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Michigan State
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This is to certify that the

thesis entitled

The Biogeochemistry of Dissolved
Organic Nitroqen in the Subtropical
North Pacific

presented by

Michelle L. Gedeon

has been accepted towards fulﬁllment
of the requirements for

M.S. degree in Environmental
Geoscience

 

 

/?;§169ﬂm¥—

gﬂ/ Major professor

 

Date 12/06/01

0—7639 MS U is an Afﬁrmative Action/Equal Opportunity Institution

THE BIOGEOCHEMISTRY OF DISSOLVED ORGANIC NITROGEN
IN THE SUBTROPICAL NORTH PACIFIC

By

Michelle Liane Gedeon

A THESIS

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Geological Sciences

2001

ABSTRACT

THE BIOGEOCHEMISTRY OF DISSOLVED ORGANIC NITROGEN
IN THE SUBTROPICAL NORTH PACIFIC

By

Michelle Liane Gedeon

In marine systems, dissolved organic nitrogen (DON) can affect the nitrogen budget,
primary production, and potentially the production of nitrous oxide. Water samples were
collected in October 1998, January 1999 and May 2000 from Stations ALOHA (22°45'N,
158°W) and Station 2 (16°N, 150°W) located in the oligotrophic North Paciﬁc
Subtropical Gyre (NPSG). The concentration and isotope value of isolated DON (>100
Da) were measured simultaneously. DON concentrations generally range between 4-7
uM in surface waters, decreasing with depth to approximately 2 uM. As DON decreases
in concentration it becomes enriched in 15 N. This inverse relationship is consistent with
microbial assimilation and degradation of DON. There is also seasonal variation in the
8'5 N values of DON. Samples collected in summer are depleted in 15N relative to those
collected in winter, suggesting a shift in nitrogen source. During the summer months
stratiﬁcation of the water column favors T richodesmium spp., a cyanobacteria that ﬁxes
15N depleted dinitrogen (0 %o). DON produced under these conditions can be injected
into deeper waters and undergo ammoniﬁcation, resulting in a large substrate pool for
nitriﬁcation. Nitrous oxide is a by-product of nitriﬁcation and may be vented to the
atmosphere via diffusion or deep mixing. Our results support relatively short time scales
for DON cycling. Evidence of DON involvement in both primary production and the

nitrous oxide pump demonstrates the key role DON can play in the NPSG.

To my extraordinarily loving and supportive husband David and the Faith we share

iii

ACKNOWLEDGEMENTS

I would like to thank my committee members Don Hall and Nathaniel Ostrom for
their reviews of this manuscript and helpful advice. A special thanks goes out to Peggy
Ostrom, a dedicated and driven advisor who always made time for me and demanded my
best. Her support and commitment to this project helped to make it a reality. I would
also like to acknowledge and thank Hasand Gandhi for his essential role in our sample
analysis.

Dr Dave Karl deserves recognition and thanks for his research, consultation and
reviews that together made a signiﬁcant contribution to this manuscript. I would also
like to acknowledge Dr Frank Sansone, Dr Brian Pope and all our University of Hawaii
collaborators as well as the captains and crew of the R/V Moana Wave and R/V Roger
Revelle.

Thanks to all my fellow geology students especially Adam Pitt, Amanda Field,
Brittany Graham, Mary Russ and Robin Sutka whose humor and help were always
appreciated. Thanks to the Geology department faculty and staff, especially Lorretta
Knutson, Jackie Bennett and Cathy Caswell for all their help and patience.

I would like to thank my amazing husband David Beloskur whose love,
encouragement and support make every day brighter. Thanks go out to my wonderful
parents Michael and Lorraine Gedeon and to all the members of my family who have
been continually loving and supportive in so many ways. I would also like to
acknowledge my friends, especially Christy Pardee, Erica Niezgoda, Katina Paron, Luba
Soskin and Megan Agy who have always been there for me despite the miles, and have

understood how important this time in my life has been. A special thanks goes out to

Mary Russ, a gifted scientist and wonderful friend who has been a constant source of

support, humor and friendship these last two years. I wish you all the very best always.

TABLE OF CONTENTS

List of Figures .................................................................................................................... vii
Introduction .......................................................................................................................... 1
Materials and Methods ......................................................................................................... 4
Results and Discussion ........................................................................................................ 6
References .......................................................................................................................... 15

vi

 

LIST OF FIGURES

Figure 1: Water column data from Station ALOHA (solid lines) and

Station 2 (dashed lines): A) October 1998, B) January and C) May 2000 ........................ 8

Figure 2: DON concentration data from ALOHA both determined by our
dialysis and sealed tube combustion technique at Michigan State University
(dark bars) and reported in the HOT database (light bars) for water samples
from the same cast. Lines represent the range of HOT data at that particular

month and depth between 1988 and 1999 .......................................................................... 9

Figure 3: DON concentration data from ALOHA (closed symbols) and
Station 2 (open symbols). Data collected in October 1998 are indicated
by triangles, January 1999 by squares and May 2000 by circles. The line

is average HOT DON data 1988-1997 from Karl et al., 2001 .......................................... 11

