[Show abstract][Hide abstract] ABSTRACT: Spawning-condition Japanese eels Anguilla japonica, fertilized eggs, and newly-hatched preleptocephali have been captured, and studies for observing spawning eels with underwater camera systems have begun. This study describes a new, less invasive, free-drifting underwater camera observation system that was deployed from the research vessel (R/V) Natsushima in June 2013. Three drifting buoy camera systems (Una-Cam) with lights-on/lights-off programmed sequencing during daytime and nighttime hours were deployed over a period of seven days at 20 locations south of a salinity front along the southern West Mariana Ridge. Live artificially matured A. japonica eels held in transparent chambers were used as an attractant source through the release of reproductive pheromones and other odors. Each system was suspended from a buoy array at a depth of 174–200 m, with four cameras and three lights pointed downward at different angles towards the eel chamber. The Una-Cam systems were stable and were effective at recording images of fish, crustaceans, and gelatinous zooplankton. Olfactory cues may have attracted male and female Derichthys serpentinus eels, which showed what seemed to be reproductive behavior and attraction to the Japanese eels in the chamber. Una-Cam systems are capable of recording images of anguillid eels, if they approach, and may be useful for observing spawning eels in their offshore spawning areas.
[Show abstract][Hide abstract] ABSTRACT: Increasing anthropogenic CO2 in the atmosphere causes global warming and subsequent environmental changes, which may lead to an increase in natural disasters jeopardizing human society. Prompt technological development for CO2 capture and sequestration is required in the international community. In this study, we performed CO2 emission and shallow-type methane hydrate decomposition experiments at the Joetsu Knoll, offshore Joetsu, Niigata, Japan, as pilot studies to test feasibility of CO2 sequestration and methane recovery using methane-CO2 replacement in shallow-type methane hydrates. An isobaric cylinder pump and probe with a built-in heater (“Heat sonde”) were developed to inject CO2 in deep-sea, high-pressure conditions. Before injecting CO2 into a methane hydrate located in deep-sea sediments, we attempted CO2 emission directly into deep-seafloor. In the experiment, liquid CO2 was emitted at the head of Heat sonde, however, the isobaric cylinder pump became clogged during operation. The result reveals that precipitates of CO2 hydrate, which are generated during mixing of inflow seawater and outflow liquid CO2, blocked flow lines of the isobaric cylinder pump and Heat sonde. This suggests that our developed instruments must be improved for future work. We also observed the collapse of an exposed methane hydrate layer at the seafloor upon contact with the Heat sonde in our experiment.
[Show abstract][Hide abstract] ABSTRACT: Pelagic cephalopods such as squid change coloration for camouflage or release ink as a defensive mechanism while being attacked by predators, which may block the view of the predator, have noxious chemical effects, or act as a warning signal for other squid (Bush and Robison 2007; Derby 2007; Wood et al. 2010). Pulsed releases of ink can also create “pseudomorphs” in the shape of squid that may serve as decoys to confuse predators about the location of the actual squid (Bush and Robison 2007; Wood et al. 2010). Some aspects of deep-sea squid behavior, including releases of their ink, have been observed using remote operated vehicles (ROV) (Hunt et al. 2000; Bush and Robison 2007), but most species have not been studied.During Hyper-Dolphin ROV (JAMSTEC) deployments over the West Mariana Ridge, observations were made of an unidentified species of pelagic squid (
[Show abstract][Hide abstract] ABSTRACT: We developed a new multi-water-sampling system, ANEMONE-11, for autonomous underwater vehicle and remotely operated underwater vehicle exploration. Water samples are continuously collected by the ANEMONE-11 sampler by an in situ water pump at 40 mL/min and are sent to a selection valve unit that consists of 128 valves connected to 40 mL sampling bottles (50 cm in length). Each valve in the unit is selected and opened at preprogrammed intervals. We also discuss the results of observations at a hydrothermal area in the Okinawa Trough.
