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Keywords:

  • Sulfate-reducing bacteria;
  • Oligonucleotide probe;
  • Sediment slurry;
  • Desulfobulbus;
  • Desulfobacter

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

The structures of prokaryotic communities are difficult to elucidate because of the apparent inability to culture most of the indigenous microorganisms. Here we report the use of 16S rRNA-targeted oligonucleotide probes to study changes in and the identities of sulfate-reducing bacterial populations present in enriched slurry microcosms from a predominantly freshwater and a predominantly marine site from the River Tama, Tokyo, Japan. Significant enrichment of signals from different oligonucleotide probes, designed to target cultured members of several SRB genera, were observed in amended slurries. Signal from a probe designed to detect Desulfobulbus spp. gave an increased response on propionate addition to slurries from both sites. The response to a probe designed to detect Desulfobacter was increased by acetate addition to slurries from the marine site. Response to a wide specificity probe also increased suggesting that uncharacterised groups were also enriched at the marine site. Our data suggest that Desulfobulbus may be an important propionate utiliser in the estuary, while Desulfobacter is responsible for acetate utilisation at the marine site. These results are compatible with the known physiology of Desulfobulbus and Desulfobacter and provide strong support for the use of oligonucleotide probes in the study of microbial communities.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

Sulfate reduction has been measured in a variety of anoxic environments including marine, salt-marsh, estuarine and freshwater lake sediments [1–3]. In high sulfate environments sulfate reduction dominates mineralisation, accounting for up to 50% of the organic matter degradation in estuarine and coastal marine sediments [3, 4]. In low sulfate environments sulfate reduction is inhibited by the lack of available sulfate for respiration and the mineralisation process is dominated by methanogenic Archaea [5, 6]. Previous studies have concentrated on processes mediated by SRB and determining which are the dominant organic substrates utilised by SRB populations [7–9]. Such studies have revealed that short-chain fatty acids (SCFA), mainly acetate and propionate, as well as hydrogen represent the major substrates for SRB within sediments [7, 8, 10].

Putative biomarkers, such as phospholipid fatty acids (PLFA), as well as polyclonal antibodies have been used to attempt to identify the dominant SRB populations within sediments [9, 11, 12]. However, it is still unclear which groups of SRB are important in the environment and what factors control the distribution and activity of these groups.

Recent developments indicate that 16S rRNA should be an ideal biomarker for microbial communities (see Amann et al. [13] and references therein). 16S rRNA-targeted oligonucleotide probes potentially allow the investigation of microbial communities without the need to cultivate microorganisms. For example, Stahl et al. [14] used oligonucleotide probes to detect transient changes in populations of rumen bacteria after perturbation with an antibiotic.

Probes which specifically target SRB genera [15] have been used to investigate the structure of SRB communities in sediment and microbial mat communities [16, 17]. The probes were designed from the sequences of SRB strains in culture [15] and, as such, may not represent the extent of genetic diversity within each genus. Conversely, they may cross-react with strains from outside their target genus that are, as yet, uncharacterised [14, 17]. Therefore signal from these probes may potentially include a proportion of non-SRB organisms. SRB-specific inhibitor controls can be used to investigate the extent of any such probe cross-reactivity.

The use of slurry microcosms allows controlled experiments to be performed under conditions that resemble those in situ, and thus allow the investigation of the response of a natural, mixed community to a specific environmental perturbation. We describe the use of experiments with slurry microcosms from two contrasting sites, one predominantly freshwater, the other predominantly marine, on the River Tama, Tokyo, Japan that were amended with SCFA, known to be important in sediments [7, 8, 10]. Controls inhibited with the SRB-specific inhibitor molybdate [18] were used to show that any response detected was due to sulfate reducers. Nucleic acids extracted from these slurries were probed with a range of probes designed to encompass most of the known Gram-negative mesophilic SRB diversity. Our aim was to determine which, if any, of these SRB genotypes were responsible for the metabolism of the added SCFA and to determine any differences in the response to substrate addition in slurries from the two sites.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

