defined, we concluded that this Suiyo Seamount caldera has functioned as a natural continuous incubator for these two phylotypes of Bacteria in an aphotic deep-sea environment.

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

ABSTRACT 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.

Abundance of Zetaproteobacteria within crustal fluids in back-arc hydrothermal fields of the Southern Mariana Trough

To extend knowledge of subseafloor microbial communities within the oceanic crust, the abundance, diversity and composition of microbial communities in crustal fluids at back-arc hydrothermal fields of the Southern Mariana Trough (SMT) were investigated using culture-independent molecular techniques based on 16S rRNA gene sequences. Seafloor drilling was carried out at two hydrothermal fields, on- and off-ridge of the back-arc spreading centre of the SMT. 16S rRNA gene clone libraries for bacterial and archaeal communities were constructed from the fluid samples collected from the boreholes. Phylotypes related to Thiomicrospira in the Gammaproteobacteria (putative sulfide-oxidizers) and Mariprofundus in the Zetaproteobacteria (putative iron-oxidizers) were recovered from the fluid samples. A number of unique archaeal phylotypes were also recovered. Fluorescence in situ hybridization (FISH) analysis indicated the presence of active bacterial and archaeal populations in the fluids. The Zetaproteobacteria accounted for up to 32% of the total prokaryotic cell number as shown by FISH analysis using a specific probe designed in this study. Our results lead to the hypothesis that the Zetaproteobacteria play a role in iron oxidation within the oceanic crust.

Hadal biosphere: Insight into the microbial ecosystem in the deepest ocean on Earth

Significance Although many microbial explorations for hadal sediments began in the 1950s, the hadal water is the least-explored microbial biosphere. In this study, unexpected microbial ecosystems associated with the hadal trench water were discovered down to a 10,257-m water depth in the Challenger Deep of the Mariana Trench, which is the deepest ocean on Earth. We found the enrichment of heterotrophic population in the hadal water (6,000 ∼10,257 m) microbial communities, whereas the chemolithotrophic populations were more abundant in the upper abyssal waters. This observation suggested that the hadal microbial biosphere was supported by the endogenous recycling of organic matter in the hadal waters associated with the trench geomorphology. 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.

Simultaneous direct counting of total and specific microbial cells in seawater, using a deep-sea microbe as target.

ABSTRACT To rapidly and accurately enumerate total and specific microbes in aquatic samples, fluorescent in situ hybridization was combined with direct counting via direct immobilization of cells on a polymer-coated Nuclepore filter. The technique, named FISH-DC, achieved almost complete recovery of total cells and reproducibility ofPsychrobacter pacificensis cells of deep-sea origin (error, ≤3%) in a mixed culture and in natural seawater. Target cells immobilized on the filter were also successfully enumerated after stringent 3-cycle hybridization and even after a 16-month preservation at −30°C.

Niche Separation of Methanotrophic Archaea (ANME-1 and -2) in Methane-Seep Sediments of the Eastern Japan Sea Offshore Joetsu

In this study, we investigated the diversity and spatial distribution of anaerobic methanotrophic archaea (ANMEs) in sediments of a gas hydrate field off Joetsu in the Japan Sea. Distribution of ANMEs in sediments was identified by targeting the gene for methyl coenzyme M reductase alpha subunit (mcrA), a phylogenetically conserved gene that occurs uniquely in methanotrophic and methanogenic archaea, in addition to 16S rRNA genes. Quantitative PCR analyses of mcrA genes in 14 piston core samples suggested that members of ANME-1 group would dominate AOM communities in sulfate-depleted sediments, even below the sulfate-methane interface, while ANME-2 archaea would prefer to populate in shallower sediments containing comparatively higher sulfate concentrations. These results suggest that, although the potential electron acceptors in sulfate-depleted habitats remain elusive, the niche separation of ANME-1 and -2 may be controlled by in situ concentration of sulfate and the availability in sediments.

Metabolically active microbial communities in marine sediment under high-CO2 and low-pH extremes

