RESEARCH ARTICLE
Prokaryotic functional diversity in di¡erent biogeochemical depth
zones in tidal sediments of the Severn Estuary, UK, revealed by
stable-isotope probing
Gordon Webster1,2, Joachim Rinna2, Erwan G. Roussel2, John C. Fry1, Andrew J. Weightman1 &
R. John Parkes2
1
Cardiff School of Biosciences, Cardiff University, Wales, UK; and 2School of Earth and Ocean Sciences, Cardiff University, Wales, UK
Correspondence: R. John Parkes, School of
Earth and Ocean Sciences, Cardiff University,
Main Building, Park Place, Cardiff, Wales
CF10 3AT, UK. Tel.: 144 29 208 70058; fax:
144 29 208 74326; e-mail:
[email protected]
Received 16 July 2009; revised 13 November
2009; accepted 26 January 2010.
Final version published online 16 March 2010.
DOI:10.1111/j.1574-6941.2010.00848.x
MICROBIOLOGY ECOLOGY
Editor: Alfons Stams
Keywords
stable-isotope probing; DNA; marine
sediments; Bacteria; Archaea; PCR-DGGE.
Abstract
Stable isotope probing of prokaryotic DNA was used to determine active
prokaryotes using 13C-labelled substrates (glucose, acetate, CO2) in sediment
slurries from different biogeochemical zones of the Severn Estuary, UK. Multiple,
low concentrations (5 100 mM) of 13C-substrate additions and short-term
incubations (7 days) were used to minimize changes in the prokaryotic community, while achieving significant 13C-incorporation. Analysis demonstrated clear
metabolic activity within all slurries, although neither the net sulphate removal nor
CH4 production occurred in the anaerobic sulphate reduction and methanogenesis
zone slurries. Some similarities occurred in the prokaryotic populations that
developed in different sediment slurries, particularly in the aerobic and dysaerobic
zone slurries with 13C-glucose, which were dominated by Gammaproteobacteria
and Marine Group 1 Archaea, whereas both anaerobic sediment slurries incubated
with 13C-acetate showed incorporation into Epsilonproteobacteria and other
bacteria, with the sulphate reduction zone slurry also showing 13C-acetate
utilization by Miscellaneous Crenarchaeotic Group Archaea. The lower potential
energy methanogenesis zone slurries were the only conditions where no 13Cincorporation into Archaea occurred, despite Bacteria being labelled; this was
surprising because Archaea have been suggested to be adapted to low-energy
conditions. Overall, our results highlight that uncultured prokaryotes play
important ecological roles in tidal sediments of the Severn Estuary, providing
new metabolic information for novel groups of Archaea and suggesting broader
metabolisms for largely uncultivated Bacteria.
Introduction
Tidal flat sediments occur at the land–sea interface in
tropical and temperate regions and are among the most
productive coastal marine ecosystems (Alongi, 1998). They
receive organic matter and nutrient input from both land
and sea, and as a result, are often characterized by intense
heterotrophic and photoautotrophic activity (Poremba
et al., 1999). As a consequence, high microbial activity in
the upper sediment layers generates steep geochemical
gradients and distinct biogeochemical zones. These biogeochemical zones enable the description of early diagenetic
reactions in sediments and represent the prokaryotic degraFEMS Microbiol Ecol 72 (2010) 179–197
dation of organic carbon using successively less energyyielding terminal electron acceptors (Froelich et al., 1979;
Canfield & Thamdrup, 2009). Biogeochemical zones have
been used to describe a wide variety of marine sedimentary
environments where the supply of labile organic matter
exceeds diffusion of oxygen into the sediment (e.g. Thomsen
et al., 2001; Mortimer et al., 2002; Parkes et al., 2005). The
depth range of each zone is determined from a characteristic
sequence of chemical changes in the sediment pore water
(Jørgensen, 1983). In estuarine sediments, typically oxygen
is depleted within the top few millimetres and anoxic
conditions prevail beneath this depth. Although nitrate, iron
and manganese are then the initial important anaerobic
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180
electron acceptors for the degradation of organic matter, it is
dissimilatory sulphate reduction that is the predominant
organic matter anaerobic degradation process in marine
sediments (Jørgensen, 1982), followed by methanogenesis
at depth when sulphate becomes depleted (Oremland &
Taylor, 1978).
The Severn Estuary is a nutrient-rich and tidally dynamic
coastal ecosystem located between England and Wales. It is a
large estuary with extensive intertidal mud-flats, sand-flats,
rocky platforms and islands. The estuary’s classic funnel
shape, unique in the United Kingdom, is a factor causing the
Severn Estuary to have the second highest tidal range in the
world (15 m, but lower than the Bay of Fundy, Canada;
Archer & Hubbard, 2003) and also one of the largest
intertidal zones in the United Kingdom. However, a few
reports suggest that the Severn Estuary as a whole supports a
rather impoverished flora and fauna, and high turbidity
means that phytoplankton productivity is generally lower
than expected due to limited light penetration (Joint, 1984).
Despite the low annual primary production, the Severn
Estuary has a high nutrient input (Morris, 1984) from the
many rivers that drain into the estuary from a large area of
England and Wales, which presumably influences the sedimentary microbial processes within this system. However,
very little is known about the prokaryotic diversity and
activity within these tidal sediments. One study of the
intertidal mudflat at Aust Warth (Wellsbury et al., 1996)
demonstrated that anaerobic sulphate reduction was the
dominant degradation process within these sediments,
accounting for 60% of the total organic matter degradation,
with methanogenesis occurring at much lower rates (o 1%)
in subsurface sediments. However, evidence was also shown
that the top 8 cm of sediment may have been recently
disturbed or deposited due to the large tidal impact encountered in the Severn Estuary. The effect of this continual
disturbance and oxygenation on the anaerobic prokaryotic
community is unknown.
Culture-independent approaches based on 16S rRNA
gene sequencing and phospholipid fatty acid (PLFA) biomarker profiles have been widely used to characterize
microbial populations in marine sediments, including those
from estuaries (e.g. Parkes et al., 1993; Purdy et al., 2001;
Roussel et al., 2009a). Stable-isotope probing (SIP) has
extended this approach by determining 13C substrate incorporation/utilization into specific biomarkers, hence enabling a direct link between substrate utilization and specific
prokaryotes (Boschker et al., 1998; Radajewski et al., 2000;
Neufeld et al., 2007b). Using this approach, we have
previously characterized acetate-, glucose- and pyruvateutilizing bacteria in an established sulphate-reducing sedimentary prokaryotic community (Webster et al., 2006b). In
the present study, we have used SIP with environmentally
relevant substrates (13C-glucose, 13C-acetate and 13CO2) to
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G. Webster et al.
investigate the active prokaryotic community in Severn
Estuary sediments from different biogeochemical depth
zones, without prior substrate enrichment, to identify
prokaryotes in addition to terminal-oxidizers. These prokaryotes may be particularly important in Severn Estuary
sediments as tidal sediment disturbance may hinder the
establishment of communities dominated by anaerobic
terminal-oxidizing Bacteria and Archaea.
Materials and methods
Pure cultures
Desulfobacter sp. DSM 2035 was grown on DSM medium
195 supplemented with either 12C-sodium acetate or 13Csodium acetate (1,2-13C2, 99%; CK Gas Products Ltd) to
provide known 13C-labelled and unlabelled DNA for use as
markers in density gradient ultracentrifugation (Webster
et al., 2006b).
Sediment slurry microcosms
Estuarine sediment cores (diameter 10 cm, depth 1 m) were
collected at low tide from tidal flats of the Severn Estuary,
Woodhill Bay, Portishead, UK (51o29 0 30.9400 N, 2o460 28.9100 W),
during two expeditions in early October 2004. The first
exploratory visit was to identify the site and sample sediments
for pore water chemical analysis and the second visit was to
collect sediment samples for SIP experiments. All sediment
samples were transported back to the laboratory for immediate
processing.
