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Applied and Environmental Microbiology, June 2003, p. 3181-3191, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3181-3191.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Analysis of the Sulfate-Reducing Bacterial and Methanogenic Archaeal Populations in Contrasting Antarctic Sediments

K. J. Purdy,1* D. B. Nedwell,1 and T. M. Embley2

Department of Biological Sciences, University of Essex, Colchester, Essex CO4 3SQ,1 Molecular Biology Unit, Department of Zoology, Natural History Museum, London SW7 5BD, United Kingdom2

Received 31 December 2002/ Accepted 24 March 2003


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ABSTRACT
 
The distribution and activity of communities of sulfate-reducing bacteria (SRB) and methanogenic archaea in two contrasting Antarctic sediments were investigated. Methanogenesis dominated in freshwater Lake Heywood, while sulfate reduction dominated in marine Shallow Bay. Slurry experiments indicated that 90% of the methanogenesis in Lake Heywood was acetoclastic. This finding was supported by the limited diversity of clones detected in a Lake Heywood archaeal clone library, in which most clones were closely related to the obligate acetate-utilizing Methanosaeta concilii. The Shallow Bay archaeal clone library contained clones related to the C1-utilizing Methanolobus and Methanococcoides and the H2-utilizing Methanogenium. Oligonucleotide probing of RNA extracted directly from sediment indicated that archaea represented 34% of the total prokaryotic signal in Lake Heywood and that Methanosaeta was a major component (13.2%) of this signal. Archaea represented only 0.2% of the total prokaryotic signal in RNA extracted from Shallow Bay sediments. In the Shallow Bay bacterial clone library, 10.3% of the clones were SRB-like, related to Desulfotalea/Desulforhopalus, Desulfofaba, Desulfosarcina, and Desulfobacter as well as to the sulfur and metal oxidizers comprising the Desulfuromonas cluster. Oligonucleotide probes for specific SRB clusters indicated that SRB represented 14.7% of the total prokaryotic signal, with Desulfotalea/Desulforhopalus being the dominant SRB group (10.7% of the total prokaryotic signal) in the Shallow Bay sediments; these results support previous results obtained for Arctic sediments. Methanosaeta and Desulfotalea/Desulforhopalus appear to be important in Lake Heywood and Shallow Bay, respectively, and may be globally important in permanently low-temperature sediments.


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INTRODUCTION
 
Investigating the ecology of bacteria and archaea is vital to understanding the functioning of global biogeochemical cycles. Key questions include the identity of dominant members of microbial communities and the determination of their different roles. Sulfate-reducing bacteria (SRB) and methanogenic archaea (MA) are important terminal oxidizers in the anaerobic mineralization of organic matter and can be seen as ecological equivalents, mineralizing organic matter to CO2 or to CO2 and CH4 in, respectively, high-sulfate and low-sulfate environments (70). SRB are thought to outcompete MA when sulfate is freely available (e.g., in marine sediments), as cultured strains have higher specific affinities for the main substrates, acetate and H2, used by both groups (4, 30, 33). However, even in marine sediments, methanogenesis still occurs, primarily via C1 compounds, such as methylamines, which SRB cannot use (69). In freshwater environments, where available sulfate is usually limited, sulfate reduction does still occur, but methanogenesis dominates (34, 64). Sulfate reduction accounts for up to 50% of organic matter degradation in coastal marine sediments, and similar proportions have been shown for methanogenesis in freshwater systems (26, 33). However, much of the understanding of the dynamics of sulfate reduction and methanogenesis in sediments is based on process measurements and inferences from pure-culture studies. Little is known of the identities of the key players in situ or the environmental factors that affect their activity and distribution.

The Southern Ocean is a region of high marine productivity, estimated at 16 g of C m-2 year-1 in the open ocean (16) and up to 300 g of C m-2 year-1 in coastal regions (67). In addition, the Antarctic region is one of the least human-impacted environments and provides a model for other low-temperature systems, such as deep-sea sediments. In keeping with the high rates of productivity in these permanently low-temperature sediments, the rates of sulfate reduction and methanogenesis are as high as those in temperate systems (42, 55). However, there is evidence to suggest that in situ temperature may affect carbon flow through these processes. Low temperatures apparently inhibit hydrogenotrophic MA and shift sedimentary metabolism toward acetogenesis and acetoclastic methanogenesis (43, 59). Although studies on temperate sediments have suggested that temperature has a substantial effect on sulfate reduction rates (1), it is not clear whether temperature can cause the kind of change in carbon flow that is seen in methanogenesis.

