Particulate Materials and Microbial Assemblages around the ...

Journal of OceanographyVol. 49, pp. 353 to 367. 1993

Particulate Materials and Microbial Assemblages around the IzenaBlack Smoking Vent in the Okinawa Trough

A. MARUYAMA1, N. MITA2 and T. HIGASHIHARA1

1Microbial Resources, National Institute of Bioscience and Human-Technology,Higashi, Tsukuba, Ibaraki 305, Japan

2Geochemistry, Geological Survey of Japan, Higashi, Tsukuba, Ibaraki 305, Japan

(Received 27 December 1991; in revised form 28 September 1992; accepted 5 October 1992)

The plume of particulate DNA (0.7–1 µg/l) was extended from a black smoker vent(c.a. 1340 m depth) to the west-southwest directions in the Izena bottom-waterregion. High concentrations of particulate materials (70–110 µg C/l; 300–570µg S/l) were also detected in the bottom-waters. Microscopic observation showedthat the bottom-waters were rich in alcian blue-stainable large amorphous par-ticles which contained coccoid and rod-shaped microbial cells mostly smaller than1 µm. These microbial matrix compounds appeared to contribute to low P-DNA/P-C ratio (0.011 ± 0.008; n = 27) in the vent environment. Sulfur was detected invarious kinds of particles in the waters, while the content varied with calcium.Microbial population in the P-DNA plume water was in the order of 105 cells/mland the most (>99.9%) were non-culturable. The composition of culturableheterotrophs differed between the bottom-waters and surface sediments sur-rounding the vent; contributions of low temperature (4°C)-culturable bacteriaand manganese-oxidizing bacteria to the total heterotrophs were higher in thesediments than in the waters. In contrast, percentages of orange-pigmentedheterotrophs and microorganisms capable of growing in thiosulfate- and ammo-nia-based media to the total heterotrophs were higher in the waters than in thesediments. These results suggest that the culturable bacterial community in thebottom-waters was nutritionally versatile. Izena hydrothermal activities seemedto have a great influence in concentrations and compositions of particulatematerials and in biomass and compositions of microbial community in the vicinityof this aphotic deep-sea environment.

1. IntroductionVarious geothermally modified compounds and ions, such as H2S, CH4, H2, NH3, CO2, Fe2+,

and Mn2+ are released from deep-sea hydrothermal vents (Corliss et al., 1979; Lilley et al., 1982;Chase et al., 1985; Karl et al., 1988). Some of these compounds are utilized by microorganismsas primary energy sources. However, the actual processes to produce organic materials and thecompositions of the microbial community in deep-sea environments are poorly understood.

Active carbon fixation by microorganisms resident in hydrothermal vent plumes has beendemonstrated by Tuttle et al. (1983) and Wirsen et al. (1986). They showed that carbon fixationactivity was enhanced by the addition of thiosulfate and organic compounds at moderatetemperatures under atmospheric pressure, suggesting that sulfur-oxidizing bacteria should be animportant primary producer around hydrothermal vents. Using warm vent water obtained fromLoihi seamount, Karl et al. (1988) found active methane oxidation coupled with uptake of radio-

354 A. Maruyama et al.

labeled nucleotides. Abundance of heterotrophic bacteria in plume waters was examined byNaganuma et al. (1989). Thermophilic sulfur-reducing bacteria (Jannasch et al., 1988), ther-mophilic methanogens (Huber et al., 1989), and manganese-oxidizing bacteria (Ehrlich, 1983)have also been isolated from vent regions. These results suggest that the microbial communityin hydrothermal environments is rather complex; consisting not only of sulfur-oxidizingbactertia, but also of other chemoautotrophs and heterotrophs.

Microbial biomass in the vent fluids has been assessed by measuring particulate ATP,lipopolysaccharide and cell abundance of microorganisms (Karl et al., 1980, 1988; Winn et al.,1986; Mita et al., 1988). These studies demonstrated larger microbial biomass in vent plumewaters than in ambient waters. Anomaly in concentration of total particulate materials has alsobeen detected around hydrothermal vents (Comita et al., 1984; Baker et al., 1985). However, thecorrelation between microbial biomass and particulate materials is poorly understood inhydrothermal environments. In vent plume waters, most of microorganisms may be resident inassociation with large particulate materials (Karl et al., 1980), and these microbe-containingparticles are expected to mediate the metal deposition (Cowen et al., 1986). Since ecosystems indeep-sea hydrothermal environments highly depend on the microbial primary production,studies on microorganisms and particulate materials are expected to give new information inbiological oceanography as well as microbial ecology.

