microbial communities associated with geological horizons in coastal subseaffoor sediments

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2003, p. 7224–7235 Vol. 69, No. 120099-2240/03/$08.00�0 DOI: 10.1128/AEM.69.12.7224–7235.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Microbial Communities Associated with Geological Horizons inCoastal Subseafloor Sediments from the Sea of Okhotsk

Fumio Inagaki,1* Masae Suzuki,1 Ken Takai,1 Hanako Oida,1 Tatsuhiko Sakamoto,2Kaori Aoki,3 Kenneth H. Nealson,1,4 and Koki Horikoshi1

Subground Animalcule Retrieval (SUGAR) Project, Frontier Research System for Extremophiles,1 and ResearchProgram for Paleoenvironment, Institute for Frontier Research on Earth Evolution (IFREE),2 Japan Marine

Science and Technology Center (JAMSTEC), Yokosuka 237-0061, and Geological Survey of Japan,AIST, Tsukuba 305-8567,3 Japan, and Department of Earth Sciences,

University of Southern California, Los Angeles, California 90089-07404

Received 16 June 2003/Accepted 29 September 2003

Microbial communities from a subseafloor sediment core from the southwestern Sea of Okhotsk were evaluatedby performing both cultivation-dependent and cultivation-independent (molecular) analyses. The core, whichextended 58.1 m below the seafloor, was composed of pelagic clays with several volcanic ash layers containing finepumice grains. Direct cell counting and quantitative PCR analysis of archaeal and bacterial 16S rRNA genefragments indicated that the bacterial populations in the ash layers were approximately 2 to 10 times larger thanthose in the clays. Partial sequences of 1,210 rRNA gene clones revealed that there were qualitative differences inthe microbial communities from the two different types of layers. Two phylogenetically distinct archaeal assemblagesin the Crenarchaeota, the miscellaneous crenarchaeotic group and the deep-sea archaeal group, were the mostpredominant archaeal 16S rRNA gene components in the ash layers and the pelagic clays, respectively. Clones of16S rRNA gene sequences from members of the gamma subclass of the class Proteobacteria dominated the ashlayers, whereas sequences from members of the candidate division OP9 and the green nonsulfur bacteria dominatedthe pelagic clay environments. Molecular (16S rRNA gene sequence) analysis of 181 isolated colonies revealed thatthere was regional proliferation of viable heterotrophic mesophiles in the volcanic ash layers, along with somegram-positive bacteria and actinobacteria. The porous ash layers, which ranged in age from tens of thousands ofyears to hundreds of thousands of years, thus appear to be discrete microbial habitats within the coastal subseafloorclay sediment, which are capable of harboring microbial communities that are very distinct from the communitiesin the more abundant pelagic clays.

The subsurface environment has been proposed to be the larg-est reservoir of biomass on Earth (39). On the basis estimates ofthe biomass in subseafloor core sediments collected by the OceanDrilling Program, more than 105 microbial cells/cm3 were consis-tently present even at a depth close to 1,000 m below the seafloor(23). However, the relationships between the microbial commu-nities on the one hand and the biogeochemical impact, sedimen-tological properties, and past geological events in subseafloorenvironments on the other hand have remained poorly defined.Recent studies of microbial communities in geologic materialshave suggested that microorganisms adapt to a variety of micro-habitats and may be considered to be indigenous to them. Forexample, the archaeal community in the black smoker hydrother-mal vent chimney collected from northeastern Papua NewGuinea consisted of hyperthermophiles and extreme halophiles,the distributions of which corresponded to the mineralogicalcharacteristics of various microhabitats in the hydrothermal de-posits (32). Similarly, the distribution of the microbial communi-ties found in a deep-sea siltstone collected from the Japan Trenchappeared to be correlated with the porosity and permeability ofthe geological matrices (11). In marked contrast, the unexpected

presence of several archaeal genera, such as Thermococcus, Sul-folobus, and Haloarcula, was reported for cold subseafloor coresediments recovered from the West Philippine Basin (9); theseorganisms may have been transferred from surrounding terres-trial acidic hot springs or hydrothermal vent fields and buried. Inthe terrestrial subsurface, loci exhibiting high rates of sulfate re-duction were observed at sandstone-shale interfaces in the deepsubsurface in central New Mexico (6, 15, 16). Recently, microbialdiversity and distribution in subsurface gold mines have beendescribed (12, 33, 34). Taken together, the information describedabove suggests that while the geological and geochemical settingsgreatly affect microbial composition in both terrestrial and marinesubsurface environments, there are many unexplained findingsthat may be related to the ability of imported microbes to survivefor long periods of time. Unexplained variation can occur even inareas that seem to be homogeneous and similar, such as methanehydrate sites, where major differences in community structurehave been reported. For example, at subseafloor methane hydratesites (Ocean Drilling Program Leg 146 core sediments from theCascadian Margin), the presence of methanogenic archaea wasreported (4, 21), while recent studies of communities in hydrate-bearing sediments in the Nankai Trough (25) revealed little sim-ilarity with the communities found in the Cascadian Margin. Itmay well be that the processes that lead to formation of methanehydrate are regionally complicated and that the roles of subsea-floor microorganisms in methane production and/or consumption

* Corresponding author. Mailing address: Subground AnimalculeRetrieval (SUGAR) Project, Frontier Research System for Extremo-philes, Japan Marine Science & Technology Center (JAMSTEC), 2-15Natsushima-cho, Yokosuka 237-0061, Japan. Phone: 81-468-67-9687.Fax: 81-468-67-9715. E-mail: [email protected].

