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This is to certify that the

dissertation entitled

STABLE NITROGEN ISOTOPES AS A TOOL FOR UNDERSTANDING
NITROGEN CYCLING PROCESSES AND MECHANISMS OF NITROUS
OXIDE PRODUCTION

presented by

Robin Leslie Sutka

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Environmental Geosciences

 

 

Major professor

7am OSWVW
CW

Date 8/? ’01

 

MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771

 

LIBRARY
Michigan State
University

 

 

 

 

STABLE NITROGEN ISOTOPES AS A TOOL FOR UNDERSTANDING NITROGEN
CYCLING PROCESSES AND MECHANISMS RESPONSIBLE FOR NITROUS
OXIDE PRODUCTION
By

Robin Leslie Sutka

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Department of Geological Sciences

2002

UMI Number: 3064317

®

UlVII

 

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Copyright 2002 by ProQuest lnfon'nation and Learning Company.

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ABSTRACT
STABLE NITROGEN ISOTOPES AS A TOOL FOR UNDERSTANDING NITROGEN
CYCLE PROCESSES AND MECHANISMS RESPONSIBLE FOR NITROUS OXIDE
PRODUCTION
By
Robin Leslie Sutka
The stable nitrogen isotopic composition of dissolved nitrate was determined at

six stations ranging from the oligotrophic North Pacific Subtropical Gyre (NPSG) to the
eutrophic Eastern TrOpical North Pacific (ETNP). Dissolved inorganic nitrogen, oxygen
and phosphate concentrations were determined in water column samples at all six
stations. In productive, oxic waters nitrate isotopically enriched in 15N (maximum 515N-
NO3' value of 12.5 0/00) was most likely the result of assimilatory nitrate reduction. In
contrast, high OlSN-Nog' values (maximum of 12.3 °/oo), associated with high nitrate
deficits and anoxic conditions, supported the interpretation of isotopic fractionation due
to denitrification. A one-dimensional vertical advection and diffusion model was used to
estimate the fractionation factor for denitrification at two stations in the ETNP. A
comparison of modeled to observed 8'5N-NO3' provided estimates of the isotopic
enrichment factor (8) of 30 at station 4 and 30 to 35 at station 5. Isotopically light nitrate
(1.1 and 3.2 °/oo) was observed in the upper 200 m of the water column at stations in the
ETNP. Tracer studies of 15NIL; and biogeochemical indicators of nitrogen fixation, such

as N/P ratios, OlsN-POM and T richodesmium abundance, supported the interpretation of

nitrification as the most plausible explanation for low 6‘5N-NO3' values observed in

upper water column samples. Nitrification rates increased across the transect from a
maximum rate of 1 nmol TI (1'1 at station 1 and 23.7 nmol 1'1 (11'1 at station 6.

The relative importance of individual microbial pathways to nitrous oxide
production is not well known. The intramolecular distribution of 15 N in nitrous oxide
provides a basis for distinguishing biological pathways. Concentrated cell suspensions of
M. capsulatus Bath and N. europaea were used to investigate the Site preference of
nitrous oxide by microbial processes during nitrification. The average site preference of
nitrous oxide formed by hydroxylamine oxidation by M. capsulatus Bath (5.5 +/- 3.1 ° 00)
and N. europaea (-1.4 +/- 1.7 °/oo) and nitrite reduction by N. europaea (-7.7 +/- 3.1 ° 00)
differed significantly (AN OVA, fags): 247.9, p=0). These results demonstrate that the
mechanisms for hydroxylamine oxidation are distinct in M. capsulatus Bath and N.
europaea. The average SRO-N20 values of nitrous oxide formed during hydroxylamine
oxidation by M. capsulatus Bath (53.1 +/— 2.9 °/oo) and N. europaea (~23.4 +/- 7.2) and
nitrite reduction by N. europaea (4.6 +/- 1.4) were also significantly different (ANOVA,
fags): 279.98, p=0) and suggests although the nitrogen isotope value of the
hydroxylamine was similar, the A15 N associated with hydroxylamine oxidation by M.
capsulatus Bath and N. europaea (-2.3 and 26.0 °/oo for M. capsulatus Bath and N.
europaea respectively) provided evidence that differences in isotopic fractionation were
associated with two mechanisms. The site preferences in this study are the first measured
values for isolated microbial processes. The differences in site preference are significant
and indicate that isotopomers provide a basis for apportioning biological processes of

nitrous oxide.

Dedicated to the memory of my friend and father, Robert Sutka.

iv

ACKNOWLEDGEMENTS

I would like to thank my mom and sister for supporting me through the last 5
years. My family has often not known what I am doing, but they support me
unconditionally. I like to acknowledge the role of Hasand Gandhi as a friend and teacher.
He is undoubtedly the most patient and kind person that I have ever known and I will
miss him very much. I would like to thank Robyn Engleright for her friendship and
support that have made the last year more pleasant.

I would like to thank my committee for support and guidance through the last five
years. Especially my advisor Peggy Ostrom for her support through the good and bad
times. I would also like to thank Nathaniel Ostrom for his creative approaches toward
solving problems. I have learned a great deal about mass spectrometry in the laboratory
from both Peggy and Nathaniel. In addition, they have become good friends that have
been a wonderful source of support. Thanks also to Dr. John Breznak for his advice on
experimental design. Through his vast expertise and great skill as a teacher he was able
to provide great options, but allow me the freedom of deciding what would meet my
experimental needs. I would like to thank Dr. David Hyndman for serving on my
committee and providing support with the modeling efforts.