2.1Description of study site and sediment sampling

The Tama River is about 100 km in length with a catchment area of some 1200 km2. It flows through the south-west part of Tokyo, Japan, and is heavily polluted with municipal sewage. Sediment samples were taken from two sites in the lower reaches of the river. Site 2, at Gasu-Bashi, 10.5 km from the river mouth, is only very occasionally inundated by the tide [3, 19] and thus is predominantly a freshwater site. Sulfate concentrations in sediment pore water (0–3 cm) were 0.1–2.8 mM over the period April 1994 to February 1995. Site 2 was sampled in February 1993. Site 4, at Haneda Airport, about 2 km from the river mouth, is essentially a marine site with sediment pore water sulfate concentrations (0–3 cm) varying between 3.5 and 32 mM over the period April 1994 to February 1995. This site was sampled in April 1993. Sediment cores were taken in perspex cores (30 cm×4.5 cm) and transported back to the laboratory for preparation of slurries.

2.2Preparation and sampling of slurry experiments

Slurry experiments were designed to investigate the response of different putative SRB genotypes detected by 16S rRNA-targeted oligonucleotide probes after addition of excess levels of various substrates. Anaerobic slurries (50% v/v with site water) of the top 5 cm of the sediment cores from each station were prepared under anaerobic conditions [9]. Aliquots of slurry from each site (100 ml) in 250 ml conical flasks sealed with rubber bungs were enriched with sulfate (10 mM) to provide adequate supplies of sulfate for development of the SRB community at the beginning of the experiments. Duplicate aliquots of slurries were then amended with either lactate, lactate+molybdate (molybdate is a potent inhibitor of sulfate reduction [18]), acetate, propionate or butyrate, all to 20 mM final concentration (only 10 mM propionate was added to the site 4 propionate-amended slurries due to an error). A duplicate set of slurries, unamended with SCFA, were used as controls. Lactate was the only substrate treatment also inhibited with molybdate as lactate is usually degraded to propionate and acetate in slurry microcosms, even in the presence of molybdate [7, 9, 20]. This single treatment acted as an inhibited control for all three substrates. Butyrate is known to accumulate on addition of molybdate to sediment slurries [8, 21] indicating that SRB can utilise this substrate.

The slurries were incubated with shaking (orbital shaker at a speed sufficient to maintain the sediment in suspension) at 30°C in the dark for 14 days. The incubation temperature was selected as typical of the in situ summer temperature for Tama River sediments and to ensure a rapid microbiological response to substrate addition. Every 2–3 days samples of slurry (10 ml) were taken while gassing the flask vigorously with oxygen-free nitrogen to maintain anaerobic conditions. These slurry samples were immediately centrifuged at 8000×g for 10 min, the supernatant removed and filtered through a glass fibre filter (GF/F, Whatman International Ltd., Maidstone, UK) to remove particulates and stored at −20°C until analysis for residual sulfate and fatty acids. The sediment pellet was washed with 120 mM sodium phosphate, pH 8.0, to remove any extracellular nucleic acid [22] and stored in 4 g samples at −20°C until nucleic acid extraction.

2.3Analysis of sulfate and fatty acid concentrations in slurry sediments

Sulfate concentrations in the supernatants from the slurry samples were determined by ion chromatography (Dionex, Sunnydale, CA, USA). Fatty acid concentrations were determined by HPLC (SCR-101H column, Shimadzu, Kyoto, Japan; L-6200 pump, Hitachi, Tokyo, Japan at 1 ml min−1; L-4000 UV detector, Hitachi, at 210 nm [23]).

2.4Extraction of nucleic acids from slurry sediment

Nucleic acids were extracted from sediment samples using the hydroxyapatite (HTP) spin-column method [24]. For nucleic acid extraction the duplicate slurry samples from each treatment were bulked to give sufficient nucleic acid for probing. Total nucleic acids were extracted from samples of site 2 slurries while rRNA was extracted separately from DNA from site 4 slurries. There is no fundamental difference in these two extraction methods [24], the only difference is in how the extracted nucleic acid is eluted from the HTP spin column. All extracts were visualised by ethidium bromide-stained gel electrophoresis to check for the presence of rRNA, and nucleic acid purity and yield was determined by scanning spectroscopy [25].