Figure 4: 6'5 N values for DON. Samples collected from Station ALOHA
in October 1998 are indicated by triangles, January 1999 by squares and
May 2000 by closed circles. Open circles represent data collected in May
from Station 2. The solid lines are least-squares regressions for each
month. The dashed line is the May least-squares regression excluding

the 50m point ..................................................................................................................... 14

vii

INTRODUCTION

Dissolved organic nitrogen (DON) is often the most abundant form of ﬁxed
nitrogen in oligotrophic marine ecosystems (Sharp, 1983). In surface waters (SlOOm) of
the North Paciﬁc subtropical gyre (NPSG) DON is usually hundreds of times more
abundant than inorganic nitrogen and frequently comprises more than 90% of the total
dissolved nitrogen pool (Karl et a1.,l993). Dissolved organic nitrogen is comprised of a
number of different compound classes including polysaccharides, nucleic acids, proteins,
humic acids and dissolved free amino acids (Keil and Kirchman 1991, Bronk and Glibert
1993, Koike and Tupas 1993). Despite the potential importance of DON, its role in
primary production and nitrogen cycling is not well understood. This is in part due to a
long-standing perception that the DON pool is predominantly refractory (Williams and
Druffel, 1988). A number of studies, however, suggest that there is an important
bioavailable component~ of the DON pool, citing turnover times on the order of days, as
opposed to hundreds or thousands of years (Toggweiler, 1989, Williams and Druffel,
1988). Bronk et a1. (1994) estimated 10:1 days for complete turnover based on 15'N
enrichment. Benner et a1. (1997) isolated high molecular weight (1-100nm) dissolved
organic matter (DOM) and estimated a turnover of 0.3-0.5% DOM per day. Given the
large size of the DON pool and the importance of nitrogen as a limiting nutrient, if even a
fraction of DON is bioavailable then nitrogen availability has been underestimated
(Fuhrman, 1992). This ﬁnding has particular relevance in oligotrophic systems.

In addition to its potential effect on the nitrogen budget, DON may be involved in
nitrogen cycling reactions which have local, regional and global scale environmental

signiﬁcance. For example, nitrogen ﬁxing organisms can produce new DON (Karl et al.,

1997) which enters the water column via microbial release or the breakdown of
particulate organic nitrogen (PON). Mineralization of this new DON to ammonium, a
substrate for nitriﬁcation, may allow DON to play a role in nitrous oxide production as
nitrous oxide is a by-product of nitriﬁcation. This is important because nitrous oxide is a
gas which contributes more to the greenhouse effect than carbon dioxide, and is
increasing at an annual rate of 0.3 % (Prinn et a1. 1990). Nitrous oxide production is of
particular interest in aerially extensive oceanographic systems due to the potentially
signiﬁcant ocean-atmosphere ﬂux. Our study sites, Station ALOHA (22°45’N, 158°W)
and Station 2 (16°N, 150°W), are located in just such a system, the large (2x107 kmz),
oligotrophic, NPSG. Data has been collected from Station ALOHA, our primary
sampling site, over an 13 year period. These data were collected as part of the Hawaii
Ocean Time-series (HOT) program and reveal that this system, once thought to be
homogeneous in space and time, exhibits dramatic change with respect to microbial
community structure and nutrient cycling on seasonal, interannual and decadal time
scales (Karl, 1999).

Station ALOHA is characterized by high DON concentrations (5-6 nM) and low
nitrate concentrations (< 10 nM) in surface waters. There is a persistent, deep
chlorophyll maximum layer centered near 115-125 m and a deep oxygen minimum at 800
m (Karl, 1999). The permanent nutricline is located between 200-600 m (Karl et al.,
2001). Nutrients needed for primary production, such as nitrate and phosphate, are
primarily supplied via recharge from deeper waters due to vertical diffusion and
horizontal transport from adjacent systems (Karl, 1999 Short term eddy-driven mixing

events are also important nutrient recharge mechanisms (Letelier et al., 2000). Nitrogen

ﬁxation is also an important source of new nitrogen, particularly when stratiﬁcation of
the water column allows for blooms of free living (e. g., T richodesmium) and
endosymbiotic (e.g., Richelia) nitrogen-ﬁxing cyanobacteria (Karl, 1997).
Measurements of variations in the stable isotopic abundances of nitrogen
contained in selected pools (e. g., 14N and 15N) have provided important insight into the
origins and transformations of organic and inorganic nitrogenous species (Saino and
Hattori, 1987; Altabet et al., 1991; Ostrom et al., 1997). Similarly, temporal shiﬁs in the
source of DON, as well as participation in nitrogen cycling reactions, should be evident
by measuring the nitrogen isotope ratios of DON. The 6'5N values of high molecular
weight marine DON isolated via ultraﬁltration suggest a major role for DON in the upper
ocean nitrogen cycle (Benner et al., 1997). F euerstein et a1. ( 1997) demonstrated that 95
% or more of DON can be effectively isolated from freshwater, quantiﬁed and measured
isotopically and that 99 % of dissolved inorganic nitrogen can be quantitatively removed
from salt water samples. Our objective is not only to isolate and quantify DON, but to

use nitrogen isotopes to examine the role of DON in nitrogen cycling within the NPSG.

