• Backarc hydrothermal system;
  • Phase-separation;
  • Methanogen;
  • Chemolithoautotroph;
  • Culturability;
  • Epsilonproteobacteria


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgement
  8. Appendix A. Supplementary data
  9. References

Phase-separation and -segregation (boiling/distillation of subseafloor hydrothermal fluids) represent the primary mechanisms causing intra-field variations in vent fluid compositions. To determine whether this geochemical process affects the formation of microbial communities, we examined the microbial communities at three different vent sites located within a few tens meters of one another. In addition to chimney structures, colonization devices capturing subseafloor communities entrained by the vent fluids were studied, using culture-dependent and -independent methods. Microbiological analyses demonstrated the occurrence of distinctive microbial communities in each of the hydrothermal niches. Within a chimney structure, there was a transition from a mixed community of mesophiles and thermophiles in the exterior parts to thermophiles in the interior. Beside the transition within a chimney structure, intra-field variations in microbial communities in vent fluids were apparent. Geochemical analysis demonstrated that different vent fluids have distinctive end-member compositions as a consequence of subseafloor phase-separation and -segregation, which were designated gas-depleted, normal and gas-enriched fluids. In comparison to gas-depleted and normal fluids, gas-enriched fluids harbored more abundant chemolithoautotrophs with gaseous component-dependent energy metabolism, such as hydrogenotrophic methanogenesis. Subseafloor phase-separation and -segregation may play a key role in supplying energy and carbon sources to vent-associated chemolithoautotrophs and subvent microbial communities.


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgement
  8. Appendix A. Supplementary data
  9. References

Deep-sea hydrothermal systems provide physically and geochemically diverse habitats for mesophilic to hyperthermophilic prokaryotes [1]. Recent researches expanded the microbial habitat to subseafloor environments of the mid-ocean ridge hydrothermal systems [2–4]. Microbial communities in a variety of hydrothermal habitats are composed of physiologically and phylogenetically diverse microorganisms [1,5–9]. To address the relationships of the microbial community structures to the environmental conditions, microbial communities occurring within the chimney structures have been intensively studied by the combined use of quantitative cultivation and culture-independent molecular techniques [10,11]. These works provided new insights into zonation and diversity of microbial communities associated with steep physical and geochemical gradients within the chimney structure. However, they mainly focused on Archaea or thermophiles. Thus, it is still unclear how the physical and geochemical parameters are associated with in situ ecophysiological functions of the overall microbial community.

In deep-sea hydrothermal systems, indigenous microbial primary production is achieved by chemolithoautotrophs utilizing H2, reduced sulfur compounds and CO2[1,12]. The energy and carbon sources are provided from magma degassing and/or from the reaction between seawater and rocks at high temperatures [13]. Even in a single hydrothermal field, the amount of energy and carbon sources in the venting fluids significantly varies from vent to vent [13]. This intra-field variation of the fluids is attributed to subseafloor phase-separation and -segregation (different extents of mixing of the vapor and liquid phase) [14]. We hypothesized that this geochemical process might have a considerable impact on the formation of the subvent microbial community and on the supply of energy and carbon sources to chemolithoautotrophs at the seafloor. Among the chemolithoautotrophs occurring in deep-sea hydrothermal environments, members of the Aquificales, Epsilonproteobacteria and Methanococcales have been predominantly detected in various deep-sea hydrothermal fields [4,7,9,15–19]. Although very few cultures of the Aquificales and Epsilonproteobacteria were reported, a number of isolates within these groups has been recently obtained and characterized as hydrogen- and/or sulfur-oxidizing chemolithoautotrophs [20–29]. Based on their metabolic characteristics, less-selective media were designed here for the quantification of the chemolithoautotrophs with versatile energy metabolism.

We investigated the chimney structures, vent fluids and colonization devices obtained from a deep-sea hydrothermal system, the Iheya North field in the Mid-Okinawa Trough. In this hydrothermal field, seven large hydrothermal mounds having hydrothermal venting or diffusing are localized in North-South direction. The hydrothermal activity center is a large sulfide mound called North Big Chimney (NBC) (approximately 30 m high) [30]. The temperature of the vent fluid is highest within NBC and generally decreases with distance from NBC. Together with the shift in the fluid temperature, variation of the Cl end-member concentration in the different vent fluids is noted. This is explained by an increasing input of vapor-phase into the hydrothermal fluid due to the subseafloor phase-separation (Chiba, H., Ishibashi, J., Kataoka, S., Umeki, Y., Kouzuma, F., Nakayama, N. and Tsunogai, U. (2002) Eos Trans. AGU Fall Meet. Suppl. 83(47), abstr. V72A-1295]. In an effort to understand how physical and geochemical parameters controlled by phase-separation and -segregation are associated with the formation of microbial communities, we performed culture-dependent and -independent analyses on the microbial communities at three vent sites in this hydrothermal system. Our results show the occurrence of distinctive microbial communities in different vent sites.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgement
  8. Appendix A. Supplementary data
  9. References

2.1Study site

Seven large hydrothermal mounds having vents and diffusing flows, including North Big Chimney (NBC), Central Big Chimney (CBC) and Event 18 (E18), were discovered in the Iheya North field in 1995 (reviewed in [31]). The hydrothermal activity occurs in a backarc volcanic ridge in a continental margin and is hosted by thick (several hundreds meters to several kilometers) organic-rich sediments. As a consequence of the interaction between the sediments and hot fluid, geochemical features of vent fluids in the Iheya North are high alkalinity and high concentrations of inline image, carbon dioxide, hydrogen sulfide and methane [32].

