![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Subground Animalcule Retrieval (SUGAR) Program, Japan Agency for Marine-Earth Science & Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan,1 Department of Earth & Planetary Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan,2 Graduate School of Engineering and Sciences, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan,3 Marine Biology and Ecology Program, Japan Agency for Marine-Earth Science & Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan,4 Department of Biology, Chemistry and Marine Sciences, Faculty of Science, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan5
Received 7 June 2007/ Accepted 26 September 2007
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Numerous shallow submarine hydrothermal activities have been investigated worldwide (2, 11, 21, 50, 51). However, little is known about entire microbial community structures in shallow submarine hydrothermal systems. A microbial community analysis based on fluorescent in situ hybridization (FISH) targeting thermophilic archaea and bacteria was reported for Vulcano Island, Italy (56). Microbiological investigations of microbial communities have also been reported for Kodakara-shima Island in Japan (21), Milos Island in Greece (6, 28, 58), Vulcano Island in Italy (16), and White Point in California (25). These studies focused on oxidation and reduction of sulfur compounds, while the distribution, diversity, and function of other chemolithotrophic microbial populations have rarely been addressed. In addition, the phylogenetic diversity of archaeal communities has been characterized for Milos in Greece (12), Vulcano in Italy (54), Tachibana Bay in Japan (63), and Tutum Bay in Papua New Guinea (49); however, the physiological diversity and ecological significance of the archaeal phylotypes remain unclear because most of the Archaea detected are still uncultivated and unidentified.
A shallow submarine hydrothermal system associated with a subtropical coral reef has been discovered off Taketomi Island in the Southern Ryukyu Archipelago, Japan. This hydrothermal system is located in and around a craterlike basin structure that is 50 to 60 m in diameter within barrier reefs. Visible fluid emissions with gas bubbles mainly consisting of CH4 and N2 occur, and the highest-temperature fluid flows out through a fissure in the bedrock at the bottom (water depth, 23 m) of the craterlike basin structure. The seafloor adjacent to the shallow submarine hot vents is partially covered with seagrass and live coral colonies (29, 41). Some of the chemical features of the fluids have been reported in previous studies (26, 45). In addition, a novel species of sulfur-oxidizing Gammaproteobacteria, Sulfurivirga caldicuralii, was previously isolated from this shallow hydrothermal system (68).
In addition to hydrothermal fluid emissions, visibly flourishing microbial communities have been observed extensively around the vent sites; these communities include white microbial mats at the deepest vent site and colored microbial mats at shallower vent sites. We have been conducting a series of geochemical and geomicrobiological surveys to elucidate the microbial ecosystem in the shallow submarine hydrothermal system off Taketomi Island. In this report, we identify bacterial and archaeal components inhabiting the hydrothermal fluid and mat formation at the deepest main vent site. Cultivation and cultivation-independent molecular techniques were combined to analyze the population size, phylogenetic diversity, main physiologies of the community, and unique microorganisms in this system.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A microbial mat sample was collected from the main vent site in December 2004. Several man-made objects, such as a sensor and steel pipes, have been planted in the seafloor near the main vent site for a long time. A large amount of a filamentous white mat was growing on the surfaces of these objects and basement rocks. A sensor found 1 m from the main vent was recovered, and the surface mat was collected. A portion of the mat (0.3 g, wet weight) collected for DNA extraction was stored at –80°C before the experiment. For the cultivation experiment, a portion of the mat was anaerobically processed on the boat as described below.
The temperature, pH, oxidation-reduction potential, and dissolved oxygen content at the sampling sites were measured in June 2005 with a portable water quality analyzer (W-23DX; Horiba).
Chemical characterization of water and gas components.
The free gas components were analyzed using gas chromatography with a thermal conductivity detector (model GC-14B; Shimadzu). Ammonium and dissolved sulfide concentrations in the water were determined by the classical indophenol method (60) and the methylene blue method (9), respectively.
