RESEARCH ARTICLE
Comparison of microbial communities associated with
phase-separation-induced hydrothermal £uids at theYonaguni
Knoll IV hydrothermal ¢eld, the Southern Okinawa Trough
Takuro Nunoura & Ken Takai
Subground Animalcule Retrieval (SUGAR) Program, Extremobiosphere Research Center, Japan Agency for Marine-Earth Science & Technology
(JAMSTEC), Yokosuka, Japan
Correspondence: Takuro Nunoura,
Subground Animalcule Retrieval (SUGAR)
Program, Extremobiosphere Research Center,
Japan Agency for Marine-Earth Science &
Technology (JAMSTEC), 2-15 Natsushimacho, Yokosuka 237-0061, Japan. Tel.: 181 46
867 9707; fax: 181 46 867 9715; e-mail:
[email protected]
Received 24 June 2008; revised 11 October
2008; accepted 8 November 2008.
First published online 14 January 2009.
DOI:10.1111/j.1574-6941.2008.00636.x
Editor: Patricia Sobecky
Keywords
deep-sea hydrothermal vent; phase separation;
hydrogen oxidizing; Okinawa Trough.
Abstract
Microbial communities associated with a variety of hydrothermal emissions at the
Yonaguni Knoll IV hydrothermal field, the southernmost Okinawa Trough, were
analyzed by culture-dependent and -independent techniques. In this hydrothermal
field, dozens of vent sites hosting physically and chemically distinct hydrothermal
fluids were observed. Variability in the gas content and formation in the
hydrothermal fluids was observed and could be controlled by the potential
subseafloor phase-separation and -partition processes. The hydrogen concentration in the hydrothermal fluids was also variable (0.8–3.6 mmol kg1) among the
chimney sites, but was unusually high as compared with those in other Okinawa
Trough hydrothermal fields. Despite the physical and chemical variabilities of the
hydrothermal fluids, the microbial communities were relatively similar among
the habitats. Based on both culture-dependent and -independent analyses of the
microbial community structures, members of Thermococcales, Methanococcales
and Desulfurococcales likely represent the predominant archaeal components,
while members of Nautiliaceae and Thioreductoraceae are considered to dominate
the bacterial population. Most of the abundant microbial components appear to
be chemolithotrophs sustained by hydrogen oxidation. The relatively consistent
microbial communities found in this study could have been because of the
sufficient input of hydrogen from the hydrothermal fluids rather than other
chemical properties.
Introduction
Recent culture-dependent and -independent microbiological surveys in various deep-sea hydrothermal vent habitats
have indicated a variety of community structures and
metabolic activities of the indigenous microbial components in the chimney structures and the potential subvent
biosphere (Harmsen et al., 1997; Takai & Horikoshi, 1999;
Reysenbach et al., 2000a, b; Takai et al., 2001; Schrenk et al.,
2003; Nakagawa et al., 2005b; Kormas et al., 2006; Pagé et al.,
2008). These studies, coupled with concurrent geochemical
and mineralogical characterizations, have led to the presumption of a potential biogeochemical interaction in the
habitat and have enabled comparison between the habitats
in a hydrothermal field (intrafield comparisons) and even
among different fields (interfield comparisons). From the
FEMS Microbiol Ecol 67 (2009) 351–370
comparisons, it is evident that members of the Thermococcales, Desulfurococcales, Aquificae and Epsilonproteobacteria
are cosmopolitan and predominant in the global deepsea hydrothermal high-temperature environments, and
members of Archaeoglobales, Methanococcales, Methanopyrales, Thermodesulfobacteriales, Deferribacteriales and Gammaproteobacteria are also major components in many
hydrothermal environments (Takai et al., 2006a; Nakagawa
& Takai, 2008).
The emerging patterns of community structures and
metabolisms in deep-sea hydrothermal ecosystems could be
associated with the physical and chemical conditions of the
habitats (Karl, 1995; Takai et al., 2006a). One potential key
factor is the concentration of gaseous compounds such as
H2, H2S and CH4 in hydrothermal fluids because they serve
as the primary energy sources for the chemolithoautotrophs.
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352
The abundance and composition of these gaseous compounds in the hydrothermal fluids are substantially controlled by complex processes related to the tectonic,
magmatic and hydrogeologic settings. Phase separation and
partition of hydrothermal fluid might also have a huge impact
on the abundance and composition of the gaseous energy
sources (Massoth et al., 1989; Lilley et al., 1993; Butterfield
et al., 1994; Nakagawa et al., 2005b; Konno et al., 2006; Takai
et al., 2008). Indeed, a phase-separation-associated intrafield
variability in functionally active microbial communities has
been demonstrated in the hydrothermal fields in the middle
Okinawa Trough (Nakagawa et al., 2005b) and in the Lau
Basin (Takai et al., 2008). The increased populations of
thermophilic and hydrogenotrophic methanogen Methanococcales members (the Iheya North field in the middle
Okinawa Trough) and Aquificales members (the Mariner field
in the Lau Basin) have been documented as potential
responses to the phase-separation-controlled variability in
the fluid chemistry.
The Yonaguni Knoll IV hydrothermal field is located at
the southwest end of the Okinawa Trough, and dozens of
black and clear smoker vent emissions have been identified
after its discovery in 2000 (Konno et al., 2006). In sediments
extending around hydrothermal vent sites, a large-scale
subseafloor liquid CO2 pool originally derived from hydrothermal fluids was also observed (Inagaki et al., 2006; Konno
et al., 2006). Multidisciplinary seafloor explorations were
conducted in 2003 and 2004 using the manned submersible
Shinkai 6500, and many hydrothermal fluid and chimney
samples were collected; deployment and recovery of in situ
colonization systems (ISCS) (Takai et al., 2003a) in hydrothermal fluids were conducted during the explorations.
Geochemical analyses of hydrothermal fluids and the chimney mineral compositions clearly demonstrated the variability in hydrothermal fluid chemistry controlled by the
subseafloor phase separation and partition (Konno et al.,
2006; Suzuki et al., 2008). In addition, unusually high
concentrations of hydrogen in hydrothermal fluids ranging
from 0.5 to 5.2 mmol kg1 were observed (Konno et al.,
2006). In this study, we present the structural and metabolic
compositions of microbial communities in the various
habitats influenced by the variation of the hydrothermal
fluid chemistry in order to determine the effects of phase
separation of hydrothermalism with a high hydrogen concentration.