Methods in Oceanography 01/2014; 8. DOI:10.1016/j.mio.2014.02.001
[Show abstract][Hide abstract] ABSTRACT: Underwater hydrothermal vents draw a lot of interest as they are the shelter for unique ecosystems, besides being a possible new opportunity for ore mining. Manganese (Mn) is found at a high concentration in hydrothermal vents, which make it possible to use this metal ion as a tracer to detect and evaluate new hydrothermal sources. Here we present a miniaturized and integrated microfluidic system for the detection of Mn in deep-sea environment, called the integrated in situ analyzer for Mn2+ (IISA-Mn). The detection system is based on the chemiluminescence reaction of Mn contained in the seawater sample with a luminol-based reagent, which offers a high sensitivity. This system is composed of a microdevice for mixing and reaction, a pumping unit, several valving units, and a photomultiplier (PMT) detector. The system is able to detect Mn concentration above 280 nM in seawater, and gives a quite linear response until 500 nM. It is also proven to be able to work continuously during the 8 h of an actual remotely operated vehicle (ROV) dive. This system has led to the discovery of a previously unknown hydrothermal site in the Okinawa Trough.
[Show abstract][Hide abstract] ABSTRACT: This paper presents a simple method to change the hydrophilic nature of the glass surface in a poly(dimethylsiloxane) (PDMS)-glass hybrid microfluidic device to hydrophobic by an extra-heating step during the fabrication process. Glass substrates bonded to a native or oxygen plasma-treated PDMS chip having microchambers (12.5 mm diameter, 110 µm height) were heated at 200°C for 3 h, and then the hydrophobicity of the glass surfaces on the substrate was evaluated by measuring the contact angle of water. By the extra-heating process, the glass surfaces became hydrophobic, and its contact angle was around 109°, which is nearly the same as native PDMS surfaces. To demonstrate the usefulness of this surface modification method, a PDMS-glass hybrid microfluidic device equipped with microcapillary vent structures for pneumatic manipulation of droplets was fabricated. The feasibility of the microcapillary vent structures on the device with the hydrophobic glass surfaces are confirmed in practical use through leakage tests of the vent structures and liquid handling for the electrophoretic separation of DNA molecules.
[Show abstract][Hide abstract] ABSTRACT: Pressure and temperature characteristics of in-situ sensors have a big
influence on measurements. A pH sensor using ISFET (Ion Sensitive Field
Effect Transistor) is regarded as a suitable sensor for measurement pH
at deep sea, however, its pressure and temperature characteristics were
seldom studied. So, in this paper, we study the pressure and temperature
characteristics of ISFET-pH sensor by the structural analysis and by the
laboratory experiments, and then consider a new structure which can
decrease the pressure effects on ISFET-pH sensor, and investigate these
effects on the actual measurement in deep sea. From experimental and
actual measurement results, we estimate the applications and efficiency
of ISFET-pH sensor. By the results of structural analysis, if the
structure of the sensor has sufficient strength for practical use,
pressure effects occur. The experimental results shows the pressure and
temperature characteristics have large individual difference. But by the
results of structural improvement analysis, the pressure effects can be
reduced by the change in construction material of the sensor. The actual
measurement in deep sea indicates that the temperature characteristics
depends on pressure, and show a problem of the present way of
calibration that is to calibrate the effects of pressure and
temperature, respectively.At the same time, it is also shown that the
sensor has high sensitivity, good responsibility, wide application and
[Show abstract][Hide abstract] ABSTRACT: In this paper, a rapid and simple method to determine the optimal temperature conditions for denaturant electrophoresis using a temperature-controlled on-chip capillary electrophoresis (CE) device is presented. Since on-chip CE operations including sample loading, injection and separation are carried out just by switching the electric field, we can repeat consecutive run-to-run CE operations on a single on-chip CE device by programming the voltage sequences. By utilizing the high-speed separation and the repeatability of the on-chip CE, a series of electrophoretic operations with different running temperatures can be implemented. Using separations of reaction products of single-stranded DNA (ssDNA) with a peptide nucleic acid (PNA) oligomer, the effectiveness of the presented method to determine the optimal temperature conditions required to discriminate a single-base substitution (SBS) between two different ssDNAs is demonstrated. It is shown that a single run for one temperature condition can be executed within 4 min, and the optimal temperature to discriminate the SBS could be successfully found using the present method.