Sediment-hosting hydrothermal systems in the Okinawa Trough maintain a large amount of liquid, supercritical and hydrate phases of CO2 in the seabed. The emission of CO2 may critically impact the geochemical, geophysical and ecological characteristics of the deep-sea sedimentary environment. So far it remains unclear whether microbial communities that have been detected in such high-CO2 and low-pH habitats are metabolically active, and if so, what the biogeochemical and ecological consequences for the environment are. In this study, RNA-based molecular approaches and radioactive tracer-based respiration rate assays were combined to study the density, diversity and metabolic activity of microbial communities in CO2-seep sediment at the Yonaguni Knoll IV hydrothermal field of the southern Okinawa Trough. In general, the number of microbes decreased sharply with increasing sediment depth and CO2 concentration. Phylogenetic analyses of community structure using reverse-transcribed 16S ribosomal RNA showed that the active microbial community became less diverse with increasing sediment depth and CO2 concentration, indicating that microbial activity and community structure are sensitive to CO2 venting. Analyses of RNA-based pyrosequences and catalyzed reporter deposition-fluorescence in situ hybridization data revealed that members of the SEEP-SRB2 group within the Deltaproteobacteria and anaerobic methanotrophic archaea (ANME-2a and -2c) were confined to the top seafloor, and active archaea were not detected in deeper sediments (13–30 cm in depth) characterized by high CO2. Measurement of the potential sulfate reduction rate at pH conditions of 3–9 with and without methane in the headspace indicated that acidophilic sulfate reduction possibly occurs in the presence of methane, even at very low pH of 3. These results suggest that some members of the anaerobic methanotrophs and sulfate reducers can adapt to the CO2-seep sedimentary environment; however, CO2 and pH in the deep-sea sediment were found to severely impact the activity and structure of the microbial community.

First cultivation and ecological investigation of a bacterium affiliated with the candidate phylum OP5 from hot springs.

ABSTRACT The phylogenetic group termed OP5 was originally discovered in the Yellowstone National Park hot spring and proposed as an uncultured phylum; the group was afterwards analyzed by applying culture-independent approaches. Recently, a novel thermophilic chemoheterotrophic filamentous bacterium was obtained from a hot spring in Japan that was enriched through various isolation procedures. Phylogenetic analyses of the isolate have revealed that it is closely related to the OP5 phylum that has mainly been constructed with the environmental clones retrieved from thermophilic and mesophilic anaerobic environments. It appears that the lineage is independent at the phylum level in the domain Bacteria. Therefore, we designed a primer set for the 16S rRNA gene to specifically target the OP5 phylum and performed quantitative field analysis by using the real-time PCR method. Thus, the 16S rRNA gene of the OP5 phylum was detected in some hot-spring samples with the relative abundance ranging from 0.2% to 1.4% of the prokaryotic organisms detected. The physiology of the above-mentioned isolate and the related environmental clones indicated that they are scavengers contributing to the sulfur cycle in nature.

The first microbiological contamination assessment by deep-sea drilling and coring by the D/V Chikyu at the Iheya North hydrothermal field in the Mid-Okinawa Trough (IODP Expedition 331)

During the Integrated Ocean Drilling Program (IODP) Expedition 331 at the Iheya North hydrothermal system in the Mid-Okinawa Trough by the D/V Chikyu, we conducted microbiological contamination tests of the drilling and coring operations. The contamination from the drilling mud fluids was assessed using both perfluorocarbon tracers (PFT) and fluorescent microsphere beads. PFT infiltration was detected from the periphery of almost all whole round cores (WRCs). By contrast, fluorescent microspheres were not detected in hydrothermally active core samples, possibly due to thermal decomposition of the microspheres under high-temperature conditions. Microbial contamination from drilling mud fluids to the core interior subsamples was further characterized by molecular-based evaluation. The microbial 16S rRNA gene phylotype compositions in the drilling mud fluids were mainly composed of sequences of Beta- and Gammaproteobacteria, and Bacteroidetes and not archaeal sequences. The phylotypes that displayed more than 97% similarity to the sequences obtained from the drilling mud fluids were defined as possible contaminants in this study and were detected as minor components of the bacterial phylotype compositions in 13 of 37 core samples. The degree of microbiological contamination was consistent with that determined by the PFT and/or microsphere assessments. This study suggests a constructive approach for evaluation and eliminating microbial contamination during riser-less drilling and coring operations by the D/V Chikyu.

Variability of subseafloor viral abundance at the geographically and geologically distinct continental margins

We studied the relationship between viral particle and microbial cell abundances in marine subsurface sediments from three geographically distinct locations in the continental margins (offshore of the Shimokita Peninsula of Japan, the Cascadia Margin off Oregon, and the Gulf of Mexico) and found depth variations in viral abundances among these sites. Viruses in sediments obtained offshore of the Shimokita and in the Cascadia Margin generally decreased with increasing depth, whereas those in sediments from the Gulf of Mexico were relatively constant throughout the investigated depths. In addition, the abundance ratios of viruses to microbial cells notably varied among the sites, ranging between 10(-3) and 10(1) . The subseafloor viral abundance offshore of the Shimokita showed a positive relationship with the microbial cell abundance and the sediment porosity. In contrast, no statistically significant relationship was observed in the Cascadia Margin and the Gulf of Mexico sites, presumably due to the long-term preservation of viruses from enzymatic degradation within the low-porosity sediments. Our observations indicate that viral abundance in the marine subsurface sedimentary environment is regulated not only by in situ production but also by the balance of preservation and decay, which is associated with the regional sedimentation processes in the geological settings.