N isotopic composition of dissolved organic nitrogen and nitrate at

Transcription

GLOBAL BIOGEOCHEMICAL CYCLES, VOL. 19, GB1018, doi:10.1029/2004GB002320, 2005
N isotopic composition of dissolved organic nitrogen and nitrate at the
Bermuda Atlantic Time-series Study site
Angela N. Knapp and Daniel M. Sigman
Department of Geosciences, Princeton University, Princeton, New Jersey, USA
Fredric Lipschultz
Bermuda Biological Station for Research, St. Georges, Bermuda
Received 18 June 2004; revised 16 November 2004; accepted 21 December 2004; published 16 March 2005.
[1] To better constrain the dynamics of the dissolved organic nitrogen (DON) pool and
the role of N2 fixation in the nitrogen cycle at the Bermuda Atlantic Time-series Study
(BATS) site, we measured the 15N/14N ratio of total nongaseous nitrogen (TN) in the
upper 250 m and of nitrate in the upper 1000 m of monthly water column profiles from
June 2000 through May 2001. The annually averaged TN d15N in the upper 100 m
is 3.9%, which is greater than thermocline nitrate (2–3% at 250 m) and similar to
literature values for shallow sinking nitrogen at BATS (3.7%). We discern no seasonal
variation in TN d15N, which suggests that most of the DON pool is recalcitrant on this
timescale. The TN data require a d15N for the sinking flux that is similar to previous
measurements, suggesting that N2 fixation is a minor component of new nitrogen at
BATS. Small but measurable differences in the concentration and 15N/14N of total organic
nitrogen (TON) between the surface and subsurface (250 m) suggest that subsurface
remineralization of 0.25 mM of the surface TON acts to lower the 15N/14N of nitrate
in the thermocline at BATS.
Citation: Knapp, A. N., D. M. Sigman, and F. Lipschultz (2005), N isotopic composition of dissolved organic nitrogen and nitrate at
the Bermuda Atlantic Time-series Study site, Global Biogeochem. Cycles, 19, GB1018, doi:10.1029/2004GB002320.
1. Introduction
[2] Although decades of study have characterized the
carbon and nitrogen budgets of the oligotrophic Sargasso
Sea [Jenkins, 1988; Michaels et al., 1994a; Bates et al.,
1996; Lipschultz et al., 2002], fundamental aspects remain
poorly understood. In particular, studies at the Bermuda
Atlantic Time-series Study (BATS) site have revealed a
drawdown of dissolved inorganic carbon (DIC) from
surface waters during the summer months for which one
cannot account in terms of either the nutrients required to
support the biological removal of the DIC or the export
production that should result from the DIC decrease
[Michaels et al., 1994a; Gruber et al., 1998]. In the
absence of significant nitrate (NO
3 ) input during this
period, N2 fixation has been proposed as a means of
fueling phytoplankton growth to remove the DIC. This
hypothesis is supported by regional geochemical observations, including the high concentration of NO
3 relative to
phosphate (PO3
4 ) in the North Atlantic thermocline
[Michaels et al., 1996; Gruber and Sarmiento, 1997]
and the low- 15N/ 14N of NO 3 in these same waters
[Altabet, 1988; Karl et al., 2002] (see also Liu et al.
[1996] for similar evidence from the Pacific).
Copyright 2005 by the American Geophysical Union.
0886-6236/05/2004GB002320$12.00
[3] Despite these regional observations, local geochemical measurements have provided arguments against N2
fixation as the missing N source for the BATS summertime
DIC drawdown. At BATS, Altabet [1988] observed a
similar 15N/14N for the N sinking into shallow sediment
traps (PNsink) and the NO
3 being supplied to the surface
from the thermocline (d15N of 3.7 and 3.5%, respectively;
d15N, in per mil versus atmospheric N2 = {[(15N/14N)sample/
(15N/14N)atm] 1} 1000). Since newly fixed N has a
d15N in the range of 3 – 0% [Hoering and Ford, 1960;
Minigawa and Wada, 1986; Carpenter et al., 1997], Altabet
[1988] concluded that there was little room in the isotope
budget for N2 fixation to be quantitatively important as a
source of N for new production at BATS.
[4] One caveat to this analysis is that dissolved organic
nitrogen (DON) was not included as a potential flux term in
the isotope budget. Since the work of Altabet [1988], it has
been shown that N2 fixers can shunt a major fraction of
newly fixed N into the DON pool, significantly increasing
the ambient DON concentration ([DON]) [Karl et al., 1992;
Capone et al., 1994; Glibert and Bronk, 1994]. Thus DON
could potentially represent a significant flux of low-d15N N
out of the surface, similar to the seasonal C concentration
dynamics of dissolved organic carbon (DOC) at BATS
[Carlson et al., 1994]. If this occurs, it could allow the
hypothesis that N2 fixation is the missing N source for the
DIC drawdown at BATS to be consistent with the isotope
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1 of 15
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KNAPP ET AL.: N ISOTOPES OF DON AND NITRATE AT BATS
data of Altabet [1988]. However, Hansell and Carlson
[2001] have shown that, unlike DOC concentration,
[DON] at BATS is remarkably stable throughout the year
and not clearly different between the mixed layer and the
underlying thermocline. Thus, if DON is important in the
15
N budget for the upper 100 m, in the absence of a
concentration gradient, there would need to be a large
DON d15N difference between the surface and subsurface
waters.
[5] The low 15N/14N of NO
3 in the thermocline of the
North Atlantic and elsewhere in the subtropics has been
interpreted as a sign that N2 fixation is occurring at rates
sufficient to cause the accumulation of a sizable NO
3
excess in the low-latitude thermocline (relative to expect3
ations of NO
3 concentration based on PO4 concentration
and Redfield ratios [Liu et al., 1996; Brandes et al., 1998;
Karl et al., 2002]). However, Altabet’s [1988] measurements indicate that not only is N2 fixation not a dominant
source of new N at BATS, but the PNsink d15N at BATS is
too high to generate the observed 15N-depletion of the
15
thermocline NO
3 . That is, while the PNsink d N is essen 15
tially equal to the NO3 d N (3.7% and 3.5%, respectively
15
[Altabet, 1988]), the low NO
3 d N at BATS ultimately
requires a low-15N N source to lower its d15N from that of
deep ocean NO
3 (5%). One might suspect that the low
d15N of thermocline NO
3 at BATS results from preferential
remineralization of low-d15N N as the sinking flux transits
through the thermocline. However, the PNsink d15N from
sediment traps at different depths does not appear to support
this hypothesis, since the PNsink d15N does not typically
increase with depth [Saino and Hattori, 1987; Altabet,
1988], and often appears to decrease with increasing depth
[Altabet et al., 1991]. Thus we lack the direct mechanism by
15
which the BATS thermocline NO
3 develops its low d N.
This is disconcerting for our efforts to make use of the NO
3
d15N data as a complement to the NO
3 concentration to
PO3
4 concentration ratio data.
[6] The goal of this manuscript is to add total N (TN)
(specifically, DON, the dominant surface TN term) and a
new suite of NO
3 isotope measurements to the N isotope
data that already exist for BATS, in order to increase the
isotopic constraints on the N cycle there. Three questions
are of central importance in this regard: (1) Does the DON
d15N at BATS and its variability indicate dynamic behavior
in the DON pool not evident in the DON concentration data
alone, in particular, in response to N2 fixation or the DIC
drawdown? (2) How does the addition of the DON and new
NO
3 data change the N isotope budget constructed by
Altabet [1988]? Specifically, does the budget now require
significant inputs from a low-d15N N source such as N2
fixation? (3) Do TN measurements indicate a means for
transporting low-d15N N from the surface to the thermocline
that can lower the d15N of NO
3 in the thermocline at BATS?
2. Sampling Site and Sampling Protocol
[7] Samples were collected monthly at the BATS site,
31500N, 64100W, between March 2000 and May 2001 on
board the R/V Weatherbird II. Samples were typically
collected at depths of 0, 40 or 60, 100, 140 or 160, 200,
GB1018
250, 300, 400, 500, 600, 800, and 1000 m; the same
hydrocasts were sampled as part of the BATS core program.