International Journal of Molecular Sciences 12/2011; 12(7):4271-81. DOI:10.3390/ijms12074271 · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, a totally integrated in situ analyzer for microbial gene detection has been developed for oceanography applications. A PDMS–glass microfluidic device that is capable of cell lysis, DNApurification, PCR, and optical detection has been utilized as the core element of the in situ analyzer. Microbial genomic DNA is purified and concentrated on glass beads packed in the microfluidic device. PCR is performed in a flow-through manner, and the amplified products are fluorescently detected using optical fibers. The sensitivity of a completed analyzer with deep-sea operation capabilities has been evaluated in a laboratory setting, and the analyzer has been operated in real deep-sea environments. Field evaluations have shown that the amplification of the eubacterial universal 16S rRNA gene and the recovery of the PCR product to the surface are successfully achieved in deep-sea environments.
[Show abstract][Hide abstract] ABSTRACT: We have developed and tested a functionally integrated in situ analyzer, the IISA-ATP system, for microbial activity assays based on a quantitative determination of the total (particulate and dissolved) ATP in ocean environments. The IISA-ATP utilizes a PDMS-glass hybrid microfluidic device as its core functional element, which can perform cell lysis and total ATP quantification by a luciferin-luciferase bioluminescence assay in situ. Transparent heaters and a temperature sensor fabricated on a glass substrate provide temperature control. As a result of the evaluation using the microfluidic device with ATP standard solutions, the bioluminescence intensity was linearly correlated with 2 × 10(-12) to 2 × 10(-8) M of ATP. A detection limit of 1.1 × 10(-11) M was determined using the completed IISA-ATP system, which includes a miniature pumping module and a control module. As a result of the evaluation using the environmental seawater sample collected from Tokyo Bay, Japan, 2.7 × 10(-10) M of total ATP was successfully determined in the laboratory by the IISA-ATP. The system was operated at a shallow submarine hot spring area in Okinawa, Japan for an in situ trial. The result shows the system was successfully operated in situ and the total ATP was determined to be 3.4 × 10(-10) M.
Lab on a Chip 08/2011; 11(20):3508-15. DOI:10.1039/c1lc20523d · 6.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hydrothermal anomalies found in the deep sea are a place where unique ecosystems exist, and may become a possible new resource for metallic ore mining. Elements such as iron and manganese are found in high concentration in hydrothermal vents. Therefore it is possible to use such metal ions as a tracer to detect and evaluate new hydrothermal sources. Here we present a miniaturized and integrated microfluidic system for the detection of manganese in deep-sea environment, called IISA-Mn (In Situ Integrated Analyzer for Mn 2+ ). The detection system is based on the chemiluminescence reaction of manganese contained in the seawater sample with a luminol-based reagent, which offers a high sensitivity. This system is composed of a microdevice for mixing and reaction, a pumping unit, several valving units, and a PMT (photomultiplier) detector. The system is able to detect manganese concentration above 100 nM in seawater, and gives a linear response until 1 µ M. It is also proven to be able to work continuously during the 8 hours of an actual dive. This system has lead to the discovery of a previously unknown hydrothermal site near Okinawa Islands.
[Show abstract][Hide abstract] ABSTRACT: Sequential operations of pre-separation reaction process by picoliter droplets and following electrophoretic separation process were realized in a single microfluidic device with pneumatic handling of liquid. The developed device consists of a fluidic chip made of PDMS, an electrode substrate, and a temperature control substrate on which thin film heater/sensor structures are fabricated. Liquid handling, including introduction of liquid samples, droplet generation, and merging of droplets, was implemented by pneumatic manipulation through microcapillary vent structures, allowing air to pass and stop liquid flow. Since the pneumatic manipulations are conducted in a fully automated manner by using a programmable air pressure control system, the user simply has to load liquid samples on each liquid port of the device. Droplets of 420 pL were generated with an accuracy of ± 2 pL by applying droplet generation pressure in the range of 40-100 kPa. As a demonstration, a binding reaction of a 15 mer ssDNA with a peptide nucleic acid oligomer used as an oligoprobe followed by denaturing electrophoresis to discriminate a single-base substitution was performed within 1.5 min. By exploiting the droplet-on-demand capability of the device, the influence of various factors, such as reaction time, mixing ratio and droplet configurations on the ssDNA-peptide nucleic acid binding reaction in the droplet-based process, was studied toward realization of a rapid detection method to discriminate rapid single-base substitution.