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are different at different sites. As more data accumulate, it maywell become possible to decipher the situation.

In the studies described here we addressed the issue ofcommunity variability in a different environment: a coastalsubseafloor sediment from a system of pelagic clays that isinterspersed with layers of volcanic ash. Analysis of the verticalprofile of microbial distribution and phylogenetic diversity at16 depths in this sediment revealed easily seen but difficult toexplain differences in these types of layers.

MATERIALS AND METHODS

Sample collection. A subseafloor sediment core, MD01-2412, was obtainedfrom the southwestern part of the Sea of Okhotsk (44°31.65�N, 145°00.25�E) offthe Shiretoko Peninsula at the eastern margin of Hokkaido at a depth of 1,225m. This sediment core (length, 58.1 m) was recovered by using a giant piston coreduring the IMAGES (International Marine Global Environmental ChangeStudy) 2001 Project (http://images-pages.org/). The core was composed mainly of(hemi-)pelagic clay and several buried volcanic ash layers. Approximately 10�12-cm3 portions of sediment were collected from the centermost part of thecore at 16 different depths (Table 1) and placed into sterile plastic tubes by usingalcohol-sterilized spatulas. The samples were kept at 4°C on board and then werestored at �80°C in the laboratory prior to analysis.

Porosity of the sediment. To determine the sediment porosity, the wet bulkdensity (WBD) was determined onboard by gamma ray attenuation of wet splitcore sediments by using a multisensor core logger (GeoTek Co., Stewartville,Minn.). The mineral grain density (MGD) was determined with a Penta PPY-12pycnometer (Quantachrome Co., Boynton Beach, Fla.) for dried sediments aspreviously described (11). For sections 12 and 13, the MGD was assumed to be2.6500 g/cm3 because the amount of sediment in each of the samples was notlarge enough for actual determination of the MGD. The fluid water density (FD)used for the porosity calculation was 1.0650 g/ml, a typical seawater value. Theporosity was estimated by using the following equation: porosity � 100[(MGD �WBD)/(MGD � FD)].

Microscopic observation. A 0.1-cm3 portion of the sediment was suspended in0.9 cm3 of sterilized MJ synthetic seawater (13) containing 3.7% (wt/vol) form-aldehyde, and the slurry was vigorously agitated for 2 min with a vortex mixer.The suspension was briefly centrifuged (�2,000 � g), and then the cells remain-ing in the supernatant were stained with acridine orange (AO) (10 �g ml�1) for15 min. The solution was filtered with a 0.22-�m-pore size polycarbonate filter(Advantec, Tokyo, Japan) and then rinsed briefly in MJ synthetic seawater. TheAO-stained cells on the filter were counted by using epifluorescence and a NikonOptishot microscope (Nikon, Tokyo, Japan). The total cell density was estimatedfrom an average cell count for 50 microscopic fields.

DNA extraction and purification. DNA was extracted from 10 g (wet weight)of sediment by using a soil DNA Mega Prep kit (Mo Bio Lab, Inc., Solana Beach,Calif.) and following the manufacturer’s instructions. Eight milliliters of anextracted DNA solution was precipitated in ethanol and reconstituted in 500 �lof TE buffer (10 mM Tris-HCl, 1.0 mM EDTA; pH 8.0) (26). Since the DNAsolution still contained some inhibitors of PCR amplification at this stage (e.g.,humic acid substances or heavy metals), a 100-�l portion was rinsed twice withcolumn and buffer solutions (S3, S4, and S5) from a soil DNA Mini Prep kit (MoBio Lab, Inc.). Finally, extracted bulk DNA for PCR amplification was concen-trated by ethanol precipitation with 20 �l of TE buffer.

Quantitative PCR analysis of archaeal and bacterial 16S rRNA genes. Quan-tification of archaeal and bacterial 16S rRNA genes in bulk extracted DNAsolutions was performed by the quantitative fluorescent PCR method by usinguniversal and domain-specific TaqMan fluorogenic probes as described previ-ously (31). The PCR and monitoring of fluorescence signals were performed byusing the GeneAmp 5700 sequence detection system (PE Applied Biosystems,Foster City, Calif.).

Construction of PCR-amplified 16S rRNA gene clone libraries. Microbial 16SrRNA genes were amplified from the extracted bulk DNA solutions by PCRperformed with LA Taq polymerase and GC buffer I (TaKaRa, Tokyo, Japan).Bacterial 16S rRNA genes were amplified by using the Bac27F and Bac927Rprimers (17). Primers Arch21F and Arch958R (5) were used for amplification ofarchaeal 16S rRNA genes. Thermal cycling was performed with the GeneAmp9600 PCR system (PE Applied Biosystems). The PCR conditions were as follows:denaturation at 96°C for 30 s, annealing at 52°C for 30 s, and extension at 72°Cfor 120 s. Bacterial 16S rRNA gene amplification was performed for 34 cycles,and archaeal gene amplification was performed for 38 cycles. PCR amplificationfrom a solution without sediment prepared as described above was processed asa negative control to check for experimental contamination.

The amplified 16S rRNA gene from each sediment sample was subjected toagarose gel electrophoresis. Approximately 850 to 950 bp of PCR product waspurified by using a Gel Spin DNA purification kit (Mo Bio Lab, Inc.) accordingto the manufacturer’s protocol. The DNA was precipitated with ethanol andcentrifuged, and the pellet was resuspended in distilled, deionized water. Thegel-purified 16S rRNA gene was then cloned in vector pCR2.1 by using anOriginal TA cloning kit (Invitrogen, Carlsbad, Calif.). Archaeal and bacterial 16SrRNA gene clone libraries were constructed from DNA obtained from each ofthe sediment samples.

