Vertical distributions of sulfate-reducing bacteria and methane-producing archaea quantified by oligonucleotide probe hybridization in the profundal sediment of a mesotrophic lake

Abstract

Vertical distributions of sulfate-reducing bacteria and methane-producing archaea were investigated in the profundal sediment of a freshwater lake using membrane-immobilized small subunit rRNA hybridization with group- and genus-specific oligonucleotide probes. The annual average of the relative abundance of small subunit rRNA hybridized with all probes for sulfate-reducing bacteria to total small subunit rRNA was 2.3% at 0–2 cm and increased with depth up to 22.9% at 8–14 cm where sulfate concentration was less than 10 nmol ml⁻¹ in interstitial water, suggesting that these bacteria may survive on alternative metabolisms. The signal of probe Dsv687 (the family Desulfovibrionaceae and some Geobacteraceae) was the main factor in this increase. The relative abundance of methane-producing archaea to total small subunit rRNA was highest (7.8%) at 8–14 cm, dominated by the order Methanosarcinales. The metabolic rates measured in the sediments demonstrated that the peaks of sulfate reduction and methane production were separated vertically, and were not linked to their small subunit rRNA distributions. Our data indicate that sulfate-reducing bacteria can coexist with methane-producing archaea from 0 to 20 cm in the freshwater lake sediment.

1Introduction

The anaerobic mineralization of organic matter in aquatic sediments is important for the global cycling of carbon and nutrients [1]. Sulfate reduction and methanogenesis are key terminal processes in deeper sediments because other kinds of electron acceptors are scavenged prior to sulfate and carbonate within the thin surface layer. The competitive and cooperative interactions between sulfate-reducing bacteria (SRB) and methane-producing archaea (MPA) have been well studied by chemical and kinetic analyses [2–4]. These interactions are believed to be mainly determined by sulfate, acetate and hydrogen concentrations in natural environments because SRB are advantageous in a high-sulfate environment for kinetic and thermodynamic reasons, while acetate and hydrogen are substrates common to both groups. However, the simultaneous occurrence of sulfate reduction and methanogenesis under non-limiting sulfate conditions [5] and the presence of SRB in sulfate-limited environments [4] have also been reported. Some SRB can use alternative metabolic pathways instead of sulfate reduction; for example, utilization of Fe(III) and nitrate as electron acceptors, disproportionation of inorganic sulfur compounds, and fermentation [6].

Although such metabolic interactions have been studied extensively in sediments, information on the distribution and composition of both microbial communities is still limited. Earlier studies [7–10] carried out by conventional culture-based methods would have provided relatively limited information, because such methods would be highly selective and would represent only a minor fraction of the actual microbial community [11]. Quantitative membrane hybridization of total small subunit (SSU) rRNA extracted from environmental samples provides a reliable estimate of the relative abundance of active populations with good sensitivity. The combined information of metabolic activities and quantitative population structure should provide a better understanding of the relationship between a microbial population and its function in anaerobic ecosystems. The aim of this study is the simultaneous characterization of the SRB and MPA communities by quantitative membrane hybridization to investigate their vertical distributions in the profundal sediment. Sulfate reduction and methane production rates were also measured under in situ conditions to examine the relationship between these activities and their generic community structures.

2Materials and methods

2.1Description of study site and sampling procedures

Lake Biwa is the largest freshwater lake in Japan. Its northern and southern basins have surface areas of 616 km² and 58 km², and mean depths of 43 m and 4 m, respectively. The northern basin is mesotrophic and monomictic, and its maximum depth is 104 m. Sampling was carried out by R/V Hakken-gou 11 times almost every month from May 1998 to July 1999 at a profundal site (35°23.4′N, 136°7.7′E, water depth of 90 m) in northern Lake Biwa. A single large sediment core (12 cm in diameter and 50 cm in length) was collected using a core-sampler (Type MT-2, Rigosha, Tokyo, Japan). Triplicate cores were then retrieved from the core-sampler with three tubes (4.5 cm in diameter and 32.5 cm in length), and were transported to the laboratory in an icebox. The cores were sliced at 0–2-cm and 3-cm intervals below 2 cm down to 20 cm depth. The dissolved oxygen and pH of bottom water were measured in the field with a CWR Fine-scale Profiler (Center for Water Research, The University of Western Australia, Crawley, Australia).