Most of the studies on the molecular ecology of microorganisms in Antarctica have focused either on the ice-covered lakes of the Antarctic Oases (8, 20) or on the microbial communities that inhabit sea ice (see Staley and Gosink [61]and references therein). These studies have indicated that Antarctic microbial communities are very diverse and that some distinctions appear to exist between Antarctic and low-latitude microbial communities (19). In addition, molecular studies on Arctic marine sediments have also suggested that the bacterial communities are very diverse and that SRB-related organisms are extremely important components of the communities (53, 54, 56). In this study, we used traditional process measurements, sediment slurry microcosms, and molecular techniques to try to identify the important SRB and MA in contrasting Antarctic sediments and to begin to investigate their functional roles in the environment.


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MATERIALS AND METHODS
 
Description of study sites and sediment sampling.
Two contrasting sites (Fig. 1), a freshwater lake (Lake Heywood) and a coastal marine site (Shallow Bay) on Signy Island, South Orkney Islands, Antarctica (60°43'S, 45°36'W), were chosen and sampled in the austral summer of 1997-1998. Lake Heywood is a mesotrophic-eutrophic freshwater lake that is ice covered for most of the year (6). When ice covered, the water column becomes stratified, with an anoxic monolimnion. After the ice melts in the austral spring, the water column becomes mixed and oxygenated, while the sediment remains anoxic to within a few millimeters of the surface. Lake Heywood was sampled at the deep spot of the lake (approximately 7 m) by using a gravity corer and yielded cores at least 15 cm long. The sediment was very flocculent, with a high water content (82% [wt/wt]). Shallow Bay is a coastal marine site that is frozen during the winter but generally ice free during the summer. Sediment cores taken from Shallow Bay were no more than 10 cm long and were very consolidated, with a low water content (28% [wt/wt]). For in situ process measurements, sediment cores were subsampled by using 5-cm-long minicores (5-ml syringes with the Luer end cut off) in oxygen-free nitrogen (OFN)-flushed gas bags. In addition, sediment from 5-cm depth horizons (0- to 5-, 5- to 10-, and 10- to 15-cm horizons) was taken for nucleic acid extraction and sediment from the top 0 to 5 cm was taken for sediment slurry microcosms.



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  		FIG. 1. Map of Signy Island, South Orkney Islands, Antarctica (60°43'S, 45°36'W), showing the positions of the two sampling sites, Lake Heywood and Shallow Bay, and the British Antarctic Survey research base.

In situ rates of sulfate reduction and methanogenesis.
Rates of sulfate reduction in 5-cm-long minicore subsamples (5-ml syringes with the Luer end cut off and sealed with Suba-Seals) taken from a larger core sample were determined by using a [35S]sulfate radiotracer (25). Five replicate subcores were taken for each 5-cm depth profile (over 0 to 15 cm for Lake Heywood cores and 0 to 10 cm for Shallow Bay cores), injected with approximately 0.2 µCi of carrier-free [35S]sulfate (specific activity, 3.7 GBq mmol-1; Amersham Biosciences, Little Chalfont, United Kingdom), and incubated at in situ temperatures (4 to 6°C) for 8 to 24 h. Lake Heywood sediment temperatures vary between 4 and 6°C over the summer (6), while the sediments in Shallow Bay are warmed by solar radiation such that in situ temperatures can reach 6°C, significantly higher than normal Antarctic seawater temperatures of -1.5 to 0.5°C (42). These samples were then fixed with 10 ml of 5% (wt/vol) zinc acetate and stored at -20°C until return to the United Kingdom, where radioactivity in tin-reducible sulfide was determined (41). Pore water was collected by centrifugation of sediments at 6,000 x g, and sulfate concentrations were determined by ion-exchange chromatography (Series Qic; Dionex Corp., Sunnyvale, Calif.).

Methanogenesis was determined by headspace accumulation of methane from subcores extruded, in an OFN-flushed gas bag, into a previously gassed universal bottle; the bottle was sealed with a Suba-Seal and incubated at in situ temperatures (4 to 6°C) for 15 h. Methane was measured by gas-liquid chromatography as described elsewhere (5).

Preparation and sampling of sediment slurry microcosms.
Anaerobic slurries (50% [vol/vol] sediment with site bottom water purged with OFN) of the top 5 cm of sediment from both sites were prepared under anaerobic conditions (50). Triplicate aliquots of the slurry from each site (15 ml in universal bottles sealed with Suba-Seals) were amended with selected substrates and specific inhibitors of key functional groups. The treatments are described in Table 1.


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  		TABLE 1. Treatments used for sediment slurries from two contrasting Antarctic sitesa

The slurries were incubated in the dark at 4 to 6°C and mixed by rotation for 1 h each day. The headspace concentration of methane was measured every 4 days, and a small sample of slurry liquid was taken. Particulates were removed from the liquid samples by filtration (0.45-µm-pore-size filter) and stored at -20°C until analysis for residual sulfate and fatty acids. Sediment samples were taken at time zero and at the end of the experiment (day 12) and centrifuged (3,000 x g for 10 min). The supernatant was filtered and stored as described above. The sediment was washed with 120 mM sodium phosphate (pH 8.0) to remove any extracellular nucleic acid (66) and stored in 4-g subsamples at -20°C until nucleic acid extraction.