My dissertation would not be possible without the financial support that I received
throught my teaching assistantships in the Department of Chemistry and the Department
of Geological Sciences. I also received additional funding from the Graduate school that
made summer research and completion of my dissertation possible. In addition, I would
like to thank Dr. David Emerson for providing me some wonderful motivation for

finishing my dissertation in order to work on what will certainly be interesting research.

TABLE OF CONTENTS

LIST OF TABLES .......................................................................... viii
LIST OF FIGURES ........................................................................ ix
INTRODUCTION ......................................................................... 1
CHAPTER 1

Stable nitrogen isotope dynamics of dissolved nitrate in a transect from the North Pacific

Subtropical Gyre to the Eastern Tropical North Pacific.

Abstract .............................................................................. 3
Introduction .......................................................................... 4
Materials and methods ............................................................. 6
Results and Discussion ............................................................ 8
General water column characteristics .................................. 8
Nitrogen isotope biogeochemistry ..................................... 9
Conclusion ......................................................................... 19
CHAPTER 2

Nitrogen isotopomer site preference of nitrous oxide produced by Nitrosomonas

europaea and Methylococcus capsulatus Bath.

Abstract ............................................................................ 37
Introduction ........................................................................ 38
Materials and Methods ........................................................... 42
Culture and media ....................................................... 42
Concentrated cell suspensions ......................................... 43

Mass spectrometric analysis of nitrous oxide and isotope units...45

vi

Standard characterization ................................................ 45

Controls ..................................................................... 46
Results and Discussion ............................................................ 47
Standard characterization ................................................ 47
Controls .................................................................... 47

Bulk nitrogen and oxygen isotopic composition of nitrous oxide.48

Nitrogen isotopomeric site preference of nitrous oxide ............. 50
Conclusion ................................................................ 53
Bibliography ................................................................................ 61

vii

LIST OF TABLES

Table 1. Summary of nitrous oxide data including concentration, site preference, 815N-
N20 and O'BO-NZO from concentrated Methylococcus capsulatus Bath cell
cultures with hydroxylamine as the substrate. Each letter (A, B, C and D)
corresponds to a separate experiment and number indicates the time course
sample. Nitrous oxide formation via hydroxylamine oxidation is stimulated in
this set of experiments. ‘Anomalous value (37.7 0/00) omitted in the calculation of
average and standard deviation of the 231.80-N20smow .......................... page 56.

Table 2. Summary of nitrous oxide data including concentration, site preference and
ONO-N20 from concentrated Nitrosomonas europaea cell cultures with
hydroxylamine as the substrate. Each letter (A, B, C and D) corresponds to a
separate experiment and number indicates the time course sample. Nitrous oxide
formation by hydroxylamine oxidation is stimulated in this set of experiments.
....................................................................................... page 57.

Table 3. Summary of nitrous oxide data including concentration, site preference and
8180-N20 from concentrated Nitrosomonas europaea cell cultures with nitrite as
the substrate. Each letter (A, B, C and D) corresponds to a separate experiment
and number indicates the time course sample. Nitrous oxide formation via nitrite
reduction is stimulated in this experiment ..................................... page 58.

Table 4. Summary of istopomeric compositions of nitrous oxide in the literature with

an emphasis on biological pathways ............................................ page 59.

viii

LIST OF FIGURES
Figure 1. Locations of sampled stations in a transect from west to east ......... page 22.
Figure 2. Dissolved nitrate concentration , oxygen concentration, nitrate deficits,
S'SN-NO3' values, nitrification rates, N/P ratios, ammonium concentration and
fluorescence of samples collected at station 1 ............................... page 23.
Figure 3. Dissolved nitrate concentration , oxygen concentration, nitrate deficits,
OlsN-NO3’ values, nitrification rates, N/P ratios, ammonium concentration and
fluorescence of samples collected at station 2 ............................... page 25.
Figure 4. Dissolved nitrate concentration , oxygen concentration, nitrate deficits,
S'SN-NO3' values, nitrification rates, N/P ratios, ammonium concentration and
fluorescence of samples collected at station 3 ............................... page 27.
Figure 5. Dissolved nitrate concentration , oxygen concentration, nitrate deficits,
815 N-NOg' values, nitrification rates, N/P ratios, ammonium concentration and
fluorescence of samples collected at station 4 ............................... page 29.
Figure 6. Dissolved nitrate concentration , oxygen concentration, nitrate deficits,
S'SN-Nog’ values, nitrification rates, N/P ratios, ammonium concentration and
fluorescence of samples collected at station 5 .............................. page 31.
Figure 7. Dissolved nitrate concentration , oxygen concentration, nitrate deficits,
SlsN-NO3' values, nitrification rates, N/P ratios, ammonium concentration and

fluorescence of samples collected at station 6 .............................. page 33.