2.5Extraction of rRNA from pure culture controls

Pure cultures of sulfate-reducing bacteria were grown as described by Widdel [26]. These cultures were Desulfovibrio vulgaris DSM 644, Desulfovibrio desulfuricans DSM 1926, Desulfobulbus propionicus DSM 2056, Desulfococcus multivorans DSM 2059, Desulfosarcina variabilis DSM 2060, Desulfobacter latus ATCC 43918, Desulfobacterium autotrophicum DSM 3382. Non-SRB controls (Escherichia coli NCTC 09001 and Bacillus subtilis NCMB 3610) were grown in nutrient broth (Oxoid, Ltd., Basingstoke, UK). Mid-exponential phase cultures (as determined by nephelometry) were sampled (50 ml) and centrifuged at 12 500×g. The resultant cell pellet was resuspended in 120 mM sodium phosphate, pH 8.0+10% (w/v) glycerol and stored in 0.5 ml aliquots at −20°C. rRNA from these aliquots was extracted using a guanidine thiocyanate method (H. McVeigh, personal communication) and purified using the HTP spin-column method [24]. Glass beads (0.5 g, 0.1 mm diameter), baked at 260°C for at least 3 h to destroy any RNAse activity [25], were placed into a 2 ml screw-capped Eppendorf tube and an aliquot (0.5 ml) of culture and 250 ml of 5 M guanidine thiocyanate (Sigma-Aldrich, Tokyo, Japan) were added. This was bead-beaten (Mikrodismembrator U, B. Braun Biotech International, Melsungen, Germany) at 2000 rpm for 30 s and centrifuged at 12 000×g for 2 min. The supernatant was collected and kept on ice. The remaining pellet was washed once with 0.75 ml of 120 mM sodium phosphate, pH 8.0, centrifuged again and the supernatant pooled with that from the first extraction. The pooled extract was then loaded onto a HTP spin column and the rRNA eluted as previously described [24]. rRNA was analysed by ethidium bromide-stained agarose gel electrophoresis and rRNA purity and yield were determined by scanning spectroscopy [25].

2.6Immobilisation of extracted nucleic acids onto nylon membranes

Known quantities of nucleic acids (site 2 either 50 ng or 900 ng total NA/slot, site 4 either 20 ng or 200 ng rRNA/slot) were slot-blotted onto nylon membrane (Hybond-N, Dupont (UK) Ltd., Stevenage) using a BioBlot manifold (Bio-Rad Laboratories Ltd., Hercules, CA, USA) after denaturation of rRNA by addition of 2% (w/v) glutaraldehyde [27]. Measured amounts of pure rRNA from pure culture positive controls specific to each probe to be utilised and negative controls were also immobilised on the nylon membranes.

2.7Oligonucleotide probes and hybridisation conditions

The oligonucleotide probes specific to most of the known mesophilic, Gram-negative SRB genera and groups were used in these experiments. A general bacterial domain probe (p338) described by Amann et al. [28] was used, along with SRB genus-specific probes described by Devereux et al. (p129, Desulfobacter; p221, Desulfobacterium; p660, Desulfobulbus; p687, Desulfovibrio; p804, Desulfobacter, Desulfobacterium, Desulfosarcina, Desulfococcus and Desulfobotulus; p814, Desulfosarcina, Desulfococcus and Desulfobotulus[15]). Oligonucleotide probes were synthesised on an automatic synthesiser (381A DNA synthesiser, Applied Biosystems, Warrington, UK) and end-labeled with [γ-32P]ATP (ICN Biomedicals, Costa Mesa, CA, USA) [25].

Hybridisations were performed as described by Stahl et al. [14]. Membranes were sealed into bags with approximately 0.1 ml of hybridisation buffer (0.9 M NaCl, 50 mM sodium phosphate, pH 7.0, 5 mM EDTA, pH 8.0, 10×Denhardt's and 0.5 mg ml−1 Poly A (Sigma-Aldrich)) per cm2 of membrane added. Membranes were prehybridised at 40°C for at least 2 h before addition of radiolabeled oligonucleotide. Incubation was continued for at least 16 h, after which the membranes were washed once with 1×SSC, 1% SDS for 30 min at 40°C and then in fresh wash buffer at the specific temperature for 30 min [15, 28]. Autoradiographs (XAR-5, Eastman Kodak Co., Rochester, NY, USA) were exposed for 1–10 days at −70°C and developed automatically (Gevamatic-60 Developer, AGFA-Gevaert, Brentford).