Analysis of headspace methane, sulfate, and fatty acid concentrations in slurry sediments.
The headspace methane concentration was measured as described above. The sulfate concentration in slurry samples was determined by ion-exchange chromatography as described above. Volatile fatty acid concentrations were analyzed by gas chromatography after extraction from acidified slurry samples with diethyl ether (4).

Extraction and quantification of nucleic acids from sediment samples and pure-culture controls and preparation of in vitro-transcribed rRNA.
DNA and RNA were extracted separately from triplicate subsamples of the stored sediments by the hydroxyapatite spin column method (48). An amalgamated DNA extract from each site was further purified on a 1x Tris-acetate-EDTA-1.4% agarose gel. Gel slices containing DNA fragments of >5,000 bp were excised from the gel, and the DNA was extracted by using a Qiaex gel extraction kit (Qiagen, Crawley, West Sussex, United Kingdom). Total rRNA was extracted from pure cultures (Table 2) as described previously (50). 16S rRNA controls for Desulfotalea and the methanogens studied in this work (Table 2) were transcribed in vitro from cloned 16S rRNA genes (46).


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  		TABLE 2. 16S rRNA-targeted oligonucleotide probes and pure-culture controls used in probing experiments with RNAs extracted from sediments from two contrasting sites on Signy Island, Antarcticaa

Total RNA extracted from sediment was quantified by UV spectrophotometry. Accurate quantification of the oligonucleotide probe response to total RNA extracted from sediment is dependent on accurate quantification of the amount of 16S rRNA obtained from pure-culture controls. It cannot be assumed that 16S and 23S rRNAs will be present in a 1:1 ratio in pure-culture extracts. Therefore, the 16S rRNA from Escherichia coli (Sigma-Aldrich) was gel purified, and its concentration was determined by UV spectrophotometry. Known volumes of total RNA extracted from pure cultures and in vitro-transcribed RNA along with pure E. coli 16S rRNA standards were run on an ethidium bromide-stained agarose gel. The band intensity of 16S rRNA was measured by using the public domain NIH Image program (developed at the National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/), and the concentrations of the samples were determined by comparison to the E. coli 16S rRNA standard curve.

Amplification and analysis of archaeal environmental 16S rRNA genes.
An archaeal 16S rRNA gene sequence clone library was produced for both sites by using a heminested approach as described previously (39) and the proofreading enzyme Pfu (Stratagene, La Jolla, Calif.). The PCR product from the secondary amplification was gel purified, blunt-end cloned into PCRscript-Amp+ (Stratagene), and transformed into XL-Gold maximum-efficiency competent cells as described by the manufacturer (Stratagene). Clones picked randomly from each library (40 clones from Lake Heywood and 60 from Shallow Bay) were sequenced by using vector-based primer T3 and an ABI automatic sequencer (Sequencing Facility, Natural History Museum, London, United Kingdom). The resulting sequences were aligned with reference taxa and available environmental clones within the Genetics Database Environment (35) and analyzed by using PAUP (63). A neighbor-joining tree was generated based on about 300 nucleotides for both forward and reverse directions, and the clones were grouped. Twenty-three representative clones were chosen (8 from Lake Heywood [5 shown on the tree] and 15 from Shallow Bay [6 shown on the tree]) and fully sequenced. An alignment of the complete sequences, reference taxa, and available environmental clones yielded 940 positions for further analysis. Pairwise distances for all sites that could be aligned were calculated by using the Logdet/paralinear distances method as described previously (49). The Logdet/paralinear distances method assumes that all sites can vary, an assumption which is not true for most data sets. Thus, we estimated the number of variable sites in the alignment by using a two-state (variable-invariable) maximum-likelihood model in PAUP. The subsequent phylogenetic analysis was limited to only variable positions (44% of sites). Bootstrap values (1,000 replicates) were determined by using PAUP. The tree was rooted with Thermoplasma acidophilum as the outgroup.

In order to determine whether crenarchaeotes could be detected in Lake Heywood sediments, crenarchaeote-specific PCR primers (27) were used to amplify sequences from these sediments, and the PCR product was cloned as described above. Twenty randomly selected clones were sequenced and aligned with reference taxa and available environmental clones as described above and analyzed by using PAUP (63). A neighbor-joining tree was generated based on 770 nucleotides.