Sediments from different depths and biogeochemical
(metabolic) zones with potentially different prokaryotic
populations were used to establish replicate sediment slurries
(see Fig. 1): aerobic zone slurry A, an aerobic sediment slurry
made from sediment from the top 3 cm including the aerobic
zone (0 to 0.5 cm); dysaerobic (Raiswell & Canfield, 1998)
zone slurry B, an anaerobic sediment slurry comprising of
sediment from a regularly disturbed (Wellsbury et al., 1996)
anoxic, but not sulphidic, sediment zone from 3 to 20 cm
depth, presumably containing the nitrate, manganese and
iron reduction zones [Canfield & Thamdrup, 2009; recent
data from fresh cores (June 2009) show active manganese
and iron reduction occurring at this site in the top 15 cm,
E.G. Roussel, M. Olivier, R.J. Parkes & H. Sass, unpublished
data] and part of the sulphate reduction zone; sulphate
reduction zone slurry C, an anaerobic sediment slurry made
from only black sulphide-rich sediments from the active
sulphate reduction zone (sulphate concentrations decrease
from 20 to 15 mM) and overlapping methanogenesis zone
(20–40 cm); and methanogenesis zone slurries D and E, two
anaerobic sediment slurries both comprising sediment from
the methanogenesis zone (40–70 cm, containing the highest
methane concentrations) and the bottom of the overlapping
FEMS Microbiol Ecol 72 (2010) 179–197
181
Stable-isotope probing of tidal sediments
(a)
(b)
Acetate (μM)
0
50
100
150
0
200
Aerobic zone
Slurry A
M
0–3
3–20 20–40 40–70
Dysaerobic zone
Slurry B
10
40
50
60
Slurries D and E
Methanogenesis zone
Slurry C
Anaerobic zone
Depth (cm)
30
Sulphate reduction zone
20
Depth (cm)
70
80
90
0
10
Sulphate (mM)
20
30
40
Methane (μmol L–1 sediment)
Fig. 1. (a) Depth profiles of sediment pore water sulphate, methane and acetate for Severn Estuary tidal flat sediment. Highlighted boxes show the
sediment depths used to make aerobic zone slurry A, dysaerobic zone slurry B, sulphate reduction zone slurry C and methanogenesis zone slurries D and
E. (b) PCR-DGGE analysis of bacterial 16S rRNA genes amplified from Severn Estuary tidal flat sediment DNA from each of the different sediment depths
shown in (a). Lane numbers represent the sample depth (cm); Lanes marked M, DGGE marker (Webster et al., 2003).
sulphate reduction zone, where rates of sulphate removal
decrease.
Sediment (250 mL) was added to 750 mL of oxic or
anoxic (reduced with 1 mM sodium sulphide) mineral salts
medium (Wellsbury et al., 1994). The anoxic media used for
the sulphate reduction zone sediment slurry C also contained 18 mM sodium sulphate. All slurries were contained
within modified 2-L screw-capped bottles (except aerobic
slurry A, which was closed with a foam bung) fitted with a
shoulder opening for gas input and a three-way stopcock at
the base for slurry sampling. The gas headspace in the anoxic
sediment slurries (slurries B, C, D and E) was replaced with
oxygen-free nitrogen. All slurries were incubated at 25oC in
the dark on an orbital shaker (150 r.p.m.).
Labelled 13C-substrates (100 mM) were added to the
slurries at time zero and subsequently each day for the first
4 days (total 5 100 mM), after which slurries were left to
incubate for a further 10 days (total 14 days). Slurry samples
FEMS Microbiol Ecol 72 (2010) 179–197
(50 mL) were taken at 0, 0.5, 1, 2, 3, 4, 5, 7 and 14 days, and
stored at 0oC until required. The substrates used were
13
C-glucose (U-13C6, 99%, CK Gas Products Ltd), slurries A
and B; 13C-acetate (1,2-13C2, 99%, CK Gas Products Ltd),
slurries C and D; and 13C-CO2 (U-13C, 99%; CK Gas
Products Ltd), slurry E.
Chemical analysis of sediment pore water
Fifty millilitres of sediment was sampled from all slurries at
regular intervals into 50-mL volume centrifuge tubes
flushed with oxygen-free nitrogen. Sediment samples were
then centrifuged for 15 min at 2300 g in a Hettich Rotanta
460R centrifuge at 10oC. The pore water supernatant was
then removed and analysed, while the remaining sediment
pellet was stored at 80oC for subsequent molecular
analysis. Sulphate, nitrate, volatile fatty acid (VFA) concentrations and other anions were determined using an
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182
ICS-2000 ion chromatography system with an AS50 autosampler (Dionex UK Ltd) fitted with two Ionpac AS15
columns in series, and an anion self-regenerating suppressor
(ASRS-ULTRA II 4-mm) in combination with a DS6 heated
conductivity cell (Dionex UK Ltd) under the conditions
described previously (Webster et al., 2009). Ammonium and
other cations were analysed using a DX-120 ion chromatography system with an AS40 autosampler (Dionex UK Ltd)
fitted with an Ionpac CS16 and a cation self-regenerating
suppressor (CSRS-300 4 mm) in combination with a DS4-1
heated conductivity cell (Dionex UK Ltd), and using 25 mM
methanesulphonic acid as an eluent.
For measuring methane, 2 cm3 of sediment was added to
20 mL 10% (w/v) KCl in 50-mL volume gas-tight serum
bottles and headspace gas analysed by GC using a modified
Perkin Elmer/Arnel Clarus 500 Natural Gas Analyser fitted
with a flame ionization detector and a thermal conductivity
detector. Headspace gas from sediment slurries was analysed
by GC directly.
DNA extraction
Genomic DNA was extracted from pure cultures of Desulfobacter sp. DSM 2035 using the FastDNA SPIN Kit (MP
Biomedicals). Sediment DNA was extracted from either 1 g
sediment or 5 1 g 13C-enriched sediment slurry using the
FastDNA SPIN kit for Soil (MP Biomedicals), with modifications as described by Webster et al. (2003). DNA extracts
were visualized by standard agarose gel electrophoresis, and
the DNA was quantified against Hyperladder I DNA marker
(Bioline) using the Gene Genius Bio Imaging System
(Syngene).
CsCl density gradient ultracentrifugation
The CsCl density gradient ultracentrifugation and DNA
fractionation into 12C- and 13C-DNA conditions were as
described by Webster et al. (2006b). DNA (5 mg) from the
13
C-enriched sediment slurries were fractionated, and 12Cand 13C-DNA fractions were removed from the CsCl gradient alongside a ‘marker’ tube containing Desulfobacter sp
DSM 2035 12C- and 13C-DNA as a visual guide (Webster
et al., 2006b). 12C- and 13C-DNA fractions were cleaned to
remove ethidium bromide and CsCl using molecular-grade
water- (Severn Biotech) saturated n-butanol, followed by
dialysis with Microcon YM-100 filters (Millipore Corporation). Purified DNA was eluted in 40 mL sterile moleculargrade water and stored at 80oC until required.
16S rRNA gene PCR-denaturing gradient gel
electrophoresis (DGGE) analysis
For PCR-DGGE analysis, bacterial and archaeal 16S rRNA
genes were amplified directly from sediment slurry DNA
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G. Webster et al.
extracts and 12C- and 13C-DNA fractions with primer pairs
357FGC-518R (Muyzer et al., 1993) and SAfGC-PARCH519R
(Øvreås et al., 1997; Nicol et al., 2003), as described previously (Webster et al., 2006a). Gels were stained with
SYBRGold nucleic acid stain (Molecular Probes), viewed
under UV and images were captured using a Gene Genius
Bio Imaging System (Syngene). DGGE bands of interest were
excised, reamplified by PCR and sequenced as described
previously (Webster et al., 2003; O’Sullivan et al., 2008).
PCR amplification, cloning and phylogenetic
analysis
Bacteria and Archaea 16S rRNA genes were amplified from
12
C- and 13C-DNA with the PCR primers 27F/907R and
109F/958R, respectively, under the conditions described in
Webster et al. (2006a). Dissimilatory sulphite reductase
(dsrA) gene sequences were also amplified using DSR1F
and DSR4R (Wagner et al., 1998).
Five PCR reactions from each sample were pooled,
cleaned and concentrated using Microcon YM 100 spin
filters (Millipore Corporation) and eluted in 40 mL steriledistilled water. Pooled PCR products were quantified and
ligated into pGEM T-easy vector, and transformed into
Escherichia coli JM109 competent cells (Promega Corporation). Clones containing the correct insert, after checking by
amplification with M13 primers, were sequenced using an
ABI 3130xl Genetic Analyzer (Applied Biosystems).