T. Nunoura & K. Takai
where the Ryukyu Arc intersects with the Taiwan Arc
(24150 0 –24151 0 N, 122151.5 0 –122152.5 0 E). Various hydrothermal fluids and chimney structures were obtained from
the ‘Tiger Chimney Mound’, having both black smoker vents
(BTC) and clear smoker vents (CTC) (24150.885 0 N,
122142.014 0 E), the ‘Lion Chimney’, having black smoker
vents (LC) (24150.938 0 N, 421020 0 E), and the ‘Swallow
Chimney’, with clear smoker vents (SC) (24150.832 0 N,
122142.013 0 13E), the manned submersible Shinkai 6500
during the cruises YK03-05 (July 2003) and YK04-05 (May
2004) of the R/V Yokosuka. The self-temperature-recording
in situ colonization systems (STR-ISCS) described previously (Takai et al., 2003a) were deployed in the hydrothermal fluid conduits of BTC and CTC vents for 5 and
7 days, respectively, during the YK04-05 cruise. The hydrothermal fluid samples were collected using both a gas-tight
fluid sampler ‘Water hydrothermal-fluid Atsuryoku tight
sampler II (WHATS II)’ equipped with a self-recording
thermometer (Saegusa et al., 2006) and a plastic bag sampler
equipped with a deep-sea impeller pump. The samples used
in this study are summarized in Table 1. At least two
portions of the chimney structures were taken, except for
the Swallow chimney site.
The chimney structures collected were divided into surface layers and interior structures as described previously
(Takai et al., 2001). The synthetic pumice stuffed in ISCS
and subsampled chimney structures were divided into three
portions. Subsamples for DNA extraction were stored at
80 1C, those for cultivation were anaerobically stored in
glass bottles under 100% N2 (200 kPa) with or without
0.05% neutralized Na2S sealed with butyl rubber stoppers
and those for cell counting were stored at 80 1C after
fixation by filtered (0.22 mm) seawater with 3% formaldehyde at 5 1C overnight. The vent emissions in WHATS gastight bottles (c. 100 mL) or plastic bags (5–10 L) were filtered
by 0.22-mm pore size cellulose acetate filters and stored at
80 1C for DNA extraction. Ten milliliters of vent emissions
were fixed with formaldehyde (final concentration, 3%) and
stored at 80 1C for direct cell counting.
Geochemistry of hydrothermal fluids
The geochemistry of hydrothermal fluids was described by
Konno et al. (2006) and Suzuki et al. (2008). Several key
features are shown in Table 2.
Microscopic observation
Materials and methods
Site description, sampling of hydrothermal
fluids and chimneys and deployment of an ISCS
The active area of the Yonaguni Knoll IV hydrothermal field
was located at the southwestern end of the Okinawa Trough,
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The chimney suspensions and hydrothermal fluids fixed
with 3% formaldehyde were stained with 4 0 ,6 0 -diamidino2-phenylindole dihydrochloride (DAPI) (Porter & Feig,
1980). After staining for 1 h at 4 1C, chimney suspensions
were centrifuged for 5 s at 2000 g. The supernatants of the
chimney suspensions and vent emissions were filtered
FEMS Microbiol Ecol 67 (2009) 351–370
353
Microbial communities associated with hydrothermal vents
Table 1. Description of the samples used in this study
Sample
ID
Vent site
Sampling (temperature
year
of vent fluids)
BTC-VE (03)
BTC-I (03)
2003
2003
BTC-S (03)
2003
BTC-VE (04)
BTC-ISCS (04)
2004
2004
BTC-I (04)
2004
BTC-S (04)
2004
CTC-VE (03)
CTC-I (03)
2003
2003
CTC-S (03)
2003
Black smoker
chimney on
Tiger chimney
mound
(330 1C)
Clear smoker
chimney on
Tiger chimney
mound
(330 1C)
CTC-VE (04)
2004
CTC-ISCS (04) 2004
CTC-I (04)
2004
CTC-S (04)
2004
LC-VE
LC-I (S)
2004
2004
LC-S (S)
2004
LC-I (F1)
2004
LC-S (F1)
2004
LC-S (F2)
2004
SC-I
2004
SC-S1
2004
SC-S2
2004
Lion chimney
(330 1C)
Swallow
chimney
(280 1C)
Description of samples
330 1C of vent emission
Subsample obtained from the
conduit surface of BTC structure
Subsample obtained from the
outer surface layer (1–3 mm) of
BTC structure
330 1C of vent emission
Deployed in the BTC emission for
5 days
Subsample obtained from the
conduit surface of BTC structure
Subsample obtained from the
outer surface layer (1–3 mm) of
BTC structure
330 1C of vent emission
Subsample obtained from the
conduit surface of CTC structure
Subsample obtained from the
outer surface layer (1–3 mm) of
CTC structure
330 1C of vent emission
Deployed in the CTC emission for
7 days
Subsample obtained from the
conduit surface of CTC structure
Subsample obtained from the
outer surface layer (1–3 mm) of
CTC structure
330 1C of vent emission
Subsample obtained from the
conduit surface of LC structure
(stem of chimney)
Subsample obtained from the
outer surface layer (1–3 mm) of LC
structure (stem of chimney)
Subsample obtained from the
conduit surface of LC structure
(flange structure)
Subsample obtained from the
outer surface layer (1–3 mm) of LC
structure (flange structure)
Subsample obtained from the
outer surface layer (1–3 mm) of LC
structure (flange structure)
Subsample obtained from the
conduit surface of SC structure
Subsample obtained from the
outer surface layer (1–3 mm) of SC
structure
Subsample obtained from the
outer surface layer (1–3 mm) of SC
structure
Cell density
(cells g1 or
mL1)
Archaeal 16S Bacterial 16S Archaeal
rRNA gene
rRNA gene
population Cultivation
amplification amplification (%)
analysis
3.3 103
2.8 105
3.2 105
1
1
1.0 103
1.2 104
1
1
ND
ND
5 104
1
ND
2.8 106
1
1
16.7
3.4 103
1.2 104
ND
ND
4.8 105
ND
3.0 103
2.0 104
ND
ND
1.0 105
1
ND
1.0 106
1
1
38.1
5.0 103
1.0 104
1
ND
ND
2.0 105
1
ND
1.0 105
ND
6.0 105
1
1
30.0
ND
1
1
1.5
1.0 105
4.2 107
1
1
7.5
ND
1
1
2.3
ND
ND
7.2
ND
ND
ND
ND
ND
ND
ND, not determined; 1, positive; , negative.
FEMS Microbiol Ecol 67 (2009) 351–370
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354
T. Nunoura & K. Takai
Table 2. Estimated chemical compositions of end-member hydrothermal fluids, assuming [Mg21] = 0 in the fluid (Konno et al., 2006)
Vent
Temperature ( 1C)
H2 (mmol kg1)
CH4 (mmol kg1)
CO2 (mmol kg1)
Cl (mmol kg1)
BTC
CTC
LC
SC
330
330
330
280
0.8
2.4–3.6
1.0–1.1
1.0–1.8
1.8
9.5–13.5
1.2–1.6
6.9–7.2
72
306–329
22–47
99–109
629
332–384
576–674
453–462
through a 0.2-mm Isopore membrane filter (Millipore,
Ireland). Then, the cells on the filters were counted under
fluorescence using an Olympus BX51 microscope. At least
50 microscopic fields for each sample were examined to
determine the microbial community density.
