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Applied and Environmental Microbiology, December 2003, p. 7224-7235, Vol. 69, No. 12
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.12.7224-7235.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

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

Subground Animalcule Retrieval (SUGAR) Project, Frontier Research System for Extremophiles,1 Research Program for Paleoenvironment, Institute for Frontier Research on Earth Evolution (IFREE), Japan Marine Science and Technology Center (JAMSTEC), Yokosuka 237-0061,2 Geological Survey of Japan, AIST, Tsukuba 305-8567, Japan,3 Department of Earth Sciences, University of Southern California, Los Angeles, California 90089-07404

Received 16 June 2003/ Accepted 29 September 2003


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


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INTRODUCTION
 
The subsurface environment has been proposed to be the largest reservoir of biomass on Earth (39). On the basis estimates of the biomass in subseafloor core sediments collected by the Ocean Drilling Program, more than 105 microbial cells/cm3 were consistently present even at a depth close to 1,000 m below the seafloor (23). However, the relationships between the microbial communities on the one hand and the biogeochemical impact, sedimentological properties, and past geological events in subseafloor environments on the other hand have remained poorly defined. Recent studies of microbial communities in geologic materials have suggested that microorganisms adapt to a variety of microhabitats and may be considered to be indigenous to them. For example, the archaeal community in the black smoker hydrothermal vent chimney collected from northeastern Papua New Guinea consisted of hyperthermophiles and extreme halophiles, the distributions of which corresponded to the mineralogical characteristics of various microhabitats in the hydrothermal deposits (32). Similarly, the distribution of the microbial communities found in a deep-sea siltstone collected from the Japan Trench appeared to be correlated with the porosity and permeability of the geological matrices (11). In marked contrast, the unexpected presence of several archaeal genera, such as Thermococcus, Sulfolobus, and Haloarcula, was reported for cold subseafloor core sediments recovered from the West Philippine Basin (9); these organisms may have been transferred from surrounding terrestrial acidic hot springs or hydrothermal vent fields and buried. In the terrestrial subsurface, loci exhibiting high rates of sulfate reduction were observed at sandstone-shale interfaces in the deep subsurface in central New Mexico (6, 15, 16). Recently, microbial diversity and distribution in subsurface gold mines have been described (12, 33, 34). Taken together, the information described above suggests that while the geological and geochemical settings greatly affect microbial composition in both terrestrial and marine subsurface environments, there are many unexplained findings that may be related to the ability of imported microbes to survive for long periods of time. Unexplained variation can occur even in areas that seem to be homogeneous and similar, such as methane hydrate sites, where major differences in community structure have been reported. For example, at subseafloor methane hydrate sites (Ocean Drilling Program Leg 146 core sediments from the Cascadian Margin), the presence of methanogenic archaea was reported (4, 21), while recent studies of communities in hydrate-bearing sediments in the Nankai Trough (25) revealed little similarity with the communities found in the Cascadian Margin. It may well be that the processes that lead to formation of methane hydrate are regionally complicated and that the roles of subseafloor microorganisms in methane production and/or consumption are different at different sites. As more data accumulate, it may well become possible to decipher the situation.

In the studies described here we addressed the issue of community variability in a different environment: a coastal subseafloor sediment from a system of pelagic clays that is interspersed with layers of volcanic ash. Analysis of the vertical profile of microbial distribution and phylogenetic diversity at 16 depths in this sediment revealed easily seen but difficult to explain differences in these types of layers.


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MATERIALS AND METHODS
 
Sample collection.
A subseafloor sediment core, MD01-2412, was obtained from the southwestern part of the Sea of Okhotsk (44°31.65'N, 145°00.25'E) off the Shiretoko Peninsula at the eastern margin of Hokkaido at a depth of 1,225 m. This sediment core (length, 58.1 m) was recovered by using a giant piston core during the IMAGES (International Marine Global Environmental Change Study) 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 the core at 16 different depths (Table 1) and placed into sterile plastic tubes by using alcohol-sterilized spatulas. The samples were kept at 4°C on board and then were stored at -80°C in the laboratory prior to analysis.