MATERIALS AND METHODS

Samples analyzed for the concentration and isotopic composition of DON were
collected from two stations within the NPSG. Water was collected at ALOHA (22°45’N,
158°W) on October 19, 1998, January 13, 1999 and May 25, 2000. Samples were also
obtained at a second station (Station 2, 16°N, 150°W) on May 28, 2000. Samples
collected in October 1998 and January 1999 were taken aboard the R/V Moana Wave in
coordination with the HOT program. Collections made in 2000 were part of a larger
Paciﬁc transect cruise on the R/V Roger Revelle. Water (0.4 to 1.0 L) was collected in
14 L (RN Moana Wave) and 20 L PVC bottles (R/V Roger Revelle), immediately
vacuum ﬁltered through acid washed, 45 mm diameter, 0.2 pm Supor polyethersulfone
ﬁlters and frozen in acid washed Nalgene bottles for subsequent processing and analysis.

In the laboratory, seawater samples were thawed, concentrated approximately 5-
fold to 80-200 mL by rotary evaporation (ca.10 mm of Hg and 38 °C), and transferred to
100 Dalton molecular weight cut off (MWCO) dialysis membranes (Spectra/For CE
Membrane). To ensure quantitative recovery, round bottom ﬂasks used during rotary
evaporation were rinsed 3 times with 4 mL of ultra-clean deionized water (Epure,
Bamstead) and this water was added to the membrane. The samples were suspended in 4
L beakers and initially dialyzed against a solution of 8 % combusted NaCl dissolved in
ultra-clean deionized water for up to 10 days (4 °C-6 °C). Beginning on the second day
of dialysis, the initial NaCl solution was diluted daily by 1-2 % until the sample was
eventually suspended in a salt-free solution. We found that initially suspending the
sample in an 8 % saline solution reduced, but did not impede, diffusion across the

membrane. Reducing the salt concentration gradually, prevented a steep concentration

gradient that can result in a high diffusion rate, expansion of the membrane pores and
potentially the loss and isotopic fractionation of the DON. Following dialysis, the
contents of each dialysis bag were removed using a combusted Pasteur pipette and
transferred to a combusted round bottom ﬂask. To assist in the quantitative transfer of
DON, the dialysis membrane was rinsed 3 times with 2 mL of Epure water and this rinse
was added to the ﬂask. Each sample was reduced to a volume of approximately 5-10 mL.
The concentrate and subsequent rinses (3, 4 mL rinses) were transferred to a pre-
combusted quartz ampoule (ca. 60 mL). Combusted quartz wool was loosely added to
the necks of the quartz ampoules to prevent sample loss under vacuum. All glassware,
quartz wool and NaCl used in the preparation and analysis of DON samples were
combusted at temperatures of at least 450 °C for 1 h, borosilicate pipettes which have a
lower heat tolerance were combusted at 375 °C for 1 h and, in accordance with F euerstein
et al., 1997, CuO was combusted at 850 °C for 4 h to ensure complete oxidation. Each
vessel was frozen in liquid nitrogen and dried in vacuo on a glass high-vacuum line.
Once the samples dried, combusted copper oxide (Aldrich, 2 g) and copper (Elemental
Microanalysis, 6 g) were added and the samples were re-evacuated. The vessels were
then sealed, combusted at 850 °C for 1 h to convert the DON to N2 and slowly cooled at a
rate of 0.4 °C/min. Nitrogen gas was separated cryogenically and the isotope value
determined on a PRISM (Micromass) mass spectrometer. Isotope ratios are expressed in
per mil (%o) notation:

5‘13 = [(Rmp../R,.,,d,,d) — 1] * 1000
where I is the heavier isotope of element E and R is the abundance ratio of the heavy to

light isotope. The standard for SISN is atmospheric nitrogen gas. The nitrogen

concentration of a sample is determined based on the mass 28 ion beam within a
calibrated volume in the dual-inlet mass spectrometer. Calibration was performed every

day that a DON sample was analyzed.

RESULTS and DISCUSSION

The concentration of DON far exceeds that of dissolved inorganic nitrogen in the
upper water column (0-100 m), however our understanding of DON cycling in marine
systems is still in its early stages. Here we present the ﬁrst data set in which all marine
DON greater than 100 Da has been isolated and isotopically characterized to further
elucidate its ecological role in the oligotrophic NPSG.

We believe our study sites in the NPSG, Station ALOHA and Station 2, are
representative of the regional habitat and therefore may contribute to the fundamental
understanding of biogeochemical processes in this major biome. The majority of our data
comes from the well-characterized Station ALOHA, however Station 2 samples have
been incorporated into our May data set due to a limited number of ALOHA samples.
There are no distinct differences in the water column data from these two stations (Figure
1C). However, DON and water column samples were collected from these sites over
three different seasons (winter, summer and fall) allowing us to explore how temporal
changes in physical and biological forcing can inﬂuence the composition and cycling of
DON.

