Antonie van Leeuwenhoek 81: 181–187, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.
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The distribution and activity of sulphate reducing bacteria in estuarine and coastal marine sediments K.J. Purdy1,∗ , T.M. Embley1 & D.B. Nedwell2 1 Department
of Zoology Natural History Museum, Cromwell Rd, London SW7 5BD, UK; 2 Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, UK (∗ Author for correspondence: Present address: School of Animal and Microbial Sciences, University of Reading, P.O. Box 228, Reading RG6 6AJ, UK. E-mail:
[email protected]) Key words: 16S rRNA, estuarine sediments, oligonucleotide probes, Sulphate reducing bacteria
Abstract Sulphate-reducing bacteria (SRB) play a vital role both the carbon and sulphur cycles and thus are extremely important components of the global microbial community. However, it is clear that the ecology, the distribution and activity of different SRB groups is poorly understood. Probing of rRNA suggests that different sediments have distinctly different patterns of SRB with complex factors controlling the activity of these organisms. The linking of community structure and function using sediment slurry microcosms suggests that certain groups of SRB, e.g., Desulfobacter and Desulfobulbus, can be linked to the use of specific substrates in situ. However, it is still unclear what environmental substrates are utilised by the majority of known SRBs. The work to date has greatly enhanced our understanding of the ecology of these organisms and is beginning to suggest patterns in their distribution and activity that may be relevant to understanding microbial ecology in general.
Introduction The study of microbial ecology is complicated by the difficulty of identifying ecologically important organisms and determining their role in the environment. Effective analysis of microbial communities requires that the problems of microbial identification be resolved and that the link between the structure and function of microbial communities be established (Fenchel 1992). The advent of molecular techniques in microbial ecology has greatly improved our ability to identify microbes and link identification with function in the environment (Amann et al. 1995). Sulphate reduction is an important process involved in both the global carbon and sulphur cycles. This process can dominate anaerobic terminal oxidation of organic matter in high-sulphate sediments, degrading up to 50% of all organic matter in coastal marine sediments (Jørgensen 1982), and plays a minor but still important role in low-sulphate sediments (Nedwell 1984; Takii & Fukui 1991). Studies have revealed that the products of fermentative organisms, primarily acetate and propionate, as well as hydro-
gen, represent the major substrates for SRB within sediments (Banat et al. 1981; Sørensen et al. 1981; Balba & Nedwell 1982). However, little is known about the identities of the important sulphate-reducing bacteria (SRB) in situ, or how community composition differs under different environmental conditions. Molecular methods can tackle these questions and provide a means of estimating the relative abundance of different SRB genotypes in situ. This paper will review the results of a series of molecular-based studies on the distribution of the ecologically important Gramnegative SRB of the δ-proteobacteria in a variety of estuarine sediments, attempt to link the degradation of a number of important substrates to specific SRB genera and offer suggestions on the implications of these studies for our understanding of the ecology and distribution of these organisms.
Diversity of the SRB community in a UK estuary SRB communities from three sites on the R. Colne estuary, Essex, UK (a tidal creek (Creek) and a marsh top
182 Table 1. 16S rRNA-targeted oligonucleotide probes (Devereux et al. 1992) and pure culture controls used in probing experiments. SRB cultures were grown according to Widdel and Bak (1992). B. subtilis was grown in nutrient broth Probe
Target group
Pure culture controls
Reference
p338 (S-D-Bact-0338-a-A-18) p129 (S-G-Dsb-0129-a-A-18) p221 (S-G-Dsbm-0221-a-A-20) p660 (S-G-Dsbb-0660-a-A-20) p687 (S-G-Dsv-0687-a-A-16)
Most bacteria
SRBs, B. subtilis NCMB 3610 D. latus ATCC 43918 D. autotrophicum DSM 3382 D. propionicus DSM 2056 D. vulgaris DSM 644
(Amann et al. 1990) (Devereux et al. 1992) (Devereux et al. 1992) (Devereux et al. 1992) (Devereux et al. 1992)
Desulfobacter Desulfobacterium Desulfobulbus Desulfovibrionaceae
(Marsh Top) site at the mouth of the estuary at Colne Point and a third site in the upper estuary at the Hythe) were analysed using 16S rRNA-targeted oligonucleotide probes. 32 P-labelled probes were hybridised to RNA extracted from environmental samples taken at intervals over a period of months. Autoradiographs were quantified using laser scanning densitometry and the results are expressed as a percentage of the signal from a general bacterial probe (Table 1; see references for detailed Materials and Methods: (Devereux et al. 1992; Purdy et al. 1996, 2001)). The Colne estuary is impacted by both treated sewage and terrestrial inputs (i.e., nitrate from agriculture). All three sites are inundated by the tide on every tidal cycle and thus are generally high salinity, high sulphate (>10 mM) marine-dominated sites and sulphate reduction was the dominant anaerobic terminal oxidation process (DBN, unpublished data). Signals from four genus-targeted probes, Desulfobacter, Desulfobacterium, Desulfobulbus and Desulfovibrionaceae (Table 1) were detected at all three sites (Fig. 1; Purdy 1997). The total detected SRB community, as determined by the combined signal for the four probes, was a greater proportion of the total bacterial community at the Creek and Hythe sites (20.8%, SEM = 1.6 (n = 7) and 24.2%, SEM = 5.5 (n = 3) respectively) compared to that detected at the Marsh Top site (8.8%, SEM = 1.2 (n = 7)). Surprisingly the Marsh Top site had rates of sulphate reduction (8.8 mols S m−2 year−1 ) more that twice those in the Creek (3.8 mols S m−2 year−1 , DB Nedwell, unpublished data). The Marsh Top site is heavily
