Farías, L., et al. Denitrification and nitrous oxide cycling within the

Limnol. Oceanogr., 54(1), 2009, 132–144
2009, by the American Society of Limnology and Oceanography, Inc.
E
Denitrification and nitrous oxide cycling within the upper oxycline of the eastern
tropical South Pacific oxygen minimum zone
Laura Farı́as,1 Maribeb Castro-González,2 Marcela Cornejo, José Charpentier, and Juan Faúndez
Laboratorio de Procesos Oceanográficos y Clima (PROFC), Departamento de Oceanografı́a y Centro de Investigación
Oceanográfica en el Pacı́fico Suroriental (COPAS), Universidad de Concepción, Casilla 160-C, Concepción, Chile
Narin Boontanon3 and Naohiro Yoshida
Department of Environmental Chemistry and Engineering, Interdisciplinary Graduate School of Science and Engineering,
Tokyo Institute of Technology, Midori-ku, Yokohama, Japan
Abstract
One of the shallowest, most intense oxygen minimum zones (OMZs) is found in the eastern tropical South
Pacific, off northern Chile and southern Peru. It has a strong oxygen gradient (upper oxycline) and high N2O
accumulation. N2O cycling by heterotrophic denitrification along the upper oxycline was studied by measuring
N2O production and consumption rates using an improved acetylene blockage method. Dissolved N2O and its
isotope (15N : 14N ratio in N2O or d15N) and isotopomer composition (intramolecular distribution of 15N in the
N2O or d15Na and d15Nb), dissolved O2, nutrients, and other oceanographic variables were also measured. Strong
N2O accumulation (up to 86 nmol L21) was observed in the upper oxycline followed by a decline (around 8–
12 nmol L21) toward the OMZ core. N2O production rates by denitrification (NO{
2 reduction to N2O) were 2.25
to 50.0 nmol L21 d21, whereas N2O consumption rates (N2O reduction to N2) were 2.73 and 70.8 nmol L21 d21.
d15N in N2O increased from 8.57% in the middle oxycline (50-m depth) to 14.87% toward the OMZ core (100-m
depth), indicating the progressive use of N2O as an electron acceptor by denitrifying organisms. Isotopomer
signals of N2O (d15Na and d15Nb) showed an abrupt change at the middle oxycline, indicating different
mechanisms of N2O production and consumption in this layer. Thus, partial denitrification along with aerobic
ammonium oxidation appears to be responsible for N2O accumulation in the upper oxycline, where O2 levels
fluctuate widely; N2O reduction, on the other hand, is an important pathway for N2 production. As a result, the
proportion of N2O consumption relative to its production increased as O2 decreased toward the OMZ core. A
N2O mass balance in the subsurface layer indicates that only a small amount of the gas could be effluxed into the
atmosphere (12.7–30.7 mmol m22 d21) and that most N2O is used as an electron acceptor during denitrification
(107–468 mmol m22 d21).
the so-called oxygen minimum zones, or OMZs (Bange et
al. 2001). Most OMZs are associated with upwelling areas
that promote high organic production and enhanced O2
consumption through the decomposition of organic matter.
These conditions, along with the presence of a subsurface
water mass with very low preexisting O2 levels, can lead to
strong O2 gradients (oxyclines) with a middepth water layer
having O2 levels of less than ,22 mmol L21. This is the case
in the eastern tropical South Pacific (ETSP), the eastern
tropical North Pacific (ETNP), the Arabian Sea, and other
less extensive regions (Helly and Levin 2004) where
heterotrophic denitrification occurs. Globally, 30–50% of
the total N loss occurs in OMZs and has been attributed to
denitrification (Codispoti et al. 2001). Anaerobic ammonium oxidation via NO{
2 to N2 (anammox) was also recently
recognized as a major sink for fixed nitrogen in the
sediments and oxygen-deficient waters (Dalsgaard et al.
2003; Kuypers et al. 2005; Hamersley et al. 2007),
reopening the debate about mechanisms of N loss and
the global N imbalance in the ocean.
In OMZs, oxyclines are frequently associated with N2O
accumulation. In the case of the ETSP, this occurs where a
very shallow and sharp oxycline impinges sometimes on the
euphotic zone. Toward the OMZ core, where NO{
2
accumulates, there is apparent N2O depletion or consumption with respect to the oxycline (Elkins et al. 1978; Farı́as
The ocean is an important natural source of N2O,
providing up to 25% of global emissions (Nevison et al.
2004); this greenhouse gas provokes well-known climatological effects on both the troposphere and the stratosphere
by influencing the Earth’s radiation budget and participating in the destruction of stratospheric ozone. A significant
fraction of this oceanic production (25–75%) comes from
1 Corresponding
author ([email protected]).
address: Departamento de Biologı́a, Universidad de
Tolima, Colombia.
3 Present address: Faculty of Environment and Resource
Studies, Mahidol University 999 Phuttamonthon 4 Road,
Phuttamonthon, Salaya, Nakhon Pathom 73170, Thailand.
2 Present
Acknowledgments
This research was financed by the Fondo Nacional de Ciencia y
Tecnologı́a (FONDECYT) grant 1050743, and additional support
was provided by Centro Oceanográfico del Pacı́fico Sur (COPAS).
We thank the captains and crews of the research vessels who
facilitated our observations and sample collections as well as J.
Moffett and B. Thamdrup for the invitations to participate in the
Knorr and Galathea-3 cruises, respectively. We acknowledge
Camila Fernández and Tage Dalsgaard for assisting (nutrient
determination) in the cruises and for critical reviews of this
manuscript. The present work was carried out as part of the
Galathea-3 expedition under the auspices of the Danish Expedition Foundation.
132
Denitrification and N2O cycling
et al. 2007). In the ocean, N2O is mainly produced by
nitrification through aerobic ammonium oxidation (AAO)
(Codispoti and Christensen 1985); moreover, certain species
of nitrifying bacteria can produce N2O by means of
ammonium oxidation to NO{
2 , which, in turn, is reduced
to N2O by a process called nitrifier denitrification (Poth and
Focht 1985). In the OMZs, however, dissimilatory NO{
2
reduction (called partial denitrification) to N2O (Codispoti
{
and Christensen 1985) or dissimilatory NO3 reduction to
ammonium could be a relatively important N2O producing
processes (Cole 1990). Complete N oxide (e.g., NO{
3 )
reduction to N2, on the other hand, can consume this gas
(Cohen and Gordon 1978; Elkins et al. 1978). It is important
to note that N2O is not an intermediate in anammox,
although NO detoxification by anammox and other bacteria
could offer a potential explanation for very low relative
percentages of N2O (Kartal et al. 2007).