Sequencing and analysis of the similarity of 16S rRNA genes. The insert of the16S rRNA gene was amplified directly by PCR from a randomly selected colonyby using M13 primers for vector pCR2.1 (20), treated with exonuclease I andshrimp alkaline phosphatase (Amersham Pharmacia Biotech, Little Chalfont,Buckinghamshire, United Kingdom), and then directly sequenced by thedideoxynucleotide chain termination method by using a dRhodamine sequencingkit (PE Applied Biosystems) according to the manufacturer’s recommendations.

TABLE 1. Sediment samples collected from Okhotsk piston core MD01-2412 for microbiological study and primarysedimentrogical characteristics

Sample Depth (mbsf)a Lithologyb Porosity (%) Remarks (volcanic events, age, lithology)c

Section 1 1.46 PC 43.00Section 2 7.50 PC 31.72Section 3 14.7 PC 46.36Section 4 18.3 Ash 38.88 Unknown tephra, light grey pumiceous sandSection 5 22.1 PC 19.48 With pumice grains, Kc-1 (Kussharo, Hokkaido)?Section 6 22.2 PC 30.52Section 7 24.8 Ash 20.80 Spfa-1 (Shikotsu, Hokkaido; 35–42 ka), light grey, fine ash patchesSection 8 30.8 PC 28.12Section 9 30.9 Ash 31.06 Unknown tephra, lower part consisting of light grey pumiceous

sand layerSection 10 37.6 PC 11.34Section 11 45.3 PC 37.64 With small pumiceSection 12 45.4 Ash 36.17 Kc-2.3 (Kussharo, Hokkaido), light grey, fine-coarse ashSection 13 45.7 Ash 33.12 Aso-4 (Aso, Kyushu; 70–90 ka), light grey, vitric ash layerSection 14 53.0 PC 22.85Section 15 57.7 Ash 35.62 Kc-4 (Kussharo, Hokkaido; 100–130 ka), yellowish ash layerSection 16 57.8 PC 14.52

a mbsf, meters below the seafloor.b PC, (hemi-)pelagic clay; Ash, volcanic ash with pumice grains.c ka, thousands of years.

VOL. 69, 2003 LITHOSTRATIGRAPHY-ASSOCIATED MICROBIAL COMMUNITIES 7225

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Single-stranded archaeal and bacterial 16S rRNA gene sequences containingapproximately 400 to 450 nucleotides were analyzed with a model ABI 3100automated sequencer (PE Applied Biosystems) by using the Arch 21F and theBac27F primers, respectively. A total of 1,210 clones (639 archaeal 16S rRNAgenes and 571 bacterial 16S rRNA genes) were selected from the clone libraries,and partial sequences of all of these clones were determined. The sequencesimilarity among the partial 16S rRNA gene sequences was analyzed by using theFASTA program equipped with the DNASIS software (Hitachi Software, Tokyo,Japan). Sequences that exhibited �97% similarity, suggesting a species levelrelationship (28), were tentatively assigned to the same phylogenetic type (phy-lotype), and a representative clone of the 16S rRNA gene was selected for eachphylotype. The 16S rRNA gene sequence of each representative clone (length,900 to 950 bp) was determined by sequencing both strands. The representative16S rRNA gene sequences of the phylotypes were subjected to similarity analysisby using the FASTA3 and gapped BLAST search algorithms with the GenBank/EMBL/DDBJ databases (1).

Phylogenetic analysis. Phylogenetic analysis of the representative archaeal andbacterial 16S rRNA gene sequences was restricted to the nucleotide positionsdescribed in the figure legends that could be unambiguously aligned in allsequences. Least-squares distance matrix analysis, based on evolutionary dis-tances, was carried out by using the correction of Kimura (14). Neighbor-joininganalysis was performed by using the DDBJ CLUSTAL-X system (36). A boot-strap analysis was performed with 100 trial replications to provide confidenceestimates for phylogenetic tree topologies. In order to discriminate the ambig-uous phylogenetic affiliations of 16S rRNA genes, if necessary, a sequence wasapplied to the SUGGEST-TREE program in Ribosomal Data Project II (19).

CFU analyses. Three solid media, MJYP medium, marine broth 2216 agar(Difco), and DSM598 medium, were used for the CFU analysis. MJYP mediumcontained 0.01% (wt/vol) yeast extract, 0.01% (wt/vol) peptone (Difco), and avitamin mixture (2) at a concentration of 0.01% (vol/vol) in MJ synthetic sea-water. This medium was used to determine the CFU counts for heterotrophs thatrequire low levels of nutrients. Marine broth 2216 agar was used for a variety ofmarine heterotrophs that prefer high nutrient concentrations (11). DSM598medium was used for cultivation of Halomonas variabilis, which enabled evalu-ation of the variable population of halophilic (or salt-tolerant) heterotrophs.DSM598 medium contained (per liter of distilled water) 95.0 g of NaCl, 81.0 gofMgSO4 � 7H2O, 1.0 g of KCl, 7.5 g of yeast extract (Difco), 2.5 g of peptone

(Difco), and 1 ml of a vitamin mixture (2). The pH values of all media wereadjusted to 7.2 with NaOH or H2SO4, and all media were solidified with 1.5%(wt/vol) agar. To prepare a slurry sample for CFU analysis, 0.1 cm3 of a sedimentsample was put into a sterilized plastic tube, and then the tube was filled with 1.0ml of MJ synthetic seawater. After the suspension was vigorously agitated for 2min with a vortex mixer, 100-�l portions of slurry were spread on solid mediumand then incubated aerobically at 5, 15, 25, 35, and 45°C for 2 weeks beforecounting.

