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Keywords:

  • sediment geochemistry;
  • sulphate reduction;
  • methanogenesis;
  • Colne;
  • DGGE ;
  • anaerobic methanotroph

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Detailed depth profiles of sediment geochemistry, prokaryotic diversity and activity (sulphate reduction and methanogenesis) were obtained along an estuarine gradient from brackish to marine, at three sites on the Colne estuary (UK). Distinct changes in prokaryotic populations [Archaea, Bacteria, sulphate-reducing bacteria (SRB) and methanogenic archaea (MA)] occurred with depth at the two marine sites, despite limited changes in sulphate and methane profiles. In contrast, the brackish site exhibited distinct geochemical zones (sulphidic and methanic) yet prokaryotic depth profiles were broadly homogenous. Sulphate reduction rates decreased with depth at the marine sites, despite nonlimiting sulphate concentrations, and hydrogenotrophic methanogenic rates peaked in the subsurface. Sulphate was depleted with depth at the brackish site, and acetotrophic methanogenesis was stimulated. Surprisingly, sulphate reduction was also stimulated in the brackish subsurface; potentially reflecting previous subsurface seawater incursions, anaerobic sulphide oxidation and/or anaerobic oxidation of methane coupled to sulphate reduction. Desulfobulbaceae, Desulfobacteraceae, Methanococcoides and members of the Methanomicrobiales were the dominant SRB and MA. Methylotrophic Methanococcoides often co-existed with SRB, likely utilising noncompetitive C1-substrates. Clear differences were found in SRB and MA phylotype distribution along the estuary, with only SRB2-a (Desulfobulbus) being ubiquitous. Results indicate a highly dynamic estuarine environment with a more complex relationship between prokaryotic diversity and sediment geochemistry, than previously suggested.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Estuarine sediments are an interface between the land and sea that receive nutrient input from both sources and consequently exhibit high levels of primary production and heterotrophic activity (Poremba et al., 1999). Characteristically, they also exhibit chemical gradients along their course, such as organic matter and nitrate concentrations decrease from the estuary head (river-end) to the estuary mouth (sea-end; Ogilvie et al., 1997), whilst the reverse is true for chloride and sulphate concentrations. In addition, estuarine sediments typically have depth-related geochemical gradients that form a distinct biogeochemical zonation of the main mineralisation processes (Canfield & Thamdrup, 2009). Oxygen is quickly depleted in surface sediments, leaving a primarily anoxic subsurface environment where prokaryotes degrade organic carbon using terminal electron acceptors of decreasing redox potential (inline image, Mn4+, Fe3+, inline image and CO2). This anaerobic organic matter degradation is controlled by groups of closely interacting microorganisms, including sulphate-reducing bacteria (SRB) and methanogenic archaea (MA). SRB obtain their energy by oxidising molecular hydrogen or organic compounds (e.g. short-chain fatty acids and aromatic compounds) whilst reducing sulphate to hydrogen sulphide. Dissimilatory sulphate reduction occurs in a physiologically and phylogenetically diverse range of prokaryotes within five bacterial and two archaeal phyla, but metabolically they fall into two groups, depending on whether or not they oxidise acetate (Muyzer & Stams, 2008). MA have three distinct methanogenic pathways: oxidation of molecular hydrogen coupled with CO2 reduction (hydrogenotrophic), utilisation of C1-compounds (methylotrophic) and utilisation of short-chain fatty acids (acetotrophic). There are seven recognised MA orders: Methanobacteriales, Methanococcales, Methanocellales, Methanomicrobiales, Methanosarcinales, Methanopyrales (Garcia et al., 2000) and Methanoplasmatales (Paul et al., 2012).

SRB and MA activity often occurs in discrete anoxic sediment niches that reflect redox zonation and availability of terminal electron acceptors (Canfield & Thamdrup, 2009). SRB generally outcompete MA in sediments for competitive substrates when sulphate is available, as the former have higher affinity and lower threshold values for hydrogen and acetate, whilst MA take over when sulphate nears depletion (Kristjansson et al., 1982; Lovley et al., 1982). Hence, the high concentration of sulphate in seawater makes sulphate reduction the major anaerobic process in marine sediments accounting for up to 50% of all organic matter degradation (Jørgensen, 1982). However, methylotrophic methanogenesis can co-occur in the presence of sulphate, as ‘noncompetitive’ methylated compounds are not metabolised by SRB (Oremland et al., 1982).

Estuaries, however, are physically dynamic, and this may have a major impact on the distribution of prokaryotes within sediments, which might not match the geochemical profiles. Despite this, it has been suggested that there are clear niches for different SRB and MA within estuarine sediments (Purdy et al., 2002; Hawkins & Purdy, 2007; Oakley et al., 2011), and this tends to be supported by sediment slurries and other experiments (Oakley et al., 2011). In this study, we further investigated these potential niches for SRB and MA in a detailed depth analysis of sediment geochemistry along an estuarine gradient at three distinct sites on the River Colne estuary (Essex, UK) and compared this with distributions of prokaryotes, particularly SRB and MA populations, and their activities. Sediment geochemistry and prokaryotic diversity were initially investigated (2005), whilst further activity measurements of the major anaerobic processes of sulphate reduction and methanogenesis (hydrogenotrophic and acetotrophic) were obtained from a separate sampling expedition (2006) to obtain additional information, along with additional geochemical measurements. The geochemical profiles obtained at the two sampling times were surprisingly similar, indicating that it was reasonable to make some comparisons between the two data sets.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Site description and sediment sampling