Membranes were stripped after autoradiography to reuse the membrane. Boiling 0.5% (w/v) SDS was poured over the membranes to be stripped and then left to cool to room temperature. The stripped membranes were autoradiographed as above to determine if all the bound probe had been removed. If any signal remained after stripping, membranes were stripped once more. If signal was still visible the membrane was discarded. Successfully stripped membranes were reprobed by the method described above.

2.8Calibration of probe signal using pure culture controls

Pure culture controls were used to calibrate the response of the target genera to the oligonucleotide probes used. Examples of calibration curves from both sites have been described previously [29]. Most pure culture standards gave a very good linear response to the probes. The accuracy of this method was assessed by calculating the apparent loading for a middle point sample using a regression line between higher and lower values. This usually gave a value close to the nominal amount loaded, with most calculated values within 10% of the nominal amount loaded.

2.9Scanning densitometry of autoradiographs and determination of signal levels

Autoradiographs were quantified using densitometry (300A Computing Densitometer, Molecular Dynamics, Kemsing, UK) and the associated ImageQuant software. Probes do not all bind equally well to target sequences and variations in labeled probe-specific activity result in different signal levels. Therefore, to standardise results of blots hybridised with different probes, densitometrically measured signals were converted to an amount of rRNA for each individual sample by comparison to the signal from known amounts of rRNA from pure culture control organisms. These standardised results were then expressed as a percentage of the signal (expressed as ng of rRNA detected) of the general bacterial probe (p338) for each individual sample.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

Slurry microcosms were used to investigate the response of a natural sediment bacterial community to the addition of excess SCFA in the presence of sulfate. Nucleic acids from samples of these slurries were extracted and probed with a suite of probes designed to detect cultured strains of SRB and the responses of the different populations targeted by these probes to substrate addition measured.

3.1Effect of substrate addition on sulfate concentration in the freshwater-dominated site 2 slurries

Added sulfate was removed only slowly in the unamended control slurries from site 2 (Fig. 1a) and not at all in slurries treated with molybdate, suggesting that sulfate removal was by indigenous SRB. In contrast, in slurries amended with either lactate, acetate, propionate or butyrate, sulfate disappeared within 5–8 days.

image

Figure 1. Effect of substrate addition on the utilisation of sulfate from (a) freshwater-dominated site 2 slurries and (b) marine-dominated site 4 slurries. Bars indicate spread of duplicates.

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3.2Utilisation of added substrate in the freshwater-dominated site 2 slurries

The unamended control slurries showed no detectable concentrations of the tested substrates (lactate, acetate, propionate and butyrate) throughout the experiment (data not shown).

In the lactate-amended slurries (Fig. 2a) lactate was rapidly removed within 2 days. There was a corresponding rise in propionate and acetate, before they both disappeared by day 14. In slurries amended with lactate+molybdate, lactate was still utilised rapidly (Fig. 2b). In this treatment, acetate and propionate concentrations rose to a peak by day 8 before beginning to fall.

image

Figure 2. Changes in substrate concentrations in freshwater-dominated site 2 slurries amended with: (a) lactate, (b) lactate+molybdate, (c) acetate, (d) propionate and (e) butyrate. Closed symbols indicate added substrate; open symbols, degradation products. Bars indicate spread of duplicates.

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In the other amended slurries (Fig. 2c–e) added substrate was utilised within 8–14 days. In the propionate- (Fig. 2d) and butyrate-amended slurries (Fig. 2e) there was a transient accumulation of acetate. In all treatments production of propionate and/or acetate corresponded stoichiometrically to added substrate utilisation.

3.3Effect of substrate addition on sulfate concentration in the marine-dominated site 4 slurries

Sulfate was utilised in the unamended control slurries from site 4 (Fig. 1b), while sulfate was not removed in slurries inhibited with molybdate. Sulfate utilisation in site 4 controls was apparently faster than in site 2 controls (Fig. 1a), suggesting a more active SRB community in the marine-dominated site. In slurries amended with either lactate, acetate, propionate or butyrate, sulfate was exhausted within 5 days.