All of these N2O cycling processes, which seem to be
strongly affected by low O2 levels, lead to an irreversible
loss of N bioavailability for most marine microorganisms
(except diazotrophs). However, the effect on global
warming differs depending on whether the denitrification
stops at N2O (partial denitrification) or N2 (complete
denitrification). In the former case, each reduction step is
carried out by several bacteria and independent enzymes
that are differentially sensitive to O2 (Bonin et al. 1989).
The regulation of all of these processes is complex when O2
concentrations are low, as is the case at the hypoxic and
suboxic boundaries of the OMZ, where the most important N 2 O production mechanisms remain unknown
(Codispoti and Christensen 1985). Measurements of the
15N : 14N ratio (d15N) found in seawater N O have been the
2
subject of controversy concerning the ocean’s N 2 O
production mechanisms. The d15N signature of N2O has
been interpreted as denitrification by Yoshinari et al.
(1997), as nitrification by Kim and Craig (1990) and Dore
et al. (1998), and as a coupling of both processes by Naqvi
et al. (1998). A useful and relatively new tool for
elucidating the mechanism of N2O production is the
determination of isotopomers in N2O, i.e., the intramolecular distribution of 15N in the linear NNO molecule of
N2O. It should be noted that the isotopomer distribution
in N2O is independent of the d15N in its precursors and is
determined by the steps of the biochemical reaction
(Toyoda et al. 2002).
The goal of this study is to identify the processes
involved in N2O cycling in the upper oxycline associated
with the OMZ of the ETSP. Specifically, we analyze the
role of denitrification in N2O cycling from oxic to suboxic
conditions found along the upper oxycline and OMZ. In
addition, we present denitrification rates for the ETSP,
considering these results in the light of new discoveries, i.e.,
the role of anammox in N2 production and even in N2O
cycling and its relative contribution with respect to
denitrification.
Methods
Sampling and field measurements—The study area is
located in the ETSP off northern Chile (Iquique ,21uS)
133
and southern Peru (,11–16uS, Fig. 1). The area off Iquique
was visited in March 2003 (Stas. CH020 and CH120; Chups
cruise on board the R/V Abate Molina) and July 2004 (Sta.
PBGQ; Prodeploy cruise on board the R/V Carlos Porter).
The area off Peru was visited twice: October 2005 (Stas.
KN008, KN020, and KN032; Peruvian cruise on board the
R/V Knorr—USA) and February 2006 (Stas. GA-4 cast 146, GA-5 cast 14-14, and GA-11 cast 14-31; Galathea-3
cruise on board the R/V Vædderen—Denmark). Hydrographic data (i.e., temperature, salinity, O2) from the water
column were obtained using a conductivity temperature
depth O2 (CTDO) probe (Seabird). Water samples for
{
chemical analyses (i.e., O2, N2O, NO{
3 , NO2 ) and for
determining isotopes and isotopomers in N2O and in
experiments were collected using Niskin bottles (10 liters)
attached to a rosette sampler. To avoid contaminating the
samples with dissolved O2 and N2O during the experiments,
N2 (at atmospheric pressure) was introduced into the
headspace overlying the Niskin bottles while withdrawing
successive samples. During the Chup and Prodeploy
cruises, the O2 sensors from the upcast CTDO were
calibrated by using discrete sample values obtained with
Winkler titration (see below). For the Knorr cruise, O2
sensors were calibrated before and after the cruise at
Woods Hole Oceanographic Institution (USA). During the
Galathea cruise, the sensor was tested in situ with new
microsensors with good agreement (Revsbech pers.
comm.).
Determination of N2O cycling rates through denitrification (N2Od)—During the Chups and Knorr cruises,
seawater from the upper oxycline (i.e., oxic to suboxic
conditions) was dispensed—avoiding oxygenation—into 1liter bottles, whereas during Knorr and Galathea seawater
was introduced directly into 2-liter bilaminated TedlarH
bags. The N2O evolution over time or the N2O cycling rate
(production and consumption) was determined under
natural O2 conditions, without any addition (control) and
with the addition of two inhibitors: 15% (v/v) acetylene and
0.086 mmol L21 (final concentration) of allylthiourea
(ATU). Acetylene is a nonspecific inhibitor of several
enzymes involved in N cycling (Wrage et al. 2004). It
inhibits N2O reductase and NHz
4 monooxygenase and,
thus, N2O production by nitrification and nitrifier denitrification and its reduction by denitrification. Recently, the
inhibitor effect of acetylene (and also ATU) on anammox
has been demonstrated by Jensen et al. (2007). Therefore,
the rate of N2O accumulation over time after inhibition
with acetylene in all the experiments corresponded to N2O
produced by denitrification (N2Opd). ATU, an NHz
4
oxidation inhibitor (Ginestet et al. 1998), was used to
evaluate net N2O cycling of denitrification (net N2Od),
which equaled production minus consumption by denitrifiers. Thus, the rate of N2O consumed by denitrification
(N2Ocd) was estimated from the rates measured in
experiments with acetylene (N2Opd) minus those quantified
in the ATU experiment (under natural O2 conditions). This
is an improved technique, since N2Ocd is typically
estimated using the difference between acetylene and
control (or net N2O cycling) treatments.
134
Farı́as et al.
For the Chups experiments, the samples were distributed
in 50-mL crimp seal bottles (GC bottle) sealed with a
rubber stopper and metallic cap under an inert atmosphere.
Acetylene and ATU were slowly injected using gastight
syringes through septa into the GC bottles in order to
determine which processes were involved in N2O production (see below). The flasks were then incubated in the dark
at the in situ temperature (11uC–15uC) for intervals of 0, 6,
and 12 h. For each incubation time, three bottles were
sacrificed for N2O and nutrient analyses. Each GC bottle
was injected with 5 mL of He, which was used to displace
5 mL of water from each flask to create headspace and then
injected with 50 mL of saturated HgCl2 in order to stop the
reactions. N2O (in duplicate) was measured from each
headspace GC bottle in the laboratory. After this analysis,
the remaining water was filtered and analyzed for NO{
2
and NO{
3 .