Amplification and analysis of SRB environmental 16S rRNA genes.
No general primer sets that exclusively target the SRB exist; therefore, a general bacterial clone library was produced with PCR primers Epsilon (an adaptation of 7f; GAGASTTGATCMTGGCTCAG) and 1541R (AAGGAGGTGATCCAGCC) (17) and the proofreading enzyme Pfu. The PCR product was gel purified and cloned as described above. Recombinants from these libraries were picked (594 Lake Heywood and 473 Shallow Bay clones) and patch plated on Luria broth agar plates containing 50 µg of ampicillin liter-1 along with colonies bearing plasmids containing the 16S rRNA genes of known SRB (Table 2). Colony lifts were prepared from these plates by using nylon membranes, and the colonies were lysed, denatured, and fixed to the membranes as described by the manufacturer (Schleicher & Schuell, Dassel, Germany). These clone libraries were then probed with the available array of SRB-targeted oligonucleotide probes (Table 2) under conditions used for the hybridization of oligonucleotides to RNA (50). However, specific wash temperatures were reduced by 4°C to account for the lower thermal stability of DNA-DNA hybrids. Colonies that were found positive were sequenced with the T3 or M13f vector-based primer (20 clones from Lake Heywood and 90 from Shallow Bay). The resulting sequences were aligned with sequences from reference taxa and available environmental clones and analyzed as described above. A neighbor-joining tree was generated based on about 300 nucleotides for both forward and reverse directions, and the clones were grouped. Representative clones were chosen, fully sequenced, and aligned with reference taxa and available environmental clones, yielding 1,139 positions for further analysis. Pairwise distances for all variable sites that could be aligned (61% of sites) were calculated by using the Logdet/paralinear distances method as described above, and bootstrap analysis was performed (1,000 replicates). The tree was rooted with E. coli as the outgroup.

Immobilization of extracted RNA on nylon membranes and hybridization conditions.
RNA extracted from sediment and slurries (either 50 or 500 ng of total RNA/slot) was slot blotted on nylon membranes (Hybond-N; Dupont Ltd., Stevenage, United Kingdom) after denaturation with 2% (wt/vol) glutaraldehyde (50) along with known amounts of rRNA from pure-culture controls and in vitro-transcribed RNA. Oligonucleotide probes (Table 2) were synthesized commercially (MWG Biotech Ltd., Milton Keynes, United Kingdom) and end labeled with 32P ([{gamma}-32P]ATP; NEN-Dupont, Hounslow, Middlesex, United Kingdom) (58). Hybridizations were performed and membranes were processed as described previously (50).

Scanning densitometry of autoradiographs and determination of signal levels.
Autoradiographs were quantified by using laser scanning densitometry (Personal Densitometer; Molecular Dynamics, Sunnyvale, Calif.). Densitometrically measured signals were converted to an amount of rRNA for each sample by comparison to a pure-culture control standard curve. The standardized results were expressed as a percentage of the combined signals of general bacterial and archaeal probes (Table 2). This standardization normalized for variations in the amount of rRNA loaded on the membranes and estimated the size of the rRNA pool for each target group relative to the size of the prokaryotic rRNA pool. It was therefore a composite measure of both the population size (number of cells) and the activity of individual cells, as expressed by intracellular 16S rRNA (50). Data are expressed as a total mean for all cores (two from Lake Heywood and three from Shallow Bay) and all depth horizons (0 to 15 cm in Lake Heywood and 0 to 10 cm in Shallow Bay).

Nucleotide sequence accession numbers.
The sequences determined here for archaeal clones have been deposited in GenBank under accession numbers AY177805 to AY177815. A representative sequence (LHC03) for BPR10 cluster clones has been deposited in GenBank under accession number AY187063. The sequences determined here for SRB-like clones have been deposited in GenBank under accession numbers AY177788 to AY177804.


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RESULTS
 
In situ rates of sulfate reduction and methanogenesis.
Significant rates of sulfate reduction and methanogenesis were detected at both sites (Table 3). Sulfate reduction dominated in the Shallow Bay sediments, and methanogenesis dominated in the Lake Heywood sediments.


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  		TABLE 3. Comparison of rates of methanogenesis and sulfate reduction in two contrasting Antarctic sedimentsa

Sediment slurry microcosm experiments.
Significant differences in headspace methane accumulation occurred in the slurries from both Lake Heywood and Shallow Bay (Fig. 2). In Lake Heywood slurries, the addition of sulfate (SLH) caused a significant decrease in methane production (Fig. 2a) (P1,6 < 0.05; one-way analysis of variance with a post hoc Tukey test [71]), as did the addition of acetate plus chloroform (ACLH) and acetate plus fluoroacetate (AFLH) (P < 0.0005 in each case). The addition of sulfate (SLH) reduced methane production in these slurries to only 63% that in control slurries (CLH). Chloroform addition stopped methane production, while the addition of fluoroacetate, a specific inhibitor of acetate metabolism (10), reduced methane production to only 10% that in the unamended control. In slurries amended with acetate (ALH), acetate plus sulfate (ASLH), and acetate plus molybdate (AMLH), methane production increased compared to that in CLH slurries, but the increases were not significant (P = 0.39 to 0.94). Methane production in ASLH and AMLH slurries was very similar to that in ALH slurries; for simplicity, these data are not shown in Fig. 2a.