3.4Utilisation of added substrate in the marine-dominated site 4 slurries

Unamended control slurries showed no detectable concentrations of the tested SCFA (data not shown).

In the lactate-amended slurries (Fig. 3a) lactate was removed within 2 days, with corresponding rises in both propionate and acetate, but, unlike site 2, these then remained fairly constant until day 11, after which the concentration of propionate fell slightly. The lactate+molybdate-amended slurries (Fig. 3b) showed fatty acid accumulation similar to that in site 2 slurries. Lactate rapidly fell below the detection limit by day 2, while acetate and propionate concentrations rose and remained elevated throughout the experiment.

image

Figure 3. Changes in substrate concentrations in marine-dominated site 4 slurries amended with: (a) lactate, (b) lactate+molybdate, (c) acetate, (d) propionate and (e) butyrate. Closed symbols indicate added substrate; open symbols, degradation products. Bars indicate spread of duplicates. (Due to an error only 10 mM propionate was added to the slurries in d.)

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In the acetate-amended slurries (Fig. 3c) added substrate decreased to 4.5 mM by day 2 and was then static until day 14. In the propionate-amended slurries, propionate disappeared by day 5 (Fig. 3d) and acetate accumulated from day 2. Butyrate was removed steadily in the butyrate-amended slurries (Fig. 3e) with a transient accumulation of acetate.

In marked contrast to site 2, the accumulation of measured acetate and propionate in the lactate- and propionate-amended slurries was not stoichiometric with added substrate utilisation (Fig. 3a,d). This indicates that the intermediate products of the added substrates were metabolised much more rapidly at site 4 than at site 2, where they accumulated transiently.

3.5Extraction of nucleic acids from slurry sediment samples

Results of gel electrophoresis showed successful extraction of rRNA from the samples (data not shown). Measured OD260/OD280 and OD260/OD230 ratios were usually >1.8 indicating relatively pure nucleic acid [25]. Yields varied, but averaged 33.5 μg NA g−1 sediment wet weight (standard error (S.E.)=2.7, n=25) for site 2 and 9.1 μg of rRNA g−1 sediment wet weight (S.E.=0.8, n=31) for site 4. The nucleic acid yield obtained from these slurry sediments was similar to the best yields reported by other researchers [22, 30, 31].

3.6Estimation of the relative abundance of SRB genotypes by probing extracted rRNA using radiolabeled oligonucleotide probes

Results of probing extracted nucleic acids with SRB-specific 16S rRNA-targeted oligonucleotide probes (see Fig. 4Fig. 5) are expressed as a percentage of the standardised signal for a general bacterial probe (p338) for that particular sample. This normalises for variation in the amounts of rRNA loaded onto the membranes and estimates the size of the rRNA pool for each SRB population, relative to the size of the general bacterial rRNA pool in each sample. It is, therefore, a composite measure of both the population size (number of cells) and the activity of individual cells as expressed by intracellular 16S rRNA [32–34]. At no point during these experiments did any probe cross-reactivity occur with any of the control organisms used, with the exception of p814 (narrow specificity). This probe is reported not to bind to Desulfobacter and Desulfobacterium spp., but under the conditions specified by Devereux et al. [15] our Desulfobacterium control gave a small but reproducible signal. This signal was not removed by washing at increased temperatures up to 50°C when signal from the target strains began to fall dramatically.

image

Figure 4. Relative abundance of SRB genotypes measured by rRNA probed with radiolabeled oligonucleotide probes in freshwater-dominated site 2 slurries amended with: (a) lactate, (b) lactate+molybdate, (c) acetate, (d) propionate, (e) butyrate and (f) unamended controls. Relative signal levels were determined after signal standardisation against known amounts of pure culture rRNA.

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image

Figure 5. Relative abundance of SRB genotypes measured by rRNA probed with radiolabeled oligonucleotide probes in marine-dominated site 4 slurries amended with: (a) lactate, (b) lactate+molybdate, (c) acetate, (d) propionate, (e) butyrate and (f) unamended controls. Relative signal levels were determined after signal standardisation against known amounts of pure culture rRNA.