The Colne estuary is a small muddy macrotidal estuary on the east coast of England, which enters the North Sea at Brightlingsea (BR), Essex (Ogilvie et al., 1997; Fig. 1). Sediment cores were collected from three sites along the Colne estuary on 25 October 2005 and 21 February 2006. Sample sites were an open mud creek at the estuary mouth (BR; 51°47.920′N, 001°01.075′E), a mid-estuarine creek [Alresford (AR); 51°50.716′N, 000°58.912′E] and a salt marsh at the estuary head [Hythe (HY); 51°52.687′N, 000°56.011′E]. Sediment temperatures were measured using a handheld thermistor thermometer model HH41 connected to a 400 series thermistor probe (Omega Engineering Ltd). Triplicate, 10 cm diameter and 30–60 cm long sediment cores were collected at each site and at each sampling time; cores were sealed with rubber bungs and transported to the laboratory on ice. All cores, with the exception of those used for measurement of rates of sulphate reduction and methanogenesis, were aseptically sectioned into 2 cm slices. Cores for molecular biological analysis were sectioned within 4 h of collection with sediment from the centre of each slice being aseptically transferred to sterile 50-mL plastic tubes for storage at −80 °C. Cores for enumeration of prokaryotic cells and biogeochemical analysis were stored at 4 °C prior to sectioning; sediment from the centre of each slice was taken using sterile 5-mL syringes with the Luer end removed.

image

Figure 1. Map showing the locations of BR, AR and HY on the River Colne estuary in Essex, UK. Scale 1 : 200 000 (PDF map), OS Strategi, Ordnance Survey UK, updated January 2011. Map created September 2011 using EDINA Digimap Ordnance Survey Service, http://edina.ac.uk/digimap.

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Enumeration of prokaryotic cells

Prokaryotic cells were enumerated with an acridine orange direct count (AODC) method (Parkes et al., 2000) for sediment samples collected in 2005. Sediment was preserved in serum vials containing filter-sterilised (0.2 μm) 4% formaldehyde in artificial seawater, followed by crimp sealing and vigorous mixing. Subsequently, subsamples were stained with a final concentration of 5 mg L−1 acridine orange, filtered onto a black polycarbonate membrane filter (0.2 μm; Nuclepore) and examined under an epifluorescence microscope (Axioskop; Zeiss, Germany). Three replicate filters were counted from each sample to minimise counting variance (Kirchman et al., 1982), and a minimum of 200 fields of view were counted per filter. Particle-associated and nonassociated cells were counted separately, and the former counts doubled to account for hidden cells (Goulder, 1977).

Geochemical analysis

Geochemical analyses were conducted on sediment collected in both 2005 and 2006. Methane gas was analysed by thoroughly mixing sediment with 10% KCl in sealed vials and storing at 20 °C overnight to allow equilibration. Headspace methane was then analysed using a modified Perkin Elmer Clarus 500 Natural Gas Analyser (NGA, Arnel Model 2101; Perkin Elmer LAS, UK), fitted with a flame ionisation detector. Gas samples were injected with a helium carrier gas and separated by three columns (two DC-200 on Chromosorb PAW and one ASAG). The system was calibrated with standard gases (Scott Speciality Gases, Plumsteadville, PA). Sediment water content was determined by transferring 10 cm3 of sediment into a preweighed glass flask, which was reweighed before and after freeze-drying (Mini Lyotrap; LTE Scientific, UK). Porewater was obtained by transferring fresh sediment into 50-mL volume plastic tubes flushed with oxygen-free nitrogen and centrifuged at 18 000 g for 20 min at 4 °C. Porewater pH was measured with a combination pH microelectrode (Mettler Toledo, UK). Filtered porewater (0.2 μm) was analysed with an ICS-2000 ion chromatography system equipped with an AS50 autosampler (Dionex®, UK) to quantify chloride, sulphate, acetate and lactate concentrations. Chromatographic separation was conducted on two Ionpac AS15 columns in series, and the determination of species was carried out using an Anion Self-Regenerating Suppressor (ASRS-ULTRA II 4-mm) unit in combination with a DS6 heated conductivity cell. The gradient programme was as follows: 6 mM KOH (38 min), 16 mM KOH min−1 to 70 mM (17 min), 64 mM KOH min−1 to 6 mM (12 min). Limits of detection for sulphate and acetate were c. 50 and c. 5 μM, respectively. A final porewater subsample (5 mL) was used for determination of dissolved inorganic carbon (DIC) concentration (for use in activity calculations) as follows: porewater was sealed in glass vials with rubber stoppers and aluminium crimps, the contents acidified with concentrated HCl to pH 2, and the headspace analysed for CO2 on the NGA.

Prokaryotic activity measurements

Potential rates of sulphate reduction and methanogenesis (acetotrophic and hydrogenotrophic) were determined for sediment collected in 2006, to supply additional information to clarify and explain the links between geochemistry and biodiversity. Sediment minicores of 2 cm diameter and 20 cm length were injected with 35S-sulphate, 14C-acetate or 14C-bicarbonate (GE Healthcare, UK). Injections were administered laterally at 2 cm depth intervals through silicone-sealed 1 mm ports. The cores were then incubated under anoxic conditions inside N2-flushed gas-impermeable sealed bags at 4 °C for 6 h (14C-acetate) or 18 h (14C-bicarbonate and 35S-sulphate), after which they were cut into 2 cm slices and transferred to glass vials. These vials contained either 10 mL 20% zinc acetate (sulphate reduction) or 10 mL 1 M NaOH (methane production) to stop activity and trap products (sulphate reduction); the vials were then sealed and mixed thoroughly. Reduced sulphur was determined using a cold distillation procedure (Kallmeyer et al., 2004). Radio-labelled methane was determined by purging the headspace with a mixture of nitrogen and oxygen (95 : 5) through a copper oxide furnace (Carbolite, UK) at 800 °C to oxidise 14C-methane to 14C–CO2. The gas was then passed through a scintillation cocktail (OptiPhase ‘HiSafe’ 3; Perkin Elmer) containing 0.7% (v/v) β-phenylethylamine (Sigma, UK) to absorb the 14C–CO2. Radioactivity was quantified in a Tri-Carb 2900TR liquid scintillation counter (Packard, UK). Rates of methanogenesis and sulphate reduction were calculated according to the proportion of radiotracer converted to products, incubation time and total sedimentary concentrations of acetate, DIC and sulphate, respectively (Parkes et al., 2005).