2.2Measurements of some characteristics of sediment samples

Redox potential in the sediment sample was measured with an ORP meter (pH/ion meter 225, Iwaki Glass, Tokyo, Japan). Ignition loss was measured after heating at 550°C for 2 h. Methane in sediments was determined by headspace analysis [12] modified by purging methane into a headspace in 15-ml vials containing 1 ml of sediment and 4 ml of saturated salt water.

Methane concentrations were measured with a gas chromatograph equipped with a flame ionization detector (GC-8A, Shimadzu Inc., Kyoto, Japan) and a Porapak Q column (80/100 mesh, 2 m×3 mm, Waters, Milford, MA, USA). Sulfate concentrations in overlaying and interstitial water were determined with an ion chromatograph (DX-120, column: AS4A, Dionex, Sunnyvale, CA, USA) after filtration with 0.22-μm membrane filters (Millipore Co., Bedford, MA, USA).

Total cell counting was performed on a single sediment sample collected on July 6, 1998, as follows. Subsamples (1.0 ml) were put into 50-ml tubes, and glutaraldehyde (4% final concentration) was added. The total number of microorganisms was determined by counting cells stained with 4′,6-diamino-2-phenylidole (DAPI; 5 μg ml⁻¹ final concentration) on randomly selected 20 fields in duplicate [13].

2.3Measurement of sulfate reduction and methane production rates

The sulfate reduction rates in the sediment were measured in triplicate for each layer by a radio-tracer method at 8°C (in situ temperature in the surface sediment) irrespective of depth [14]. The methane production rates were measured in triplicate at 8°C as described previously [15]. To remove methane in the sediment slurry in a 15-ml vial, degassing and displacement of the headspace with nitrogen gas were repeated five times, then nitrogen gas was introduced into the slurry for 90 s while mixing vigorously. Preliminary experiments showed that increase in methane concentration was negligible during incubation in the sediment slurries added with NaOH solution (final concentration 0.8 M) after the gassing procedure.

2.4RNA extraction

Sediment samples were washed with 120 mM sodium phosphate buffer (pH 8.0) three times to remove extracellular nucleic acids. Two grams (wet weight) of the washed sediments was distributed (0.5 g each aliquot) into four screw-cap tubes together with 0.5 g of glass beads (diameter 0.1 mm: 0.05 mm=1:1, w/w). Subsamples were stored at −80°C after the addition of 0.9 ml of lysing reagent (50 mM Tris–HCl pH 8.0, 0.1 mM ethylenediamine tetraacetic acid (EDTA) pH 8.0, 25% sucrose and 10 mM sodium pyrophosphate) until nucleic acid extraction. RNA extractions from the sediment samples were carried out by the hydroxylapatite (HTP) (Bio-Gel HTP Gel, Bio-Rad Laboratories Ltd., Hercules, CA, USA) spin-column method [16]. DNA in the extracts was digested with DNase I until DNA was not observed in ethidium bromide-stained agarose gel. The concentrations of nucleic acids were measured with a spectrophotometer (GeneSpec III, Hitachi, Tokyo, Japan).

RNA extraction from the pure cultures of reference microbes was carried out by the low-pH, bead-beating method [17]. The microbial strains used here were cultivated as follows. SRB (Desulfobacter latus DSM3381, Desulfovibrio desulfuricans DSM642, Desulfobulbus propionicus DSM2032, Desulfobacterium autotrophicum DSM3382, Desulfococcus multivorans DSM2059) were grown on the sulfide-reduced bicarbonate-buffered defined media described by Widdel and Bak [6]. MPA (Methanosarcina barkeri DSM804, Methanosaeta concilii DSM3671, Methanogenium thermophilum DSM2373, Methanococcus voltae DSM1537, Methanobrevibacter smithii DSM861) were grown on the media prepared according to the procedure on the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) web site. Escherichia coli (IAM1264) and Saccharomyces cerevisiae (IAM4206) were cultured with nutrient broth (Oxoid Ltd., Hants, UK) and malt extract broth (Difco, Detroit, MI, USA), respectively.