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  		FIG. 2. Headspace accumulation of methane in sediment slurries from Lake Heywood (a) (methane production in ASLH and AMLH slurries was very similar to that in ALH slurries, and so these data are not shown) and Shallow Bay (b) (methane production in SSB, ASB, ASSB, and AFSB slurries was very similar to that in control slurries, and so these data are not shown). Symbols: •, control; {circ}, acetate; {square}, sulfate; {triangledown}, acetate plus chloroform; {triangleup}, acetate plus fluoroacetate; {diamond}, acetate plus molybdate. The results are the means for triplicate slurries with each treatment; error bars represent 1 SEM. Note the smaller scale on the y axis of the Shallow Bay plot (b).

In Shallow Bay slurries, methane accumulated, but to concentrations that were 100-fold lower than those attained in Lake Heywood slurries (Fig. 2). Only the addition of the inhibitors chloroform and molybdate (acetate plus chloroform [ACSB] and acetate plus molybdate [AMSB]) had any significant effect on the production of methane in these slurries (Fig. 2b). The addition of ACSB decreased methane production to zero compared to that in control slurries (CSB) (P < 0.05), while the addition of AMSB significantly increased methane production (P < 0.001). Methane production in slurries amended with sulfate (SSB), acetate (ASB), acetate plus sulfate (ASSB), and acetate plus fluoroacetate (AFSB) was not significantly different from that in the unamended control, and so these data are not shown in Fig. 2b.

Sulfate and fatty acid concentrations in both sets of slurry experiments showed little or no change over the period of the experiments, with the exception of the ALH slurries, in which acetate fell from an initial concentration of 13 mM (standard error of the mean [SEM], 1.92; n = 3) to 6.3 mM (SEM, 0.5) by day 12. This slurry also produced the most methane, which accounted for 22% of the acetate consumed. Oligonucleotide probing of RNA extracted from these slurries indicated that no detectable shifts in the SRB or MA community had taken place over the period of the experiments (data not shown).

Phylogenetic analysis and relative abundance of MA in Lake Heywood and Shallow Bay sediments.
The phylogenetic relationships of the archaeal clones from both Lake Heywood and Shallow Bay to reference taxa and other environmental clones are shown in Fig. 3a. There was a distinct lack of diversity in clone libraries, with only three clusters of clones being detected at each site. With a 97% similarity cutoff value (60), clone library coverages were estimated at 97% for Lake Heywood and 100% for Shallow Bay, indicating that the libraries were very well sampled for the diversity that they contained (23, 38). Clone identifications for both sites are given in Table 4.



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  		FIG. 3. Inferred phylogenetic relationships among archaeal environmental sequences, reference taxa, and other environmental clones and relative abundance of MA in RNAs extracted from the two Antarctic sites (EHB, 1MT*, 2MT*, and 2C* are clones from a United Kingdom estuary [39, 49]). (a) Logdet/paralinear distances tree based on 44% (the estimated number of variable sites) of 940 nucleotides that could be aligned. Bootstrap (1,000 replicates) values of >50% are shown. (b) Relative abundance of MA based on hybridization of MA-targeted oligonucleotide probes (Table 2) to rRNAs extracted from sediments from the two sites. Data are expressed relative to the combined signals for general bacterial and archaeal probes. Signal level means were determined after arcsine transformation of the data and then back-transformed to give the results shown (71). Results are the means of 18 extractions; error bars represent 1 SEM. M', Methano; LH, Lake Heywood; SB, Shallow Bay.


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  		TABLE 4. Identification and abundance of methanogen-related clones from general euryarchaeotal clone libraries from both Lake Heywood and Shallow Bay

In order to determine whether crenarchaeotes could be detected in Lake Heywood sediments, crenarchaeote-specific PCR primers (27) were used to amplify sequences. However, sequences amplified by these primers were not crenarchaeotes but were related to a group of environmental sequences and the endosymbiont of the protozoan Plagiopyla nasuta that were not detected in the initial archaeal libraries. These sequences are not included in the MA tree (Fig. 3a), as they were substantially shorter (850 bp) than the fragments from the euryarchaeotal clone library; however, the group in which they clustered, the BP R10 cluster, is shown.