Sequence chromatographs were analysed using the
CHROMAS LITE software version 2.01 (http://www.technely
sium.com.au/chromas.html). Partial sequences and their
closest relatives were identified by NCBI BLASTN (http://
www.ncbi.nlm.nih.gov/). All nucleotide sequences were
aligned using CLUSTALX (Thompson et al., 1997) with sequences retrieved from the database. Alignments were edited
manually using BioEdit Sequence Alignment Editor version
5.0.9 (Hall, 1999) and regions of ambiguous alignment
were removed. Phylogenetic trees were constructed using
neighbour-joining with the Jukes and Cantor correction
algorithm in MEGA 4 (Tamura et al., 2007).
The new sequences reported here have been submitted to
the EMBL database under accession numbers FN424302
–FN424337 for 16S rRNA gene sequences and FN424338–
FN424349 for dsrA gene sequences.
Results
Sampling site geochemistry
The sediments at the Severn Estuary sampling site were
homogeneous silty-clay (Mclaren et al., 1993) down to the
bottom of the core (100 cm). Methane and sulphate profiles
were measured throughout the core. Sulphate concentrations at
the surface were 29 mM and decreased with depth to 10 mM
FEMS Microbiol Ecol 72 (2010) 179–197
183
Stable-isotope probing of tidal sediments
at 60 cm below the sediment surface (Fig. 1a). In contrast,
methane concentrations increased with depth from zero in the
surface 10 cm to concentrations of 16–25 mmol L1 sediment
below 50 cm. Acetate (Fig. 1a) and other VFAs (lactate and
formate; data not shown) were consistently present in the
sediment pore water. Acetate concentrations decreased with
depth (103 to 63 mM acetate), while both lactate and formate
varied in concentration between 8–34 mM lactate and
90–127 mM formate. Nitrate (10 mM) was only detected in the
sediment surface (data not shown). Samples from a recent core
taken in June 2009 had an in situ temperature of 22oC down to
10 cm and a porosity of 51–77%.
Bacterial 16S rRNA gene PCR-DGGE analysis of DNA
extracted from sediments representative of each of the four
zones used to prepare the slurries clearly demonstrated that
each zone had a different bacterial community structure
(Fig. 1b), indicative of different geochemical conditions at
each depth.
Sediment slurry geochemistry and prokaryotic
activity
Pore water from all sediment slurries incubated with 13Csubstrates was analysed at specific time points throughout
the SIP experiment.
Aerobic zone sediment slurry A
During and after the repeated addition of 13C-glucose
(5 100 mM) to the aerobic zone sediment slurry A, no
increases in VFAs occurred, suggesting complete oxidation
of glucose; acetate slowly decreased from 30 mM at 1 day
to 19 mM at 14 days, lactate remained relatively constant
between 36 and 40 mM and formate decreased with time (6
to o 0.2 mM), with a slight peak in concentration (14 mM)
at 3 days. Sulphate concentrations remained constant
throughout the incubation at 4.5 mM. Nitrite and nitrate
concentrations were below detection until 14 days, when
the values were 175 and 300 mM, respectively, coupled with
a steady decrease in ammonia from 1 mM at 24 h to
below detection at 14 days, indicative of aerobic ammonia
oxidation.
Sulphate reduction zone sediment slurry C
Repeated 100 mM additions of 13C-acetate to the sulphate
reduction zone sediment slurry C caused a steady increase in
acetate from time zero (57 mM) to 3 days (460 mM), and then
by 4 days, acetate had been rapidly utilized, decreasing to
50 mM. After 4 days, acetate utilization was slower, with
concentrations decreasing to only 30 mM by 14 days.
Formate concentrations remained low (0–7.2 mM) throughout the incubation and lactate was undetectable until 4 days
(45 mM) and then remained constant until 14 days.
Sulphate concentrations remained relatively stable throughout the incubation (fluctuating between 22 and 23 mM),
which suggests that no or very little net sulphate reduction
occurred during the experiment.
Methanogenesis zone sediment slurries D and E
In contrast to slurry C, the repeated addition of 13C-acetate
to the methanogenesis zone sediment slurry D demonstrated that acetate was being rapidly removed from the
slurry as increased acetate concentrations were only observed at 1 day (78 mM), after which acetate remained low
and subsequent 13C-acetate additions were not detected.
Acetate concentrations fluctuated between 22 and 29 mM
over the remaining 14-day incubation period. The acetate
concentration in the 13CO2-amended methanogenesis zone
sediment slurry E increased to 289 mM after 1 day and then
rapidly decreased to 24 mM by 2 days, remaining relatively
constant thereafter until 14 days, suggesting initially possible
acetogenesis, followed by acetate utilization or removal. In
both methanogenesis zone sediment slurries, formate was
low (0–7.2 mM). Lactate increased from zero at 24 h to
40 mM at 2 days and remained relatively constant for 14
days in the 13C-acetate slurry D and was below detection in
the 13CO2 slurry E at most time points, with the exception of
a peak at 3–4 days (52 and 36 mM). In both sediment slurries
D and E, sulphate slightly increased from 4 to 6 mM and
no significant increase in methane above the background
occurred during the 14-day incubation.
Interestingly, acetate concentrations detected in all the
slurries after the 14-day incubation were mostly similar to
those in the original sediment pore water, suggesting that
acetate may have reached a steady state (Fig. 1a).
Dysaerobic zone sediment slurry B
In contrast to slurry A, addition of the same concentration
of 13C-glucose to the anoxic sediment slurry B resulted in a
clear increase in acetate (175 mM) after 1 day, suggesting
fermentation of glucose. This acetate was rapidly utilized,
decreasing to o 27 mM by 4 days. Lactate and formate
remained low or below detection (0–7 mM), with the
exception that the lactate concentration peaked at 4 days
(36 mM). Sulphate remained constant at 6 mM.
FEMS Microbiol Ecol 72 (2010) 179–197
Changes in the prokaryotic community structure
of sediment slurries over time as assessed by 16S
rRNA gene PCR-DGGE
Changes in the archaeal and bacterial community structures
of the sediment slurries were monitored throughout the
incubations with 13C-substrates using PCR-DGGE analysis
of 16S rRNA genes. During the 14-day incubation, some
clear changes in the bacterial 16S rRNA gene DGGE profiles
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184
G. Webster et al.
were observed for all 13C-substrates with time compared
with the time zero community structure, before 13C-substrate addition. Figure 2a, for example, clearly demonstrates
the enrichment of new bacterial species (marked with an
asterisk) in slurry C, 1 day after addition of 13C-acetate,
which, when sequenced, belonged to the Arcobacter cluster
of the Epsilonproteobacteria. Archaeal communities were
more difficult to interpret because banding patterns varied
slightly at different time points, with some bands disappearing and reappearing with time (see Fig. 2b for example). It
seems likely that there was little change in the overall
archaeal population with time and the observed banding
pattern fluctuations are probably due to stochastic amplification within the PCR, possibly due to the low numbers of
archaeal cells within the sediment. Although, between 0.5and 1-day incubation, with all substrates, there was some
evidence of a change in the band intensity for some bands
compared with time zero, suggesting the stimulation and
(a)
M
0
0.5
1
2
3
4
5
7
14
M
∗
growth of some archaeal species. For example, Fig. 2b shows
the stimulation of Archaea (marked with asterisk) belonging
to the Marine Benthic Group D (MBG-D) in sediment
slurry C amended with 13C-acetate.
CsCl density gradient ultracentrifugation for
separation of 12C- and 13C-labelled sediment DNA
Separation of 13C-DNA from the unlabelled 12C-DNA was
carried out by CsCl-density gradient ultracentrifugation on
DNA extracted from the 13C-substrate sediment slurries after
7 days of incubation (Webster et al., 2006b). This incubation
time was selected as previously we determined that it provided sufficient incorporation of 13C from low concentrations
of substrate into sediment prokaryotic DNA to enable separation of 13C-DNA from the bulk 12C-DNA. DNA extracted
from all 13C-labelled sediment slurries after centrifugation in a
CsCl–ethidium bromide density gradient was visible as two
faint diffuse bands (Fig. 3a). This suggested that the sediment
DNA was comprised of unlabelled, partially labelled and fully
13
C-labelled DNA, similar to that observed in other SIP
experiments (e.g. Morris et al., 2002; Hutchens et al., 2004;
Webster et al., 2006b). Two DNA fractions (12C- and 13CDNA fractions labelled 1 and 2, respectively; Fig. 3a) were
collected from the CsCl gradients guided by a ‘marker’ density
gradient, containing Desulfobacter sp. 12C- and 13C-DNA.
