Adenosine Triphosphate Measurement in Deep Sea Using a Microfluidic Device.

Total ATP (adenosine triphosphate) concentration is a useful biochemical parameter for detecting microbial biomass or biogeochemical activity anomalies in the natural environment. In this study, we describe the development and evaluation of a new version of in situ ATP analyzer improved for the continuous and quantitative determination of ATP in submarine environments. We integrated a transparent microfluidic device containing a microchannel for cell lysis and a channel for the bioluminescence L⁻L (luciferin⁻luciferase) assay with a miniature pumping unit and a photometry module for the measurement of the bioluminescence intensity. A heater and a temperature sensor were also included in the system to maintain an optimal temperature for the L⁻L reaction. In this study, the analyzer was evaluated in deep sea environments, reaching a depth of 200 m using a remotely operated underwater vehicle. We show that the ATP analyzer successfully operated in the deep-sea environment and accurately quantified total ATP within the concentration lower than 5 × 10-11 M.

Defining boundaries for the distribution of microbial communities beneath the sediment-buried, hydrothermally active seafloor.

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 δ13C 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.

Comparative Analysis of Microbial Communities in Iron-Dominated Flocculent Mats in Deep-Sea Hydrothermal Environments.

UNLABELLED: It has been suggested that iron is one of the most important energy sources for photosynthesis-independent microbial ecosystems in the ocean crust. Iron-metabolizing chemolithoautotrophs play a key role as primary producers, but little is known about their distribution and diversity and their ecological role as submarine iron-metabolizing chemolithotrophs, particularly the iron oxidizers. In this study, we investigated the microbial communities in several iron-dominated flocculent mats found in deep-sea hydrothermal fields in the Mariana Volcanic Arc and Trough and the Okinawa Trough by culture-independent molecular techniques and X-ray mineralogical analyses. The abundance and composition of the 16S rRNA gene phylotypes demonstrated the ubiquity of zetaproteobacterial phylotypes in iron-dominated mat communities affected by hydrothermal fluid input. Electron microscopy with energy-dispersive X-ray microanalysis and X-ray absorption fine structure (XAFS) analysis revealed the chemical and mineralogical signatures of biogenic Fe-(oxy)hydroxide species and the potential contribution of Zetaproteobacteria to the in situ generation. These results suggest that putative iron-oxidizing chemolithoautotrophs play a significant ecological role in producing iron-dominated flocculent mats and that they are important for iron and carbon cycles in deep-sea low-temperature hydrothermal environments. IMPORTANCE: We report novel aspects of microbiology from iron-dominated flocculent mats in various deep-sea environments. In this study, we examined the relationship between Zetaproteobacteria and iron oxides across several hydrothermally influenced sites in the deep sea. We analyzed iron-dominated mats using culture-independent molecular techniques and X-ray mineralogical analyses. The scanning electron microscopy-energy-dispersive X-ray spectroscopy SEM-EDS analysis and X-ray absorption fine structure (XAFS) analysis revealed chemical and mineralogical signatures of biogenic Fe-(oxy)hydroxide species as well as the potential contribution of the zetaproteobacterial population to the in situ production. These key findings provide important information for understanding the mechanisms of both geomicrobiological iron cycling and the formation of iron-dominated mats in deep-sea hydrothermal fields.

Evaluation of the Effect on Temperature Conversion of pHT at 25°C in the Temperature Range 0 - 40°C Due to Incorrect Estimations of Salinity, Alkalinity, and Phosphate and Silicate Concentrations.

Under conditions where carbonate and oceanic parameters such as alkalinity (A(T)), salinity (Sp), and phosphate (P(T)) and silicate (SiT) concentrations cannot be obtained, the use of fixed values to calculate total pH (pHT) at 25°C was demonstrated. For temperatures of 20 - 30°C, these values caused a maximum error of ±0.001 in pHT. For temperatures of 0 - 40°C, using fixed values with correct SP values caused a maximum error of approximately +0.0021.

Colorimetric pH measurement for seawater samples using a three light-emitting diodes detector and a calibration method for temperature dependence.

A colorimetric pH measurement of seawater samples using a light source comprising a three light-emitting diodes (TLED) detector and meta-cresol purple (mCP) as an indicator was investigated. The molar absorption ratios (e1, e2, and e3/e2) for mCP using the TLED detector at 25°C were determined to be 0, 1.9994, and 0.1010, respectively. Next, the pH values of 2-amino-2-hydroxymethyl-1,3-propanediol (TRIS) and 2-aminopyride (AMP) seawater buffers were determined. Notably, the raw pH of TRIS buffer (∼8.1) agreed with the reference value, while that for the AMP buffer (∼6.8) had an error of +0.004 due to the small absorption ratio (R) and being out of the lower adequate pH range (> 7.2) for mCP. pHT measurements obtained for seawater samples using the present colorimetric method agreed with those obtained using a glass electrode. These results demonstrate that this low-cost TLED detection system with a short cell length, 5 cm, can be used for seawater pHT analysis.

Fast measurement of dissolved inorganic carbon concentration for small-volume interstitial water by acid extraction and nondispersive infrared gas analysis.

We developed a system for measuring the total dissolved inorganic carbon (DIC) concentrations in interstitial water and hydrothermal fluid, which are hard to obtain in large volumes. The system requires a sample volume of only 500 µL, and it takes only 150 s per one sample. The detection limit of this system was estimated to be 66.6 µmol/kg with repeated analysis of CO(2)-free ultrapure water (n = 9). The precision of this nondispersive infrared (NDIR) system was ±3.1% of the relative standard deviations (2σ) by repeated CRM batch 104 (n = 10). This result is much larger than the required precision for oceanographic studies, but is comparable to a previous result of interstitial water analysis. An on-site trial showed a significant DIC enrichment in interstitial water of hydrothermally altered sediment, and is considered to occur by the mixing of hydrothermal fluid. This procedure will achieve carbon dioxide flux calculations from hydrothermal activities, and will bring a more accurate feature on the global carbon cycle.

Disturbance of deep-sea environments induced by the M9.0 Tohoku Earthquake.

The impacts of the M9.0 Tohoku Earthquake on deep-sea environment were investigated 36 and 98 days after the event. The light transmission anomaly in the deep-sea water after 36 days became atypically greater (∼35%) and more extensive (thickness ∼1500 m) near the trench axis owing to the turbulent diffusion of fresh seafloor sediment, coordinated with potential seafloor displacement. In addition to the chemical influx associated with sediment diffusion, an influx of (13)C-enriched methane from the deep sub-seafloor reservoirs was estimated. This isotopically unusual methane influx was possibly triggered by the earthquake and its aftershocks that subsequently induced changes in the sub-seafloor hydrogeologic structures. The whole prokaryotic biomass and the development of specific phylotypes in the deep-sea microbial communities could rise and fall at 36 and 98 days, respectively, after the event. We may capture the snap shots of post-earthquake disturbance in deep-sea chemistry and microbial community responses.

Spatial distribution of viruses associated with planktonic and attached microbial communities in hydrothermal environments.

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.

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.