Limnol. Oceanogr., 54(2), 2009, 577–589
2009, by the American Society of Limnology and Oceanography, Inc.
E
Production of nitrogen gas via anammox and denitrification in intact sediment cores
along a continental shelf to slope transect in the North Atlantic
Mark Trimmer and Joanna Claire Nicholls
School of Biological and Chemical Sciences, Queen Mary University of London, London E1 4NS, United Kingdom
Abstract
We measured the production of N2 gas from anammox and denitrification simultaneously in intact sediment
cores at six sites along a transect of the continental shelf (50 m) and deeper slope (2000 m) in the North Atlantic.
Maximum rates of total N2 production were measured on the shelf and were largely due to denitrification, with
anammox contributing, on average, 33% of this production. On the continental slope, the production of N2 gas
decreased but the proportion due to anammox reached a maximum of 65%. This change in both amount and
dominant pathway of N2 production could be explained largely by the concentration of organic carbon at each
site. With increasing carbon the total production of N2 increased rapidly while the response of anammox was not
significant. On the continental slope, total N2 production fell below 2 mmol N m22 h21 and anammox was
strongly related (r 5 0.95) to denitrification but the relative magnitude of anammox to denitrification (1.65 : 1)
suggested that anammox could not be fuelled by NO {
2 from denitrification alone. On the shelf, however, where
total N2 production was predominantly greater than 2 mmol N m22 h21, no relationship between anammox and
denitrification was found and anammox remained constant at 1.4 mmol N m22 h21. Despite the constancy and
greater availability of NO {
3 and lower temperatures on the continental slope, the significance of anammox to the
total production of N2 appears primarily controlled by the overall rate of N2 production.
Nitrogen (N) is generally considered to be limiting for
the assimilation of carbon by primary production in the
marine environment and the balance between N fixation
and its removal via ‘denitrification’ is key to the regulation
of primary production (Nixon et al. 1996; Falkowski 1997;
Karl et al. 2002). Trends of increasing CO2 in the
atmosphere are predicted to contribute to global warming
and climate change, the effects of which appear to alter the
balance between N fixation and N loss in coastal systems
(Fulweiler et al. 2007) which, as such, necessitate a more
comprehensive understanding of the removal of N in
aquatic environments.
Denitrification was thought to be the major pathway of
N removal in the environment, but the discovery of
anaerobic ammonium oxidation (anammox) in the laboratory in 1995 (Mulder et al. 1995) and the subsequent
quantification of its potential contribution to N2 production in aquatic sediments, has altered the way in which we
view the nitrogen cycle in such systems (Dalsgaard et al.
2005). Anammox is the formation of N2 gas from the
oxidation of ammonium coupled to the reduction of NO {
2
at a 1 : 1 ratio under anaerobic conditions and is mainly
thought to be an autotrophic metabolism (Van de Graaf et
al. 1995; Güven et al. 2005). The biogeochemical significance of anammox is that it enables an anaerobic aquatic
without the classic
system to lose some of its NH z
4
coupling of aerobic nitrification to denitrification and it
certainly helps explain the absence of NH z
4 in the anoxic
parts of the world’s ocean (Devol 2003).
The potential for anammox has now been reported for
sediments from estuaries (Trimmer et al. 2003; RisgaardPetersen et al. 2004; Meyer et al. 2005) and coastal seas
(Dalsgaard and Thamdrup 2002; Thamdrup and Dalsgaard
2002; Rysgaard et al. 2004) and, in addition, several anoxic
bodies of water (Dalsgaard et al. 2003; Kuypers et al. 2003,
2005). The contribution of anammox to N2 production has
been found to be very high in some sediments, accounting
for up to 80% of N removal (Engström et al. 2005). The
precise regulation of anammox in sediments is, as yet,
unclear, though the current dogma is that denitrification
decreases relative to anammox with increasing water depth
offshore because of the associated decrease in availability
of organic carbon needed to drive sediment mineralization
and, thus, denitrification (Thamdrup and Dalsgaard 2002;
Dalsgaard et al. 2005; Engström et al. 2005). In addition,
anammox may be favored in colder regions and–or those
{
systems where NO {
3 and its reduction to NO 2 are both
high and consistent (Rysgaard et al. 2004; Meyer et al.
2005; Trimmer et al. 2005). Despite this progress the
majority of studies on anammox and denitrification in
sediment are potentials based on anaerobic slurry incubations.
Even before the advent of anammox, very few direct
measurements of ‘denitrification’ or N2 production had
been made in sediments beyond the estuarine or immediate
coastal zone and our knowledge of N2 production in
sediments in deeper (.100 m) offshore waters is scant
(Devol 1991; Nowicki et al. 1997; and review by
Steingruber et al. 2001). The majority of measurements of
‘denitrification’ in whole sediment cores over the past 25 yr
have been made using either the isotope pairing technique
(IPT; Nielsen 1992) or acetylene blockage (Sørensen 1978).
Acknowledgments
We thank the crew and technicians on board the R.R.S.
Charles Darwin and R.V. Pelagia and also I. Sanders, A. Voak,
C. Rooks, and A. Jaeschke for help with sampling and analysis
at sea. We also thank the anonymous reviewers and the Associate Editor for their helpful suggestions and comments to
improve this manuscript. This research has been supported by a
Natural Environment Research Council grant (NER/A/S/2003/
003) to M. T.