DNA extraction and purification

Total community genomic DNA was extracted from sediment collected in 2005 using the FastDNA Spin Kit for Soil (MP Biomedicals) incorporating the procedural modifications described by Webster et al. (2003). DNA extractions were performed on every other 2 cm samples using c. 0.75 g sediment in duplicate; this DNA was then pooled prior to purification by dialysis in Microcon YM-100 centrifugal filter devices (Millipore, MA) and elution in 100 μL sterile molecular biology grade water (Severn Biotech, Kidderminster, UK). Extracted sediment DNA was quantified using an Ultrospec® 2100 pro (Amersham Pharmacia Biotech) spectrophotometer.

PCR conditions

All PCR reactions were performed under aseptic conditions and included appropriate positive (+DNA) and negative (−DNA) control reactions. When nested PCRs were performed, 1 μL of the first round product was used as template for the second-round PCR (including negative control), prior to visualisation by 1.2% (w/v) agarose gel electrophoresis. PCR amplifications were carried out using a Primus 96 plus (Eurofins MWG Operon), a PTC200 DNA Engine (MJ Research, Boston) or a Dyad DNA Engine (MJ Research) thermal cycling machine. All PCR primers and cycling parameters for 16S rRNA gene, dissimilatory sulphite reductase (dsrA/B) and methyl coenzyme M reductase (mcrA) genes are described in Supporting Information, Table S1. Primers were synthesised at Eurofins MWG Operon Biotech; and standard 50-μL PCR reactions contained 1.5 mM MgCl2, 200 μm each dNTP (Promega), 200 nM each primer, 2 U Taq DNA polymerase (Promega) and 1 μL DNA template in 1X PCR buffer (Promega). PCR reactions with environmental sediment DNA template also contained 10 μg bovine serum albumin (BSA; Promega).

Group 1–6 SRB-specific 16S rRNA gene PCRs (Daley et al., 2000; Scholten et al., 2005) targeted Desulfotomaculum (group 1), Desulfobulbus (group 2), Desulfobacterium (group 3), Desulfobacter (group 4), Desulfonema, Desulfosarcina and Desulfococcus (group 5) and Desulfovibrio and Desulfomicrobium (group 6). MA-specific 16S rRNA genes PCRs (Banning et al., 2005) targeted the orders Methanomicrobiales (Mm), Methanosarcinales (Ms), Methanobacteriales (Mb) and Methanococcales (Mc). Note the SRB group 3 PCR was not utilised further in this study because a product could not be amplified from Desulfobacterium macestii DSM 4194-positive control DNA. In addition, although the SRB group 6 and Mc PCRs amplified products from Desulfovibrio desulfuricans DSM1924 and Methanocaldococcus jannoshii DSM 2661, respectively, only nontarget DNA was amplified from sediment DNA presumably due to low concentration or absence of these groups in the sediments analysed.

DGGE analysis

DGGE analysis was used to profile bacterial and archaeal sediment populations to investigate diversity changes (with depth) between the three sampling sites. 16S rRNA gene PCR-DGGE was conducted as previously described (Webster et al., 2006), with all gels run under identical conditions with appropriate standardised markers on each gel. General bacterial DGGE profiles were produced from PCR products generated directly with primers 357F-GC and 518R (Muyzer et al., 1993; Table S1). General archaeal DGGE profiles were produced from nested PCRs with primers 109F and 958R (Delong, 1992; Grosskopf et al., 1998) followed by SAf and PARCH519r (Øvreas et al., 1997; Nicol et al., 2003; Table S1). Group 1–6 SRB-specific profiles were produced from a variation on the three-step nested-PCR-DGGE strategy described by Dar et al. (2005). A two-step strategy was employed to produce SRB-specific DGGE PCR products as follows: SRB-specific PCRs (group 1–6; Daley et al., 2000) were performed directly on diluted sediment DNA, and the resultant product was used as template with primers 357F-GC and 518R. Mm/Ms-specific DGGE profiles were produced from nested PCRs with primers 355F and 1068R (Banning et al., 2005) followed by 355F-GC and PARCH519r (Table S1). Mb-specific DGGE profiles were produced from nested PCRs with primers 109F and 1401R (Banning et al., 2005) followed by SAf and PARCH519r. Mc-specific DGGE profiles were generated from nested PCRs with primers 344F and 1202R (Banning et al., 2005) followed by 344F-GC and PARCH519r (Table S1). Bacterial and archaeal DGGE profiles were analysed as described by Fry et al. (2006), and agglomerative cluster analysis was carried out using the software community analysis package version 3.1 (Pisces Conservation Ltd). Dendrograms were constructed by average linkage and average distance method (Webster et al., 2007).

Sequencing and phylogenetic analysis

Individual bands were excised from DGGE gels, re-amplified and sequenced (O'Sullivan et al., 2008). SRB DGGE bands were re-amplified with primers 357F-GC-M13R and 518R-AT-M13F, Mm/Ms bands with 355F-GC-M13R and PARCH519r-AT-M13F, and Mc bands with SAf-GC-M13R and PARCH519r-AT-M13F (Table S1). Re-amplified bands were sequenced with primer M13F (Table S1) and grouped into phylotypes at the ≥ 98% sequence similarity level. Sequencing of DGGE bands was carried out on an ABI PRISM 3130xl Genetic Analyser (Applied Biosystems, Foster City, CA), using BigDye terminator chemistry. Chromatographs were viewed and edited using Chromas lite software version 2.01 (http://www.technelysium.com.au). DGGE band closest 16S rRNA gene sequence matches were identified using the nucleotide blast (blastn) program at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov).

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Sediment along a gradient from brackish (HY) to marine (BR and AR) on the Colne estuary (UK) was analysed for prokaryotic diversity and activity to depths (> 30 cm) greater than in previous studies, which mainly focused on the top 0–5 cm depth horizon (Hawkins & Purdy, 2007; Kondo et al., 2007; Oakley et al., 2010, 2011). Cluster analysis of DGGE depth profiles at the individual sites (Fig. S1) indicated that at BR and AR, there were clear changes in the community composition with depth for both Bacteria and Archaea, demonstrating the importance of analysing sediment depth profiles rather than only near-surface sediments. By contrast, HY showed no major changes in bacterial or archaeal DGGE profiles with sediment depth.