Figure 1. Hybridisation of SRB targeted oligonucleotide probes to rRNA extracted from sediment from three contrasting sites on the R. Colne, Essex, UK. Data expressed relative to signal from a general bacterial probe (p338). Annual relative signal means were determined after arcsine transformation of the data, then back-transformed and statistically analysed using a one-way analysis of variance (ANOVA) with a post-hoc Tukey test (Zar 1996). Results are means of three or seven extractions, error bars = 1 standard error.
vegetated and has more total organic carbon (as a percent of dry weight) than the Creek (Munson et al. 1997) and it may be that the SRB community at the Marsh Top site represented a smaller proportion of a more active total bacterial community. As can be seen clearly in Figure 1 these three sites had two distinct patterns of SRB distribution. The SRB genera were similarly distributed at both Colne Point sites (Marsh Top and Creek sites) with Desulfovibrio the largest group. However, in the Hythe sediments while all four groups were detected, the distribution pattern was different with the community dominated by Desulfobacterium.
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Figure 2. Hybridisation of SRB targeted oligonucleotide probes to rRNA extracted from sediment from two contrasting sites on the R. Tama, Tokyo, Japan. Annual data expressed and analysed as in Figure 1. Results are means of three or four extractions.
Diversity of the SRB community in a Japanese estuary The SRB community in two marine dominated sites on the River Tama estuary in Tokyo, Japan were studied over a seasonal cycle (Purdy et al. 2001). The Tama flows through Tokyo and is heavily polluted with municipal sewage. Site 3 is marine dominated but has occasional low sulphate concentration (0.1–25.7 mM) while site 4 is characterised by more consistently high sulphate concentrations (3.5–32 mM, see the following References for a more detailed site description: Takii and Fukui 1991; Purdy et al. 2001). Signals from Desulfobacter, Desulfobacterium, Desulfobulbus and Desulfovibrio were detected in RNA extracted from samples from these marinedominated sites (Figure 2). The total SRB signal accounted for 10.0% (SEM = 1.4%, n = 3) of the bacterial signal at site 3 and 11.4% (SEM = 0.8%, n = 4) at site 4 (Purdy et al. 2001)). More than 50% of the total detected SRB signal in these sites came from the acetoclastic Desulfobacter and we would hypothesise that this genus was the dominant SRB in these sediments. The distribution patterns of SRB in the marinedominated sediments from this Japanese estuary was distinctly different from those seen in the UK estuary, with Desulfobacter the dominant SRB and Desulfovibrio detected only intermittently (Figure 2). Desulfobulbus was again detected as a significant proportion of the bacterial community (1.5 – 5%) in both sites at all sampling times as it has been in all the studies we have performed (Purdy 1997; Trimmer et al. 1997; Li et al. 1999; Purdy et al. 2001).
In the five marine-dominated estuarine sites we have studied sulphate reduction was the dominant anaerobic terminal oxidation process, yet there were three distinctly different SRB distribution patterns which would suggest SRB ecology is very complex. The reasons for this could be many. It is possible that the differential distribution of these organisms is due to gross environmental factors that distinguish the different sites. The two estuaries in this study are very different. The R. Tama in Tokyo has a metropolitan catchment while the R. Colne is much more rural, although both are polluted with sewage effluents. These differences may underlie the different distributions of SRB between the two sites. However, either localised environmental differences or some other factors must be responsible for the difference between the Colne Point sites and the Hythe on the R. Colne. It is also possible that the differences in SRB distribution between the sites is not due to gross environmental differences but in how microbial communities assemble and then alter over time. It has been suggested that microbial communities may be assembled randomly and may then fall into a number of potentially different final stable states (Miskin et al. 2001). This could explain why the SRB communities at these sites vary to such an extent. Alternatively it may be that the stable-state for all these communities is very similar but that certain selective pressures cause temporary perturbations to the community. These could be selective cell death caused by bacteriophage or protozoan grazing activity or an enrichment of a specific population that may be associated with a particular event. We have shown previously that peaks in Desulfovibrio activity in a UK estuary are associated with unseasonal peaks in sulphate reduction (Trimmer et al. 1997) and hypothesised that this may have been caused by increased organic matter entering the estuary from sugar beet processing plants.