577
6.8
5.9
9.7
5.5
8.5
3.7
7.3
6.3
7.7
6.5
7.1
7.0
7.1
7.0
0.727
217
221
244
135
96
40
182
371
466
321
365
393
367
381
0.001
0.39
0.36
0.26
0.13
0.09
0.04
0.25
0.55
0.68
1.02
1.07
1.00
0.94
0.88
0.000
0.74
0.75
0.56
0.53
0.49
0.42
0.61
0.71
0.71
0.85
0.90
0.81
0.81
0.80
0.024
mud
mud
mud
sand and mud
mud and fine sand
1.41
1.49
1.69
1.77
1.77
1.64
1.63
1.53
1.52
1.23
1.31
1.29
1.28
1.36
0.021
mud and clay
20.4
20.8
15.8
16.8
9.8
11.4
15.4
6.1
1.6
5.4
6.0
1.1
4.7
4.0
0.001
50
100
100
05.79
05.92
32.00
35.56
34.84
34.16
6
6
5
5
5
5
13.15
13.08
52.00
52.98
07.22
07.15
51
51
53
53
54
54
Mean
p
6
autumn
spring
autumn
spring
autumn
spring
5
3
2
Mean
4
500
1000
3.9
3.9
9.5
9.6
10.6
10.4
7.5
10.6
8.6
11.7
8.3
14.4
7.5
10.2
0.183
2000
51.22
51.16
42.31
42.38
42.38
42.18
9
9
9
9
9
9
03.61
03.73
10.96
10.74
16.11
14.74
Autumn
Spring
Autumn
Spring
Autumn
Spring
1
Latitude
48
48
48
48
48
48
Organic C
(mmol cm23)
Organic C
(% dry wt)
Porosity
Sediment type
Bulk
density
(g cm23)
Ambient
NO {
3
(mmol L21)
Cruise
Sampling methods and sites—The study sites (Fig. 1;
Table 1) represent a depth transect from the North Atlantic
continental slope (2000 m, Site 1), to Dundrum Bay in the
Site
Methods
Bottom
water temp.
(uC)
Because neither of these key methods is suitable for
measuring production of N2 by sediments where anammox
and denitrification coexist, the precise roles and regulatory
factors of the two pathways of N2 production in intact
sediment are currently unknown. Risgaard-Petersen et al.
(2003) proposed the revised-IPT (r-IPT), with the addi{
15
tional parameter r14 (the ratio of 14NO {
x : NO x in the
sediment), to distinguish between N2 production (p14) via
anammox and denitrification. Subsequently we developed a
method for determining r14 based on the distribution of 15N
in N2O and, arguably, produced the first measurements of
N2 production via anammox and denitrification in whole
sediment cores (Trimmer et al. 2006).
The aim of this study was to use the r-IPT to measure
anammox and denitrification in intact sediment cores at six
sites along a depth transect, from 2000 m on the
continental slope in the North Atlantic, to 50 m on the
shelf in the Irish Sea to improve our understanding of
anammox and denitrification in the marine environment.
Depth
Longitude (m)
Fig. 1. Station positions for both the spring and
autumn cruises.
C : N (atom)
Trimmer and Nicholls
Table 1. Decimal coordinates for each site plus some water and sediment characteristics for both the autumn and spring cruises. Mean values and significance values for a
t-test comparison between sites grouped on either the continental slope (1, 2 and 3) or the shelf (4, 5 and 6) are given.
578
Anammox in shelf sediments
Irish Sea (50 m, Site 6), and were selected to give a broad
distribution of ambient NO {
3 and organic carbon but were,
on the whole, muddy sediments. Sampling took place
aboard the R.R.S. Charles Darwin from 12 September 2005
to 02 October 2005 (referred to hereon as the autumn
cruise) and the R.V. Pelagia from 13 March 2006 to 03
April 2006 (referred to hereon as the spring cruise).
Sediment samples were collected using a Netherlands
Institute for Sea Research cylindrical box corer (50 cm in
diameter) and sub-sampled on deck as whole sediment
cores with overlying water into Plexiglass cores (25 cm long
3 3.4 cm inside diameter [i.d.]), sealed at one end with a
bung. Cores were equilibrated to atmospheric pressure and
incubation temperature (8uC at all sites) in storage barrels
full of aerated site water, collected with a rosette water
sampler fitted with Seabird Electronics sensor package
(salinity, depth, temperature, conductivity, oxygen, and
fluorescence).
Sediment characteristics—Vertical sub-samples (5 cm) of
sediment were collected from the box cores using truncated
syringes (5 mL), transferred to small plastic bottles (Bijou;
Bibby Sterilin) and immediately frozen on board. Later, the
samples were defrosted before weighing, then drying and
reweighing to calculate porosity. The sediment samples
were then homogenized using a mortar and pestle, and a
sub-sample (50–100 mg) placed into a preweighed scintillation vial (5 mL) and treated with HCl (2 mL, 1 mol L21
organic-free grade) as per Hedges and Stern (1984). Carbon
and nitrogen content were analyzed using an elemental
analyzer coupled to a continuous flow isotope ratio mass
spectrometer ([CF/IRMS] Thermo-Finnigan; Delta Matt
Plus) calibrated against known quantities of urea.
Rates of anammox, denitrification, and dissimilatory
nitrate reduction to ammonium (DNRA) in whole
sediment cores—At each site two core experiments were
conducted; a time-series experiment, designed to assess
whether the mixing of 15N and 14N at the zone of reduction
had reached equilibrium during the experiment; and a
concentration-series experiment, designed to show changes
in the distribution of 15N in the N2O and N2 pools with
changes in 15N concentration. In total, 32 cores were used
in these experiments.
Time-series core experiment—After the equilibration
period (,12 h), 14 cores were removed from the storage
barrels and placed in a single holding tank (Trimmer et al.
2006). The overlying water in each core was then enriched
21 (50 mL of 116.3 mmol L21
with 15NO {
3 to ,50 mmol L
Na15NO3 [99.3 15N atom%] Sigma-Aldrich) and carefully
stirred before being bubbled gently with air through
individual pieces of silicon (2 mm i.d.) tubing connected
to a distributor and pump. The rate of bubbling was
sufficient to fully oxygenate the water throughout the preincubation period but not enough to disturb the sediment
surface. The duration of the pre-incubation period was
driven by an estimate of time taken by 15NO {
3 to reach the
nitrate reduction zone based upon oxygen penetration
profiles (M. Trimmer unpubl.) and the diffusivity of NO {
3
579
based on porosity and temperature as per Li and Gregory
(1974). The estimations agreed well with the lack of a
significant relationship between r14 and time at all sites (see
Results).