In the present study we attempted to elucidate qualitative and quantitative characters ofparticulate materials, which should associate with the microbial life in seawater, around a newlydiscovered black smoker in the Okinawa trough. Components of particulate materials werecharacterized by microscopic observation and analysis. Microbial biomass plume was estimatedfrom DNA and carbon concentrations. Culturable microbial communities in seawater andsurface sediments were also examined using various media for heterotrophs and chemoautotrophs.

2. Materials and Methods

2.1 SamplingDuring a dive of the Japanese submersible “Shinkai 2000” in June of 1989, a black smoking

vent was discovered at the depth around 1340 m in the Izena basin (Nakamura et al., 1990), whichis located in the mid-Okinawa trough and about 100 km north-west of Okinawa Island (Fig. 1).In the following geochemical study (Sakai et al., 1990), large amounts of CO2, H2S, CH4 and H2

discharges were detected in the immediate smoker fluid (maximum temperature; 320°C).In the GH 89-3 cruise of the R/V Hakurei-maru from July to September, 1989, topography

of north-east part of the Izena basin was surveyed by in situ black-and-white and color videomonitors. The smoker vent position was determined using a transponder system with an accuracyof less than 30 m. Water sampling was carried out at the vent location (Stn. W55 & W60: collectedtwice in the same location) and at the stations located approximately 1 km from the vent (Stns.W56, W57, W58 & W59, W61, W62, and W65; Fig. 1). Sampling positions in the bottom regionwere also monitored by echo-sounding using a pinger (Benthos Inc.) attached to near the top ofwire and corrected by comparison with the bottom topographic map (Kato et al., 1989). Samplingdepths were also corrected by reversing thermometers attached to 5 l Go-Flo samplers (GeneralOceanogr. Co.). The teflon-coated samplers were cleaned by rinsing thoroughly with 70%ethanol. Water samples were collected aseptically in 1 liter glass bottles and immediately storedat 4°C.

Sediment samples were collected with a GSJ-type grab sampler (c.a. 50 × 50 cm) or a core

Particles and Microbes around the Izena Black Smoker 355

sampler (φ15 cm). Surface sediments (upper 1 cm) were subsampled aseptically from thesesamples and kept at 4°C until they were processed. In some of the sediment samples, elementalsulfur granules were found. Most of the surface sediments were brown-colored, suggestingoxidized conditions, though some portions under the surface were white-colored and light blue-colored, apparently a result of geothermal modification.

2.2 Particulate DNA, carbon and sulfurParticulate materials in seawater were collected by vacuum filtration (<200 mm Hg) on

sterile membrane filters (Gelman, Supor-200; pore size, 0.2 µm) or on glass fiber filters (Whatman,GF/F; precombusted overnight at 450°C) and stored at –20°C. Estimation of particulate DNAconcentration in seawater was from the membrane filter samples according to the method of Pauland Myers (1982). Experimental blanks were run to confirm that background fluorescense waslow. Microbial cells were decomposed by intermittent sonication on ice (Tomy UD-201sonicator; probe φ14.5 mm, 20 kHz, 160 W, 2 min.). The sonication condition was determinedby monitoring destruction of Pseudomonas aeruginosa cells under a microscopy. Fluorescenceresulting from binding of solubilized DNA with Hoechst 33258 dye (Sigma) was measured witha Simazu RF-5000 fluorescence spectrometer (excitation: 350 nm, emission: 450 nm; bandwidth: 10 nm). DNA concentration was calibrated using a calf thymus DNA (Type I, Sigma) asa standard. Carbon and sulfur contents were measured by a C/S analyzer (Horiba, EMIA-520).

Fig. 1. Location of sampling stations in the Izena basin. A black smoker is situated at the location of Stn.W55(60).


After drying particulate materials on the GF/F filters, the samples were combusted, and theevolved CO2 and SO2 were quantified by infrared detection. Data obtained by the same mannerswith samples from the Mariana trough (20–21°N, 143–144°E) and the Kaikata seamount(hydrothermal vent region; 26°42′ N, 141°05′ E) during the GH 89-1 cruise of the R/V Hakurei-maru (April–May, 1989) were used as references.