Molecular analysis of 12C- and 13C-DNA fractions
PCR-DGGE analysis of bacterial and archaeal 16S
rRNA genes
Time (days)
Bacterial and archaeal 16S rRNA genes from the 12C- and 13CDNA fractions from each of the 13C-labelled sediment slurries
were amplified by PCR and analysed by DGGE (Figs 3 and 4).
(b)
M
0
0.5
1
2
3
4
5
7
14
M
Aerobic zone sediment slurry A
∗
∗
Time (days)
Fig. 2. Examples of PCR-DGGE analysis of bacterial and archaeal 16S
rRNA genes amplified from DNA extracted from the sulphate reduction
zone sediment slurry C incubated with 13C-acetate for 14 days. (a)
Bacterial 16S rRNA genes (b) archaeal 16S rRNA genes. Lane numbers
represent the sample time points in days; Lanes marked M, DGGE marker
(Webster et al., 2003). Bands labelled with asterisk were excised and
sequenced.
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c
Analysis of the 13C-glucose-amended slurry A (Fig. 3b)
showed that the 13C-DNA fraction was a subset of the total
bacterial community (12C-DNA fraction), suggesting that
the 13C-DNA DGGE profile represented the bacterial population able to utilize 13C-glucose and/or glucose degradation
products. Sequencing of two of the most intensely stained
DGGE bands from the 13C-DNA fraction revealed that some
of the sediment bacteria able to incorporate glucose under
oxic conditions belonged to the Gammaproteobacteria and
were related to Vibrio species and Idiomarina baltica (both
with 97% sequence similarity; Table 1).
In contrast, the DGGE pattern for the archaeal 13C-DNA
fraction (Fig. 4a) was very similar to the 12C-DNA
pattern, suggesting that all Archaea identified in the aerobic
sediment slurry A by PCR-DGGE were able to incorporate
13
C-glucose or its degradation products. All dominant
FEMS Microbiol Ecol 72 (2010) 179–197
185
Stable-isotope probing of tidal sediments
(b)
(a)
M
(c)
1
M
2
1
Marker
tube
2
Fb6
Fb2
Ab3 Ab1
Fb3
∗1
Fb4
Ab2
Fb5
∗2
(d)
(e)
M
1
Sb1
2
(f)
1
M
2
M
1
2
Mb1
Sb2
Mb4
Mb3
Mb5
Sb3
Mb2
Fig. 3. Separation of sediment slurry DNA by CsCl/ethidium bromide
density gradients after equilibrium centrifugation and subsequent PCRDGGE analysis of 12C and 13C-DNA fractions. (a) DNA extracted from the
Severn Estuary sulphate reduction zone sediment slurry C incubated with
13
C-acetate for 7 days (asterisks highlight the DNA fractions removed)
alongside a marker tube containing DNA extracted from the SRB,
Desulfobacter sp. DSM 2035 (grown on either 12C- or 13C-acetate as
the sole carbon source). PCR-DGGE analysis of bacterial 16S rRNA genes
from (b) aerobic zone slurry A and (c) dysaerobic zone slurry B incubated
with 13C-glucose, (d) sulphate reduction zone slurry C and (e) methanogenesis zone slurry D incubated with 13C-acetate, (f) methanogenesis
zone slurry E incubated with 13CO2. Lanes marked 1, 12C-DNA fraction;
lanes marked 2, 13C-DNA fraction; lanes marked M, DGGE marker
(Webster et al., 2003). Labelled DGGE bands represent bands that were
excised and sequenced (see Table 1).
archaeal bands that were excised and sequenced belonged to
the largely uncultivated Archaea group Marine Group 1
(MG1) and had 97–99% sequence similarity to archaeal 16S
rRNA gene sequences retrieved from a marine sponge,
seawater and marine sediments (Table 2).
Dysaerobic zone sediment slurry B
PCR-DGGE analysis of the bacterial 16S rRNA genes in the
13
C-DNA fraction from the 13C-glucose-amended slurry B
incubated under anaerobic conditions showed that the 13CFEMS Microbiol Ecol 72 (2010) 179–197
DNA bacterial community structure was different from the
12
C-DNA bacterial population. However, unlike the aerobic
slurry A (Fig. 3b), the 13C-DNA bacterial population in
slurry B (Fig. 3c) was more complex and dominated by a
number of distinct DGGE bands, suggesting that under
anaerobic conditions, a more diverse population of sediment bacteria are able to utilize glucose and/or its degradation products. Sequencing a number of DGGE bands
revealed that 13C was incorporated by bacteria within the
phyla Gamma- and Epsilonproteobacteria. Gammaproteobacterial sequences were related to Vibrio species (97–99%
sequence similarity) and Marinobacter aquaeolei (97%
sequence similarity), while the sequence (band Fb6) belonging to the Epsilonproteobacteria had 99% sequence similarity
to clone MZ-53.NAT from a coastal near-surface sediment
(Table 1) and was also closely related (99% sequence
similarity) to the nitrogen-fixing Arcobacter nitrofigilis isolated from a salt marsh plant root (McClung et al., 1983)
and Arcobacter sp. strain NA105 isolated from a tidal flat
sediment of the Wadden Sea (Freese et al., 2008).
Similar to slurry A, all Archaea sequences identified in the
anoxic slurry B, which were able to incorporate 13C from
glucose, belonged to the MG1 (Fig. 4b; Table 2). However, in
contrast to slurry A, the MG1 within slurry B were an active
subset of the total archaeal population. All MG1 sequences
were closely related (97–100%) to clone sequences identified
previously in other marine environments and to sequences
identified in the aerobic slurry A.
Sulphate reduction zone sediment slurry C
Analysis of the bacterial 16S rRNA genes from the 12C- and
13
C-DNA fractions from the sulphate reduction zone slurry C
amended with 13C-acetate showed a considerable similarity in
their DGGE profiles (Fig. 3d), suggesting that all members of
the dominant bacterial community were able to incorporate
acetate under these conditions. Both 12C- and 13C-DNA
DGGE profiles were dominated by one brightly stained band
(bands Sb1 and Sb2) related to the seawater clone VH-FL6-38
(97–98% sequence similarity) within the Arcobacter cluster of
the Epsilonproteobacteria. In addition, a small number of
other bands in the 13C-DNA DGGE profile (e.g. band Sb3)
were brighter than their corresponding bands in the 12C-DNA
profile. Band Sb3 was most similar to sequences belonging to
the Deltaproteobacteria order Desulfuromonadales and was
96% similar to the Fe(III)- and Mn(IV)-reducing bacterium,
Geoalkalibacter subterraneus (Greene et al., 2009).
Archaeal sequences shown to be active and incorporating
13
C-acetate or metabolites within sediment slurry C were a
discrete subset of the 12C diversity and these belonged to
members of the C3 (Inagaki et al., 2006) subgroup of the
uncultivated Miscellaneous Crenarchaeotic Group (MCG;