Phylogenetic analysis of the colony isolates. A total of 181 colonies wereselected from incubated solid media from the CFU assays based on the differentcultivation conditions (medium and incubation temperature) and colony mor-phology (color and size). Each colony was grown in 1 ml of the same liquidmedium from which it was isolated. The cultures were incubated for 3 days at theisolation temperature. Cells were harvested by centrifugation (3,500 � g) for 10min, and the genomic DNA of each pellet was then extracted with a soil DNAMini Prep kit (Mo Bio Lab, Inc.) used according to the manufacturer’s suggestedprotocol. A 16S rRNA gene fragment of each isolate was amplified by PCR byusing the Bac27F and Uni1492R primers (17) and was purified with a Gel SpinDNA purification kit (Mo Bio Lab, Inc.). A single-stranded 16S rRNA genesequence of each isolate that was 400 to 450 bp long was directly sequenced byusing the Bac27F primer, and the representative isolates of the phylotypes weredetermined by the clone library analysis procedure described above. The 16SrRNA gene sequences of the representative isolates were determined by usingboth strands, and then the similarity and phylogenetic analyses were carried outas described above.

Nucleotide sequence accession numbers. All 16S rRNA gene sequences de-termined in this study have been deposited in the GenBank/EMBL/DDBJ da-tabases. The accession numbers of 16S rRNA gene sequences of isolates, OHKAclones, and OHKB clones are AB094456 to AB094472, AB094513 to AB094561,and AB094795 to AB094962, respectively.

RESULTS

Sample characteristics. The length of sediment core MD01-2412 recovered was 58.1 m. The sediment core was found to becomposed of (hemi-)pelagic clay with several volcanic ash lay-ers containing pumice grains. A total of 16 samples of inner-most core sediments were collected from the pelagic clays andash layers at different depths (Table 1). The ages and deriva-tions of the volcanic ash layers were determined by using therefractive indices of volcanic glass shards and minerals (collec-tively referred to as tephra). In addition, preliminary results ofan analysis of diatoms and volcanic tephra by using a 7.7-msediment core recovered from the same site suggested that thesedimentation rate in the sampling field was approximately 100cm/103 years (27) and that potentially recent environmentalchanges, such as sea ice coverage and volcanic eruptions, wererecorded. These estimates are consistent with the dates of thevolcanic events identified as responsible for the formation ofthe ash layers (the sediment at a depth of 57 m is approxi-mately 100,000 years old), assuming that there was compactionof the sediments with depth (Table 1) (27). As Table 1 shows,the porosity values of pelagic clay generally decreased withincreasing depth by a factor of approximately 2 to 3 (except forsections 5 and 11 containing small pumice grains), while thoseof ash layers were relatively constant at approximately 35%.

Direct cell counting and quantitative PCR analysis of 16SrRNA genes. Epifluorescence microscopic observation of AO-stained cells indicated that the microbial population in ashlayers was slightly larger than that in the pelagic clay environ-ments. Approximately 4 � 10 6 cells were present in 1 cm3 ofpelagic clay at depths below 10 m below the seafloor, whereasapproximately three to four times more cells were present inthe ash layers (Fig. 1A). The results of a quantitative PCRanalysis of archaeal and bacterial 16S rRNA genes were con-

FIG. 1. Profiles of total cell density (A) and 16S rRNA gene con-centration (B) in the MD01-2412 core sediments. (A) Total cell den-sities in pelagic clay samples (E) and ash layer samples (F) wereestimated by direct counting of AO-stained cells. (B) Archaeal (�) andbacterial (■ ) concentrations of the 16S rRNA gene were determinedby quantitative PCR by using domain-specific fluorogenic probes.mbsf, meters below the seafloor; rDNA, ribosomal DNA.

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sistent with the cell count data, showing that the amounts ofbacterial 16S rRNA genes in bulk DNA solutions extractedfrom ash layers were 2 to 10 times larger than the amounts inbulk DNA solutions extracted from pelagic clays (Fig. 1B). Forexample, the concentrations of the bacterial 16S rRNA genesin ash layers of section 13 (Ash-4) and section 5 (Kc-1) wereestimated to be 8.2 � 104 and 4.3 � 104 fg ml�1, respectively,whereas the concentrations in pelagic clays of sections 3 and 6were 5.3 � 103 and 6.4 � 103 fg ml�1, respectively. In contrast,the amount of archaeal 16S rRNA genes decreased as thedepth increased (Fig. 1B).

Archaeal 16S rRNA gene clone library analyses. A total of639 partial archaeal 16S rRNA gene sequences were deter-mined for clone libraries constructed from all 16 layers. Asimilarity analysis of all sequences indicated that 49 differentrepresentative clones of the archaeal 16S rRNA gene werepresent. A comparison of the archaeal 16S rRNA gene phylo-types in the clay and ash layers showed that they were easilydistinguishable, with the deep-sea archaeal group (DSAG)dominating the pelagic clays and the miscellaneous crenar-

chaeotic group (MCG) dominating the ash layers (Fig. 2 and3).