Prokaryotic cell numbers

Sediment depth distributions of AODC showed that total cell numbers generally decreased from the estuary head (HY) to the estuary mouth (BR) and also decreased with depth at all sites (Figs 2-4). Cell distributions at the marine sites BR and AR were within the range reported for many marine sediments (Parkes et al., 2000), whereas those at the brackish site, HY were substantially higher reaching a maximum of 1.6 × 1010 cells cm−3 at 6 cm depth (Figs 2-4). These high cell numbers could be explained by the position of HY near the head of the hyper-nutrified Colne estuary, where sediments receive high organic matter input from the surrounding land (Ogilvie et al., 1997).

image

Figure 2. Depth profiles of prokaryotic cell numbers, geochemical data and prokaryotic activity for BR sediment. (a) Prokaryotic cell numbers (AODC) – the solid and dotted lines, respectively, show a general model for prokaryotic cell distributions in marine sediments with associated 95% prediction limits (Parkes et al., 2000); (b) temperature and porewater chloride concentration; (c) wet sediment methane concentration and porewater sulphate concentration; (d) porewater acetate and lactate concentrations; (e) potential rates of sulphate reduction and methanogenesis from acetate and H2/CO2. Black and grey plots relate to data obtained from sediment collected in 2005 and 2006, respectively.

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image

Figure 3. Depth profiles of prokaryotic cell numbers, geochemical data and prokaryotic activity for AR sediment. All other details are as per legend for Fig. 2.

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image

Figure 4. Depth profiles of prokaryotic cell numbers, geochemical data and prokaryotic activity for HY sediment. All other details are as per legend for Fig. 2.

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Sediment geochemistry

Depth profiles of chloride, sulphate, methane, acetate and lactate concentrations, and temperature are shown in Figs 2-4. Mean sediment chloride and sulphate concentrations decreased from the estuary mouth (BR) to the estuary head (HY) as expected. Chloride concentrations decreased only slightly with depth at BR and AR, but were more variable at HY and even increased slightly with depth in 2006. Sulphate concentrations decreased with depth at all three sites, and at HY sulphate, concentrations were reduced to < 1 mM between c. 20 and 25 cm, nearing the potentially limiting concentration for sulphate reduction (Boudreau & Westrich, 1984; Lovley & Klug, 1986). Methane concentrations were consistently very low at all depths analysed at BR (Fig. 2c) and AR (Fig. 3c), but were much higher at HY for all depths analysed (Fig. 4c). There was no sulphate-methane transition zone (SMTZ) at either BR or AR, with low levels of CH4 being present even in the sulphate-containing zone, and sulphate was not depleted at the depths studied. By contrast, at HY in October 2005, there was a diffuse SMTZ between c. 10 and 20 cm where sulphate and methane profiles intersected (Fig. 4c), and at HY in February 2006, there was a defined SMTZ at c. 22–24 cm (Fig. 4c). Differences between the geochemical parameters observed in sediments collected at these two time points were surprisingly small, despite an average temperature difference of 8 °C (Figs 2b, 3b and 4b), suggesting that it was reasonable to compare the data sets in terms of understanding prokaryotic processes influencing geochemical profiles.

Prokaryotic sulphate reduction and methanogenesis activities

Depth profiles of sulphate reduction rates were similar for BR (Fig. 2e) and AR sediment (Fig. 3e); however, rates at HY were generally higher despite the fact that sulphate concentrations at this brackish site were significantly lower (Fig. 4e). At HY, there was a peak in sulphate reduction rate in the first 2 cm, and at c. 30 cm sulphate reduction rates increased to a maximum of 132.6 nmol cm−3 day−1 and remained elevated to the bottom of the core. Measurements of high rates of sulphate reduction in low-sulphate sediments at the estuary head compared with lower rates in high-sulphate sediments at the estuary mouth were previously noted in the Colne estuary (Nedwell et al., 2004; Kondo et al., 2007). This was attributed to lower availability of labile carbon in BR and AR or could be due to freshwater-adapted SRB having a lower threshold concentration for sulphate uptake than marine-adapted SRB (Ingvorsen & Jørgensen, 1984). Although it is widely considered that sulphate does not limit sulphate reduction at concentrations > 3 mM (Boudreau & Westrich, 1984), some studies suggest that sulphate reduction can occur without restriction at much lower concentrations (< 1 mM; Wilms et al., 2007), even down to a threshold of 30 μM sulphate in freshwater sediments (Lovley & Klug, 1986).

Depth profiles of rates of acetotrophic and hydrogenotrophic methanogenesis indicated relativity low levels of activity for BR (Fig. 2e) and AR (Fig. 3e), consistent with this being the sulphate reduction zone. Rates of methanogenesis at HY were comparable with those observed at BR and AR from 0 to 20 cm, but rates increased significantly at depths > 20 cm (Fig. 4e), to maxima of 1063.6 pmol cm−3 day−1 (acetotrophic) and 194.9 pmol cm−3 day−1 (hydrogenotrophic), which was consistent with increased methane concentrations. The position of the 2006 HY SMTZ (c. 22–24 cm) is consistent with the relatively high rates of sulphate reduction together with extremely low rates of methanogenesis down to c. 25 cm, followed by high rates of methanogenesis below c. 25 cm.

Interestingly, at BR and AR, where the subsurface peaks in rates of sulphate reduction were not evident, there were distinct subsurface peaks of active methanogenesis, particularly hydrogenotrophic methanogenesis (Figs 2e and 3e), again despite the presence of nonlimiting sulphate concentrations. By contrast, at the more freshwater HY site, acetotrophic methanogenesis was stimulated in deeper layers (Fig. 4e). This dominance of acetotrophic methanogenesis in more freshwater anoxic sediments and hydrogenotrophic methanogenesis in marine sediments is a general observation suggested by the 13C content of generated CH4 (Whiticar, 1999). This difference is usually explained by the activity of more competitive SRB in marine systems making surviving organic matter less prone to produce acetate on further degradation. However, our results for the more freshwater HY site do not fit with this explanation, as near-surface sulphate reduction rates were comparable with those at BR and AR (Fig. 4e), whereas surviving organic matter still supported the dominance of acetotrophic over hydrogenotrophic methanogenesis in deeper sediment. This observation suggests that factors other than absence of sulphate reduction are resulting in subsurface acetotrophic methanogenesis in more freshwater sediments.