In the Knorr and Galathea experiments, subsamples for
{
N2O, NO{
3 , and NO2 were removed at different times
from 2-liter black two-laminated TedlarH bags (one bag per
experiment). Each bag had a hose and valve with a septum,
through which all the different treatments were injected.
The bags were also incubated under controlled laboratory
conditions. The time selected for the end of the incubation
was based on an experiment carried out during the first
cruise, in which the incubation was extended for 48 h;
linearity was observed between 0 and 24 h. Preservation and
storage procedures were as used for the Chups experiments.
{
cycling
The net rates of N 2O, NO{
3 , and NO2
(measured in three replicates per incubation time) were
calculated from the slopes of the linear regression of
concentration as a function of time for each treatment.
Positive slope values represented N2O or nutrient accumulation over time, and negative values, consumption. The
rate uncertainty was calculated directly from standard
errors from the slope in the case of net N2O, N2Od, and
N2Opd cycling (control, ATU, and acetylene treatments,
respectively). In the case of N2Ocd, which was estimated as
the difference between the N2O cycling rate in ATU and
acetylene treatments, the rate uncertainty (standard error)
was estimated using the propagation error from the
standard deviations of the slopes, if and only if the slopes
were statistically significant. Student’s t-test was used to
evaluate the significance of differences between the slopes.
Chemical and geochemical analyses—Dissolved O2
(125 mL, triplicate samples) was analyzed only in the
Chups and Prodeploy cruises, following a modified
Winkler method. N2O was determined by He equilibration
in the vial, followed by quantification with a Varian 3380
gas chromatograph using an electron capture detector
maintained at 350uC (Cornejo et al. 2006). The dissolved
N2O concentration was calculated in accordance with
Weiss and Price (1980). Dissolved NO{
2 was measured
immediately after collecting the seawater sample (both
seawater and water from experiments), whereas the water
{
for dissolved NO{
3 from experiments, as well as NO3 and
3{
PO4 from both seawater and experiments, was filtered
(0.7 mm, GF-F glass filter) on board and stored frozen until
analysis using standard colorimetric techniques following
Grasshoff et al. (1983) with an automatic analyzer
(Alpkem, Flow Solution IV). In Galathea-3, the concen{
tration of NO{
3 + NO2 was measured as NO (NOx
analyzer model 42C, Thermo Environmental Instruments)
after reduction by vanadium chloride (Braman and
{
Hendrix 1989). Coefficient variations for NO{
3 , NO2 ,
3{
and PO4 were better than 610%, 63%, and 60.7%,
respectively. N2O isotopic and isotopomer determinations
(duplicate samples) were carried out at the Tokyo Institute
of Technology using a Finnigan MAT 252 mass spectrometer (Toyoda et al. 2002). For this, N2O was extracted from
the samples and introduced into a preconcentration–gas
chromatograph–isotopic ratio mass spectrometry system;
d15N (N2O) and d18O (N2O) were determined in relation to
atmospheric nitrogen and Vienna standard mean ocean
water, respectively. N2O isotopomers were determined
based on the analysis of ionic mass fragments (NO+ and
N2O+) formed by electron bombardment of N2O. This
determination is possible since NO+ fragments contain the
central nitrogen (a), which allows the calculation of
fragment ratios from isotopic ratios of 14N15NO and
15N14NO. Although a rearrangement reaction occurs
during the ionic fragmentation process, its magnitude can
be corrected for. Site preference (SP) is the notation of the
difference in N isotope composition between the center and
end position N in N2O, namely, d15Na and d15Nb.
Reproducibility (duplicate samples) was better than 0.2%.
Data analysis—The Brunt–Vaisälä frequency (BVF) was
determined using temperature and salinity data. For better
data interpretation, BVF profiles were visually fitted to an
eight-term Gaussian model included in Matlab software.
Equilibrium concentrations of dissolved N2O in the water
column were calculated with the Weiss and Price (1980)
equation and the apparent N2O production–consumption
(DN2O) value was obtained by the difference between the
N2O saturation concentration and its measured concentration in the seawater. Using salinity (S) and potential
temperature (h), T-S analyses were employed to identify
water mass distributions. The mixed layer depth (MLD)
was identified using density profiles. The base of the MLD
was estimated to be the upper limit of the oxycline. The
base of the oxycline was delimited from NO{
2 and O2
profiles, where the vertical distribution of NO{
2 increased
abruptly and dissolved O2 reached constant values. N2O
inventories were estimated by linear integration over a
depth range in which a N2O peak was clearly observed. In
addition, the NO-h index (Naqvi and Sen Gupta 1985) used
as a NO{
3 deficit parameter in oxygen-deficient water was
calculated in order to delineate the possible vertical pattern
of N loss.
Results
Hydrographic and oceanographic features—Temperature,
salinity, dissolved O2 and N2O, and dissolved inorganic
{
nitrogen compounds (NO{
2 and NO3 ) measured in the
water column during the Chups, Knorr, and Galathea
cruises, where the experiments were performed, are shown
in Fig. 2. Below a narrow layer of 10–20 m, which could be
Denitrification and N2O cycling
135
there was a pronounced depletion of N2O with levels of
about 9–30 nmol L21 at the oxycline base. A N2O
minimum, with subsaturated concentrations lower than
9 nmol L21, was observed within the OMZ core during the
Knorr and Galathea cruises.
Vertical NO{
2 profiles revealed two distinct maxima
(Fig. 2b): a sparser and less pronounced (up to 0.5 mmol
L21) primary NO{
2 maximum was located between the ML
base and the upper part of the oxycline, and a secondary
21
NO{
2 maximum (SNM), with levels up to 10 mmol L ,
was found from the oxycline base (60 m) to 400-m depth;
the SNM was more compressed (narrower) at the
also
northernmost station. The distribution of NO{
3
showed an intense gradient along the oxycline and in the
OMZ (Fig. 2b). Below the ML, NO{
3 increased gradually
to maximum values that were associated with the same
depth of the N2O maximum and then decreased to
minimum values that coincided with the upper OMZ
boundary embedded in the SNM. The NO-h index shows
that there is significant N loss (except at Sta. CH020), not
only in the SNM but also along the upper oxycline
(Fig. 2b).