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

Porosity of the sediment.
To determine the sediment porosity, the wet bulk density (WBD) was determined onboard by gamma ray attenuation of wet split core sediments by using a multisensor core logger (GeoTek Co., Stewartville, Minn.). The mineral grain density (MGD) was determined with a Penta PPY-12 pycnometer (Quantachrome Co., Boynton Beach, Fla.) for dried sediments as previously described (11). For sections 12 and 13, the MGD was assumed to be 2.6500 g/cm3 because the amount of sediment in each of the samples was not large 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. The porosity 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 in 0.9 cm3 of sterilized MJ synthetic seawater (13) containing 3.7% (wt/vol) formaldehyde, and the slurry was vigorously agitated for 2 min with a vortex mixer. The suspension was briefly centrifuged (<2,000 x g), and then the cells remaining in the supernatant were stained with acridine orange (AO) (10 µg ml-1) for 15 min. The solution was filtered with a 0.22-µm-pore size polycarbonate filter (Advantec, Tokyo, Japan) and then rinsed briefly in MJ synthetic seawater. The AO-stained cells on the filter were counted by using epifluorescence and a Nikon Optishot microscope (Nikon, Tokyo, Japan). The total cell density was estimated from 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 an extracted DNA solution was precipitated in ethanol and reconstituted in 500 µl of TE buffer (10 mM Tris-HCl, 1.0 mM EDTA; pH 8.0) (26). Since the DNA solution still contained some inhibitors of PCR amplification at this stage (e.g., humic acid substances or heavy metals), a 100-µl portion was rinsed twice with column and buffer solutions (S3, S4, and S5) from a soil DNA Mini Prep kit (Mo Bio Lab, Inc.). Finally, extracted bulk DNA for PCR amplification was concentrated by ethanol precipitation with 20 µl of TE buffer.

Quantitative PCR analysis of archaeal and bacterial 16S rRNA genes.
Quantification of archaeal and bacterial 16S rRNA genes in bulk extracted DNA solutions was performed by the quantitative fluorescent PCR method by using universal and domain-specific TaqMan fluorogenic probes as described previously (31). The PCR and monitoring of fluorescence signals were performed by using the GeneAmp 5700 sequence detection system (PE Applied Biosystems, Foster City, Calif.).

Construction of PCR-amplified 16S rRNA gene clone libraries.
Microbial 16S rRNA genes were amplified from the extracted bulk DNA solutions by PCR performed with LA Taq polymerase and GC buffer I (TaKaRa, Tokyo, Japan). Bacterial 16S rRNA genes were amplified by using the Bac27F and Bac927R primers (17). Primers Arch21F and Arch958R (5) were used for amplification of archaeal 16S rRNA genes. Thermal cycling was performed with the GeneAmp 9600 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°C for 120 s. Bacterial 16S rRNA gene amplification was performed for 34 cycles, and archaeal gene amplification was performed for 38 cycles. PCR amplification from a solution without sediment prepared as described above was processed as a negative control to check for experimental contamination.

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

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

Single-stranded archaeal and bacterial 16S rRNA gene sequences containing approximately 400 to 450 nucleotides were analyzed with a model ABI 3100 automated sequencer (PE Applied Biosystems) by using the Arch 21F and the Bac27F primers, respectively. A total of 1,210 clones (639 archaeal 16S rRNA genes and 571 bacterial 16S rRNA genes) were selected from the clone libraries, and partial sequences of all of these clones were determined. The sequence similarity among the partial 16S rRNA gene sequences was analyzed by using the FASTA program equipped with the DNASIS software (Hitachi Software, Tokyo, Japan). Sequences that exhibited >=97% similarity, suggesting a species level relationship (28), were tentatively assigned to the same phylogenetic type (phylotype), and a representative clone of the 16S rRNA gene was selected for each phylotype. The 16S rRNA gene sequence of each representative clone (length, 900 to 950 bp) was determined by sequencing both strands. The representative 16S rRNA gene sequences of the phylotypes were subjected to similarity analysis by using the FASTA3 and gapped BLAST search algorithms with the GenBank/EMBL/DDBJ databases (1).