Density proﬁles show that there is distinct seasonal variation in the depth of the
mixed layer (Figure 1). Historically this layer reaches 40 to 100 m at ALOHA (Karl,
1999). Deepening of the mixed layer is primarily the result of winter cyclones which
cross the NPSG on a weekly basis (Karl, 1999). Our water column data show that the
mixed layer reaches depths of approximately100 m in January, 50 m in May and 75 m in
October. These depths are consistent with previous observations during these months

(Karl, 1999). Temperatures, however, remain relatively constant in the upper 50 m (24-

25 °C)i The concentration of chlorophyll and the depth of the chlorophyll maximum vary
seasonally. In October the chlorophyll concentration reached 0.23 pg/L and extended
from 85 to 105 m. The maximum concentration in January was 0.16 11g chlorophyll/L
between 85 and 105 m decreasing to a low 0.02 ug/L at 175 m. The chlorophyll

concentration in May peaked sharply, reaching 0.29 ug/L at 125 m (Figure l).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   

Figure 1
Chlorophyll a (mg/L) Temperature (°C) Potential Density (kym’)
A) 0 0.1 02 03 04 0 10 20 so 22 14 26 23
o l A n o A A o A L
30 300 ‘ 3W ‘
,.,
E 100 l
.5 600 ‘1 m <
g 150 «
900 .
200 -l 90° '
250 1200 1200
0 0.102 03 04 0 10 20 30 22 24 26 20
B) o J, . + o L 4 0 - . .
so I 300 -l 300 .
A
s 1001
600 600 q
E" [50 -l
900 . 900 .
200 .
250 1200 , 1200
0 or 01 03 04 o 22
so . ‘
300 q 300 -
E 100
.1
V 000 .
5 600 .
g 150 «
900 «
250 1200 1200

 

 

 

 

 

 

 

 

 

 

Water column data from Station ALOHA (solid lines) and Station 2 (dashed lines): A) October 1998, B)
January 1999 and C) May 2000.

Understanding the trend in DON concentration at our sampling sites is one of the keys to
deciphering the reactivity and cycling of DON in the NPSG. The ﬁrst step in assessing
these data was to compare our DON concentration values to those determined by the
HOT core measurement program for the same cruise and/or with the range of data for the
corresponding month between 1988 and 1999 (Figure 2). Comparative concentration

data for our Station 2 samples were not available, as this station is not afﬁliated with the

 

   
 

 

 

 

 

 

 

 

 

 

 

HOT program.
Figure 2
g 8
3 7 4 October 1998 Januan/ 1999 May 2000
.5
g I
g UHOT
o
0
Z
O
Q

100 450 550 2000 l 10 120 300 450 400 600

Depth (m)

DON concentration data from ALOHA both determined by our dialysis and sealed tube combustion
technique at Michigan State University (dark bars) and reported in the HOT database (light bars) for water
samples from the same cast. Lines represent the range of HOT data at that particular month and depth
between 1988 and 1999.

Our samples underwent sealed tube combustion and estimates of DON concentration
were based on the ion beam produced within a calibrated volume of the mass '
spectrometer. Dissolved organic nitrogen concentrations available in the HOT database
were estimated as the difference between total dissolved nitrogen (TDN) and nitrate +
nitrite (N+N) concentrations (Karl et al., 2001). Total dissolved nitrogen concentrations

were determined by UV oxidation (Armstrong et al., 1966) followed by measurements of

N+N and ammonium as hydrolysis products (Walsh, 1989). Nitrate + nitrite
concentrations were determined using a four-channel Technicon Autoanalyzer II
continuous ﬂow system. The N+N analysis method is based on recommendations of
Technicon Industrial Systems (1979) with modiﬁcations from Armstrong et al. (1967).
The excellent agreement between our data and those of the HOT database validates both
our DON isolation technique and our method for determining DON concentration.

In the development of our dialysis method it became apparent that a salt gradient
was imperative in order to achieve adequate diffusion. Dialysis of a concentrated marine
sample against ultra-pure deionized water can result in rapid difﬁrsion of water into the
100 Da MWCO membrane, expanded pore size and subsequent fractionation of the DON.
Several lots of 100 Da MWCO membrane were used in the course of our method
development and sample analysis. According to the manufacturer, while pore size is
constant across lots, porosity can vary substantially, causing individual lots to differ in
their diffusion rates and consequently in their integrity when exposed to high salt
concentrations. We explored using larger pore size membranes. When a Spectra/For CE
3500 Da MWCO membrane was used to dialyze our salt solution against ultra—pure
deionized water no expansion was detected. This indicates that when 3500 Da MWCO
or larger pore size membranes are used the membrane is not compromised during dialysis
and therefore it is not necessary to dialyze against a saline gradient.