2.2Sample collection and subsampling

All samples used in this study were obtained by means of manned submersible Shinkai 2000 in the cruise of April–May 2002. Bulks of chimney structures (100 g) were obtained from the main vent orifices at the summits of NBC and CBC. Each chimney sample was aseptically subsampled into three sections on board as previously described [11]. Each subsample was further divided and subjected to nucleic acid extraction, microscopic observation and liquid serial dilution culture. For liquid serial dilution culture experiments, the subsamples of the chimneys (10 g) were slurried with sterile MJ synthetic seawater (30 ml) [33] containing 0.05% (wt/vol) of Na2S under N2 atmosphere, and stored at 4 °C in the dark until they were used. For microscopic observation, the subsamples (5 g) were fixed with sterile MJ synthetic seawater (30 ml) containing 3.7% (wt/vol) of formaldehyde. The remaining subsamples were stored at −80 °C for nucleic acid extraction.

In addition to analyzing naturally occurring chimney structures, three in situ colonization systems (ISCSs) [26] were directly deployed into vent orifices in the cruise of June 2000 and recovered in April–May 2002 (Table 1). During both sampling cruises, the maximum temperatures of the vent fluids at NBC and CBC were stable, being 311 and 247 °C, respectively. The temperature of the vent fluids at E18 in 2000 was measured to be maximally 70 °C. The ISCS consisted of a stainless-steel pipe with many small holes filled with porous substratum, natural pumice, for microbial colonization [26]. For a control, one ISCS designated Cnt-ISCS was attached to the submersible during a dive and subjected to a series of the experiments. For a series of experiments, the substrata of ISCSs (150 g) were prepared and stored as described above. The total cell count (per wet g) in each hydrothermal sample was obtained by direct cell counting of formaldehyde-fixed slurries with DAPI (4′, 6-diamidino-2-phenylindole) [34] and SYBR Green I (Molecular Probes, Eugene, OR) [35]. At least 100 fields/filter, containing 20–30 cells/field, were counted.

Table 1.  Summary of the hydrothermal samples used in this study
Sample codesVent site (temperature of vent fluids)Cell density (cells g (wet wt]−1)Properties
  1. aIn width from exterior surface.

NBC-1NBC (311 °C)8.1 × 107Black exterior surface layer, 0–3 mma
NBC-24.3 × 104Gray middle intermediate part, 4–15 mma 
NBC-32.1 × 104Gray and crystalline surface of conduit, 16–50 mma 
NBC-ISCS2.4 × 104Deployed into the main hydrothermal conduit of NBC for 2 years 
CBC-1CBC (247 °C) (30 m SE from NBC)4.9 × 107Gray exterior surface layer, 0–3 mma
CBC-27.9 × 107Black and crystalline internal part, 4–19 mma 
CBC-34.0 × 105Black and crystalline surface of conduit, 20–30 mma 
CBC-ISCS2.5 × 107Deployed into the main hydrothermal conduit of CBC for 2 years 
E18-ISCSE18 (70 °C) (50 m NNE from NBC)2.3 × 107Deployed into the main hydrothermal conduit of E18 for 2 years
Cnt-ISCS  Attached to submersible during a dive (about 6.5 h)

2.3Geochemical analyses of vent fluids

Vent fluid samples for geochemical analyses were collected with the ORI pump water sampling system (750 ml in volume) [36] or with WHATS (150 ml in volume) [37]. At least two fluid samples were obtained from the same vent for geochemical analysis. The temperatures of each vent fluid were monitored during the sampling using a Pt resistant temperature probe equipped at the intake of sampling systems. Chemical compositions listed in Table 3 were analyzed using identical methods as described in Gamo et al. [38]. End-member geochemical compositions of vent fluids were estimated by the conventional method, that is extrapolation to Mg = 0 of linear relationship of concentration of each species to Mg among the obtained samples [39].

Table 3.  End-member chemical composition of vent fluids
 VentAmbient seawater
  1. ND, not determined.

  2. aActual measurement value. Range of Mg values measured for multiple fluid samples from a same vent is shown.

H2S (mM)a0.184.00ND0.00
Mg (mM)a38.1–41.325.2–45.243.0–46.652.7
Cl (mM)864511338540
Alkalinity (mM)ND1.812.782.30
Si (μM)32104210ND0.18
K (mM)
Na (mM)745405288483
Ca (mM)19.918.111.910.2
Li (mM)1.3451.2220.90.026
Mn (μM)460669445<0.001

2.4Liquid serial dilution cultures

To estimate the abundance of culturable microorganisms, a series of serial dilution cultures was performed under a total of 39 different cultivation conditions (Table 2). We designed less-selective media (MMJHS, MMJHS-2 and MMJHS-3), containing a mixture of electron donors and electron acceptors for hydrogen/sulfur-oxidizing chemolithoautotrophs (Table 2). 200–500 μl of the each slurry was inoculated to tubes containing 3 ml of each liquid medium, and then 500 μl of the medium was serially transferred to the tube of the next dilution step and incubated in the dark at defined temperatures (Table 2). In every dilution step, the tubes were vigorously shaken on a vortex mixer to disaggregate cell clumps and to dislodge cells from particles. The presence or absence of cell growth was determined by daily microscopic observation for at least ten days. The highest positive dilution tube was subjected to dilution-to-extinction to obtain pure cultures. The purity was confirmed routinely by microscopic examination. The minimum density of culturable cells in the original sample was calculated from the wet weight of the sample and the dilution factor.