Nucleic acid extraction and 16S rRNA gene amplification.
Genomic DNA was extracted from environmental samples (vent fluid, microbial mat, and reference seawater) and isolates with an UltraClean soil DNA kit (Mo Bio Laboratories, Inc.) by following the manufacturer's instructions. Bacterial and archaeal 16S rRNA genes were amplified by PCR as previously described (20), using primers Bac27F and Uni1492R (30) for bacterial rRNA genes and primers Arch21F and Arch958R (13) for archaeal rRNA genes.
Cloning and sequencing of 16S rRNA genes from environmental samples.
The PCR products of 16S rRNA genes from the environmental samples were cloned into the vector pCR2.1 (Invitrogen) to construct libraries, and sequences of single strands that were approximately 0.6 kb long were determined with an Applied Biosystems 3130xl genetic analyzer as described previously (20). Similarity among the clone sequences was analyzed using the FASTA program with DNASIS software (Hitachi Software Engineering), and sequences showing 
97% identity were assigned to the same phylogenetic clone type (phylotype). Sequences (approximately 1 kb) of both strands were further determined for representative phylotypes. Chimeric sequences were detected with the CHECK-CHIMERA program from RDP II (http://rdp8.cme.msu.edu/html/) and by comparison of the neighbor-joining trees constructed from the first and second halves of each sequence using ARB software.
Sequence analysis of the isolates.
For the microbial isolates, partial sequences (approximately 0.6 kb) of the amplified 16S rRNA gene products were directly determined. Similarity among the sequences was analyzed as described above, and isolates showing 98% sequence identity were assigned to the same species. Sequences (0.9 to 1.4 bp) of both strands were further determined for representative isolates that were phylogenetically different from extant species.
Phylogenetic analysis.
The 16S rRNA gene sequences obtained were first compared with the sequences in the nonredundant DDBJ/EMBL/GenBank databases by using the FASTA and gapped-BLAST search programs. The sequences were subsequently imported into the ARB software program and automatically aligned using the fast aligner utility, and the alignment was manually edited. Unambiguously aligned nucleotide positions were used for construction of phylogenetic trees with the neighbor-joining algorithm using CLUSTALX, version 1.83. Bootstrap analysis was performed to assign confidence levels to tree topology.
Cultivation by the liquid serial dilution method.
For cultivation experiments, a 30-ml subsample of the main vent fluid was placed into a glass vial with a butyl rubber cap, and the headspace in the vial was filled with 100% N2. A portion of the filamentous white mat (0.1 g) was suspended in 30 ml of sterilized seawater with or without 0.05% (wt/vol) neutralized sodium sulfide in a vial under the 100% N2 gas phase (200 kPa). The mat in the vial was suspended by vigorous shaking before inoculation. The total number of cells in the slurry was determined by 4',6-diamidino-2-phenylindole (DAPI) staining. To calculate the number of culturable microbial cells, samples were diluted using a liquid decimal dilution series with media targeting a wide range of metabolisms. The cultivation experiment was performed using 3 ml of medium in a 15-ml tube in triplicate.