At T0, a water sample (5 mL) was collected from each
core, filtered (0.2 mm Minisart PlusTM; Sartorius UK) and
{
kept in the refrigerator until analysis for NH z
4 , NO 2 , and
{
NO 3 concentrations by automated segmented wet chemistry later that day (see below). The cores were then sealed
and incubated in the dark at 8uC, with gentle stirring (,60
revolutions per minute [rpm]) of the water column. At Tf
(time final) a further sample of water (5 mL) was taken and
treated as above before the core was slurrified by gently
mixing the sediment and overlying water with a Plexiglas
rod. A slurry sample (20 mL) was carefully drawn off in a
large syringe, and allowed to overflow into a gas-tight vial
(Exetainer, 12 mL; Labco), fixed using formaldehyde
solution (100 mL, 38% w/v) and sealed for later 15N-N2
and 15N-N2O analysis. A further slurry sample (5 mL) was
carefully transferred into a bijou bottle and frozen
immediately on dry-ice for 15NH z
4 analysis later in the
home laboratory. The time-series cores were sacrificed from
the second hour of incubation, every hour, for 14 h.
Concentration-series whole core experiment—Fourteen
cores were pre-incubated (as above) with 8-mmol L21
15N concentraincrements of 15NO {
3 (approximate final
tions of 8, 16, 24, 32, etc., mmol L21) for at least 24 h
before T0. At T0, the cores were treated as above. The
concentration-series cores were sacrificed, as above, after a
12-h incubation period. An additional four cores were left
unamended as references to determine the background
abundance of 15N gases; these were sacrificed immediately,
as above, at the beginning of the experiment.
Traditional confirmation of anammox and additional
slurry experiments with formate—In line with other studies
on anammox in sediments we used anaerobic sediment slurries
to confirm the presence of the anammox metabolism
(Dalsgaard and Thamdrup 2002; Trimmer et al. 2003;
Risgaard-Petersen et al. 2004). Briefly, working in a portable
anaerobic glove box (Belle Technologies), sediment (20 mL
from the top 2–4 cm of sediment from a box core) was
homogenized with degassed site water (14 mL) and distributed
into serum bottles (37 mL) before sealing with butyl rubber
stoppers and aluminum crimps. The slurries were mixed on
rollers (Spiramix; Thermo-Finnigan) overnight at 8uC to
remove any traces of remaining oxygen or NO {
x before
(80
mL of
addition, through the septa, of 15N labeled NH z
4
120 mmol L21 15NH4Cl [99.4 15N atom%] Sigma-Aldrich) and
1 4 NO { (final concentrations 500 mmol L 2 1 and
3
100 mmol L21, respectively). The slurries were returned to the
rollers overnight before taking a sample of the headspace
(1 mL) and transferring it to a water filled gas-tight vial
(12 mL; Exetainer). The samples were then stored upside-down
before direct analysis of the headspace as described below.
At Sites 2 (continental slope, 1000 m) and 6 (shelf, 50 m)
additional slurry experiments were designed to show the
potential effect of carbon supplements on the relative
contributions of anammox and denitrification to the
580
Trimmer and Nicholls
production of N2. Briefly, at each site, 64 sediment slurries
were created as above with four left unamended as
references. The remaining serum bottles were injected with
14NH z (final concentration 500 mmol L21) and then
4
divided into the four treatments: 15NO {
+ formate;
3
15NO { + formate; 15NO { ; and 15NO { . Half of the
2
3
2
experimental bottles (30) were injected with sodium
formate (final concentration 500 mmol L21); 15 of these
were then injected with increasing aliquots of 15NO {
3 (final
concentrations 1–15 mmol L21), and the remaining 15
injected with increasing aliquots of 15NO {
2 (final concentrations 1–15 mmol L21). The other 30 serum bottles were
treated in the same way but without the addition of formate
and after incubation overnight the serum bottles were
treated as above. The amounts of 15N gas per unit of slurry
and the relative contribution of anammox and denitrification to the production of N2 were calculated according to
Dalsgaard and Thamdrup (2002).
15N-N
and 15N-N2O gas analyses—On return to the
home laboratory, a headspace (1 mL analytical grade He)
was introduced into the gas-tight vials from the core
experiments using a two-way valve and a syringe. The vials
were then shaken vigorously and stored upright at 22uC to
allow the gases to equilibrate between the water phase and
headspace. For N2 analysis, samples of the headspace
(40 mL) for both the core and slurry experiments were then
injected using an auto-sampler into an elemental analyzer
(which merely served as an interface), but bypassing the
reduction and oxidation columns so that 15N labeled N2O
would not be reduced to 15N-N2. Gases were separated on
the elemental analyzer’s gas chromatograph (GC) column
prior to passing to the CF/IRMS. Calibration was
performed with N2 in helium over air-equilibrated water
at 22uC and the mass charge ratios for mass charge ratios
(m/z) 28, m/z 29, and m/z 30 nitrogen (28N2, 29N2, and 30N2)
measured. For N2O isotope ratio analysis, samples of the
headspace were sub-sampled (10–100 mL depending on the
concentration of N2O in the headspace predetermined on a
GC fitted with a m-electron capture detector) using a gastight syringe (Vici Precision Sampling) into an air filled gastight vial. The entire content of the gas-tight vial was then
swept, using a two-way needle and analytical grade He, to a
trace gas preconcentrator (Cryo-Focusing; PreCon,
Thermo-Finnigan), where the gases are dried and scrubbed
of most of the CO2 before being cryo-focused twice in
liquid N2 and final separation of N2O from CO2 on a
PoraPLOT Q capillary column. The sample then passes to
the CF/IRMS via an interface (ConFlo III Interface,
Thermo-Finnigan) and the mass charge ratios for m/z 44,
m/z 45, and m/z 46 (44N2O, 45N2O, and 46N2O) measured.
Calibration was performed with known amounts of N2O
(98 mL L21; Scientific and Technical Gases) over the range
0.41–13.25 nmol N2O (S 44N2O, 45N2O, and 46N2O) and
was linear between 0.8 pmol and 99 pmol for 45N2O and
46N O. Here, for clarity, we refer to 15N labeled N O as
2
2
45N O and 46N O and not 29N 16O and 30N 16O.