Seawater samples were collected in 60-mL HDPE bottles
that had been soaked overnight in acid and rinsed with
deionized water. Each bottle was rinsed with sample water
three times before being filled. Samples were frozen at
20C until analysis. NO
3 concentration ([NO3 ]) and NO3
d15N measurements were made on samples collected between 0 and 1000 m. The concentration and d15N of TN was
measured on samples from 0 to 250 m, and also 300-m
samples for August 2000 and October 2000, when 250-m
samples were not collected.
3. Methods
3.1. Nitrate
[8] The sum of the concentration of NO
3 and nitrite
(NO
2 ) was measured by reduction to nitric oxide (NO) in
a heated solution of acidic V(III), followed by chemiluminescent detection of the NO [Braman and Hendrix, 1989].
With the exception of rare wintertime events [Lipschultz et
al., 1996, Lipschultz, 2001], NO
2 is typically scarce in the
water column at BATS and is thus subsequently disre15
garded. The NO
3 d N was analyzed by quantitative bacterial reduction of NO
3 to nitrous oxide (N2O) using a
strain of denitrifier that lacks N2O reductase activity,
followed by automated extraction, purification, and analysis
of the N2O product by an isotope ratio mass spectrometer
(the ‘‘denitrifier method’’ [Sigman et al., 2001; Casciotti et
al., 2002]). Individual analyses are referenced to injections
of N2O from a pure N2O gas cylinder and then standardized
using an internationally accepted NO
3 isotopic reference
material (IAEA-N3, with a d15N of 4.7% [Bohlke and
Coplen, 1995]). Replicate analyses are generally consistent
with a standard deviation for analysis of ±0.2%. One
difficulty with analysis of samples with 1 – 2 mM NO
3
was producing standards uncontaminated with NO
3 ; at this
low concentration, a 0.1 mM NO
3 background in the solvent
water can yield a substantial isotopic difference from the
NO
3 standard.
3.2. Total Dissolved N
[9] We describe here a new method for the natural
abundance-level isotopic analysis of total dissolved N
(TDN, the sum of NO
3 , NO2 , ammonium, and DON) in
seawater. The method is a straightforward coupling of the
commonly used ‘‘persulfate oxidation’’ method for TDN
conversion to NO
3 [Solorzano and Sharp, 1980; Bronk et
al., 2000] with the denitrifier method for the isotopic
analysis of NO
3 described above.
3.2.1. Oxidation of TDN to NO
3
[10] To oxidize TDN to NO
3 , 12 mL of sample is added
to a boro-silicate glass test tube with Teflon lined cap. To
this sample is added 2 mL of a persulfate oxidizing reagent
(POR), which is made up daily with 6 g certified ACS-grade
NaOH dissolved in 100 mL of deionized water (DIW),
followed by the addition of 6 g certified ACS-grade K2S2O8
(potassium persulfate) (POR recipe adapted from Solorzano
and Sharp [1980]). The potassium persulfate is recrystallized three times following a procedure described by
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Table 1. DON Standard Oxidation Yields
Compound
Glycine
Urea
ACA
EDTA
Antipyrine
Standard in DIWa
c
102 ± 14%
100 ± 7.5%
102 ± 6%
105 ± 0.5%
42 ± 2.3%
Standard in DIW (Others’)b
Standard in 0.5 M NaCl Solution
Standard in SW (Others’)b
92.9 – 98.5%
98.7 – 105.6%
N/R
N/R
45 – 68.1%
97 ± 5%
109 ± 25%
N/R
N/R
N/R
N/Rd
87.5 – 100%
N/R
N/R
N/R
a
Standard in DIW is as calculated from a 0.0, 2.5, 5.0, 7.5, and 10.0 mM concentration series of each standard, in DIW and SW
or 0.5M NaCl solution where applicable.
b
Standard in DIW is from Bronk et al. [2000, and references therein].
c
Reported values reflect the 95% confidence level.
d
N/R denotes: not reported.
Grasshoff et al. [1999]. Immediately after adding the POR
to the sample, screw caps are closed tightly, and samples are
autoclaved for 55 min on a slow vent setting. Finally, in
each batch of samples, three test tubes with 12 mL of the
POR are also autoclaved with the samples to determine the
concentration and d15N of the N contamination associated
with the POR (subsequently referred to as the ‘‘reagent
blank’’).
[11] After the samples cool, the sum of the NO
3 and NO2
concentration ([NO
]
+
[NO
])
of
the
samples
and
of
the
3
2
persulfate reagent is measured by chemiluminescence (fur
ther referred to as [NO
3 ], since [NO2 ] was typically
undetectable). For the samples, [TDN] is calculated by mass
balance by subtracting the [NO
3 ] of the autoclaved POR
]
of
the
post-oxidized
sample, taking dilution
from the [NO
3
into account. [DON] is calculated by subtracting the [NO
3]
of the seawater sample before the persulfate treatment from
the [TDN].
[12] We found it necessary to be vigilant about contamination at virtually every step and in the use of every
reagent, and to monitor the reagent/procedural blanks for
each ‘‘batch’’ of samples [Hopkinson et al., 1993]. Glassware was washed with copious DIW, then soaked in soap
and 10% HCl baths, and washed again with DIW after all
steps. All nonvolumetric glassware was then combusted at
450C for 4 hours. Volumetric glassware and black phenolic
screw caps with Teflon liners were rinsed in DIW, then in a
10% HCl bath overnight, rinsed with DIW again, and dried
at 50C. Our water deionization system includes an UV
lamp for oxidizing organic matter. Persulfate oxidation of
our DIW indicated that its [TDN] was typically 0.5 mM.
[13] In the surface waters of the Sargasso Sea and of many
other subtropical and tropical regions, dissolved inorganic
nitrogen (DIN, the sum of NO
3 , NO2 , and ammonium) is
sufficiently scarce to be considered absent for our purposes.
The [NO
3 ] + [NO2 ] for surface water samples (100 m)
collected for this study was consistently below the detection
limit of our methods (<0.1 mM), except in August 2000,
March 2001, and April 2001, when the concentrations of
some samples were between 0.1 and 1.0 mM. In shallow
thermocline waters (300 m), where the [NO
3 ] is typically
less than the [DON], dual analyses of TDN and NO
3 allow
the concentration and isotopic composition of DON to be
calculated by mass balance.
[14] Since none of the BATS samples were filtered, the
concentration and isotopic analyses of ‘‘TDN’’ also include
suspended particulate organic N (PNsusp) and are thus
truly TN measurements. Because PNsusp concentrations
([PNsusp]) represent <10% of the total N concentration
([TN]) at BATS [Michaels et al., 1994b; Michaels and
Knap, 1996], we often refer to our analyses as being of
TDN (or of DON in the surface). Nevertheless, it will be
suggested in section 5.4 that variations in [PNsusp] and
PNsusp d15N with depth are apparent in our data, and we
are careful to use the term ‘‘TN’’ (rather than ‘‘TDN’’)
when PNsusp is relevant. In the upper 100 m, where [DIN] is
extremely low, TN is essentially equivalent to total organic
nitrogen (‘‘TON,’’ the sum of DON + PNsusp). In the
subsurface, the concentration and d15N of TON is estimated
by differencing NO
3 from TN, as would be done to
estimate DON from TDN and NO
3 measurements in
samples devoid of particles.
3.2.2. TDN D15N Analysis
[15] After the oxidation of TDN to NO
3 , the samples and
aliquots of autoclaved POR were converted to N2O using
the denitrifier method [Sigman et al., 2001; Casciotti et al.,
2002]. Because the POR that is added to the DON sample
creates an extremely alkaline solution, the pH of the sample
must be lowered into a range that is suitable for bacterial
conversion by adding 6 N HCl (made from certified ACSgrade HCl) until the sample pH is between 3 and 6. The
volume of sample aliquots for bacterial reduction to N2O
was adjusted to yield either 10 or 20 nmols N. The bacterial
medium was prepared with three times the amount of
potassium phosphate buffer described by Sigman et al.
[2001] to provide extra buffering of the medium pH, given
the high normalities of NaOH and HCl involved in the
oxidation protocol.
[16] As with the concentration measurements, mass balance calculations are required to (1) correct for the effect
of the POR blank on the sample TDN d15N and (2) extract
the DON d15N from the measurements of TDN d15N and
15
NO
3 d N in a given sample. These calculations require
concentration and d15N analysis of (1) the NO
3 in the
unoxidized sample, when it is measurable, (2) the POR
alone, and (3) the oxidized sample.