Phylogenetic analysis.
Phylogenetic analysis of the representative archaeal and bacterial 16S rRNA gene sequences was restricted to the nucleotide positions described in the figure legends that could be unambiguously aligned in all sequences. Least-squares distance matrix analysis, based on evolutionary distances, was carried out by using the correction of Kimura (14). Neighbor-joining analysis was performed by using the DDBJ CLUSTAL-X system (36). A bootstrap analysis was performed with 100 trial replications to provide confidence estimates for phylogenetic tree topologies. In order to discriminate the ambiguous phylogenetic affiliations of 16S rRNA genes, if necessary, a sequence was applied 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 medium contained 0.01% (wt/vol) yeast extract, 0.01% (wt/vol) peptone (Difco), and a vitamin mixture (2) at a concentration of 0.01% (vol/vol) in MJ synthetic seawater. This medium was used to determine the CFU counts for heterotrophs that require low levels of nutrients. Marine broth 2216 agar was used for a variety of marine heterotrophs that prefer high nutrient concentrations (11). DSM598 medium was used for cultivation of Halomonas variabilis, which enabled evaluation 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 g ofMgSO4 · 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 were adjusted 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 sediment sample was put into a sterilized plastic tube, and then the tube was filled with 1.0 ml of MJ synthetic seawater. After the suspension was vigorously agitated for 2 min with a vortex mixer, 100-µl portions of slurry were spread on solid medium and then incubated aerobically at 5, 15, 25, 35, and 45°C for 2 weeks before counting.

Phylogenetic analysis of the colony isolates.
A total of 181 colonies were selected from incubated solid media from the CFU assays based on the different cultivation conditions (medium and incubation temperature) and colony morphology (color and size). Each colony was grown in 1 ml of the same liquid medium from which it was isolated. The cultures were incubated for 3 days at the isolation temperature. Cells were harvested by centrifugation (3,500 x g) for 10 min, and the genomic DNA of each pellet was then extracted with a soil DNA Mini Prep kit (Mo Bio Lab, Inc.) used according to the manufacturer's suggested protocol. A 16S rRNA gene fragment of each isolate was amplified by PCR by using the Bac27F and Uni1492R primers (17) and was purified with a Gel Spin DNA purification kit (Mo Bio Lab, Inc.). A single-stranded 16S rRNA gene sequence of each isolate that was 400 to 450 bp long was directly sequenced by using the Bac27F primer, and the representative isolates of the phylotypes were determined by the clone library analysis procedure described above. The 16S rRNA gene sequences of the representative isolates were determined by using both strands, and then the similarity and phylogenetic analyses were carried out as described above.

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


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RESULTS
 
Sample characteristics.
The length of sediment core MD01-2412 recovered was 58.1 m. The sediment core was found to be composed of (hemi-)pelagic clay with several volcanic ash layers containing pumice grains. A total of 16 samples of innermost core sediments were collected from the pelagic clays and ash layers at different depths (Table 1). The ages and derivations of the volcanic ash layers were determined by using the refractive indices of volcanic glass shards and minerals (collectively referred to as tephra). In addition, preliminary results of an analysis of diatoms and volcanic tephra by using a 7.7-m sediment core recovered from the same site suggested that the sedimentation rate in the sampling field was approximately 100 cm/103 years (27) and that potentially recent environmental changes, such as sea ice coverage and volcanic eruptions, were recorded. These estimates are consistent with the dates of the volcanic events identified as responsible for the formation of the ash layers (the sediment at a depth of 57 m is approximately 100,000 years old), assuming that there was compaction of the sediments with depth (Table 1) (27). As Table 1 shows, the porosity values of pelagic clay generally decreased with increasing depth by a factor of approximately 2 to 3 (except for sections 5 and 11 containing small pumice grains), while those of ash layers were relatively constant at approximately 35%.

Direct cell counting and quantitative PCR analysis of 16S rRNA genes.
Epifluorescence microscopic observation of AO-stained cells indicated that the microbial population in ash layers was slightly larger than that in the pelagic clay environments. Approximately 4 x 10 6 cells were present in 1 cm3 of pelagic clay at depths below 10 m below the seafloor, whereas approximately three to four times more cells were present in the ash layers (Fig. 1A). The results of a quantitative PCR analysis of archaeal and bacterial 16S rRNA genes were consistent with the cell count data, showing that the amounts of bacterial 16S rRNA genes in bulk DNA solutions extracted from ash layers were 2 to 10 times larger than the amounts in bulk DNA solutions extracted from pelagic clays (Fig. 1B). For example, the concentrations of the bacterial 16S rRNA genes in ash layers of section 13 (Ash-4) and section 5 (Kc-1) were estimated to be 8.2 x 104 and 4.3 x 104 fg ml-1, respectively, whereas the concentrations in pelagic clays of sections 3 and 6 were 5.3 x 103 and 6.4 x 103 fg ml-1, respectively. In contrast, the amount of archaeal 16S rRNA genes decreased as the depth increased (Fig. 1B).