Aerobic to microaerobic, hydrogen- and/or sulfur-oxidizing chemolithoautotrophs were cultivated at 30, 37, and 55°C in MMJHS medium (65) with four types of headspace gas: (i) H2-CO2-O2 (80:19:1; 200 kPa); (ii) H2-CO2-O2 (75:15:10; 200 kPa); (iii) N2-CO2-O2 (80:19:1; 200 kPa); and (iv) N2-CO2-O2 (75:15:10; 200 kPa). For aerobic methane oxidizers, MJmet medium was used. MJmet medium was prepared by adding 6 mM NaHCO3 and 1 µM CuSO4 to MJ medium consisting of (per liter) 30 g NaCl, 0.14 g K2HPO4, 0.8 g CaCl2, 3.4 g MgSO4·7H2O, 4.18 g MgCl2·6H2O, 0.33 g KCl, 0.25 g NH4Cl, 0.25 g NaNO3, 0.5 mg NiCl2·6H2O, 0.5 mg Na2SeO3·5H2O, 0.1 mg Na2WO4, 20 mg Fe(NH4)2(SO4)2·6H2O, 10 ml of a trace mineral solution (4), and 1 ml of a vitamin solution (4). MJmet medium was prepared using the procedure previously described (19) with headspace gas containing CH4, N2, CO2, and O2 (45:40:10:5; 200 kPa). Methanotrophs were cultivated at 37 and 50°C. For anaerobic, fermentative, sulfur-reducing thermophiles, MJYPS medium (70) and incubation at 55, 70, 85, and 95°C were used. For anaerobic dissimilatory Fe(III) and/or sulfate reducers, MMJHFe medium was used with headspace gas containing H2 and CO2 (80:20; 200 kPa) or N2 and CO2 (80:20; 200 kPa), and it was incubated at 30, 55, 70, and 85°C. MMJHFe medium is MMJ medium (66) supplemented with 0.005% (wt/vol) yeast extract, 5 mM formate, 5 mM acetate, 5 mM pyruvate, 5 mM lactate, 0.2% (wt/vol) sodium sulfate, 10 mM Fe(III) citrate, and 10 mM ferrihydrite (27) and reduced with 0.02% (wt/vol) sodium sulfide. For aerobic and anaerobic heterotrophs, MJYPV medium (57) was used with air or N2-CO2 (80:20; 200kPa) as the headspace gas at cultivation temperatures of 30 and 55°C. For cultivation of strictly anaerobic photoautotrophic microorganisms, MJHS-photo medium was employed; this medium consisted of (per liter) 20 g NaCl, 0.09 g K2HPO4, 0.07g KH2PO4, 1.25 g NH4Cl, 0.8 g CaCl2, 7 g MgCl2·6H2O, 0.33 g KCl, 0.05 g FeCl3, 0.01 g Fe(NH4)2(SO4)2·6H2O, 5 mg NiCl2·6H2O, 5 mg Na2SeO3·5H2O, 1 mg Na2WO4·2H2O, 0.5 mg of resazurin, and 10 ml each of a trace mineral solution (4) and a vitamin solution (4), as well as 0.2% (wt/vol) NaHCO3, 0.1% (wt/vol) Na2S2O3·5H2O, and 0.1% Na2S·9H2O. The final pH was adjusted to 7.0, and the gas phase for this medium was H2-CO2 (80:20; 200 kPa). Cultivation was carried out at 30 and 55°C with a photosynthetic photon flux density of 30 µmol/m2·s.
Some of the microorganisms cultivated in the most diluted media were also isolated subsequently by the dilution-to-extinction method. Partial sequences of the 16S rRNA gene (approximately 700 to 1,400 bp) of the isolates were determined as described below.
FISH and cell counting.
The fixed cells collected on a poly-L-lysine-coated 0.2-µm-pore-size polycarbonate Nuclepore filter were subjected to direct cell counting and phylogenetic quantification by FISH and catalyzed reporter deposition FISH as previously described (48, 61). In addition to the previously reported rRNA-targeted oligonucleotide probes specific for each phylogenetic group, two probes newly designed for bacterial isolates obtained in this hydrothermal system were employed, as shown in Table 1.