3.2.3. DON Oxidation Efficiency
[17] Table 1 lists the degree of completeness to which
common DON standards were oxidized to NO
3 in both
DIW and seawater (‘‘SW’’) and/or 0.5 M NaCl solution
using persulfate oxidation and compares our results with
other published values. Standards include: glycine, urea,
6-amino caproic acid (ACA), EDTA, and 4-aminoantipyrine
(Sigma part A1-4328), most of which were selected for
3 of 15
GB1018
comparison because they have been used previously for
testing the efficacy of [DON] methods [Bronk et al., 2000,
and references therein]. In all cases, recoveries of DON
standard as NO
3 were equal to or greater than other
published values, with the exception of 4-aminoantipyrine,
a cyclic compound with three nitrogen atoms, which has
been noted previously as a difficult compound to oxidize
(Table 1) [Walsh, 1989; Hopkinson et al., 1993; Bronk et
al., 2000]. Differences in reported oxidation efficiencies of
4-aminoantipyrine may result from the use of different
compounds marketed under the common name ‘‘antipy-
GB1018
rine’’ (e.g., compare work of Suzuki et al. [1985] with that
of Bronk et al. [2000]).
3.2.4. DON D15N Precision and Accuracy
[18] The ability of the persulfate and denitrifier methods
to yield precise and accurate DON d15N data was tested
by measuring the d15N of the model compounds glycine
and ACA at a range of concentrations (0 –10 mM) in
deionized water and/or 0.5M NaCl solution. The isotopic
composition of these model compounds was determined
by direct combustion and isotopic analysis of the undiluted compound salts in the laboratory of M. A. Altabet,
yielding d15N values of 9.6% and 4.6% for the glycine
and ACA standards, respectively. In the tests reported
here, the measured d15N follows the expected trend with
compound concentration when the reagent blank is taken
into account (Figure 1). The agreement between the
measured and calculated TDN d15N indicates that (1) the
oxidation of these model compounds is adequately complete and/or nonfractionating (preventing significant preferential loss of either the light or heavy isotope during
the oxidation), (2) the blank in this method is due almost
entirely to N in the POR, and (3) the reagent blank is
constant and characterizable within a set of oxidations.
Figure 1. Accuracy of DON d15N method tested using
laboratory standards and reagent blank characterization.
(a) The d15N versus N concentration ([N]) added for ACA
concentration series (0, 2.5, 5, 7.5, and 10 mM) in DIW, with
ACA d15N = 4.6% (dashed line). Curved line represents the
calculated TDN d15N using the known ACA and measured
[N] and d15N of the POR (0.3 mM when diluted by sample,
and 3.6%, respectively). That the measured TDN d15N
(pluses) fall along the expected TDN d15N trend indicates
that (1) the oxidation of these model compounds is complete
and/or nonfractionating, (2) the ‘‘blank’’ in this method is
largely N in the POR, and (3) the concentration and isotopic
value of the blank is constant and accurately characterized.
(b) d15N versus [N] added for filtered seawater amended
with a concentration series (0, 2.5, 5, 7.5, 10 mM) of glycine,
d15N = 9.6% (dashed line). Curved line represents TDN
d15N calculated as above (blank [N] when diluted by
sample = 0.4 mM, blank d15N = 1.1%). The measured TDN
d15N (asterisks) fall along the calculated TDN d15N line. The
background N in this experiment includes N in the POR as
well as natural DON in the filtered seawater matrix. (c) The
d15N versus quantity of POR added (as a factor of the
standard POR addition) for analyses of 5 mM ACA in DIW.
The solid and dashed lines represent POR d15N (6.7%)
and ACA d15N (4.6%), respectively. Crosses are the
measured TDN d15N, and open circles represent the
corrected d15N after correction for the reagent blank. As
the quantity of the reagent is increased, the d15N approaches
the d15N of the reagent. It should be noted that the [N] of the
POR in this experiment, 9.1 mM in the undiluted reagent, is a
value typically four times greater than any POR used to
generate the BATS data shown in Figures 2 and 3. The large
isotopic difference between the sample and POR also
contributed to the large blank correction in this experiment.
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Table 2. Replicate BATS DON Analyses
Depth, m
Feb. 2001
July 2000
July 2000
Jan. 2001
Mediane
100
60
0
100
[DON], mM
3.80, 3.79
4.95, 4.37
4.28, 4.32
4.13, 4.08, 4.20
Standard Deviation [DON], mM
a
0.01
0.41b
0.02
0.06
0.2
DON d15N, %
Standard Deviation DON d15N, %
n
3.35, 3.57
3.59, 3.63
4.30, 4.32
4.68, 4.08, 3.91
0.16
0.03
0.01c
0.41d
0.2
2
2
2
3
a
Smallest standard deviation for [DON] for entire data set was measured on February 2001 100-m samples.
Largest standard deviation for [DON] for entire data set was measured on July 2000 60-m samples.
c
Smallest standard deviation for DON d15N for entire data set was measured on July 2000 0-m samples.
d
Largest standard deviation for DON d15N for entire data set was measured on January 2001 100-m samples.
e
Median standard deviations for entire data set are listed for both [DON] and DON d15N analyses.
b
It must be stressed that the POR [N] and d15N may vary
from one ‘‘batch’’ of reagent to another (Figure 1), so
that the [N] and d15N of the reagent blank must be
measured with each batch of oxidations.
[19] Since the tests shown in Figure 1 were conducted,
significant improvements have been made in blank reduction, blank characterization, and the mechanics of the
oxidation step, and these were in place for all of the BATS
measurements reported below. Previously, the reagent blank
[N] was occasionally very high (as in Figure 1c, for which
the POR [N] was 9.1 mM), necessitating a large blank
correction. Subsequently, the POR [N] has been reduced
during the recrystallization process by drying the potassium
persulfate crystals in the presence of indicating Drierite in a
vacuum dessicator. Since this step was taken, no data have
been generated using a POR with a [N] > 4 mM. Currently,
the POR [N] ranges from 1.2 to 2.5 mM (effective concentration of 0.2 – 0.4 mM when diluted by the sample), and the
POR d15N ranges between 14 and 10%. The oxidation
step has been improved by using 15 mL test tubes (Fisher
part 14-933-1A) which reliably maintain a seal during
autoclaving. During the tests run in Figure 1, test tubes of
different sizes and brands would occasionally leak or vent
during the autoclaving step, causing ‘‘fliers’’ in [DON]
and/or DON d15N.
[20] The reproducibility of the DON d15N method can be
assessed by repeatedly analyzing a given seawater sample.
Shown in Table 2 are the results of replicate [DON] and
DON d15N analyses of the four BATS samples that yield
the smallest and largest standard deviations for all of the
[DON] and DON d15N measurements reported here.
Additionally, the median [DON] and DON d15N standard
deviations for all analyses made on samples collected at
one depth are reported in Table 2. Frequently, standard
deviations include not only replicate analyses on a single
sample but also analyses of duplicate samples collected at
the same depth.
[21] Our [DON] measurements for BATS surface (upper
100 m) samples are within the same range as those
previously made at the same site by UV oxidation; our
typical range is from 3.8 to 5.0 mM, with total range of
3.4 – 5.9 mM, whereas Hansell and Carlson [2001] report
a concentration range of 4 – 5.5 mM TON. Although there
is no external check for the [DON] or DON d15N of the
samples reported here, a low median standard deviation
for replicate [DON] and DON d15N analyses of a single
sample (0.2 mM and 0.2%, respectively, Table 2) suggests
that oxidation is complete and/or nonfractionating and
thus yields an accurate assessment of the DON d15N of
these seawater samples.
4. Results
4.1. Nitrate
15
[22] At depths of 800 m, NO
3 d N is close to the
nominal mean ocean value of 5% [Sigman et al., 2000]
and decreases upward from 800 m to 2.5% at 250 m,
while the [NO
3 ] decreases from 22 mM at 800 m to
15
2.5 mM at 250 m (Figures 2 and 3). At 250 m, NO
3 d N
appears to change gradually during the sampling period.