Table 2.  Cultivation conditions used to evaluate the abundance of microorganisms represented by various physiological characteristics
Medium designationIngredienta,bGas phaseMain target organismpHTemperatureReference
  1. aMJ synthetic seawater [33] was used as the saline base.

  2. bThe concentration (wt/vol) of each ingredient was shown in parenthesis.

MJYPYeast extract (0.1%) and tryptone (0.1%)Atmospheric air; 100 kPaThermales and Aeropyrum7.070 and 85[40]
MJYP-2Yeast extract (0.1%) and tryptone (0.1%)N2/O2, 99/1; 200 kPaThermales7.055 and 70This study
MJAISS0 (3%), NaHCO3 (0.1%), Na2S (0.05%), resazurin (0.0001%) and vitamin solution [41]H2/CO2, 80/20; 350 kPaDesulfurobacterium group, Desulfurococcales, and Epsilonproteobacteria6.525, 55, 70, 85, and 95This study
MMJaNaHCO3 (0.1%), CaCl2 (0.07%), Na2S· 9H2O (0.05%), cysteine-HCl (0.05%), resazurin (0.0001%) and vitamin solution [41]H2/CO2, 80/20; 350 kPaMethanococcales and Methanopyrus7.037, 55, 70, 85, and 95[42]
MJYPGSYeast extract (0.2%), tryptone (0.2%), glucose (0.02%), S0 (3%), and Na2S· 9H2O (0.05%)N2; 200 kPaThermococcales and Thermotogales7.070, 85, 95, and 102This study
MMJHSNaNO3 (0.1%), Na2S2O3· 5H2O (0.1%), S0 (3%), NaHCO3 (0.1%), and vitamin solution [41]H2/CO2, 80/20; 350 kPaAquificales and Epsilonproteobacteria7.025, 37, 55, 70, 85, and 95[26]
MMJHS-2NaNO3 (0.1%), Na2S2O3· 5H2O (0.1%), S0 (3%), NaHCO3 (0.1%), and vitamin solution [41]H2/CO2/O2, 79/20/1; 350 kPaAquificales and Epsilonproteobacteria7.025, 37, 55, 70, 85, and 95[26]
MMJHS-3NaNO3 (0.1%), Na2S2O3· 5H2O (0.1%), S0 (3%), NaHCO3 (0.1%), and vitamin solution [41]H2/CO2/O2, 75/15/10; 350 kPaAquificales and Epsilonproteobacteria7.025, 37, 55, 70, 85, and 95[26]
MJYSYeast extract (0.1%), acetate (0.025%), pyruvate (0.025%), lactate (0.025%), Na2SO4 (0.2%), NaHCO3 (0.1%), resazurin (0.0001%) and vitamin solution [41]H2/CO2, 80/20; 350 kPaThermodesulfobacteria and Archaeoglobales6.555, 70, and 85This study

2.5Phylogenetic analysis of pure cultures

Genomic DNA was extracted from cell pellet of each isolate with Soil DNA Kit Mini prep (MO BIO Laboratories, Inc., Solana Beach, CA), according to the manufacturer’ s instructions. The 16S rRNA gene was amplified by PCR with Eubac27F and 1492R [43] for the bacterial rRNA gene or Arch21F [44] and 1492R [43] for the archaeal rRNA gene. The PCR conditions were as previously described [11]. Using approximately 1.5 kb of PCR product as a template, partial sequence of the approximately 900 bp of PCR product was directly determined in both strands with Arch21F, Eubac27F, 515R, 515F, 927R and Arch958R [43,44]. The identities between the sequences obtained in this study were determined with DNASIS software (Hitachi Software, Tokyo, Japan). Isolates having 16S rRNA gene identity of 97% were assigned to the same species. Representative sequences were applied to sequence identity analysis with databases by the FASTA algorithm in DNA Data Bank of Japan (DDBJ) [45].

2.616S rRNA gene clone library

Microbial DNA was directly extracted from each hydrothermal sample (approximately 10–50 g (wet weight]), using the Soil DNA Kit Mega prep (MO BIO Laboratories). As a routine control to check for experimental contamination, a blank sample was subjected to the same process. The oligonucleotide primers used for PCR were Eubac27F or Arch21F [44] and 1492R [43] as described above. Thermal cycles were performed under the following conditions: denaturation at 96 °C for 20 s; annealing at 50 °C for 45 s; and extension at 72 °C for 120 s, for the bacterial 16 S rRNA gene amplification of 30 cycles and the archaeal 16 S rRNA gene amplification of 40 cycles.

