RESEARCH ARTICLE
The depth-specific significance and relative abundance of
anaerobic ammonium-oxidizing bacteria in estuarine sediments
(Medway Estuary, UK)
Christine Rooks1, Markus C. Schmid2,3, Wahida Mehsana1 & Mark Trimmer1
1
School of Biological and Chemical Sciences, Queen Mary, University of London, London, UK; 2Department für Mikrobielle Ökologie, Universität
Wien, Vienna, Austria; and 3Institute for Water and Wetland Research, Department of Microbiology, Radboud University Nijmegen, Nijmegen,
The Netherlands
Correspondence: Mark Trimmer, School of
Biological and Chemical Sciences, Queen
Mary, University of London, Mile End Road,
London E1 4NS, UK. Tel.: +44 (0)
207 882 3007; fax: +44 (0) 207 882 7732;
e-mail: [email protected]
Received 27 July 2011; revised 11 November
2011; accepted 19 November 2011.
Final version published online 16 January
2012.
DOI: 10.1111/j.1574-6941.2011.01266.x
MICROBIOLOGY ECOLOGY
Editor: Gary King
Keywords
anammox; estuarine sediments; DNRA;
fluorescence in situ hybridization.
Abstract
Variations in the overall and depth-specific significance of anammox were measured using 15N isotope experiments in both bioirrigated and undisturbed sediments of the Medway Estuary, UK. This was performed over two surveys,
alongside FISH experiments, to identify and track shifts in the relative abundance of anammox organisms with depth. In Survey 1 (initially screening for
the presence of anammox), the potential for anammox (ra) decreased from
32% upstream to 6% downstream. In Survey 2, depth-specific values of ra varied between a maximum of 37% upstream and a minimum of 4% downstream.
This was linked to a small population of anammox organisms accounting for
< 1–8% of total bacteria with depth in Survey 1 and < 1–3% in Survey 2. The
relationship between the relative abundance of anammox cells and the potential
contribution of anammox to total N2 production did not however correlate. In
Survey 2, infaunal disruption of the sediment substrata, and concomitant fluctuations of O2 over depth, did not appear to inhibit the potential for anammox, even at the most bioturbated site. Moreover, deficits detected in the
retrieval of 15N gas from denitrification in Survey 2 may imply potential links
between dissimilatory nitrate reduction to ammonium and anammox in estuarine sediments.
Introduction
The availability of fixed forms of nitrogen is an important
factor in regulating primary production (Vitousek &
Howarth, 1991). In aquatic environments, NHþ
4 derived
from the decomposition of organic matter is oxidized to
NO
3 (via NO2 ) in nitrification (Painter, 1970). The
produced
in this process subsequently diffuses into
NO
3
the suboxic zone of the sediment, where it is denitrified
to N2 gas in coupled denitrification (Zumft, 1997). Until
recently, the removal of fixed nitrogen was almost entirely
attributed to coupled denitrification (Devol, 1991). The
discovery of anaerobic ammonium oxidation (a process
whereby NHþ
4 is anaerobically oxidized with NO2 to
form N2 gas), however, has redefined this concept
(Thamdrup & Dalsgaard, 2002). Consequently, anammox
represents an alternative N removal pathway that circumFEMS Microbiol Ecol 80 (2012) 19–29
vents the critical aerobic nitrification phase, typically
associated with coupled denitrification (Mulder et al.,
1995; van de Graaf et al., 1995).
Environmental studies have confirmed the presence of
anammox in a diverse range of geographically and biogeochemically distinct environments such as oxygen minimum zones (Kuypers et al., 2003, 2005; Lam et al., 2009),
freshwater (Zhang et al., 2007; Yoshinaga et al., 2011),
tropical freshwater (Schubert et al., 2006; Amano et al.,
2011), Arctic sea ice (Rysgaard & Glud, 2004) and Arctic
(Rysgaard et al., 2004; Gihring et al., 2010), marine
(Thamdrup & Dalsgaard, 2002; Amano et al., 2007; Schmid et al., 2007; Engström et al., 2009; Jaeschke et al.,
2010) and estuarine sediments (Trimmer et al., 2003,
2005; Meyer et al., 2005; Risgaard-Petersen et al., 2005;
Tal et al., 2005; Rich et al., 2008; Dong et al., 2009).