In RNA extracted from Lake Heywood sediments, the archaeal community represented 34% (SEM, 1.7%; n = 18) of the total prokaryotic community (combined signals of general bacterial and archaeal probes). Probes that targeted the MA detected a substantial proportion of the Lake Heywood prokaryotic community (Fig. 3b). The combined signal of the Methanomicrobiales- and Methanosarcinales-targeted probes was 10% (SEM, 0.5%) the signal for the total community (31% [SEM, 1.5%] of the archaeal community). Within these groups, the Methanogenium-targeted probe detected only a small proportion of the prokaryotic community detected by the Methanomicrobiales-targeted probe (<1%), despite the fact that all of the Methanogenium-related clones in the Lake Heywood clone library contained the target site for this probe. The Methanosaeta-targeted probe, which is nested within the general Methanosarcinales-targeted probe, did detect a significant component of the prokaryotic community (4.3% [SEM, 0.2%]; 13.2% of the archaeal community); hence, this component was a significant proportion of the total prokaryotic community in these sediments. The signal from the Methanosaeta-targeted probe was not significantly different from that from the Methanosarcinales-targeted probe (P1,34 = 0.068). Members of the obligate C1 compound-utilizing Methanococcoides were not detected in Lake Heywood sediments by probes or in the clone libraries.

The archaeal community was a much smaller component of the total prokaryotic community (0.2% [SEM, 0.02%]; n = 18) in the marine sediments of Shallow Bay than in Lake Heywood. However, signals were detected from three MA-targeted probes (Fig. 3b): the general Methanomicrobiales-targeted probe, the Methanosarcinales-targeted probe, and the Methanococcoides-targeted probe. Signals from the Methanomicrobiales- and Methanosarcinales-targeted probes accounted for 62% (SEM, 7.4%) of the archaeal signal in these sediments. No signals were detected by the Methanosaeta- and Methanogenium-targeted probes.

Phylogenetic analysis and relative abundance of SRB in Lake Heywood and Shallow Bay sediments.
The Lake Heywood clone library had only two clones that were closely related to any known SRB (Fig. 4a, group 3). The rest of the clones that were positive (usually weakly positive) with the SRB-targeted oligonucleotide probes either were not {delta}-proteobacteria or were very distinct from any known SRB clusters. The {delta}-proteobacterial clones were related to the metal- and sulfur-oxidizing Pelobacter-Geobacter and Desulfuromonas lineages (groups 3 and 4). No clones related to gram-positive Desulfotomaculum, a known freshwater SRB, were detected. For the Shallow Bay clone library, 10.3% of the sampled clones (473 clones) were SRB related and fell within a number of distinct SRB-related clusters (Fig. 4a and Table 5). No clones related to gram-positive Desulfovibrio or Desulfotomaculum were detected.



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  		FIG. 4. Inferred phylogenetic relationships among SRB-related environmental sequences from the two Antarctic sites, reference taxa, and other environmental clones and relative abundance of SRB in RNAs extracted from the two Antarctic sites (Sva clones were from Arctic sediments ([54]). (a) Logdet/paralinear distances tree based on 61% (the estimated number of variable sites) of 1,139 nucleotides that could be aligned. Bootstrap (1,000 replicates) values of >50% are shown. Known psychrophilic and psychrotolerant organisms are underlined. (b) Relative abundance of SRB based on hybridization of SRB-targeted oligonucleotide probes (Table 2) to rRNAs extracted from sediments from the two sites. Data are expressed relative to the combined signals for general bacterial and archaeal probes. Signal level means were determined after arcsine transformation of the data and then back-transformed to give the results shown (71). Results are the means of 18 extractions; error bars represent 1 SEM. D', Desulfo; LH, Lake Heywood; SB, Shallow Bay.


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  		TABLE 5. Identification and abundance of SRB-related clones from general bacterial clone libraries from both Lake Heywood and Shallow Bay

Four probes, those for Desulfobacterium, Desulfotalea/Desulforhopalus, Desulfobulbus, and Desulfobacteriaceae, produced positive responses with RNA extracted from Lake Heywood sediments (Fig. 4b), while a signal from Desulfobacter was not detected. None of the SRB detected represented >0.3% of the total prokaryotic community; in total, the SRB accounted for only 0.9% (SEM, 0.01%; n = 18) of the prokaryotic community.

In Shallow Bay sediments, the SRB represented a substantial proportion of the prokaryotic community (14.6% [SEM, 0.5%]; n = 18) (Fig. 4b). The probe for the largely psychrophilic Desulfotalea/Desulforhopalus cluster produced the dominant signal detected (10.7% [SEM, 0.3%]). In addition, signals from Desulfobacterium (4.1% [SEM, 0.2%]), Desulfobulbus (0.3% [SEM, 0.05%]), and Desulfobacteriaceae (1.3% [SEM, 0.06%]) were also detected. No signal from Desulfobacter was detected in RNA from Shallow Bay sediments.


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DISCUSSION
 
Despite the importance of microbial activities in permanently low-temperature sediments in the global ecosystem, little quantitative work has been performed on these sediments. In this study, we aimed to investigate the distributions and activities of SRB and MA in Antarctic sediments in order to gain an understanding of the dynamics of these ecologically important organisms in permanently low-temperature systems.