ix

Figure 8. Comparison of modeled to observed nitrate concentrations and 5'5N values of
dissolved nitrate at station 4. Parameters used at station 4 include a K of 7.65E-5
m2 sec", R0 of 0.85E-7 pmol L" see“, (I) of1.0E-3 m" and t1 ofO.76E-2 m".
...................................................................................... page 35.
Figure 9. Comparison of modeled to observed nitrate concentrations and S'SN values of
dissolved nitrate at station 5. Parameters used at station 5 include a K = 7.65E-5
m2 sec", R0 = 1.35E-7 nmol L" sec", 0) = 1.0E-3 m'1 and p = 0.76E-2 m'l.
....................................................................................... page 36.
Figure 10. Keeling plot approach to characterizing the site preference of the nitrous
oxide standard where Qa = moles of 98 % 15N"‘-nitrous oxide and Qm = total
moles of nitrous oxide ............................................................ page 55.
Figure 11. Proposed pathway for the formation of nitrous oxide from the reduction of
nitrite showing involvement of an enzyme-bound intermediates modified from

Weeg-Aerssens et al. (1988) ..................................................... page 60.

INTRODUCTION

Increases in the atmospheric concentration of the important greenhouse gas
(nitrous oxide) N20, have stimulated an interest in identifying its sources and sinks and
characterizing mechanisms of production of this important greenhouse gas. The ocean
environment is a net source of N20, but the relative importance of the oceans compared
to other environments is not well known (Yoshinari, 1997). In the oceans, two microbial
processes, nitrification and denitrification, produce N20. Isotopic data have suggested
that in oxic waters of the Pacific Ocean, nitrification is the dominant source of the trace
gas N20 (Kim and Craig, 1990; Dore and Karl, 1996; Ostrom et al., 2000). However, in
anoxic environments, denitrification likely plays an important role in the flux ofN20
(J orgenson et al., 1984). In addition, N20 can be produced during methanotrophic
nitrification (Yoshinari, 1985). The first objective of this dissertation research was to
delineate nitrogen cycling in oceanic environments and develop methods to identify
specific nitrogen cycle process that produce N20. The second objective was to define the
isotopomeric composition of N20 formed during distinct microbial pathways.

The first chapter details research that is part of a collaborative effort between
Michigan State University and the University of Hawaii to investigate the controls on the
production of N20 in the oceans. Nitrogen isotope biogeochemistry of dissolved nitrate
was examined in a west to east transect extending from the NPSG to the ETNP, where a
change in environmental conditions from oligotrophy to eutrophy occurs. While N20
was not investigated in this study, delineation of microbial processes in the water column
provides insight into the production mechanisms for N20 in related studies. In the

second chapter, laboratory studies were done to characterize the isotopomeric

composition of N20 produced during hydroxylamine oxidation by N. europaea and

M. capsulatus Bath and nitrite reduction by N. europaea. The isotopomeric data were the
first reported values for autotrophic nitrification, methanotrophic nitrification, and
nitrifier denitrification. Applications of this data include understanding in situ sources of

N20 in oceanic and soil environments.

CHAPTER 1
STABLE NITROGEN ISOTOPE DYNAMICS OF DISSOLVED NITRATE IN A
TRANSECT FROM THE NORTH PACIFIC SUBTROPICAL GYRE TO THE
EASTERN TROPICAL NORTH PACIFIC
ABSTRACT
The stable nitrogen isotopic composition of dissolved nitrate was determined at
six stations ranging from the oligotrophic North Pacific Subtropical Gyre (NPSG) to the
eutrophic Eastern Tropical North Pacific (ETNP). Dissolved inorganic nitrogen, oxygen
and phosphate concentrations were determined in water column samples at all six
stations. In productive, oxic waters nitrate isotopically enriched in 15N (maximum
OlsN-NO3' value of 12.5 0/00) was most likely the result of assimilatory nitrate reduction.
In contrast, high SUN-N03” values (maximum of 12.3 °/oo) in association with high nitrate
deficits and anoxic conditions supported the interpretation of isotopic fractionation due to
denitrification. A one-dimensional vertical advection and diffusion model was used to
estimate the fractionation factor for denitrification at two stations in the ETNP. A
comparison of modeled to observed SlsN-NO3' data indicated an isotopic enrichment
factor (a) of 30 at station 4 and 30 to 35 at station 5. Isotopically light nitrate (1.1 and
3.2 0/00) was observed in the upper 200 m of the water column at stations in the ETNP.
Tracer studies of 1"NFL; and biogeochemical indicators of nitrogen fixation, such as N/P

ratios, OlsN-PN and T richodesmium abundance, supported the interpretation of

nitrification as the most plausible explanation for low 5'5 N-N03' values observed in water
column samples. The nitrification rates increased along the transect from a maximum

rate of 1 nmol l'1 d'1 at station 1 to 23.7 nmol l‘1 d'1 at Station 6.

INTRODUCTION

Oceans account for approximately half of the world’s primary production (Karl,
1999). This productivity is fueled by recharge of nutrients from deep waters and in most
of the world’s oceans, the availability of fixed nitrogen limits productivity (Howarth,
1988). The availability of nitrogen is not homogeneous and this results in variations in
ecosystem productivity. Two systems with contrasting nitrogen dynamics include
subtropical/tropical gyres and coastal upwelling zones. Subtropical/tropical gyre
ecosystems are characterized by oligotrophic conditions and were once thought to be
marine deserts (Karl, 1999). However, given their large size (40 % of the Earth’s surface
area), gyre ecosystems can contribute substantively to global primary productivity (Karl,
1999). In contrast, the fraction of the world’s oceans characterized by coastal upwelling
zones is small, but eutrophic conditions support a disproportionately large amount of
primary productivity.