Amplified rRNA gene was purified, and cloning and sequencing were followed by the previously described procedure [11]. Eubac27F or Arch21F primer was used for partial sequencing analysis (approximately 400 bp) to determine the phylogenetic clone type (phylotype) of the bacteria or archaea, respectively. The rRNA gene sequences with 95% of identity were assigned to the same clone type in bacterial libraries, and 97% in archaeal libraries. Approximately 900 bp of the sequence of each representative rRNA gene clone were determined from both strands as described above. Some of the non-representative clones were also sequenced in order to ascertain the grouping. The chimeric sequences were searched by checking secondary structure anomalies, by the CHIMERA_CHECK program of the Ribosomal Database Project II (RDP-II) [46] and by fractional treeing [47]. One sequence was identified to be chimeric and eliminated from subsequent analysis. To estimate the representation of the phylotypes, coverage was calculated by Good’ s equation [48] with the formula (1 − (n1/N)] × 100, where n1 is the number of single-occurrence phylotypes within a library and N is the number of clones examined.

2.7Construction of the phylogenetic tree

In order to determine the phylogenetic positions of the representative isolates and clone types, the sequences were compiled using ARB software version 20030822 [49] and aligned with the database of Phil Hugenholtz (, updated with sequences from the DDBJ. Resulting alignments were manually verified against known secondary structure regions. Using a 50% conservation filter of ARB, phylogenetic analyses were restricted to nucleotide positions (approximately 550 bp) that could be unambiguously aligned. Phylogenetic trees were generated with distance and parsimony methods implemented in PAUP* 4.0b10 [50]. Distances were estimated with the Jukes and Cantor correction. Bootstrap analysis was used to provide confidence estimates for the tree topologies.

2.8Quantification of archaeal rRNA genes

Quantification of archaeal and bacterial rRNA genes in whole microbial rRNA gene assemblages was performed using the real-time PCR technique with TaqMan probes as previously described [51]. A dilution series of each of the DNA samples was prepared, and the samples were assayed with the universal rRNA gene mixture and the archaeal rRNA gene mixture [51] as the standards for quantification of whole microbial rRNA genes and archaeal rRNA genes, respectively.

2.9Species richness estimation

To evaluate richness, diversity statistics were calculated using the program EstimateS 6b1 (R. K. Colwell, University of Connecticut (]). Each cloned sequence was treated as a separate sample, and 100 randomizations were carried out for all tests. Both bacterial and archaeal richness were estimated for each hydrothermal sample using the Chao1 [52], abundance-based coverage estimators (ACE), Shannon diversity index [53] and the reciprocal of Simpson’ s index [53].

2.10Comparison of clone libraries

The Morisita index (IM) of community similarity was calculated with the following formula [54]:

  • image

where λ is Simpson’ s index of dominance (calculated separately for each library), ni is the number of phylotype i, and N is the total number of clones sequenced. The Morisita index ranges from 0 to 1, with 0 indicating that no phylotypes are shared between the two communities and 1 indicating complete identity. This community similarity index refers to the probability that individuals randomly drawn from each of the two communities will belong to the same phylotype, relative to the probability of randomly selecting a pair of individuals of the same phylotype from one of the communities.

Bacterial and archaeal 16S rRNA gene libraries were also compared statistically using the LIBSHUFF software [55]. Sequences were randomly shuffled 999 times between samples, prior to the distance between the curves being calculated using the Cramér-von Mises test statistic [56]. The DNADIST program of PHYLIP [57], using the Jukes-Cantor model for nucleotide substitutions, was used to generate the distance matrix for inserting data into LIBSHUFF.

2.11Nucleotide sequence Accession Numbers

The 16S rRNA gene sequences obtained in this study are available from the DDBJ/EMBL/GenBank nucleotide sequence databases and have been assigned the following Accession Nos.: AB175498 to AB175520, AB189149, and AB189150 (for isolates); AB175521 to AB175609, AB189150 and AB189151 (for clones).


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgement
  8. Appendix A. Supplementary data
  9. References

3.1Sampling and cell counting

Two chimney structures and four colonization devices (ISCSs) were used in this study (Table 1). The chimney sample from NBC, approximately 10 cm in length and up to 15 cm in width, allowed the definition of three sections. The subsamples were designated NBC-3 (interior) through NBC-1 (exterior) and are referred to by these designations (Table 1). Likewise, the chimney sample from CBC, approximately 6 cm in length and up to 3 cm in width, was subsampled into three sections. The subsamples were also designated CBC-3 (interior) through CBC-1 (exterior) (Table 1). Each sample was subjected to total cell counting, serial dilution cultures and nucleic acid extraction. Since CBC-1 was too small, it was subjected to only cultivation and cell counting. The total cell count in each sample varied in the range of 2.1 × 104 (in NBC-3) to 8.1 × 107 (in NBC-1) cells g−1 (wet weight) (Table 1). Within both chimney structures, the total cell counts in the exterior parts were higher than those in the interior parts. These cell counts and distribution profiles in chimney structures were similar with those reported previously [10,11,19].

3.2Geochemical characteristics of vent fluids

Geochemical analysis revealed that different vent fluids had distinctive end-member compositions (Table 3). Based on the classification of Massoth et al. [14], vent fluids from E18, NBC and CBC can be referred to Cl-depleted, normal, and Cl-enriched fluids, respectively. The linear relationships between Cl and cation (K+, Ca2+ and Na+) concentrations indicated that those fluids had the same source [58]. Although we could not determine the H2S concentration in E18 vent fluid because of the high extent of entrainment of ambient seawater (Table 3), the fluctuation in the Cl, metal and gas concentrations clearly represented that the fluids have experienced phase-separation and -segregation in the subseafloor as reported in other hydrothermal fields of Juan de Fuca Ridge [14,58]. E18, NBC and CBC vent fluids represented the gas-enriched, -normal and -depleted compositions, respectively [14].