2.3 Optical and electron microscopic observationSeawater samples were fixed immediately after collection with glutaraldehyde (final 2%;

Kanto-kagaku) or formaldehyde (final 2%; Wako) for microscopic observation. After stainingwith 4′6-diamidino-2-phenylindole (final 0.01–0.05 µg/ml; DAPI; Aldrich), microbial cellswere collected on 0.2 µm-pore-sized Nuclepore filters stained with irgalan black (final 0.2%;Ciba-Geigy) and observed with a fluorescence microscope (Nikon, FX-RFLPh), using anexcitation filter at 365 nm and a barrier filter DM400. The microscope was equipped with a NikonFluor 100/Ph4DL lens, which allowed observation of the samples under both fluorescence andphase-contrast conditions. Samples were also stained with alcian blue (final 0.1%; Fluca), whichis known to dye poly-anions as acidic polysaccharides (Ghiorse and Hirsch, 1979), and observedunder the phase-contrast condition. Hitachi X-650 scanning electron microscope (SEM) equippedwith a Kevex micro-X 7000 energy dispersive X-ray fluorescence analyzer (EDX) was used forelemental analysis of particulate materials. Particles collected on Nuclepore filters (0.2, 2 and 10µm) from 10 to 15 ml of the fixed near-vent water samples were coated with carbon, and overa hundred particles were examined. Direct counting of microbial cells was carried out with DAPI(Porter and Feig, 1980), after dispersing aggregates in the samples through mild sonication andvortex mixing.

2.4 Viable counting of microorganismsA portion of the surface sediment sample was added to a glass bottle containing 9 ml of

Kester’s artificial seawater (KSW; Kester et al., 1967) to give total 10 ml volume. The KSWsolution had been autoclaved and kept on ice prior to use. Several hundred ml of water sampleswere aseptically filtered with a Nuclepore filter (0.2 µm) under vacuum (<200 mm Hg), and theconcentrated particles were resuspended in a glass bottle containing 5 ml of sterilized and cooledKSW. After serial dilution, each sample was inoculated on agar plates for colony counting (PC;duplicate) and also in test tubes for enumerating most probable number (MPN; triplicate). Fiveculture media were used (g/l), (1) 1/2 TZ: Polypepton (Daigo), 2.5; Bacto-yeast extract (Difco),0.5; N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (Hepes; Dojin), 4.77 in 90% KSW,at pH 7.5, (2) 1/2 TZMn: supplemented 1 mM MnSO4 to 1/2 TZ, manganese solution was filter-sterilized with Millipore GV filters, (3) 1/100 TZ: 1/50 nutrient strength of 1/2 TZ, (4) NS:Na2S2O3·5H2O, 0.82; NaCl, 25.16; (NH4)2SO4, 0.26; MgCl2·6H2O, 1.02; K2HPO4, 0.034; NaHCO3,0.26; CaCl2·2H2O, 0.33; KCl, 0.74; Hepes, 4.77, 1 ml of SL-8 trace metal mixture (Pfenning,1978) in DW, at pH 7.5, thiosulfate solution was filter-sterilized, (5) N: excluded Na2S2O3·5H2Ofrom NS.

To make agar plates, Purified agar (Difco) was added to the N, NS and 1/100 TZ liquidmedia, and Bacto-agar (Difco) was added to others at the concentration of 1.5% (w/v). Samplesin test tubes were incubated on board in the dark at 20°C, and the agar plates were incubated atboth 20°C and 4°C. The agar plates incubated at 20°C were checked for colonies on the 15th dayand then stored at 4°C. Samples for 4°C-culturable bacteria were kept below 10°C throughoutthe experiment to maintain the growth activity of psychrophilic bacteria (Morita, 1975). Colony


counting was carried out after two months, and the microbial growth in the N and NS liquid mediawas checked for a period of four months and portions of each culture were streaked onto the Nand NS agar media to confirm the growth. Thiosulfate concentration was determined by themethod of Sörbo (1957). Nitrate and nitrite concentrations were measured colorimetrically usingan Alpkem rapid flow analyzer (RFA-300; USA) equiped with an open tublar cadmium reductioncolumn. Bacterial colonies grown on the 1/2 TZMn plates at 20°C were further transfered to thefresh 1/2 TZMn plates. After incubation at 20°C, manganese-oxidizing bacteria were detectedby the use of benzidineacetic acid (Gregory and Staley, 1982).