Diversity of SRB and MA in Colne estuary sediment

Colne sediment was analysed using taxon-specific 16S rRNA gene and functional gene primers for sulphate reducers (dsrA and dsrAB) and methanogens (mcrA; Tables 1 and 2). Amplification of dsrA/B gene sequences was successful from nearly all sediment samples, and most samples gave a positive result for at least one SRB-specific 16S rRNA gene PCR (Table 1). The mcrA gene was amplified from most sediment samples, and all samples gave a positive result for at least one MA-specific 16S rRNA gene PCR (Table 1). SRB- and MA-specific 16S rRNA gene DGGE depth profiles (0–30 cm) were also obtained at each sampling site for SRB groups 2 (Fig. 5a), 4 (Fig. 5b), 5 (Fig. 5c) and Mm/Ms (Fig. 6). PCR products and DGGE profiles were not obtained for SRB group 1, SRB group 6, Mb and Mc due to their apparent low abundance (or absence) in sediment samples. Results obtained via specific PCR screening (Table 1) and DGGE profiling (Figs 5 and 6) were very similar with only a few minor differences, probably explained by different primer pair combinations used for PCR screening (footnotes for Table 1) and DGGE ('Materials and methods').

Table 1. Presence or absence of SRB and MA in Colne estuary sediment, as inferred by amplification of marker genes (16S rRNA, dsrA and mcrA) by direct and nested PCR
Target geneColne estuary sampling site and sediment depth (cm)
BrightlingseaAlresfordHythe
261014182226302610141822263026101418222630
  1. image_n/fem12106-gra-0001.png, detected by direct PCR; image_n/fem12106-gra-0002.png, detected by nested PCR; , detected but products subsequently shown to be non-specific; , not detected.

  2. Note, the order of primer pairs utilised in this table is not the same as those used to generate PCR-DGGE profiles (see Figs 5 and 6, Fig. S1).

  3. a

    Direct PCR with 357F-GC/518R.

  4. b

    Direct PCR with DFM140/DFM842; nested PCR with 27F/1492R and DFM140/DFM842.

  5. c

    Direct PCR with DBB121/DBB1237; nested PCR with 27F/1492R and DBB121/DBB1237.

  6. d

    Direct PCR with DSB127/DSB1273; nested PCR with 27F/1492R and DSB127/DSB1273.

  7. e

    Direct PCR with DCC305/DCC1165; nested PCR with 27F/1492R and DCC305/DCC1165.

  8. f

    Direct PCR with DSV230/DSV838; nested PCR with 27F/1492R and DSV230/DSV838.

  9. g

    Direct PCR with DSR-1F/DSR-4R or DSR-1F+/DSR-R; nested PCR with DSR-1F/DSR-4R and DSR-1F+/DSR-R.

  10. h

    Direct PCR with 109F/958R.

  11. i

    Direct PCR with 355F/1068R; nested PCR with two rounds of primers 355F/1068R.

  12. j

    Direct PCR with 109F/1401R; nested PCR with two rounds of primers 109F/1401R.

  13. k

    Direct PCR with 344F/1202R; nested PCR with two rounds of primers 344F/1202R.

  14. l

    Direct PCR with ME1f/ME2r or MLf/MLr; nested PCR with primers ME1f/ME2r and MLf/MLr.