3.3Culture-dependent analysis

Distinctive culturable community was detected in each hydrothermal niche. The overall culturable populations varied between undetectable (Cnt-ISCS) and 8.0 × 107 cells g−1 (wet weight) (NBC-1), which accounted at most for 69.4% of the total cell count. The phylogenetic characteristics of the representative isolates are summarized in Table 4. Although isolates obtained from NBC- and CBC-portions under identical cultivation conditions represented the similar phylogenetic affiliation (Supplementary Table 1), there were remarkable differences in the composition and abundance of the culturable microbial community in each sample as follows.

Table 4.  Phylogenetic analysis of representative isolates obtained from terminal positive tubes on the basis of partial 16S rRNA gene sequences, including the closest relatives as identified by using the FASTA program in the DDBJ databases
Phylogenetic groupType isolatesMediaSample, temperatureOther samples from which closely related isolates were obtainedTop match (Accession No.]aIdentity (%)
  1. aenv. denotes sequences detected as PCR-amplified products in complex microbial communities.

  2. bPhylogenetic grouping of Epsilonproteobacteria were based on Corre et al. [60] and Takai et al. [26].

AquificalesNS70-1MMJHSNBC-1, 70NBC-2, CBC-1, NBC-ISCS, and CBC-ISCSPersephonella hydrogeniphila (AB086419)98.1
 NS85-1MMJHSNBC-1, 85NBC-2Aquifex aeolicus (AE000709)98.2
DeulfurobacteriumE9S70-DMJAISCBC-1, 70E18-ISCS and NBC-3Balnearium lithotrophicum (AB105048)99.4
 CBC70-2MMJHS-2CBC-ISCS, 70Desulfurobacterium crinifex (AJ507320)98.7
Group ACBC55-2MMJHS-2CBC-ISCS, 55env. A1 B010 (AF420342)94.5
 NS55-2MMJHS-2NBC-1, 55CBC-1 and CBC-2env. A1 B010 (AF420342)90.8
 MI55-1MMJHSNBC-2, 55env. A1 B010 (AF420342)89.2
Group BNS25-1MMJHSNBC-1, 25NBC-2, CBC-2, CBC-3, NBC-ISCS,Sulfurimonas autotrophica (AB088431)99.0
    CBC-ISCS, and E18-ISCS  
Group DNS55-1MMJHSNBC-1, 55NBC-2Epsilonproteobacterium strain 18.2 (AF357196)92.1
Group FE9I37-1MMJHSCBC-3, 37env. A2 B009 (AF420348)96.6
 NM25-1MMJHSNBC-2, 25NBC-ISCSenv. A2 B004 (AF420345)99.0
 E9S37-1MMJHSCBC-1, 37NBC-1, NBC-2, and CBC-1env. A1 B030 (AF420346)96.4
Group GE1825-1MMJHSE18-ISCSenv. S17sBac16 (AF299121)93.7
MethanococcalesE1885-MMMJE18-ISCS, 85NBC-1, NBC-2, and CBC-ISCSMethanocaldococcus jannaschii (U67517)98.6
 E1855-MMMJE18-ISCS, 55NBC-1, NBC-2, CBC-2, and CBC-ISCSMethanothermococcus okinawensis (AB057722)99.4
ThermococcalesNS85-TMJYPGSNBC-1, 85NBC-2, NBC-3, CBC-1, CBC-2, CBC-3,Thermococcus celer (M21529)99.2
    NBC-ISCS, CBC-ISCS, and E18-ISCS  
 NS102-TMJYPGSNBC-1, 102NBC-2, CBC-ISCS, and E18-ISCSPyrococcus horikoshii (AP000001)99.9
ArchaeoglobalesNS70-AMJYSNBC-1, 70NBC-2Archaeoglobus fulgidus (AE000965)99.5
 NI85-AMJYSNBC-3, 85Archaeoglobus profundus (AF322392)97.7
ThermalesE9S70-OMJYP-2CBC-1, 55Oceanithermus profundus (AJ430586)99.3
ClostridialesNS55-AMJYSNBC-1, 55Tepidibacter thalassicus (AY158079)98.9
3.3.1Chimney structures

In the NBC structure, the ratio of overall culturable population of the total cell count increased from interior to exterior parts (Fig. 1). There was a transition from a mixed community of mesophiles and thermophiles in the exterior parts to thermophiles in the interior. Throughout the NBC structure, the most abundantly recovered population consisted of heterotrophic and hyperthermophilic archaea, members of the genus Thermococcus (Fig. 1). The number of culturable Thermococcus cells comprised 42.7% of total cell count in NBC-1. Together with the Thermococcus population, chemolithoautotrophic and mesophilic to hyperthermophilic hydrogen/sulfur oxidizers were also predominantly cultured. They were members of the Persephonella, Aquifex or Epsilonproteobacteria (Fig. 1), and related to environmental clones retrieved from various deep-sea hydrothermal fields [4,7,8,17] (Fig. 2). The composition of culturable community in NBC-2 was similar with that in NBC-1 (Fig. 1). In NBC-3, culturable population consisted of only thermophiles, i.e., sulfate-reducing prokaryotes (SRP) (Archaeoglobus), heterotrophic sulfur reducers (Thermococcus) and chemolithoautotrophic, hydrogen-oxidizing S0 reducers (Balnearium) (Fig. 1).