Although the occurrence of anammox is relatively
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Published by Blackwell Publishing Ltd. All rights reserved
C. Rooks et al.
20
widespread, the significance of this process is highly variable and frequently associated with a small population of
anammox bacteria (< 1–9%) (Schmid et al., 2007). The
potential contribution of anammox to total N2 production, in marine, coastal and estuarine sediments, varies
between 24% and 67% (Thamdrup & Dalsgaard, 2002)
and < 1–35% (Rysgaard et al., 2004; Nicholls & Trimmer,
2009), yet the specific factors that govern this variation
require further clarification.
þ
Whilst the presence of NO
2 and NH4 represent prerequisites for anammox activity, the position of the oxic–
suboxic interface may also serve as a governing factor in
the spatial distribution of anammox organisms throughout marine and estuarine sediments. Previous studies
have shown that anammox organisms are reversibly
inhibited by as little as 1 lM oxygen in enrichment culture and by < 10 lM oxygen in the environment (Strous
et al., 1999; Jensen et al., 2008). This would suggest that
the formation of a stable anammox community can only
occur in the suboxic zone of the sediment substrata. The
disruption of the sediment substrata, however, by bioturbating infauna, along with any subsequent bioirrigation,
strongly influences sediment processes by altering the
sources and distribution of nutrients and oxygen to
microorganisms, causing an extension of oxic and suboxic
zones into otherwise highly reduced, anoxic sediment
(Henriksen et al., 1980). A potential consequence of bioturbation is an increase in the abundance of NO
2 -producing organisms and hence the overall availability of
NO
2 (Mermillod-Blondin et al., 2004). Whilst this may
be of benefit to the anammox community, active bioturbation exposes the sediment substrata to concomitant
spatial and temporal fluctuations in oxygen. Given the
slow growth rate (maximum doubling time, 11 days) and
the sensitivity of anammox organisms to oxygen, this
process may in fact perturb the formation of an anammox community, in any given environment (Strous et al.,
1999).
Nevertheless, an increasing number of studies have
demonstrated that anammox activity may occur under
seemingly unfavourable conditions. Woebken et al.
(2007) described the presence of anammox in oxygendepleted microniches (within planktonic snow) at oxygen
concentrations of up to 25 lM in the surrounding bulk
water body. Essentially, in such environments, a protective layer of microorganisms (consuming oxygen) shields
anammox bacteria from ambient O2 (Nielsen et al., 2005;
Woebken et al., 2007). In view of this, the distribution of
anammox organisms may not necessarily be constrained
by natural fluctuations in dissolved oxygen.
The purpose of this study was to determine whether
variations in the significance of anammox are linked to
changes in the size of the anammox community. The
ª 2011 Federation of European Microbiological Societies
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zone of anammox activity was characterized by 15N
isotope labelling experiments, oxygen profiles and depthspecific rate measurements of CO2 production (as measurements of total carbon mineralization). Fluorescence
in situ hybridization and 4′6-diamino-2-phenylindole
dihydrochloride (DAPI) were used alongside these techniques to determine shifts in the abundance of anammox
organisms with depth, thus potentially linking the significance of anammox to deviations in the anammox community. This was explored both at sites in the presence
and absence of bioturbating invertebrates, along a transect
of the Medway Estuary, UK.
Materials and Methods
Site locations and sediment sampling
The Medway Estuary lies to the south of the Thames
Estuary in South East England, Kent. It is connected to
the Thames Estuary via the Isle of Grain and drains into
the North Sea through tidal channels that divide large
islands of salt marsh (Aldridge & Trimmer, 2009). Sediment samples were collected at low tide from the intertidal mudflats of five sites in Survey 1 (April 2005,
initially screening for the presence of anammox) and
three sites in Survey 2 (September 2007, re-examination
with depth). In Survey 1, samples were retrieved (as per
Nicholls & Trimmer, 2009) and collected in a seaward
direction from Medway Bridge Marina (landward site), to
Strood, Upnor, Stoke Saltings and Grain (coastal site).
The sediments follow a typical gradient for estuaries in
this region, from muddy with high porosity towards the
land, to coarser grain, sandier sediments with lower
porosity at the coast (Nicholls & Trimmer, 2009). In both
surveys, site water salinity was measured using a handheld refractometer. Sediment porosity and organic carbon
content were measured as part of Survey 2 as per Nicholls
& Trimmer (2009) (Table 1).