Fig. 1. Eastern tropical South Pacific (ETSP) region and
location of sampling stations for the different cruises.
defined as the mixed layer (ML), the temperature decreased
with depth from ca. 16uC to ca. 13uC at 50–70-m depth
(Fig. 2a), except at the southernmost station (off Chile),
where the temperature declined from 20uC to 13uC. Salinity
decreased gradually with depth (Fig. 2a), except at the
stations located off Chile (,21uS) where the salinity profile
showed a subsurface minimum salinity layer (MSL)
between 20- and 60-m depth, previously described for this
region by Schneider et al. (2003). This is a quasi-permanent
characteristic off northern Chile and southern Peru (see
Fig. 2a). Extremely high stability, estimated through the
BVF, was observed at the pycnocline, and it was always
associated with the presence of the MSL (see Fig. 2a).
The vertical distribution of dissolved gases is shown in
Fig. 2c. At all stations, vertical O2 gradients (oxyclines)
were extremely sharp and shallow and were always found
below the ML. There, the O2 concentration varied greatly,
from oxic (220 mmol L21) at the ML base to suboxic (less
than 11 mmol L21) at the oxycline base, with O2 gradients
from 1 to 2 mmol L21 m21. Below the oxycline, O2
concentrations reached constant and sometimes nearly
anoxic levels (1.8 mmol L21) at 70–400 m. This zone is
called the OMZ core and is clearly associated with a
preexisting oxygen-deficient equatorial subsurface water
(ESSW 26.2 kg m23, isopycnal) off southern Peru and
northern Chile (Farı́as et al. 2007). The northeast station,
located on the continental shelf (KN032), however, seems
to be influenced by other oxygen-poor water masses, fed by
a type of equatorial or Cromwell undercurrent (Kessler
2006). The N2O maxima, with levels fluctuating from 37 up
to 86 nmol L21, was always located at the oxycline,
particularly at its lower part (where dissolved O2 concentrations ranged from 5 to 30 mmol L21). Below these peaks,
Isotope and isotopomer signals in N2O in the water
column—Vertical profiles of d15N and d18O in dissolved
N2O, d15Na, and d15Nb, along with some hydrographic
data obtained during the Prodeploy cruise (Sta. PBGQ) are
shown in Fig. 3. The d15N values in the dissolved N2O at
the oxycline changed drastically, i.e., they were more
depleted in 15N (8%) at maximum N2O levels, with respect
to the layers immediately above (around 12% d15N) and
below (up to 15% d15N at 100-m depth) as N2O was
consumed (Fig. 3d). At the oxycline, d15Na and d15Nb
showed opposite trends (decreasing and increasing, respectively), with a maximum difference between the two (SP
maxima) where the N2O maximum was observed (see
Fig. 3e). On the other hand, the d18O in dissolved N2O
increased strongly (62.9–86.3%) with depth (30–100 m),
but a break in the slope was observed in the upper oxycline
(Fig. 3d).
N2O and nutrient cycling rates associated with denitrification—Table 1 shows the results of experiments carried
out along the oxycline and OMZ, together with other
environmental conditions measured at the time of sampling. At each depth, the results of the various treatments
that were performed were significantly different (p ,
0.005), allowing the estimation of N2Opd and N2Ocd rates.
Figure 4 shows a representative time course for the
accumulation or depletion of N2O during an experiment
performed at Sta. KNO20 (50-m depth) using different
treatments, i.e., control (without any addition), acetylene,
and ATU. With acetylene, we presume that this technique
only measures N2O production by denitrification activity
based on preexisting NO{
3 . N 2 O was produced by
denitrification, with N2Opd rates fluctuating between 2.25
and 50.0 nmol L21 d21; the N2Opd rates obtained from the
experiments off northern Peru (KN032) were the highest.
The rate of N2O reduction to N2 (N2Ocd) fluctuated
between 2.63 and 70.1 nmol L21 d21. Thus, N2Ocd proved
136
Farı́as et al.
Fig. 2. Vertical profiles of oceanographic variables from sampling stations sorted in columns from the south (,20uS off Iquique)
toward north (,11uS), station name are indicated in the lower right corner of the each panel of first row. First row (a): temperature
(dashed line), Buoyancy frequency (solid black line), and salinity (solid gray line). Second row (b): Nitrate (open circles), nitrite (closed
circle), and NO{
3 h (gray bars). Third row (c): N2O (open circles) and O2 (solid gray line).
Denitrification and N2O cycling
137
{
15
Fig. 3. Vertical profiles of (A) temperature and salinity; (B) N2O and O2; (C) NO{
3 and NO2 ; (D) isotopic composition of N and
in N2O; and (E) isotopomer composition at a Sta. PBGQ (Prodeploy cruise) located off northern Chile. The shaded areas represent
the lower part of the oxycline or upper boundary of the OMZ (light gray) and the OMZ (dark gray). Horizontal line indicates the peak in
N2O. Reproducibility (duplicate samples) was better than 0.2%.
18O
to be more dependent on O2 levels than N2Opd. Figure 5
shows the N2O production or consumption ratio as a
function of the dissolved O2 concentration. The N2O
production or consumption ratio varied from 6.55 to 0.36,
decreasing as the O2 declined.
{
The NO{
3 and NO2 cycling rates measured in the
control experiments are also presented in Table 1. NO{
3
cycling rates shifted from production (8.9 mmol L21 d21) to
consumption (210.2 mmol L21 d21) in a depth trend that
coincided with O2 levels from the upper to lower part of the
oxycline, or the oxycline base, whereas NO{
2 cycling rates,
one order of magnitude lower than NO{
3 cycling rates,
varied from production to consumption (0.01 to 4.2 mmol
L21 d21) (see Table 1).
Discussion
In the ETSP, N2O always accumulates in the oxycline
(up to 86 nmol L21), particularly in the layer defined in a
previous study as the upper OMZ boundary (Farı́as et al.