There is a distinct decrease in DON concentration down water column. With the
exception of our 150 m sample collected in May 2000, concentrations generally range
from 4-7 uM in near surface waters to approximately 2 uM below 800 m (Figure 3). The

down water column trend in our data is consistent with that observed for the average

10

DON concentrations obtained from the HOT database (Karl et al., 2001). There is an
inherent variability in DON concentrations particularly near the surface that can be
observed in our data but is not obvious from the historical average. Concentrations
reaching almost 10 uM have been measured several times over the course of the HOT
program. This variability is a result of seasonal and temporal changes in the

environmental conditions of the NPSG (Karl, 1999).

 

 

Figure 3
DON concentration (11M)
0 2 4 6 8 10 12
O 1 J I
O
400 a
A 800 A
E
.t:
*5. 1200 .
O
G
1600 ~
2000 a A

 

 

 

DON concentration data from ALOHA (closed symbols) and Station 2 (open symbols). Data collected in
October 1998 are indicated by triangles, January 1999 by squares and May 2000 by circles. The line is
average HOT DON data 1988-1997 from Karl et al., 2001.

Depth related trends in DON concentration reﬂect the dynamic nature of this large
organic reservoir. The elevated DON concentrations in the upper water column are a
result of local production, including passive release from photosynthetic microorganisms
as well as lysis of phytoplankton and bacterial cells due to heterotrophic grazing, cell

death and virus-induced lysis (Karl et al., 1998; Bronk and Glibert, 1993). In certain

11

oceanic, coastal and estuarine environments an average of 25-41 % of dissolved
inorganic nitrogen taken up by phytoplankton can be released as DON (Bronk et al.,
1994)

The decrease in DON concentration down water column is evidence of net
assimilation and remineralization to nitrate (Figure 3). According to our data, at depths
of only 400 m DON concentrations were reduced by 50 % or more relative to those in the
upper 200 m. This relates to the observation that dissolved organic nitrogen is utilized
quickly, predominantly by bacteria (Karl et al., 2001). Utilization of low molecular
weight DON compounds, such as dissolved combined amino acids and dissolved DNA,
can fuel up to 50 % of bacterial growth (Keil and Kirchman 1991; Jorgensen et al., 1994).
Bacteria are considered the predominant consumers of DON, however there is evidence
that some phytoplankton have cell-surface enzymes that may allow them to utilize more
DON than previously thought (Bronk et al., 1994).

The relative contributions of labile and refractory components to the DON pool is
still uncertain, however we do know that DON not assimilated by microbes can aggregate
into colloids and particles which can then sink and undergo mineralization (Fenchel et al.,
1998). Some of this sinking material is inherently refractory. For example, structural
macromolecules such as peptidoglycans, which are important components in bacterial
membranes, have been identiﬁed as major contributors (~64 %) to the high molecular
weight ( >1000 Da) fraction of DON (McCarthy et al., 1998). However, it is important to
note that this molecular weight ﬁ'action only accounts for approximately 25 % of the
DOM pool (Benner et al., 1997). Recalcitrant biomolecules likely contribute to a deep

ocean pool of oxidation resistant, lower water column DON with a radiocarbon age of

12

over 4000-6000 years old (Williams and Druffel, 1988). The observation that the
reservoir of DON in deep waters retains refractory components may explain why DON

concentrations do not drop to zero but rather appear to stabilize at 2 uM. Historically

DON concentrations in the NPSG have been reduced to 2 uM at depths as shallow as 350
m, but more commonly this stabilization occurs somewhere between 500-800 m and
remains relatively constant to at least 2000 m (Karl et al., 2001).

While our concentration data suggest that DON is actively involved in NPSG
nitrogen cycling , isotopic analyses provide more detailed insight into the dominant
processes that contribute to the formation and utilization of this abundant nitrogen source.
A number of interpretations can be made from the trends found in our DON isotopic data.
There is a depth-dependent trend in each data set which demonstrates that DON isolated
from surface waters is depleted in 15 N relative to samples taken at depth (Figure 4).

The data from January (R2=O.96, p=5.0 x 10“) and May (R2=0.80, p=.02) are
signiﬁcantly correlated. When the 50 m May sample is excluded from the regression the
data are signiﬁcant at the 99 % conﬁdence level (R2 = 0.93, p=7.7 x 103). Although the
DON concentration of this sample is appropriate to the season and depth from which it
was taken, the observation that the isotope value is enriched, rather than depleted, in 15N
relative to data from 150 m suggests that the nitrogen transformation processes within the
mixed layer may vary from those taking place below the pycnocline. The elevated SISN
value could result from phytoplankton uptake of a residual pool of 15N enriched nitrate or
ammonium (our 5‘5N values for nitrate from ALOHA are >4.5 %o) and subsequent
production of DON. This is consistent with the observation that kinetic isotope effects

result in mass discrimination when 14N is preferentially incorporated into the product of

13

the reaction leaving the residual substrate enriched in lsN (Mariotti et al., 1981).
Alternatively, this sample may reﬂect a 1"’N enriched pool of residual DON that could be
achieved by intense recycling of DON in the mixed layer. The October concentration
data are not as highly correlated (R2=0.71, p=0.l6) as the other two seasons and may
represent a transitional period between seasons.

Figure 4

 

200 a

400 4

600 a

800 ~

Depth (m)

1000 .
§§
2000 .