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3.6.1Results of probing nucleic acid extracts from the freshwater-dominated site 2 slurries

Signals from all SRB probes were detected in the unamended control slurries (Fig. 4f). The standardised signal from each specific probe was always <10% of that of the general bacterial probe signal suggesting that the target genotypes comprised a relatively minor part (10–12%) of the total detected bacterial community.

The standardised probe signals in a treated slurry were statistically compared with those for the same probe in the control slurry. All time samples, except day 0, were pooled for each treatment, and treatments were compared by the non-parametric Mann-Whitney U test after arcsine transformation of the data [35]. Standardised signal from p660 (‘Desulfobulbus’) showed a significant increase, compared to the unamended control (Fig. 4), in the two slurries which contained large amounts of propionate (P<0.05). In contrast, no significant increase in p660 signal (P>0.05) occurred in the lactate+molybdate-amended slurries (Fig. 4b) where sulfate reduction and SRB growth was inhibited. Furthermore, there was no significant increase in p660 signal in either acetate- or butyrate-amended slurries, where no propionate was detected (Fig. 4c,e).

None of the other probes targeted against the other SRB genera showed any significant increases in standardised signal compared to the unamended controls in the site 2 slurries (P>0.05). Results from probe p804 (wide specificity) indicated that none of the genotypes targeted by this probe, including ‘Desulfobacter’ and ‘Desulfobacterium’, was enriched in any of these slurries (Fig. 4). This was corroborated by results from p129, whose standardised signal remained only 1–5% of the standardised signal from the bacterial probe in the unamended controls and in the amended slurries (Fig. 4). Surprisingly, the probe which targeted Desulfovibrio spp. (p687) also showed no significant enrichment in any of the treatments compared to the unamended controls.

3.6.2Results of probing extracts from the marine-dominated site 4 slurries

Signals were detected from all the probes in the unamended control slurries from site 4 (Fig. 5f). The relative signals from p129 (‘Desulfobacter’) and p804 (wide specificity) were usually greater than in site 2 slurries, while the other probes were at levels similar to those in site 2 controls. During the experiment the standardised signals from all probes fell to <2% of the total bacterial signal by day 14 in the control slurries. Except where molybdate was present, addition of substrates resulted in a significant stimulation of some of the target genotypes relative to the unamended control (Fig. 5, P<0.05).

As at site 2, p660 (‘Desulfobulbus’) gave a significantly increased signal in site 4 in lactate- and propionate-amended slurries, where large amounts of propionate were detected (Fig. 5a,d, P<0.05). No significant increase in p660 signal was detected in lactate+molybdate, acetate- or butyrate-amended slurries (Fig. 5b,c,e).

In contrast to site 2, standardised signal from the putative Desulfobacter-specific probe, p129, increased significantly (P<0.05), compared to the control, in all slurries from site 4 in which acetate was detected, with the exception of the butyrate-amended slurries and the molybdate-inhibited slurries (Fig. 5).

The signals from p221 (‘Desulfobacterium’) and p687 (‘Desulfovibrio’) showed no significant increase in any of the amended slurries compared to the unamended controls (Fig. 5).

The wide specificity probe p804 showed, as would be expected of a probe that included Desulfobacter, significantly increased standardised signal in those slurries where Desulfobacter was apparently enriched (P<0.05) but its signal was usually slightly higher than that of p129 (‘Desulfobacter’, Fig. 5a,c,d). Standardised signal from p804 was significantly higher than signal detected from p129 in propionate- and butyrate-amended slurries, although the actual difference in signal magnitude was much greater in the butyrate-amended slurries (Fig. 5d,e) where there was no increase in p129 (‘Desulfobacter’) signal. Use of the more specific p814 probe, which is designed to exclude Desulfobacter and Desulfobacterium, did not show any enrichment. This indicates that genera that bind p814, i.e. ‘Desulfosarcina, Desulfococcus and Desulfobotulus’, were probably not the groups enriched in the butyrate-amended slurries.