3. Results

3.1 Particulate materialsConcentrations of particulate DNA (P-DNA), carbon (P-C) and sulfur (P-S) were determined

in the water column from 1000 to 1340 m depth of the vent station: Stn. W55(60) (Fig. 2). P-DNAconcentration increased with depth and the highest value (1.02 µg/l) was obtained in the deepestsample. In contrast, P-C and P-S concentrations were lower in increasing depth at the vent station,33–50 µg C/l and 175–284 µg S/l in the 1330–1340 m layer. Temperature and salinity were about4.0°C and 34.4‰ in the 1330–1340 m layer of the vent station, respectively (Fig. 2); 4.0–4.2°Cand 34.4–34.5‰ in the layer below 1100 m of the surrounding stations.

By plotting the data into a three dimensional Izena topographic map, P-DNA plume whichextended to the west-southwest directions from the vent area was revealed (Fig. 3(A)). P-DNAconcentration higher than 0.9 µg/l was detected in the 1340 m layer of Stn. W55(60) and the 1310m layer of Stn. W58(59), and the concentration higher than 0.7 µg/l was in bottom-waters (c.a.1300–1570 m) of Stns. W57, W58(59), W65 and W56. Showing similar extension to the P-DNAplume, high concentrations of P-C and P-S (>70 µg C/l, >300 µg S/l) distributed in the 1310 mlayer of Stn. W58(59) and in bottom-waters (c.a. 1300–1520 m) of Stns. W57 and W65 (Fig.3(B)). P-C concentration was also high in bottom-waters (c.a. 1190–1220 m) of Stns. W56, W61and W62 and upper-waters (c.a. 880–1030 m) of Stns. W55(60), W56, W61 and W57. In bottom-waters (c.a. 1220–1250 m) of Stns. W55(60) and W61 and upper-waters (c.a. 880–1030 m) of

Fig. 2. Vertical profiles of temperature, salinity and particulate DNA, carbon and sulfur at the vent station(Stn. W55(60)). Data were obtained from W55 samples (�, �) and W60 samples (�, �).


Fig. 3. Distributions of particulate DNA (A) and particulate carbon and sulfur (B) in the Izena bottom-water region. The plume of P-DNA appears to extend to the west-southwest directions from the ventposition (Stn. W55(60)).


Fig. 4. Correlations of carbon with DNA (A) and sulfur (B) in particulate materials obtained fromhydrothermal environments (�: Izena, �: Kaikata), Mariana deep-sea (�: 3700–5200 m) and surface(�: Izena, �: Mariana; 0–50 m).

Fig. 5. Microorganisms (A; fluorescence condition) arrested in large amorphous particles (B; phase-contrast condition) in the plume waters. Microphotographs were taken in the same field after stainedwith both alcian blue and fluorescent DAPI. Bar shows 10 µm.


Stns. W56 and W57, P-S concentration was also higher than the surroundings.Relationship between the concentrations of P-DNA and P-C in all samples from the Izena

basin was shown in Fig. 4(A). The ratio of P-DNA to P-C in the bottom-waters averaged 0.011(SD = 0.008; n = 27), whereas 0.040 (SD = 0.010; n = 5) in the surface waters. In the Kaikatahydrothermal vent samples, P-DNA/P-C ratios were almost in the same level as the Izena ventsamples; mean 0.010 (SD = 0.003; n = 6). The ratios were not so different between the surfaceand the deep waters (3700–5200 m) from the Mariana region; mean 0.023 (SD = 0.006; n = 2)in the surface, 0.019 (SD = 0.009; n = 30) in the deep; because of low P-C level in the deep waters(Fig. 4(A)).

Fluorescence microscopic observation of the DAPI-stainable particles in the Izena bottom-waters showed that small coccoid and rod-shaped microbial cells dominated in poorly-stainedmatrices. The contribution of microbial cells smaller than 1 µm (cell diameter) to the total wasbeyond 95% in the matrices. When the samples on Nuclepore filters were double-stained withDAPI and alcian blue, large blue-colored amorphous particles were revealed by the phase-contrast microscopy (Fig. 5(B)). Under the fluorescence condition, most of microbial assem-blages were observed within the blue amorphous particles (Fig. 5(A)). These microbial matrices(mostly >10 µm) were a major component of particulate materials. Large amounts of the matrix

Fig. 6. SEM photograph and SEM-EDXA spectra of sulfur-rich particles in near-vent waters of Stn.W55(60). Particles (c.a. φ2–6 µm) appears to contain considerable amount of calcium, not only ofsulfur.


compounds probably explain low P-DNA/P-C ratios in the bottom-water samples.Concentrations of P-S in the bottom-waters surrounding the Izena and Kaikata hydrothermal

vents were in the same level, 300–600 µg S/l, and were higher than in the Mariana deep andsurface waters (Fig. 4(B)). By the SEM-EDX analysis, major sulfur-rich particles were specifiedto cristal-formed minerals with high calcium content (Fig. 6), and few elemental sulfur granuleswere found. Iron-containing particles were rarely detected and did not correlate closely withsulfur. Few manganese-rich particles were found in the bottom-water samples of Stn. W55(60).