Inagaki et al., 2003), whereas DGGE bands excised and
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186
G. Webster et al.
Table 1. Bacterial 16S rRNA gene sequence matches to excised DGGE bands from sediment slurries incubated for 7 days with different 13C-substrates
% Sequence
similarity
(alignment
length, bp)
DGGE
band
Sediment slurry (DNA
fraction)
Nearest match by BLASTN
search (accession number)
Ab1
Slurry A with 13C-glucose
(13C-DNA)
Slurry A with 13C-glucose
(13C-DNA)
Slurry A with 13C-glucose
(12C-DNA)
Slurry B with 13C-glucose
(13C-DNA)
Slurry B with 13C-glucose
(13C-DNA)
Slurry B with 13C-glucose
(13C-DNA)
Slurry B with 13C-glucose
(13C-DNA)
Slurry B with 13C-glucose
(13C-DNA)
Slurry C with 13C-acetate
(12C-DNA)
Slurry C with 13C-acetate
(13C-DNA)
Slurry C with 13C-acetate
(13C-DNA)
Slurry D with 13C-acetate
(13C-DNA)
Slurry D with 13C-acetate
(13C-DNA)
Slurry D with 13C-acetate
(13C-DNA)
Slurry D with 13C-acetate
(12C-DNA)
Slurry D with 13C-acetate
(12C-DNA)
Uncultured Vibrio sp. clone
97 (151)
6-268 (AY374408)
Idiomarina baltica strain SS-01 97 (146)
(EU624441)
Uncultured Vibrio sp. clone
99 (149)
6-268 (AY374408)
Vibrio lentus isolate 42
98 (172)
(EF178477)
Uncultured Vibrio sp. clone
97 (151)
KR80_O05 (AM183765)
Uncultured Vibrio sp. clone
99 (160)
6-268 (AY374408)
Marinobacter aquaeolei
97 (171)
isolate OC-8 (AY669168)
Uncultured bacterium clone
99 (169)
MZ-53.NAT (AJ810559)
Uncultured bacterium clone
97 (169)
VH-FL6-38 (EF379678)
Uncultured bacterium clone
98 (142)
VH-FL6-38 (EF379678)
Geoalkalibacter subterraneus
96 (172)
strain Red1 (EU182247)
Uncultured bacterium clone
98 (138)
VH-FL6-38 (EF379678)
Uncultured bacterium clone 4- 98 (140)
UMH 22% pond (AF477875)
Vibrio sp. FALF307
100 (151)
(EU655386)
Uncultured bacterium clone
99 (165)
C13S-27 (EU617763)
Uncultured bacterium DGGE
99 (166)
band NN5
Ab2
Ab3
Fb2
Fb3
Fb4
Fb5
Fb6
Sb1
Sb2
Sb3
Mb1
Mb2
Mb3
Mb4
Mb5
Phylogenetic affiliation
Isolation environment of
nearest sequence match
Gammaproteobacteria
Seawater, Barnegat Bay, NJ
Gammaproteobacteria
Marine sediment
Gammaproteobacteria
Seawater, Barnegat Bay, NJ
Gammaproteobacteria
Seaweed surface
Gammaproteobacteria
Gammaproteobacteria
Estuarine water, Karnaphuli
River, Bangladesh
Seawater, Barnegat Bay, NJ
Gammaproteobacteria
Marine sediment
Epsilonproteobacteria
Surface sediment, Milazzo
Harbour, Italy
Seawater, Victoria Harbour,
Hong Kong
Seawater, Victoria Harbour,
Hong Kong
Petroleum reservoir
Epsilonproteobacteria
Epsilonproteobacteria
Deltaproteobacteria
Epsilonproteobacteria
Alphaproteobacteria
Gammaproteobacteria
Gammaproteobacteria
Gammaproteobacteria
Seawater, Victoria Harbour,
Hong Kong
Solar saltern
Bacterioplankton, Plum Island
Sound, MA
Marine sediment, Yellow Sea
Tidal flat sediment, Wadden
Sea
Bold DGGE band names highlight sequences retrieved from 13C-DNA fractions (see Fig. 3).
sequenced from the DGGE profile of the 12C-DNA fraction
demonstrated that the total archaeal community within this
slurry also contained members of the MBG-D in addition to
MCG (identified by band position; see Fig. 4c).
Methanogenesis zone sediment slurries D and E
Sediment slurries from the methanogenesis zone (Fig. 1) were
individually incubated with the substrates 13C-acetate (slurry
D) and 13CO2 and (slurry E). PCR-DGGE analysis of bacterial
16S rRNA genes demonstrated that a large diversity of bacterial
species were able to use 13CO2 or metabolites (Fig. 3f) in slurry
E, and a more specific community was able to utilize 13Cacetate (Fig. 3e) in slurry D. Interestingly, a different subset of
Bacteria seemed to be able to utilize acetate under low-sulphate
conditions (4 mM) than those in the high-sulphate sediment
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c
slurry C (22 mM), with the exception of band Mb1. Band
Mb1 was identical to the Epsilonproteobacteria (Arcobacter-like)
sequence identified in slurry C (Table 1). It should be noted
that because there was a high diversity of bacteria able to
incorporate 13C from 13CO2, represented by many faint DGGE
bands (Fig. 3f), it was difficult to excise DGGE bands and
obtain good sequence information, and therefore no sequence
data are presented for slurry E.
However, in contrast to Bacteria, Archaea (neither methanogens nor uncultivated lineages) were not detected in the
13
C-DNA fraction of either slurry D or E, suggesting that
Archaea identified in the 12C-DNA fractions (Fig. 4d and e)
were not active and/or were unable to incorporate 13CO2 or
13
C-acetate under the conditions or the incubation time
used in this experiment. Nevertheless, presumed active
methanogenic Archaea were readily identified in these
FEMS Microbiol Ecol 72 (2010) 179–197
187
Stable-isotope probing of tidal sediments
(a)
(b)
M
1
2
1
(c)
2
M
(d)
1
2
M
(e)
1
2
1
2
Fig. 4. PCR-DGGE analysis of archaeal 16S rRNA
genes from sediment slurry 12C and 13C-DNA
fractions of (a) aerobic zone slurry A and (b)
dysaerobic zone slurry B incubated with
13
C-glucose, (c) sulphate reduction zone slurry C
and (d) methanogenesis zone slurry D incubated
with 13C-acetate and (e) methanogenesis zone
slurry E incubated with 13CO2. Lanes marked 1,
12
C-DNA fraction; lanes marked 2, 13C-DNA
fraction; lanes marked M, DGGE marker
(Webster et al., 2003). Labelled DGGE bands
represent bands that were excised and
sequenced (see Table 2).
sediments. For example, methanogen mcrA functional genes
were amplified from Severn Estuary DNA extracted from the
same sediment zone, Euryarchaeaota 16S rRNA genes were
detected in slurries D and E (data not shown) and methanogens have been enriched from the same site using a range
of substrates including acetate and H2/CO2 (A.J. Watkins,
H. Sass & R.J. Parkes, unpublished data).
Phylogenetic analysis of bacterial and archaeal
16S rRNA genes, and dsrA genes
Because of the importance of sulphur cycling and sulphate
reduction within marine sediments in general (Jørgensen,
1982; Muyzer & Stams, 2008) and that documented in a
previous study on Severn Estuary sediments (Wellsbury
et al., 1996), further investigation of sediment slurry C
(predominantly from a zone associated with sulphate reduction; Fig. 1) was carried out. Gene libraries (Bacteria and
Archaea 16S rRNA genes, and dsrA genes) were constructed
from the 12C- and 13C-DNA fractions.
Analysis of the bacterial 16S rRNA gene libraries
(Fig. 5) did not identify any sequences related to known
sulphate-reducing bacteria (SRB), although sequences related to other sulphur cycling bacteria were present. For
example, in the 13C-DNA library (n = 29), a large number of
sequences fell within the Arcobacter cluster of the Epsilonproteobacteria (52%), some of which are known to oxidize
sulphide to sulphur (Telang et al., 1999; Gevertz et al., 2000;
Wirsen et al., 2002), and 31% fell within the Deltaproteobacteria order Desulfuromonadales, which contains known
sulphur- and metal-reducing bacteria. Representatives of
these dominant groups of sequences (e.g. clones 13CSRZB1/13CSRZ-B2 and 13CSRZ-B17/13CSRZ-B19; Fig. 5) were
also closely related (96–97% and 93–100% sequence
similarity) to sequences identified by PCR-DGGE (Fig. 3d;
Table 1). In addition, other sequences included members
FEMS Microbiol Ecol 72 (2010) 179–197
of the sulphur-oxidizing Epsilonproteobacteria family
Thiovulgaceae (10%; Campbell et al., 2006) as well as
members of the Bacteroidetes and a novel group related to
Chlorobi.
The majority of 13C-DNA archaeal 16S RNA gene
sequences (95%, n = 20; Fig. 6) from slurry C fell within the
Crenarchaeota MCG, with 65% grouping within the Marine
Benthic Group C (MBG-C) and 30% within the C3 (Fig. 6a).
Additionally, one sequence belonged to the Euryarchaeota
group MBG-D (Fig. 6b). Such a limited diversity of archaeal
phylotypes that utilized 13C-acetate and were active within
sediment slurry C is interesting considering that a much
higher diversity of archaeal sequences (n = 23) was obtained
from the 12C-DNA. For example, the archaeal community
identified from the 12C-DNA also contained sequences
belonging to a number of other clusters within the MCG
along with members of the MG1 (Crenarchaeota Group
1.1a), Marine Hydrothermal Vent Group and the Crenarchaeota Group 1.1b. Similar to the bacterial 16S rRNA gene
library, some archaeal sequences identified by PCR cloning
of the 13C-DNA were closely related to sequences detected by
PCR-DGGE (e.g. clone 13CSRZ-A15 was 91–94% similar to
bands Sa1–Sa3; Figs 4c and 6; Table 2).