2
2
2
2
2
15NH z
analysis for estimation of net DNRA—The
frozen slurry samples from the time-series incubations were
4
defrosted and immediately mixed with KCl (6 mL of
2 mol L21 KCl). The samples were then mixed on rollers
(as above) in the dark at 4uC for half an hour, before
centrifuging. The supernatant was then sub-sampled into
two gas-tight vials (3 mL Exetainer; 1 mL in each), before
being bubbled with oxygen-free nitrogen (British Oxygen
Company) for 10 min to remove any 15N gases dissolved in
solution. A further sample of the supernatant was frozen
for later measurement of NH z
4 concentration by wet
chemistry (see nutrient analyses below). The 15NH z
4
content of the sample was then quantified using the
hypobromite oxidation method of Rysgaard and Risgaard-Petersen (1997). The lids of both of the gas-tight
vials were removed, and to one of the vials a blocked
hypodermic needle was inserted and 50 mL of prepared
hypobromite solution added to the cap of the needle. The
other vial (reference sample) contained only the sample
liquid. The lids were then replaced and sodium hydroxide
(50 mL of 12 mol L21) was injected through the septa into
the sample liquid of all the vials. The samples were then
agitated gently and left for at least 24 h before analysis of
15N-N gas in the headspace by mass spectrometry as
2
described above. The 15N labeling in the sediment due to
15NH z was determined by subtracting the signal of the
4
reference sample from the signal of the sample with the
added hypobromite, generating a proportion of 15N in the
sample. This was then multiplied by the concentration of
NH z
4 determined from the nutrient sample (as described
below), taking into account the dilution by KCl. Ambient
rates of net DNRA based on the reduction of 14NO {
3 were
calculated according to
DNRA ~ r14 | 15 NHz
4
ð1Þ
Where r14 is calculated from the distribution of 15N in the
N2O from each core (see below) and 15NH z
4 is the rate of
15NH z production (mmol m22 h21).
4
{
Nutrient analyses—Concentrations of NO {
3 , NO 2 , and
z
NH 4 were measured using a segmented flow auto-analyzer
(Skalar) and standard colorimetric techniques. The limit of
and NO {
was
detection and precision for NO {
3
2
21 6 5%.
0.2 mmol L21 6 1%, and for NH z
0.5
mmol
L
4
Calculation of anammox and denitrification—The revised
isotope pairing technique (r-IPT) was used to estimate the
genuine total production of N2 (p14 N2 as N from both
anammox and denitrification) using the theory and
methods described in Risgaard-Petersen et al. (2003) and
Trimmer et al. (2006). The r-IPT estimates total N
production as follows:
r-IPT p14 ~ 2r14 | p29 N2 z p30 N2 | ð1 { r14 Þ
ð2Þ
and anammox (p14 anammox) as follows:
p14 anammox ~ 2r14 | p29 N2 { 2 | r14 | p30 N2
ð3Þ
Denitrification is then taken as the difference between Eq. 2
and Eq. 3. p29N2 and p30N2 are the production of 29N2 and
30N after 15NO { amendment and r
2
14 is the ratio between
3
Anammox in shelf sediments
Table 2. Pearson correlation analysis between water depth,
water (temperature and NO {
3 ), and sedimentary organic matter
for all six sites across the shelf and continental slope. Top value in
each row is the correlation coefficient (R), the bottom value the
significance (p), n 5 12.
Depth
Temperature
(uC)
NO {
3
(mmol L21)
R
p
20.744
0.006
0.940
0.000
Organic C Organic C
(% dry wt) (mmol cm23)
20.545
0.067
20.480
0.114
14NO {
{
and 15NO {
3 in the NO 3 reduction zone. In Eqs. 2
30
and 3
2 and p N2 are directly quantifiable using mass
spectrometry, and r14 can be calculated from the distribution of 15N in the N2O pool, according to Eq. 4 (Trimmer et
al. 2006).
3
p29N
r14 ~
p45 N2 O
2 | p46 N2 O
ð4Þ
The respective contributions of p14 supported by either
NO {
3 from the overlying water (p14w) or that coupled to
nitrification in the sediment (p14n) as per Nielsen (1992)
were calculated according to Risgaard-Petersen et al.
(2003):
r14 w
r14
ð5Þ
p14 n ~ p14 { p14 w
ð6Þ
p14 w ~ p14 |
15NO { in the
where r14w is the ratio of 14NO {
3 to
3
overlying water determined by the difference in concentration of NO {
3 between the reference and experimental cores.
The two parameters p14w and p14n were further allocated to
anammox and denitrification as Aw, An, Dw, and Dn, by
substitution into Eqs. 5 and 6, respectively.
The term r14 can be converted into a more familiar
parameter, which is useful when interpreting these calculations (i.e., q). The term q is the proportion of 15N in the
produced N gas pool and is directly related to r14:
q~
1
r14 z 1
ð7Þ
Strictly this definition should only be used to describe the
proportion of 15N in the produced gases if they were
generated from random isotope pairing (Hauck et al. 1958).
Here we use it to describe the proportion of 15N for both
15N gas species (i.e., q’ N and q N O) and look for
2
2
deviations from 1 : 1 to indicate the presence of anammox
(Trimmer et al. 2006). Hence, if anammox is present, by
definition, q’ N2 cannot reflect random isotope pairing
because the total N2 produced will contain 14N from both
{
the NH z
4 and NO 3 pools.
Results
Site characteristics—The coordinates for each station
along with some characteristics for the bottom water and
sediment are reported in Table 1 and a correlation analysis
581
between some of the measured variables and depth is
presented in Table 2. The temperature of the bottom water
was negatively correlated with depth, decreasing from
14.4uC on the shelf at 50 m to 3.9uC at 2000 m. In addition,
the temperature was more consistent at Sites 1, 2, and 3 on
the continental slope, changing by ,2% between the two
seasons, but it changed by .18% at Sites 4, 5, and 6 on the
shelf (48% at Site 6). Counter to this, bottom water NO {
3
was positively correlated with depth, peaking at
20.8 mmol L21 at 2000 m and was, overall, higher on the
continental slope compared to on the shelf.
The majority of the sites had muddy sediment with
porosity .0.7 but it was lower at Site 2 (0.53, 0.56) and
particularly low at the partly sand and mud Site 3 (0.42,
0.49). The organic carbon content of the sediment,
expressed as either a percentage of dry weight or as a
concentration, was, on average, greater on the shelf
compared to on the continental slope but there was no
overall simple relationship with water depth (Tables 1 and
2).