Of 340 archaeal 16S rRNA gene clones from pelagic clays,262 (77.0%) were affiliated within the DSAG. SequenceOHKA2.33 was the most frequently detected phylotype (126related clones) (Fig. 3), and this sequence is closely related tothe CRA8-27 sequence (97.3%) detected in deep-sea coastalmarine sediments (37). The second most abundant archaealphylotype in the DSAG lineages was OHKA10.11 (47 related16S rRNA gene clones) (Fig. 3), which exhibited 98.9% simi-larity with the MA-A1-3 sequence from methane hydrate-bear-ing subseafloor sediments from the Nankai Trough (25). WhileFig. 2 shows that there were some differences in phylotypecomposition as the depth of the core increased, these differ-ences were often ascribed to small numbers of clones of spe-cific phylotypes.

In contrast, of the 299 archaeal clones analyzed from clonelibraries constructed from volcanic ash layers, 211 (70.6%)belonged to the MCG group (Fig. 2 and 3). This cluster waspreviously designated the terrestrial miscellaneous crenarchae-

FIG. 2. Profiles of archaeal and bacterial communities in 16S rRNA gene clone libraries constructed for various depths of pelagic clay andvolcanic ash layers. The numbers of clones examined are indicated in parentheses. The percentages of the cloned sequences affiliated with thephylogenetic groups are indicated by the bar graphs. Sec., section; mbsf, meters below the seafloor.


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pGrfC26 (U59986)

OHKA15.20 (AB094560)HTA-B10 (AF418925)

OHKA9.8 (AB094554)OHKA4.59 (AB094541)

OHKA13.26 (AB094558)

OHKA4.46 (AB094539)OHKA4.12 (AB094536)

OHKA5.23 (AB094546)VAL11 (AJ131312)

Arc.9 (AF005756)MA-C1-5 (AY093451)OHKA5.34 (AB094548)

pSL123 (U63345)OHKA1.1 (AB094513)

JTB173 (AB015273)MA-B1-3 (AY093447)

MA-A1-1 (AY093446)MA-C1-3 (AY093450)

OHKA12.82 (AB094557)OHKA1.5 (AB094517)

OHKA15.43 (AB094561)

Arc.1 (AF005752)OHKA5.24 (AB094547)

OHKA4.18 (AB094537)OHKA4.25 (AB094538)OHKA4.47 (AB094540)AT-R021 (AF419653)

Arc.171 (AF005765)OHKA1.9 (AB094519)OHKA1.16 (AB094522)

OHKA1.27 (AB094524)APA4-0cm (AF119138)

OHKA3.34 (AB094533)

OHKA11.45 (AB094556)

Mariana clone No.1 (D87348)Cenarchaeum symbiosum (U51469)

SAGMA-V (AB050227)SCA11 (U62820)

Pyrolobus fumarii (X99555)pJP89 (L25300)

Sulfolobus metallicus (X90479)

OHKA2.33 (AB094532)OHKA7.31 (AB094553)OHKA1.2 (AB094514)

CRA8-27cm (AF119128)OHKA1.4 (AB094516)OHKA1.43 (AB094528)OHKA1.15 (AB094521)OHKA7.26 (AB094552)

OHKA1.8 (AB094518)

OHKA2.14 (AB094531)OHKA6.39 (AB094550)

OHKA6.41 (AB094551)MA-A1-3 (AY093448)

OHKA10.11 (AB094555)OHKA14.11 (AB094559)

OHKA1.18 (AB094523)

APA3-11cm (AF119137)OHKA4.94 (AB094544)

pMC2A308 (AB019721)

pMC2A256 (AB019717)

pMC2A15 (AB019718)OHKA1.28 (AB094525)

pJP27 (L25852)

JTB167 (AB015275)

0.02

Miscellaneus CrenarchaeoticGroup (MCG)

Marine HydrothermalVent Group (MHVG)

Deep-Sea Archaeal Group(DSAG)

Marine Group I(MGI)

(A)

Aquifex pyrophilus (M83548)

FIG. 3. Phylogenetic relationships of archaeal 16S rRNA gene sequences of representative clones from the Okhotsk core sediments and ofrelated pure cultures and environmental clones in the kingdoms Crenarchaeota (A) and Euryarchaeota (B). The trees were inferred by neighbor-joining analysis by using restricted homologous positions of 16S rRNA gene sequences. The solid circles at nodes indicate positions where theconfidence value for 100 bootstrap trial results is less than 40%. The sequences of representative clones determined in this study are indicated byboldface type. The numbers in parentheses are accession numbers of sequences. Scale bar � 0.02 nucleotide substitution per sequence position.


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otic group, but given the recent reports of marine isolates inthis group (25), we suggest that it should be designated theMCG. The most predominant phylotype in the MCG from ashlayers (79 related clones) was OHKA4.47, which exhibited90.1% similarity to pSL123, obtained from a hot spring inYellowstone National Park (3). OHKA4.12, OHKA4.18, andOHKA5.34 comprised 77 closely related phylotypes with highlevels of similarity to the MA-C1-5 sequence from methanehydrate-bearing subseafloor sediments from the NankaiTrough (Fig. 3) (25). The 16S rRNA gene sequences related toHTA-B10 were also predominant archaeal 16S rRNA genecomponents in ash layers (Fig. 2A). An identical sequence wasobtained from metal-rich particles in a terrestrial freshwaterreservoir (29). Another prominent phylotype was OHKA4.4,which was detected in all ash layers and exhibited similarity toa group called the South African gold mine euryarchaeoticgroup (33).

With the exception of a few minor phylotypes, there was verylittle overlap between the communities found in the clay andash layers (Fig. 2). For example, a total of 33 archaeal 16S

rRNA gene clones were members of marine benthic group D(37), whose members were detected in both pelagic clays andash layers, and the OHKA1.1 sequence was a unique repre-sentative phylotype detected throughout the core sediments(Fig. 2 and 3B); the closest relative of this phylotype wasJTB173 from deep-sea anoxic cold seep sediments from theJapan Trench (18, 10) (Fig. 3B).