Nucleic acid extraction
The DNA assemblage was extracted from chimney structures, ISCS substrata (pumice) and filtered microbial cells in
hydrothermal fluids using either the Ultra Clean Soil DNA
Purification Mega Kit or the Ultra Clean Soil DNA Purification Kit (Mo Bio Laboratories Inc., Solana Beach, CA). The
extracted DNA was further purified by MagExtractor-PCR
and Gel Clean up (TOYOBO, Osaka, Japan) if necessary. The
DNA of isolates from the cultivation test was also extracted
using the Ultra Clean Microbial DNA Kit (Mo Bio Laboratories).
Amplification of archaeal and bacterial 16S rRNA
gene and clone analysis
The archaeal and bacterial 16S rRNA genes were amplified
from extracted DNA by PCR using LA Taq polymerase with
GC buffer (Takara Bio, Otsu, Japan). The oligonucleotide
primers for PCR amplification were Arch21F (TTCCGGTT
GATCCYGCCGGA) and Arch958R (YCCGGCGTTGAMT
CCAATT) or U1492R (ASGGNTACCTTGTTACGACTT)
for archaeal 16S rRNA gene, and B27F (AGAGTTTGAT
CCTGGCTCAG) and U1492R for the bacterial 16S rRNA
gene (Lane, 1991; DeLong, 1992). PCR amplification was
performed by a thermal cycler GeneAmp 9600 (PE Applied
Biosystems, Foster City, CA), and the DNA amplification
conditions were 30–40 cycles of 96 1C for 25 s, 50 1C for 45 s
and 72 1C for 120 s for the archaeal 16S rRNA gene and
20–30 cycles of 96 1C for 25 s, 54 1C for 45 s and 72 1C for
120 s for the bacterial 16S rRNA gene. The PCR cycle
numbers represent the minimum cycle numbers required
to provide sufficient amplified products for the cloning
based on the preliminary PCR amplification experiments
using the same templates.
The amplified gene fragments of the 16S rRNA gene were
cloned into pCRII vector (Invitrogen, Carlsbad, CA), and
then clone libraries were constructed. The inserts were directly
sequenced by the dideoxynucleotide chain-termination meth2009 Federation of European Microbiological Societies
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od using a dRhodamine sequencing kit (PE Applied Biosystems) according to the manufacturer’s recommendations.
The primers Arch21F and Bac27F were used for the initial
single-strand sequencing of the archaeal and the bacterial
rRNA gene, respectively.
The sequence similarity among all of the single-strand
gene sequences of the 16S rRNA gene, c. 0.5–0.7 kb long,
was analyzed by the FASTA-composing algorithm run by
DNASIS software (Hitachi Software, Tokyo, Japan). The
sequences in the clone analysis showing Z97% and 96%
identities of the archaeal and the bacterial 16S rRNA gene,
respectively, on DNASIS analysis were assigned to the same
phylogenetic clone type (phylotype). The sequences of the
16S rRNA gene from isolates showing Z97% identity on
DNASIS analysis were tentatively assigned to the same species.
The representative rRNA gene clones from each clone type
and 16S rRNA gene from each species were further subjected
to sequencing, and c. 0.8–1.0 kb of sequences were determined from both strands. Both archaeal and bacterial 16S
rRNA gene clone libraries were compared by principal
component analysis (PCA) and Jackknife Environment
Clusters analysis in UNIFRAC program (http://bmf2.colorado.
edu/unifrac/index.psp).
Quantification of the 16S rRNA gene
Quantification of the archaeal and all prokaryotic 16S rRNA
genes in the whole microbial DNA assemblages was performed by a quantitative fluorescent PCR method using the
7500 Real Time PCR System (Applied Biosystems) as
described previously (Takai & Horikoshi, 2000). The copy
number of the 16S rRNA gene in each sample was determined by the average of the triplicate analyses.
Nucleotide sequence accession numbers
The 16S rRNA gene sequences of cultured and uncultured
organisms determined in this study were deposited at the
DDBJ/EMBL/GenBank nucleotide sequence databases under
the following accession numbers: AB235309–AB235325,
AB235326–AB235395 and AB464784–AB464836, respectively.
Cultivation analysis
The abundance of viable microorganisms represented by a
variety of physiological and metabolic characteristics was
FEMS Microbiol Ecol 67 (2009) 351–370
355
Microbial communities associated with hydrothermal vents
determined by a series of serial dilution cultures using each
of the hydrothermal fluids and chimney subsamples under
the various cultivation conditions. For extremely thermophilic to hyperthermophilic fermentative sulfur-reducing
heterotrophs, MJYPS medium (Takai et al., 2000) was used
at 70, 85 and 95 1C; for extremely thermophilic to
hyperthermophilic methanogens, MMJ medium (Takai
et al., 2002) was used at 70, 85 and 95 1C; for thermophilic
to hyperthermophilic sulfate reducers, MMJSO medium
(Nunoura et al., 2007b) was used at 55, 70, 85 and 95 1C;
for mesophilic to hyperthermophilic, strictly anaerobic and
autotrophic sulfur reducers, MMJS medium (Nunoura
et al., 2008b) was used at 37, 55, 70, 85 and 95 1C; for
mesophilic to extremely thermophilic, anaerobic to microaerophilic autotrophs (nitrate-reducing and microaerophilic hydrogen oxidizers and sulfur oxidizers), MMJHS
medium (Takai et al., 2003a) with three types of head space
gases of 80% H2 and 20% CO2 (2 atm), 79% H2, 20% CO2
and 1% O2 (2 atm) and 75% H2, 20% CO2 and 5% O2
(2 atm) was used at 37, 55 and 70 1C; and for strictly
anaerobic thermophilic mixotrophs, MMJYPS medium
(Nunoura et al., 2007a) was used at 55 1C. The microorganisms present in the most diluted series of the medium at
each temperature were isolated by the subsequent extinction–dilution method (Takai et al., 2000). PCA and cluster
analysis of viable populations were conducted using the
BLACK-BOX program (http://aoki2.si.gunma-u.ac.jp/BlackBox/
BlackBox.html).
Results and discussion
Variability in hydrothermal fluid chemistry
As reported previously (Konno et al., 2006; Suzuki et al.,
2008), the chemical composition of the hydrothermal fluids
in the Yonaguni Knoll IV field is variable among the
chimney sites (Table 2). The chlorinity of the potential
end-member hydrothermal fluids ranges between 332 and
674 mmol kg1. In contrast to the chlorinity of the fluid,
and the concentrations of gas species such as H2, CH4 and
CO2 are increased in the Cl-depleted hydrothermal fluids
(Table 2). The variability in the chlorinity and the gas
content could be associated with the subseafloor phaseseparation and -partition processes (Konno et al., 2006).
Among the chimney sites studied, the black smoker fluids
are Cl-enriched (gas-depleted) and the clear smoker fluids
are Cl-depleted (gas-enriched) (Table 2). Interestingly, the
Tiger chimney mound has both black (e.g. BTC) and clear
(e.g. CTC) smoker vents in an area several meters in
diameter, while the black and clear smoker fluids in the
Tiger chimney mound show a clear differentiation into
Cl-depleted and -enriched fluids (Table 2).