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  		FIG. 1. Profiles of total cell density (A) and 16S rRNA gene concentration (B) in the MD01-2412 core sediments. (A) Total cell densities in pelagic clay samples ({circ}) and ash layer samples (•) were estimated by direct counting of AO-stained cells. (B) Archaeal ({square}) and bacterial ({blacksquare}) concentrations of the 16S rRNA gene were determined by quantitative PCR by using domain-specific fluorogenic probes. mbsf, meters below the seafloor; rDNA, ribosomal DNA.

Archaeal 16S rRNA gene clone library analyses.
A total of 639 partial archaeal 16S rRNA gene sequences were determined for clone libraries constructed from all 16 layers. A similarity analysis of all sequences indicated that 49 different representative clones of the archaeal 16S rRNA gene were present. A comparison of the archaeal 16S rRNA gene phylotypes in the clay and ash layers showed that they were easily distinguishable, with the deep-sea archaeal group (DSAG) dominating the pelagic clays and the miscellaneous crenarchaeotic group (MCG) dominating the ash layers (Fig. 2 and 3).



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




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  		FIG. 3. Phylogenetic relationships of archaeal 16S rRNA gene sequences of representative clones from the Okhotsk core sediments and of related 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 the confidence value for 100 bootstrap trial results is less than 40%. The sequences of representative clones 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.

Of 340 archaeal 16S rRNA gene clones from pelagic clays, 262 (77.0%) were affiliated within the DSAG. Sequence OHKA2.33 was the most frequently detected phylotype (126 related clones) (Fig. 3), and this sequence is closely related to the CRA8-27 sequence (97.3%) detected in deep-sea coastal marine sediments (37). The second most abundant archaeal phylotype in the DSAG lineages was OHKA10.11 (47 related 16S rRNA gene clones) (Fig. 3), which exhibited 98.9% similarity with the MA-A1-3 sequence from methane hydrate-bear-ing subseafloor sediments from the Nankai Trough (25). While Fig. 2 shows that there were some differences in phylotype composition as the depth of the core increased, these differences were often ascribed to small numbers of clones of specific phylotypes.

In contrast, of the 299 archaeal clones analyzed from clone libraries constructed from volcanic ash layers, 211 (70.6%) belonged to the MCG group (Fig. 2 and 3). This cluster was previously designated the terrestrial miscellaneous crenarchaeotic group, but given the recent reports of marine isolates in this group (25), we suggest that it should be designated the MCG. The most predominant phylotype in the MCG from ash layers (79 related clones) was OHKA4.47, which exhibited 90.1% similarity to pSL123, obtained from a hot spring in Yellowstone National Park (3). OHKA4.12, OHKA4.18, and OHKA5.34 comprised 77 closely related phylotypes with high levels of similarity to the MA-C1-5 sequence from methane hydrate-bearing subseafloor sediments from the Nankai Trough (Fig. 3) (25). The 16S rRNA gene sequences related to HTA-B10 were also predominant archaeal 16S rRNA gene components in ash layers (Fig. 2A). An identical sequence was obtained from metal-rich particles in a terrestrial freshwater reservoir (29). Another prominent phylotype was OHKA4.4, which was detected in all ash layers and exhibited similarity to a group called the South African gold mine euryarchaeotic group (33).

With the exception of a few minor phylotypes, there was very little overlap between the communities found in the clay and ash 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 and ash 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 was JTB173 from deep-sea anoxic cold seep sediments from the Japan Trench (18, 10) (Fig. 3B).