 

 

 

 

2200

8"N values for DON. Samples collected from Station ALOHA in October 1998 are indicated by triangles,
January 1999 by squares and May 2000 by closed circles. Open circles represent data collected in May
from Station 2. The solid lines are least-squares regressions for each month. The dashed line is the May
least-squares regression excluding the 50m point.

The roughly inverse relationship between concentration and 815N is consistent
with down water column degradation of DON. However, correlations between the 6'5N
values of DON and the natural log of the DON concentration were not signiﬁcant (F-test,

p=0.05) indicating that degradation does not adhere to a Rayleigh model (Mariotti et al.,

14

1988; Ostrom et al., 2002). This suggests that the isotopic composition of DON is not
controlled by a single unidirectional reaction. This is not surprising for an actively
cycling pool of organic nitrogen likely comprised of a variety of compounds. In the
upper water column where production, and hence concentrations, of DON are high the
nitrogen isotope ratios are correspondingly low, reﬂecting the selective release of ”N
depleted DON from microbial cells. As this DON is assimilated and degraded by
microbial activity the residual pool becomes progressively more enriched in 15N with
depth. Although fractionation during degradation is the most likely cause of the increase
in BUN values of DON with depth, it is important to stress that there are a number of
degradative processes that can result in isotopic enrichment of DON. Enrichment of the
DON pool can occur as microbes preferentially assimilate isotopically depleted DON.
This occurs either as low molecular weight organics are directly assimilated by bacteria
or, in the case of larger proteins and other nitrogenous polymers, when membrane bound
hydrolytic enzymes ﬁrst break down the compounds until they are suitable for
assimilation (F enchel et al., 1998). The microbial process of ammoniﬁcation which
converts DON to NH: (and eventually to NO3' via nitriﬁcation) can also result in the
degradation and the consequent enrichment of DON with depth. In addition to
degradation reactions the high 6'5N values of DON (>12 %o), collected well below the
euphotic zone in both the fall and winter, may be a result of bacterial uptake of
regenerated, ‘5 N enriched, ammonium with subsequent production of enriched DON
(McCusker et al., 1999). The low concentrations of ammonium in the NPSG (<1 pM)

are consistent with a residual pool that has undergone bacterial utilization.

15

While increasing 815 N values of DON is a trend seen in all three data sets, the
range that these values span varies with season, reﬂecting the physical conditions and
succession of primary producers within the NPSG. The distinct seasonal difference
among the three data sets is most evident in the comparison of winter and summer
samples (Figure 4). In May, the DON pool has isotope values ranging from 1.6 to 11.0
%o. The lower end of this range suggests the inﬂuence of nitrogen-ﬁxing organisms in
upper water column DON production (atmospheric N2=0 %o). Samples collected in
January have DON that is relatively more enriched in 15N, with values ranging from 8.3
to 14.0 %o. These values are consistent with DON produced by nitrate utilizing primary
producers. Nitrate isotope values from samples collected at Station ALOHA were
analyzed in our laboratory and found to be 4.5-9.5 %o (Sutka et al., in preparation).
Similar to the samples collected in January, the isotope values from the October DON
samples span 8.9 to 15.0 %o, again consistent with DON production predominantly fueled
by nitrate or ammonium.

Recent studies provide evidence that nitrogen ﬁxing cyanobacteria are an
important seasonal source of new nitrogen and, hence, DON (Karl et al., 1997). There
are at least three key groups of nitrogen-ﬁxing cyanobacteria in the NPSG including: (1)
the large ﬁlamentous, non-heterocystous, bloom-forming Trichodesmium spp. (Capone et
al., 1997), (2) the background population of small (3-5 pm) unicellular
nanocyanobacteria (Zehr et al., 2001), and (3) endosymbiotic associations between the
unicellular heterocystous cyanobacterium Richelia and several species of diatoms

(Villareal,1999) which also form aperiodic blooms.

l6

At Station ALOHA nitrogen ﬁxation is most prevalent during the summer
months when stratiﬁcation of the water column allows for the selection of the bloom-
forrning, nitrogen ﬁxing microbes (Karl et al., 1997). In the case of T richodesmium, up
to 50 % of their ﬁxed nitrogen may be released as dissolved new nitrogen (Glibert and
Bronk, 1994). Interestingly, concentrations of DON are slightly, although not
statistically, higher during the fall and summer months as compared to winter and spring
(Karl et al., 2001). In addition there is evidence that during El Niﬁo Southern Oscillation
(ENSO) events, prolonged stratiﬁcation of the water column allows for extended periods
of nitrogen ﬁxation leading to even greater DON production (Karl et al., 1997). During
one such episode, a dramatic increase in DON concentration coincided with an increase
in Trichodesmium spp. at Station ALOHA (Karl et al., 1997). These seasonal and
interannual changes in DON production are supported by our work and by the work of
Karl et al. (1997). The data suggest a direct relationship between DON and the nitrogen
ﬁxing organisms of the microbial loop and further support the labile nature of DON.