4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

The results of these slurry experiments indicate that sulfate reduction by SRB at both sites in the Tama River was limited by the availability of labile carbon so long as sulfate was in excess. Control slurries with added sulfate but no added carbon source showed steady but slow sulfate reduction at both sites. Where substrates were added, initial rates of sulfate reduction, as determined by the rate of sulfate utilisation, increased dramatically. The rate of sulfate utilisation in the marine-dominated site 4 slurries was apparently faster than in the freshwater-dominated site 2 slurries. This suggested a SRB community with a smaller capacity for sulfate at site 2, where sulfate was normally present only in low concentrations, than the more marine-dominated, high sulfate sediment at site 4.

Differences in the communities at the two sites were apparent on addition of added substrates, and in the effect of the addition of molybdate. Addition of molybdate to lactate-amended slurries from site 2 inhibited sulfate reduction (Fig. 3b), however, signal from the targeted genotypes (Fig. 4f) did not disappear. In contrast, in site 4 slurries signal from all probes fell in the presence of molybdate (Fig. 5b). Molybdate inhibits sulfate respiration [18], and these results suggest that SRB at site 4 may have been more physiologically dependent on sulfate respiration than the SRB at site 2. In addition, site 2 slurries continued to utilise added SCFA after complete depletion of the added sulfate (Fig. 1a, Fig. 2), while slurries from site 4 effectively stopped using available organic carbon after sulfate depletion (Fig. 1b, Fig. 3). SRB in low sulfate environments may have a higher affinity for sulfate than those in high sulfate sediments [36, 37], making them much more effective at sequestering available sulfate. In low sulfate environments SRB may also survive either by acting as proton reducers [20] or by nitrate respiration [38–40]. It is not known whether this physiological difference between high and low sulfate SRB is reflected in phylogenetic differences in such SRB communities. However, the difference in the genotypes enriched within these two different sites would suggest that there is a difference in the composition of these two communities as detected by the available oligonucleotide probes.

In unamended slurries from the freshwater-dominated site 2 (Fig. 4f) SRB populations were relatively static or falling by the end of the experiment, but the total contribution from the detected SRB populations was still substantial (>10% of the total bacterial signal on day 14). In contrast, the SRB populations in unamended slurries from the marine-dominated site 4 showed a marked and almost continuous fall from their initial levels (Fig. 5f). Signal from p129 (‘Desulfobacter’), which represented the largest measured proportion of the bacterial rRNA pool in these sediments at day 0, was undetectable by day 14. This difference may be due to either the depletion of sulfate (Fig. 2b) or possibly the exhaustion of the available organic matter in the site 4 slurries.

In slurries amended with SCFA enrichment of several putative SRB genotypes using rRNA-targeted oligonucleotide probes was successfully detected. ‘Desulfobulbus’ was apparently significantly enriched, as judged by increased signal with p660, in slurries from both sites when propionate was available. This is compatible with the capacity of cultured members of this genus to utilise propionate [26]. Standardised signal from the probe which targets known Desulfobacter spp. (p129) and the wide specificity probe (p804) increased significantly only at the marine-dominated site 4 (Fig. 5). The increase in p129 (‘Desulfobacter’) signal in acetate-containing slurries from site 4 was compatible with the known physiology of Desulfobacter as an acetate-utilising SRB [26]. Acetate is one of the most abundant organic intermediates in aquatic sediments and is considered an important substrate for SRB in marine sediments [7, 8, 10, 41]. Generally, about 50% of sulfate reduction in coastal marine sediments appears to be driven by acetate [7, 8, 10] and the oxidation of acetate to CO2 is important in completing the pathway of anaerobic carbon mineralisation [42]. The apparent enrichment of Desulfobacter by acetate in site 4 slurries suggests that it may be an important acetate utiliser in these sediments.