In this study, all 15N and FISH labelling experiments
were performed with homogenized samples taken from
the first 2 cm of the sediment (as per Trimmer et al.,
2003; Nicholls & Trimmer, 2009). Samples from Survey 2
were collected from Medway Bridge Marina, Upnor and
Grain using five identical Perspex core tubes (sediment
depth 6 cm, i.d. 9 cm). In this survey, 15N isotope labelling experiments were performed using subsamples of 1-cm
sections, over a depth of 5 cm. In addition, standard FISH
labelling experiments were performed using 0.5-cm sections
of subsampled sediment over a depth of 2.5 cm.
In both surveys, sediment samples were returned to the
laboratory within two hours of collection. In Survey 1,
the samples were processed as described by Nicholls
& Trimmer (2009). In Survey 2, sediment cores were
FEMS Microbiol Ecol 80 (2012) 19–29
21
Abundance and significance of anammox in estuarine sediments
Table 1. Sediment characteristics (Survey 2, September 2007), average site water salinity and grid references from Surveys 1 (April 2005) and 2
(September 2007) of the Medway Estuary, UK
Site
Medway Bridge Marina
Strood
Upnor
Stoke Saltings
Grain
Organic carbon
(% of dry weight)
Porosity
Salinity (&)
Grid reference
3.38
0.81
3.41
0.73
0.41
0.53
6
5
15
27
32
51°
51°
51°
51°
51°
transferred to a water bath containing aerated low-nutrient sea water (LNS), diluted to site salinity using ultra
high-purity water (UHP) (Trimmer et al., 2006). Filtered
LNS was collected from Lowestoft on the North Sea coast
of the UK and used during experiments throughout
Surveys 1 and 2. The advantage of using LNS is that it
contains far less ambient nitrate compared to actual site
water. The cores were re-equilibrated overnight and
maintained at an average in situ temperature of 12 °C,
using a Grant thermo circulator (Grant instruments,
Cambridgeshire, UK).
Porewater oxygen profiles and infaunal
sampling
To determine the position of the oxic–suboxic interface
in Survey 2, porewater oxygen profiles were measured
using a Clark-type oxygen microsensor with an outer tip
diameter of 40–60 lm (OX50; Unisense, Aarhus, Denmark) (Revsbech, 1989). Oxygen microelectrodes were
positioned perpendicular to the sediment surface and driven into the sediment at 100-lm intervals. Readings from
the microelectrode were displayed on a picoammeter (PA
2000; Unisense) and logged after 4 s when the signal had
stabilized. The overall shape of the profile was taken to
indicate the transport of oxygen into the sediment either
by simple diffusion (a steady decrease in oxygen with
depth) or by bioirrigation (disruption of the profile with
spikes in oxygen). Following sectioning, the remaining
sediment in each core was sieved (mesh size, 0.4 mm) to
identify the infaunal community composition and corresponding population density.
The relative contribution of anammox to total
N2 production – 15N labelling ‘end-point’
incubation experiments
The relative contribution of anammox to total N2 production was determined according to methods adapted
from Risgaard-Petersen et al. (2003). In Survey 1, anaerobic sediment slurries were prepared with LNS. In brief,
14 mL of degassed LNS (diluted with UHP according to
FEMS Microbiol Ecol 80 (2012) 19–29
22′
23′
24′
26′
27′
42.02′′N,
37.66′′N,
20.38′′N,
30.89′′N,
03.83′′N,
0°
0°
0°
0°
0°
28′
30′
31′
37′
43′
48.62′′
29.01′′
36.28′′
40.67′′
22.46′′
E
E
E
E
E
site salinity) was added to 20 mL of homogenized sediment, sealed within a 37-mL serum bottle (Alltech Associates) and shaken vigorously to create anoxic slurry. To
limit exposure to oxygen, all sediment slurries were prepared inside an anoxic glove box filled with oxygen-free
nitrogen (Belle Technology, Dorset, UK). Preincubation,
slurry enrichment (final concentration, 200 lM 15 NO
3)
and ‘end-point’ incubation gas headspace sampling were
performed according to the methods described by Trimmer et al. (2003) and Risgaard-Petersen et al. (2003). In
Survey 2, sectioned sediment (1 g) was distributed into
3-mL, gas-tight glass vials (Exetainer, Labco Ltd, High
Wycombe, UK). In total, one unamended reference and
two end-point incubation samples were collected from
each depth in five replica cores (Risgaard-Petersen et al.,
2003) and weighed. The glass vials were then transferred
into an anoxic glove box (as above). Each vial was subsequently filled with 1 mL of LNS (diluted to site salinity
and degassed with oxygen-free nitrogen), sealed and shaken vigorously. This was followed by a preincubation period of 24 h to remove all background NO
x and O2
(Risgaard-Petersen et al., 2003). During this preincubation period, the vials were placed on rotating rollers
(Spiromix, Denley) and maintained at constant temperature (12 °C) in the dark. The samples were then injected
with degassed (oxygen-free nitrogen) stock solutions con15
Natm
taining an isotopic mixture of Na15 NO
3 (99.2
þ
14
%) and NH4 Cl using a Hamilton syringe (SigmaAldrich, Poole, UK). The final concentrations of 15 NO
3
and 14 NHþ
4 were 200 and 500 lM, respectively, in each
slurry. The vials were then placed on rollers for a further
24 h and incubated at 12 °C in a constant temperature
room (as above). At the end of the incubation period, the
samples were injected with 50% ZnCl2 (w/v) to inhibit
further microbial activity. On this occasion, we did not
include standard controls in the presence and absence of
15
NHþ
4 , as we have previously shown good agreement in
29
15
N2 production with either 15 NHþ
NO
3 . This has
4 or
been confirmed at this site and in other estuaries in the
region (Nicholls & Trimmer, 2009).