FEMS Microbiol Ecol 67 (2009) 351–370
Culture-independent analyses
The compositions of both archaeal and bacterial 16S rRNA
gene phylotypes among four different chimney sites are
shown in Tables 3 and 4, respectively, and the phylogenetic
positions of the archaeal and bacterial phylotypes are
indicated in Figs 1 and 2, respectively. The results of 16S
rRNA gene amplification are presented in Table 1. Except for
the Tiger clear chimney, we could construct two 16S rRNA
gene clone libraries from each chimney surface structure.
In archaeal 16S rRNA gene clone analysis, the clonal
predominance of Thermococcales phylotypes was observed
commonly in the Tiger chimney black smoker fluid, the
ISCS deployed in both the Tiger black and clear smokers and
the internal structures of all the chimneys. Among these
samples, only the Tiger black and clear smoker chimney
habitats (BTC-I and CTC-I) hosted potentially thermophilic
chemolithoautotrophic phylotypes of the Desulfurococcales
and/or Methanococcales other than the Thermococcales phylotypes. On the other hand, in the chimney surface habitats,
the abundance of Thermococcales was relatively less compared with that in the habitats directly associated with the
high temperatures of hydrothermal fluids (Table 3). Instead,
potential thermophilic chemolithoautotrophic archaeal
phylotypes such as the Methanococcales, Methanopyrales
and Desulfurococcales, and a phylotype belonged to previously uncultured Deep-sea Hydrothermal Vent Euryarchaeotic Group II (DHVEG-II) subgroup 8 (Nercessian et al.,
2003) showed an increase in their populations (Fig. 1, Table
3). The Archaeoglobales clone was only detected on the
surface of the Lion chimney [LC-S (F2)] as a minor
population by the 16S rRNA gene clone analysis, although
the Archaeoglobales phylotypes have always been identified
as one of the dominant archaeal phylotypes in the hydrothermal fields around Japan such as the Iheya North field in
the Middle Okinawa Trough (Nakagawa et al., 2005b), and
the Myojin Knoll and the Suiyo Sea-mount in the Izu-Bonin
Arc (Takai & Horikoshi, 1999; Higashi et al., 2004). The
DHVEG-II subgroup 8 was found to be one of the major
components in the Tiger clear smoker chimney surface
(CTC-S), together with the hyperthermophilic lineages of
the Thermococcales, Methanococcales and Desulfurococcales
(Table 3). As far as we know, this DHVEG-II subgroup 8 has
always been detected along with other hyperthermophilic
phylotypes such as the Thermococcales, Methanococcales,
Methanopyrales and Archaeoglobales in high-temperature
habitats such as the 131N in the East Pacific Rise, the Suiyo
Seamount and the Iheya North field (Nercessian et al., 2003;
Higashi et al., 2004; Nakagawa et al., 2005b). Thus, although
the DHVEG II subgroup 8 members are uncultivated and
their physiology is still unclear, the (hyper)thermophily
could be predicted as a key physiological trait of the DHVEG
II subgroup 8 based on its habitational preference. Only one
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c
Crenarchaeota
Desulfurococcales
pYK04-7A-34
pYK03-5A-3N
pYK04-8A-2
pYK04-8A-10
pYK04-14A-15
pYK04-10A-13
pYK04-10A-26
pYK04-18A-1
pYK04-18A-2
pYK04-18A-12
pYK04-18A-15
pYK04-18A-31
Thermoproteales
pYK04-18A-19
MCGI
pYK03-3A-4
pYK03-3A-15
pYK04-8A-1
DSAG
pYK04-19A-7
Euryarchaeota
Thermococcales
pYK04-1A-1N
pYK03-12A-3
pYK04-19A-19
Methanococcales
pYK03-5A-5N
pYK04-8A-3
pYK04-18A-21
23
24
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19
1
7
14
BTC-S
(03)
5
18
2
1
1
1
1
1
BTC-S
(04)
24
LC-I
(S)
24
LC-S
(S)
15
2
LC-S
(F1)
LC-S
(F2)
1
1
23
6
12
1
CTC-I
(04)
CTCISCS (04)
BTC-I
(04)
BTC-VE
(04)
BTC-ISCS
(04)
Cl-depleted vent fluids
Cl-enriched vent fluids
Table 3. Distribution of representative archaeal 16S rRNA gene phylotypes in the vent fluid, ISCSs and chimney structures
3
14
1
1
3
CTC-S
(04)
1
1
8
1
1
1
1
1
1
1
1
SC-S1
19
1
SC-S2
356
T. Nunoura & K. Takai
FEMS Microbiol Ecol 67 (2009) 351–370
Methanopyrales
pYK04-14A-14
Archaeoglobales
pYK04-19A-30
ANME II
pYK04-19A-43
DHVE group II
DHVE3
pYK04-18A-26
pYK04-19A-46
PYK04-19A-49
DHVE4
pYK04-19A-8
DHVE8
pYK04-10A-1
pYK04-20A-4
DHVE group I
DHVE1
pYK04-19A-1
pYK04-19A-5
pYK04-19A-20
pYK04-19A-34
DHVE2
pYK04-8A-26
pYK04-14A-26
pYK04-18A-7
pYK04-19A-6
pYK04-19A-18
pYK04-19A-37
pYK04-19A-45
pYK04-19A-48
pYK04-20A-7
Unclear affiliation
pYK04-8A-8
pYK04-19A-39
Total
Table 3. Continued.