Bacteria 16S rRNAa + + + + + + + + + + + + + + + + + + + + + + + +
SRB group 1 16S rRNAb
SRB group 2 16S rRNAc + + + + + + + + + + + + + +
SRB group 4 16S rRNAd + + + + + + + + + + + + + + +
SRB group 5 16S rRNAe + + + + + + + + + + + + + + + + + + + + + +
SRB group 6 16S rRNAf
dsrA/dsrAB g + + + + + + + + + + + + + + + + + + + + + +
Archaea 16S rRNAh + + + + + + + + + + + + + + + + + + + + + + + +
Mm/Ms 16S rRNAi + + + + + + + + + + + + + + + + + + + + + + + +
Mb 16S rRNAj
Mc 16S rRNAk
mcrA l + + + + + + + + + + + + + + + + + + + +
Table 2. Closest cultured and overall nucleotide sequence matches to excised 16S rRNA gene DGGE bands grouped into phylotypes at the ≥ 98% sequence similarity level, as predicted by the NCBI nucleotide blast program
PhylotypeNumber of sequencesSequence length (bp)Closest cultured relative (accession number)% SimilarityClosest 16S rRNA gene sequence (accession number); environmental origin% Similarity
SRB group 2 (Fig. 5a)
SRB2-a12194Desulfobulbus mediterraneus (NR_025150)93Clone Red_74 (DQ345703); deep mine99
SRB2-b4194Desulfobulbus mediterraneus (NR_025150)98Desulfobulbus mediterraneus (NR_025150)98
SRB2-c4194Desulfobulbus mediterraneus (NR_025150)96Clone 31 EDB2 (AM882629); contaminated coastal sediment97
SRB2-d2194Desulfobulbus rhabdoformis (AB546248)94Clone GoM_SMBush4593_Bac56 (AM404380); marine sediment97
SRB2-e1194Desulfobulbus rhabdoformis (AB546248)95Clone 31 EDB2 (AM882629); contaminated coastal sediment100
SRB2-f1194Desulfobulbus rhabdoformis (AB546248)96Clone cs28 (DQ088251); moat sediment100
SRB2-g1194Desulfobulbus rhabdoformis (AB546248)95Clone HDBW-WB29 (AB237692); deep subsurface groundwater98
SRB2-h1195Desulfobulbus japonicus (AB110550)95Clone GUP4D10 (HQ178753); sediment treated with glycerol99
SRB group 4 (Fig. 5b)
SRB4-a4194Similarity ≤ 91%Clone SIMO-2089 (AY711455); salt marsh100
SRB4-b3194Desulfobacter postgatei (NR_028830)99Clone F5K2Q4C04JVUG3 (GU912884); activated sludge98
SRB4-c2194Desulfobacter hydrogenophilus (FR733673)97CloneB8_10.4_2 (FJ717001); marine sediment99
SRB4-d2194Desulfobacula phenolica (NR_025350)97Clone 70mos_0d_F12 (GQ261767); deep sea sediment97
SRB4-e1194Desulfotignum toluenicum (EF207158)97Isolate JS_SRB250Ace (AM774316); tidal flat sediment98
SRB4-f1194Desulfotignum phosphitoxidans (AF420289)96Clone Fe_B_121 (GQ356938); methane seep sediment100
SRB4-g1194Desulfobacter postgatei (NR_028830)96Clone 13C16S1-53 (AM118004); tidal sediment slurry100
SRB4-h1194Desulfobacter postgatei (NR_028830)97Clone OXIC-015 (JF344285); marine oil polluted sediments99
SRB4-i1194Desulfobacula phenolica (NR_025350)95Desulfobacula sp. clone CB29 (DQ831540); estuarine sediment100
SRB4-j1194Desulfobacter halotolerans (NR_026439)95Desulfobacter halotolerans (NR_026439); salt lake95
SRB4-k1194Desulfobacula phenolica (NR_025350)94Desulfobacula clone GF1-21 (FN423830); brackish sediment96
SRB4-l1194Desulfobacter curvatus (NR_041851)95Clone NZ_309_Bac88 (JF268415); marine methane seep sediment97
SRB4-m1194Desulfobacter curvatus (NR_041851)95Clone GoC_Bac_63_D1_C0_M0 (FN820309); mud volcano sediment97
SRB4-n1194Desulfobacter curvatus (NR_041851)96Desulfobacter curvatus (NR_041851)96
SRB group 5 (Fig. 5c)
SRB5-a7194Desulfosarcina variabilis (NR_044680)93Clone TRAN-057 (JF344473); polluted marine sediment97
SRB5-b4194Desulfosarcina ovata (NR_037125)95Clone H105aug-118 (AB429835); marine sediment98
SRB5-c4194Desulfosarcina ovata (NR_037125)92Clone OXIC-137 (JF344407); polluted marine sediment100
SRB5-d4194Desulfosarcina cetonica (NR_028896)94Clone BS1-0-78 (AY254948); tidal flat sediment96
SRB5-e3194Desulfosarcina variabilis (NR_044680)94Clone ANOX-143 (JF344705); polluted marine sediment100
SRB5-f3194Desulfonema magnum (NR_025990)92Clone bOHTK-74 (FJ873365); methane rich cold seep sediment99
SRB5-g2194Desulfosarcina variabilis (NR_044680)94Clone SIMO-1690 (AY711056); salt marsh sediment99
SRB5-h2194Desulfobacterium indolicum (NR_028897)96Clone SIMO-1708 (AY711074); salt marsh sediment98
SRB5-i1194Desulfosarcina ovata (NR_037125)95Clone B1-86 (FJ175523); methane seep sediment99
SRB5-j1194Desulfofaba gelida (NR_028730)97Desulfofaba gelida strain PSv20 (NR_028730); arctic marine sediment97
SRB5-k1193Desulfobacterium indolicum (NR_028897)93Clone Pink_2G10 (GQ483980); intertidal thrombolites95
SRB5-l1194Desulfonema magnum (NR_025990)92Clone 1′C10 (AM889191); lagoon sediment100
SRB5-m1195Desulfobacterium indolicum (NR_028897)95Desulfobacteraceae clone ZLL-A87 (JF806819); river sediment98
SRB5-n1194Deltaproteobacterium strain AK-P (AY851291)94Clone ANOX-081 (JF344643); polluted marine sediment100
SRB5-o1194Desulfosarcina variabilis (NR_044680)94Clone H105aug-118 (AB429835); marine sediment97
SRB5-p1194Desulfosarcina cetonica (NR_028896)93Clone SIMO-1439 (AY710879); salt marsh sediment98
Mm/Ms (Fig. 6)
MmMs-a5137Similarity ≤ 91%ANME2a clone 2MT7 (AF015991); estuary sediment99
MmMs-b5137Similarity ≤ 91%CSG-1 clone MOBKM43T9523 (AM942133); subsurface marine sediment99
MmMs-c4135Similarity ≤ 91%RCV clone LAa32.25 (EU750852); meromictic arctic lake91
MmMs-d1137Methanococcoides methylutens (FR733669)96Clone SBC12BA090 (AB561491); deep sea sediment96
MmMs-e1137Methanococcoides methylutens (FJ477324)94Clone GUAY_37enr_Arch24 (FR682486); marine sediment94
MmMs-f1137Methanococcoides methylutens (FJ477324)98Clone GUAY_37enr_Arch24 (FR682486); marine sediment98
MmMs-g1137Methanospirillum hungatei (AB517987)93Clone VE07-10-ARC (GQ340380); reservoir water94
image

Figure 5. DGGE profiles of prokaryote 16S rRNA gene diversity in BR, AR and HY sediment on the Colne estuary: (a) SRB group 2 (Desulfobulbus), (b) SRB group 4 (Desulfobacter) and (c) SRB group 5 (Desulfonema, Desulfosarcina and Desulfococcus). The order of primer pairs utilised to generate the DGGE profiles shown here (see 'Materials and methods') is not always the same as the order used for PCR screening (see Table 1 footnotes). DGGE band labels are described in Table 2. DGGE depth profiles are shown in the following order from left to right: BR, AR and HY. Sediment depth in centimetre is shown above the lane (2 cm depth refers to the sediment section 0–2 cm).

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image

Figure 6. DGGE profiles of Methanomicrobiales and Methanosarcinales (Mm/Ms) 16S rRNA gene diversity in BR, AR and HY sediment on the Colne estuary. All other details are as per legend for Fig. 5.

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Visual inspection of SRB- and MA-specific DGGE profiles revealed very obvious differences in community structure, both with depth and between sites. Population changes with depth (Figs 5 and 6) were broadly consistent with the prokaryote community depth changes shown by cluster analysis (Fig. S1). At BR, SRB groups 2 (Fig. 5a) and 4 (Fig. 5b) were detected above 6 cm but not below, which corresponded with a general change in the bacterial and archaeal community (Fig. S1). The general community composition of the top 10 cm (Archaea) to 14 cm (Bacteria) at AR was distinct from deeper sediment layers, and this discontinuity was also clearly seen for SRB groups 2 (Fig. 5a), 4 (Fig. 5b), 5 (Fig. 5c) and Mm/Ms (Fig. 6). However, at HY, there were no major depth-related population shifts for SRB, MA, Bacteria or Archaea (Figs 5 and 6 and Fig. S1).