Concentrations of 28N2, 29N2 and 30N2 were measured directly from the headspace of each vial using a
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Published by Blackwell Publishing Ltd. All rights reserved
C. Rooks et al.
22
continuous flow isotope ratio mass spectrometer,
calibrated with N2 in He over air-equilibrated water
(Delta Matt plus; Thermo-Finnigan, Bremen, Germany).
The potential contribution of anammox to total N2
production (ra%) was calculated accordingly (Thamdrup
& Dalsgaard, 2002; Risgaard-Petersen et al., 2003).
Rates of CO2 production with sediment depth –
an indicator of total carbon mineralization
Aliquots of 2.5 g of sediment from each section (1 cm)
were distributed into gas-tight, 12-mL glass vials (as
above) from five replica cores in Survey 2. Each vial was
sealed with an anoxic headspace, and the wet weight was
recorded prior to further processing. The accumulation of
CO2 was measured directly from the headspace of each
sealed vial by gas chromatography–flame ionization detection after hot catalytic reduction of CO2 to CH4 (hot
nickel NiCat, Agilent Technologies) (see Sanders et al.,
2007 and Nicholls & Trimmer, 2009). Samples were
injected into the gas chromatograph at intervals of
84 min. The rate of CO2 production was calculated by
the linear regression of CO2 production, plotted against
time.
Fluorescence in situ hybridization (FISH)
In Surveys 1 and 2, FISH was performed according to the
protocols described by Schmid et al. (2000, 2003). Briefly,
sediment (1 g) was fixed with 4% (v/v) paraformaldehyde
in PBS solution and incubated at room temperature
for 2 h. Each sample was then washed twice with PBS
and resuspended in a 50% (v/v) PBS–ethanol solution.
All samples were subsequently stored at 20 °C until
required.
In Survey 1, the anammox-specific probes BS 820
(Kuypers et al., 2003) and Scabr 1114 (Schmid et al.,
2003) were used in combination with Pla 46 (Neef et al.,
1998) during hybridization experiments. Until recently, it
was understood that whilst anammox activity was relatively widespread, the diversity of these organisms was
extremely limited (Kuypers et al., 2003; Schmid et al.,
2007; Woebken et al., 2008). As a consequence, probes
specifically targeting members of the genus Scalindua
were the focus of Survey 1. An increasing number of
studies, however, indicate that this no longer the case
(Amano et al., 2007, 2011; Dale et al., 2009; Yoshinaga
et al., 2011). As Amx 0368 facilitates the identification of
a broader range of anammox organisms, a combination
of Pla 46, Amx 0368 (Schmid et al., 2000) and Sca 1309
(Schmid et al., 2003) was therefore utilized during Survey
2. To the best of our knowledge, the use of Amx 368
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accounts for the full diversity of anammox organisms
described to date.
Following hybridization, the sediment samples were
counterstained with DAPI (fluorochrome 4′6-diamino2-phenylindole dihydrochloride) and viewed using an
epifluorescence microscope (Leica microsystems, UK).
Images were captured from 20 visual fields with a blackand-white digital camera (Leica microsystems). A total of
five FISH labelling experiments were performed across
the first 2 cm of homogenized sediment in Survey 1. In
Survey 2, five FISH experiments were performed at each
depth interval per core. All labelled oligonucleotide
probes were purchased as Cy3-, Cy5- and 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester (Fluos)-labelled
derivatives from MWG-Biotech (London, UK) and
Thermo Electron (Ulm, Germany).