BTC-S
(03)
BTC-S
(04)
LC-I
(S)
LC-S
(S)
LC-S
(F1)
LC-S
(F2)
FEMS Microbiol Ecol 67 (2009) 351–370
23
24
20
21
c
31
1
24
24
25
1
7
1
35
11
1
2
1
1
2
1
1
1
2
1
1
1
2
3
23
19
CTC-I
(04)
CTCISCS (04)
BTC-I
(04)
BTC-VE
(04)
BTC-ISCS
(04)
Cl-depleted vent fluids
Cl-enriched vent fluids
29
1
1
7
CTC-S
(04)
21
1
1
SC-S1
33
12
1
SC-S2
Microbial communities associated with hydrothermal vents
357
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T. Nunoura & K. Takai
Table 4. Distribution of representative bacterial 16S rRNA gene phylotypes in the chimney surface habitats
Cl-enriched vent fluids
BTC-S (03)
Desulfurobacteriaceae
pYK03-3B-3
2
Thermodesulfobacteriaceae
pYK03-5B-47
1
pYK04-20B-52
Thermosulfidibacteriaceae
pYK04-20B-66
Deltaproteobacteria & relatives
pYK03-5B-40
7
pYK03-5B-89
1
pYK03-5B-96
1
pYK04-10B-25
pYK04-14B-1
pYK04-18B-7
pYK04-18B-12
pYK04-18B-20
pYK04-19B-33
pYK04-19B-72
pYK04-20B-26
pYK04-20B-51
Epsilonproteobacteria
Hydrogenimonaceae (Group A)
pYK03-3B-2
10
pYK03-5B-31
1
pYK04-18B-5
pYK04-19B-56
Thiovulgaceae (Group B)
pYK03-3B-19
1
pYK03-9B-46
pYK04-14B-17
pYK04-18B-6
pYK04-19B-2
pYK04-19B-49
pYK04-19B-64
Thiovulgaceae (Group F)
pYK03-8B-1
1
pYK03-8B-4
pYK04-7B-10
pYK04-14B-37
pYK04-18B-1
pYK04-18B-2
pYK04-18B-4
pYK04-18B-8
pYK04-18B-13
pYK04-19B-6
pYK04-19B-38
pYK04-19B-50
pYK04-19B-55
Nautiliales (Group D)
pYK03-3B-6
2
pYK03-3B-10
1
pYK03-3B-11
pYK04-7B-19
pYK04-14B-6
pYK04-14B-10
BTC-S (04)
LC-S (F1)
LC-S (F2)
1
CTC-S (04)
SC-S1
3
SC-S2
1
1
1
4
1
1
3
1
1
2
1
1
1
3
1
10
1
12
7
2
1
10
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Cl-depleted vent fluids
2
1
1
1
3
1
2
1
1
2
3
1
2
3
5
6
3
1
2
2
1
12
1
1
3
1
1
3
1
7
5
1
6
2
2
6
1
1
FEMS Microbiol Ecol 67 (2009) 351–370
359
Microbial communities associated with hydrothermal vents
Table 4. Continued.
Cl-enriched vent fluids
BTC-S (03)
Thioreductoraceae (Group G)
pYK03-3B-2
10
pYK03-5B-67
2
pYK04-10B-32
Campylobacteraceae
pYK04-19B-53
pYK04-20B-58
Alphaproteobacteria
pYK03-5B-2
1
pYK03-5B-14
4
pYK03-5B-44
1
pYK03-5B-65
1
pYK04-19B-5
pYK04-19B-59
pYK04-19B-63
Gammaproteobacteria
pYK03-5B-28
2
pYK04-14B-5
pYK04-14B-16
pYK04-19B-16
pYK04-19B-67
pYK04-19B-69
pYK04-19B-71
Bacteroidetes
pYK03-5B-45
1
pYK03-5B-53
1
pYK04-19B-70
pYK04-20B-61
Planctomycetes
pYK04-19B-4
Actinobacteria
pYK04-19B-7
Acidobacteria
pYK04-20B-64
Chloroflexi
pYK04-20B-34
Deferribacteres
pYK04-20B-36
OP8
pYK04-20B-60
pYK04-20B-62
TM7
pYK04-19B-68
pYK04-20B-13
Total
39
BTC-S (04)
Cl-depleted vent fluids
LC-S (F1)
CTC-S (04)
7
1
1
SC-S1
SC-S2
12
1
1
1
3
1
2
1
2
1
6
1
1
1
2
1
1
1
1
1
1
1
33
39
of the samples from the Lion chimney surface structure [LCS (F2)] showed that the DHVE subgroups 1 and 2 predominated the archaeal community. However, considering the
growth temperature of Aciduliprofundum boonei, the only
isolates in the DHVE 2 that ranged from 55 to 75 1C
(Reysenbach et al., 2006), the in situ temperature of the
sample might be lower than that of other samples and likely
influenced the archaeal community. On the basis of the
FEMS Microbiol Ecol 67 (2009) 351–370
LC-S (F2)
41
29
29
1
31
composition and abundance of the dominating phylogenetic groups, the archaeal rRNA gene community structures
in the hydrothermal fluid and chimney habitats in the
Yonaguni Knoll IV field more closely resemble those in the
Central Indian Ridge (CIR) Kairei field (Takai et al., 2004)
than those in the Iheya North field (Nakagawa et al., 2005b),
which is geographically closer to the Yonaguni Knoll IV
field. Nevertheless, it is also evident that the abundance of
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360
T. Nunoura & K. Takai
(a)
(b)
Fig. 1. Phylogenetic analysis of 16S rRNA
gene sequences of the representative strains
and phylotypes of (a) Crenarchaeota and
(b) Euryarchaeota based on neighbor-joining
method with 529 and 530 homologous positions,
respectively. The boldface type indicates the rRNA
gene obtained in this study. MCG I, Marine
Crenarchaeotic Group I; DHVEG, Deep-sea
Hydrothermal Vent Euryarchaeotic Group.
hyperthermophilic methanogenic phylotypes in the ISCS
and chimney inside structures is much less in the Yonaguni
Knoll IV field than in the CIR Kairei field (Takai et al., 2004).
In bacterial 16S rRNA gene clone analysis, we could
not obtain indigenous bacterial rRNA genes from high2009 Federation of European Microbiological Societies
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temperature habitats such as hydrothermal fluids, ISCS
deployed in the fluids and chimney interior structures.
Other than the Swallow chimney surface habitat (SC-S)
samples and one of the samples from the Lion chimney
surface habitat [LC-S (F2)], generally similar bacterial 16S
FEMS Microbiol Ecol 67 (2009) 351–370
361
Microbial communities associated with hydrothermal vents
(a)
(b)
Fig. 2. Phylogenetic analysis of 16S rRNA gene sequences of the
representative strains and
phylotypes of (a) Aquificae,
(b) Epsilonproteobacteria,
and (c) Alpha- and Gammaproteobacteria and (d)
Deltaproteobacteria based
on neighbor-joining method
with 711, 603, 603 and 603
homologous positions,
respectively. The boldface
type indicates the rRNA
gene sequences obtained in
this study.
rRNA gene community structures were obtained from the
chimney surface habitats (Table 4). In the surface habitats of
the Tiger black and clear smoker chimneys and the Lion
black smoker chimney, the most predominant bacterial
phylotypes were affiliated with the thermophilic epsilonproteobacterial family Nautiliaceae and the mesophilic family
Thioreductoraceae (group D and G Epsilonproteobacteria)
(Nakagawa et al., 2005a; Takai et al., 2005). In addition,
bacterial phylotypes related to the genera Thermodesulfobacterium and Balnearium, and the deltaproteobacterial genera
were commonly detected in the clone libraries (Table 4 and
FEMS Microbiol Ecol 67 (2009) 351–370
Fig. 2). The rRNA gene clones within the Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes groups were also
found as minor fractions in some chimney surface habitats
(Table 4 and Fig. 2). By contrast, in the Swallow chimney
surface (SC-S), the epsilonproteobacterial phylotypes of
the mesophilic family Thiovulgaceae including the genera
Sulfurimonas (Group B), Sulfurovam (Group F) and Nitratifractor (Group F) (Inagaki et al., 2003, 2004; Nakagawa et al.,
2005d; Campbell et al., 2006; Takai et al., 2006b) were
predominant (Table 4, Fig. 2). In one of the samples from
the Lion chimney surface [LC-S (F2)], the predominance of
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362
T. Nunoura & K. Takai
(c)
(d)
Fig. 2. Continued.