3.2 Microbial communityTotal microbial cell numbers in Izena bottom-waters were in the order of 108 cells/l (Table

1). In the sample closest to the vent (W55-1338), microbial population was slightly higher thanin the upper (W55-1134) and the southwestern samples (W59). Culturable heterotroph numbersobtained with the 1/2 TZ plate at 20°C (1/2 TZ-PC.20) were three orders of magnitude higher inthe surface sediments (103 cfu/cm3) than those in the surface and bottom-waters (103 to 104

cfu/l) (Fig. 7). Numbers of bacterial colonies in the NS plate at 20°C were in the same order as1/2 TZ-PC.20 in the bottom-waters, while they were over a hundred-fold lower than the 1/2 TZ-PC.20 in the surface sediments.

In the surface water samples, plate counts at 4°C (PC.4) in all media were approximately 10to 40% of PC.20 values. The ratio of PC.4/PC.20 decreased with depth (Fig. 7). In the bottom-water samples, the ratio decreased to below 1%. In the surface sediments, however, the PC.4/PC.20 ratios were high (c.a. 10–100%). A difference in the culturable bacterial componentsbetween the water and the sediment was also seen in the occurrence of manganese-oxidizingbacteria. In the benzidine test, some of the colonies obtained from the surface sediments turnedblue (positive reaction), but no evidence of manganese-oxidizing bacteria was found in culturesfrom any water samples (Fig. 7). In addition, over 90% abundance of small, round, smooth-shaped and orange colony-forming bacteria in the near-vent water (W55-1338 m) was observedin all TZ plates incubated at 20°C (Fig. 7). The same shaped and colored colonies were also foundin low frequency in the 1603 m sample of Stn. W59, but not in any other water or sedimentsamples. Although no colored colony was found in NS plate samples, quite similar shaped and

Station-Depth (m) W55-1338 W55-1134 W59-1603 W59-1308

DC* (×108) 1.7 (±0.8) 1.2 (±0.5) 1.1 (±0.5) 1.4 (±0.6)

Plate count1/2 TZ 6.5 × 104 3.6 × 103 5.0 × 103 1.8 × 104

NS 4.0 × 104 6.0 × 102 3.5 × 103 1.8 × 104

Most probable number1/2 TZ 5.5 × 104 2.3 × 103 2.3 × 104 1.2 × 104

NS 6.0 × 104 1.2 × 103 7.5 × 104 2.3 × 105

N 1.2 × 104 5.5 × 103 1.2 × 104 2.3 × 104

Table 1. Direct and viable counts of bacteria in seawaters obtained around the Izena black smoking vent(cells/liter).

*Data show mean (±SD) for 30 fields (n = 30).


colored colonies appeared in the 1/ 2 TZ replica plate which had been reinoculated from the NSplate sample of W55-1338 m. After purifying these orange-pigmented bacteria with the 1/2 TZplates, however, they did not grow in either NS or N plate, suggesting that certain chemoautotrophshad grown with them in the initial NS plate.

With the ammonia-containing liquid media (N, NS), viable microbial numbers of thebottom-water samples were estimated in the order of 103 to 105 MPN/l, similar to the 1/2 TZ-

Fig. 7. Culturable microbial community in the Izena basin. Relative abundance was shown comparingwith the plate counts with the 1/2 TZ incubated at 20°C. Solid black bars show frequency of 4°C-viablebacteria (<: below 1%, —: not examined). Dotted area represents orange-pigmented bacteriadominated in the near-vent water (W55-1338). Numbers in the 1/2 TZ and 1/2 TZMn rows show viablecounts (×104 cfu/l seawater, ×104 cfu/cm3 sediment) and percentage of manganese-oxidizers, respec-tively. RS: grab sediment samples obtained from the area within a radius of about 1 km from the ventsite, P: core sediment samples from without the area in the basin.