2.5Quantitative membrane hybridization and data analysis

Quantitative membrane hybridization was carried out according to the general method described previously [17–19] with the following modification. An aliquot (7.7–59.4 ng) of sample RNA and a dilution series of reference RNA were immobilized in duplicate onto a nylon membrane (Hybond-N⁺ membrane, Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA) using a slot-blotter (Bio-Dot SF, Bio-Rad Laboratories Ltd., Hercules, CA, USA) after denaturation of the RNA by the addition of 2% (v/v) glutaraldehyde. Then RNA was fixed to the membrane by heating at 80°C for 10 min and 50 mJ of ultraviolet (UV) exposure. The concentrations of all RNA samples and reference RNA were normalized with the universal probe (Univ1390) [19] and the dilution series of E. coli RNA. A concentration of E. coli RNA for normalization was predetermined with a spectrophotometer.

Three general eukarya-, bacteria-, and archaea-specific oligonucleotide probes (Euca502, Bact338 and Arch915) were used along with the dissimilatory, mesophilic, Gram-negative SRB group-specific probes (Dsb129, Dsv687, Dsbb660, Dsbm221 and Dscoc814) described by Devereux et al. [20] and the MPA group-specific probes (Msar821, Msae825, Msar860, Mmic1200, Mcoc1109 and Mbac310) described by Raskin et al. [18]. All oligonucleotide probes were synthesized by the Invitrogen Co. (Carlsbad, CA, USA), end-labeled with [γ-³²P]ATP (ICN Biomedicals, Costa Mesa, CA, USA), and purified using columns (NucTrap Probe Purification Columns, Stratagene, La Jolla, CA, USA) according to the manufacturer's protocol.

For the universal probe, 1 μl of RNA samples from each layer was applied to each slot. For the group-specific probes, RNA samples extracted from the layers below 2 cm were combined every two layers because the amount of SSU rRNA was not sufficient for over 15 hybridizations. The RNA samples were prehybridized at 40°C and washed at the washing temperature given for each probe. The hybridization signals were measured using a phosphorimager (Bio-Imaging Analyzer BAS2000, FUJIX, Tokyo, Japan). The signal intensity of each sample was the average of the duplicate signals. The total SSU rRNA amount was calculated by summing up signals of three domain-specific probes since the total signal occasionally exceeded the signal by the universal probe as described by Alm et al. [21]. The universal probe was used when a vertical profile of the total yield of RNA abundance was determined (Fig. 1D).

Figure 1. Vertical profiles of ignition loss (A), oxidation reduction potential (ORP) (B), total bacterial count (C), and total yield of SSU rRNA amounts (D). Ignition loss and total yield of SSU rRNA were represented by the average of 11 measurements throughout the sampling period. Horizontal bars indicate the standard errors (n=11). ORP and total bacterial counts were measured on the single sample obtained on 6.7.1999. ORP was derived from a single measurement. Total cells were counted on randomly selected 20 fields in duplicate. Horizontal bars indicate spread of duplicates.

3Results

3.1Properties of bottom water and sediment

The temperature of the surface sediment was constant at 8°C all year. The pH of bottom water was also constant at 7.0. The thermocline was recognized at a depth of 15–25 m from May in 1998 to January in 1999 during the sampling period. Circulation occurred between February and March in 1999. The minimal dissolved oxygen (3.4 mg l⁻¹) in the bottom water was recorded in the survey on September 30, 1998.

Surface sediment had a brownish color, indicating the presence of oxidized compounds, such as ferrous oxide and manganese oxide, but the color of the sediment was black below 5 cm. A dense mat of trichomes identified as Thioploca spp. was observed on the surface sediment throughout the year [22]. Vertical profiles of some properties of the sediment samples are shown in Fig. 1. All profiles showed decreases with depth. In particular, the redox potential and total bacterial count markedly declined between 0–2 and 2–5 cm, and were relatively stable at deeper layers below 2 cm. The ignition loss steadily decreased from 13.8 to 8.4% with increasing depth. The water content of the sediment also decreased from 88.5 to 70.2% (data not shown).

3.2Vertical profiles of sulfate and methane concentrations and measurements of microbial activity

Average sulfate concentrations in the interstitial water fell steeply from 108.7±14.4 to 31.5±6.6 nmol ml⁻¹ (means±standard errors, n=9) between 0–2 and 2–5 cm, and were stable at about 10 nmol ml⁻¹ at greater depths (Fig. 2A). Sulfate concentrations of the interstitial water at 0–2 cm sometimes exceeded those of the bottom water (around 115 nmol ml⁻¹). Average sulfate reduction rates (SRR) were 11.1±4.6 and 13.1±3.1 nmol ml⁻¹ day⁻¹ (mean±standard errors, n=9) at 0–2 and 2–5 cm, respectively, and decreased gradually below 5 cm. SRR fell under the detection limit below 14 cm depth.