Detecting anammox using differences in the 15 N
gas pools—Here we use r14 calculated from the distribution
of 15 N in the N 2 O as a proxy for the ratio of
14NO { : 15NO { (r ) in the NO { reduction zone and,
14
x
x
3
ideally, this ratio should be constant throughout the
incubation. The values for r14 calculated from the 15NN2O (i.e., ‘true’ r14) and 15N-N2 (i.e., potentially ‘false’ r14:
anammox contributes more 14N and increases r14 in the N2
pool) in the time-series experiments were essentially
constant with time. Examples of the maximum and
minimum differences in r14 for N2O and N2 from Site 1
(continental slope) and Site 4 (shelf), respectively, are given
in Fig. 2A and B. Clearly there was a marked difference in
the two derivations of r14 at Site 1, whereas they were more
similar at Site 4. Additionally, the relationship between r14
as a function of 15NO {
3 concentration in the overlying
water showed r14 decreasing, predictably, with increasing
15NO { at all sites on both cruises and again examples for
3
Sites 1 and 4 are given in Fig. 2C and D. Again there was
clear separation between r14 for the N2O compared to that
for the N2 at Site 1 but not so much at Site 4.
To visualize more clearly the effect of anammox on the
distribution of 15N in the two respective gas pools r14 (as a
function of 15NO {
3 ) was converted into the term q.
Regression analysis of q’ N2 vs. q N2O showed a significant
linear relationship between the two distributions throughout the study (for example, Fig. 2E and F) at all sites (p ,
0.05). Further, the intercept was not significantly different
from zero at any site (p . 0.05), that is, had no 15NO {
3
been added there would indeed be no 15N in either the N2
or N2O gas pools. Deviations from a slope of 1 : 1 indicate
significant anammox activity (Trimmer et al. 2006) and all
of the slopes of q’ N2 vs. q N2O were significantly different
to 1 : 1 (t-test, p , 0.05) at all sites; though no significant
production of 15N gases could be measured at the more
sandy Site 3 in the spring.
Total N2 production, anammox, and denitrification in
whole sediment cores—Our estimates of the rates of N2
582
Trimmer and Nicholls
Fig. 2. Selected examples showing the maximum and minimum differences between r14 derived from 15N2 and 15N2O as a function
of time for (A) Site 1 (continental slope) and (B) Site 4 (shelf), and as a function of 15NO {
3 addition to the overlying water for (C) Site 1
and (D) Site 4. (E) and (F) are q’ N2 vs. q N2O plots derived from the r14 data at Sites 1 and 4, respectively. The solid line represents a
first-order linear regression through the data, the dashed line represents a slope of 1 : 1 (i.e., where q’ N2 5 qN2O), which was taken as no
significant anammox activity. Regression coefficients (r2 and b1 [i.e., slope], p , 0.05 in all cases) are included to illustrate the difference
between the measured slope and 1 : 1 and the respective differences between the two sites. All data from the spring cruise.
Anammox in shelf sediments
583
Fig. 3. Production of 14N2 gas (as N) in whole sediment cores as a function of water depth at all sites on both cruises: (A) total
production (anammox plus denitrification) and broken down into (B) anammox, and (C) total denitrification. Open and closed symbols
indicate spring and autumn cruises, respectively, with circles and squares indicating the continental slope and shelf sites, respectively.
Numbers indicate sites. Strength of the relationship between depth and each respective process on the continental slope is also indicated
(r; points are means 6 SE, n 5 14). (D) Total production of N2 and that by anammox as a function of the concentration of organic
carbon in the sediment. Data are mean values for both cruises at each site, except Site 3 where there was no spring data.
production by either anammox or denitrification are based
on the mean values from the concentration-series experiments, where n 5 14 in each case. Comparisons across the
two regions are based on the means for each set of three
sites from both cruises (e.g., Sites 1, 2, and 3 on the
continental slope, n 5 6). Total production of N2 gas
(anammox plus denitrification) ranged from 0.31 mmol N
m22 h21 to 6.84 mmol N m22 h21 and was greater in the
autumn compared to the spring at all sites (Fig. 3A).
Overall, production was significantly greater on the shelf
(Sites 4, 5, and 6, mean 5 4.82 6 0.83 SE, n 5 6) compared
to on the continental slope (1, 2, and 3 mean 5 1.02 6 0.36
SE, n 5 5, p 5 0.004) and increased significantly (r 5 0.9, p
, 0.05) with depth on the continental slope. Across the six
sites total N2 production was positively correlated with the
organic carbon content of the sediment, when expressed as
either a percentage (org C% dry wt: r 5 0.678, p 5 0.022, n
5 11) or as a concentration (mmol cm23: r 5 0.698, p 5
0.017). The averages for the two cruises for total N2
production as a function of the concentration of organic
carbon are given in Fig. 3D; note the site numbers and how
this was not simply a function of depth.
Production of N2 gas from anammox ranged from
0.11 mmol N m22 h21 to 2.51 mmol N m22 h21 and
increased significantly (r 5 0.9, p , 0.05) with depth on
the continental slope but, on average, there was no
significant difference in anammox between the shelf and
the continental slope (1.38 6 0.24 SE to 0.61 6 0.26 SE, n
5 6 and 5 respectively; Fig. 3B). In addition, compared to
differences in total N2 production, anammox was relatively
constant between autumn and spring at the majority of
sites (Fig. 3A and B). Anammox was not significantly
584
Trimmer and Nicholls
related to the concentration of organic carbon in the
sediment (r 5 0.508, p 5 0.111, n 5 11), though the
relationship held for the carbon content of the sediment as
a percentage (r 5 0.754, p 5 0.007, n 5 11). Regardless of
the expression of sediment carbon content, the important
point is the steepness of the relationship for total N2
production relative to that for anammox (Fig. 3D). The
production of N2 gas from denitrification was 0.20–
5.83 mmol N m22 h21 across all sites (Fig. 3C) and was
significantly greater on the shelf compared to on the
continental slope (3.44 6 0.76 SE to 0.42 6 0.11 SE,
respectively; p 5 0.01). There was a marked increase in
denitrification between the two seasons, especially on the
shelf. Denitrification tended to decrease with depth (r 5
20.608, p 5 0.047) but followed a similar pattern as for
total N2 production as a function of the concentration of
organic carbon (r 5 0.676, p 5 0.022; i.e., denitrification is
the difference between the two sets of data in Fig. 3D).