15
NO
3 d N is typically <2.5% from May through August of
2000 and >2.5% from November 2000 through February
2001 (Figures 2 – 4). This variation appears to be associated
with a slight increase in [NO
3 ] at the same depth (Figures 2 –
4). [NO
3 ] decreases to <0.1 mM typically by or somewhat
below 100 m. In many of the shallow sections of the
15
profiles, there is also evidence for an increase in NO
3 d N
from 200 m to 100 m, presumably due to isotope
fractionation during NO
3 assimilation by phytoplankton.
15
Generally, there is more variability in the NO
3 d N near
the top of the water column, which is to be expected from
the large relative variations in [NO
3 ] at this depth and
from the various degrees to which the isotope effect of
NO
3 assimilation will be expressed when NO3 supply and
NO3 consumption are occurring in a complex sequence.
4.2. TN Concentration and D15N
[23] As also observed by Hansell and Carlson [2001], the
range of measured [TON] in the upper 100 m reported here
is quite limited, with a mean concentration of 4.2 mM and
standard deviation for the entire data set in the upper 100 m
of 0.5 mM, with no distinct seasonal or depth related trends
(Table 3, Figures 2 – 4). The TON d15N in the upper 100 m
ranges from 3.5 to 4.5%, and, as with [TON], no seasonal
trends in TON d15N are recognized (Figures 2 – 4). The only
sample deviating from these values is the 0-m January 2001
sample, with a [TON] of 6 mM and TON d15N of 2%.
This sample yielded similar values when it was analyzed
four times, twice each from two sample bottles collected
from the same hydrocast bottle, indicating that the values
are real, although perhaps the result of hydrocast bottle
contamination.
[24] The annually averaged [TON] decreases by 0.25 mM
from 100 to 250 m, with an average [TON] concentration of
5 of 15
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15
Figure 2. At BATS, (a) [NO
3 ] (pluses) and [TON] (solid triangles) and (b) NO3 d N (pluses) and TON
data
are
from
March
2000
through
May
2001.
TON
data are from June 2000
d15N (solid triangles). NO
3
through May 2001. Altabet’s [1988] PNsusp d15N (0.2%) (solid circle) and PNsink d15N (3.7%) (solid
circle) are shown for reference. Replicate analyses were performed on individual samples. In addition, at
roughly half the sampling depths, replicate samples were collected. Plotted values are averages of all
analyses at a given depth. See color version of this figure at back of this issue.
3.93 mM at 250 m (0.38 mM, 1 SD) (Table 3). The KruskalWallis test for nonparametric data [Desu and Raghavarao,
2003] indicates that the difference in [TON] between the
upper 100 m and 250 m is significant beyond the 99% level.
Between 100 and 250 m, the TON d15N increases slightly
but significantly from a mean of 3.9% between 0 and 100 m
to a mean of 4.1% at 250 m (0.39%, 1 SD). The KruskalWallis test for nonparametric data indicates that the 0 –
100 m and 250 m TON d15N data are significantly different
from each other above the 93% confidence level.
[25] While there are no other bulk marine DON/TON
d15N values in the literature that can be directly compared
with those reported here, we can compare our results with
those of related analyses. Benner et al. [1997] report a d15N
of 6.6% for ultrafiltered DON from surface waters at BATS,
which is higher than our average surface water bulk DON
d15N of 3.9%. The ultrafiltered material generally represents 30% of total DON in open ocean waters, implying that
the remaining smaller size fractions of DON have a d15N of
roughly 2.7%. Feuerstein et al. [1997] report bulk DON
d15N of 1.2 to +5.8% in Great Lakes water, interestingly
different from our marine data. In a meeting abstract, Abell
et al. [1999] report bulk DON d15N of 1 – 2% for surface
water samples from the Pacific Ocean, lower than the results
reported here.
5. Discussion
5.1. Stability of DON D15N at BATS
[26] The lack of seasonal change in [TON] in the upper
100 m at BATS, observed previously by Hansell and
Carlson [2001], implies that the bulk surface DON pool
6 of 15
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15
Figure 3. (a) [TON], (b) TON d15N, (c) [NO
3 ], and (d) NO3 d N at BATS from June 2000 through
May 2001. See color version of this figure at back of this issue.
is not dynamic on the timescale of seasons. However, this
measure, by itself, leaves open the possibility that there
are large but balanced inputs to and losses from the
surface DON pool. The stability of the DON d15N in the
upper 100 m at BATS observed in this study argues
against this possibility, in that large inputs and outputs
would need to be isotopically balanced. For instance, if
1 mM N were simultaneously added to and removed from
a 4 mM DON pool, and the d15N of the DON removed
was 3% lower than the DON added, then the d15N of the
DON pool would increase by 0.75%. Although we
cannot rule out the possibility of similar isotope compositions for DON that is produced and consumed, no
processes or feedbacks have yet been described that
would maintain this isotopic similarity for DON inputs
and outputs. Thus we infer from the isotopic stability that
the rates of input and output are small.
[27] The refractory nature of DON revealed by its
isotopic stability is perhaps expected given the previous
[DON] measurements [Hansell and Carlson, 2001], as
well as recent bulk dissolved organic matter (DOM) D14C
age measurements [Loh et al., 2004]. If DON is present
and stable at such relatively high concentrations at BATS,
it is hard to imagine that much of it could be labile,
unless the system was limited by something else (e.g., P).
Of course, while the stability of both its concentration
and d15N makes a strong case for the generally recalcitrant nature of the bulk DON at BATS, it does not rule
out the possibility of large inputs of a class of DON that
is immediately and completely consumed. Ammonium is
a good example of such dynamic behavior: It is an
important N source for phytoplankton at BATS, but its
concentration is so low that it is not important in our TN
analyses [Lipschultz, 2001].
[28] The static behavior of DON at BATS represents a
constraint on the potential N sources for the biological
removal of DIC during the summer at BATS. A 2.5 mM
N decrease is required to fuel the observed DIC removal
over the upper 150 m from April through December
[Michaels et al., 1994a], and calculations for the DIC
removal restricted to the upper 25 m require as much as
6 mM N to remove the commensurate DIC, taking into
account additional DIC sources such as advection
[Gruber et al., 1998]. Since there is no appreciable
DIN in the surface waters or identified fluxes of DIN
that could be immediately consumed during this time
period [Lipschultz, 2001], we consider other sources of N.
While it is clear that some phytoplankton can use certain
kinds of DON as a primary N source [Palenik and Morel,
1990; Capone et al., 1994; Mullholland et al., 2002;
Berman and Bronk, 2003], no changes in [DON] of this
magnitude are observed at BATS. The lack of change in
[DON] through the summer season would thus require
that N must be added to the DON reservoir to offset this
loss if DON were fueling the DIC drawdown. The
isotope data provide the additional constraint that the N
added to and removed from the DON pool would need to
have very similar d15N. Revisiting the calculation above,
if 2.5 mM DON is consumed from the bulk 4 mM DON
pool and the DON consumed is 3% higher than the
7 of 15
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Figure 4. (top) The d15N and (bottom) [N] versus month (June 2000 through May 2001). Top panel
shows 0 m TON d15N (solid triangles), 40 m TON d15N (solid diamonds), 100 m TON d15N (solid
15
squares), and 250 m NO
3 d N (solid circles). Bottom panel shows 0 m [TON] (open triangles), 40 m
[TON] (open diamonds), 100 m [TON] (open squares), and 250 m [NO
3 ] (open circles). See color
version of this figure in the HTML.
2.5 mM DON that must be simultaneously added back to
the DON pool, then the bulk DON d15N would decrease
from 4% to 2%, which is clearly not observed.