SRB diversity

SRB of the families Desulfobulbaceae (SRB group 2, 31% of SRB determined sequences) and Desulfobacteraceae (SRB groups 4 and 5, 69% of determined SRB sequences) were detected at all three sites and seem to be ubiquitous members of the SRB populations in Colne sediment. All SRB group 2 phylotypes were most closely related to the genus Desulfobulbus as expected (Fig. 5a; Table 2). Desulfobulbus was previously found to have a wide distribution in Colne estuary sediment (Nedwell et al., 2004; Kondo et al., 2007; Oakley et al., 2011), perhaps reflecting their physiological flexibility and ability to use a range of organic substrates and electron acceptors, disproportionate sulphur and grow by fermentation (Purdy et al., 2001). SRB group 4 phylotypes (Fig. 5b; Table 2) were most closely related to the genera Desulfobacter, Desulfobacula and Desulfotignum. SRB group 5 phylotypes (Fig. 5c; Table 2) were most closely related to the genera Desulfosarcina, Desulfofrigus, Desulfofaba, Desulfosalina, Desulfoluna, Desulfonema, Desulfococcus and Desulfobacterium. Desulfobacteraceae were also previously found to be important members of the SRB sediment community in the Colne (Nedwell et al., 2004; Kondo et al., 2007) and other estuaries (Purdy et al., 2001; Leloup et al., 2006). SRB group 1 was not detected in any of the marine and brackish sites in this study, possibly due to the low salt concentrations required for optimal growth of many Desulfotomaculum species (Widdel, 2006). Similarly, Kondo et al. (2007) only detected Desulfotomaculum in Colne freshwater sediments (East Hill Bridge and the Weir). Leloup et al. (2006) also found Desulfotomaculum dominant in freshwater, but not marine (mixing-zone) mudflats of the Seine estuary. SRB group 6 (Desulfovibrio and Desulfomicrobium) was not detected in this study and was only previously detected to a limited extent in the Colne (Nedwell et al., 2004; Kondo et al., 2007) and other estuaries (Purdy et al., 2001; Leloup et al., 2006).

MA diversity

The diversity of MA in Colne sediment at all sites studied was relatively low. Methanococcales and Methanobacteriales were apparently absent, as previously reported by Purdy et al. (2002). Four of the seven Mm/Ms DGGE phylotypes (Fig. 6) were affiliated with the orders Methanosarcinales (MmMs-d, e and f; Table 2) and Methanomicrobiales (MmMs-g; Table 2). Methanosarcinales phylotypes were related to the genus Methanococcoides containing obligately methylotrophic methanogens (Kendall & Boone, 2006) and were found only sporadically at BR and AR (Fig. 6). Methanomicrobiales phylotype MmMs-g was related to the H2/CO2 or formate-utilising genus Methanospirillum (Garcia et al., 2006). Presence of MmMs-g only at 30 cm at HY corresponded with an increase in methanogenic activity below c. 30 cm sediment (Fig. 4e). The remaining three Mm/Ms phylotypes were affiliated with Euryarchaeota not known to be methanogens. MmMs-a affiliated with anaerobic methanotrophs ANME2a and appeared to be present at all three Colne estuary sites (Fig. 6). Anaerobic methanotrophs (ANME) conduct anaerobic oxidation of methane (AOM) with sulphate as the final electron acceptor and are often active at the SMTZ where both sulphate and methane are present (Knittel & Boetius, 2009). Phylotype MmMs-b affiliated with a deep branching Euryarchaeota cluster designated the Coastal Sediment group-1 (CSG-1; Roussel et al., 2009) and were sporadically detected at BR and AR (Fig. 6). Phylotype MmMs-c was novel having only 91% sequence similarity to its closest environmental match (EU750852) and probably belonged to the unclassified order Rice Cluster V (RCV). It dominated at AR below 16 cm (Fig. 6) and coincided with peaks of hydrogenotrophic methanogenesis (Fig. 3e).

Therefore, MA in Colne sediments above 30 cm depth were mainly populations of methylotrophic Methanococcoides that probably exist by utilising noncompetitive simple substrates such as methanol and methylated amines that most SRB cannot use (Oremland et al., 1982). Hydrogenotrophic and acetotrophic MA were probably outcompeted by SRB for common substrates above 30 cm where sulphate was readily available. Parkes et al. (2012) found that low methane and high-sulphate concentrations also occurred in near-surface sediments in Arne Peninsula salt marsh creek sediments, and this was attributed to the dominance of methylamine-utilising Methanosarcinales. Overall, our study suggests less MA diversity than reported by Purdy et al. (2002). Likely reasons for this difference are as follows: (1) differences in PCR primer sets used between the two studies; (2) only the most dominant phylotypes are detected by DGGE analysis; and (3) the sites investigated in the two studies were not identical. Despite this, Methanococcoides sequences were common to both studies, suggesting an important role for this methylotrophic methanogen.

SRB and MA phylotype distribution along the estuary

There were also differences in the distribution of phylotypes between the three Colne estuary sites (Figs 5 and 6; Table 3). This was most obvious for SRB group 2 (Desulfobulbus) where phylotype SRB2-a were present at all three sites, indicating a ubiquitous distribution, whilst SRB2-b, SRB2-c and SRB2-d were only found in HY, AR and BR, respectively. Also, phylotypes SRB5-e and SRB5-f were restricted to BR; SRB4-c, SRB5-g and MmMs-c were restricted to AR; whilst SRB4-a, SRB4-b, SRB4–d, SRB5-a and SRB5-d were restricted to HY. Discrete Desulfobulbus genotypes were also noted in other studies (Hawkins & Purdy, 2007; Oakley et al., 2010), and it was proposed that the genus Desulfobulbus populations are distributed according to salinity-driven niche separation along the Colne estuary. Thus, our results further support the hypothesis that distribution of some prokaryotic species (and speciation) is subject to similar factors controlling the distribution of metazoan and higher organisms and hence, that environmental niche partitioning and biogeography may also be applied to microbial communities (Oakley et al., 2010).