Bacterial 16S rRNA gene clone library analyses. Bacterial16S rRNA gene clone libraries were constructed from eightsediment layers (four ash layers and four clay layers), and the571 partial sequences of the bacterial 16S rRNA gene thatwere analyzed (Fig. 2) revealed considerable diversity (167representative bacterial phylotypes). Like the archaeal 16SrRNA gene community members, the compositions of bacte-rial phylotypes showed that there were distinct differences be-tween the pelagic clay and volcanic ash layers, with the claylayers containing mainly members of the candidate divisionOP9 (8) and the green nonsulfur bacteria and the ash layersdominated by the members of the gamma and alpha subclassesof the Proteobacteria (Fig. 2 and 4).

FIG. 3—Continued.


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A total of 249 bacterial 16S rRNA gene sequences frompelagic clay samples were analyzed (Fig. 2); 144 (57.8%) and54 (21.7%) of the clones from pelagic clay samples were affil-iated with the OP9 candidate division and the green nonsulfurbacteria, respectively. The OHKB2.44 (47 related clones) andOHKB6.20 (70 related clones) sequences were the most pre-

dominant bacterial phylotypes in the OP9 candidate division,and these sequences were very similar to the 16S rRNA se-quence of clone JTB138 from cold seep sediments from theJapan Trench (10, 18) (Fig. 4B). A variety of phylotypes be-longing to green nonsulfur bacteria were detected, primarily inthe topmost clay layer, where they were the dominant group

FIG. 4. Phylogenetic relationships of bacterial 16S rRNA gene sequences of representative clones from the Okhotsk core sediments and relatedpure cultures and environmental clones belonging to the class Proteobacteria (A) and to the Dehalococcoides group of the green nonsulfur bacteriaand the candidate OP9 division (B). The NT-B3 and NT-B4 clusters in panel B correspond to the clusters described by Reed et al. (25). The treeswere inferred by neighbor-joining analysis by using restricted homologous positions of 16S rRNA gene sequences. The solid circles at nodesindicate positions where the confidence value for 100 bootstrap trial results is less than 40%. 16S rRNA gene sequences of representative clonesand isolates determined in this study are indicated by boldface type. The numbers in parentheses are accession numbers of sequences. Scale bar� 0.02 nucleotide substitution per sequence position.


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(Fig. 2). The phylotype most similar to any of these sequenceswas the Dehalococcoides ethenogenes phylotype (22). Anothersmall cluster, represented by OHKB2.37, was most closely re-lated to the MB-A2-101 sequence from the Nankai Trough(25) (Fig. 4B). Small numbers of other proteobacterial phylo-types were found in the clay layers and accounted for a fewpercent of the total (Fig. 2).

In marked contrast, the OP9 candidate division and greennonsulfur bacterial phylotypes were nearly absent from theclone libraries from the ash layers (3 and 10 clones, respec-tively), which were dominated by members of the gamma sub-class of the Proteobacteria (Fig. 2). Of 322 bacterial 16S rRNAgenes that were analyzed, 264 (82.0%) grouped with thegamma subclass of the Proteobacteria. The predominant gam-ma-proteobacterial 16S rRNA gene components were the gen-era Halomonas, Methylophaga, and Psychrobacter (Fig. 4A).The Halomonas relatives included 147 clones (45.7%) and twolarge groups that were closely related to the described speciesHalomonas variabilis and Halomonas meridiana (Fig. 4A). Inthe two ash layers from the bottom that were analyzed, abun-dant clones grouped with Methylophaga, a type I methanotroph(7) (Fig. 4A). Several other type I methanotrophs were foundin various ash layers, but with the exception of Methylophagaonly small numbers were found. The alpha subclass of theProteobacteria was represented by small numbers of Sulfito-bacter and Octadecabacter (Fig. 2 and 4A).

Isolation and characterization of bacteria. CFU assays wereperformed by using three different solid media at several tem-peratures. For the ash layers, growth was seen on all media attemperatures between 5 and 35°C, but no colonies grew at45°C. Both MJYP medium and marine broth 2216 agar yielded�4 � 104 growing cells/cm3, while slightly higher numbers (�2� 105 cells/cm3) were obtained with DSM598 medium de-signed for halophilic bacteria (Fig. 5). Several pelagic claylayers (sections 1, 2, 3, 6, 14, and 16) yielded no viable colonieson any of the media tested, and in general, the numbers ofCFU were lower in the pelagic clay layers than in the ash layers(Table 1 and Fig. 5). As determined by comparing the numbersof CFU to the total cell counts obtained by AO analysis, 1% orfewer of the total colonies were cultivated from the ash layers,and for the clay layers much less than 0.1% of the total colonieswere grown.

A total of 181 colonies (selected on the basis of colonymorphology, sample depth, and cultivation conditions) wereanalyzed by partial sequencing of the 16S rRNA gene, and theresults revealed that 93.3% of isolates were members of theProteobacteria and 7.7% were gram-positive bacteria (Table 2).Strain DSM25.14, the most predominant colony phylotype,accounted for 82 isolates that formed cream-colored colonieson DSM598 medium and marine broth 2216 agar. The 16SrRNA gene sequence of DSM.25.14 exhibited 97.9% similarity

FIG. 4—Continued.