Phylogenetic analysis of 10 partial sequences from the
13
C-DNA fraction of sediment slurry C revealed a very low
diversity of novel dsrA genes (data not shown). All 10 novel
dsrA gene sequences were similar to each other (98–100%
sequence similarity) and were related (82–83% sequence
similarity) to a clone sequence (clone DSR-W) retrieved
from a deep-sea hydrothermal sediment from the Rainbow
(Mid-Atlantic Ridge) vent field (Nercessian et al., 2005),
whereas 10 sequences analysed from the 12C-DNA fraction
showed a much higher level of diversity and included
sequences that were related (84–90% sequence similarity)
to known SRB within the family Desulfobacteraceae and to
other novel dsrA genes (93–99% sequence similarity)
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188
G. Webster et al.
Table 2. Archaeal 16S rRNA gene sequence matches to excised DGGE bands from sediment slurries incubated for 7 days with different 13C-substrates
% Sequence
similarity
(alignment
length, bp)
Phylogenetic
affiliation
Isolation environment
of nearest sequence
match
97 (122)
MG1
Antarctic sponge
98 (123)
MG1
Amsterdam Mud Volcano,
Eastern Mediterranean
98 (86)
MG1
98 (96)
MG1
MG1
Water column above gas
hydrate, Gulf of Mexico
Methane hydrate bearing
subseafloor sediment,
Peru Margin
Antarctic sponge
MG1
Altamira Cave, Spain
MG1
MG1
Deep-sea whale-fall in
Monterey Canyon, CA
Altamira Cave, Spain
MG1
Antarctic sponge
MG1
Marine surface sediment,
East Sea
Antarctic sponge
DGGE
band
Sediment slurry
(DNA fraction)
Nearest match by BLASTN
search (accession number)
Aa1
Slurry A with 13C-glucose
(12C-DNA)
Slurry A with 13C-glucose
(12C-DNA)
Slurry A with 13C-glucose
(12C-DNA)
Slurry A with 13C-glucose
(13C-DNA)
Uncultured archaeon clone 2
(AY320199)
Uncultured archaeon clone
Amsterdam-MN13BT4-177
(AY593283)
Uncultured archaeon clone
M400-45 (EU791562)
Uncultured archaeon clone
ODP1230A18.06 (AB177102)
Slurry A with 13C-glucose
(13C-DNA)
Slurry B with 13C-glucose
(12C-DNA)
Slurry B with 13C-glucose
(12C-DNA)
Slurry B with 13C-glucose
(13C-DNA)
Slurry B with 13C-glucose
(13C-DNA)
Slurry B with 13C-glucose
(13C-DNA)
Slurry B with 13C-glucose
(13C-DNA)
Slurry B with 13C-glucose
(13C-DNA)
Slurry B with 13C-glucose
(13C-DNA)
Slurry C with 13C-acetate
(13C-DNA)
Slurry C with 13C-acetate
(13C-DNA)
Uncultured archaeon clone 2
99 (113)
(AY320199)
Uncultured archaeon clone 937
99 (105)
(EF188780)
Uncultured archaeon clone
100 (110)
R33_10d_G7 (EU084520)
Uncultured archaeon clone 937
97 (125)
(EF188748)
Uncultured archaeon clone 2
98 (124)
(AY320199)
Uncultured archaeon clone EU1-3 99 (116)
(EU332076)
Uncultured archaeon clone 2
99 (123)
(AY320199)
Uncultured archaeon clone
98 (128)
Napoli-1A-25 (AY592458)
Uncultured archaeon clone
99 (105)
ANT33-07(AB240747)
Uncultured archaeon clone
97 (84)
K8MV-C21-07(AB362542)
Uncultured archaeon clone
100 (110)
ODP1230A33.09 (AB177118)
Sa3
Slurry C with 13C-acetate
(13C-DNA)
Uncultured archaeon clone
ODP1230A33.09 (AB177118)
C3
Sa4
Slurry C with 13C-acetate
(12C-DNA)
Slurry C with 13C-acetate
(12C-DNA)
Slurry C with 13C-acetate
(12C-DNA)
Uncultured archaeon clone
96 (128)
C10_1C (EU570139)
Uncultured archaeon clone
96 (128)
C10_1C (EU570139)
Uncultured archaeon clone Hua0- 96 (130)
s19 (EU481602)
MBG-D/Thermoplasmatalesrelated
MBG-D/Thermoplasmatalesrelated
MBG-D/Thermoplasmatalesrelated
Slurry C with 13C-acetate
(12C-DNA)
Uncultured archaeon clone
CAVMV301A980 (DQ004669)
MBG-D/Thermoplasmatalesrelated
Aa2
Aa3
Aa4
Aa5
Fa1
Fa2
Fa4
Fa5
Fa6
Fa7
Fa8
Fa9
Sa1
Sa2
Sa5
Sa5
Sa7
98 (109)
95 (123)
MG1
MG1
MG1
C3
C3
Napoli Mud Volcano,
Eastern Mediterranean
Cold-seep sediment,
Nankai Trough
Methane-seep sediment,
Nankai Trough
Methane hydrate bearing
subseafloor sediment,
Peru Margin
Methane hydrate bearing
subseafloor sediment,
Peru Margin
Hypersaline microbial mat,
Guerrero Negro, Baja, CA
Hypersaline microbial mat,
Guerrero Negro, Baja, CA
High altitude saline
wetland, Salar de Huasco,
Chile
Captain Arutyunov Mud
Volcano, Eastern
Mediterranean
Bold DGGE band names highlight sequences retrieved from 13C-DNA fractions (see Fig. 4).
previously identified in estuarine (Joulian et al., 2001;
Leloup et al., 2006), hydrothermal (Dhillon et al., 2003)
and salt marsh sediments (Bahr et al., 2005), as well as a
sulphate-reducing sediment slurry (Webster et al., 2006b).
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Discussion
The repeated addition of low concentrations of 13C-substrates (5 100 mM) to marine sediment slurries (Severn
FEMS Microbiol Ecol 72 (2010) 179–197
189
Stable-isotope probing of tidal sediments
Fig. 5. Phylogenetic tree showing the diversity of Bacteria 16S rRNA gene sequences from the 12C- and 13C-DNA fractions extracted from the Severn
Estuary sulphate reduction zone sediment slurry C incubated with 13C-acetate for 7 days. Bootstrap support values over 50% (1000 replicates) are
shown. Representative sequences of Deinococcus–Thermus were used as outgroups; Thermus aquaticus (L09663), Meiothermus ruber (L09672) and
Deinococcus radiodurans (M21413). , 12C-DNA 16S rRNA gene clones; , 13C-DNA 16S rRNA gene clones.
FEMS Microbiol Ecol 72 (2010) 179–197
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190
G. Webster et al.
(a)
Fig. 6. Phylogenetic tree showing the diversity of Archaea 16S rRNA gene sequences from the 12C- and 13C-DNA fractions extracted from the Severn
Estuary sulphate reduction zone sediment slurry C incubated with 13C-acetate for 7 days in the (a) Crenarchaeota and (b) Euryarchaeota. Bootstrap
support values over 50% (1000 replicates) are shown. Representative sequences of the Korarchaeota were used as outgroups; hot spring clone pBA5
(AF176347), hot spring clone pJP27 (L25852) and hot spring clone SRI-306 (AF255604). , 12C-DNA 16S rRNA gene clones; , 13C-DNA 16S rRNA
gene clones.
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FEMS Microbiol Ecol 72 (2010) 179–197
191
Stable-isotope probing of tidal sediments
(b)
Fig. 6. Continued.
Estuary tidal flat), without the use of prior enrichment of
active prokaryotes as used previously in SIP of sediment
slurries by Webster et al. (2006b), resulted in clearly detectable 13C-incorporation. This was despite using relatively
short-term incubations (up to 7 days) under a variety of
prokaryotic metabolic conditions and with sediment slurries
from different biogeochemical zones. Analysis of prokaryotic 16S rRNA genes in all sediment slurries demonstrated
rapid enrichment (within 1 day) of the original community
after incubation with 13C-substrates, with limited changes in
the total prokaryotic diversity. Therefore, the repeated
addition of low concentrations of 13C-substrates for up to 4
days, followed by further 10 days of incubation caused very
little changes in the prokaryotic diversity, and hence the
prokaryotes detected by SIP should reflect the active prokaryotic community of the sediment zone under the conditions
used. The results of geochemical analysis of the slurries
demonstrated that the desired conditions were achieved in
some cases [e.g. lack of fermentation products and an
increase in nitrite and nitrate in the aerobic zone slurry A
with 13C-glucose; fermentation products in the anaerobic
(dysaerobic zone) slurry B with 13C-glucose and no sulphate
removal]. However, in the sulphate reduction zone sediment
slurry C and methanogenesis zone slurries D and E no net
sulphate removal or methane production occurred, demonstrating that in these slurries, terminal-oxidizing processes
did not dominate and that activities could have been similar
to those occurring in situ (Wellsbury et al., 1996) and/or that
any sulphide or methane produced may have been reoxidized. This, however, provided an opportunity to detect
nonterminal-oxidizing prokaryotes that are active under the
incubation conditions and/or those prokaryotes involved
FEMS Microbiol Ecol 72 (2010) 179–197
when sedimentary zones are impacted by sediment disturbance, preventing the dominance of anaerobic terminaloxidizing prokaryotes, which is common in the dynamic
Severn Estuary (Yallop & Paterson, 1994).