The North Pacific Subtropical Gyre (NPSG) is the largest open ocean gyre (area
of 2 x 107 kmz) (Karl, 1999). In this ecosystem, nitrogen and phosphorus are supplied to
the surface by diffusion across the thennocline and horizontal transport fi'om adjacent
waters (Karl, 1999; Reid et al., 1978). However, the observation that the amount of
primary productivity in the NPSG cannot be accounted for by the supply of nutrients by
diffusion from deep waters led to a reevaluation of nitrogen cycling (Letelier and Karl,
1998). The periodic appearance of T richodesmium blooms at station ALOHA and an
increase in the N/P ratio of dissolved and particulate matter provided evidence for a

significant role of nitrogen fixation in the NPSG (Karl et al., 1997). Quantitative

estimates indicate that diazotrophic microbes could fuel up to half of the new production
in the NPSG ( Karl et al., 1997).

In the ETNP, nutrients are supplied by upwelling off the Central American coast.
The large flux of sinking organic carbon from primary production increases oxygen
demand. The result of this increased respiration is a large lens of oxygen deficient water
in the ETNP. The suboxic conditions and upwelled nitrate provides the necessary
environment for water column denitrification. The associated loss of fixed nitrogen is
significant in the region (10 x 1012 to 30 x 1012 g N per year, Cline and Kaplan, 1975) and
variations in denitrification rates may contribute to glacial-interglacial changes in the
atmospheric concentration of carbon dioxide. In addition to effects on past climate
change, the production of nitric oxide and N20 at the oxic/anoxic interface in these
waters has important consequences on future climate change (Ward, 2000).

Studies of ecosystems that differ with respect to nutrient supply provide an
important framework for understanding nitrogen cycle controls on primary productivity
and climate change (Yoshida et al., 1984 Ganeshram et al., 1995; Dore et al., 1998; Karl,
1999). The current study contributes additional information on nitrogen cycling through
the analysis of samples across a west to east transect. The stable nitrogen isotopic
composition of dissolved nitrate and l5N-NI-L, oxidation studies are used make
interpretations of predominant microbial processes. This suite of data allows the
evaluation of nitrogen cycling and primary production in two contrasting systems, the

NPSG and ETNP.

MATERIALS AND METHODS
Water column samples were collected during a May-June 2000 sampling cruise that was
part of the Eastern Pacific Redox Experiment (EPREX) aboard the RN Roger Revelle.
Six stations were sampled during the cruise along a west to east transect from the
oligotrophic North Pacific gyre to the eastern tropical North Pacific (ETNP) (Station 1:
22.7°N, 158°W; Station 2: 16.0°N, 150°W; Station 3: 16.0°N, 136°W; Station 4: 15.8°N,
119.1°W; Station 5: 16.3°N, 107.2°W; Station 6: 15.6°N, 98°W) (Figure 1). The water
column was characterized at each station by determining temperature, salinity, dissolved
oxygen and chlorophyll fluorescence. Water samples were collected to determine nitrate
concentration and stable nitrogen isotope values.

Nitrate was extracted from the seawater samples by a distillation method (Ostrom
et al., 1997). During the distillation NH] was converted to NH3 by addition of 4 mL of
distilled 5 N NaOH to adjust the pH above 10. Finely ground Devarda’s alloy (0.3 g)
was added to the sample to reduce N03' to NH3 for a second distillation. Approximately
250 mL of condensate was collected in a flask containing 30 mL of 0.084 N HCl. The
sample was bound to 100 mg of zeolite molecular sieve in two successive bindings and
dried at 40 °C. Zeolite bound samples were placed in a quartz tube to which excess
copper and copper oxide were added, evacuated, sealed and heated to 850 °C for 1 hour.
and slowly cooled (Macko, 1981). Nitrogen (N2) was purified cryogenically on a
vacuum line and analyzed on a Prism isotope ratio mass spectrometer (Micromass,
Manchester, UK). Samples were corrected for background associated with addition of
Devarda’s alloy using an isotope mass balance equation (Ostrom et al., 1998). Accuracy

and precision of replicate samples for stable nitrogen isotopic analysis of dissolved nitrate

are typically better than 1 °/oo. The stable nitrogen isotopic composition is expressed in
per mil notation (°/oo):

5‘5N = [(‘5N/‘4N,,m,../ 'SN/‘4N,,,,.d,,d) - 1] x 1000 (Equation 1)
The isotopic standard for 6'5 N is atmospheric N2.

The concentration of dissolved ammonium in samples was determined during
shipboard analyses using a solvent extraction techniques (Brzezinski, 1988). Dissolved
oxygen concentrations were determined by Winkler titration (Carpenter, 1965). Analyses
of dissolved nitrate, nitrite and phosphate concentrations were determined at the
University of Washington (Unesco, 1994; Valderrama, 1981).