Figure 2. Vertical profiles of sulfate and methane concentrations (A), and measurements of sulfate reduction and methane production rates (B). Data were the mean values of measurements throughout the sampling period. Horizontal bars indicate the standard errors derived from nine (sulfate concentration and SRR) or 11 (methane concentration and MPR) mean values calculated individually on each date. Measurements of sulfate concentration and SRR were not performed on 30.9.1998 and 1.12.1998 samples. Sampling dates are indicated in the legend of Table 1.

Methane concentrations in the sediment increased with depth. The average concentration at 17–20 cm was 10 times greater than that at 0–2 cm (Fig. 2B). The methane production rate (MPR) was negligible at 0–2 cm during the sampling period. Below 0–2 cm it increased drastically until 5–8 cm and then gradually until 17–20 cm.

3.3Depth profiles of SSU rRNA abundance and domain composition

Total SSU rRNA was highest (10 912±1514 ng ml⁻¹ mean±standard error, n=11) at 0–2 cm, decreased steeply with depth, and was relatively stable below 11–14 cm (Fig. 1D). Table 1 shows the vertical distribution of the relative three domain SSU rRNA. The relative bacterial SSU rRNA gradually decreased from 92.4% at 0–2 cm to 80.6% at 8–14 cm, and was stable at approximately 80% in the deeper layers. The relative archaeal SSU rRNA gradually increased from 2.6% at 0–2 cm to 11.7% at 8–14 cm, and was constant at approximately 12% below 8–14 cm. The relative eukaryal SSU rRNA slightly increased with depth from 5.0% at 0–2 cm to 8.0% at 14–20 cm.

Table 1.  Vertical profiles of relative abundance of bacteria, archaea, and eukarya to total SSU rRNA

Values are the means throughout the experimental period. Sampling was carried out 11 times (6 May 1998 (6.5.98), 9.7.98, 10.8.98, 1.9.98, 30.9.98, 27.10.98, 1.12.98, 28.1.99, 18.3.99, 6.5.99 and 6.7.99). Numbers in parentheses represent standard errors (n=11). ^aThe total SSU rRNA amount was calculated by summing up the signals of three domain-specific probes.
Layer (cm)	Relative abundance (% of total SSU rRNA)^a
	Eukarya	Bacteria	Archaea
0–2	5.0 (0.5)	92.4 (0.4)	2.6 (0.2)
2–8	5.2 (0.6)	89.3 (1.0)	5.5 (0.5)
8–14	7.8 (1.0)	80.6 (1.5)	11.7 (0.8)
14–20	8.0 (1.1)	79.8 (1.6)	12.1 (0.6)

3.4Depth profiles of SRB and MPA community structures

The vertical profiles of SSU rRNA in individual groups of SRB and MPA are shown in Table 2A and B, respectively. The dominant SRB SSU rRNA was of the microorganisms detected by probe Dsv687. The high proportion occurred at 8–14 and 14–20 cm, where its SSU rRNA amounts were 278 and 127 ng ml⁻¹, accounting for 19.5 and 17.2% of total SSU rRNA, respectively. Desulfobulbus (Dsbb660) was detected at a constant ratio (approximately 1% of total SSU rRNA) throughout the vertical profile. Its SSU rRNA amount decreased from 97 to 6 ng ml⁻¹ with depth. Desulfococcus group (Dscoc814) was not detected at 0–2 cm but increased with depth to 2.2% at 8–14 cm. The sum of SSU rRNA hybridized with SRB-targeting probes used in this study accounted for 2.3 and 5.1% of total SSU rRNA at 0–2 and 2–8 cm, respectively. However, the proportion rose to approximately 20% in the layers below 8 cm. SSU rRNA hybridized with probe Dsv687 was the main factor in this increase. Desulfobacter (Dsb129) was not detected (the detection limit varied from 0.1 to 1.0%) in any case.