[29] N2 fixation is considered a plausible explanation
for the summertime DIC drawdown at BATS [Bates et
al., 1996; Gruber et al., 1998]. Again, it would seem
unlikely for a hypothetical input of N to the DON pool
via N2 fixation, with its presumably low-15N N, to have
the resulting change in d15N of the bulk DON pool be
closely balanced by DON consumption. While fractionation during degradation/consumption of DON is certainly
possible [Macko et al., 1987; Waser et al., 1998], it
would be highly fortuitous if it led to loss of N with
the same d15N as the input of DON from N2 fixers. Thus,
if N2 fixation is responsible for the summertime drawdown of DIC, it seems unlikely that it is supplying the
needed N through the bulk DON pool. Given earlier
evidence for DON production by N2 fixers [Karl et al.,
1992; Capone et al., 1994; Glibert and Bronk, 1994], we
are left with three possible conclusions: (1) The fraction
of DON produced by N2 fixers at BATS is completely
consumed as rapidly as it is produced, so that it never
becomes a significant fraction of the resident TON pool
measurable by monthly sampling, although it would
appear in the N exported from the surface (see below);
(2) the link between N2 fixation and DON production
observed elsewhere [Karl et al., 1992; Capone et al.,
1994; Glibert and Bronk, 1994] does not apply at BATS;
Table 3. Average Concentration and d15N for NO
3 , TN, and TON
at BATS
N Pool
Depth,
m
Average
[N], mM
Standard
Deviation
[N],a mM
Average
d15N, %
Standard
Deviation
d15N, %
TN
TN
NO3
TONb
0 – 100
250
250
250
4.18
6.32
2.39
3.93
0.51
0.52
0.57
0.38
3.92
3.57
2.65
4.13
0.48
0.24
0.32
0.39
a
Standard deviations apply to entire suite of measurements made on
samples collected between June 2000 and May 2001.
b
TON is calculated by differencing NO
3 from TN.
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Table 4. Estimated N Fluxes to Surface Watersa
N Source
Flux, mol N m2 yr1
Wet and dry deposition
NO
3 from below
N2 fixation
N2 fixation
N2 fixation
N flux required for DIC drawdown
0.01
0.84
0.015
0.045
0.072
0.16c
0.25d
0.33e
N2 Fix/Sum New Nb
Reference
2%
5%
8%
16%
22%
28%
Prospero et al. [1996]
Jenkins and Doney [2003]
Orcutt et al. [2001]
Hansell et al. [2004]
Gruber and Sarmiento [1997]
Gruber et al. [1998]
Ono et al. [2001]
Michaels et al. [1994a]
a
Table is after Lipschultz et al. [2002].
Estimates of N2 fixation are divided by the sum of the estimate of the flux of NO
3 from below from Jenkins and Doney [2003] and the
estimate of N2 fixation being considered.
c
Amount of N required to close the DIC imbalance takes the DIC drawdown of 40 mmol/kg that occurs over the upper 25 from 9 May to 16
October [Gruber et al., 1998]. This N flux is calculated by converting the units to mol C m2, and dividing by the Redfield C/N ratio of 6.6.
This value assumes DIC is only removed during the 160-day period considered, and thus does not multiply by (365/160), so the flux is not
‘‘per year,’’ but ‘‘per period.’’
d
Value denotes amount of N required to account for the Ono et al. [2001] estimate of shallow remineralization (between 100 and 250 m) of
1.53 mol C m2 period1 (240 days) at BATS, calculated by dividing the C flux term by the Redfield C/N ratio of 6.6. This value only refers
to the time period considered (April 16 through December 12), and is thus not an annual rate.
e
Value denotes amount of N required to fuel a DIC drawdown of 2.16 mol C m2 from April to December [Michaels et al., 1994a]. The N
flux is calculated by dividing the DIC value by 6.6 and represents a flux ‘‘per period’’ (275 days).
b
or (3) N2 fixation rates are not sufficient to fuel the DIC
drawdown at BATS, as suggested by in situ measurements
[Orcutt et al., 2001] (Table 4).
5.2. Absolute Values of [DON] and DON D15N
[30] As is observed elsewhere in the oligotrophic open
ocean [Abell et al., 2000], DON constitutes the largest pool
of fixed N at BATS. Because of the high concentration of
this N pool in this otherwise N-scarce region, determining
the d15N of the bulk DON pool has been a priority for
researchers [Druffel and Williams, 1992; Capone, 2001].
The data in Figure 2 show that at BATS, TON d15N is
higher than the d15N of all other N pools and fluxes at
BATS. Altabet’s [1988] measurements of the average PNsusp
d15N (0.2%) and of the average PNsink d15N (3.7%) are
lower than the average TON d15N in the upper 100 m and at
250 m (3.9% and 4.1%, respectively, Table 3). Moreover,
the TON d15N is higher than the d15N of NO
3 in the shallow
thermocline (2 – 3% at 250 m).
[31] More work will be required before we understand the
processes that set the absolute values of [DON] and DON
d15N, both in the surface and subsurface. One useful albeit
overly simplistic hypothesis is that there is little fractionation in the production or consumption of DON, so that
newly produced DON is essentially isotopically identical to
the new N supplied to the surface ocean. Even in this
simplest case, the interpretation of the bulk surface DON
d15N is complicated by the observation that at BATS, there
is only a small vertical gradient in [DON] between the upper
100 m and 250 m, which implies that a large fraction of the
surface DON is simply DON that is mixed up from below. If
most of the surface DON in the open ocean comes from the
upward mixing of subsurface DON, then the surface DON
d15N will have a long response time to surface processes
and/or changes in the N cycle.
[32] Since very little work has been done on DON d15N
[Feuerstein et al., 1997; Benner et al., 1997; Abell et al.,
1999] or on the isotope dynamics of DON transformations
[Macko et al., 1987; Hoch et al., 1996; Waser et al., 1998],
we cannot at this point rule out the possibility of multiple
isotope fractionations associated with DON production and
consumption; indeed, we expect them. Given this situation,
it remains unclear what the absolute d15N of surface or
subsurface DON signifies when considered in isolation, as
has been pointed out previously for other forms of organic
N [Altabet, 1988; Karl et al., 2002]. Consequently, we focus
on what can be learned by including DON in the N isotope
budget at BATS, which does not require such an understanding of DON isotope systematics.
5.3. N Isotope Budget at BATS
[33] Building on the conceptual model of Altabet
[1988], we develop an isotope budget for the upper
100 m at BATS that includes only N fluxes from the
underlying thermocline. This represents a test of the
premise that N2 fixation is not a prominent fraction of
the new N supplied to the euphotic zone at BATS
(Figure 5, Table 4). The 0 – 100 m depth interval is
examined because it is the typical euphotic zone depth
throughout the year at BATS, and is thus the depth range
over which the DON pool should be the most dynamic.
A typical maximum winter mixed layer depth is 250 m
[Michaels et al., 1994b; Michaels and Knap, 1996;
Hansell and Carlson, 2001], and is thus a reasonable
choice to represent the TN flux up from below. The
vertical (one-dimensional) N mass and isotope budget
includes the following three fluxes: TN mass flux up
from 250 m, TN mass flux down from the upper 100 m
to 250 m, and the PNsink flux, represented by the terms
ND, NS, and P, respectively, in equation (1), where Q is
the flux term representing the annually averaged vertical
exchange of water,
ðND NS Þ Q ¼ P:
ð1Þ
Note that ND and NS represent TN concentrations and thus
include NO
3 , DON, and PNsusp. A similar equation can be
9 of 15
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Figure 5. N fluxes and transformations at BATS. The water column is divided into the upper 100 m and
100– 250 m. Of the 6.3 mM TN pool with d15N = 3.6% at 250 m, 3.9 mM is a recalcitrant DON pool with
d15N = 4.1% and cycles essentially unchanged between upper 100 m and 250 m. The remaining 2.4 mM
15
of the 250-m TN pool is NO
3 (d N = 2.8%), which mixes up to the surface waters, where it is
completely consumed, and then partitioned into two fluxes. The larger of the two N fluxes out of the
upper 100 m is PNsink, which has a calculated d15N of 3.0%, based on the values reported here. Altabet’s
[1988] measured d15N for PNsink (3.7%) is also shown for reference. The other, much smaller N flux out
of the upper 100 m is from a labile TON pool (0.25 mM, 1.3%) that is respired back to NO
3 at 250 m
after being mixed out of the upper 100 m; this flux is most likely composed of PNsusp (see text).
applied to the 15N budget at BATS, approximated here using
d15N rather than 15N/14N,
d15 ND ND d15 NS NS Q ¼ d15 NP P;
ð2Þ
where d15ND, d15NS, and d15NP represent the d15N of TN at
250 m, the d15N of TN in the upper 100 m, and the d15N
of PNsink, respectively. Substituting equation (1) into
equation (2) yields the following expression for PNsink d15N:
d15 NP ¼
d15 ND ND d15 NS NS =ðND NS Þ:
ð3Þ
[34] From June 2000 through May 2001, the average
[TN] between 0 and 100 m is 4.2 mM with a standard
deviation of 0.5 mM, and the average TN d15N is 3.9% with
a standard deviation of 0.5% (Table 3). The average [TN]
from June 2000 through May 2001 at 250 m is 6.3 mM with
a standard deviation of 0.5 mM, and the average d15N of TN
is 3.6%, with a standard deviation of 0.2% (Table 3). The
standard deviations for these annual averages include not
only errors associated with the measurements, but also any
systematic (e.g., seasonal) changes that may occur. From
equation (3), we calculate an expected PNsink d15N of 3.0%.