For determination of nitrification rates via 15N-ammonia oxidation, seawater
samples were collected at the depth of intended in situ incubation. Typically, samples
were taken at the nitracline, chlorophyll maximum, upper edge of the chlorophyll
maximum and at a depth 150 m below the chlorophyll maximum. To determine
nitrification rates 4 L of seawater was collected at selected depths below the chlorophyll
maximum in the water column. Isotopically enriched (99.9%15N-NH4CI) was added to
the seawater that was transferred into glass bottles. The 4 L bottles were lowered back to
the depth that they were taken and incubated in situ for 24 or 48 hours. At the end of the
incubation period, the bottles were brought to the surface, filtered, transferred to Nalgene
bottles and frozen until analysis. For determination of the S‘SN value of the dissolved
nitrate in the incubation samples, the nitrate was first extracted by distillation. A sample
volume of 1 L was distilled following a modification of the distillation technique
described by Ostrom et al. (1997). The modification was necessary to ensure complete

removal of residual 15N-NH4 from the seawater and distillation glassware. To remove

lSN-NH4, the sample was distilled three times. To remove trace NH4 from the distillation
apparatus, the glassware was washed in 1 N HCl and heated to 500°C for 2 hours. Tests
of this technique before sample analysis proved that the modifications successfully
removed residual ‘5 N from the distillation apparatus. The calculation of nitrification rates
from the oxidation of 15NH4 was based on the equations described in Glibert and Capone
(1993)
Nitrate deficits were calculated using equations of Cline and Richards (1972).

The amount of nitrate used as a substrate during denitrification is the difference between
the expected nitrate concentration and the observed concentration of nitrate plus nitrite.

N03'(¢xp¢c,cd) = 14.8 x P04+ (measured) (Equation 2)

N03' deficit = N03'(cxp¢ctcd) — (N 03'(m¢a,u,cd) + N02'(mcasu,cd)) (Equation 3)
Nitrogen to phosphorus ratios of inorganic dissolved nutrients were calculated according
to Fanning (1992).

N/P = ([N02'] + [N0;'] + [NH4+])/ [POX] (Equation 4)

RESULTS AND DISCUSSION
General Water Column Characteristics
Stations along the transect (Figures 2 to 7) capture the gradual broadening, both

horizontally and vertically of anoxic water from west to east and concomitant change
from oligotrophic conditions in the NPSG to eutrophic conditions in the ETNP (Cline and
Kaplan, 1975; Ward and Zafiriou 1988; Karl, 1999). The oxygen minimum zone occurs
at an increasingly shallow depth from west to east with a change from 700 m at station 1
to 65 m at station 6 (Figures 2 and 7). Furthermore, minimum oxygen concentrations at

station 1 are 30 uM and decrease to <1 pM at Station 6. The decreasing depth of the

oxygen minimum zone is associated with a change in depth of the chlorophyll maximum
from 140 m at station 1 to 40 m at Station 6 (Figures 2 and 7). The fluorescence
increases from west to east with a maximum of 0.25 RFU at Station 1 to 0.95 RFU at
station 6 (Figures 2 and 7). Ammonium concentrations in surface waters at station 1 are
between 0 and 20 nM and at stations 4, 5 and 6 increase to 100 to 300 nM (Figures 2, 5, 6
and 7). This is expected due to the enhanced productivity and subsequent mineralization
of organic matter in the eastern portion of the transect.

Nitrogen Isotope Biogeochemistry

The primary microbial processes that influence the nitrogen isotopic composition
of nitrate include denitrification, assimilatory nitrate reduction, nitrogen fixation and
nitrification (Cline and Kaplan, 1975; Mariotti et al., 1981; Liu et al., 1996). During
these microbial-driven nitrogen transformations, isotopic discrimination can result due to
a slight difference in the enzymatic reaction rate between 15 N and l4N containing
compounds (Mariotti et al., 1981). The result is an accumulation of 14N in the product
and 15N in the residual substrate (Mariotti et al., 1981).

Nitrification and nitrogen fixation can result in nitrate that is depleted in '5 N. The
fractionation factor associated with nitrogen fixation is small and produces fixed nitrogen
with a nitrogen isotopic composition approximately equal to that of atmospheric nitrogen
0 0/00 (Hoering and Ford, 1960; Minagawa and Wada, 1978). Isotopically light nitrate
can be produced by magnification of fixed nitrogen and subsequent nitrification. In
Kuroshio waters, l4N enriched nitrate (1 to 3 °/oo) was attributed to the activity of
unusually high numbers of a nitrogen fixer, T richodesmium (Liu et a1, 1996). In contrast

to nitrogen fixation, laboratory studies reveal that microbial nitrification has a relatively

large isotopic enrichment factor (a of -35 °/oo) (Mariotti et al., 1981; Yoshida, 1988),
where 8 is defined by:
s,,,,=(or-1)103 (Equation 5)
Where:
a = k('4N)/k(‘5N)
k(l4N) = the reaction rate constant for molecules containing 14N

k(15 N) = the reaction rate constant for molecules containing 15N

In Conception Bay, nitrification was proposed as the likely cause of low 515 N values of
dissolved nitrate (515 N-NO3') (average 0.2 °/oo) (Ostrom etal., 1997).