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with the 16S rRNA gene sequence of H. variabilis (Table 2 andFig. 4A). Fifty-eight colony isolates were closely related to thegenus Psychrobacter, and the sequences of representativestrains MJYP.15.12 and 2216.25.11 were closely related to Psy-chrobacter pacificensis (99.5%) and Psychrobacter submarinus(99.0%), respectively (Table 2 and Fig. 4A). The third-most-abundant colony type, represented by strain MJYP.25.10, wasrelated to Sulfitobacter mediterranneus in the alpha subclass ofthe Proteobacteria (96.2% similarity) (Table 2 and Fig. 4A).Gram-positive bacteria represented only 7.7% of the total col-ony isolates, and they consisted of 10 different phylotypes.

DISCUSSION

The sediments which we examined in this study ranged fromsediments which were freshly deposited at the surface to sed-iments that were about 100,000 years old at the bottom of thecore. The core was composed primarily of grayish (hemi-)pe-lagic clay interspersed with layers of volcanic ash deposited as

a result of various eruptions (Table 1). Several questions wereaddressed in this study. First, how do the total numbers ofbacteria vary as a function of depth (time) in the sedimentarycolumn, and how do the total numbers in the clay and ashlayers differ? It is clear from the data shown in Fig. 1 that thenumber of cells decreased with depth; both direct cell counting(Fig. 1A) and quantitative PCR estimates (Fig. 1B) showedthat the number of cells decreased with depth. For the claysamples, both methods indicated that the number of cells re-mained constant with depth after about 15 m below the sea-floor. The ash layers contained higher numbers of cells, by afactor of about 4. The uppermost ash layer (18 m) containedthe highest numbers of cells, and in each subsequent layer thenumber of cells decreased, until near the bottom the numberwas nearly identical to the number in the clay layers. Theseobservations are consistent with what has been observed inother subsurface systems, in which the higher numbers at thesediment surface decreased to constant numbers (on the orderof 106 cells per cm3 of sediment). In contrast, the population

FIG. 5. CFU assay of MD01-2412 subseafloor sediments. Three kinds of solid plates were incubated at 5, 15, 25, 35, and 45°C for 2 weeks. Nocolonies were observed after incubation at 45°C. Sec., section; PC, pelagic clay.


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sizes decreased with depth in the ash layers, while high porosityremained relatively constant throughout the core column.Many factors account for the levels of microbes in variousniches, but one factor that may be important in our column isporosity. The pore space and the geohydrogical flow of inter-stitial water in ash layers are substantially higher than those inclay environments, and such regional sedimentogical charac-teristics may affect the mass and distribution of microbial com-munities in coastal subseafloor environments.

Second, were the microbial community structures in the ashand clay environments qualitatively different, and did the com-positions (as well as total numbers) change with the age of thesediment layer? Given the long burial times, it might have beenreasonable to expect that the microbial communities of the twosedimentary regimens, if they were different when they weredeposited, might have been able to shift and adapt with ambi-ent environments during the sedimentation process. Also, un-less the communities were very different when the layers weredeposited, it might be expected that the robust bacteria wouldbe the same in both the ash and clay layers and that they mightbecome more similar with depth. With regard to the first issue,it is very clear that the phylotype compositions in the twoenvironments (ash and clay) were very different throughout thesubseafloor sediment core. Figure 6 shows a summary pie chartof the differences between the two layers; in a sense, this is asummation of the differences shown for the individual layers inFig. 2.

The differences with depth appeared to be qualitative as wellas quantitative for both the clay layers and the ash layers. AsFig. 1 shows, the clay samples included one rather shallowsample, and the bacterial population in the shallow layer wasdifferent from the populations in the deeper layers. Althoughthe molecular analysis did not reflect the actual microbial com-munities, the abundance of green nonsulfur phylotypes in theupper sample and the abundance of their relatives in deeperlayers may indicate that as a group, the members of this mi-crobial community change with depth or are not robust survi-vors. For the ash layers, the members of the alpha subclass of

the Proteobacteria appeared to be less robust, disappearingwith depth, while the members of the gamma subclass becamedominant. However, since virtually nothing is known about thecompositions of the initial microbial communities in these ashlayers, one must consider the possibility that the events leadingto the shallow layer simply resulted in a qualitatively differentcommunity during the sedimentation process (Fig. 2).

Are there any reasonable explanations for the qualitativedifferences in the microbial communities that we observed inthis study? With regard to the archaea, the clay layers weredominated by the DSAG, while the ash layers were dominatedby the MCG (previously designated the terrestrial miscella-neous crenarchaeotic group). In the absence of successful cul-tivation of these archaea, it is not possible to assign physiolog-ical and metabolic properties to the archaeal assemblages.However, it is now becoming clear that the DSAG and MCGlineages have been detected most often in marine and terres-trial environments, respectively (30, 33). One possible inter-pretation is that the microbial communities in coastal subsea-floor environments are strongly influenced by the geologicaland geochemical settings. Indeed, 16S rRNA gene sequencesof the OP9 group have been detected so far in various reducingenvironments (8, 10, 18, 24, 35). The abundance of OP9 phy-lotypes in the deeper clay layers might be associated withanoxic subseafloor clay environments.