Linking structure and function in SRB communities The distribution of SRB genera is only one aspect of their ecology: an equally important aspect is the actual function of these communities in the environment (i.e., the electron acceptors and donors utilised). However linking a process to a specific organism in situ is extremely difficult. Brock suggested that one approach would be to utilise model systems that can be easily manipulated in the laboratory (Brock 1987).
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Figure 3. (a) Autoradiograph of the hybridisation of a Desulfobacter targeted probe to extracted rRNA from marine-dominated site 4 short-chain fatty acid amended slurries. (b) Signal from the Desulfobacter targeted probe relative to signal from a general bacterial probe over the time period of the slurry experiment.
In this case we used sediment slurry microcosms to investigate which groups of SRB were capable of utilising substrates that are known to be significant in sediments, such as acetate, propionate and hydrogen (Sørensen et al. 1981; Balba & Nedwell 1982; Parkes et al. 1993). Certain SRB genera, i.e., Desulfobacter have a fairly limited substrate range while others, such as Desulfobulbus are physiologically flexible but have a defining phenotype, which for Desulfobulbus is the incomplete oxidation of propionate. Thus the relatively simple metabolism of many of SRB genera makes them ideal candidates for use of sediment slurry experiments. The addition of acetate (and sulphate) and other fatty acids that produced acetate as an intermediate product (lactate and propionate) to sediment slurries from the R. Tama, Japan, led to a significant increase in signal from a Desulfobacter-targeted probe in slurries from marine-dominated site 4, but not from the freshwater-dominated site 2 (Figures 3 and 4a; Purdy et al. 1997). This response was extremely rapid, with signal in the site 4 acetate-amended slurries rising from approximately 10% of the total bacterial signal on Day 0 to 65% on Day 2 (Figure 3b; Purdy et al.
Figure 4. Slurry experiments from two contrasting sites on the R. Tama, Tokyo, Japan. (a) The effect of acetate + sulphate addition on signal from a Desulfobacter targeted probe. (b) The effect of propionate + sulphate addition on signal from a Desulfobulbus targeted probe. Signal level means were determined after arcsine transformation of the data, then back-transformed (see Purdy et al. (1997) for more detailed results).
1997). This response matches the know physiology of this genus as an acetate utiliser (Widdel & Bak 1992), indicating that the Desulfobacter population at Site 4 was active and supports our hypothesis that this genus was the dominant acetoclastic SRB at this site.
185 We have similar results from slurry experiments on the River Colne estuary in the UK (KJP, unpublished data). Substantial Desulfobacter signal was detected for this group in the Colne estuary sediments in the UK (Figure 1). Addition of acetate caused a significant increase in signal from Desulfobacter in marine-dominated sediments from Colne Point. Thus it would appear that Desulfobacter represent an important group of SRB in marine-dominated estuarine sediments. The addition of propionate (and sulphate) led to significantly increased signal from a Desulfobulbus targeted probe in both the marine and freshwater ends of the R. Tama estuary (Figure 4b; Purdy et al. 1997). Again the increase in signal was rapid with substantial increases in signal occurring within 2 days and, as with Desulfobacter above, these results correlate with the known physiology of this genus as incomplete oxidisers of propionate (Widdel & Bak 1992). This suggests that where sulphate is available Desulfobulbus utilise propionate even if the site is usually a low-sulphate, freshwater environment. In slurries from the Colne estuary (which were not amended with propionate) the addition of molybdate, an inhibitor of sulphate reduction led to a reduction in the signal from Desulfobulbus in slurries from the marine-dominated sediments of Colne Point, indicating a dependence on sulphate reduction by Desulfobulbus in this site. However, at the freshwater-dominated end of the estuary the addition of H2 /CO2 led to a significant increase in signal from a Desulfobulbus targeted probe (KJP, unpublished data). No similar increase was detected in slurries amended with H2 /CO2 + sulphate. We have previously suggested that Desulfobulbus are ubiquitous in sediments (Purdy et al. 2001) and that this ubiquity is maintained by members of this genus having flexible phenotypes that are capable of utilising alternative electron acceptors to sulphate, such as nitrate or nitrite (Widdel & Pfennig 1982; Dalsgaard & Bak 1994), which these slurry data support. The slurry data from the R. Tama would suggest that Desulfobulbus are capable of utilising propionate where sulphate is freely available and the R. Colne slurries suggest that the freshwater Desulfobulbus population in the Colne appear to be physiologically adapted to life in a low sulphate environment and capable of utilising alternative electron acceptors to sulphate. It is not yet possible for us to determine whether the physiological flexibility of Desulfobulbus is due to the members of this genus all being flexible generalists or whether the flexibility is due to selec-