Nitrate enriched in 15N has been associated with the processes of denitrification
and assimilatory nitrate reduction by phytoplankton (Cline and Kaplan, 1975; Brandes et
al., 1998 and Voss et al., 2001). During the reduction of N03' to N2, the residual pool of
N03' becomes increasingly enriched in 15 N. This can be substantive due to a large 8
associated with denitrification of approximately -30 in previous studies in the ETNP
(V 055 et al., 2001; 8 = -27 +/- 3 °/oo, Brandes et al., 1998). In anoxic regions of the
ocean, denitrification has resulted in OISN-NO3' values as high as 18.7 ° 00 (V 085 et al.,
2001). In oxic waters, preferential assimilation Of '4NO3' by phytoplankton can also
result in high SUN-N03] (Cline and Kaplan, 1975). However, the fractionation due to
assimilatory nitrate reduction, 8 of O to -19, is not as large as that associated with
denitrification (Wada and Hattori, 1978; Wu et al., 1997; Sigman et al., 1999; Altabet et

al., 1999).

10

High 515 N-NO3' values observed in samples from suboxic (oxygen concentrations
equal to or less than 1 11M) waters at our stations suggest denitrification (Figures 4 to 7).
Maximum B'SN-N03' values of 11.8, 12.3 and 10.6 °/.,0 were observed in samples taken
from stations 4, 5 and 6 respectively. Nitrate deficits within anoxic waters are generally
high with maximum values of 14.0, 16.1 and 16.0 pM at stations 4, 5 and 6 and indicate
active denitrification (Figure 5, 6 and 7). In the ETNP, enrichment in 15’N of nitrate in
association with suboxic waters and high nitrate deficits has previously been attributed to
denitrification (Cline and Kaplan, 1975; Brandes et al., 1998; Voss et al., 2001).

The isotopic enrichment factor for denitrification was estimated using a model
that incorporated the vertical advection and diffusion of waters in and out of the suboxic
waters at stations 4 and 5. The typical model for estimating the fractionation factor for a
single step reaction is derived from the Rayleigh equation (Mariotti et al., 1981; Macko et
al., 1986). An important assumption in the Rayleigh model is that after the reaction is
initiated, there are no additional inputs of substrate and that the reaction is unidirectional.
Thus, estimating the fractionation factor for denitrification from a Rayleigh model in
open ocean systems is inappropriate because there is an influx of nitrate from water
bodies below the zone of denitrification. In open-systems, studies have employed a one-
dimensional vertical advection and diffusion model to describe the concentration and
isotopic composition of nitrate as a function of depth in the water column (Cline and
Kaplan, 1975). The current study applied a vertical advection-diffusion model to
estimate a for denitrification using water column data from 150 m to 700 m at stations 4
and 5 (Cline and Kaplan, 1975; Velinsky et al. 1991). Boundary conditions included a

depth interval with a linear temperature and salinity relationship and apparent absence of

11

microbial processes other than denitrification on the nitrogen isotopic composition of
dissolved nitrate. The first step is to find an analytical solution of the following
differential equation (Equation 6) subject to the boundary conditions [N]z= z] = [N]1, [N]z
= 22 = [N12-

d2[N]/d22 + wd[N]'/dz — (Roe'”)/K = 0 (Equation 6)

Where:
2 = depth (m)

[N] = concentration of nitrate (uM)

K = vertical eddy diffusion coefficient (m2 sec“)

R0 = rate of denitrification (pmol L'l sec")

00 = mixing parameter (Vz/K) (m'l)

Vz = vertical velocity (m see")

p = describes the attenuation of microbial activity with depth (m")
The solution of Equation 6 is used to determine the values for the parameters that provide
the best comparison of modeled to observed nitrate concentrations. The parameters
evaluated in the first step include K, R0, (0 and u. The second step is to obtain a
numerical solution of the isotope portion of our model. Using an implicit finite
difference approximation to Equation 7 produces a tridiagonal matrix. The system of
equations was inverted and solved using the Thomas algorithm (Roache, 1998). We
increased the computational mesh until successive refinements produced no additional
change in the results. A typical step size used in the computations is ~ 0.5 m.

dZC/dzz + to dC/dz —(1/a)C/[N] (Roo'W/K)

+ ((Ot-l)/Ot)( Room/K) 1000 = 0, (Equation 7)

12

Where:
C = 5 ‘ [N]
. o = o‘SN—No; (°/,,)
or = isotopic fractionation factor
A comparison of modeled to observed nitrate concentrations at stations 1 to 3 indicated
that denitrification was not active at the time that we sampled the water column. The
parameters used at station 4 that provided a reasonable comparison of modeled to
observed nitrate concentrations include K = 7.65E-5 m2 sec", R0 = 0.85E-7 umol L'l
sec", 0) = 1.0E-3 m‘1 and p. = 0.76E-2 rn'I (Figure 8). At station 5, the values for K, 00
and u were the same, but a value of R0 of 1.35E-7 pmol L'l sec'l was used (Figure 9).
Despite a good comparison of modeled to observed nitrate concentrations at station 6, e
was not estimated due to insufficient isotopic data in the modeled depth interval. At
station 4, the model estimate was an e of -30. Since estimates of a were very sensitive to
changes in 5N-N03' near the boundary, we define our estimates of 8 based on data near
the center of the modeled depth interval. A comparison of modeled to observed
SISN-NO3' at station 5 suggest an e of -30 to -35 (Figure 9). The non-ideal fit of the
modeled to observed data at station 5 is likely due to the low number of observed
OlSN-N03' values. A comparison of the results from station 4 to those at station 5 suggest
that the isotopic fractionation factor is Similar despite a change in the rate of
denitrification (R) from 0.85E-7 umol L'l sec“1 at station 4 to 1.35E-7 umol L'l sec'1 at
station 5. Our estimates of the isotopic enrichment factor are within the range of previous

studies in the ETNP (a = -27 to -60) (V OSS et al., 2001; Brandes et al., 1998; Cline and

Kaplan, 1975) and closely resemble the value for majority of the estimates (a of -30).