{
2007), where NO{
3 consumption (and a NO3 deficit) but
{
not NO2 accumulation occurred. However, strong N2O
depletion was seen in the OMZ core, as was a huge
accumulation of NO{
2 (see Fig. 2). This distribution is
typical of other OMZs (Cohen and Gordon 1978; Elkins et
al. 1978; Bange et al. 2001), but the mechanisms involved in
such distributions have yet to be resolved. The ETNP and
Arabian Sea have well-defined oxyclines, although never as
shallow and compressed as that described for the ETSP
(Farı́as et al. 2007). In the case of the ETSP, the oxycline is
located in the euphotic zone in the layer just above a
preexisting oxygen-poor layer of equatorial origins (i.e.,
ESSW). This water mass defines the OMZ core itself.
According to the h-S diagrams (not shown), the oxycline
off northern Chile (21uS) coincided with the presence of a
permanent low-salinity layer associated with Eastern South
Pacific intermediate water (40–60 m; T 5 12.5uC; S 5
34.25; see Figs. 2a, 3a) (Schneider et al. 2003) that produces
strong stratification (see Fig. 2a) and isolates the mixed
layer from the subsurface layer. The oxycline seems to be
maintained, under conditions of reduced vertical mixing, by
the strong aerobic respiration of settled organic matter. In
fact, Pantoja et al. (2003) found that more than 80% of the
fresh organic matter produced at the surface can be
respired in the upper part of the oxycline. This process is
coupled to nitrification (Molina et al. 2005) and is also
responsible for N2O production at the upper oxycline.
Denitrification and its role as an important process
involved in both N2O production and consumption
processes are discussed here, using an experimental
approach and following the natural isotopic-isotopomeric
signatures of dissolved N2O.
N2O cycling by denitrification along the upper oxycline—
Although open ocean NO{
3 concentrations are 1000 times
higher than those of NO{
2 , NO, and N2O, in the OMZ,
8.9061.68
KN008
GA11
KN020
4.2762.01
0.060
nd
20.0860
nd
26.7069.78 22.0061.88
nd
0.460.02
2.9760.32 10.362.87 5.9562.01
24.3262.16 20.2660.05
18.967.56
3.1462.6
7.8165.85
14.863.94
7.1463.03
3.8462.88
3.1260.55
3.9460.53
210.260.
nd
nd
0.060
2.4362.19 19.360.48
10.0861.84 10.46460
20.1660.08 20.2460.11 21.0360.10 20.1560.02
6.2561.17
2.2761.17
4.0864..5
9.664.32
70.0865.2
50.0562.02
28.3764.80
nd
nd
22.2560.13 20.4860.06 20.4360.09
5.6160.9
10.860.07
b
10u599S, 78u99
KN032
51
Oct 2005
omz
30
40
11.6*
1.8*
84.2
61.7
17.9
13.8
0.41
3.41
5.5260.9 220.1664.8
* Treatment with ATU inhibitor to avoid ammonium oxidation; m, middle oxycline; b, oxycline base; OMZ, oxygen minimum zone; nd, not determined.
{ Oxygen values taken from upcast of CTDO.
{ The error in N2Ocd was calculated using the propagation error, which was estimated from the standard deviations of the slopes of the ATU and acetylene experiments.
L21 d21)
Net NO{
3 (mmol
L21 d21)
42.5613.3
GA4
20u16.99S,
20u 03.39S, 70u45.279W
15u559S, 74u359W
14u 14.139S, 76u 36.479W 13u189S,
72u17.29W
76u599W
220
64
61
43
67
May 2003
Feb 2007
Oct 2005
Feb 2007
Oct 2005
omz
m
m
m
m
90
30
50
60
30
40
50
30
50
50
12.8
44.14
1..32
1.19
72.2{
65.0*
47.2*
101
3.7
28.6*
12.5
28.6
41.8
25.8
29.6
37.0
40.3
19.5
46.6
45.3
11.3
19.5
24.6
21.0
nd
nd
nd
21.1
28.4
19.6
0.22
0.78
0.38
0.25
1.04
0.74
0.85
0.17
0.16
5.58
8.7565.6
11.260.23 8.2861.23 1.6860.06 23.9860.01 7.0564.33 23.3060.94 2.6461.2 4.2760.48 5.2460.89
CH120
22.30610.32 27.63612.95 11.560.23
37
May 2003
omz
40
80
25.3
7.7
71.7
30.8
17.8
8.9
1.5
4.8
14.6260
220.1667.92
m
20u19.09S, 70u36.99W
CH020
N2Opd (nmol L21 17.2560
d21)
2.6360
N2Ocd (nmol L21
d21){
0.0860.05
Net NO{
2 (mmol
L21 d21)*
km from coast
Date
Depth (m)
O2 (mmol L21)
N2O (nmol L21)
21)
NO{
3 (mmol L
21)
NO{
2 (mmol L
Net N2O (nmol
Location
Stations
{
Table 1. N2O, NO{
2 , and NO3 recycling rates (mean 6 standard error) measured at natural O2 levels at the study stations. N2Opd : N2O production by denitrification;
N2Ocd : N2O consumption by denitrification; net N2Od : net N2O by denitrification. Stations set in order of latitudinal coordinates.
138
Farı́as et al.
Fig. 4. Examples of changes in concentration (accumulation
or depletion) during incubations carried out under natural oxygen
conditions at a representative station (KN020) at 50-m depth.
Top: nitrous oxide, nitrite, and nitrate time courses in controlled
incubations. Middle: nitrous oxide time course in ATU amended
incubations. Bottom: nitrous oxide time course in acetylene
amended incubation. Nitrate represents one discrete subsample,
whereas nitrous oxide and nitrite concentrations represent
duplicate subsamples. The lines represent linear regressions (only
for N2O data).
{
NO{
2 and NO3 concentrations have the same order of
magnitude. This situation could result from the use of
{
different substrates as electron acceptors (NO{
3 and NO2 )
but mainly depends on the extent of the denitrification
reaction (whether partial to N2O or NO or total to N2). The
use of different electron acceptors by bacteria (assuming
that the reaction is not ‘‘canonical’’) seems to be dependant
Denitrification and N2O cycling
Fig. 5. N2O production : consumption ratio obtained from
natural N2Opd and N2Ocd experiments at oxygen concentrations
(expressed as natural logarithm scale) observed in the open sea off
northern Chile and southern Peru. Horizontal line represents ratio
equal 1 (i.e., N2O production equals its consumption).
on their enzymatic battery and activity, which vary highly
with ambient O2 levels.