Table 2.  Vertical profiles of SSU rRNA composition of sulfate-reducing bacteria (A) and methane-producing archaea (B)

Values are the means throughout the experimental period. Numbers in parentheses represent standard errors (n=9 (A), n=11 (B)). Errors were derived from nine (A) or 11 (B) mean values calculated individually on each date. Hybridizations for SRB were not performed on 30.9.98 and 1.12.98 samples. Sums for MPA detected were determined by adding up the results obtained with probe Mmic1200 and Msar860. The signals of probe Dsb129, Mcoc1109, and Mbac310 were under the detection limit.
Layer (cm)	Desulfovibrionaceae (Dsv687)		Desulfobulbus (Dsbb660)		Desulfobacterium (Dsbm221)		Desulfococcus group (Dscoc814)		Sum for SRB detected
	RNA conc. (ng ml^−1)	% of total RNA	RNA conc. (ng ml^−1)	% of total RNA	RNA conc. (ng ml^−1)	% of total RNA	RNA conc. (ng ml^−1)	% of total RNA	RNA conc. (ng ml^−1)	% of total RNA
A
0–2	149	1.4 (0.1)	97	0.9 (0.1)	1	0	0	0	247	2.3
2–8	137	2.6 (1.7)	78	1.5 (0.3)	11	0.2 (0.1)	46	0.9 (0.7)	271	5.1
8–14	278	19.5 (0.5)	12	0.9 (0.1)	5	0.4 (0.4)	32	2.2 (0.1)	327	22.9
14–20	127	17.2 (1.4)	6	0.8 (0.1)	0	0	15	2.0 (0.3)	147	20.0
Layer (cm)	Methanosaetaceae (Msae825)		Methanosarcina (Msar821)		Methanosarcinales (Msar860)		Methanomicrobiales (Mmic1200)		Sum for MPA detected
	RNA conc. (ng ml^−1)	% of total RNA	RNA conc. (ng ml^−1)	% of total RNA	RNA conc. (ng ml^−1)	% of total RNA	RNA conc. (ng ml^−1)	% of total RNA	RNA conc. (ng ml^−1)	% of total RNA
B
0–2	23	0.2 (0.0)	0	0	76	0.7 (0.1)	34	0.3 (0.1)	111	1.0
2–8	42	0.8 (0.1)	4	0.1 (0.0)	115	2.2 (0.3)	37	0.7 (0.1)	152	2.9
8–14	23	1.6 (0.2)	0	0	80	5.6 (0.5)	33	2.3 (0.2)	112	7.8
14–20	10	1.3 (0.1)	0	0	38	5.2 (0.6)	9	1.3 (0.2)	48	6.5

The dominant SSU rRNA of methane-producing archaea was of the order Methanosarcinales, including Methanosaeta and Methanosarcina (aceticlastic methanogen), detected by probe Msar860 (Table 2B). SSU rRNA of the family Methanosaetaceae (Msae825) was 10–42 ng ml⁻¹ accounting for about 30% of the order Methanosarcinales (Msar860) throughout the layers, and Methanosarcina (Msar821) was almost negligible through the year and layers. The sum of signals of the family Methanosaetaceae (Msae825) and Methanosarcina (Msar821) did not reach the signal level of the order Methanosarcinales (Msar860). The order Methanomicrobiales (Mmic1200) (hydrogenotrophic methanogen) peaked at 8–14 cm, accounting for 2.3±0.2% of total SSU rRNA. There was a peak in SSU rRNA amount of methanogen (sum of Methanosarcinales and Methanomicrobiales) at 2–8 cm. The signals by probes Mcoc1109 and Mbac310 were not detected in any occasion.

4Discussion

4.1SRB and MPA community structures revealed by membrane hybridization

The average SSU rRNA index of total SRB detected by all SRB probes used in this study was 2.3–5.1 and 20.0–22.9% of total SSU rRNA in the upper layers (0–2 and 2–8 cm) and the lower layers (8–14 and 14–20 cm), respectively (Table 2A). The values in the upper layers are comparable to those reported for other freshwater sediments. For instance, Gram-negative mesophilic SRB detected by probe SRB385 was 4.2% of all SSU rRNA detected by probe Univ1400 in the surface sediment (0–3 cm) in Lake Kizaki [23], and the combined SRB SSU rRNA was 1.6% of bacterial SSU rRNA in the freshwater site of the River Tama estuary [24].