The net production of DON that occurs during ENSO episodes would result in
local storage and eventual export of DON. Furthermore, increases in NPSG DON over
the course of the HOT program have been attributed to the inﬂuence of nitrogen ﬁxation
(Church et al., in press; Karl et al., in press). In the NPSG, ENSO enhanced nitrogen
ﬁxation can result in a supply of up to 51 mmole’zy" (Karl et al., 1997). Fixed
nitrogen released from microbes can accumulate as DON in the euphotic zone. This
DON is then exported to the lower water column most likely due to mixing events and
the sinking of aggregated DON. Mineralization of the exported DON results in a

substantial substrate pool for nitriﬁcation, the predominant mechanism for the production

17

of nitrous oxide in this region, according to studies conducted at ALOHA (Dore et al.,
1996; Ostrom et al., 2000). Nitrous oxide produced in the deep waters of the NPSG
could ultimately be vented to the atmosphere by diffusion and deep mixing events,
contributing to the increasing atmospheric concentration of this greenhouse gas. This
chain of events, beginning with ENSO induced stratiﬁcation of the water column and
resulting in the production of nitrous oxide, would effectively create a climatically driven
nitrous oxide pump and place DON in a key intermediary position amidst the ecosystem

changes that occur in the NPSG.

18

REFERENCES

Altabet M.A., Deuser W.G., Honjo S., Stienen C. (1991) Seasonal and depth-related
changes in the source of sinking particles in the North Atlantic. Nature 354, 136-138.

Armstrong F.A., Williams P.M., Strickland J DH. (1966) Photo-oxidation of organic
matter in seawater by ultraviolet radiation, analytical and other applications. Nature 211,
481-483.

Armstrong F .A.J ., Sterns C.R., Strickland J DH (1967) The measurement of upwelling
and subsequent biological processes by means of the Technicon Autoanalyzer and
associated equipment. Deep-Sea Res. 14, 381-389.

Benner R., Biddanda 8., Black B., McCarthy M. (1997) Abundance, size distribution,
and stable carbon and nitrogen isotopic compositions of marine organic matter isolated
by tangential-ﬂow ultraﬁltration. Mar. Chem. 57, 243-263

Bronk DA. and Glibert PM. (1993) Contrasting patterns of dissolved organic nitrogen
release by two size fractions of estuarine plankton during a period of rapid NH4+
consumption and N02' production. Mar. Ecol. Prog. Ser. 96, 291-299.

Bronk, D.A., Glibert, P.M., Ward, BB, 1994. Nitrogen uptake, dissolved organic
nitrogen release, and new production. Science. 265, 1843-1846.

Capone D.G., Zehr J .P., Paerl H.W., Bergman B., Carpenter E.J. (1997) Trichodesmium a
globally signiﬁcant marine cyanobacterium. Science. 276, 1221-1229.

Church M.J., Ducklow H.W., Karl D.M. Multi-year increases in dissolved organic matter
inventories at Station ALOHA in the North Paciﬁc Subtropical Gyre. In Press.

Dore J .E. and Karl D.M (1996) Nitriﬁcation in the euphotic zone as a source for nitrite,
nitrate and nitrous oxide at Station ALOHA. Limnol. Oceanogr. 4(18), 1619-1628.

Fenchel T., King G.M., Blackburn TH. (1998) Bacterial Biogeochemistry-The
Ecophysiology of Mineral Cycling. Academic Press, California.

19

Feuerstein T.P., Ostrom P.H. Ostrom, NE. (1997) Isotopic biogeochemistry of dissolved
organic nitrogen: A new technique and application. Org. Geochem. 27, 363-370.

Fuhrman J. (1992) Bacterioplankton roles in cycling of organic matter: The microbial
food web. In Primary Productivity and Biogeochemical Cycles in the Sea (eds. P.G.
F alkowski and AD. Woodhead). Plenum Press, New York. pp. 361-379.

Glibert PM. and Bronk DA. (1994) Release of dissolved organic nitrogen by marine
diazotrophic cyanobacteria, Trichodesmium spp. Appl. Environ. Microbiol. 60, 3996-
4000.

Jergensen N.O.G., Kroer N., Cofﬁn RB. (1994) Utilization of dissolved nitrogen by
heterotrophic bacterioplankton: Effect of substrate C/N ratio. Appl. Environ. Microbiol.
60, 4124-4133.

Karl D.M., Tien G., Dore J ., Winn CD. (1993) Total dissolved nitrogen and phosphorous
concentrations at US-J GOF S Station ALOHA: Redﬁeld reconciliation. Mar. Chem. 41,
203-208.

Karl D., Letelier R., Tupas L., Dore J ., Christian J., Hebel D. (1997) The role of nitrogen
ﬁxation in biogeochemical cycling in the subtropical North Paciﬁc Ocean. Nature 388,
533-538.

Karl D.M. (1999) A sea of change: Biogeochemical variability in the North Paciﬁc
subtropical gyre. Ecosystems 2(3), 181-214.

Karl D.M., Bjorkman K.M., Dore J .E., Fujieki L., Hebel D.V., Houlihan T., Letelier
R.M., Tupas L.M. (2001) Ecological nitrogen-to-phosphorous stoichiometry at station
ALOHA. Deep-Sea Res. 48, 1529-1566.