Although there was a positive relationship (r 5 0.73, p 5
0.011, n 5 11) between depth and the contribution from
anammox (ra) to the total production of N2 gas across the
six sites, it was much stronger for the continental slope on
its own (r 5 0.96; Fig. 4A). In addition, the contribution
from anammox to the production of N2 was markedly
different between the spring and autumn on the shelf but
more consistent on the continental slope. Regardless of
depth, greater contributions from anammox to the total
production of N2 gas were associated with lower rates of
total production; though the data are suggestive of an
optimum for the significance of anammox at around
2 mmol N m22 h21 of total N2 production (Fig. 4B). Total
production of N2 gas was dominated by pw on the
continental slope (mean for Sites 1–3, 68%) and pn on the
shelf (mean for Sites 4–6, 81%).
DNRA—Evidence for DNRA (i.e., the presence of
4 in the slurrified whole core sample), was only
found at Sites 4 and 6 (both on the shelf). The data were
noisy but the enrichment in terms of excess signal for 29N2
over total signal (sum of 28N2 and 29N2) was significant.
For example, at Site 6 we measured a mean signal for
excess 29N2 over total of 6.07 3 1024 6 SE 1.9 3 1024 and
at Site 4, 4.9 3 1024 6 SE 1.29 3 1024, which gave
respective t-values of 3.8 and 3.2 (t-critical 5 1.771, n 5
14). Whereas at Site 5, for example, we had 5.4 3 1026 6
SE 1.1 3 1025 and a t-value of only 0.5 and the same was
also true for Sites 1, 2 and 3. Our best estimate for the rates
22 h21 and
of net 14NH z
4 production were 0.001 mmol N m
0.005 mmol N m22 h21 for Sites 4 and 6, respectively.
15NH z
Traditional confirmation of anammox and additional
slurry experiments with formate—In line with other studies
on anammox in sediments, we confirmed its presence at all
six sites with measurable production of 29N2 gas from
15NH z and 14NO { in anaerobic slurries (data not shown;
3
4
one-tailed t-test against zero production, p , 0.05). With
the additional slurry experiments there was no difference in
{
the gas yield between NO {
3 and NO 2 and the data
presented in Fig. 5 are the mean values for the two as
NO {
x . The addition of formate had no significant effect on
Fig. 4. Contribution of anammox to total N2 production (%)
in whole sediment cores as a function of (A) depth (same symbols
as Fig. 3, panel A), and (B) rate of total N2 production (points are
means, n 5 14).
the yield of gas from denitrification at either Site 2 or Site 6
(Fig. 5A and B) but it caused a significant suppression in
the yield of gas by anammox at both sites (Fig. 5C and D;
ANCOVA, p , 0.001).
Discussion
Application of the revised isotope pairing technique (rIPT)—We have used a revised version of a widely applied
technique (IPT; Nielsen 1992) and it is worthwhile
explaining its application and the validity of our data (rIPT; Risgaard-Petersen et al. 2003; Trimmer et al. 2006).
Anammox interferes with the original IPT and causes an
overestimate of N2 production, but it can be accounted for
{
15
if the ratio of 14NO {
3 : NO 3 (r14) in the nitrate reduction
zone is known. Assuming that denitrification makes N2O
and N2, while anammox makes only N2, we can use the
Anammox in shelf sediments
585
Fig. 5. Additional sediment slurry experiments with the yield of N2 gas from denitrification as a function of amended 15NO {
x
concentration at (A) Site 2 (continental slope) and (B) Site 6 (shelf) and the yield of N2 gas from anammox at (C) Site 2 and (D) Site 6,
with and without the addition of formate, on the spring cruise only. The separation between the yield of N2 gas from anammox for both
with and without formate was highly significant at both sites (p , 0.001).
ratio of 15N to 14N in the N2O pool as a proxy for r14
(Trimmer et al. 2006). In the presence of anammox the
proportion of 15N in the N2 pool (q’) will be diluted relative
to the N2O pool (q) and q’ , q. As an example we can use
the data from slurries in Fig. 5 to estimate the potential
contribution of anammox to the production of N2 (ra) at
each site and use this to predict the offset in q’ relative to q
(as in Fig. 2E and F) in our intact core experiments
according to (Trimmer et al. 2006):
ra ~
2{2|
2{
q0
q
q0
q
ð8Þ
With an ra from the slurries of 0.28 and 0.12 for Sites 2 and
6 respectively, we would predict an average ratio for q’ : q of
0.85 : 1 and 0.95 : 1 for the intact cores at the two sites. We
actually measured a ratio for q’ : q of 0.71 : 1 and 0.83 : 1,
which gives higher estimates for ra in the cores of 0.45 and
0.29, respectively. This increase in ra in cores relative to
slurries is consistent with our initial measurements in fjord
and estuarine sediments (Trimmer et al. 2006) and can be
explained by the anammox bacteria having to compete for
any NO {
x with far more of the facultative anaerobes in a
slurry, relative to that in an intact sediment core.
Our measurements agree with our predictions for the
offset in q’ : q across a range of anammox activity and we
have also demonstrated that r14 is constant with time (i.e.,
{
14
that the 15NO {
3 and NO 3 are uniformly mixed), which is
the same criterion required of the original IPT (Nielsen
1992). In addition, taking all of the data together for the six
sites revealed no significant dependency between our
estimates of anammox (p14A) and our additions of
15NO { (r2 5 0.092). Taken individually, however, and at
3
Site 5 only, the estimate of p14A did decrease significantly
21 and 110 mmol L21 at
with 15NO {
3 between 20 mmol L
21 (r2 5 0.44,
20.009 mmol N m22 h21 per 15NO {
3 mmol L
586
Trimmer and Nicholls
p 5 0.019) but removal of the first two concentrations of
15NO { (22 and 29 mmol L21) removed this dependency,
3
which may have been due to inadequate mixing at these
lower concentrations. Overall we are confident that the
revised-IPT provides a robust estimate of genuine N2
production by both anammox and denitrification.
