Yasuo Furushima

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.

Methylomarinum vadi gen. nov., sp. nov., a methanotroph isolated from two distinct marine environments.

Two aerobic methane-oxidizing bacterial strains were isolated from distinct marine environments in Japan. Strains IT-4(T) and T2-1 were Gram-stain-negative, aerobic, motile, plump short rods or oval-shaped bacteria with a single polar flagellum and type I intracytoplasmic membranes. They were obligate methanotrophs that grew only on methane or methanol. Each strain possessed the particulate methane monooxygenase (pMMO). The ribulose monophosphate pathway was operative for carbon assimilation. The strains grew best at 37 °C, and did not grow at 45 °C. NaCl was required for growth within a concentration range of 1-8 % (w/v). The major phospholipid fatty acids were C16 : 0, C16 : 1ω7c, and C16 : 1ω5t. The major isoprenoid quinone was MQ-8. The DNA G+C content was 50.9-51.7 mol%. The 16S rRNA gene sequences of the strains showed 99.4 % similarity to each other, and DNA-DNA hybridization analysis indicated that the strains were representatives of the same species. The 16S rRNA gene sequences were highly similar to some marine environmental sequences (94.0-97.7 % similarity), but did not show similarities more than 94 % with sequences of members of other related genera, such as Methylomicrobium, Methylobacter, Methylomonas and Methylosarcina. Phylogenies based on 16S rRNA gene sequences and deduced partial PmoA sequences, and the physiological and chemotaxonomic characteristics revealed that strains IT-4(T) and T2-1 represent a novel species of a new genus in the family Methylococcaceae, for which the name Methylomarinum vadi gen. nov., sp. nov. is proposed. The type strain is IT-4(T) ( = JCM 13665(T) = DSM 18976(T)).

Isolation and characterization of a thermophilic, obligately anaerobic and heterotrophic marine Chloroflexi bacterium from a Chloroflexi-dominated microbial community associated with a Japanese shallow hydrothermal system, and proposal for Thermomarinilinea lacunofontalis gen. nov., sp. nov.

A novel marine thermophilic and heterotrophic Anaerolineae bacterium in the phylum Chloroflexi, strain SW7T, was isolated from an in situ colonization system deployed in the main hydrothermal vent of the Taketomi submarine hot spring field located on the southern part of Yaeyama Archipelago, Japan. The microbial community associated with the hydrothermal vent was predominated by thermophilic heterotrophs such as Thermococcaceae and Anaerolineae, and the next dominant population was thermophilic sulfur oxidizers. Both aerobic and anaerobic hydrogenotrophs including methanogens were detected as minor populations. During the culture-dependent viable count analysis in this study, an Anaerolineae strain SW7T was isolated from an enrichment culture at a high dilution rate. Strain SW7T was an obligately anaerobic heterotroph that grew with fermentation and had non-motile thin rods 3.5–16.5 μm in length and 0.2 μm in width constituting multicellular filaments. Growth was observed between 37–65°C (optimum 60°C), pH 5.5–7.3 (optimum pH 6.0), and 0.5–3.5% (w/v) NaCl concentration (optimum 1.0%). Based on the physiological and phylogenetic features of a new isolate, we propose a new species representing a novel genus Thermomarinilinea: the type strain of Thermomarinilinea lacunofontalis sp. nov., is SW7T (=JCM15506T=KCTC5908T).

Methylomarinovum caldicuralii gen. nov., sp. nov., a moderately thermophilic methanotroph isolated from a shallow submarine hydrothermal system, and proposal of the family Methylothermaceae fam. nov.

A novel methane-oxidizing bacterium, strain IT-9(T), was isolated from a shallow submarine hydrothermal system occurring in a coral reef in Japan. Strain IT-9(T) was a Gram-negative, aerobic, motile, coccoid or oval-shaped bacterium with the distinctive intracytoplasmic membrane arrangement of a type I methanotroph. Strain IT-9(T) was a moderately thermophilic, obligate methanotroph that grew on methane and methanol at 30-55 °C (optimum 45-50 °C). The strain possessed the particulate methane monooxygenase (pMMO). The ribulose monophosphate pathway was operative for carbon assimilation. NaCl was required for growth within a concentration range of 1-5 % (optimum 3 %). The hao gene encoding hydroxylamine oxidoreductase (HAO) involved in nitrification was detected by a PCR experiment. The major phospholipid fatty acids were C16 : 0 and C18 : 1ω7c. The major isoprenoid quinone was Q-8. The DNA G+C content was 66.0 mol%. The 16S rRNA gene sequence of strain IT-9(T) was only moderately related to the sequences of members of the closest genera Methylohalobius (94.1 % similarity) and Methylothermus (91.7-91.9 % similarity); however, those sequences formed a deeply branching monophyletic group within the order Methylococcales. Phylogenies based on 16S rRNA gene sequences, deduced partial PmoA sequences and deduced partial Hao sequences and physiological and chemotaxonomic characteristics revealed that strain IT-9(T) represents a novel species of a new genus, for which the name Methylomarinovum caldicuralii gen. nov., sp. nov. is proposed. The type strain of Methylomarinovum caldicuralii is IT-9(T) ( = JCM 13666(T) = DSM 19749(T)). In addition, we propose a new family, Methylothermaceae fam. nov., in the order Methylococcales, to accommodate the genera Methylothermus, Methylohalobius and Methylomarinovum. The genera Methylothermus and Methylohalobius have been recognized as being distinct from other genera in the methane-oxidizing order Methylococcales in the class Gammaproteobacteria. These genera form a distinctive monophyletic lineage within the order on the basis of 16S rRNA gene sequence phylogeny. This seems consistent with their distinctive physiological traits; the genus Methylothermus includes the most thermophilic species, and the genus Methylohalobius includes the most halophilic species, within the order. Although these two genera include only three species at the time of writing, similar sequences of 16S rRNA genes and pmoA genes encoding pMMO have been detected in a geothermal area or deep-sea hydrothermal vent fields by studies using culture-independent techniques. This suggests that unknown methanotrophs of this lineage inhabit various extreme environments.