[Show abstract][Hide abstract] ABSTRACT: Hydrothermal fluids contain high concentration of anoxic chemical
species, i.e. methane and hydrogen sulfide, helium-3, and heavy metals
derived from the rock-water interaction. During the hydothermal plume
spreading, it is known that several chemical species are oxidized which
include available energy source for microorganism, however, few results
have been reported on the spatial variation of both of chemical and
microbiological concentration and species. In the southern Mariana
Trough, some site surveys have been conducted with CTD hydrocasts, the
manned submersible, and ROVs since 2003. In this field, three
hydrothermal vent sites were discovered within the small area, where the
chemistry of each hydrothermal fluid was different from each other.
These differences of chemistry are prospected to affect the individual
plume evolution. In order to discuss the each hydrothermal plume
evolution, we conducted high-resolution plume mapping by the AUV
"URASHIMA" with some chemical sensors. Additionally, we loaded 24
bottles of water sampler for the geochemical and microbial analysis.
During this cruise, we detected hydrothermal plume anomalies derived
from each hydrothermal site with the highly precise topographic results.
Based on the results, we will discuss the relationships between the
spreading of hydrothermal plume (geochemical evolution) and the ecology
of plume microbes.
[Show abstract][Hide abstract] ABSTRACT: An integrated in situ analyzer for microbial ATP (IISA-ATP) has been developed with a microfluidic device as its core component to realize a compact and fully integrated system. In the system, a bioluminescence (luciferin---luciferase) reaction is conducted for ATP quantification. The microfluidic device has a coil-shaped microchannel for highly sensitive photo intensity measurement. In this paper, the concept of the IISA-ATP and optimization of the microchannel design to enhance sensitivity are presented. As a result of the optimization, linear correlation of the luminescence intensity with the ATP concentration in the range of 2 to 2 × 104 pM was achieved.
IEEJ Transactions on Sensors and Micromachines 01/2009; 129(3):73-76. DOI:10.1541/ieejsmas.129.73
[Show abstract][Hide abstract] ABSTRACT: This paper presents design, fabrication and evaluation of a polydimethylsiloxane (PDMS) ---glass microfluidic device for quantitative determination of manganese ion (Mn(II)) in oceanic environments. The microfluidic device can perform luminol-hydrogen peroxide chemiluminescence (CL) reaction in a flow-through manner for highly sensitive Mn (II) quantification. A chelate resin column was integrated in the microfluidic device for removal of coexisting metal ion such as Fe(II). The developed device could quantify Mn(II) in nM level under coexistence of muM level of Fe(II). The microfluidic device developed here will be used as a core element of a totally integrated in situ chemical analyzer that we have been developing for oceanography applications.
IEEJ Transactions on Sensors and Micromachines 01/2009; 129(3):69-72. DOI:10.1541/ieejsmas.129.69
[Show abstract][Hide abstract] ABSTRACT: This paper describes the study of the DNA markers of uncultured microorganisms in order to develop the technology for detecting methane leakage in the deep sea. Methane oxidizing bacteria called methanotrophs consume methane as their sole energy and carbon source and are distributed at methane producing sites. Aerobic methanotrophic bacteria use soluble methane monooxygenases (sMMO) or particulate methane monooxygenases (pMMO) to convert methane to methanol. The anaerobic oxidation of methane mediated by anaerobic methanotrophs (ANME) and sulphate-reducing bacteria plays a critical role in anoxic sediments of cold seeps. Methanotrophs are ubiquitous in soil, fresh water and the open ocean but have not been well characterized in deep-sea hydrocarbon seeps and gas hydrates, where methane is unusually abundant. In the last few decades, cultivation-independent molecular methods have been applied widely to investigate microbial diversity and quantify predominant organisms in natural microbial community. Methanotroph diversity has been studied in different environments using the polymerase chain reaction (PCR). Our goal in this study was to compare the composition of bacterial and archaeal communities associated with gas hydrates with those of bacterial and archaeal communities in normal marine sediments in the Nankai Trough of Japan in order to identify specific DNA markers (16S RNA and pmoA genes) for methane leakage in the deep sea. The development of in-situ detection of DNA markers associated with methane leakage is now ongoing with the application of a totally integrated and automated system for in-situ gene analysis of microbes (IISA-gene system).