Planctomycete and anammox-like cells were identified
on the basis of the presence of doughnut-like internal
compartmentalization and probe binding patterns. Planctomycete-like cells were identified according to positive
hybridizations with the probe Pla 46. These cells typically
emitted green fluorescence. Anammox-like cells were
identified according to the triple hybridization of Pla 46
in combination with anammox-specific probes. These
cells emitted white fluorescence. Only anammox-like cells
that simultaneously hybridized with all three probes were
included in anammox-specific counts. The abundance of
anammox bacteria was expressed relative to the corresponding total of DAPI-stained cells. The total number of
bacteria per micrograph was calculated using the software
IMAGE PRO PLUS version 5.0 (Media Cybernetics, Inc.). The
identification and abundance of anammox cells was manually evaluated.
Results
Survey 1 (April 2005)
In situ detection and relative significance of
anammox in the Medway Estuary
Anammox organisms were detected at all sites in Survey
1 of the Medway Estuary (April 2005). The fluorescently
labelled probes Scabr 1114 (Cy3) and BS 820 (Cy5) were
used in combination with Pla 46 (Fluos). This resulted in
the triple hybridization of probes with target organisms,
pointing towards the presence of ‘Scalindua-like’ cells.
Overall, the cells depicted were typically ‘doughnutshaped’ and fell within the reported size range for anammox organisms.
The relative abundance of anammox organisms
decreased in a seaward direction from 8% at Strood
(landward) to < 1% at Grain (seaward). This is in good
FEMS Microbiol Ecol 80 (2012) 19–29
23
10
35
ra (%)
rel. abundance (%)
30
8
25
6
20
15
4
10
2
5
0
0
MBM
S
Up
Site
SS
Gr
The relative abundance of anammox bacteria to total bacteria (%)
The potential contribution of anammox to total N2 production (%)
Abundance and significance of anammox in estuarine sediments
(a)
(b)
Fig. 1. The potential contribution of anammox to total N2 production
(black symbols) and the relative abundance of anammox organisms
(white symbols) at Medway Bridge Marina (MBM), Strood (S), Upnor
(Up), Stoke Saltings (SS) and Grain (Gr) along the Medway Estuary
(April 2005). Each data point represents the mean of 5 (ra) and 20
replicates (relative abundance of anammox organisms), respectively.
Sediment samples were collected from the top 2 cm of the sediment.
agreement with values of ra, where the potential contribution of anammox to total N2 production decreased in
a seaward direction from 19% to 6% (Fig. 1). Interestingly, a discrepancy between the relative abundance of
anammox organisms and values of ra (32%) was evident
at the most landward site, Medway Bridge Marina. Here,
anammox organisms formed just 2% of the total prokaryotic population. Consequently, this led to the detailed
re-examination of three sites (Medway Bridge Marina,
Upnor and Grain) from the Medway Estuary. The results
of this investigation are described below.
(c)
Survey 2 (September 2007)
Porewater oxygen profiles
The position of the oxic–suboxic interface was on average
9.9, 3.6 and 3.5 mm at Medway Bridge Marina, Upnor
and Grain, respectively (Fig. 2). The dissolved oxygen
profiles measured at Medway Bridge Marina indicated
both significant bioturbation and bioirrigation within the
top 0–1.5 cm of sediment strata by macrofauna (Fig. 2a).
This was associated with the presence of the polychaete,
Nereis diversicolor, and the amphipod, Corophium volutator, at mean densities of 14 (SE = ± 2) and 45 (SE = ± 9)
per m2, respectively.
In contrast, the dissolved oxygen profiles measured at
Upnor and Grain were more typical of diffusional
exchange, demonstrating a clear separation of the sediFEMS Microbiol Ecol 80 (2012) 19–29
Fig. 2. Multiple measurements of dissolved oxygen profiles in the
presence and absence of bioturbating/bioirrigating macrofauna during
Survey 2 (September 2007). (a) Medway Bridge Marina, (b) Upnor
and (c) Grain. Each profile corresponds to an individual core.
ment into oxic and suboxic strata (Fig. 2b and c). In the
absence of major bioirrigation, the oxic zone was constrained to the upper 3.5 mm of the sediment and was
only one-third of that measured at Medway Bridge Marina. The position of the oxic–suboxic interface and the
shape of the oxygen profiles were almost identical at the
latter two sites.
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C. Rooks et al.