Spatial Distribution of Viruses Associated with Planktonic and Attached Microbial Communities in Hydrothermal Environments

ABSTRACT 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.

A basic experiment of coral culture using sexual reproduction in the open sea

Coral larvae, produced from a mass spawning event, were successfully settled on special stone settlement sticks and raised in situ for eventual transport to other reefs. The test area, Sekisei lagoon, Okinawa Prefecture, Japan, is located close to the warm Kuroshio current and is surmised to be the source from which major Japanese corals are derived. A total of 131 settlement sticks, with small holes in their sides to increase protection from grazing (4 mm in diameter and approximately 5 mm deep), were deployed in the lagoon the day before the coral’s mass spawning. After 3 months, 61 sticks were recovered containing 71 corals, mostly in the holes. After 1 year, three corals were confirmed to be growing well and extending outside the holes of the three sticks out of 70 sticks left in the water at the lagoon site. They survived two potentially lethal conditions, that is, high water temperatures with associated extensive coral bleaching and continuous grazing pressure from predators. This procedure is applicable for large-scale coral transplantation, not only in Japan but also in other tropical countries.

Development of ceramic settlement devices for coral reef restoration using in situ sexual reproduction of corals

A ceramic coral settlement device (CSD) was developed to raise juvenile corals in situ in preparation for transplantation. The CSD is designed to be deployed in the water before mass spawning of corals, allowing larvae to settle and grow on the CSD before it is fixed to the reef structure. CSDs were aggregated and fixed on 0.2-m2 frames (either 648 or 720 CSDs per frame) and deployed in the Sekisei Lagoon around the full moons of April and May in 2002. Seven frames were collected in August 2002 and settled corals were counted in the laboratory. The maximum number of coral colonies settled on CSDs in one frame was 617, with 94% belonging to the genus Acropora. The aggregated CSDs successfully functioned as a coral settlement substratum, with more appropriate arrangement of CSDs on the frame, enabling greater exposure to sunlight and currents, which is expected to increase the amount of settlement.

The scyphomedusan fauna of the Japan Trench: preliminary results from a remotely-operated vehicle

A series of dives was conducted during April/May 2002 in a variety of water masses over the Japan Trench using the remotely-operated vehicle ROV HyperDolphin. The scyphomedusan fauna was catalogued, resulting in the first records of Atolla vanhoeffeni and Atolla russelli from Japanese waters. A new genus and species referable to the family Paraphyllinidae was also recognized. The ulmarid scyphomedusa Poralia rufescens is by far the most common scyphomedusa in these waters. Its distribution was determined by water mass structure and it was not present in subducted Oyashio Current-derived waters occurring within its usual depth range. Temperature and salinity affected its distribution more than dissolved oxygen concentrations. Distributional information, data on the physico-chemical parameters of the water column, and notes on biological associations with scyphomedusae are introduced.

Evaluation of coral bleaching condition in situ using an underwater pulse amplitude modulated fluorometer

Photosynthetic activities of hermatypic corals (28 genera, c. 68 species and c. 92 specimen) were measured in November 2000 at Sekisei lagoon, Okinawa, Japan, using an underwater pulse amplitude modulated fluorometer. Maximum quantum yield (Fv/Fm) ranged from 0.61 to 0.70 and had an average value of 0.664 (standard deviation [SD], 0.0289). Following the measurement of Fv/Fm, quantum yield (Fm′-F)/Fm′ at eight-leveled actinic-light intensities were obtained from all specimens. Values of electronic transport rates (ETR) were nearly the same under low photosynthetic active radiation (PAR; μmol quanta/m2 per s) condition and varied with increasing PAR. The value of ETR at 100 μmol quanta/m2 per s ranged from 21.3 to 30.0 and had an average of 27.8 (SD, 2.09). In August 2001, a coral bleaching event was observed in Sekisei Lagoon. During the event, photosynthetic activities of 21 specimens of eight species (seven genera) were measured. By applying two indicators, Fv/Fm and ETR at 100 μmol quanta/m2 per s obtained in August 2000, 18 of 21 specimens were suggested under the bleaching process. In case of Porites lutea, seven colonies were visually evaluated for their condition of bleaching and were found to have two ‘normal’, three ‘slightly bleached’ and two ‘heavily bleached’. From the density of zooxanthellae, colonies evaluated ‘normal’ or ‘slightly bleached’ did not show an apparent difference. However, they were clearly identified by Fm/Fv and ETR at 100 μmol quanta/m2 per s.

Periodic Behavior of the Bubble Jet (Geyser) in the Taketomi Submarine Hot Springs of the Southern Part of Yaeyam aA rchipelago, Japan

The periodicity of the bubble jet that spouts intermittently from the Taketomi submarine hot spring in Yaeyama archipelago, Japan, was measured for the first timeusinganacousticcurrentmeter.Thetimeseriesanalysisoftheupwardvelocity for the data, without a spiky signal, indicates that the cycle fluctuates between 38 and 85 s. Focusing on the period of high and low tide, the dominant time cycles were 66 and 41 s, respectively. These results show that the fluctuation of pressure with tide affected the eruption period of the bubble jet. In accordance with the vertical tube theory, estimations of thermal source and recharge water temperatures were carried out by taking into account the boiling point change due to the tidal variation in hydrostatic pressure. The result indicated that if the heat source temperature was higher than 200.0 °C and recharge water temperature was preheated to 117.96 °C, the observed eruption time cycles at high and low tide were stable.