In contrast, the lack of response of p129 and the wide specificity probe, p804, in the lower sulfate environment of the freshwater-dominated site 2 would imply that genotypes targeted by these probes may be limited to the higher sulfate, marine-dominated site 4. That the signal from this probe did not increase in acetate-containing slurries from the freshwater-dominated site 2 is in agreement with the previously reported importance of SRB as acetate utilisers in marine but not freshwater sediments [6, 10, 43]. Differences in the distribution of SRB genotypes from high and low salinity environments have been shown previously [44] where distinct populations were found in site waters of differing salinities from oil fields. However, acetate was utilised in site 2 slurries in conjunction with sulfate. This indicates that undetected acetate-utilising SRB were present at site 2, although they are probably not the major acetate users at this site under in situ conditions, due to low sulfate concentrations. It has been demonstrated previously [43] that acetate turnover in low sulfate sediments apparently does not involve SRB, but probably methanogenic Archaea.

The increased signal of p804 (wide specificity) in slurries from site 4 amended with butyrate (Fig. 5e), along with the rapid utilisation of sulfate (Fig. 1b), but without corresponding increases from more specific probes, implies that unknown SRB may have been enriched in these slurries. Standardised signal from p804 began to fall after day 8, which corresponded to the complete depletion of added butyrate (Fig. 3e), indicating that the unknown genotype probably used this substrate.

No increase in p804 signal was seen in the site 2 slurries, thus excluding this probe's target genotypes as utilisers of any of the added substrates at this site. Also, there was no increase in probe signal in slurries from either site of p687, putative probe to Desulfovibrio. The apparent absence of Desulfovibrio from both sites is surprising as enrichment isolations have, to date, indicated this to be a common and widespread genus [45–47]. Our data indicate that Desulfovibrio may not be abundant in the Tama River sediment and its apparent ubiquity in enrichment isolations may reflect that this genus is a copiotrophic opportunist which dominates selective isolations. Neither p221 (‘Desulfobacterium’) nor p814 (‘Desulfosarcina, Desulfococcus or Desulfobotulus’) was significantly enriched in site 4 slurries. This suggests either that targeted members of these genera could not use the substrates available in these slurries or that they were out-competed for the available substrates by other groups, both detected and undetected, that were enriched.

Previous researchers [9, 48], using PLFA as biomarkers, failed to detect changes in Desulfobacter biomass in slurries from sea lochs despite enrichment with acetate. They did, however, detect changes in putative biomarkers for Desulfovibrio and Desulfobulbus in slurries amended with propionate and lactate. The use of polyclonal antibodies raised against specific SRB strains to detect cells from sediment samples has been reported [12], however it has been suggested that such antibodies are often extremely specific and of limited use in investigating complex microbial communities [49]. Risatti et al. [16] showed that the populations of SRB, as defined by signal from the probes used in this study, were found at different depths in a microbial mat, while Devereux et al. [17] correlated a peak in signal to SRB-targeted probes with a peak in mercury-methylation activity in sediment. However, probes designed to cultured members of a genus may not include the true extent of genetic diversity of that genus, or may cross-react with strains from outside their target genus that are, as yet, uncharacterised [14, 17]. Therefore signal from probes represents the extent of binding of target-identical species which will include a proportion of the targeted genera. In this study the significant difference in probe-signal levels between molybdate-inhibited slurries and uninhibited treatments implies that the responses detected were due to true SRB populations.

These experiments can be used to draw some important conclusions about the sediments of the Tama River and to show some important differences between the two sites. There are apparent differences in the composition of responsive SRB populations in the predominately marine site 4 compared to the predominately freshwater site 2. Desulfobulbus (as defined by p660) was enrichable by propionate at both sites, while Desulfobacter (as defined by p129) and an unknown group, or groups, which binds p804 were enriched by either acetate or butyrate at site 4. The apparent enrichment of Desulfobulbus and Desulfobacter is in agreement with their known physiologies as propionate- and acetate-utilising SRB respectively and suggests they may be important components of the in situ community. Furthermore, the failure to enrich the apparently ubiquitous Desulfovibrio; those groups targeted by p804 at site 2; or p221 (‘Desulfobacterium’) and p814 (‘Desulfosarcina, Desulfococcus and Desulfobotulus’) at both sites, with any of the added substrates, would imply that these genera may not be significant parts of the fatty acid-utilising population of SRB in these environments. Most Desulfovibrio and Desulfobacterium isolates have been reported to be able to use hydrogen [26], and it is possible that these genera use substrates in situ that we did not test.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References
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