24
Fig. 3. Depth-specific measurements CO2 production (black symbols),
ra (white symbols) and the relative abundance of anammox
organisms to total bacteria (grey symbols) at Medway Bridge Marina
(a), Upnor (b) and Grain (c) during Survey 2 (September 2007). The
dotted line represents the position of the oxic–suboxic interface.
Values of CO2 production are 1 ± SEM (n = 5). Each data point
represents the mean of 5 (ra) and 20 replicates (relative abundance of
anammox organisms). Sediment samples were collected from the first
5 cm of the sediment.
(a)
Depth-specific rates of CO2 production – an
indicator of total carbon mineralization
The production of CO2 was consistently linear (r2 > 0.8,
on average) with time but varied in magnitude between
sites and according to depth. The greatest depth-specific
rates of CO2 production were measured at Medway
Bridge Marina where they peaked within the first cm of
the sediment (1.6 nmol CO2 g1 wet sediment h1) and
decreased to a depth of 4.5 cm (1.2 nmol CO2 g1 wet
sediment h1) (Fig. 3a). In general, there was no depthspecific trend in CO2 production at Upnor (Fig. 3b).
Similarly, there was little variation in the depth-specific
rates of CO2 production at Grain (Fig. 3c). The average
rates of CO2 production were 1.4 nmol CO2 g1 wet sediment h1, 0.2 nmol CO2 g1 wet sediment h1 and
0.3 nmol g1 wet sediment h1 at Medway Bridge Marina, Upnor and Grain, respectively.
(b)
The significance of anammox relative to
denitrification with sediment depth
The relative contribution of anammox to total N2 production (ra) was on average 28%, 10% and 6% at Medway
Bridge Marina, Upnor and Grain, respectively. Across all
sites, the greatest potential for anammox was localized to
the upper 2 cm of the sediment. The greatest depth-specific
values of ra were recorded at Medway Bridge Marina, where
ra peaked at 37% and then subsequently decreased to 17%
at 3.5 cm into the sediment (Fig. 3a). Similarly, a peak in
ra (15%) was evident in the subsurface at Upnor (Fig. 3b),
followed by a decrease to 7% at 4.5 cm. The depth-specific
values of ra were lowest at Grain, where the potential for
anammox decreased to 4% at 4.5 cm (Fig. 3c).
(c)
Depth-specific rates of CO2 production relative
to the potential for anammox
The depth-specific values of ra increased as a function of
CO2 production (Fig. 4b), with a particularly clear pattern
at Medway Bridge Marina. Here, the maximum rate of
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FEMS Microbiol Ecol 80 (2012) 19–29
25
Abundance and significance of anammox in estuarine sediments
(a)
(a)
(b)
Fig. 5. (a) Combined epifluorescence micrograph of a sample from
Medway Bridge Marina (sediment layer: 0.75 to 1.25 cm), Survey
2 (September 2007). The scale bar represents 20 lm. (b) An
enlargement of panel a showing a white, ‘doughnut-like’ anammox
cell (white circle). The scale bar represents 7 lm.
(b)
these data indicated that proportionately less 15 NO
3 was
recovered as 15N-labelled gas for denitrification (Fig. 4a).
At maximum rates of CO2 production, the yield of 15Nlabelled gas from anammox was 22%, relative to just 36%
for denitrification at Medway Bridge Marina.
In situ detection and the depth distribution of
anammox organisms
Fig. 4. (a) The yield of 15N-labelled gas from anammox (black
symbols, y = 67x + 32, r2 = 0.54) and denitrification (white symbols,
y = 26x + 53, r2 = 0.82). (b) The relative contribution of anammox
(black symbols, y = 17612x + 2.9, r2 = 0.816) and denitrification
(white symbols, y = 17612x + 97, r2 = 0.82) to total N2 production
as a function of CO2 production. Values are a scatter of data points
across all sites from Survey 2 (September 2007).
CO2 production (1.6 nmol CO2 g1 wet sediment h1)
coincided with a peak in ra (37%) (Fig. 4b). In contrast,
the depth-specific potential for denitrification decreased in
response to CO2 production (Fig. 4b). When the yield of
15
N-N2 from anammox and denitrification was plotted as a
function of sediment respiration (CO2 production), a similar relationship was apparent; however, regression through
FEMS Microbiol Ecol 80 (2012) 19–29
The presence of anammox organisms was verified by
FISH at all sites. The fluorescently labelled probes Amx
0368 (CY3), Sca 1309 (fluos) and Pla 46 (CY5) clearly
hybridized with anammox cells (Fig. 5). Probe binding
patterns therefore pointed towards the presence of ‘Scalindua-like’ bacteria. The cells depicted were typically
‘doughnut-shaped’ and fell within the reported size range
for anammox organisms (Fig. 5).