|
Phytoplankton were identified and counted by using red autofluorescence with green light excitation, and total cell density was determined by counting DAPI-stained cells. The microbial cells on a polycarbonate filter were immobilized by dipping the filter in a 0.2% low-gelling-temperature agarose gel and were dried at 45°C. The cells were then incubated with lysozyme (10 mg per ml in 0.05 M EDTA and 0.1 M Tris [pH 7.5]) and proteinase K (4 U per ml in 0.05 M EDTA and 0.1 M Tris [pH 7.5]) for 1 h at 37°C to increase cell wall permeability and treated with 0.01 M HCl for 20 min to deactivate these enzymes and endogenous peroxidase. The microbial cells on a filter section were hybridized for 3.5 h at 35°C in a hybridization solution (0.9 M NaCl, 20 mM Tris-HCl [pH 7.5], 10% dextran sulfate, 0.02% sodium dodecyl sulfate, 1% blocking reagent, 0.05% salmon sperm DNA, 0.05% Escherichia coli tRNA) containing 0.1 pmol per µl of each probe labeled with horseradish peroxidase at its 5' end and the appropriate formamide concentration (Table 1). The filter section was then washed in wash buffer I (5 mM EDTA [pH 8.0], 20 mM Tris-HCl [pH 7.5], 0.01% sodium dodecyl sulfate) containing the appropriate salt concentration for the probe for 10 min at 37°C and subsequently in wash buffer II (1x phosphate-buffered saline with 0.05% Triton X-100) for 15 min at room temperature. The hybridized cells were reacted in 1/50 Cy3-labeled tyramide solution (TSA direct; PerkinElmer) according to the manufacturer's instructions and then washed in wash buffer II and dehydrated with ethanol. The cells were observed with a fluorescence microscope (model BX61; Olympus, Tokyo, Japan) using the WU (excitation at 360 nm and emission at 460 nm) and WIG (excitation at 546 nm and emission at 585 nm) fluorescent filter sets for DAPI- and Cy3-stained microbial cells, respectively. Microbial cell densities were determined by counting more than 5,000 DAPI-stained cells in at least 30 microscopic fields.
Nucleotide sequence accession numbers.
The 16S rRNA gene sequences of the phylotypes and representative isolates and the sequences of the particulate methane monooxygenase gene pmoA of the methanotrophs have been deposited in the DDBJ/EMBL/GenBank databases. The accession numbers for bacterial phylotypes are as follows: pItb-HW, AB294888 to AB294922; pItb-vmat, AB294923 to AB294979; and pItb-RF, AB294980 to AB295005. The accession numbers for archaeal phylotypes are as follows: pIta-HW, AB301857 to AB301881; pIta-vmat, AB301882 to AB301898; and pIta-RF, AB301899 to AB301906. The accession numbers for 16S rRNA genes of the isolates are shown in Table 3. The accession numbers for pmoA of isolates IT-4 and IT-9 are AB302947 and AB302948, respectively.
|
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
|
At the two hydrothermal vent sites, the temperature, pH, dissolved oxygen content, and salinity of the vent fluids were markedly different even 1 m above the vents due to dilution with ambient seawater (Table 2). Only the oxidation-reduction potential values were still low 1 m above the vents. The free gas compositions were similar at the two hydrothermal sites; the gas consisted mainly of CH4 (68.2 to 69.5%) and N2 (28.1 to 30.1%) and there were small amounts of CO2, O2, and H2, which is consistent with the previous results (26). The main vent fluid contained dissolved H2S (162 to 188 µM) in addition to CH4, CO2, H2, and NH4+. The abundant CH4 in this Taketomi system contrasted with the findings for other shallow hydrothermal systems, where CO2 has been reported to be the primary gas component and CH4 has been reported to be a rather minor component (2, 11, 21, 50, 51).
Phylogenetic diversity of 16S rRNA gene clones.
To investigate the phylogenetic diversity of the main vent communities, bacterial and archaeal 16S rRNA gene clone analyses were performed for the vent fluid, white mat, and reference seawater.
(i) Bacterial diversity.