In the upper sediment layer of Lake Biwa where sulfate reduction was relatively active (Fig. 2B), microorganisms detected by probe Dsv687 were most dominant in SRB SSU rRNA, followed by Desulfobulbus (Dbb660). Although the results were similar to those for freshwater lake sediments examined by the culture-dependent method [10,25], Desulfobulbus was also revealed to be predominant in freshwater sediments by the membrane hybridization method [23,24]. In any event, these results indicate that the incompletely oxidizing SRB contribute mainly to sulfate-reducing activity, resulting in the production of acetate in freshwater sediments. Maeda and Kawai [26] reported that acetate in the northern Lake Biwa sediment was predominant at all depths and had accumulated with the concomitant production of sulfides after the addition of various organic substrates.

The abundance of SSU rRNA hybridized to probe Dsv687 increased greatly at deep layers where no sulfate reduction was observed (Table 2A, Fig. 2B). A similar result has been reported by Sham et al. [27] for the Antarctic marine sediment in which the target microorganisms of this probe increased with depth to 36.0% of the bacterial SSU rRNA in the 25–28-cm layer. However, they could not detect Desulfovibrionaceae in the sediment either by MPN culture or a clone library [28]. This probe hybridizes to some species of Geobacteraceae in addition to Desulfovibrionaceae[27,29]. The clones hybridized to the probe in their clone library were mostly related to Desulfuromonas palmitatis[28]. All those clones, however, contained one mismatch, yet yielded a positive signal by the probe [28]. Moreover, we could not obtain the sequences from Desulfovibrionaceae in the deep sediment of Lake Biwa by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of reverse transcribed products from rRNA (Koizumi, unpublished data). Thus, the most abundant microorganism hybridized to this probe would not actually be SRB in the deep sediment. Further phylogenetic analysis such as by clone library will be needed to examine what kind of microorganisms were detected with probe Dsv687. However, other SRB SSU rRNAs (Desulfobulbus, Desulfobacterium and Desulfococcus) were still detected in the deep layers (Table 2A). These probes are highly specific as long as their specificities were evaluated by Probe_Match [30] and BLAST [31]. Desulfobulbus and Desulfobacterium can survive by using alternative electron acceptors such as nitrate and Fe(III) or by fermentation under low-sulfate conditions [6]. These results suggest that some kinds of SRB survive without sulfate in the deep sediments of freshwater lakes.

The sum of methanogens detected in this study (Msar860 and Mmic1200) accounted for approximately 40–70% of Archaea quantified by probe Arch915 (Table 2B). Archaea consists of three kingdoms, called Euryarchaeota, Crenarchaeota, and Korarchaeota[32]. The distributions of Euryarchaeota other than mesophilic MPA and Korarchaeota are limited to only extremely salty or high-temperature environments [32,33]. Therefore, the remaining 30–60% of Archaea might be Crenarchaeota, since the wide diversity of this group in non-thermal freshwater environments has been reported in several studies [34–36].

SSU rRNA of the family Methanosaetaceae and the order Methanomicrobiales were the most abundant MPA among total archaeal SSU rRNA, accounting for 8.1–14.5 and 10.1–19.8% (20.5–27.6 and 18.8–30.6% of total MPA SSU rRNA), respectively. Falz et al. [37] detected two major clusters related to M. concilii and to a protozoan endosymbiont (the order Methanomicrobiales) in an archaeal clone library from the sediment of Rotsee (Switzerland). They also showed that Methanosaeta spp. represented on average 91% of the archaeal population by fluorescent in situ hybridization (FISH) using newly designed probes targeting the above clusters. Go et al. [38] also obtained 11 clones affiliated with Methanosaeta spp. along with 26 clones clustered around the order Methanomicrobiales from 40 archaeal SSU rDNA clones from the sediment of Lake Soyang (South Korea). The dominance of Methanosaeta was also consistent with a report on rice paddy soil [39]. Methanosarcina was hardly detected throughout the layers in the present study, in accordance with the above reports. Most likely Methanosarcina is outcompeted by Methanosaeta in low acetate concentration environments since the acetate K_m value estimated for Methanosarcina (barkeri) (about 5 mM) [40] is much higher than that for Methanosaeta (0.5 mM) [41]. On the other hand, in Lake Michigan the dominant MPA were the order Methanomicrobiales and the family Methanococcaceae, whereas the order Methanosarcinales was less than 0.25% of total SSU rRNA throughout the sediment [21].