The relative abundance of anammox organisms
detected varied between 0% and 3% at all sites and at all
depth intervals, which is in line with the previous observations (Schmid et al., 2007). The overall trend with
depth at Grain was an increase from 0.7% at 5 mm to
3.3% at 20 mm (Fig. 3c). Here, the maximum depthspecific value of ra (5%) coincided with a peak in the
abundance of anammox organisms (3.3%). This was not
the case, however, at Upnor where the greatest number of
anammox organisms accumulated within the first 1.5 cm
(2.9%) (Fig. 3b). Anammox organisms were observed
within the oxic zone at the Medway Bridge Marina and
increased from 1% (at 0.25 cm) to 3% (at 2.25 cm).
Discussion
The purpose of this study was to determine whether variations in the significance of anammox were linked to
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
26
changes in the size of the anammox community. The relative contribution of anammox to total N2 production, in
the Medway Estuary, was in line with the previous findings where ra has been shown to be positively correlated
with sediment organic carbon (Trimmer et al., 2003;
Nicholls & Trimmer, 2009). The importance of anammox
in N removal at Medway Bridge Marina was however
substantially elevated in relation to comparative systems
(Trimmer et al., 2003; Nicholls & Trimmer, 2009). In the
Thames Estuary, ra does not exceed 8% (in sediment
slurries) yet the contribution of anammox to total N2
production at Medway Bridge Marina was, on average,
28% in September 2007 and 32% in April 2005. According to current knowledge, this places the results amongst
the highest values of ra reported for estuarine sediments
(maximum ra = 30%) (Dong et al., 2009).
Overall, values of ra peaked at the sediment subsurface
across all sites (Fig. 3). In sediments where there is a sufficient supply of organic matter, NO
2 accumulates within
the upper cm of the sediment as a result of NO
3 reduction (Stief et al., 2002). At such depths, and in the
absence of oxygen, this process could provide a sufficient
supply of NO
2 to maintain a stable anammox community. In this study, the capacity for anammox decreased
below the top 0.5–1.5 cm of the sediment. This could be
linked to a decrease in the availability of porewater NO
2,
as NO
3 reduction declines (Meyer et al., 2005). Consequently, this may have lead to an overall reduction in the
capacity for N2 formation via anammox (Fig. 3).
Depth-specific rates of CO2 production were positively
correlated with the formation of N2 via anammox
(Fig. 4). High rates of CO2 production represent elevated
rates of sediment metabolism. In environments where
there is an abundant supply of organic matter, this
increases sediment metabolism by stimulating the growth
of heterotrophic, denitrifying bacteria (Sloth et al., 1995).
As a result, this could enhance the supply of freely diffusible NO
2 to the anammox community and therefore promote close coupling between these processes (Thamdrup
& Dalsgaard, 2002; Kuypers et al., 2003). This is further
corroborated by the broadscale positive correlation
between ra and the availability of organic carbon observed
across many estuaries (Nicholls & Trimmer, 2009).
The recovery of 15N-labelled gas, following the addition
of 15 NO
3 , indicates that denitrification is perhaps not the
only significant process enabling the delivery of NO
2 to
anammox organisms. In terms of the overall yield of
15
N-labelled gas, from anammox and denitrification, it
was evident that whilst anammox increased as a function
of CO2 production, the corresponding recovery of
15
N-labelled gas from denitrification decreased by a factor
of 4 (Fig. 4a). This deficit could perhaps be explained, in
part, by dissimilatory nitrate reduction to ammonium
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
C. Rooks et al.
(DNRA). Although this should be viewed with caution, if
DNRA is active in these sediments, this could potentially
reduce the pool of 15 NO
3 available for denitrification
(Revsbech et al., 2006).
Nevertheless, it is also important to consider the probability of producing a false signal of denitrification when
enriching with 15 NO
3 , in the presence of both anammox
and DNRA. Essentially, this is because 15 NO
3 may be
as
a
result
of
this
process.
Consereduced to 15 NHþ
4
generated
by
DNRA
could
therefore
be
quently, 15 NHþ
4
,
thus
resulting
in
an
overproduccombined with 15 NO
3
tion of 30N2. As it is assumed that 30N2 is derived from
denitrification, this could lead to an overestimation of
denitrification (Thamdrup & Dalsgaard, 2002). However,
according to Nicholls & Trimmer (2009), the probability
of this occurring under the exact same conditions is estimated to be approximately 1.4%.