This is somewhat lower than, although perhaps not significantly different from, the annually averaged d15N of 3.7%
measured for sediment trap materials collected at 100 m
at BATS in 1986 [Altabet, 1988] (Figure 2). From this
calculation, we see that our isotope budget requires no
additional input of low-d15N new N to the surface water at
BATS beyond the terms included in this budget. If N2
fixation were an important source of new N at BATS, our
calculated PNsink d15N should be greater than the measured
value to reflect a missing N source with a low d15N not
included in our fixed N balance. Thus our work supports
the conclusions of Altabet’s [1988] budget that the NO
3
flux up from below is the dominant source of new N at
BATS and that N2 fixation is too small an input to be
apparent in the isotope budget. That our budget yields an
estimated PNsink d15N that is less than the measured value
of 3.7% results from the d15N of the NO
3 that we measure
at 250 m (a component of the 250 m TN term), which is
1% lower than that reported by Altabet [1988].
[35] The result of our budget is consistent with in situ
measurements of N2 fixation at BATS [Orcutt et al., 2001],
which, when compared with flux estimates of NO
3 to the
mixed layer, are on the order of 2% of new N on an annual
basis (Table 4). The higher geochemical estimates of N2
fixation [i.e., Gruber and Sarmiento, 1997] represent 8%
of the new N flux to BATS (Table 4), which might also be
difficult to resolve within the surface ocean N isotope
budget described here. For example, assuming a d15N of
1% for newly fixed N [Minigawa and Wada, 1986;
Carpenter et al., 1997], an 8% contribution from N2
fixation to the annual N budget would lower the PNsink
10 of 15
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d15N by 0.3% relative to the PNsink d15N that one
estimates on the basis of N supply solely from NO
3 from
below. Since the fluxes of NO
3 from below and N2 fixation
should be temporally separated at BATS [Michaels et al.,
1994a; Bates et al., 1996; Lipschultz et al., 2002], the
seasonal invariance of [DON] and DON d15N may be more
sensitive indicators of the limited importance of N2 fixation
than the annually integrated N isotope budget described in
this section. However, this only applies if N2 fixation
measurably augments the DON pool (see section 5.1).
[36] Returning to the summertime DIC drawdown at
BATS, we consider what corresponding fluxes of N, in
the form of N2 fixation, are required to account for the DIC
removal. Gruber et al. [1998] estimate that 40 mmol C kg1
are removed between April and October in the upper 25 m
at BATS. On the basis of the Redfield ratio N equivalent,
this magnitude of C removal requires a N2 fixation flux of
0.16 mol N m2 between April and October. This is roughly
16% of the new N on an annual basis at BATS, is an order
of magnitude greater than in situ N2 fixation flux measurements made at BATS [Orcutt et al., 2001], and is more than
twice the geochemical N2 fixation estimate of Gruber and
Sarmiento [1997] (Table 4). Nevertheless, the amount of N2
fixation required to resolve the Gruber et al. [1998] DIC
imbalance may be a minimum estimate, for two reasons.
First, N2 fixers may have a lower C:N than Redfield ratio
values [McCarthy and Carpenter, 1979; Carpenter et al.,
2004], thus requiring a larger flux of N than that calculated
in Table 4 to remove a given amount of DIC. Second,
Gruber et al. [1998] consider DIC removal over only the
upper 25 m, even though the mixed layer is often deeper
than 25 m throughout that time period, and DIC may be
removed below this depth during portions of the time period
considered [Michaels et al., 1996]. Ono et al. [2001]
estimate a C flux of 1.53 mol C m2 period1 (240 days)
between 100 and 250 m from April to December at BATS,
which, when applying Redfield C/N ratios, corresponds to a
N flux of 0.25 mol N m2 period1 (Table 4). This estimate,
which is greater than that of Gruber et al. [1998], may also
represent a lower bound, as it does not detect any exported
N that sinks below 250 m.
[37] An upper estimate of the amount N2 fixation contributes to new production at BATS is provided by the
DIC drawdown estimate of Michaels et al. [1994a] of
2.16 mol C m2 period1, which is calculated over the
upper 150 m from April through December. This amount
of DIC removal requires a N2 fixation flux of 0.33 mol
N m2 period1, equal to almost 40% of the NO
3 flux up
from below (Table 4), which was clearly not detected in
the isotope budget.
[38] In summary, given that the various DIC drawdown
estimates require a N input equivalent to 20% of the
annual new N supply at BATS, a N2 fixation flux large
enough to reconcile the summertime DIC drawdown
appears to be inconsistent with the N isotope budget results
presented here, unless extreme deviations from Redfield
C:N ratios for export production are invoked [Orcutt et al.,
2001; Anderson and Pondaven, 2003]. This sense of deviation is not apparent in sinking particles collected at BATS
[Conte et al., 2001] or in the C:N ratios of N2 fixers
GB1018
[McCarthy and Carpenter, 1979; Carpenter et al., 2004].
The differences in the mean concentrations and seasonal
cycles of DOC and DON imply a non-Redfieldian stoichiometry for summertime DOM at BATS [Hansell and
Carlson, 2001], and previous work has shown that DOC
is effectively exported from the euphotic zone during winter
mixing [Carlson et al., 1994]. However, this C loss term is
not adequately large to explain the C drawdown at BATS
[Michaels et al., 1994a].
15
5.4. Organic N and NO
3 D N at BATS
15
[39] The low NO3 d N in the thermocline of the Sargasso
Sea and other subtropical regions should be relevant for
understanding the oligotrophic N cycle. As mentioned
previously, the low d15N of NO
3 in the thermocline, both
at BATS and elsewhere, has been interpreted as evidence of
N2 fixation [Liu et al., 1996; Brandes et al., 1998; Karl et
15
al., 2002]. Working to increase the low NO
3 d N in the
thermocline are both diapycnal and isopycnal mixing, which
15
introduce NO
3 into the thermocline with a d N close to
5%, typical of NO3 in the deep ocean [Sigman et al.,
2000]. Thus we expect a significant source of low-d15N N to
the BATS thermocline, such that a combination of this N
15
source and the input of deep ocean NO
3 yield a NO3 d N
15
of 2 – 3% at 250 m depth. However, the d N of shallow
PNsink measured at BATS is 3.7% [Altabet, 1988], which is
somewhat greater than (not less than) the d15N of NO
3 in the
shallow subsurface at BATS. Moreover, data from sediment
traps at multiple depths show no evidence for preferential
remineralization of 14N as the sinking flux passes through
the water column [Saino and Hattori, 1987; Altabet, 1988;
Altabet et al., 1991]. Thus, sinking N and its remineralization do not appear to represent the vehicle by which the lowd15N N is accumulated in the shallow thermocline.
[40] Given the large isotope effect of nitrification (15–
35% [Mariotti et al., 1981; Casciotti et al., 2003]), it has
been postulated that nitrification can significantly lower the
d15N of NO
3 in subsurface ocean waters [Sutka et al.,
2004]. However, nitrification can only have this effect if the
ammonium (NH+4 ) released into the thermocline does not
+
eventually end up in the thermocline NO
3 pool. NH4
concentrations are low throughout the year at BATS
(30 nM), indicating that direct NH+4 export by circulation
is not significant [Lipschultz, 2001]. Assimilation of NH+4 is
perhaps the most feasible means for nitrification to affect
15
15
N-rich
the NO
3 d N, by providing a pathway by which
+
NH4 could be diverted away from nitrification. However,
the organic N resulting from this assimilation of NH+4 must
be exported from the shallow thermocline; otherwise, this
putative high-15N organic N would eventually end up in the
thermocline NO
3 pool. As mentioned above, there is no
evidence for the creation or export of high d15N particles
from the thermocline. While we cannot rule out the loss of
15
N-rich DON from the thermocline to the deep ocean, this
seems unlikely. Moreover, NH+4 assimilation by microbes
may have an isotope effect comparable to that of nitrification, as is the case for assimilation by eukaryotic phytoplankton [Waser et al., 1998], in which case, NH +4
assimilation would not even represent a possible conduit
for the escape of high-d15N N from conversion to NO
3.