Existing knowledge on N2 gas production—The discovery
of anammox has altered our fundamental understanding of
the N cycle in the marine environment and beyond
(Dalsgaard et al. 2005). The data presented here, are to
our knowledge, the first simultaneous measurements of
anammox and denitrification in intact sediment cores from
along a continental shelf to slope transect. Rysgaard et al.
(2004) did report areal rates for anammox and denitrification measured in intact cores of arctic sediment (to 100 m),
but their approach relied on an indirect correction for
anammox derived from slurries, rather than being measured directly in cores as reported here. Even before
anammox, data for N2 production in coastal and more
offshore sediments were scarce. Data reported for coastal
seas using the N2 flux method give a range for production
of 10–141 mmol N m22 h21 but represent an integral for
production by both anammox and denitrification (Devol
1991; Devol et al. 1997; Nowicki et al. 1997). Alternatively,
estimates of N2 gas production made using the IPT of
Nielsen (1992) typically range from 0 mmol N m22 h21 to
300 mmol N m22 h21 (Steingruber et al. 2001) but, besides
being for shallow coastal waters, are likely to have been
overestimated if significant anammox was unaccounted for
(Risgaard-Petersen et al. 2003; Trimmer et al. 2006).
Comparison in the current study between N2 production
calculated with either the IPT or r-IPT showed a significant
overestimation using the original IPT of between 18% and
188% (paired t-test, p , 0.001) at all but Sites 2 and 3
during the autumn cruise.
Regulation of N2 gas production—Along our gradient the
total production of N2 gas was positively related to the
concentration of organic carbon in the sediment (Fig. 3D),
which is consistent with that measured in transects from
estuaries and coastal regions to deeper waters in both
sediment cores and slurries (Nowicki et al. 1997; Dalsgaard
and Thamdrup 2002). In contrast, the relationship between
anammox and the concentration of organic carbon was not
significant, which suggested other factors maybe key in
regulating anammox activity (Fig. 3D). The increasing
separation between each line is a measure of the increasing
contribution of denitrification to N2 production and,
whereas denitrification was able to exploit the increase in
available carbon, anammox could not (Dalsgaard et al.
2005). Despite the likely greater availability of NH z
4 to
potentially supply anammox as organic carbon increased,
the increased demand for electron acceptors (NO {
3 and
{
NO {
2 ) may have limited the availability of NO 2 for
anammox (Dalsgaard et al. 2005; Trimmer et al. 2005).
On the shelf, the majority of N2 production was coupled
to nitrification in the sediment (pn 5 An + Dn), as would be
expected away from riverine influences (Seitzinger 1988)
and, in contrast, on the continental slope, fuelled by NO {
3
Fig. 6. Anammox as a function of denitrification for all sites
on both cruises. Filled symbols are data from Sites 1, 2, and 3 on
the continental slope (Site 1 is at the top) and the open symbols
are data from Sites 4, 5, and 6 on the shelf. A first-order regression
has been fitted through the data for the continental slope sites,
forced through the origin because the intercept was not
significantly different to zero and the regression coefficients r2
and b1 (i.e., slope [p 5 0.03]) are included.
from the overlying water column (pw 5 Aw + Dw), in
agreement with previous measurements of N2 flux at 630 m
(Devol 1991) and the respective difference in bottom water
NO {
across the two regions. Rysgaard et al. (2004)
3
reported a strong positive correlation between water
column NO {
3 and potential rates of anammox but, overall,
we found no such relationship here (r 5 20.43, p . 0.05).
Maximal anammox activity, measured at Site 5, was
actually sustained by nitrification in the sediment (An),
rather than NO {
3 in the overlying water (Aw), and such a
correlation would not be expected. Overall, our trend
agrees very well with that reported formerly in the
literature, that anammox can be a highly significant, if
not the major pathway of N2 production, in less reactive
sediments (Thamdrup and Dalsgaard 2002; Rysgaard et al.
2004; Engström et al. 2005).
Coupling anammox to a supply of NO {
2 —Three common
reduction
have
been
suggested
to supply
pathways of NO {
3
{
NO {
to
anammox,
either
directly
as
NO
to
NO {
2
3
2
{
through NO 3 respiration (Nicholls et al. 2007), or
indirectly via leakage of NO {
2 as part of the denitrification
{
(NO {
R
NO
R
NO
R
N2O R N2; Dalsgaard and
3
2
Thamdrup 2002; Trimmer et al. 2003; 2005) or DNRA
z
{
processes (NO {
3 R NO 2 R NH 4 ).
{
If the source of NO 2 for anammox was denitrification,
the two could be coupled, as has been suggested previously
(Rysgaard et al. 2004). Plotting anammox as a function of
denitrification revealed a very strong positive correlation
between the two processes at Sites 1, 2, and 3 on the
continental slope but not at Sites 4, 5, and 6 on the shelf
(Fig. 6). This alone suggested different mechanisms of
Anammox in shelf sediments
regulation of anammox across the regions, although there
was overlap between Site 5 and the continental slope when
denitrification was ,1 mmol N m22 h21. In this analysis,
which considers the dataset as a whole, the ratio of
anammox to denitrification on the continental slope was
1.65 : 1 (b1 in Fig. 6), which is equivalent to 62% of N2
production coming from anammox, relative to a mean of
52% by site (Fig. 4A). In contrast, the average contribution
of anammox to N2 production on the shelf was 33%.
Dalsgaard et al. (2003) presented an eloquent argument for a tight coupling between anammox and denitrification in the water column of the Golfo Dulce, based on
the combined stoichiometry for the two reactions and a
common Redfield ratio of 6.6 : 1 for the organic matter
being mineralized. Indeed, they predicted that 29% of
N2 production would be due to anammox which, on
average, agreed very well with their measurements (19–35%
mean ,27%). This is interesting because our similar value
of 33%, on the shelf, suggested anammox could be fuelled
z
by NO {
2 and NH 4 coming from denitrification in this
region, though the two were not related. On the shelf,
conditions for denitrification were more favorable and
total production of N2 maximal (Fig. 3C). The relationship on the continental slope holds up to ,2.5 mmol N
m22 h21 (total N2 production) or the lower rate of
denitrification measured at Sites 4 and 6. It may be that
the rate of denitrification at Sites 4 and 6 was always
{
sufficient to ‘leak’ enough NO {
2 to avoid NO 2 limitation
of anammox but that an alternative factor kept anammox
constant.