Glucose utilization in marine sediment slurries
The addition of 13C-glucose to both aerobic (slurry A) and
anaerobic (slurry B) sediment slurries showed that similar
sediment bacteria, Vibrio species, were able to incorporate
13
C-glucose and/or its degradation products under both these
conditions. It is well documented that Vibrio species are
facultative anaerobes that are often isolated from marine
environments (Freese et al., 2009) and readily incorporate
glucose under both anaerobic and aerobic conditions (Alonso
& Pernthaler, 2005). It has been suggested that because of the
facultative nature of Vibrio species, they are perfectly adapted
to survive in the oxic–anoxic zones of tidal sediments (Alonso
& Pernthaler, 2005). The demonstration in this study that
these bacteria can be stimulated quickly and incorporate
added glucose carbon under both oxic and anoxic sedimentary conditions supports this suggestion. Interestingly, a 16S
rRNA gene phylotype was also detected in the aerobic
sediment slurry A, which was similar to I. baltica, a strict
aerobe that can grow poorly on glucose, but can grow better
on acetate (Brettar et al., 2003; Martı́nez-Cánovas et al.,
2004). Therefore, it is likely that a bacterium related to
Idiomarina species is incorporating 13C from glucose/or
metabolites and conducting a similar metabolism in slurry
A. Similarly, under anaerobic conditions (dysaerobic zone
slurry B), one sequence was also obtained that was related to
Marinobacter, a ubiquitous marine bacterial genus, capable of
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192
anaerobic growth. Some pure cultures of Marinobacter species are known to utilize glucose under these conditions (e.g.
Marinobacter salsuginis; Antunes et al., 2007) and other
species are known to grow on glucose fermentation products
such as acetate and lactate (Sass et al., 2001; González &
Whitman, 2006).
The dominance of Gammaproteobacteria within the bacterial population of these two 13C-glucose-amended sediment slurries (aerobic zone slurry A and dysaerobic zone
slurry B) is consistent with this subphyla often being
dominant in the surface of coastal and tidal sediments
(Wilms et al., 2006; Edlund et al., 2008; Kim et al., 2008).
However, the dominance of the archaeal group MG1 within
the active archaeal population from these slurries is somewhat surprising because there are no reports of this group of
largely uncultured Archaea being able to utilize glucose,
despite reports that MG1 Archaea are phylogenetically
diverse and ubiquitous in marine sediments and the overlying water column (Vetriani et al., 1999; Francis et al., 2005;
Teske & Sørensen, 2008; Roussel et al., 2009b). The only
cultured representative of the MG1 is an aerobic, chemolithoautotrophic ammonia-oxidizing archaeon (Könneke
et al., 2005), although the ability of some marine Crenarchaeota to actively take up amino acids (Ouverney & Fuhrman, 2000; Herndl et al., 2005) and evidence that the carbon
isotopic composition of MG1 lipids shows some degree of
organic carbon assimilation suggest that some members of
MG1 have a heterotrophic or a mixotrophic metabolism
(Ingalls et al., 2006). Alternatively, rapid assimilation of 13Cglucose by Gammaproteobacteria within these sediment
slurries could be providing metabolites, including 13CO2,
which might be fixed by autotrophic MG1 species, particularly those in the aerobic slurry A, where there was evidence
of ammonia oxidation.
Acetate utilization in anaerobic marine
sediment slurries
As sulphate reduction is a very important process within
marine sediments (Muyzer & Stams, 2008), including
Severn Estuary sediments (Wellsbury et al., 1996), a greater
focus in terms of molecular analysis was on the sulphate
reduction zone sediment slurry C incubated with
13
C-acetate, an important substrate for sulphate reduction
in marine sediments (Parkes et al., 1989). Consistent with
the short incubations times and the absence of net sulphate
removal, no incorporation of 13C-acetate into known terminal-oxidizing SRB was detected. This may be expected as
even in active sulphate-reducing sediment zones, SRB only
represent a relatively small proportion of the total bacterial
population (e.g. up to 11%, Mußmann et al., 2005; average
of 13%, Leloup et al., 2009) and are sometimes not even
detected by molecular approaches (Parkes et al., 2005).
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G. Webster et al.
However, they can be detected by SIP in sediment slurries
pre-enriched for sulphate reduction (Webster et al., 2006b),
but under these conditions, the pre-enrichment results in
considerable changes in the prokaryotic community. In our
nonenriched sulphate reduction zone slurry C, SRB were
only detected using the more selective and sensitive dsrA
functional gene, including sequences related to known SRB
in the 12C-DNA fraction and some novel dsrA sequences
within the active 13C-DNA fraction. In addition, other
sulphur cycle prokaryotes were also found to have incorporated 13C-acetate. For example, sequences belonging to the
Deltaproteobacteria order Desulfuromonadales were detected
in the 13C-DNA by both DGGE and 16S RNA gene library
analysis. Pure cultures of the genus Desulfuromonas, such as
Desulfuromonas acetoxidans and Desulfuromonas palmitatis
are known sulphur reducers that are capable of oxidizing
acetate and using it as a sole source of carbon. It is intriguing
that a large number of sequences that belong to the
epsilonproteobacterial Arcobacter and Thiovulgaceae clusters
were also found (Fig. 5). Epsilonproteobacteria have been
increasingly recognized as important bacteria involved in
sulphur-dependent biogeochemical cycles and are globally
ubiquitous in marine and terrestrial environments (Campbell et al., 2006). For example, deep-sea hydrothermal fields
(Nakagawa et al., 2005), sulphidic caves (Porter & Engel,
2008) and symbiotic associations (Urakawa et al., 2005)
have epsilonproteobacterial populations. Representative
cultured members of the Arcobacter cluster and the Thiovulgaceae are often chemolithoautotrophic and oxidize sulphide to sulphur under microaerophilic conditions and/or
with nitrate as the electron acceptor (Gevertz et al., 2000;
Wirsen et al., 2002; Kodama & Watanabe, 2004). Our results
suggest that within the sulphate reduction zone sediment
slurry C sulphur cycling may be occurring between populations of novel sulphate reducers, novel sulphur/sulphideoxidizing Epsilonproteobacteria, sulphur-reducing Deltaproteobacteria and other bacteria similar to the sulphur cycling
occurring in sulfureta (Postgate, 1979) and defined mixed
cultures (Biebl & Pfennig, 1978; Telang et al., 1999). This is
consistent with no net sulphate removal occurring in this
slurry.
Many cultured sulphur-oxidizing members of the Arcobacter cluster do not utilize acetate under microaerophilic or
anaerobic conditions (Gevertz et al., 2000; Wirsen et al.,
2002), and therefore, it seems unlikely that the Arcobacterlike sequences detected in this study have the same metabolic restrictions as these previously cultured species. This is
because the Arcobacter species in sediment slurry C seem to
be the dominant members of the community (by 13C-DNA
16S rRNA gene libraries and PCR-DGGE) and that they
are quickly stimulated during incubation (within 1 day; Fig.
2a), suggesting that they are actively utilizing 13C-acetate
and not relying on 13CO2 produced from other bacteria
FEMS Microbiol Ecol 72 (2010) 179–197
193
Stable-isotope probing of tidal sediments
within the sediment slurry. It is possible that these uncultured Arcobacter group members are able to utilize acetate
for sulphur, metal or nitrate reduction and/or incorporate
acetate carbon for cell synthesis. For example, some strains
of the nitrogen-fixing bacterium A. nitrofigilis are able to
utilize acetate and grow anaerobically with nitrate (McClung
et al., 1983), and sequences belonging to uncultured members of the Arcobacter group have been implicated in
manganese reduction in Black Sea shelf sediments coupled
with oxidation of acetate (Thamdrup et al., 2000). Interestingly, low concentrations of dissolved Fe and Mn were
shown to increase with time (3–7 days of incubation) in
slurry C (data not shown), indicating that very low rates
of metal reduction had occurred. However, significant
Arcobacter biomass had already been observed by 1 day
(see Fig. 2a) and therefore the detected metal reduction
within the slurry may have been carried out by other
prokaryotes, such as members of the Desulfuromonadales
(Lovley, 1993).