Thiovulgaceae and sulfur-oxidizing Gammaproteobacteria
was observed (Table 4, Fig. 2). None of the phylotypes
related to the Aquificales were found from the chimney
habitats of the Yonaguni Knoll IV field (Table 4 and Fig. 2).
The microbial community density was enumerated by
DAPI-stained, direct cell counting. As has always been
demonstrated in the previous investigations (e.g. Takai
et al., 2008), the community density was much larger on
the chimney surface than on the inside portion of the same
chimney and in the hydrothermal fluid hosted by the
chimney (Table 1). On comparing the cell densities among
the surface layers of different chimneys, the Swallow chimney (SC-S) had the largest density (4.0 107 cells g1) and
the Lion chimney (LC-S) had the smallest density
(6.0 105 cells g1) (Table 1). The proportion of archaeal
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16S rRNA gene in the all prokaryotic small subunit rRNA
gene assemblages was found to be 7.2% and 16.7% in the
Tiger black smoker chimney (BTC-S) samples, 38.1% in the
Tiger clear smoker chimney (CTC-S) sample, 30.0% and
1.5% in the the Lion black smoker chimney samples, LC-S
(F1) and LC-S (F2), respectively, and 7.5% and 2.3% in the
Swallow chimney (SC-S) samples (Table 1). These results
imply that the archaeal rRNA gene proportion decreases in
the Swallow chimney surface habitat that hosts the largest
microbial community density. Based on the bacterial rRNA
gene community structure in the Swallow chimney surface
samples (SC-S) and one of the Lion chimney surface
samples (Table 4), mesophilic Thiovulgaceae phylotypes are
likely the populations particularly enriched in the Swallow
chimney surface and part of the Lion chimney surface. Thus,
FEMS Microbiol Ecol 67 (2009) 351–370
363
Microbial communities associated with hydrothermal vents
the small proportion of archaeal rRNA gene could be
explained by increasing populations of mesophilic bacterial
components. Furthermore, the dominant members in the
archaeal community in LC-S (F2) likely grow at lower
temperatures compared with Thermococcales species. The
uniqueness of bacterial and archaeal community structures
in these samples was reflected in PCA (Fig. 3). Consequently, the variation of the microbial community density
and the archaeal rRNA gene proportion may be associated
with the variation of the temperature in the Yonaguni Knoll
IV field.
(a)
Cultivation analysis
The samples used for cultivation analysis are summarized in
Table 1. We did not use LC-S (F2) and SC-S2 samples for
cultivation analysis, although their 16S rRNA gene communities were presented. We could not obtain any successful
cultures from all hydrothermal fluid samples, and the ISCS
samples deployed in the Tiger clear smoker and the Lion
black smoker fluids as well (Tables 5 and 6). These samples
represent the typical habitats consistently exposed to high
temperatures, although the 16S rRNA gene clone analysis
(b)
Fig. 3. UNIFRAC PCA analysis of 14 archaeal (a) and seven bacterial (b) 16S rRNA gene clone libraries. Blue and red squares indicate Cl-enriched and depleted fluids vent sites, respectively. 1, BTC-VE (04); 2, BTC-ISCS (04); 3, BTC-I (04); 4, BTC-S (03); 5, BTC-S (04); 6, LC-I (S); 7, LC-S (S); 8, LC-S (F1); 9,
LC-S (F2); 10, CTC- ISCS (04); 11, CTC-I (04); 12, CTC-S (04); 13, SC-S1; 14, SC-S2.
Table 5. The viable population size of Methanococcales, Thermococcales, Aquificales, Thermodesulfobacteriales, Nautiliales and chemolithoautotrophic Epsilonproteobacteria
Viable population (cells g1 chimney structure or pumice)
Sample
category
BTC-ISCS
BTC-I
BTC-S
CTC-ISCS
CTC-I
CTC-S
LC-I
LC-S (S)
SW-I
SW-S
Thermodesulfobacteriales
Nautiliales
Autotrophic
Epsilonproteobacteria
0–3 103
0–2 10
7 103
6 100–2 10
3 103–2 107
0–2 103
7 103–1 105
7–2 103
3 103–7 107
1 104
1 104
1 10–2 104
2 102
2 104
2 104
Methanococcales
Thermococcales
Aquificales
0–6 100
3 102–9 102
4 10
4 100–8 100
2 104–4 105
0–3 10
0–4 100
2 103–2 104
0–1 10
3 10
3 10
1 102–8 104
2 103
1 105
2
3 10
3
The viable population was obtained by serial dilution cultivation analysis that conducted for each two samples for one category except for BTC-ISCS,
CTC-ISCS and the Swallow chimney site.
FEMS Microbiol Ecol 67 (2009) 351–370
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c
MMJYPS
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MMJHS O2 1%
MMJHS
MMJS
MMJ
95
Medium
1.0 10
2.0 10
Pyrococcus sp.
str. 95-12-1
8.0 104
Thermococcus
sp. str. 83-5-2
2.0 10
Thermococcus
sp. str. 70-4-2
LC-S
Lebetimonas
sp. str. 55S121
2.0 10
Thermococcus
sp. str. 83-5-2
2.0 103
Thermococcus
sp. str. 83-5-2
CTC-I
2.0 104
Thermococcus
sp. str. 83-5-2
2.0 104
Thermococcus
sp. str. 70-4-2
CTC-S
6.0 100
Persephonella
sp. str. 70-8-1
2.0 10
Sulfurimonas
sp. str. 37-8
37
70
6.0 100
Persephonella
sp. str. 70-8-1
55
70
1.0 103
Nitratiruptor
sp. str. 55-12-4
1.0 107
Sulfurimonas
sp. str. 37-8
2.0 102
1.0 104
Persephonella
sp. str. 70-8-1
1.0 104
Nitratiruptor
sp. str. 55-12-4
1.0 10
Persephonella
sp. str. 70-8-1
1.0 102
Nitratiruptor
sp. str. 55-12-4
2.0 103
Sulfurimonas
sp. str. 37-8
7.0 106
Sulfurimonas
sp. str. 37-8
2.0 10
Balnearium sp.
str. 70-12-3
2.0 104
Nitratiruptor
sp. str. 55-12-1
2.0 104
9.0 102
5.0 104
Thermococcus
sp. str. 83-5-2
4.0 105
Thermococcus
sp. str. 70-4-2
BTC-S
55
6.0 100
Thermococcus
sp. str. 70-4-2
BTC-I
Clear smoker (Cl-depleted vent fluids)
1.0 103
8.0 10
Thermococcus
sp. str. 83-5-2
8.0 10
Thermococcus
sp. str. 70-4-2
BTC-ISCS
Black smoker (Cl-enriched vent fluids)
70
70
83
70
83
Temperature
( 1C)
Table 6. Distributions and viable numbers of representative strains obtained by serial dilution count cultivation analyses in each category of samples
Hydrogenivirga
okinawensis LS12-2T
2.6 102
Thiomicrospira sp. str.