Bacterial 16S rRNA gene clone library analyses.
Bacterial 16S rRNA gene clone libraries were constructed from eight sediment layers (four ash layers and four clay layers), and the 571 partial sequences of the bacterial 16S rRNA gene that were analyzed (Fig. 2) revealed considerable diversity (167 representative bacterial phylotypes). Like the archaeal 16S rRNA gene community members, the compositions of bacterial phylotypes showed that there were distinct differences between the pelagic clay and volcanic ash layers, with the clay layers containing mainly members of the candidate division OP9 (8) and the green nonsulfur bacteria and the ash layers dominated by the members of the gamma and alpha subclasses of the Proteobacteria (Fig. 2 and 4.




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  		FIG. 4. Phylogenetic relationships of bacterial 16S rRNA gene sequences of representative clones from the Okhotsk core sediments and related pure cultures and environmental clones belonging to the class Proteobacteria (A) and to the Dehalococcoides group of the green nonsulfur bacteria and 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 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 the confidence value for 100 bootstrap trial results is less than 40%. 16S rRNA gene sequences of representative clones and 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.

A total of 249 bacterial 16S rRNA gene sequences from pelagic clay samples were analyzed (Fig. 2); 144 (57.8%) and 54 (21.7%) of the clones from pelagic clay samples were affiliated with the OP9 candidate division and the green nonsulfur bacteria, respectively. The OHKB2.44 (47 related clones) and OHKB6.20 (70 related clones) sequences were the most predominant bacterial phylotypes in the OP9 candidate division, and these sequences were very similar to the 16S rRNA sequence of clone JTB138 from cold seep sediments from the Japan Trench (10, 18) (Fig. 4B). A variety of phylotypes belonging to green nonsulfur bacteria were detected, primarily in the topmost clay layer, where they were the dominant group (Fig. 2). The phylotype most similar to any of these sequences was the Dehalococcoides ethenogenes phylotype (22). Another small cluster, represented by OHKB2.37, was most closely related to the MB-A2-101 sequence from the Nankai Trough (25) (Fig. 4B). Small numbers of other proteobacterial phylotypes were found in the clay layers and accounted for a few percent of the total (Fig. 2).

In marked contrast, the OP9 candidate division and green nonsulfur bacterial phylotypes were nearly absent from the clone libraries from the ash layers (3 and 10 clones, respectively), which were dominated by members of the gamma subclass of the Proteobacteria (Fig. 2). Of 322 bacterial 16S rRNA genes that were analyzed, 264 (82.0%) grouped with the gamma subclass of the Proteobacteria. The predominant gamma-proteobacterial 16S rRNA gene components were the genera Halomonas, Methylophaga, and Psychrobacter (Fig. 4A). The Halomonas relatives included 147 clones (45.7%) and two large groups that were closely related to the described species Halomonas variabilis and Halomonas meridiana (Fig. 4A). In the two ash layers from the bottom that were analyzed, abundant clones grouped with Methylophaga, a type I methanotroph (7) (Fig. 4A). Several other type I methanotrophs were found in various ash layers, but with the exception of Methylophaga only small numbers were found. The alpha subclass of the Proteobacteria was represented by small numbers of Sulfitobacter and Octadecabacter (Fig. 2 and 4A).

Isolation and characterization of bacteria.
CFU assays were performed by using three different solid media at several temperatures. For the ash layers, growth was seen on all media at temperatures between 5 and 35°C, but no colonies grew at 45°C. Both MJYP medium and marine broth 2216 agar yielded ~4 x 104 growing cells/cm3, while slightly higher numbers (~2 x 105 cells/cm3) were obtained with DSM598 medium designed for halophilic bacteria (Fig. 5). Several pelagic clay layers (sections 1, 2, 3, 6, 14, and 16) yielded no viable colonies on any of the media tested, and in general, the numbers of CFU were lower in the pelagic clay layers than in the ash layers (Table 1 and Fig. 5). As determined by comparing the numbers of CFU to the total cell counts obtained by AO analysis, 1% or fewer of the total colonies were cultivated from the ash layers, and for the clay layers much less than 0.1% of the total colonies were grown.



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  		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. No colonies were observed after incubation at 45°C. Sec., section; PC, pelagic clay.