The bacterial phylotypes obtained from volcanic ash layerswere dominated by psychrophilic or mesophilic, aerobic het-erotrophs belonging to the gamma and alpha subclasses of theProteobacteria. Among this group, sequence analysis indicatedthat the members of the genus Halomonas were a major com-ponent, which may have been an indication of their ability tosurvive in the presence of a wide range of salt concentrations.Since these aerobes have been isolated, they may be active, andthe nutrients required for growth, such as organic substratesand oxygen, may be present in deeply buried ash layers. Thedetection of these bacterial types, along with Sulfitobacter andthe type I methanotrophs, might permit reconstruction of someof the metabolic interactions that occurred or are still occur-

TABLE 2. 16S ribosomal DNA sequence similarity analysis of representative colony isolates from MD01-2412 core sediments

Representative strain Color Section Most closely related organism indatabases Accession no. %

Similarity Phylogenetic groupNo. ofrelatedisolates

OHKMJYP.25.10 White 7 Sulfitobacter mediterraneus Y17387 96.2 -Proteobacteria 26OHKDSM.25.14 Cream 13 Halomonas variabilis AJ306893 97.9 -Proteobacteria 82OHK2216.25.11 White 4 Psychrobacter submarinus AJ309940 99.0 -Proteobacteria 48OHKMJYP.15.12 White 8 Psychrobacter pacificensis AB016058 99.5 -Proteobacteria 9OHKMJYP.25.32 Orange 11 Psychrobacter pacificensis AB016059 99.1 -Proteobacteria 1OHK2216.25.25 Orange 9 Erythrobacter citreus AF118020 97.6 -Proteobacteria 3OHK2216.15.5 Yellow 4 Flexibacter aggregans AB078039 98.6 CFB groupa 1OHKMJYP.25.24 Clear 9 Marinolactobacillus psychrotolerans AB083413 99.1 Low-G�C-content gram-positive bacteria 10OHK2216.15.16 Lemon 9 Paenibacillus amylolyticus D85396 99.5 Low-G�C-content gram-positive bacteria 2OHK2216.25.2 Cream 1 Bacillus pseudofirmus X76439 97.1 Low-G�C-content gram-positive bacteria 2OHK2216.25.27 Orange 9 Bacillus marinus AJ237708 98.1 Low-G�C-content gram-positive bacteria 2OHK2216.15.2 Clear 1 Sporosarcina psychrophila D16277 95.6 Low-G�C-content gram-positive bacteria 1OHK2216.25.22 Lemon 9 Citrococcus muralis AJ344143 97.8 Actinobacteria 2OHK2216.35.28 Orange 9 Geogenia muralis X94155 99.4 Actinobacteria 2OHK2216.25.15 Orange 7 Rhodococcus erythropolis AF001265 98.6 Actinobacteria 1OHK2216.35.9 Orange 4 Dietzia maris X79290 100.0 Actinobacteria 1OHK2216.35.31 Red 9 Kocuria erythromyxa Y11330 98.3 Actinobacteria 1

a CFB group, Cytophaga-Flavobacterium-Bacterioides group.


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ring. However, no pore water chemistry data were collectedduring this project cruise, so any such reconstruction could notbe based on environmental data.

A final question is related to the value of the informationobtained from the cultivation studies (and characterization ofthe cultivars). Figure 5 shows that when three media were usedat several temperatures, only two major groups of bacteriawere cultivated, both from the ash layers. Even in these layers,only �1% of the total populations could be cultivated. Ofthese, virtually all belonged either to the genus Halomonas inthe gamma subclass of the Proteobacteria (and they were me-sophiles), to the gram-positive group, or to the Actinobacteria(Table 2). The first group was also found to be abundant byanalysis of 16S rRNA gene sequences (Fig. 4A), while theother two groups, both renowned for formation of restingstages, were not found by molecular analysis. It seems likely

that the Halomonas group is a very robust group with regard tosurvival and that members of this group were probably im-ported into the ash layer specifically from the outcrop of thecoastal wedge, as these organisms were essentially absent inthe clay layers as determined by either technique. The samemay be said of the gram-positive bacteria and the actinobac-teria; they were probably brought to or buried with the ashlayers and are very good survivors.

A final point that is still under debate is the use of 16S rRNAgene clone analysis for determining microbial diversity. For avariety of reasons (copy number, bias during DNA extraction,PCR, cloning), the frequency of 16S rRNA gene clone appear-ance does not always reflect the in situ microbial communitystructure (38). Despite these reservations, however, it is clear thatthe molecular ecological methods revealed diversity far greaterthan that of previously isolated microorganisms and in some

FIG. 6. Summary pie charts of archaeal and bacterial phylotype compositions for 16S rRNA gene clone libraries constructed from pelagic clayand volcanic ash layers in the MD01-2412 Okhotsk core sediments. MHVG, marine hydrothermal vent group; MG1, marine group 1; MBG-D,marine benthic group D; SAGMEG, South American gold mine euryarchaeotic group; , alpha subclass of the class Proteobacteria; , gammasubclass of the class Proteobacteria; �, delta subclass of the class Proteobacteria.


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cases, such as the OP9 group, revealed a major bacterial compo-nent that could never have been seen by cultivation. If there is abias in the methods, examination of various levels of ash and claysuggested that it is consistent, and as the methods improve, pre-viously unrecognized biases or artifacts should be revealed.

In conclusion, the data presented here demonstrate that inthis environment, two very different geohydrological settings(grayish pelagic clays and volcanic ash layers) contained decid-edly different microbial communities and that the differencespersisted through a series of layers spanning approximately100,000 years. Whether these communities are active, just sur-viving, or dead remains unknown, but it is clear that the coastalsubseafloor sediments are a reservoir of prokaryotic biologicaldiversity and that these reservoirs maintain their genetic prop-erties over long periods of time.

ACKNOWLEDGMENTS

We are very grateful to the R/V Marion Du Frence operation teamand to Minoru Ikehara, Tadamichi Ohba, and Hotaka Kawahata forhelping us collect the subseafloor sediment samples. We also thank allmembers of the MD01-2412 Okhotsk Core Scientific Party for usefuldiscussions.

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