37-SI-2
Hydrogenimonaceae str.
37-1%-8-3
Thermodesulfobacterium
sp. str. 70-S-12
Methanocaldococcus sp.
str. 70-8-3
Methanocaldococcus sp.
str. 70-8-3
Methanocaldococcus sp.
70-8-3
3.0 10
Methanocaldococcus sp.
str. 70-8-3
2.0 103
Thermococcus sp. str. 835-2
SW-I
2.6 102
Persephonella
sp. str. 70-8-1
2.6 102
Lebetimonas
sp. str. 55S121
1.7 104
3.0 10
2.6 102
Pyrococcus sp.
str. 95-12-1
1.4 105
Thermococcus
sp. str. 83-5-2
SW-S
364
T. Nunoura & K. Takai
FEMS Microbiol Ecol 67 (2009) 351–370
365
1.7 104
Lebetimonas
sp. str. 55S121
1.0 105
Lebetimonas
sp. str. 55S121
4.0 10
Lebetimonas
sp. str. 55S121
7.0 106
FEMS Microbiol Ecol 67 (2009) 351–370
55
MMJYPS
Sulfurimonas
sp. str. 37-8
Numbers above representative strain names indicate cell numbers g1 chimney structure or pumice.
2.0 102
Lebetimonas
sp. str. 55TY-94
2.0 10
Lebetimonas
sp. str. 55S121
2.0 10
37
Sulfurimonas
sp. str. 37-8
Sulfurimonas
sp. str. 37-8
2.0 10
Nitratiruptor
sp. str. 55-12-4
55
Nitratifractor
sp. str. 37-SO2
7.0 103
Caminibacter
sp. str. 55YT-84
1.0 104
9.0 102
2.0 103
1.0 10
Nitratiruptor
sp. str. 55-12-4
1.0 10
Nitratiruptor
sp. str. 55-12-4
3.0 10
Nitratiruptor
sp. str. 55-12-4
2
CTC-S
CTC-I
BTC-I
BTC-ISCS
Medium
Temperature
( 1C)
Table 6. Continued.
Black smoker (Cl-enriched vent fluids)
BTC-S
2
LC-S
3
Clear smoker (Cl-depleted vent fluids)
SW-I
Hydrogenimonaceae str.
37-1-8-3
SW-S
2.6 102
Nitratiruptor
sp. str. 55-12-4
Microbial communities associated with hydrothermal vents
detected a population of the Thermococcales rRNA genes
(Table 3). However, from similar samples, such as the ISCS
sample deployed in the Tiger black smoker (BTC-ISCS) and
the interior samples of the Tiger black and clear smoker
chimneys and the Swallow chimney, the Thermococcales
members were found to be one of the most predominantly
cultivated populations (Tables 5 and 6). From the ISCS
sample of the Tiger Black smoker chimney, only the
Thermococcus spp. (strains 83-5-2 and 70-4-2) were detected
as a viable population, while the Methanocaldococcus sp.
(strain 70-8-3) was also obtained from the Swallow chimney
internal habitat (Tables 5 and 6). Not only hyperthermophilic archaeal populations but also thermophilic and
mesophilic bacterial members such as Persephonella sp.
(strain 70-8-1), Lebetimonas spp. (strains 55S12-1 and
55TY-9-4), Nitratiruptor sp. (strain 55-12-4) and Sulfurimonas sp. (strain 37-8) were obtained from the internal
habitats of the Tiger black and clear smoker chimneys
(Tables 5 and 6). Among these species, the Thermococcus
and Lebetimonas members could be derived from the
indigenous microbial components potentially inhabiting
the chimney inside structures. Considering their obligate
anaerobic and thermophilic traits based on a culturability
test, it is difficult to assume that these members are simply
contaminated from the exterior habitats during the sample
recovery and the subsampling. In contrast, the abundance of
the Sulfurimonas sp. strain 37-8 in the interior structure of
the Tiger clear smoker chimney (Tables 5 and 6) could be
attributed to the contamination from the more abundant
population in the exterior habitat of the chimney because of
their mesophilic and facultatively aerobic features.
The microbial components from the surface habitats of
the chimney showed high culturability and diversity (Tables
5 and 6). The cultivated microbial community structures of
the four chimney surface habitats were generally similar to
each other (Table 5); the heterotrophic Thermococcales, the
hydrogenotrophic methanogen Methanocaldococcus and the
hydrogen- and/or sulfur-oxidizing autotrophic Epsilonproteobacteria (Sulfurimonas, Hydrogenimonaceae and Nitratiruptor) species were the dominant microbial components
commonly recovered from all the chimney surface habitats
(Fig. 3). In the cultivation test for the S0-reducers at 55 1C,
the Nautiliales members such as Lebetimonas and Caminibacter species were always obtained from the highest dilution cultures under both heterotrophic (mixotrophic)
(MMJYPS medium) and autotrophic (MMJS and MMHJS
media) conditions. The 16S rRNA gene sequence analysis of
the strains isolated from the autotrophic and heterotrophic
(mixotrophic) cultures indicated that both autotrophic and
heterotrophic (mixotrophic) strains were phylogenetically
related to each other. Therefore, it seems likely that most of
the Nautiliales members isolated from the Yonaguni Knoll
IV field are able to grow under both autotrophic and
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366
heterotrophic (mixotrophic) conditions, although all the
previously isolated and reported strains of Nautiliaceae do
not utilize organic carbon as both energy and carbon
sources (Alain et al., 2002; Miroshnichenko et al., 2002,
2004; Takai et al., 2005; Voordeckers et al., 2005). These
potentially mixotrophic Nautiliales members were also predominant in the chimney surface habitats, except for the
Lion chimney in the Yonaguni Knoll IV field.
The potentially mixotrophic Nautiliales members and the
hydrogen- and/or sulfur-oxidizing autotrophic Aquificales
members (Persephonella spp. and Hydrogenivirga) (Nakagawa et al., 2003; Nunoura et al., 2008a) were also present in
abundance in the cultivated microbial communities in most
of the chimneys (Tables 5 and 6). Meanwhile, considerable
populations of the thermophilic H2-oxidizing S0 or SO2
4 reducing autotrophs such as Balnearium and Thermodesulfobacterium spp., respectively (Jeanthon et al., 2002; Takai
et al., 2003b), were detected only in the Tiger clear smoker
chimney surface habitat (Table 6). A population of the
Nitratifractor sp. (strain 37-SO-2) within the Thiovulgaceae
was obtained as the Group F Epsilonproteobacteria only from
the Swallow chimney surface, in which the predominance of
the Group F epsilonproteobacterial phylotypes was shown
by the 16S rRNA gene clone analysis (Table 4). Because of
the relatively less culturability of Group F Epsilonproteobacteria (Sulfurovam and Nitratifractor) compared with the
high culturability of Group B (Sulfurimonas) reported in
various hydrothermal vent habitats by Nakagawa et al.