If the values in this study are representative of denitrification, then this would suggest that whilst anammox
perhaps benefits from the release of NO
2 (derived as an
intermediate of DNRA), denitrifying organisms may be
outcompeted for the NO
3 reduced in this process. Studies addressing the potential relationship between anammox and DNRA are nevertheless scarce. Dong et al.
(2009) recently documented high rates of DNRA in the
presence of anammox in estuarine sediments, however, it
is only in the Peruvian OMZ and Arabian Sea that these
processes have been directly linked (Lam et al., 2009; Jensen et al., 2011). Lam et al. (2009) imply that in the
absence of denitrification, the NO
2 produced during
þ
NO
3 reduction, in combination with NH4 derived from
DNRA, is the principal source of NO2 and NHþ
4 for
anammox. In the context of the Medway Estuary, NO
2
delivered by both denitrification and DNRA may be
responsible for maintaining anammox activity in this system. Potential links between anammox and DNRA may
therefore expand our current knowledge of the complexity of N transformations in estuarine sediments.
In the top layer (0–1.5 cm) of the sediment, the depthspecific values of ra reached 37%, yet anammox cells
represented just 1–2% of the microbial community.
Differences between the relative abundance of anammox
organisms and ra, however, were less marked at Upnor and
Grain (Fig. 3b and c). Large discrepancies, between the relative abundance of anammox cells and values of ra, were
also evident during an initial survey of Medway Bridge
Marina (April 2005) where anammox accounted for as
much as 32% of the total N2 production and just 2% of the
microbial population (Fig. 1). This was in contrast to the
reduction in anammox cells (from 8% to < 1%)
downstream, in line with a decrease in ra (19% to 6%)
(Fig. 1). Whether this indicates that the relative abundance
of anammox cells is perhaps underrepresented by a
FEMS Microbiol Ecol 80 (2012) 19–29
27
Abundance and significance of anammox in estuarine sediments
combination of nonspecific probes remains unknown. To
resolve this discrepancy would require the specific enumeration of anammox cells linked to cell-specific activity.
With respect to bioturbation and associated fluctuations
in the sediment oxygen regime, this seems to have little
impact on the formation of a stable anammox community.
The effect of bioirrigating macrofauna on the sediment
strata is shown in Fig. 2a, where the multiple peaks in the
dissolved oxygen profiles suggest ventilation of the sediment substrata with surface water. Whilst we cannot claim
that anammox organisms are aerotolerant, it is clear that
the movement of anammox cells through the sediment
substrata by bioturbating infauna does not perturb the
potential for anammox nor the formation of an anammox
community. Moreover, invertebrate burrowing could in
fact enhance the significance of anammox by extending the
zone of NO
3 reduction, and thus, the availability of NO2
(Henriksen et al., 1980; Meyer et al., 2005).
This study confirms the presence of anammox organisms in the Medway Estuary and therefore adds to knowledge of anammox in estuarine environments. Anammox
organisms were detected at all sites and throughout the
sediment substrata; however, the distribution of these
organisms and depth-specific values of ra do not correlate. To determine whether this is the result of a low
number of ‘highly active’ anammox organisms, or even
perhaps an elusive member of the anammox group,
future studies should focus on resolving the depth-specific
rate measurements per cell and the specificity of the available probes. This could be achieved by quantitative FISH
and measurements of depth-specific anammox activity.
Given the nontranslucent properties of sediment samples,
however, volume-based quantification of the total organisms may prove challenging. Measurements of CO2 production verified a positive correlation between the
significance of anammox and sediment metabolism. The
yield of 15N-labelled gas, from 15 NO
3 , revealed a fourfold
decrease in denitrification relative to an increase in anammox (at higher sediment metabolism). This could implicate DNRA as an important pathway for the delivery of
NO
2 to the anammox community, thus complementing
our current understanding of N-cycling pathways. Studies
addressing the role of DNRA in the regulation of anammox are however required to specifically confirm this in
estuarine sediments. Furthermore, little is known regarding the effect of bioturbation and associated fluctuations
in oxygen concentration on the formation of a stable
anammox community.
Acknowledgements
This project was funded by a PhD scholarship from
Queen Mary University of London and the Short Term
FEMS Microbiol Ecol 80 (2012) 19–29
Scientific Mission, COST 856. We would like to give
thanks to Andrew Voak for his technical assistance and
the anonymous reviewers who contributed to this manuscript.
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