Archaea usually represent no more than a few percent of the total prokaryotic community (13, 18), with occasional reports of far higher proportions (up to 34%) in planktonic communities (14, 36). However, in the sediments of Lake Heywood, the archaeal community represented 34% of the total prokaryotic community, the largest proportion ever detected in sediments; 30% of this archaeal signal could be linked to known methanogens. The controls used in these probing experiments were in vitro-transcribed 16S rRNA fragments (approximately 1,400 bp long) and not native 16S rRNA; this factor may have affected the quantification (37). However, in vitro-transcribed RNA was the standard for all of the MA-targeted probes, and our method of calculation allows for differences in probe responses, a factor which would mitigate against a systematic error in the quantification. The unidentified archaea did not appear to be crenarchaeotes, as we detected none by using crenarchaeote-specific primers, nor did they appear to be known MA. It is possible that a proportion of MA present in Lake Heywood sediments did not hybridize to the group-specific MA-targeted probes that we used and that we underestimated the communities of Methanosarcinales and Methanomicrobiales. Sequence analysis of the Lake Heywood Methanosaeta clones indicated that all should have hybridized to the probe for the Methanosarcinales. It was not possible to check the Methanogenium-like clones, as our sequences did not include the site of the probe for the Methanomicrobiales.

Methanogenesis was the dominant process in Lake Heywood sediments, with in situ rates (127 mmol m-2 day-1) similar to the maximum rates measured (95 to 112 mmol m-2 day-1) (40, 65) in temperate freshwater lakes. Fluoroacetate inhibition of methane production in Lake Heywood sediment slurry experiments indicated that acetate accounted for 90% of methanogenesis in these sediments, corroborating previous work with 14C-labeled acetate and CO2 (6) and in agreement with other work on low-temperature methanogenesis (18, 43, 59). Similarly, the most abundant MA clones in an archaeal 16S rRNA clone library were closely related to Methanosaeta concilii, an obligate acetate utilizer. Oligonucleotide probing of directly extracted RNA showed that Methanosaeta RNA represented a substantial proportion (4.3%) of the prokaryotic RNA in these sediments. Together, these results suggest that Methanosaeta organisms are important methanogens in Lake Heywood sediments. It was suggested previously (49) that the apparent ubiquity of clones related to Methanosaeta in freshwater sediments indicates that members of this group may be globally dominant acetoclastic MA, and the data presented here provide further support for the widespread importance of these organisms.

Acetoclastic methanogenesis is not the only pathway by which methane can be produced from acetate. It has been reported that syntrophic acetate degradation, in which acetate is oxidized with the production of H2, which is then utilized by hydrogenotrophic MA, can be responsible for methane production in slurries amended with acetate (44). However, in sediments where syntrophic acetate degradation occurs, no acetoclastic methanogens were detected in situ (44), in contrast to the results for the Antarctic sediments studied here. Furthermore, hydrogenotrophic methanogenesis, which is linked to syntrophic acetate degradation, is inhibited at low temperatures (43, 59). Thus, it seems unlikely that the methanogenic activity that we have detected is due to syntrophic acetate degradation.

The other MA detected in the Lake Heywood sediments were related to Methanogenium, a group of organisms that are known to utilize H2, and it is possible that the remaining 10% of methanogenesis in Lake Heywood sediments is produced through H2. Probing experiments revealed no supporting evidence of an active Methanogenium community in Lake Heywood sediments; therefore, the putative H2-utilizing MA in these sediments have not been identified.

Methanogenesis in Shallow Bay sediments represented only a small proportion (approximately 2%) of the total carbon flow through the system, which was matched by the low proportion of archaeal RNA detected in these sediments (0.2%). Methanogens (Methanomicrobiales, Methanosarcinales, and Methanococcoides) were detected, but they represented only 0.1% of the total prokaryotic community. The detection of Methanosarcinales and Methanococcoides links activity in these sediments to the degradation of C1 compounds, while the detection of a signal from the general probe for the Methanomicrobiales also suggests that hydrogenotrophic methanogenesis can occur in these sediments. This suggestion is supported by results from the slurry experiments, where the inhibition of sulfate reduction by the addition of molybdate led to a rapid and substantial increase in methane production. These results suggest that substrates used by SRB in these sediments can be instantly utilized by a standing MA community, most likely H2-utilizing Methanogenium. A signal from the probe for the Methanosarcinales has been linked to the use of trimethylamine in slurries from a coastal salt marsh in Essex, United Kingdom, while the data presented here would suggest that members of the Methanococcoides play a significant role in C1 compound degradation in these Antarctic marine sediments.