Figure 1. Total cell count and minimum viable count determined by using DAPI staining cell count and serial dilution culture techniques, followed by phylogenetic analysis of the isolates.

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Figure 2. Phylogenetic relationships of 16S rRNA sequences of the representative isolates and clones as determined by Neighbor-joining analysis. Trees A, B and C were constructed from 507, 515 and 655 sites of the rRNA gene sequence that could be unambiguously aligned. The numbers at the nodes are the bootstrap values. Bootstrap values are based on 100 replicates each and are shown for branches with more than 50% support. Sequences obtained in this study are marked in bold, and the remaining sequences were obtained from DDBJ. The numbers in parentheses are the DDBJ/EMBL/GenBank accession numbers. The scale bar represents the expected number of changes per nucleotide position. (A) A tree indicating the phylogenetic relationship among Epsilonproteobacteria. (B) A tree indicating the phylogenetic relationship among the Aquificales and the Desulfurobacterium group. (C) A tree indicating the phylogenetic relationship among Euryarchaeota.

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Compared to the NBC structure, subsamples of CBC exhibited significantly low culturability (Fig. 1), indicating that most of the microorganisms observed within CBC were not viable or unable to grow under the cultivation conditions used in this study. Similar to the condition in the NBC structure, members of the genus Thermococcus represented the most abundant culturable population in CBC-1 and -3 (Fig. 1). However, abundances of the culturable Persephonella and Epsilonproteobacteria within the CBC structure were significantly lower than those in the counterparts of the NBC structure (Fig. 1). In addition, neither hyperthermophilic SRP (such as Archaeoglobus) nor hyperthermophilic hydrogen/sulfur oxidizers (such as Aquifex) were cultured from CBC.

3.3.2Colonization devices

In the substrata of NBC- and CBC-ISCSs, the culturable communities significantly differed from those in the adjacent chimney parts (i.e., NBC-3 and CBC-3), indicating that the microbial communities observed in the ISCSs were not contaminants from chimney structures. A control ISCS (without exposure to vent fluid) gave no positive enrichment. Therefore, the ISCSs appeared to gather the potentially indigenous microbial components of the subvent biosphere entrained by the hydrothermal fluids. Although the microbial colonization process is potentially dependent on the surface composition of substrata, no specificity of microorganisms for a particular substratum was reported in deep-sea hydrothermal environments [59]. The culturable communities in NBC- and CBC-ISCSs were mainly composed of members of the Thermococcus and Epsilonproteobacteria (Fig. 1). In contrast, the most abundant culturable population in E18-ISCS was thermophilic and hydrogenotrophic methanogen (Fig. 1). Takai et al. [4] reported the predominance of a methanogen in the ISCS exposed to vent emission in a Central Indian Ridge hydrothermal field. These results clearly indicated that different microbial communities colonized to the ISCSs exposed to physically and geochemically different hydrothermal fluids.

3.4Culture-independent molecular analyses

DNA was successfully extracted and used to construct bacterial and archaeal 16S rRNA gene clone libraries. Control tubes with no samples and two subsamples with low total cell counts, i.e., NBC-2 and -3, showed no amplification of the PCR reactions.

3.4.1Molecular phylogenetic characterization of bacteria

A total of 48 different phylotypes was identified from the 9 bacterial libraries on the basis of classification with 95% of identity (Supplementary Table 2). The coverage values were above 80% (Table 5). Many 16S rRNA gene sequences of the frequently retrieved clones were closely related to those of the isolates obtained in this study at species or higher taxonomic levels (Fig. 2). The rRNA gene clones affiliated to the Epsilonproteobacteria were detected in all hydrothermal samples (18–95% in clonal frequencies) (Fig. 3). These clones were interspersed to all subgroups of the Epsilonproteobacteria, based on the classification of Corre et al. [60]. Clones of the Epsilonproteobacteria Group A were detected in both chimney structures, which were affiliated with clusters represented by the strain MI55-1 or strain CBC55-2 (Fig. 2). Although clones of the Epsilonproteobacteria Group G were scarcely detected in both chimney structures, they represented the most dominant phylotype in E18-ISCS (Fig. 3). Clones of the Epsilonproteobacteria Group G clustered with strain E1825-1 (Fig. 2). Members of the order Aquificales were detected only in NBC-1. The Aquificales clones detected were closely related to strain NS70-1 and Persephonella hydrogeniphila[25] (Fig. 2).

Table 5.  Comparison of diversity indices of deep-sea hydrothermal vent communities
SampleCoverageSaShannon indexACEbChao11/Dc
  1. aPhylotype richness.

  2. bAbundance-based coverage estimator.

  3. cReciprocal of Simpson’ s index.