13

In the upper water column (<200 m depth), multiple processes influence the
distribution of inorganic nitrogen including assimilatory nitrate reduction, nitrogen
fixation and nitrification. This leads to the potential for overlap of active processes
within similar depth intervals. For example, in an incubation study using 15N substrates,
Ward et a1. (1989) demonstrated that nitrification occurs simultaneously with the
assimilation of nitrate by phytoplankton. Interactions between microbial-driven nitrogen
transformations are further complicated in the ETNP where a shallow oxygen minimum
zone promotes denitrification at the base of the euphotic zone. The potential for
interaction between nitrification and denitrification at oxic/anoxic interfaces in the
environment has been noted by Ward (1996). At the boundary of the interface, a flux of
substrates and products can promote a coupling of the oxidation and reduction of
different inorganic nitrogen species (Ward, 1996). As a consequence of the coupling and
Spatial overlap of processes, interpretations of the isotopic composition of inorganic
nitrogen in the upper water column often requires additional geochemical information
such as nitrogen to phosphorus ratios, fluorescence and dissolved oxygen concentration.

In productive, oxic waters high S'SN-N03‘ values that occur at stations 2, 5 and 6
are suggestive of assimilatory nitrate reduction (Figures 3, 6 and 7). In our data, this is
most evident at station 6 where high SUN-N03” values of 8.3 and 10.0 ° 0, at 28 and 44 m,
respectively, are coincident with the chlorophyll maximum (as interpreted from
fluorescence data) (Figure 7). In addition, localized isotopic maxima in oxic surface
waters at stations 2 and 5 likely reflect an influence of assimilatory nitrate reduction
(Figures 3 and 6). This is consistent with previous interpretations of high SUN-N05

found in near-surface waters of the ETNP (Cline and Kaplan, 1975).

14

Low S'SN-N03' values (1.1-3.2 °/oo) in the upper 200 m depth are Observed in our
samples from stations 3, 4, 5 and 6 (Figures 4 to 7). The processes of nitrogen fixation or
nitrification could be invoked as the plausible cause of this isotopically light nitrate. In
near—surface waters of the ETNP, low 515 N-N03' values (5 °loo) were attributed to
nitrogen fixation (Brandes et al., 1998). Estimates from an isotope mass balance model
indicated that as much as 20 % of the nitrate was derived from nitrogen fixation (Brandes
et al., 1998). However, isotopic data alone cannot distinguish nitrate derived from
nitrification and nitrogen fixation. Other lines of evidence indicative of nitrogen fixation
include the 615 N value of particulate organic nitrogen (PON), nitrogen to phosphorus
ratios and the abundance of nitrogen fixing organisms (Saino and Hattori, 1987; Liu et
al., 1996; Karl, 1999).

Studies in the Western Pacific and Sargasso Sea have suggested that isotopically
light PON (f)15 N of -3 °/oo to 2 °/oo) is the result of nitrogen fixation activity (Saino and
Hattori, 1987; Altabet et al., 1991). These low OISN values of PON occur in waters
where T richodesmium, a nitrogen fixing organism, are present (Saino and Hattori, 1987;
Altabet et al., 1991). The observation that a culture of T richodesmium had a BISN value
of -l.6 °/,,o provides further evidence that low 5'5 N values of PON (SUN-PON) can result
from nitrogen fixation (Carpenter et al., 1997). Within the same depth interval where we
observed isotopically light nitrate (ETNP), 8'5N-P0N values reported by Voss et al.
(2001) between 4.4 and 8.2 ° 00 are not indicative of nitrogen fixation.

In addition to the nitrogen isotopic composition of PON, the nitrogen to
phosphorus ratio of dissolved and particulate matter is also used to indicate nitrogen

fixation. This is based on the observation that cells of nitrogen fixing organisms are

15

characterized by nitrogen to phosphorus ratios in excess of Redfield (16:1 and 44:1 for
Redfield and nitrogen fixing cells respectively) (Carpenter, 1983). Nitrogen to
phosphorus ratios of dissolved inorganic nitrogen and phosphorus were demonstrated to
be significantly higher than Redfield (40:1) in Kuroshio water northeast of Taiwan where
unusually abundant populations of nitrogen fixers were found (Liu et al., 1996). In
previous studies at station 1, an increase above the Redfield ratio to 20:1 or higher in the
nitrogen to phosphorus ratio of the dissolved and particulate pools was interpreted by
Karl et al. (1997) as evidence for the importance of nitrogen fixation. Our data indicate
that maximum nitrogen to phOsphorus ratios of total dissolved inorganic nitrogen to total
phosphorus were never higher than 12.1 :1 in the upper 200 m of all stations (Figures 2 to
7). In the region of our transect where we observed isotopically light nitrate the nitrogen
to phosphorus ratios are sub-Redfield and not consistent with a significant input of
nitrogen via nitrogen fixation. Station 1 (ALOHA) was demonstrated by Karl et al.
(1997) to have seasonal (July to October) and ENSO period increases in the significance
of nitrogen fixation. However, we sampled station 1 in May and an El Nino event did not
take place in 2000.