[Show abstract][Hide abstract] ABSTRACT: Many metal ions that can be found at trace or ultratrace level in ocean are coming from deep-sea hydrothermal vents. In particular, manganese (Mn<sup>2+</sup>) can be assimilated by phytoplankton and so can be found in all the food chain. It is then of interest to be able to detect manganese to determine its spatial distribution and/or to find new hydrothermal sources. Usually, determination of manganese concentration is done by sampling seawater in bottles, followed by analysis in lab onboard a ship or on the ground. Major drawbacks of that method are that only a few samples can be taken during each dive and that there is a risk of contamination of the sample. The use of microfabrication techniques to produce an in-situ microanalyzer that can be installed in an autonomous underwater vehicles (AUVs) or remotely operated vehicles (ROVs) can allow a higher sampling rate, with experiments over a longer time, a reduced risk of contamination. We propose here a microdevice designed for in situ detection of manganese ion in deep-sea environment. Detection is based on chemiluminescence of luminol triggered by the H<sub>2</sub>O<sub>2</sub> decomposition catalyzed by manganese ion. The set-up is composed of pressure-proof micropumps for handling of the reagents, a 6-way valve for samples introduction, a PDMS (poly(dimethylsiloxane)) microchip made by soft lithography, and a photomultiplier for photon detection. The limit of detection of this system is about 10 nanomolar at the moment.
[Show abstract][Hide abstract] ABSTRACT: In this study, "Integrated in situ analyzer-ATP (IISA-ATP)" which is to estimate microbial activities in deep-sea environments has been developed. On the system, a microfluidic device is used as a core element to perform a bioluminescent (luciferin-luciferase) reaction for ATP quantification. The microfluidic device has a coil-shaped microchannel for photo intensity measurement. In this paper, concept of the IISA-ATP and optimization process of the microchannel design are presented. As a result of the microchannel design and flow rate optimization, 2 pM to 2 x 10<sup>4</sup> pM (20 nM) of ATP was successfully quantified.
[Show abstract][Hide abstract] ABSTRACT: An integrated and miniaturized device for in situ quantitative analysis of microbial ATP was developed in this study. The device can perform extracellular ATP elimination, cell lysis and ATP quantification in a microfluidic chip. A pattern of a microchannel has been optimized to measure luminescence caused by luciferin-luciferase reaction. As a result of evaluation of the developed microfluidic device, 10 nM to 100 muM of ATP was successfully quantified. Environmental samples and bacterial culture media were also analyzed by the microfluidic device. Towards in situ calibration of the ATP quantitative analysis device, a prototype of caged ATP based online calibration component was also fabricated and evaluated.
Underwater Technology and Workshop on Scientific Use of Submarine Cables and Related Technologies, 2007. Symposium on; 05/2007
[Show abstract][Hide abstract] ABSTRACT: Manganese is a metal ion that can be found at ultra trace level in the ocean. Mainly, manganese comes into the ocean through hydrothermal vents. Usually, determination of manganese concentration is done by sampling seawater in bottles and measuring afterward onboard a ship or in a lab on the ground. Major drawbacks of that method are that only a few samples can be taken at a time and that there is always a risk of contaminations. The use of microfabrication techniques to produce an in situ microanalyzer that can be installed in Autonomous Underwater Vehicles (AUVs) or Remotely Operated Vehicles (ROVs) can overcome these issues. Miniaturization is an asset for mounting an analyzer in AUVs or ROVs due to low total volume (less reagents) and electrical consumption, and for increasing spatial and temporal resolution (higher rate of sampling) to make 3D manganese cartography possible. Here we show the development of an integrated in-situ analyzer for manganese based on chemiluminescence detection. Manganese catalyzes the decomposition of H<sub>2</sub>O<sub>2</sub> into radicals OHldr that react with luminol to produce light, which intensity is proportional to Mn<sup>2+</sup> concentration. Reagents are brought by syringe pumps or pressure-proof mieropumps to a reaction chamber made of polydimethylsiloxane (PDMS) material.
Underwater Technology and Workshop on Scientific Use of Submarine Cables and Related Technologies, 2007. Symposium on; 05/2007