Table 2. Microbial growth, thiosulfate consumption and nitrate production in MPN bottles.

MPN estimates (Table 1). Active thiosulfate oxidation was found in lower dilution bottles of theNS-MPN, while the activity was weak in higher dilution bottles (Table 2). Nitrate arised in theN-MPN bottles during the culture, and the production corresponded to the growth in the N-MPNbottles.

4. DiscussionIn the bottom-water region around the Izena black smoking vent in the Okinawa trough, the

P-DNA plume extended to the west-southwest directions was clearly detected. Microbialbiomass in the plume is assessed to be at least two to three times higher than in ambient seawater,which is almost the same biomass extension level as observed in other vent areas (Karl et al., 1980,1988; Winn et al., 1986; Mita et al., 1988; Straube et al., 1990). Although we could not detectanomaly in temperature or salinity in the present observation, the elevated P-DNA concentra-tions near the black smoker vent suggest that microorganisms in the hydrothermal plumeoriginate from warm or hot temperature environments (Straube et al., 1990). Particulate carbonand sulfur concentrations were also relatively high in the Izena bottom-water region, while theprocess to produce these components remained to be elucidated. The anomaly in P-C concen-tration has been also observed in the East Pacific Rise; 100–200 µg C/l around the vents (Comitaet al., 1984).

The P-DNA/P-C ratios in the non-hydrothermal vent samples were in the range of 0.02 to0.05, while the mean ratio in the vent samples was 0.01. In the previous studies, the P-DNA/P-C ratios in estuary and offshore waters were assessed to be around 0.05 (Paul et al., 1985) andthe ratio of 0.03 was used for calculation of natural bacterial genome size (Fuhrman and Azam,1982). By using the P-DNA/P-C ratios of 0.05 and 0.03, therefore, living carbon concentrationsat the P-DNA level from 0.35 to 1.02 µg/l in the Izena bottom-waters were supposed to be in theranges of 7 to 20 µg/l and 12 to 34 µg/l, respectively. From total cell number (1.1–1.7 × 108

cells/l) and assumptive cell volume (0.1–0.3 µm3), microbial cell carbon was also assessed to be2 to 10 µg/l by using the conversion factor of 0.2 mg C/mm3 (Kogure and Koike, 1987). Con-tributions of the living carbon to the total P-C in the bottom-waters are then assessed to be in the

*Numbers in NS-MPN columns show extent of thiosulfate consumption in the culture, 1: 75–100%,2: 50–74%, 3: 25–49%, 4: 0–24%.

*Numbers in N-MPN columns show nitrate concentration (mg/l) arised in the culture, 1: 0.50<, 2:0.30–0.50, 3: 0.05–0.29, 4: <0.05, —: not tested. Nitrite concentration was below 0.06 mg/l in the samplestested.

*Circle means positive growth.


range of 10 to 46% (mean 20 ± 10%; n = 27). From the above estimation, we assume thatsignificant portions of the P-C components in the Izena bottom-waters (at least 50% of the totalP-C) consist of non-living carbon.

The microscopic observation and analysis showed that the Izena bottom-waters were richin large amorphous particles containing microbial cells. This indicates that most of the nonlivingcarbon components can be attributed to the microbial matrix compounds as mucopolysaccha-rides. Similar microbe-containing large amorphous particles have been observed in seawaterfrom the Galapagos vents (Karl et al., 1980) and the southern Juan de Fuca Ridge (Cowen et al.,1986). Therefore, these particles rich in non-living organic carbon seem to be characteristic inhydrothermal deep-sea waters.

Microbial extracellular production of organic polymers as mucopolysaccharides has beendemonstrated to mediate particle-aggregation in the euphotic zone (Biddanda, 1986; Biddandaand Pomeroy, 1988). Although the process to produce particulate organic carbon in hydrothermalplume waters is not so clear, chemoautotrophic organic production may contribute in the earlyformation process (Seki and Naganuma, 1989). Aggregated organic particles are supposed to bea good nutrient source for filter-feeding animals resident in the hydrothermal environment. In afar-vent plume, furthermore, these microbial extracellular compounds are expected to mediatethe deposition of manganese and iron (Cowen et al., 1986).