Highly diverse bacterial 16S rRNA gene community structures were found in the vent fluid (35 phylotypes in 85 clones; the library was designated pItb-HW), the mat (57 phylotypes in 78 clones; pItb-vmat), and the reference seawater (29 phylotypes in 62 clones; pItb-RF) (Fig. 2a). The dominant phyla in the vent fluid and adjacent microbial mat were identified as Gamma-, Epsilon-, and Deltaproteobacteria, and then the phylotypes within these groups were phylogenetically characterized (Fig. 3a to e).
|
|
The phylotypes in the methane-oxidizing family Methylococcaceae were recovered only from the mat sample (Fig. 3c). Three phylotypes showed the highest similarity (>94.0%) with methanotrophic isolate IT-4 that was obtained in this study, while one phylotype was similar to the genus Methylomonas. Most of the rest of the gammaproteobacterial phylotypes were previously uncultivated groups of microorganisms, although some of them were phylogenetically associated with environmental clones obtained from deep-sea hydrothermal vent sites.
(b) Epsilonproteobacteria.
The phylotypes in the Epsilonproteobacteria were detected only in the vent fluid (Fig. 2a). The pItb-HW-11 phylotype, the second most dominant phylotype in the fluid (clonal frequency, 12.9%), was affiliated with the thermophilic hydrogen-oxidizing genus Caminibacter (Fig. 3d). One of the phylotypes belonging to the family "Thiovulgaceae" (8), pItb-HW-17, showed 99.9% sequence similarity with the novel mesophilic hydrogen-oxidizing isolate Ho30-mm.
(c) Deltaproteobacteria.
The Deltaproteobacteria was the second most abundant group in the mat (clonal frequency, 26.9%) and the third most abundant group in the fluid (11.7%) (Fig. 2a). This anaerobic group was not detected in the oxidative reference seawater. The deltaproteobacterial phylotypes in the libraries from the fluid and mat were quite different (Fig. 3e). Within the families Desulfobulbaceae and Desulfobacteraceae, some of the phylotypes obtained from the mat library, including the predominant pItb-vmat-4 phylotype (clonal frequency, 16.7%), were closely related to the sulfate-reducing endosymbionts of the marine oligochaetes Olavius spp.
(d) Deferribacterales.
Only one phylotype, phylotype pItb-HW-19, in the thermophilic Deferribacterales was found to be a relatively abundant component of the fluid (clonal frequency, 8.3%; accession no. AB294899). This phylotype was distantly related to the genus Deferribacter, and the data indicated a closest relationship to Flexistipes sp. strain vp180 (92.7% sequence similarity) obtained from a high-temperature offshore oil field (47), whereas we isolated one Deferribacter sp. strain from the same fluid sample.
(ii) Phylogenetic diversity of archaeal 16S rRNA genes.
Phylogenetic analysis of archaeal 16S rRNA gene clone sequences revealed that the archaeal community structures in the fluid, mat, and reference seawater might differ greatly (Fig. 2b). In the fluid community (25 phylotypes in 55 clones; pIta-HW), two predominant phylotypes were found: phylotype pIta-HW-4 (clonal frequency, 20.0%) affiliated with anaerobic methane oxidation group 1 (ANME-1) (Fig. 4a) and phylotype pIta-HW-1 (clonal frequency, 18.2%) assigned to the miscellaneous crenarchaeotic group (MCG) (Fig. 4b). Members of ANME-1, which is known as a potential anaerobic methane-oxidizing group, have frequently been detected in various methane-rich marine sediment environments, including methane seeps (44, 46), and even in deep-sea hydrothermal habitats (62, 74). Our results revealed the predominant occurrence of ANME-1 in a shallow submarine hydrothermal system for the first time. The MCG is known to occur in a wide range of marine and terrestrial environments, although the physiological traits remain unknown (73). The phylotypes belonging to the deep-sea hydrothermal vent euryarchaeotic group (DHVEG) and the Archaeoglobales, groups which are specifically found at hydrothermal vent sites (38, 64), were retrieved only from the fluid. The recent successful cultivation of a thermophilic DHVEG archaeon from actively venting deep-sea sulfide structures (52) strongly supports the assumption that the DHVEG consists of thermophiles.
|
Quantitative cultivation and isolation of representative microorganisms.