Unlike the diverse archaeal communities seen in clone libraries from the Colne River estuary in Essex, United Kingdom (39, 49), the archaeal libraries from the two Antarctic sites investigated here were very limited, with only 5 distinct groups detected across both sites compared to 12 from the temperate sediments of the Colne River. It has been suggested that diversity in communities in physically or chemically stressed environments is likely to be lower than that in communities which are not physically stressed and where diversity is regulated primarily by interspecies competition (11); our data support this hypothesis. However, we detected additional MA clones related to BP R10 (Fig. 3a) when we used a different set of PCR primers, suggesting that the original archaeal analysis did not fully sample the archaeal diversity in Lake Heywood. Bias and selection can occur in PCR (9, 45, 62), although evidence suggests that they result in the overrepresentation of minor components rather than the loss of diversity (62). Sequences from the BP R10 cluster can be detected by the method that we used (49). It is possible that the BP R10 cluster sequences from Lake Heywood were poorly amplified under the conditions that we used or that the BP R10-like population did not represent a substantial part of the Lake Heywood methanogen community and we did not sample enough clones to detect similar sequences in our original archaeal clone library.

Sulfate reduction dominated the terminal oxidation processes in the Shallow Bay sediments. Signal from the probe for Desulfotalea/Desulforhopalus represented a substantial proportion of the prokaryotic community in the Shallow Bay sediments, and these organisms were the most commonly detected SRB in the Shallow Bay clone library (Fig. 4a, groups 1 and 2); these results suggest that these organisms were extremely important in sulfate reduction in these coastal marine sediments. Desulfotalea isolates are psychrophiles, originally isolated from Arctic marine sediments from Svalbard in Norway (29, 56), while Desulforhopalus isolates have been isolated from coastal marine sediments (24). All members of this cluster are incomplete oxidizers, and many are also psychrophiles. The substrate profile of members of this group is similar to that of Desulfovibrio (lactate and H2 utilizers), a finding which led Knoblauch et al. (29) to hypothesize that these two groups may be ecological equivalents. In both this study and those on the Arctic Svalbard sediments, Desulfovibrio isolates were not detected in bacterial clone libraries (54). Sahm et al. (57) also did not detect this group in probing studies on the Arctic sediments. Thus, it appears that while Desulfovibrio isolates have been reported to be important components of the SRB community in a variety of temperate sediments (31, 47), they may be either absent or inactive in permanently low-temperature sediments, with psychrophiles from the Desulfotalea/Desulforhopalus group occupying the ecological niche of the mesophilic Desulfovibrio.

Knoblauch et al. (28) found very low numbers of acetate-utilizing SRB in MPN counts in Arctic sediments, and the acetate-utilizing SRB that they did isolate grew extremely slowly. Previous work linked the acetoclastic Desulfobacter to acetate degradation in marine-dominated temperate estuarine sediments (50, 51), but no signal from a Desulfobacter-targeted probe was detected in these Antarctic sediments. It has been suggested that acetate may be utilized by Desulfotomaculum in marine sediments (7), although we detected no Desulfotomaculum in either the Shallow Bay or the Lake Heywood clone libraries. Ravenschlag et al. (54) found a large number of clones in a general bacterial clone library derived from primers different from ours that were related to the sulfur and Fe- or Mn-reducing Desulfuromonas cluster. We found clones very closely related to these Svalbard clones in our Shallow Bay sediments (Fig. 4a, group 4). It has been hypothesized that acetate in the Arctic sediments is not utilized primarily by SRB but by sulfur-reducing Desulfuromonas or by acetate-utilizing Pelobacter or Geobacter (54). The data from our study are not inconsistent with this hypothesis.

It appears that organisms closely related to M. concilii are ubiquitous in freshwater sediments, and these organisms may be globally important in the degradation of acetate in freshwater systems (18, 22, 49), including permanently low-temperature systems. The detection of a number of clones within the Desulfotalea/Desulforhopalus cluster and the relative abundance of this group in the sediments of Shallow Bay suggest that this group is important in sulfate reduction in the Antarctic marine sediments of Shallow Bay. The previous discovery of this group in Arctic sediments (29) and in a permanently low-temperature deep-sea sediment (32) suggests that this group is ubiquitous in low-temperature marine sediments and may also be globally important. The data presented here and the work of others (28, 54) have indicated that the process of anaerobic terminal oxidation in low-temperature sediments is poorly understood and requires further study.


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ACKNOWLEDGMENTS
 
This work was funded by NERC grant GR3/10738.

Special thanks are due to Matt Edworthy and Cynan Ellis-Evans for scientific and field technical support. Thanks are also due to the British Antarctic Survey, particularly people on board the RRS Bransfield and at Signy Island Research Base, for their logistical support during this project.


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FOOTNOTES
 
* Corresponding author. Present address: School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading RG6 6AJ, United Kingdom. Phone: 44 (0)118-9318892. Fax: 44 (0)118-9316671. E-mail: K.J.Purdy{at}Reading.ac.uk. Back


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Applied and Environmental Microbiology, June 2003, p. 3181-3191, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3181-3191.2003
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