Figure 3. Composition of the microbial population based on taxonomic grouping of 16S rRNA gene clone sequencing. See Fig. 1 for phylogenetic groups. Abbreviations are as follows: MG I, Marine Crenarchaeotic Group I [44]; DHVEG, Deep-sea Hydrothermal Vent Euryarchaeotic Group [11]. “n” indicates the number of clones examined. *The ratio of Bacteria/Archaea of the total microbial community was determined by using real-time PCR.

Download figure to PowerPoint

3.4.2Quantification and molecular phylogenetic characterization of archaea

E18-ISCS represented the highest proportion of archaeal rRNA gene (Fig. 3). Except for E18-ISCS and CBC-3, the proportions of archaeal rRNA gene were stable and varied from 1.8 to 4.2%. Previous studies showed high variation (1.3–76.7%) in the archaeal proportion within a single chimney structure [10,11,19].

In total, 37 different phylotypes were identified throughout the archaeal rRNA gene libraries on the basis of classification with 97% of identity (Supplementary Table 3). The coverage values were above 80% (Table 5). The archaeal community structures markedly differed from each other, although Thermococcales clones were detected in almost all the libraries (Fig. 3). Most clones within the Archaeoglobales, Methanococcales and Thermococcales were closely related to the isolates obtained through liquid serial dilution culture experiments (i.e., strain NS70-A, E1855-M and NS85-T) (Fig. 2). Methanococcales clones were frequently detected only in E18-ISCS (94% in clonal frequency) (Fig. 3), which were clustered with strain E1855-M isolated from the same sample (Fig. 2).

3.5Diversity indices and comparison of clone libraries

Values of diversity indices of microbial communities in the chimney portions were generally higher than those in the ISCSs (Table 5). The 1/D value of the bacterial community in NBC-1 was relatively low (Table 5), indicating a relatively higher extent of dominance [61]. Among the ISCSs deployed into different hydrothermal conduits, E18-ISCS had the lowest values of diversity indices.

Phylogenetic analysis suggested that each of the clone libraries was unique (Fig. 3). This notion was supported by statistical library comparisons using LIBSHUFF software and Morisita index. 40 of 42 library-pairs exhibited a small (<0.5) Morisita index, indicating that microbial community structures differ in different habitats. Although the Morisita index is calculated from the number of phylotypes shared by two clone libraries compared, LIBSHUFF performs sequence-based comparisons [55]. In LIBSHUFF analysis, two libraries are considered significantly different when P < 0.05 (see [55] for details). LIBSHUFF analysis demonstrated significant differences among the libraries (P < 0.05 in 83 of 84 pairs) (Supplementary Table 4). Although only one pair (i.e., bacterial community of NBC-ISCS vs CBC-2) exhibited a high P value, the reverse comparison exhibited low P value (Table 6). None of the library-pairs exhibited high P value (>0.05) and high Morisita index (>0.5). These results clearly indicated that microbial community structures inferred from the rRNA gene libraries significantly differ from each other.

Table 6.  Comparisons of 16S rRNA gene clone libraries. Comparisons exhibiting P values larger than 0.05 or Morisita index larger than 0.5 are shown. All comparisons are provided in the supplementary material online
ComparisonPaMorisita index
  1. aProbability values (P) of LIBSHUFF analysis mean the differences between homologous and Heterologous coverages in a reciprocal comparison as a function of evolutionary distance. See [55] for details.

CBC-2 (A) and NBC-ISCS (B)0.001 (A vs B)0.003 (A vs B)0.150.02
 0.415 (B vs A)0.001 (B vs A)  
CBC-ISCS (A) and NBC-ISCS (B)0.017 (A vs B)0.001 (A vs B)0.790.00
 0.032 (B vs A)0.001 (B vs A)  
CBC-ISCS (A) and CBC-3 (B)0.001 (A vs B)0.001 (A vs B)0.550.35
 0.001 (B vs A)0.001 (B vs A)  


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgement
  8. Appendix A. Supplementary data
  9. References

In this study, the variability in microbial community associated with the physical and geochemical variations of the vent fluids was evaluated. A considerable variation of microbial community in a single hydrothermal field (intra-field variation) was for the first time demonstrated using culture-dependent and -independent techniques. Since both microbiological techniques have limitations and biases, the combined use of culture-dependent and -independent methodologies is effective to characterize the microbial ecosystem (reviewed in [62]). So far, a few investigations adopted the polyphasic approach to the microbial communities in deep-sea hydrothermal systems [4,10,11,18]. Compared to these previous researches, more cultivation conditions with less-selective media were employed in this study. The culturable microorganisms accounted for up to 69.4% of the total cell count. The extension of the phylogenetic diversity of the isolates was roughly equivalent to that of the frequently retrieved rRNA gene clones on the species or higher taxonomic levels. Successful cultivation of the predominant but previously uncultivated Epsilonproteobacteria members demonstrated the effectiveness of liquid serial dilution culture experiments using less-selective media.

4.1Microbial community in different vents

In different vent fluid-associated niches, the microbial communities differed in terms of abundance, culturability, diversity and composition.