Table 3. Relative distribution of major SRB and MA phylotypes at three sites along the Colne estuary
PhylotypeNumber of sequences at Colne estuary site
BRARHY
  1. A phylotype refers to DGGE band sequences grouped at ≥ 98% similarity.

  2. Phylotypes with only one representative band/sequence were not included in this table.

  3. Filled boxes illustrate the presence of a phylotype at a particular sediment site.

SRB group 2 (Desulfobulbus)
SRB2-a246
SRB2-b  4
SRB2-c 4 
SRB2-d2  
SRB group 4 (Desulfobacter)
SRB4-a  4
SRB4-b  3
SRB4-c 2 
SRB4-d  2
SRB group 5 (Desulfonema, Desulfosarcina and Desulfococcus)
SRB5-a  5
SRB5-b22 
SRB5-c 22
SRB5-d  4
SRB5-e3  
SRB5-f3  
SRB5-g 2 
Mm/Ms (Methanomicrobiales and Methanosarcinales)
MmMs-a3 2
MmMs-b32 
MmMs-c 4 

Increased rates of both methanogenesis and sulphate reduction below c. 25 cm at HY

At HY, there was a surprising and substantial increase in the rates of not only methanogenesis, but also sulphate reduction, below c. 25 cm (Fig. 4e) where sulphate concentrations were low (< 1 mM) and methane concentrations increased. MA and SRB were both detected in the sediment at this depth, although the only obvious change in DGGE profiles was the appearance of phylotype MmMs-g (Fig. 6; Table 2). SRB and MA can share niches in anoxic habitats rich in organic matter (Oremland et al., 1982), but the increased activity below 25 cm correlated with a general reduction in volatile fatty acid concentrations (Fig. 4d). Four possible explanations for the increased activities are as follows: (1) sulphate might be provided by the anoxic oxidation of sulphides (cryptic sulphur cycle), with the resultant sulphate rapidly being consumed by SRB so that it remains at low concentrations in the porewater (Blazejak & Schippers, 2011), but it is unclear what the oxidants might be and why they are present below 25 cm; (2) vertical transport of oxygen into the sediment via plant roots could facilitate rapid sulphide oxidation and thus sulphate reduction (Sand-Jensen et al., 1982); (3) SRB in situ might be using alternate electron acceptors such as Fe(III) (Coleman et al., 1993); and (4) HY sediments are subject to periodic penetration of tidal water and, hence, an increased supply of sulphate (Nedwell et al., 2004) or possibly causing both sulphide and ferrous oxidation. Although chloride concentrations were low in 2006 (Fig. 4b), there was a slight increase with depth, consistent with previous seawater penetration. Such regular events could maintain and stimulate the SRB population resulting in significant 35S-sulphate reduction in assays, despite low sediment porewater sulphate concentrations at the time of sampling (Fig. 4c).

Interestingly, the HY SMTZ located at c. 22–24 cm in 2006 (Fig. 4c) had the typical concave distribution that is characteristic for AOM (Knittel & Boetius, 2009). Therefore, it is also possible that AOM coupled to sulphate reduction by ANME and SRB consortia may explain co-occurrence of sulphate reduction and methanogenesis via CH4 cycling (Parkes et al., 2007). Both ANME-2a (MmMs-a; Fig. 6) and SRB group 5 (Fig. 5c) were prominent in DGGE profiles at HY, with the latter containing the key genera Desulfosarcina and Desulfococcus commonly associated with ANME-2 (Knittel & Boetius, 2009).

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Various factors may influence the complex inter-related biodiversity of sedimentary prokaryotic communities along the Colne estuary, including microbial physiology, nutrient availability, salinity gradients, geochemistry and sedimentology. Consistent with this, we found clear changes in the sediment prokaryote community (Bacteria, Archaea, SRB and MA) both with depth and between-sampling sites (Table 3, Figs 5 and 6, Fig. S1). Population shifts occurred at c. 6 cm at BR and c. 10–14 cm at AR, despite limited changes in sulphate and methane profiles. The community change at AR corresponded with a change in activity from high rates of sulphate reduction to hydrogenotrophic methanogenesis (Fig. 3e), whilst the change at BR corresponded with a significant decrease in the total cell numbers (Fig. 2a). Notably, although the brackish site HY had distinct sulphate and methane zones with clear depth-related changes in activities (Fig. 4), it was also the most homogenous site in terms of community depth profiles (Figs 5 and 6). These results may reflect a highly dynamic estuary environment subject to regular perturbation, where geochemical profiles are re-established more quickly than diversity profiles at some locations. Frequently disturbed estuary sediment is less likely to exhibit the clear links between geochemical zones and specialist prokaryotic groups, often seen at less tidally dynamic sites (Banning et al., 2005; Fry et al., 2006; Parkes et al., 2007, 2012; Webster et al., 2007). Perhaps there are more complex interactions between, and environmental controls on, competitive groups of anaerobic terminal oxidisers in dynamic estuarine sediments, than indicated by geochemistry or activity profiles.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was funded through the Natural Environment Research Council (NERC) Post Genomics and Proteomics thematic programme (Aquatic Microbial Metagenomes and Biogeochemical Cycles – NE/C507929/1). The authors would like to thank Steven Hope from the Cardiff School of Biosciences molecular biology support unit (Cardiff University, Wales, UK) for sequencing. Thanks are also given to Ashley Houlden, Mark Osborne, Cindy Smith, Liang Dong, Emily Flowers and Henrik Sass for productive sampling trips.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
fem12106-sup-0001-FigureS1.pptapplication/ppt465KFig. S1. Cluster analysis of DGGE profiles of bacterial and archaeal 16S rRNA gene diversity in Brightlingsea, Alresford and Hythe sediment on the Colne estuary.
fem12106-sup-0002-TableS1.docWord document86KTable S1. PCR primers and cycling parameters used in this study.

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