tion of phylogenetically and phenotypically distinct species or sub-species of Desulfobulbus. However, the data presented do leave a number of unanswered questions. We have, to date, failed to determine a specific environmental substrate for Desulfovibrio, an important SRB in the UK estuarine sediments. Similarly none of the substrates used has successfully enriched for Desulfobacterium. Isolated strains of Desulfobacterium are known to be able to utilise a wide range of substrates including short-chain fatty acids, hydrogen, alcohols and, in several cases, aromatic compounds and are able to utilise a range of alternative electron acceptors to sulphate, including nitrate and nitrite. Therefore it is possible that this group of flexible generalists use minor components within the carbon pool in sediments rather than compete for the more common substrates such as acetate or hydrogen. There is ample evidence to suggest that co-metabolism of a range of substrates can be competitively advantageous especially under nutrient-limited conditions (see Egli (1995) and References therein). Therefore it may be that versatile organisms like Desulfobacterium co-metabolise a range of substrates and that this explains why they often represent a significant proportion of the standing community but do not appear to respond to the single substrate additions discussed here. Boschker et al. (1998, 2001), using stable isotope labeling of phospholipid fatty acids (PLFA), have suggested that Desulfobacter are not responsible for acetate utilisation in marine sediments, linking this activity instead to Desulfotomaculum and Desulfofrigus, an acetate-utilising SRB related to Desulfococcus (Boschker et al. 1998, 2001; Knoblauch et al. 1999, 2001). Further work, preferably using both approaches on the same samples, is necessary to explore the basis for this apparent incongruence and to determine if it is biological or methodological. In addition to the above points we have yet to begin determining the ecological role of the large number of new SRB genera.
Conclusions The distribution of SRB populations appears to be very complex with a number of different distributions in a variety of sites. As stated above, the reasons for this could be many. A deterministic viewpoint would suggest that the differential distribution of these organisms is due the interaction of a wide variety of their phenotypic characteristics with the gross envir-
186 onmental factors that distinguish the different sites. However, Miskin et al. (2001) have argued that microbial communities may be assembled randomly with localised environmental factors substantially affecting the final steady state or perturbing a community from a previous steady state, therefore making the final composition of any community far less predictable. Determining how communities come together and then alter over time is a fundamental question in microbial ecology and this study highlights some of the issues that surround this idea. Linking specific groups of organisms to the use of particular substrates in the environment is an important step in understanding the link between microbial community structure and function. Here we show evidence linking the activity of Desulfobacter to sulphate-dependent acetate consumption in marinedominated estuarine sediments. Thus we hypothesise that this genus is the dominant acetoclastic SRB in high-sulphate estuarine sediments. Desulfobulbus appeared to require sulphate for growth in marinedominated sediments but not in freshwater-dominated sediments. This flexibility within a genus adds more complexity to the study of SRB, and by extension microbial distribution in general. Physiological flexibility within a genus will make the assignment of an ecological role to a detected organism extremely difficult. It is clear that SRB distribution and ecology is complex in terms of both the distribution of the different genera across different sites and their specific roles in the environment. However, the importance of these organisms to the global ecosystem and the relatively limited physiologies of many of the known SRB suggests that they do represent a good model for studying microbial ecology and may eventually allow us to derive valid theories about microbial distribution and activity in the environment. References Amann RI, Binder BJ, Olson RJ, Chisholm R, Devereux R & Stahl DA (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analysing mixed microbial populations. Appl. Environ. Microbiol. 56: 1619–1625. Amann RI, Ludwig W & Schleifer K-H (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59: 143–169. Balba MT & Nedwell DB (1982) Microbial metabolism of acetate, propionate and butyrate in anoxic sediments from the Colne Point saltmarsh, Essex, UK. J. Gen. Microbiol. 128: 1415–1422. Banat IM, Lindstrøm EB, Nedwell DB & Balba MT (1981) Evidence for coexistence of two distinct functional groups of
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