Karl D.M., Bidigare R.R., Letelier R.M. Sustained and aperiodic variability in organic
matter production and phototrophic microbial community structure in the North Paciﬁc
subtropical gyre. In Press.

20

Keil R.G., Kirchman D.L. (1991) Contribution of dissolved free amino acids and
ammonium to the nitrogen requirements of heterotrophic bacterioplankton. Mar. Ecol.
Prog. Ser. 73, 1-10.

Koike 1., Tupas L. (1993) TOtal dissolved nitrogen in the Northern North Paciﬁc assessed
by a high-temperature combustion method. Mar. Chem. 41, 209-214.

Letelier R.M., Karl D.M., Abbott M.R., Flament P., Freilich M., Lukas R., Strub T.
(2000) Role of late winter mesoscale events in the biogeochemical variability of the

upper water column of the North Paciﬁc Subtropical Gyre. J. Geophys. Res. 105, 723-
728, 739.

Mariotti A., Lancelot C., Billen G. (1981) Natural isotopic composition of nitrogen as a
tracer of origin for suspended matter in the Scheldt estuary. Geochim. Cosmochim. Acta
48, 549-555.

Mariotti A., Landreau A., Simon B. (1988) 15N isotope biogeochemistry and natural
denitriﬁcation process in groundwater: Application to the chalk aquifer of northern
France. Geochim. Cosmochim. Acta. 52, 1869-1878.

McCarthy M.D., Hedges J .I., Benner R. (1998) Major bacterial contribution to marine
dissolved organic nitrogen. Science 281, 231-234.

McCusker E.M., Ostrom P.H., Ostrom N.E., Jeremaison J., Baker J. (1999) Seasonal
variation in the biogeochemical cycling of seston and PCBs in Grand Traverse Bay, Lake
Michigan. Org Geochem. 30, 1543-1557.

Ostrom N.E, Macko S.A., Deibel D., Thompson R. (1997) Seasonal variation in the
stable carbon and nitrogen isotope biogeochemistry of a coastal cold ocean environment.
Geochimica et Cosmochimica Acta. 61, 2929-2942.

Ostrom N.E., Russ M.E., Popp B., Rust T.M., Karl D.M. (2000) Mechanisms of nitrous
oxide production in the subtropical north Paciﬁc based on determinations of the isotopic
abundances of nitrous oxide and di-oxygen. Chemosphere 2, 281-290.

21

Ostrom N.E., Hedin L.O, von Fisher J .C., Robertson G.P. Nitrogen transformations and
nitrate removal at a soil-stream interface: a stable isotOpe approach. Ecological
Applications. In press.

Prinn R., Cunnold D., Rasmussen R.A., Simmonds P., Alyea F., Crawford A., Fraser P.,
Rosen R. (1990) Atmospheric emissions and trends of nitrous oxide deduced from 10
years of ALE-GAGE data. J. Geophys. Res. 59, 18369-18385.

Saino T. and Hattori A. (1987) Geographical variation of the water column distribution of
suspended particulate organic nitrogen and its 15N natural abundance in the Paciﬁc and its
marginal seas. Deep-Sea Res. 34 (5/6), 807-827.

Sharp J .H. (1983) The distribution of inorganic nitrogen and dissolved and particulate
organic nitrogen in the sea. In Nitrogen in the Marine Environment (eds. E.J. Carpenter
and DC. Capone). Academic Press, New York. pp. 1-35.

Sutka R.L., Ostrom P.H., Ostrom N.E. Stable nitrogen isotopic composition of nitrate as
an indication of water column microbial processes of the Eastern Tropical North Paciﬁc
Ocean. In Preparation.

Technicon Industrial Systems (1979) Nitrate and nitrite in water and seawater.
Autoanalyzer 11 Industrial Method No. 158- 71 W. Technicon Ind. Systems, Tarrytown,
New York.

Toggweiler IR. (1989) Is the downward dissolved organic matter (DOM) ﬂux important
in carbon transport? In Productivity of the ocean: Present and past (eds. W. H. Berger,
V.S. Smetacek and G. Wefer). J. Wiley and Sons, New York. pp. 65-84.

Villareal T.A., Pilskaln C., Brzezinski M., Lipschultz F., Dennett M, Gardner GB.
(1999) Upward transport of oceanic nitrate by migrating diatom mats. Nature. 397, 423-
425.

Walsh T.W. (1989) Total dissolved nitrogen in seawater: A new high temperature
combustion method and a comparison to photo-oxidation. Mar. Chem. 26, 295-311.

Williams PM. and Druffel E.R.M. (1988) Dissolved organic matter in the ocean:
Comments on a controversy. Oceanogr. 1, 14-17.

22

Zehr J .P., Mellon M.T., Zani S. (1998) New nitrogen-ﬁxing microorganisms detected in
oligotrophic oceans by ampliﬁcation of nitrogenase (niﬂ-I) genes. Appl. and Environ.
Microbiol. 64, 3444-3450.

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