4.2NBC: hydrothermal activity center

The exterior parts of the NBC structure contained mesophilic to hyperthermophilic microorganisms. Although the exterior surface of the chimney structure was exposed to cold ambient seawater, abundant and diverse thermophiles were cultured and detected from the exterior part of the NBC structure (0–3 mm from exterior surface and approximately 5 cm away from the conduit of 311 °C fluid). The culturable thermophilic population consisted of a heterotrophic sulfur-reducer (Thermococcales), hydrogen/sulfur oxidizers (Epsilonproteobacteria Group A and D, Persephonella and Aquifex), SRP (Archaeoglobus) and fermenters (Tepidibacter). Together with Thermococcus, the predominant population in NBC-1 was hydrogen/sulfur-oxidizing and thermophilic chemolithoautotroph, being Persephonella members accounting for 14.2% and 31.9% of the total microbial population as determined by culture-dependent and -independent analyses, respectively. The result represented a striking contrast to the dominance of non-thermophilic candidates in the exterior wall of a huge chimney structure [19]. This discrepancy probably reflects that the thin wall of the NBC structure had steeper physical and geochemical gradients as suggested by previous reports using geochemical modeling [64]. The occurrence of diverse SRP populations in the exterior surface of the NBC structure was recently suggested by the cloning and sequencing of the dissimilatory sulfite reductase (DSR) gene [63]. Furthermore, the exterior part of NBC was characterized by the high total cell count, high culturability and great diversity of the microbial community. These results demonstrate that the exterior wall, the interface between effluent hot fluid and ambient seawater, provides beneficial habitats for physiologically and phylogenetically diverse microorganisms.

4.3CBC: sulfide mound venting brine rich fluid

As compared to the NBC structure, the microbial communities occurring in the CBC structure highly varied across the structure and differed from those in the counterparts of NBC. The diversity and abundance of the culturable microorganisms in CBC were significantly less than those in NBC. Molecular analyses of the exterior region (4–19 mm from the exterior surface) of the chimney structure suggested the dominance of the heterotrophic SRP (Deltaproteobacteria) rather than chemolithoautotrophic hydrogen/sulfur oxidizers (Epsilonproteobacteria). The Deltaproteobacteria population was rarely detected in any samples obtained from the NBC site. The variability in the microbial communities is probably attributed to the differences in fluid chemistry between NBC and CBC. The vent fluid from CBC represented a gas-depleted and Cl- and metal-enriched composition. The brine rich fluid from CBC contains relatively low amounts of gas components such as H2, H2S and CO2, which are required to support the active chemolithoautotrophic populations.

4.4Distinctive subseafloor populations

Microbial populations specifically found in the colonization device exposed to hydrothermal fluids potentially represent the indigenous inhabitants of the subvent biosphere entrained by hydrothermal fluids [3,4,18]. Both culture-dependent and -independent analyses demonstrated the occurrence of unique microbial communities in each of the colonization devices, which might reflect the variability in microbial communities occurring beneath hydrothermal mounds in the Iheya North field.

In subseafloor environments of active hydrothermal fields, one of the most important microbial processes might be hydrogenotrophic methanogenesis, since the process is independent of seawater-derived oxidants such as O2 or nitrate, but on the other hand depends on hydrothermal fluids-derived gaseous substrates such as H2 and CO2[4]. Methanogens are probably the most important primary producers in the subvent biosphere [18]. In this study, abundance of the hydrogenotrophic, thermophilic methanogens in the ISCSs was significantly different among the vent sites. The hydrogenotrophic and thermophilic methanogens were predominantly cultured and detected from only one colonization device that deployed into the hydrothermal conduit of the E18 mound. It may be an explanation that the methanogens became dominant during the deployment, since the temperature of the E18 vent fluid fell within the range of growth temperatures of the methanogens [42]. However, a habitat with a similar temperature range could occur within the chimney structures of NBC and CBC as well, from which methanogens were scarcely detected and cultured. Rather than temperature, the amount of hydrothermal fluid-derived H2 and CO2 could be a more decisive parameter controlling the abundance of hydrogenotrophic methanogens [4]. Although we could not measure the actual amounts of these gases, fluid chemistry clearly indicated the enrichment of the gas components in E18 vent fluids. The gas-enriched fluids must supply higher amounts of energy and carbon sources for the hydrogenotrophic methanogens in the subvent and seafloor habitats of the E18 vent site and lead to the formation of a unique microbial community when compared to the gas-depleted and -normal hydrothermal fluids. Takai et al. [4] suggested that formation of a hydrogen-based, methanogen-dominating subseafloor lithoautotrophic microbial ecosystem might strongly depend on the tectonic setting of the hydrothermal field. However, in this study, it was also suggested that phase-separation and -segregation could locally generate the gas-enriched habitats for the methanogen-dominating subseafloor lithoautotrophic microbial ecosystem. In addition to the inter-field comparison of the microbial communities, further geochemical and microbiological characterizations of the intra-field variation will be necessary for future microbiology of deep-sea hydrothermal vents.


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgement
  8. Appendix A. Supplementary data
  9. References

We thank the captain and the crew of R/V Natsushima and Shinkai 2000 for helping us to obtain deep-sea hydrothermal vent samples. We also thank two anonymous reviewers for comments on an early draft. This work was partially supported by a Grant-in-Aid for Science Research (No. 12460093) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. S.N. was supported by the Research Fellowship of the JSPS.

Appendix A. Supplementary data

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgement
  8. Appendix A. Supplementary data
  9. References

Supplementary data associated with this article can be found, in the online version at doi:10.1016/j.femsec.2005.03.007.


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgement
  8. Appendix A. Supplementary data
  9. References
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