A total of 181 colonies (selected on the basis of colony morphology, sample depth, and cultivation conditions) were analyzed by partial sequencing of the 16S rRNA gene, and the results revealed that 93.3% of isolates were members of the Proteobacteria 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 colonies on DSM598 medium and marine broth 2216 agar. The 16S rRNA gene sequence of DSM.25.14 exhibited 97.9% similarity with the 16S rRNA gene sequence of H. variabilis (Table 2 and Fig. 4A). Fifty-eight colony isolates were closely related to the genus Psychrobacter, and the sequences of representative strains MJYP.15.12 and 2216.25.11 were closely related to Psychrobacter 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, was related to Sulfitobacter mediterranneus in the alpha subclass of the Proteobacteria (96.2% similarity) (Table 2 and Fig. 4A). Gram-positive bacteria represented only 7.7% of the total colony isolates, and they consisted of 10 different phylotypes.


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


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DISCUSSION
 
The sediments which we examined in this study ranged from sediments which were freshly deposited at the surface to sediments that were about 100,000 years old at the bottom of the core. The core was composed primarily of grayish (hemi-)pelagic clay interspersed with layers of volcanic ash deposited as a result of various eruptions (Table 1). Several questions were addressed in this study. First, how do the total numbers of bacteria vary as a function of depth (time) in the sedimentary column, and how do the total numbers in the clay and ash layers differ? It is clear from the data shown in Fig. 1 that the number of cells decreased with depth; both direct cell counting (Fig. 1A) and quantitative PCR estimates (Fig. 1B) showed that the number of cells decreased with depth. For the clay samples, both methods indicated that the number of cells remained constant with depth after about 15 m below the seafloor. The ash layers contained higher numbers of cells, by a factor of about 4. The uppermost ash layer (18 m) contained the highest numbers of cells, and in each subsequent layer the number of cells decreased, until near the bottom the number was nearly identical to the number in the clay layers. These observations are consistent with what has been observed in other subsurface systems, in which the higher numbers at the sediment surface decreased to constant numbers (on the order of 106 cells per cm3 of sediment). In contrast, the population sizes decreased with depth in the ash layers, while high porosity remained relatively constant throughout the core column. Many factors account for the levels of microbes in various niches, but one factor that may be important in our column is porosity. The pore space and the geohydrogical flow of interstitial water in ash layers are substantially higher than those in clay environments, and such regional sedimentogical characteristics may affect the mass and distribution of microbial communities in coastal subseafloor environments.

Second, were the microbial community structures in the ash and clay environments qualitatively different, and did the compositions (as well as total numbers) change with the age of the sediment layer? Given the long burial times, it might have been reasonable to expect that the microbial communities of the two sedimentary regimens, if they were different when they were deposited, might have been able to shift and adapt with ambient environments during the sedimentation process. Also, unless the communities were very different when the layers were deposited, it might be expected that the robust bacteria would be the same in both the ash and clay layers and that they might become more similar with depth. With regard to the first issue, it is very clear that the phylotype compositions in the two environments (ash and clay) were very different throughout the subseafloor sediment core. Figure 6 shows a summary pie chart of the differences between the two layers; in a sense, this is a summation of the differences shown for the individual layers in Fig. 2.



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  		FIG. 6. Summary pie charts of archaeal and bacterial phylotype compositions for 16S rRNA gene clone libraries constructed from pelagic clay and 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}, alpha subclass of the class Proteobacteria; {gamma}, gamma subclass of the class Proteobacteria; {delta}, delta subclass of the class Proteobacteria.

The differences with depth appeared to be qualitative as well as quantitative for both the clay layers and the ash layers. As Fig. 1 shows, the clay samples included one rather shallow sample, and the bacterial population in the shallow layer was different from the populations in the deeper layers. Although the molecular analysis did not reflect the actual microbial communities, the abundance of green nonsulfur phylotypes in the upper sample and the abundance of their relatives in deeper layers may indicate that as a group, the members of this microbial community change with depth or are not robust survivors. For the ash layers, the members of the alpha subclass of the Proteobacteria appeared to be less robust, disappearing with depth, while the members of the gamma subclass became dominant. However, since virtually nothing is known about the compositions of the initial microbial communities in these ash layers, one must consider the possibility that the events leading to the shallow layer simply resulted in a qualitatively different community during the sedimentation process (Fig. 2).