11 of 15
GB1018
Thus it seems safe to conclude that nitrification is not an
15
important driver of the low NO
3 d N observed at BATS.
[41] Here we consider the implications of our TN measurements for the missing source of the low-d15N NO
3 in
the thermocline at BATS, focusing on our observation of
differences between the TON (calculated by differencing the
TN and NO
3 measurements) in the upper 100 m (‘‘surface’’) and at 250 m. The difference in [TON] between the
surface and at 250 m describes the amount of TON
generated in the upper 100 m (subsequently referred to as
‘‘semi-labile’’) that is eventually mixed downward and
respired back to NO
3 at 250 m,
½TONS ½TOND ¼ NO
3 remin ;
ð4Þ
where the average [TON] in the upper 100 m, the average
[TON] at 250 m, and the [NO
3 ] at 250 m resulting from
remineralization of the downward-mixed surface TON are
represented by the terms [TONS], [TOND], and [NO
3 remin],
respectively. The same expression can be written for 15N,
again approximating 15N/14N as d15N,
d15 N-TONS ½TONS d15 N-TOND ½TOND ¼ d15 N-NO
3 remin NO3 remin ;
ð5Þ
where d15N-TONS is the d15N of TON in upper 100 m,
d15N-TOND is the d15N of TON at 250 m, and d15N15
NO
3 remin is the d N of the component of surface TON
that is remineralized to NO
3 at 250 m. Substitution of
equation (4) into equation (5) yields an expression for
the d15N of the remineralized semi-labile surface TON,
15
15
d15 N-NO
3 remin ¼ ðd N-TONS ½TONS d N-TOND
½TOND Þ=ð½TOND ½TONS Þ:
ð6Þ
[42] [TONS] and [TOND] are 4.18 mM and 3.93 mM,
respectively, which indicates a net addition of 0.25 mM
TON in the upper 100 m (Table 3, Figure 5). The d15N of
TON in the upper 100 m and at 250 m are 3.9% and 4.1%,
respectively (Table 3, Figure 5). This indicates that the
0.25 mM TON added in the upper 100 m has an isotopic
composition of 1.3% (Figure 5). Upon the annual vertical
mixing of this upper 100 m TON pool down into the
subsurface (250 m), the TON added in the upper 100 m
is respired back to NO
3 (equation (4)), adding 0.25 mM
15
NO
3 with a d N of 1.3% to the thermocline. Thus the
annual net production of low-d15N TON in the upper 100 m
and subsequent oxidation of this semi-labile TON in the
subsurface appears to provide a vehicle for the transport of
low-d15N N into the subsurface at BATS.
[43] The difference in [TON] and TON d15N between the
upper 100 m and at 250 m, while subtle, is consistent with
the previously observed depth variations in PNsusp. Between
0 and 100 m, [PNsusp] is 0.3– 0.4 mM, and is 0.1 –0.2 mM
at 250 m [Michaels et al., 1994b; Michaels and Knap,
1996; Michaels et al., 1996]. Additionally, our calculated
d15N of the remineralized NO
3 , 1.3%, is generally
consistent with the d15N of the N that appears to be lost
from PNsusp as it is mixed down from the upper 100 m and
GB1018
respired in the subsurface (1%, calculated from data of
Altabet [1988, 1989]). Thus the low-d15N TON being
remineralized to NO
3 in the subsurface is likely the PNsusp
that is generated in the upper 100 m.
[44] Given the subtle month-to-month variations of the
data in hand, we tentatively note that the surface-to-subsurface gradients in both [TON] and TON d15N are weakest in
the late winter (January – April 2001; Figure 3). This is
roughly consistent with the interpretation that winter mixing
supplies new semi-labile TON (i.e., PNsusp) to the subsurface, with subsurface remineralization causing a subtle
decrease in [TON] and increase in TON d15N at 250 m
during the rest of the year. Temporal variations in subsur15
face NO
3 d N are roughly consistent with this interpreta15
tion, as the highest NO
3 d N appears to occur from
January to April, as would be expected if vertical mixing
homogenizes the upper water column. The June-to-August
15
minimum in NO
3 d N observed here is slightly early
relative to the period of peak N2 fixation, late summer
and early fall [Orcutt et al., 2001], which raises some doubt
about the role of N2 fixation-driven N export in generating
the low d15N of thermocline NO
3 . However, we are
discouraged from close interpretation of the temporal
15
changes in NO
3 d N because there is a strong correlation
15
in depth between NO
3 d N and [NO3 ] (Figures 2 and 3),
15
and we observe a shoaling of high-[NO
3 ], high-d N water
at 400– 600 m depth during the winter and spring of 2001
15
(Figure 3), which may erode the low NO
3 d N from below.
[45] The degree to which the mixing-driven flux of semilabile TON from the surface to the subsurface can influence
the d15N of the NO
3 in the thermocline depends on the rate
at which the remineralized NO
3 accumulates in the subsurface relative to the timescale on which NO
3 communicates
with the higher latitude surface waters and with the deep
sea. By combining the N flux balance (equation (1)) with
literature data for the sinking flux [Michaels et al., 1994a]
and with our data on the vertical gradient in [TN], we
estimate a vertical mixing rate between 250 m and the upper
100 m (Q in equation (1)). This value is combined with the
vertical gradient in [TON] to estimate a rate of oxidation of
1
PNsusp to NO
3 in the shallow thermocline of 0.1 mM yr
15
(calculation not shown). This rate is low if the low- N
NO
3 of the thermocline is generated entirely since the water
last left the surface (over a period of 1 – 8 years [Jenkins,
1980]). However, the water in the Sargasso Sea thermocline
may be recycled multiple times through the North Atlantic
thermocline circulation [Jenkins and Doney, 2003], so the
low-d15N NO
3 may have a much longer time to diverge
from the high d15N of deeper water NO
3 . Thus the net
production of low-15N TON in the upper 100 m and
subsequent oxidation of this semi-labile TON in the subsurface may accumulate at a rate capable of explaining the
low-d15N of the NO
3 in the shallow thermocline at BATS.
This question could be addressed more directly with a water
column transect that sampled thermocline water as it ages
away from its site of subduction, analogous to the use of N*
by Gruber and Sarmiento [1997].
[46] The recognition of the semi-labile PNsusp flux as the
form in which low d15N-N is transported from the surface
into the thermocline does not address the ultimate question
12 of 15
GB1018
of what the low-d15N N source is to the surface. It has been
posited that N2 fixation is this source. As described above,
the flux of N2 fixation required to fit observations of
elevated N* in the Sargasso Sea thermocline is sufficiently
small that it is not clearly in conflict with the N isotope
budget for the euphotic zone at BATS that we present here,
which does not require any input from N2 fixation. While
the vertical N mass and isotope budget we have constructed
for the shallow thermocline at BATS is in balance for the
temporal and spatial scales considered above, a N isotope
imbalance remains when considering the fluxes of N
between the thermocline and the deep ocean. At 1000 m,
the d15N of the NO
3 being incorporated into the thermocline from the deep ocean (5%, Figures 2 and 3) is
significantly higher than the d15N of the sinking N (3%
[Altabet et al., 1991]), such that an input of low-d15N N to
the upper ocean is required.
[47] While N2 fixation occurring elsewhere in the basin is
the most straightforward explanation for this perceived
imbalance, there are other sources of low-d15N N to the
subtropical Atlantic surface waters that may contribute to
the low d15N of thermocline NO
3 . Specifically, while
precipitation fluxes of fixed N are small, they are of the
same order of magnitude as the in situ N2 fixation rates
measured at BATS (Table 4). More importantly for the
isotopic budget, the d15N of NO
3 in Bermuda rain varies
seasonally (range of 14 to 2%) but has a low annual
mean, flux-weighted d15N (5% [Hastings et al., 2003]).