The sum of the two aceticlastic populations targeted with probe Msar821 and Msae825 did not reach the level of Methanosarcinales detected by probe Msar860 (Table 2B). There are non-aceticlastic genera in Methanosarcinaceae (e.g. Methanococcoides, Methanolobus, and Methanohalophilus), which utilize methanol, methylamines, and other C-1 compounds. However, these genera have been isolated so far exclusively from saline environments [42]. Of six clones related to M. concilii obtained from the lake sediment by Falz et al. [37], four contained one mismatch to probe Msae825, but were perfectly matched to probe Msar860. A similar analysis of Methanosaeta-related clones obtained from other sediments also supports this observation [38,39,43]. Thus, Methanosaeta spp. with SSU rRNA containing few mismatches to probe Msae825 might dominate in freshwater sediments.

Although 11 probes specific for five SRB and six MPA were used for comprehensive understanding of the depth-related community structure changes of SRB and MPA, many more specific and selective probes are being developed based on the extensive SSU rRNA data bank. There is still possibility of existence of other kinds of SRB and MPA that could not be recognized by the probes used in this study. In order to choose probes for further species-specific analysis and to design new probes for uncultured groups, phylogenetic analysis (e.g. clone library) is needed.

4.2Specific cellular sulfate reduction and methane production rates

Vertical distribution patterns of SSU rRNA of SRB and MPA were different from those of SRR and MPR, although in general cellular rRNA content positively correlates with growth rate [44]. The relationship between rRNA content and the cellular metabolic rate (e.g. SRR and MPR) may differ according to the bacterial species and growth conditions. Moreover, SSU rRNA recovery from sediment may not be constant. However, cellular SRR and MPR were roughly estimated in order to compare other studies and evaluate growth conditions. This estimation was conducted on the assumption that all microorganisms have the same abundance of rRNA. Cell numbers of SRB and MPA were estimated from the relative contribution of SRB or MPA rRNA to the total prokaryotic rRNA (Bact338 and Arch915) and direct cell counts derived from the sample of July 6, 1999. The cellular SRR was between 0.84 and 0.01 fmol cell⁻¹ day⁻¹, and decreased with depth (Fig. 3). The low value in the deep layer might be due to an overestimation of the Desulfovibrio proportion by probe Dsv687 as described above. These rates were comparable to the previous reports (0.01–0.09 fmol cell⁻¹ day⁻¹ (0–8.5 cm depth) by Sahm et al. [45], and 0.03–0.14 fmol cell⁻¹ day⁻¹ (0–10 cm depth) by Ravenchlag et al. [46] determined with the similar method.

Figure 3. Depth profiles of the apparent specific cellular SRR and MPR. SRR and MPR derived from the single sample of 6.7.1999 were used for calculation. Cell numbers of SRB and MPA were roughly estimated from the data of direct cell counts and the SSU rRNA index on 6.7.1999. The SSU rRNA indices for SRB and MPA were the summation of ratios of specific SRB probe signals (Dsv687, Dsbb660, Dsbm221 and Dscoc814) and MPA probe signals (Msar860 and Mmic1200) to the total of prokaryote target probe signals (Bact338 and Arch915), respectively.

The cellular MPR was negligible at 0–2 cm but relatively constant at about 0.2 fmol cell⁻¹ day⁻¹ below 2 cm. These rates are much lower than the previous data [47] obtained from pure culture experiments, manure digestor, bovine rumen, and salt marsh sediment based on MPN enumeration (480–960, 24–72, 384, and 2.4–7.2 fmol cell⁻¹ day⁻¹, respectively). The apparent cellular MPR of the pure culture and in bovine rumen were approximately 10 000 times higher than the rate in the present freshwater sediment because these rates were obtained under nearly optimum conditions, with abundant substrates and optimum temperature (30–37°C). In addition, culture-dependent cell counts seem to underestimate MPA cell numbers. The accumulation of such data would be important in estimating the number and contribution of SRB and MPA in natural environments by culture-independent methods like SSU rRNA membrane hybridization.

Acknowledgements

We wish to thank the entire crew of the R/V Hakkengou, Lake Biwa Research Institute, for their valuable help in sampling, and Dr. M. Fukui and Mr. H. Kojima for their helpful suggestions and comments. This work was partly supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Sumitomo Foundation to S.T.