In contrast, this was not likely to be the case on the
continental slope with a contribution to the production of
N2 from anammox of up to 65%. Dalsgaard et al. (2003)
argued that .29% of N2 coming from anammox could be
due to the mineralization of organic matter with a lower
C : N ratio (i.e., more N released per mole of carbon
oxidized with NO {
3 ). Our measurements of C : N for the
organic matter in the sediment do not necessarily support
this, because the ratio tended to be higher (autumn) when
the contribution of anammox to N2 production was
greatest. Even if NH z
4 was in excess in the sediment,
anammox would still require a source of NO {
2 which, at
contributions of up to 65% from anammox to N2
production (Fig. 4), would require denitrification to be
very inefficient and ‘lose’ 46% of its NO {
2 .
Alternatively, such a tight coupling on the continental
{
slope may be via an ‘upstream’ supply of NO {
2 from NO 3
respiration, which fuelled both anammox and denitrification when total N2 production was low (i.e., ,2 mmol N
m22 h21) and organic C ,220 mmol C cm23 sediment. This
certainly helps explain the pattern in the contribution of
anammox to N2 production at Sites 1, 2, and 3, because
although the rates of anammox and denitrification were
different across the two seasons, the difference was
proportional and the contribution to N2 production via
each source remained constant at each site respectively.
Blaszczyk (1993) measured a significant accumulation of
{
NO {
2 (up to 70% of NO 3 reduction) from Paracoccus
denitrificans growing on minimal medium (ethanol, acetate,
{
or methanol) with NO {
3 , but no accumulation of NO 2
587
with growth on nutrient broth. Hence, here, under similar
carbon limitation, the first step of N removal stalls after
{
NO {
3 reduction and liberates NO 2 which, in turn, is taken
by both anammox and denitrification. This continued
because the availability of carbon increased with both
anammox and denitrification increasing. Eventually conditions became more favorable for ‘complete’ denitrification (.2 mmol N m22 h21 total N2 production), NO {
2 was
consumed and, despite the availability of NH z
4 in the
sediment, anammox could not proceed without NO {
2 and
became proportionally less significant. Given that anammox and denitrification appear to have similarly very
21
high affinities for NO {
2 , possibly as low as 0.1 mmol L
(Dalsgaard and Thamdrup 2002), it is the availability of
organic carbon to sustain heterotrophic respiration that
enables denitrification to eventually win out over anammox
for a common source of NO {
2 .
DNRA and its effect on anammox—An additional factor
to consider is the potential for DNRA found in the intact
sediment cores at Sites 4 and 6, because the production of
15NH z from the reduction of 15NO { could potentially
3
4
enable anammox to generate 30N2, which would undermine
the central assumptions of anammox 15N techniques
(Thamdrup and Dalsgaard 2002; Trimmer et al. 2006;
Kartal et al. 2007). The net (because some of the 15NH z
4
will be oxidized to NO {
x or potentially directly to N2 via
anammox) rates of DNRA were very low compared to the
gross rates of anammox and denitrification, representing
,0.04% and 0.13% of N2 gas production at Sites 4 and 6,
respectively. We can conclude from this that unless gross
DNRA was directly linked to anammox as suggested by
15NH z
Kartal et al. (2007; i.e., metabolism of 15NO {
3 to
4
and N2, coupled to the oxidation of simple organics), the
z
contribution of 15NH 4 to the anammox N2 gas pool would
pool was
be negligible, because the ambient 14NH z
4
relatively large (,30 mmol L21 data not shown). At Site
6, however, the slurry experiment showed that the addition
of organic carbon suppressed the production of N2 by
anammox, as it did also at Site 2, but without a
concomitant rise in denitrification (Fig. 5). If there were
15NH z
an entirely intracellular metabolism of 15NO {
3 to
4
and N2 then we may have expected an increase in the
15NH z ), which
production of 30N2 (15NO {
x pairing with
4
would have appeared as an increase in denitrification, but
we did not (Fig. 5). The potential for DNRA does
represent an alternative sink for NO {
x in the sediments
z
and
NH
and an alternative source of NO {
2
4 for anammox
but it appears of minor importance and does not appear to
be directly part of the anammox metabolism itself.
Maintenance of anammox—Several researchers have
argued that anammox is more successful where environmental conditions are relatively constant and that denitrification is more flexible in response to environmental
change (Risgaard-Petersen et al. 2004; Rysgaard et al. 2004;
Dalsgaard et al. 2005). In addition, it has been suggested
that both the availability and consistency in a supply of
NO {
3 maybe essential for maintaining anammox (Rysgaard et al. 2004; Risgaard-Petersen et al. 2005; Trimmer et
588
Trimmer and Nicholls
al. 2005) and anammox has been shown to be favored at
lower temperatures relative to denitrification (Dalsgaard
and Thamdrup 2002; Rysgaard et al. 2004). Certainly
anammox was consistently more significant at Sites 1, 2, 3,
21 in the
and 5 where NO {
3 never drops below 5 mmol L
overlying bottom water and, at Sites 1, 2, and 3,
temperatures were lower. Sites 1–3 are in deep upwelling
water in the North Atlantic and Site 5 is in the gyre system
of the western Irish Sea (Trimmer et al. 1999; Horsburgh
et al. 2000). In contrast, Sites 4 and 6 are more variable
21 in the spring and
and NO {
3 can reach ,1 mmol L
summer (Gowen et al. 2000). This was further corroborated
by the relative change in bottom-water NO {
3 between the
two seasons, where the difference was greatest at Sites 4
and 6 (74% and 343%, respectively), but less marked
(,16%) at Sites 1, 2, 3, and 5. Despite this, the dominant
factor in the significance of anammox to the production of
N2 along our transect was the overall metabolic rate of N2
production.
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Edited by: Bo Thamdrup
Received: 27 June 2008
Accepted: 18 October 2008
Amended: 13 November 2008