Based on the analyses of chemical characteristics and 16S rRNA gene communities, sulfur- and methane-oxidizing thermophiles and mesophiles were expected to be the predominant organisms in the communities associated with the fluid emission at the main vent site. We estimated the culturability and population density of potentially predominant microbial components in the vent fluid and mat using the liquid serial dilution cultivation technique. Microorganisms showing a variety of types of energy and carbon metabolism were successfully cultivated (Fig. 5). Each representative isolate obtained from the most diluted tube was subjected to a comparative 16S rRNA gene sequence analysis (Table 3 and Fig. 3a to e).
|
(ii) Methane-oxidizing microorganisms.
Methane oxidizers were estimated to be less abundant than sulfur oxidizers in both samples (Fig. 5); however, novel mesophilic and thermophilic gammaproteobacterial type I methanotrophs were successfully isolated. Methane oxidizers that grew at 50°C were successfully cultivated only from the vent fluid. The thermophilic, methane-oxidizing isolate IT-9 showed the highest sequence similarity (94.0%) with the mesophilic organism Methylohalobius crimeensis (18) (Fig. 3c). Although a methanotrophic strain was cultivated by incubation of the vent fluid at 37°C, the rRNA gene sequence of the strain was identical to the sequence of strain IT-9. IT-9 was the most abundant methanotroph in the vent fluid at both cultivation temperatures (37 and 50°C), whereas thriving mesophilic methanotrophs were found in the mat. Five mesophilic isolates were obtained from the mat at different cultivation temperatures (30 to 42°C), and sequencing analysis revealed that these isolates were phylogenetically identical. The representative isolate IT-4 was moderately related to Methylobacter marinus (92.6% sequence similarity), which was its closest relative (Fig. 3c). The optimum growth temperatures of IT-4 and IT-9 were found to agree with the in situ temperatures of their habitats. The gene encoding particulate methane monooxygenase (pmoA) was successfully amplified from the DNA of IT-4 and IT-9 using the A189/A682 primer set (22) in conditions previously described (20), while soluble methane monooxygenase genes were not detected in either of the isolates when the previously reported primers and conditions were used (34, 35). Electron microscopic analysis of ultrathin sections of IT-4 and IT-9 cells showed the presence of a stack of intracellular membrane disks typical of type I methanotrophs (data not shown).
(iii) Hydrogen-oxidizing microorganisms.
Hydrogen oxidizers were cultivated only from the mat at 30°C, and the population density was relatively low (Fig. 5). The obtained epsilonproteobacterial isolate Ho30-mm was moderately related to Sulfurimonas paralvinellae (92.1% sequence similarity) (71) (Fig. 3d). No thermophilic hydrogen oxidizers were cultivated in this study. Molecular hydrogen was a minor component of the free gas (Table 2), which might be the reason that culturable hydrogen oxidizers were less abundant at the main vent site. In contrast, rRNA gene clone analysis demonstrated the presence of thermophilic, hydrogen-oxidizing, anaerobic to microaerobic Caminibacter in the vent fluid (Fig. 3d).
(iv) (Hyper)thermophilic microorganisms.
In addition to the potential primary producers described above, iron-, sulfur-, and sulfate-reducing, (hyper)thermophilic heterotrophs were also cultivated from the fluid and/or mat, even though their population densities were lower than those of the chemolithotrophic primary producers (Fig. 5). Cultivation of hyperthermophilic sulfur reducers at 70 to 95°C resulted in isolation of seven Thermococcus isolates showing 98 to 100% sequence similarity from both samples. The representative isolate Tc85 showed 100% sequence similarity with Thermococcus kodakaraensis (3). An iron-reducing Deferribacter sp. showing 99.3% sequence similarity with Deferribacter desulfuricans (67) was isolated only from the fluid. Thermococcus spp. have often been cultivated from other shallow hydrothermal systems with high-temperature fluid emissions (21, 23, 56, 63). In contrast, Deferribacter spp. have previously been obtained only from deep-sea hydrothermal habitats (36, 67); therefore, in this study Deferribacter was cultivated for the first time from a shallow hydrothermal habitat.