In spite of the global biological and climatological
importance of denitrification, it has been mainly estimated
through indirect stoichiometric and N2 : Ar ratio calculations (Codispoti et al. 2001; Devol et al. 2006); currently,
there are very few direct measurements of denitrification in
the open ocean. Measurements of denitrification have been
made through the production of N2 (spiked 15 NO{
3 ) or
from N2O accumulation (acetylene blockage).
However, from a methodological point of view, it is
possible to underestimate this process since the currently
available methods rely on N2 formation. This underestimation could occur if nitrate is not being used solely as an
electron acceptor or if complete denitrification is not
taking place, leading to the formation of N2O instead of
N2.
Oceanic denitrification rates measured by different
methods vary enormously, going from , 0.1 to 300 nmol
L21 d21 (see Table 2). The highest rates (including data
from this study; see Sta. KN032, Table 2) mostly occur in
continental shelf areas (Naqvi et al. 2006). Indeed,
denitrification rates measured in this study (i.e., those
estimated through N2O accumulation, after acetylene
addition minus ATU as a control) fluctuated between
2.27 and 70 nmol L21 d21 and are slightly higher than
those previously measured as N2 production in the study
area (see Table 2).
A further complication in the global estimates of N loss
comes from anammox, which also drives N loss. To date,
only two anammox studies have been carried out in this
area, i.e., Thamdrup et al. (2006) and Hamersley et al.
(2007). Off Chile, the anammox rates fluctuated from 0.2 to
5 nmol L21 d21 (Thamdrup et al. 2006), whereas off Peru
(over the continental shelf), rates ranged from 2 to 30 nmol
L21 d21 (Hamersley et al. 2007). These rates were of the
same magnitude or slightly lower than our measured N2
139
denitrification rates. These results raise an important
question: if anammox is working in the area, is it involved
in the strong N2O cycling observed (both production and
consumption)? At present, no clear evidence exists that
anammox is mediating N2O cycling. Based on a metagenomic study, Strous et al. (2006) sequenced the Candidatus
‘‘Kuenenia stuttgartiensis’’ genome. They did not find
evidence that this bacterium has the N2O reductase gene,
{
but it has two genes that encode for NO{
3 -NO2 oxide
reductase and for NO reductase, a well-recognized enzymatic pathway in denitrification. Strous and Jetten (2004)
describe NO as an intermediary of anammox and, more
recently, Kartal et al. (2007) mentioned that N2O could be
produced (although in very small proportions) during NO
detoxification. So far, there is no direct and forceful
evidence that anammox is producing N2O. Under the
current assumption that only denitrification is able to
consume N2O, it is most likely that denitrification is the
main process responsible for N2O consumption in the
studied area. This is confirmed by the isotopic signatures of
NO{
3 and N2O (see below) in the ETSP.
Moreover, the occurrence of anammox in the absence of
denitrification, as found by Kuypers et al. (2005), could be
due to a methodological bias. Anammox is always
measured under degassing conditions (the sample is flushed
with helium), mainly in order to eliminate the high
abundance of 14N14N gas (Dalsgaard et al. 2003). This
procedure logically results in a removal of the N2O pool,
which affects the last step in denitrification, but also results
in anoxic conditions that modify the rate of N2O : N2
produced during denitrification. It has been shown that, as
water becomes anoxic, NO{
2 accumulates and N2O is
consumed (this may be due to a differential sensitivity of
enzymes involved in the denitrification). Thus, the lack of
15N15N in incubated samples after the addition of 15 NO{
3
or 15 NO{
2 may be a methodological artifact. In this sense,
our denitrification rates were measured under natural
conditions, i.e., with O2 levels quite similar to those
found at the sampling time and after a short incubation
time. This calls into question the co-occurrence of
anammox and denitrification, particularly in the layer
where O2 drops to levels below ,11 mmol L21, which is
precisely where the NO{
2 accumulates and N2O decreases
(i.e., the OMZ core).
Regarding the regulation of denitrification by O2, rates
of NO{
2 reduction to N2O or N2Opd were not equal to
those of N2O reduction to N2 or N2Ocd at the same depth.
Rather, the latter varied from 2.6 to 70 nmol L21 d21,
tending to be progressively higher as O2 declined toward
the base of the oxycline. In general, production rates were
higher than consumption rates, but these differences
decreased toward the base of the oxycline and even more
toward the OMZ core. This pattern was found by CastroGonzalez and Farı́as (2004) in the same study area.
Figure 5 shows a clear inverse relationship between the
natural log of the N2Opd : N2Ocd ratio and the decline in
O2 (expressed as ln); the ratio was less than 1 (i.e., N2O
consumption higher than its production by denitrification)
at very low O2 concentrations. Differences between N2Opd
and N2Ocd and even in the rates of denitrification in the
140
Farı́as et al.
Fig. 6. Vertical profiles of dissolved oxygen (solid black line), apparent nitrous oxide production (DN2O, dotted line), and saturated
equilibrium N2O concentration (dashed line) at stations located along the eastern tropical South Pacific from 20 to 13uS. Two way arrows
represent the depth range at which DN2O was integrated at a well-defined N2O peak where the oxycline is developed (Table 3, third
column). Net N2O cycling (from experiments) is indicated as bars. Negative rates indicate that consumption is higher than production by
denitrification. Station names are indicated in the lower right corner of each panel.
Denitrification and N2O cycling
Table 2.
141
Literature and present estimates of denitrification and anammox rates in the principal oceanic OMZs.