A range of metabolisms within this slurry may be
associated with the physical mixing of the original sediments, bringing previously spatially separated electron donors and acceptors into close proximity, facilitating their
utilization by fast-growing, acetate-oxidizing/incorporating
Arcobacter species. In addition, the closely related Arcobacter
species strain NA105, isolated from tidal flat sediments of
the Wadden Sea (Freese et al., 2008), can reduce dimethylsulphoxide and trimethylamine oxide (TMAO) (H. Sass,
unpublished data), and TMAO and dimethylsulphoxide
reductases are also found in the Arcobacter butzleri genome
(Miller et al., 2007). Moreover, recently, several SIP studies
indicate that syntrophic acetate oxidation in some environments (soil and lake sediment) can be carried out by
nonacetogenic bacteria such as Geobacter, Syntrophus, other
Deltaproteobacteria, Betaproteobacteria and Nitrospira when
electron acceptors are limited (Chauhan & Ogram, 2006;
Schwarz et al., 2007).
As was observed in the aerobic (slurry A) and dysaerobic
(slurry B) zone sediment slurries with 13C-glucose, the
active archaeal population determined by analysis of the
13
C-DNA from the sulphate reduction zone sediment slurry
C comprised of sequences derived entirely from uncultivated groups of Archaea. Both PCR-DGGE and PCR-cloning
of archaeal 16S rRNA genes showed that the active Archaea
able to utilize 13C-acetate belonged to members of the
diverse MCG. The MCG are one of the predominant
archaeal groups in 16S rRNA gene libraries of deep subsurface sediments (Fry et al., 2008; Teske & Sørensen, 2008),
although they have also been found in other environments
including terrestrial (Chandler et al., 1998) and coastal
marine surface sediments (Roussel et al., 2009a). The term
‘miscellaneous’ within the name reflects the diverse habitat
range and phylogenetic diversity of sequences that make up
FEMS Microbiol Ecol 72 (2010) 179–197
the MCG (e.g. see Fig. 6a). Given the MCG’s substantial
sequence diversity, the identification of distinct MCG subgroups and their dominance in marine sediments, it is not
unreasonable to suggest that some members of this group
can incorporate 13C-acetate under anaerobic conditions,
considering the importance of acetate as an anaerobic
substrate. In addition, carbon isotopic signatures of archaeal
cells and polar lipids from MCG-dominated subsurface
sediments suggest that these archaeal populations are able
to utilize buried organic carbon (Biddle et al., 2006), and
hence, that most members of the MCG are heterotrophic, an
inference directly supported by our results. Interestingly,
although MCG sequences were abundant in the 13C-DNA
16S rRNA gene libraries from the sulphate reduction zone
slurry, no sequences belonging to MG1 Archaea were
detected in this fraction despite their presence in the 12CDNA library (Fig. 6a). This suggests that MG1 are not able
to utilize 13C-acetate carbon or were not active within the
sulphate reduction zone sediment slurry C.
As with sediment slurry C, no terminal-oxidizing prokaryotes, in this case methanogens, were identified in the
methanogenesis zone sediment slurries D and E by 16S
rRNA gene analysis incubated with 13C-acetate or 13CO2,
respectively. However, a diverse range of Bacteria were
rapidly stimulated (within 1 day) and had incorporated 13C
by 7 days (Fig. 3e and f). Also, like sediment slurry C with
13
C-acetate, Arcobacter-related sequences were present in the
methanogenesis zone sediment slurry D, but interestingly,
they did not dominate the 13C-DNA DGGE profile. This
suggests that Arcobacter were less active in the methanogenesis zone sediment slurry, possibly as their electron acceptors
were less abundant in deeper sediments and/or sulphur
cycling was less prevalent in this low-sulphate sediment
slurry. Interestingly, as the lower potential energy conditions
of these methanogenesis zone slurries were the only conditions where no 13C incorporation into Archaea occurred,
this may suggest that Archaea were not as active under these
conditions compared with the other, potentially higher
energy slurry conditions (slurries A, B and C). This was
surprising because Archaea have been suggested to be lowenergy specialists (Valentine, 2007). However, it would be
interesting to know whether 13C-incorporation would have
occurred with increased incubation times and/or under
methane-producing conditions.
Implications of DNA-SIP for the study of
sedimentary processes
This study shows that DNA-SIP is a very useful tool to study
uncultivated groups of prokaryotes involved in different
biogeochemical sedimentary processes, without the need
for prior enrichment of specific prokaryotic populations.
However, under these conditions, it is difficult to detect
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Published by Blackwell Publishing Ltd. All rights reserved
c
194
terminal-oxidizing SRB and methanogens, probably due to
their low cell numbers and activities, unless more selective
functional genes are used. It should also be noted that PCRDGGE and analysis of only a limited number of 16S rRNA
gene clones were used in this study and, therefore, it is
probable that other less abundant prokaryotes including
terminal-oxidizers would have been detected with more
extensive sampling (Quince et al., 2008), for example
through use of high-throughput sequencing technologies
(Tringe & Hugenholtz, 2008) such as pyrosequencing
(Huber et al., 2007).
Multiple, low concentrations of 13C-substrate additions
and relatively short incubations (7 days) seemed successful
in restricting changes in the prokaryotic community, while
achieving significant 13C incorporation (confirmed by 13C
analysis of PLFA; J. Rinna, G. Webster, A.J. Weightman &
R.J. Parkes, unpublished data). This is important because
longer incubation times are often cited as a disadvantage of
DNA-SIP because of cross-feeding from original 13C-substrates and changes in the community structure (Neufeld
et al., 2007b). Some studies have, therefore, used very short
incubation times and reported 13C incorporation into DNA
and SSU rRNA of some sedimentary bacteria within 1–2 h
(Gallagher et al., 2005; MacGregor et al., 2006). However,
such very short incubations may not have resulted in 13Clabelling of some of the uncultured Bacteria and Archaea
detected in this study. The use of 15N-labelling techniques as
used for slow-growing anaerobic methane-oxidizing communities (Krüger et al., 2008), in combination with
DNA-SIP (Buckley et al., 2007) and/or RNA-SIP (Manefield
et al., 2002), may further improve our approach. Also, in
DNA-SIP, it has been shown to be difficult to completely
separate 12C- and 13C-DNA fractions with confidence (Neufeld et al., 2007a). In the present study, the absence of
archaeal PCR products in the 13C-DNA fractions of the
methanogenesis zone sediment slurries D and E (Fig. 4d and
e) demonstrates that there was no smearing of 12C-archaeal
DNA into the 13C-DNA fraction, and hence, that our
procedures enabled good 13C incorporation and 12C- and
13
C-DNA separation.
This study demonstrates that although it may be a
difficult challenge to use DNA-SIP to understand complex
prokaryotic communities and processes in marine sediments, it is a very useful tool to identify the activities of
uncultured groups of sediment Archaea and Bacteria, the
substrates that they incorporate and the conditions under
which they are active. Our results highlight that several
groups of uncultured prokaryotes play important ecological
roles in carbon and sulphur cycling of tidal sediments of the
Severn Estuary, specifically providing new metabolic information for uncultured groups of Archaea (e.g. MG1, MCG)
and suggesting broader metabolisms for Bacteria with
limited cultured representatives (e.g. Arcobacter species).
2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
G. Webster et al.
Acknowledgements
G.W. and J.R. were funded by the NERC Marine and
Freshwater Microbial Biodiversity programme research
grant numbers NER/T/S/2000/636 and 2002/00593. The
work was also supported by the European Union contract
number EVK3-CT-1999-00017, and NERC NE/F018983/1
and NE/F00477X/1. The authors would like to thank Mr
Stephen Hope (Cardiff University) for technical support
with DNA sequencing, Professor Richard Evershed and
Dr Richard Pancost (University of Bristol) for advice
and kind use of their analytical facilities and Dr Henrik
Sass for helpful comments during the preparation of this
manuscript.
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