(v) Other microorganisms.
Anaerobic sulfate-reducing Desulfovibrio spp. were cultivated only from the mat sample, indicating that there were anaerobic microhabitats inside the mat. Aerobic heterotrophs isolated from the fluid and mat were affiliated with Pseudoalteromonas and Vibrio, respectively. The obligately photoautotrophic, strictly anaerobic, sulfur-oxidizing isolate 5H2 was cultivated as a minor component of the mat community. Isolate 5H2 exhibited the closest relationship to a marine photosynthetic bacterium, Prosthecochloris aestuarii in the Chlorobi group (98.1% sequence similarity). The detection of a few phototrophic microorganisms at the main vent site was consistent in both culture-dependent and culture-independent analyses.
Cell densities determined by FISH.
The total cell density was greater in the main vent fluid than the ambient seawater 1 m above the vent and the reference seawater (Table 4). Quantitative FISH analyses using several group-specific oligonucleotide DNA probes indicated that the number of Bacteria cells (EUB338 positive) was much greater than the number of Archaea cells (ARCH915 positive) in the vent fluid and reference seawater, whereas the number of Archaea cells slightly exceeded the number of Bacteria cells in the seawater 1 m above the vent (Table 4). At the reference site, probes EPS402 and DELTA495 did not show any signature or showed only a slight signature, indicating that no detectable epsilonproteobacterial cells and few deltaproteobacterial cells were present in the reference seawater. In contrast, at the main vent site, all of the targeted bacterial groups were detected with the group-specific probes, and only the Epsilon- and Deltaproteobacteria populations were considerably larger in the vent fluid. In addition, unclassified cells that were positive with the EUB338 probe but negative with all other probes used were abundant (16.1% of the EUB338-positive cells) in the vent fluid. These results indicate that the Epsilon- and Deltaproteobacteria and the unclassified bacteria prefer the hot vent fluid. The unclassified components may contain additional epsilonproteobacterial populations, because probe EPS402 had 2- or 3-base mismatches specifically with members of the family Nautiliaceae in the Epsilonproteobacteria. Phylotype pItb-HW-11 of the genus Caminibacter was abundant in the main vent fluid (Fig. 3d), and pItb-HW-11 also had a 3-base mismatch with EPS402. Consequently, the presence of Epsilonproteobacteria could have been underestimated in this FISH experiment.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Implication for subseafloor communities.
In addition to ANME-1 archaea, anaerobic to microaerobic (hyper)thermophilic microorganisms, such as Caminibacter, Deferribacter, Thermococcus, and DHVEG archaea, were found to be abundant components of the microbial community in the main vent fluid (52°C). Some inconsistencies among the results of different experiments were found in this study. The phylotype of Caminibacter was abundant in the vent fluid; nevertheless, no thermophilic hydrogen oxidizer was obtained in cultivation experiments. Thermococcus strains were successfully cultivated from the fluid and mat samples, but the sequence of Thermococcus was not retrieved by 16S rRNA gene clone analysis. For Deferribacter and Thermococcus species, the in situ temperature at the main vent site was markedly lower than the optimum growth temperatures of these organisms. Some of the microorganisms detected in the vent fluid were anaerobic to microaerobic despite the presence of a detectable amount of oxygen in the fluid. These inconsistencies suggest that there are more reductive and/or hotter subseafloor habitats and that some microorganisms from these habitats were entrained by hydrothermal fluid flow.
Epsilonproteobacteria habitats expanded to a shallow hydrothermal system.