Area
Denitrification rates
(nmol L21 d21)
Anammox rates
(nmol L21 d21)
3.2
5.4–22
nd
nd
2.6–70
48–2700
nm
nm
4.8–16.8
1.5–106
nm
72–600
0.2–4.8
9.9–15
5–207
up to 300
nd
ETSP open sea
ETSP (5–25uS) (110–330 m{)
ETSP (21uS) open sea (55–150 m{)
ETSP 8.9–12uS (25–400 m{)
ETSP 12–21uS open sea (30–80 m)
ETNP Golfo Dulce Coastal bay (120–
200 m{)
AS Central Arabian 15–20uN open
sea (125–300 m{) AS and IO
15–21uS open sea (150–300 m{)
10.7–17.9uS coastal sea (20–72 m)
AS continental shelf off India
(15.4uN) (20–125 m)
BS Benguela shelf 23uS (50–200 m)
Method
Sources
ABT*
ETS{
15N-N production1
2
15N-N production1
2
Modified ABT
15N-N production1
2
Elkins et al. (1978)
Codispoti and Packard (1980)
Thamdrup et al. (2006)
Hamersley et al. (2007)
This study
Dalsgaard et al. (2003)
nd
15N-N
2O
Nicholls et al. (2007)
nm
15N-N
2
nm
ABT*
12.5–180
15N-N
production1
production||
Devol et al. (2006)
Naqvi et al. (2000)
2
production
Kuypers et al. (2005)
ETSP, eastern tropical South Pacific; ETNP, equatorial tropical North Pacific; AS, Arabian Sea; IO, Indian Ocean; BS, Benguela System; SNM,
secondary nitrite maxima; nd, not detected; nm, not measured.
* ABT, acetylene block technique.
{ Depths where a clear SNM was observed.
{ ETS, electron transport activity converted to respiration rates using NO{
3 .
15
15
1 Rates measured as 14N15N production after the anaerobic incubation with 15 NHz
NO{
NHz
3 addition; higher rates were measured with
4 and
4 +
14
NO{
2 addition (data not shown).
{
15
|| Rates measured as 14N15N production after NO3 addition using isotope labeling technique.
same study area may be indicative of O2 levels differentially
regulating N2O cycling through denitrification. Several
laboratory studies have shown O2 to be the principal
regulator of denitrifying reductases (Körner and Zumft
1989), and N2O reductase is the most sensitive to O2
(Betlach and Tiedje 1981). Bonin et al. (1989) found that, in
pure cultures, increases in the O2 levels above ca. 16 mmol
L21 could inhibit N2O reductase, producing partial
denitrification. In field studies carried out in suboxic areas,
N2O production through partial denitrification has only
been reported at ,22 mmol L21 O2 for the Baltic Sea
(Rönner and Sörensson 1985) and the Indian Shelf (Naqvi
et al. 2006). In the Arabian Sea, N2O production by
denitrifiers (measured by 15 NO{
2 addition) was predominant for N2O accumulation at the oxic–suboxic interface
(from 20 to 4 mmol O2 L21) (Nicholls et al. 2007). In our
case, N2O production through partial denitrification took
place even at O2 levels as high as 50 mmol L21.
Furthermore, net NO{
2 cycling (i.e., differences between
production and consumption) does not occur at the same rate
as NO{
3 cycling (the former is much slower, see Table 1);
{
clear differences were observed in the NO{
3 and NO2 cycling
rates between the upper oxycline and OMZ core (see
Table 1), leading to concomitant NO{
2 accumulation as
observed in the OMZ (Codispoti et al. 2001). This
accumulation is not, however, observed in the upper oxycline,
where nitrifier–denitrification process, AOA (Ammonium
Oxidation Archae), AOB (Ammonium Oxidation Bacteria),
and even anammox bacteria can coexist using NO{
2 as a
substrate for their metabolism. In this same sense, Nicholls et
al. (2007) found that N2 production with 15 NO{
2 additions
did not occur at the same rates throughout the water column,
also indicating different O2 regulation. Nevertheless, net
NO{
3 consumption was observed at high O2 levels, supporting the idea that dissimilative NO{
3 reduction is a facultative
process that can act in the presence of O2 (Bonin et al. 1989).
Table 3. Inventories of N2O and DN2O at the N2O maximum located in the upper oxycline. Integrated N2O production and
consumption by denitrification based on rates obtained from experiments (see Table 1); estimated air–sea exchange is included. Negative
values indicate consumption or efflux of N2O at the N2O maxima.
Sta.
Oxycline
boundaries
(m)
Inventory
N2O
(mmol m22)
Inventory
DN2O
(mmol m22)
Integrated*
net N2O
(mmol m22 d21)
Integrated
N2Opd
(mmol m22 d21)
Integrated
N2Ocd
(mmol m22 d21)
Air–sea
N2O flux{
(mmol m22 d21)
CH020
CH120
GA4
GA11
KN008
KN020
KN032
9–80
25–75
12–70
10–60
9–75
12–80
6–60
3611
2271
1626
1787
1786
2971
3287
2991
1837
1116
978
1235
2126
2799
599.42
nm
163.64
26.28
295.68
267.24
59.28
707.25
nm
432.82
511.68
279.98
553.35
528.65
2107.83
nm
2307.61
2459.10
2376.26
2286.11
2468.72
230.50
213.73
212.78
229.41
226.84
213.11
230.79
{ Wanninkhof parameterization; nm, not measured.
* Integrated rates on well-defined N2O peak based on experimental measurements taken from Table 1.
142
Farı́as et al.
Isotope and isotopomer composition in N2O at the
subsurface N2O maximum—The determination of N
isotopes and their isotopomer ratio or SP is a direct way
to elucidate N2O production mechanisms (Toyoda et al.
2002). Whereas d15Nbulk distribution is dependent upon the
extent of the reaction and the substrate that is being
consumed, the isotopomer distribution in N2O must be
independent of the d15N of the precursors and be
determined only by the biochemical reaction step since
precursors contain only one N atom (NO{
2 , NO, NH2OH),
thereby excluding the possibility that different chemical
species combine to form N2O (Toyoda et al. 2002). Thus,
the observed vertical pattern in d15Nbulk and SP (see Fig. 3)
indicate different mechanisms of N2O cycling in the upper
oxycline.
The surface mixed layer had homogeneous d15Nbulk and
SP values (near 8% and 9%, respectively), with the values
for the former close to that expected for equilibrium with
the atmosphere (7.0 6 0.6%); the upper oxycline,
nonetheless, showed strong d15Nbulk and SP variations. In
the oxycline, the negative shifts of d15Nbulk between 20 and
40 m (see Fig. 3d) could be explained partially by a
depletion in the 15N content of theN2O produced, relative
to the initial NHz
4 substrate (Popp et al. 2002). In this
depth range, the SP showed an increasing trend from
9.45% to 14.2% (see Fig. 3e) that could be attributed to the
enhancement of AAO at lower O2 values according to the
mechanism proposed by Toyoda et al. (2002), in which two
precursor molecules unite to a hyponitrite-type intermediary (-ONNO-); consequently, the NO bond is broken where
the lightest isotopes are found, based on the zero point
energy values given by Yung and Miller (1997), thus
determining the selectivity for 15N in the central position.