In the P-DNA plume water samples (e.g. W55-1338 m, W59-1308 m), active thiosulfateoxidation was found in the lower dilution bottles of the inorganic-based NS-MPN. This resultsuggests that thiosulfate-oxidizing bacteria distributed in the plume water. The N-MPN estimationalso showed that the growth-corresponding nitrate production occurred in the plume watersample. In addition, we have detected relatively high concentration of dissolved ammonia (about30 µg-at N/l) in the bottom-water samples obtained at Stns. W55(60), W61 and W62. Althoughno pure strain of ammonia-oxidizing autotrophic bacteria has been obtained from deep-seahydrothermal environments, the present data suggest that the chemoautotrophic organic productiondepending on the ammonia discharge is possible in the Izena hydrothermal environment, as wellas being suggested in other vent regions (Jannasch and Wirsen, 1981; Lilley et al., 1982).

In the P-DNA plume water samples, the culturable bacterial numbers in the inorganic-basedN and NS media corresponded approximately to those in the polypeptone-containing TZ media.Almost the same result was obtained in the non-plume water samples (e.g. W61; Fig. 7). Theratios of these culturable bacterial numbers to the total microbial cells were lower than 0.1%,corresponding to the ratios in general open sea (Kogure et al., 1979). Although it is uncertaineither the culturable bacteria were mainly in a free-living form or an attached form, as arrestedin the large amorphous particles, the present viable counting and the colony observation revealthat the compositions of culturable microbial community differ depending on habitat.

First, the occurrence of the 4°C-culturable bacteria was extremely low in the bottom-waterregion (PC.4/PC.20 ratios: <1%), although they were common (PC.4/PC.20 ratios: >10%) in thesurface seawater and the surface sediments around the vent (Fig. 7). In the Mariana deep-seawater and sediment samples (3700–5200 m), the PC.4/PC.20 ratios varied 10 to 80% without anycorrelation with habitat (unpublished data). We have also observed high PC.4/PC.20 ratios (10–40%) in samples obtained vertically from surface to 6000 m depth of the Japan Trench (33°20′–34°20′ N, 141–142°E; unpublished data). These ratios were similar to those in the Izena surfaceseawater. We suspect that the occurrence of 4°C and atmospheric pressure-culturable bacteria(psychrophilic and psychrotrophic bacteria) was anomalously low in the Izena bottom-waterregion.


In the second, the number of bacteria grown on the NS agar plates was very low in the surfacesediments around the vent in comparison with the heterotrophic bacterial number (1/2 TZ-PC.20), while it was abundant as the heterotrophs in the water column. Although the NS platesallow the growth of sulfur-oxidizing and ammonia-oxidizing autotrophs as well as oligo-heterotrophs, the present result indicates that the bacterial community in the sediments were richin heterotrophic bacteria.

The third difference was that manganese oxidization was not found in the bacterial coloniesfrom the bottom-water samples, but was frequently observed in those from the surface sediments.This result is consistent with the observations showing that few manganese deposites were foundin association with the microbial aggregates in the SEM-EDX analysis and that the particulatemanganese concentration was very low in the Izena dissolved manganese plume (almost1 mg/l) extended to the southwest direction (Dr. K. Shitashima, personal communication). Inaddition, the orange-pigmented heterotrophic bacteria dominated in the plume water of W55-1338 m and distributed also in the water of W59-1603 m, while they could not be found in anyother water and sediment samples.

These differences in the microbial community among the different habitats seem to originatenot only from the environmental difference between water and sediment, but also from the activehydrothermal discharge in this bottom-water region. Since the Izena basin is located in the middleof the Okinawa back-arc region, it is also possible that the hydrothermal products in other basinsmay be flowing in the Izena bottom-water region. Further studies on the origin and the fate of themicrobe-containing particles and their components will help to elucidate not only carbon-fixingand bio-production processes, but also microbial community characters in the aphotic deep-seahydrothermal environments.

AcknowledgementsWe would like to thank Drs. T. Urabe, Y. Okuda, K. Nakamura and A. Usui, Geological

Survey of Japan; K. Shitashima, Central Research Institute of Electric Power Industry; K.Kamimura, Government Industrial Research Institute, Cyugoku; and the officers and crew of theR/V Hakurei-maru for their help in the cruises. Thanks are also due to Drs. Y. Suwa, NationalInstitute for Resources and Environment; H. Iguchi, Toray Research Center, for technicalsupports and to C. Arnosti, Woods Hole Oceanographic Institution, for her valuable comments.This work was supported by a grant of large-scale project (R&D on Fine Chemicals from MarineOrganisms) from Ministry of International Trade and Industry, Japan.

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