Although the cultivation analysis did not show prominent culturability of the Epsilonproteobacteria, culture-independent molecular analyses suggested that such organisms were abundant in the hydrothermal fluid and the proximal habitats. Many previous studies have stressed the abundance, phylogenetic and metabolic diversity, and potential ecophysiological significance of the Epsilonproteobacteria in deep-sea hydrothermal environments (8, 38, 39, 53, 61, 64, 65). However, the ecological features of these organisms in shallow submarine hydrothermal systems are very poorly understood. Our results demonstrated their predominance in the shallow Taketomi system. In addition, not only members of the Thiovulgaceae previously cultivated from coastal oxic-anoxic interface zones (5) but also members of the Nautiliaceae that had previously been believed to occur only in deep-sea hydrothermal systems were identified for the first time in a shallow hydrothermal system. This may imply that there is unseen propagation of deep-sea Epsilonproteobacteria and adaptation of these organisms to other habitats outside the deep sea.
Predominance of chemolithoautotrophs and methanotrophs.
Our results indicate that chemolithoautotrophs and methanotrophs, including members of the Epsilonproteobacteria and unique members of the Gammaproteobacteria, predominated at the main vent site in the Taketomi system. There have been few comparable previous investigations of entire microbial communities in shallow submarine hydrothermal systems, although Sievert et al. (59) demonstrated in a study of the Milos shallow hydrothermal system that there were more heterotrophs than autotrophs in the sediment community despite the fact that high numbers of chemolithoautotrophic sulfur-oxidizing bacteria were cultivated from the hydrothermal sediments. Energy sources could be supplied in different ways in sediment and fluid, which should affect microbial community structures. More complex distribution of energy sources is assumed to occur in the sediment environments. Energy and carbon sources, such as H2, H2S, CO2, CH4, and NH4+ derived from geothermal activities, are dissolved in and transferred by the hydrothermal fluid flows, and thus the predominance of chemolithoautotrophs and methanotrophs in the hydrothermal fluid of the Taketomi system is consistent with the expected flow of chemical inputs. In the Taketomi system, analyses of hydrothermal sediment communities around the main vent site are now in progress in our laboratory. Hence, we will soon be able to compare the sediment communities in the Taketomi and Milos systems.
Photosynthetic contribution to shallow hydrothermal systems.
The other important energy source for microbial primary production in a shallow-water submarine ecosystem is sunlight. Tarasov et al. (72) revealed that photosynthesis is conspicuously stimulated by the high input of nutrients from hydrothermal fluid in shallow-water hydrothermal environments. In this study, however, phototrophic microorganisms were scarce components of the communities around the main vent site at a depth of 23 m. This may have been the result of less energy input from sunlight into the deepest point of the craterlike basin compared with the greater input of inorganic energy sources by the hydrothermal fluid. In contrast, thick green and brown microbial mats developed at the bubbling vent site at a depth of 13 m. In this report, the community structure of the green and brown microbial mats around the bubbling vent site is not described in detail, but a single phylotype closely related to the phototrophic isolate 5H2 obtained from the main vent site accounted for 95% of the bacterial clone library in the colored mat sample (data not shown). This implies that the microbial mats flourishing around the bubbling vent site are likely dominated by a strictly anaerobic, sulfur-oxidizing photoautotroph belonging to the Chlorobi. The difference in microbial community structures in the mats at different depths might be associated with the photosynthetic photon flux densities in the two habitats.
Numerous reports of microorganisms associated with shallow submarine hydrothermal systems have been published, although none of these reports presented a comprehensive characterization of bacterial and archaeal communities in the systems. In this study, we combined cultivation-based and molecular analytical techniques to obtain an overview of active microbial communities in the Taketomi system. At the moment, it is difficult to provide key microbiological characteristics to differentiate the microbial ecosystems in shallow and deep-sea hydrothermal systems other than the light energy input. This is partially due to the limited information available for shallow submarine hydrothermal systems despite the much greater accessibility of these systems. Nevertheless, this study could undoubtedly contribute to our understanding of unique characteristics of variety of global shallow hydrothermal systems and similarities between shallow-water and deep-sea hydrothermal systems.
|
FOOTNOTES |
|---|
Published ahead of print on 5 October 2007.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