Are there any reasonable explanations for the qualitative differences in the microbial communities that we observed in this study? With regard to the archaea, the clay layers were dominated by the DSAG, while the ash layers were dominated by the MCG (previously designated the terrestrial miscellaneous crenarchaeotic group). In the absence of successful cultivation of these archaea, it is not possible to assign physiological and metabolic properties to the archaeal assemblages. However, it is now becoming clear that the DSAG and MCG lineages have been detected most often in marine and terrestrial environments, respectively (30, 33). One possible interpretation is that the microbial communities in coastal subseafloor environments are strongly influenced by the geological and geochemical settings. Indeed, 16S rRNA gene sequences of the OP9 group have been detected so far in various reducing environments (8, 10, 18, 24, 35). The abundance of OP9 phylotypes in the deeper clay layers might be associated with anoxic subseafloor clay environments.

The bacterial phylotypes obtained from volcanic ash layers were dominated by psychrophilic or mesophilic, aerobic heterotrophs belonging to the gamma and alpha subclasses of the Proteobacteria. Among this group, sequence analysis indicated that the members of the genus Halomonas were a major component, which may have been an indication of their ability to survive in the presence of a wide range of salt concentrations. Since these aerobes have been isolated, they may be active, and the nutrients required for growth, such as organic substrates and oxygen, may be present in deeply buried ash layers. The detection of these bacterial types, along with Sulfitobacter and the type I methanotrophs, might permit reconstruction of some of the metabolic interactions that occurred or are still occurring. However, no pore water chemistry data were collected during this project cruise, so any such reconstruction could not be based on environmental data.

A final question is related to the value of the information obtained from the cultivation studies (and characterization of the cultivars). Figure 5 shows that when three media were used at several temperatures, only two major groups of bacteria were cultivated, both from the ash layers. Even in these layers, only ~1% of the total populations could be cultivated. Of these, virtually all belonged either to the genus Halomonas in the gamma subclass of the Proteobacteria (and they were mesophiles), to the gram-positive group, or to the Actinobacteria (Table 2). The first group was also found to be abundant by analysis of 16S rRNA gene sequences (Fig. 4A), while the other two groups, both renowned for formation of resting stages, were not found by molecular analysis. It seems likely that the Halomonas group is a very robust group with regard to survival and that members of this group were probably imported into the ash layer specifically from the outcrop of the coastal wedge, as these organisms were essentially absent in the clay layers as determined by either technique. The same may be said of the gram-positive bacteria and the actinobacteria; they were probably brought to or buried with the ash layers and are very good survivors.

A final point that is still under debate is the use of 16S rRNA gene clone analysis for determining microbial diversity. For a variety of reasons (copy number, bias during DNA extraction, PCR, cloning), the frequency of 16S rRNA gene clone appearance does not always reflect the in situ microbial community structure (38). Despite these reservations, however, it is clear that the molecular ecological methods revealed diversity far greater than that of previously isolated microorganisms and in some cases, such as the OP9 group, revealed a major bacterial component that could never have been seen by cultivation. If there is a bias in the methods, examination of various levels of ash and clay suggested that it is consistent, and as the methods improve, previously unrecognized biases or artifacts should be revealed.

In conclusion, the data presented here demonstrate that in this environment, two very different geohydrological settings (grayish pelagic clays and volcanic ash layers) contained decidedly different microbial communities and that the differences persisted through a series of layers spanning approximately 100,000 years. Whether these communities are active, just surviving, or dead remains unknown, but it is clear that the coastal subseafloor sediments are a reservoir of prokaryotic biological diversity and that these reservoirs maintain their genetic properties over long periods of time.

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ACKNOWLEDGMENTS
 
We are very grateful to the R/V Marion Du Frence operation team and to Minoru Ikehara, Tadamichi Ohba, and Hotaka Kawahata for helping us collect the subseafloor sediment samples. We also thank all members of the MD01-2412 Okhotsk Core Scientific Party for useful discussions.


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FOOTNOTES
 
* Corresponding author. Mailing address: Subground Animalcule Retrieval (SUGAR) Project, Frontier Research System for Extremophiles, Japan Marine Science & Technology Center (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan. Phone: 81-468-67-9687. Fax: 81-468-67-9715. E-mail: inagaki{at}jamstec.go.jp. Back


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Applied and Environmental Microbiology, December 2003, p. 7224-7235, Vol. 69, No. 12
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.12.7224-7235.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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