(2005c), the discrepancy between the culture-dependent
and -independent analyses may have substantially arisen
due to the different extents of difficulty in cultivation among
the groups of Epsilonproteobacteria. Similar situations may
occur in the archaeal populations; the Thermococcales population could be detected by cultivation relatively easily,
while the Desulfurococcales members were always identified
by molecular analyses.
Intrafield and interfield comparison of the
microbial communities in the Yonaguni Knoll IV
field
Both culture-dependent and -independent analyses showed
that several chimney structures in the Yonaguni Knoll IV
field hosted functionally active microbial communities
potentially consisting of archaeal and bacterial components
such as Thermococcales, Methanococcales, Methanopyrales,
Desulfurococcales, Aquificales, Thermodesulfobacteriaceae,
Desulfurobactericeae, Gammaproteobacteria and Epsilonproteobacteria. The microbial community structures inferred
from either culture-dependent or -independent analysis
were different in the composition, for instances, the relative
abundance of Group F epsilonproteobacterial phylotypes
and species in the Swallow chimney site and the active
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T. Nunoura & K. Takai
Fig. 4. PCA of viable populations in each chimney surface samples. Blue
and red squares indicate Cl-enriched and -depleted fluids vent sites,
respectively. 1, BTC-S (03); 2, BTC-S (04); 3, LC-S (S); 4, LC-S (F1); 5, CTCS (03); 6, CTC-S (04); 7, SC-S1.
Thermodesulfobacterium and Balnearium populations specifically in the Tiger clear smoker chimney site. In fact,
bacterial 16S rRNA gene populations in each chimney
samples were relatively diverse (Fig. 3); however, the composition and diversity of the abundant archaeal rRNA gene
phylotypes and the cultivated populations were generally
similar among all the chimney sites studied, with a few
exceptions (Figs 3 and 4).
In the Yonaguni Knoll IV hydrothermal field, it is already
known that the hydrothermal fluids are chemically variable
among the chimney sites and the variation of the fluid
chemistry could be controlled by the relatively shallow
subseafloor phase-separation and -partition processes (Konno
et al., 2006; Suzuki et al., 2008). In addition, the phaseseparation-associated intrafield variability in the microbial
community has been studied intensively in the hydrothermal
fields in the middle Okinawa Trough (Nakagawa et al., 2005b)
and in the Lau Basin (Takai et al., 2008). The thermophilic
and hydrogenotrophic methanogen Methanococcales members in the Iheya North field and of hydrogen- and/or sulfuroxidizing Aquificales members in the Mariner field were
particularly abundant in the habitats associated with the
Cl-depleted (gas-enriched) hydrothermal fluids in these fields
(Nakagawa et al., 2005b; Takai et al., 2008). In both cases,
it was suggested that the phase-separation-induced H2 enrichment might be a major factor responsible for the increased
populations of hydrogenotrophic chemolithoautotrophs.
The potential phase-separation-induced H2 concentration
FEMS Microbiol Ecol 67 (2009) 351–370
367
Microbial communities associated with hydrothermal vents
(a)
(b)
Fig. 5. Cluster analyses of clone libraries. The
trees were created with Jackknife Environment
Clusters analysis of the UNIFRAC program. Jackknife
with 100 permutations was performed. Jackknife
values over 50 are given at corresponding
branches. Comparisons of archaeal 16S rRNA
gene clone libraries from all samples (a) and from
chimney surface samples (b), and of bacterial
16S rRNA gene clone libraries (c).
(c)
(a)
Fig. 6. Cluster analyses of viable populations in
chimney surface samples. Comparisons of viable
populations in chimney surface samples (a) and
that of maximum viable populations in each vent
sites (b).
(b)
anomaly (0.8–3.6 mmol kg1) is also observed in the hydrothermal fluids of the Yonaguni Knoll IV field. If the H2
enrichment in the hydrothermal fluids were to have an impact
on the hydrogenotrophic components of the microbial communities as in the cases of the Iheya North and the Mariner
fields, some of the Methanococcales, Aquificales and Epsilonproteobacteria phylotypes and species should be more abundant in the community in the Tiger clear smoker chimney (the
most H2-enriched) than in the Tiger black smoker chimney
(the least H2-enriched). Neither culture-dependent nor independent characterization in this hydrothermal field supFEMS Microbiol Ecol 67 (2009) 351–370
ports this assumption. Neither 16S rRNA gene community
structures nor viable microbial communities show any difference between Cl-enriched or -depleted vent sites in PCA and
cluster analysis (Figs 5 and 6), and some of the differences in
16S rRNA gene clone analysis may be explained by the in situ
temperature of each sample as described above. The specific
viable population of hydrogenotrophic Thermodesulfobacterium and Balnearium species and the most abundant occurrence of the potentially mixotrophic Nautiliales members in
the Tiger clear smoker chimney may represent a possible
response of the microbial community to the H2 enrichment.
2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
368
However, it is still unclear, without further detailed biogeochemical and microbiological characterizations, whether the
increased culturability of these thermophilic hydrogenotrophs
is a response to the enriched H2 in the hydrothermal fluid or
to other physical and chemical conditions of the habitat. It can
be said that the chemical variability in the phase-separationcontrolled hydrothermal fluid has much less impact on the
composition and function of the microbial communities
among the chimney sites in the Yonaguni Knoll IV field than
in the Iheya North (Nakagawa et al., 2005b) and Mariner
(Takai et al., 2008) fields. The variation of the H2 concentration in the end-member hydrothermal fluids is 45–
96 mmol kg1 and 12–130 mmol kg1 in the Mariner field
(Takai et al., 2008) and the Iheya North field (K. Takai et al.,
unpublished data), respectively. The variation of H2 in these
fields occurs at a one magnitude lower concentration than in
the Yonaguni Knoll IV field (0.8–3.6 mmol kg1). Thus, one
possible explanation for the relatively less intrafield variability
in the microbial community may be the sufficient supply of
H2 and other energy sources to the chimney habitats from the
hydrothermal fluids even in the Cl-enriched (gas-depleted)
vent sites for the Yonaguni Knoll IV field. Nevertheless, the
geological and geochemical mechanisms behind why the
hydrothermal fluids in the Yonaguni Knoll IV field are highly
enriched with H2 are still unclear, even though the field is
located in the typical Ryukyu Arc – Backarc system (Suzuki
et al., 2008). Further investigation of geographically and
geologically distinct hydrothermal systems and interfield
comparisons may possibly provide key insights into the link
between geological, physical, chemical and microbiological
settings of the deep-sea hydrothermal systems.
Acknowledgements
We thank R/V Yokosuka and Shinkai 6500 operation teams
during the cruises YK03-05 and YK04-05 (JAMSTEC) for
their assistance in collecting samples.
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