The mechanism proposed by Toyoda et al. (2002) has been
corroborated in experiments by Sutka et al. (2006), who
found SP values reaching 14.9% and 33.5% for Nitrosomonas europaea and 32.5% for Nitrosospira multiformis in
cultures with hydroxylamine as a substrate for nitrifying
activity.
The SP values in N2O greatly increase from the middle of
the oxycline (40–50 m) to the OMZ core (ca. 100 m),
indicating a change in the dominant N2O cycling processes.
At the middle of the oxycline, two N2O producing
processes (i.e., AAO and NO{
2 reduction to N2O) seem
to be acting (although the proportion of N2O produced by
each process is not yet known).
On the other hand, N2O is being progressively reduced
(consumption) to N2 as O2 declines toward the OMZ core,
producing a marked enrichment of 15N, which is used as an
electron acceptor during total denitrification. This pattern
is also reflected in the d18O distribution. In the surface layer
(0 to 20 m), d18O gradually increases as oxygen decreases.
This observation supports the idea that the O atom of N2O
produced by NHz
4 oxidation comes from dissolved O2,
which is isotopically enriched as it is consumed by aerobic
respiration (Ostrom et al. 2000). In the intermediate layer
(20 to 50 m), coincident with the NO{
3 maximum, an
abrupt change is observed in the d18O profile indicating a
change in the N2O production mechanism. This change
may be accounted for by several different processes,
including the incorporation of O atoms from water to
N2O, which has been observed for some specific NO
reductases (Ye et al. 1991). However, we have no conclusive
evidence to support this or other hypotheses. Below 50 m,
very similar profile shapes are observed for d18O and NO{
2
(Fig. 3), coincident with very low O2 concentrations. Since
the O atom of N2O produced during NO{
2 reduction comes
{
from NO{
2 (Averill 1996), and NO2 is produced reducing
18
isotopically enriched NO{
3 , the highly correlated d O and
profiles
indicate
that
N
O
is
produced
mainly
by
NO{
2
2
reduction.
Furthermore,
N
O
consumption
observed
NO{
2
2
close to 100-m depth also can yield a high d18O in the N2O.
We think that, at least at this depth, both processes (NO{
2
and N2O reduction) contribute to the d18O signal on N2O.
Similar patterns were observed by Yamagishi et al.
(2005) in the OMZ located in the ETNP but in a deeper
water column (500 m). Thus, the fact that the observed
15 bulk
N2O minimum coincides with SP, NO{
2 , and d N
maxima supports the conclusion that denitrification is the
main process reducing N2O to N2 in the OMZ and is
progressively important as O2 decreases below the center of
the oxycline. Data from the same study area (21uS)
reported herein (see above) and in Molina et al. (2005)
and Farı́as et al. (2007) as well as the vertical distribution of
d15N of NO{
3 fluctuating from ,11%, just below the
pycnocline to ,18% at 100 m (De Pol-Holz et al. in press)
all support the idea of the presence of a nitrifying layer and
an increasing tendency for denitrification in N2O cycling
toward the OMZ core.
N2O mass balance in the upper oxycline of the ETSP—
Finally, a mass balance was estimated in order to assess the
relative contributions of other production or consumption
processes beside denitrification as N2O sources or sinks in
the subsurface N2O maximum. Figure 6 shows vertical
distributions of oxygen N2O, saturated equilibrium N2O
concentration, net N2O production, and the N2O peak
limits that were used to estimate the balances. Table 3
shows the inventory of N2O and DN2O estimated at the
well-defined subsurface peak, as well as the integrated rates
of N2O production : consumption and its fluxes across the
air–sea interface. It has been assumed that vertical
advection by coastal upwelling is the main outgassing
mechanism from the subsurface maximum to the surface
layer and that diapycnal mixing is insignificant. Thus,
fluxes across the air–sea interface and N2O consumption by
denitrification are the main mechanisms of N2O loss. N2O
released into the atmosphere, as estimated using compiled
daily mean wind velocities and the Wanninkhof (1992)
equation (see Table 2), ranged from 12.7 to 30.7 mmol m22
d21, confirming our study area as an important source of
N2O toward the atmosphere, with values similar to those
reported for areas of high biological productivity, e.g., the
Arabian Sea (Law and Owens 1990). These effluxes,
however, represent a small percentage (6–27%) of reduction
due to consumption by denitrification (107–468 mmol m22
d21, see Table 3). Certainly, the shape of the N2O
maximum reflects strong N2O consumption at the base of
the oxycline. Unlike the upper limits of the oxycline, where
strong stratification restricts the diffusion of N2O (see BVF
Denitrification and N2O cycling
parameter; Fig. 2a), there is no diffusion barrier at the base
of the oxycline where the shape of the profiles should be
smoother. On the other hand, some stations showed net
N2O production that must lead to transient N2O accumulation, whereas others showed net consumption (see
Table 3). This means that other N2O production processes
must exist besides denitrification—perhaps AAO by bacteria and archaea or even nitrifier denitrification—to allow
the observed N2O accumulation at the subsurface peak. In
fact, AAO (previously discussed) could occur in this layer.
The N2O pool fluctuated from 1790 to 3610 mmol m22.
Such variation may depend on the oceanographic variability in the study area (seasonal and interannual), perhaps
related to changes in primary production rates and in the
oxygenation of the water produced by seasonal upwelling
or ENSO (El Niño Southern Oscillation) events. Residence
time (t) calculated from de rates of loss are 46–160 d based
on the N2O efflux, but only 3.3–31 d based on integrated
N 2O consumption rates. The difference stresses the
importance of N2O as an acceptor for denitrifier microorganisms.
This study shows that partial denitrification takes place
under a range of O2 conditions, contributing to the
formation of the subsurface N2O peak, a typical feature
of OMZs. The results indicate that N2O cycling and the
ratio of N2O : N2 produced by denitrification along the
upper oxycline off northern Chile and Peru are variable and
mainly regulated by O2 levels, which determine the partial
or total reduction of N2O at different depths.
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Edited by: Mary I. Scranton
Received: 22 January 2008
Accepted: 3 September 2008
Amended: 19 September 2008