The d15N of TN in the Bermuda rain appears to have similar
characteristics, with a low annual average, flux-weighted
d15N (0% (A. N. Knapp and M. G. Hastings, manuscript
in preparation, 2005)). Thus the d15N of these precipitation
fluxes clearly mimics the d15N signal of N2 fixation and
may contribute significantly to the low d15N of NO
3
observed in the thermocline at BATS.
[48] Above, we indicate that the concentration and isotopic
imbalances in the upward and downward annual fluxes of
TON in the upper 250 m at BATS could be explained by the
PNsusp term. According to this view, which borrows heavily
from Altabet [1988], NO
3 supplied to the surface produces
two downward fluxes of N. The first is a small PNsusp flux
transported by annual mixing; the PNsusp at the surface has a
low d15N because of the uptake by phytoplankton of the lowd15N NH+4 that is excreted by plankton and cycles within
the euphotic zone [Checkley and Miller, 1986]. The second,
much larger flux is as PNsink, which has a higher d15N
[Checkley and Miller, 1986]. Both of these components are
eventually reoxidized to NO
3 in the thermocline (Figure 5). If
this view of the PNsusp component of the TON flux is correct,
it suggests that essentially all of the DON in the surface
originates in the subsurface, since the surface DON would be
indistinguishable in concentration and d15N from the DON
at 250 m (Figure 5). These results suggest that at BATS,
any labile DON that is produced is completely and rapidly
consumed and incorporated into PN. However, the lack of
any variation in [DON] or DON d15N in this oligotrophic
region begs the question, under what conditions will the
resident surface ocean DON pool show dynamic behavior?
[49] Here we have only considered a one-dimensional N
mass and isotope balance for the upper 100 m at BATS.
GB1018
This balance does not address a number of potentially
important processes that may affect our measurements, such
as lateral advection, isopycnal transport, and/or communication between the deeper ocean and the upper 250 m. As
described by Hansell et al. [2004], much of the N* signal
observed at BATS may be generated to the south and east of
BATS by the remineralization of N2 fixer biomass and
transported along isopycnals to the Sargasso Sea. This
process would also be expected to lower the d15N of
thermocline NO
3 at BATS. Additionally, Jenkins and
Doney [2003] describe a ‘‘nutrient spiral’’ in the Sargasso
Sea in which low-d15N N would be entrained in the upper
thermocline waters and cycled on timescales longer than
those examined here. Consequently, it is likely impossible
15
to understand the origin of high N* and low NO
3 d N in
the thermocline at BATS solely by studies at this one
location. The same concerns apply to DON in the surface;
given its apparently low degree of reactivity at BATS, its
properties at any given site may well be influenced by
transport within the gyre circulation. Transect studies should
help to clarify the interaction of transport with N biogeochemistry in the subtropical and tropical Atlantic.
6. Conclusions
[50] We report a robust method for TDN d15N analysis
that can be used for DON d15N measurement in NO
3 -free
and low-NO
3 waters. This method yields [DON] and DON
d15N measurements that converge upon the concentration
and d15N of lab standards, as well as previously published
[DON] measurements made at BATS by UV oxidation.
Blanks are small and well characterized, allowing for
accurate measurement of DON d15N down to 1 – 2 mM
DON. With its relatively broad applicability, this TDN
d15N method can be used to help constrain nitrogen
budgets, both in freshwater and marine systems. Future
work to improve this method might focus on quantitative
removal of NO
3 prior to the DON oxidation step, so that
DON can be measured without the differencing of TDN
and NO
3 ; any such removal step must be tested for
contamination and for alteration of the sample DON.
[51] At BATS, the new method was used to measure
TON d15N in the upper 250 m over a year, and revealed
a relatively static surface TON pool, with shallow TON
d15N of 4% throughout the year. The TN concentration
and isotope data require a d15N for the sinking flux that
is similar to previous measurements of sediment trap
materials [Altabet, 1988], suggesting that N2 fixation is
too small a fraction of the annual new N supply to be
apparent in the N isotope budget for the upper 100 m at
BATS. While BATS is often taken as a typical example
of an oligotrophic subtropical environment, its relatively
high latitude leads to conditions that promote significant
exchange between the euphotic zone and the shallow
thermocline, such that the upward supply of NO
3 from
below is much higher at BATS than it is farther south. As
a result, even the relatively high N2 fixation rate suggested by recent geochemical studies [Gruber and
Sarmiento, 1997] would represent less than 10% of the
new N supply to the BATS euphotic zone over the course
13 of 15
GB1018
of a year. Thus it is perhaps expected that N2 fixation is
not a dominant term in the N isotope budget at BATS,
and it is not clear that the isotope budget is in direct
conflict with geochemical estimates of N2 fixation. Looking forward, we are motivated to study lower latitude,
more stratified regions, where a given rate of N2 fixation
should represent a larger fraction of the annual new N
supply [Lipschultz et al., 2002].
[52] While our results are not necessarily in conflict with
N*-based estimates of N2 fixation, they do appear to pose a
problem for the hypothesis that N2 fixation fuels the
summertime DIC drawdown at BATS. As discussed above,
the rate of N2 fixation required to account for the apparent
DIC drawdown is quite large and probably should have
been apparent in our N isotope budget. Moreover, the lack
of variation in both [TON] and TON d15N throughout the
year indicates that bulk DON does not accumulate at BATS
due to N2 fixation, nor is it the major source of N for
phytoplankton during the period of DIC drawdown. If
isotope approaches for estimating N2 fixation are to be
pursued further at BATS, it is recommended that the d15N of
PNsink be revisited; this was last measured by Altabet [1988]
using sinking particle collection techniques that have subsequently been improved. Moreover, vertical migration at
BATS may play an important role in the N isotope budget
and requires attention [Villareal et al., 1999; Steinberg et
al., 2002]. Finally, N fluxes other than N2 fixation and NO
3
from below, such as precipitation [Hastings et al., 2003],
should also be considered in the context of N mass and
isotope budgets at BATS as well as in other oligotrophic
regions.
[53] We report evidence for the transfer of low-d15N N
from the euphotic zone to the thermocline, via the downward mixing of a small, low-d15N fraction of TON that is
remineralized in the shallow thermocline (250 m). The
depth gradients in TON concentration and d15N are consistent with those of PNsusp [Altabet, 1988, 1989; Michaels et
al., 1994b; Michaels and Knap, 1996; Michaels et al.,
1996], suggesting that PNsusp is the low-15N N being mixed
down and then remineralized. If so, this suggests that DON
is invariant throughout the year (in concentration and d15N)
over the upper 250 m at BATS, and that this pool is
recalcitrant on the timescale of a few years.
[54] Acknowledgments. We thank Deborah Bronk and Bess Ward
for methodological advice, and Michael Bender, Matt Reuer, and Jorge
Sarmiento for discussions. The BATS staff kindly collected all samples.
This work was funded by a DOD ASEE/NDSEG graduate fellowship to
A. N. K., by U.S. NSF Biocomplexity grants OCE-9981479 (to D. M. S.,
through the MANTRA project) and DEB-0083566 (to Simon Levin), and by
British Petroleum and Ford Motor Company through the Carbon Mitigation
Initiative at Princeton University. This is BBSR contribution 1653. Figure 3
was generated by Peter DiFiore with the application ‘‘Ocean Data View,’’
provided by R. Schlitzer of AWI, Bremerhaven, Germany.
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Figure 2. At BATS, (a) [NO
3 ] (pluses) and [TON] (solid triangles) and (b) NO3 d N (pluses) and
15
TON d N (solid triangles). NO3 data are from March 2000 through May 2001. TON data are from June
2000 through May 2001. Altabet’s [1988] d15N PNsusp, (d15N = 0.2%) (solid circle) and d15N PNsink
(d15N = 3.7%) (solid circle) are shown for reference. Replicate analyses were performed on individual
samples. In addition, at roughly half the sampling depths, replicate samples were collected. Plotted values
are averages of all analyses at a given depth.
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Figure 3. (a) [TON], (b) TON d15N, (c) [NO
3 ], and (d) NO3 d N at BATS from June 2000 through
May 2001.
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N isotopic composition of dissolved organic nitrogen and nitrate at

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