The abundance of T richodesmium is potentially indicative of the significance of
nitrogen fixation in marine environments. In the western Pacific, isotopically light nitrate
(1 to 3 0Am) is observed in waters characterized by a cell density of T richodesmium as
high as 4.9 x 106 trichomes m'3 (Liu et al., 1996). At station 1, Karl et al. (1997) noted
increases in T richodesmium abundance in association with seasonal increases in N to P
ratios of particulate and dissolved matter. East of 130 °W in the North Pacific

T richodesmium has not been observed (Carpenter, 1983). Thus, the locations where

16

isotopically light nitrate were observed in our current study fall outside the easterly range
of T richodesmium. Although the influence of nitrogen fixation from other genera (e.g.
Synechococcus, Cyanothece and Erythrosphaera) in these waters cannot be discounted,
T richodesmium is the dominant diazotroph in tropical and subtropical oceans (Carpenter
et al., 1997 Zehr et al., 2000). Thus we conclude that aside from isotopically light
nitrate, OISN values of PON, nitrogen to phosphorus ratios and T richodesmium abundance
do not suggest nitrogen fixation as a source of isotopically light nitrate. Thus, we
consider nitrification as a likely mechanism.

Previous incubation studies have found nitrification rates of 1 to
137.4 nmol L'1 d'1 at ALOHA (station 1) and a maximum of 20 nmol L'l d'l in the ETNP
(Dore and Karl, 1996; Ward and Zafiriou, 1988). Our incubation studies document that
nitrification occurs at every station in this study (Figures 2 to 7). At station 1, maximum
nitrification rates of 1 nmol L'l (II are within the range of previous studies
(1 - 137.4 nmol L'1 d") (Dore and Karl, 1996) (Figure 2). The large variation in
nitrification rates at station 1 could be due to differences in sampling depth or possible
temporal dynamics of nitrification rates. The data for station 6 (maximum of
23.7 nmol L'1 d") are similar to the rate of 20 nmol L'1 d'1 from a study in the ETNP by
Ward and Zafiriou (1988) (Figure 7). There was an increase in nitrification rates from
west to east in the transect with a maximum rate at station 6 about 20 times greater than
station 1. High ammonium concentrations (100 toi300 nM) at stations 5 and 6 indicate
ample substrate for nitrification. The data also indicate that the highest rates of
nitrification generally occur at the base of the chlorophyll maximum (Figures 2 to 7).

The depth distribution of rate measurements could be explained by light inhibition of

17

nitrifiers and competition for ammonium with phytoplankton. Culture studies have
indicated that nitrifiers are inhibited by high light (Horrigan et al., 1981). At the depth
that phytoplankton are typically most active high light levels and are inhibitory to
nitrification. As light intensity decreases with depth, nitrifiers are less inhibited and
phytoplankton becomes less active. The data demonstrate that minima in SlsN-N03'
occurs where nitrification was active. Most notably at station 4, the maximum
nitrification rate of 15.8 nmol L’1 d'1 and the minimum SUN-N05 of 3.2 °/oo coincide
(Figure 5). However, maximum nitrification rates do not always coincide with isotopic
minima. For example at station 5, the nitrification rate is highest at a depth of 65 m, but
the 515N-N03’ of approximately 9 °/00 at a depth of 75 m suggests a predominance of
assimilatory nitrate reduction (Figure 6). This is consistent with a study by Ward et al.,
(1989) that demonstrated that nitrification and assimilatory nitrate reduction activity at
the same depths. The influence of assimilatory nitrate reduction can obscure the isotopic
signature of nitrification. The decrease in the 6'5N-N03' with depth at stations 5 and 6
above 150 suggests that the influence of assimilatory nitrate reduction on the isotopic
composition of nitrate decreases with depth and the importance of nitrification increases.
Thus, isotopically light nitrate observed in the data at stations 5 and 6 (128 and 104 m,
respectively) reflects the predominant influence of nitrification processes despite
maximum nitrification rates at a depth of 65 m (Figures 6 and 7). In combination with
knowledge of isotopic fractionation during nitrification, the incubation studies support
the interpretation that low SlSN-N03 (1.1-3.2 °/oo) in the data reflect the influence of

nitrification rather than nitrogen fixation.

18

CONCLUSIONS

Variations in the relative importance of microbial processes of the nitrogen cycle
as a function of ecosystem conditions have implications for trace gas production and
primary productivity. Low O‘SN-N03' values in the ETNP have been interpreted to be
indicative of nitrogen fixation (Brandes et al., 1998). In the past, inputs of nitrogen via
fixation and the abundance of diazotTOphic organisms in the open ocean were considered
insignificant (Ward, 2002; Paerl and Zehr, 2000). More recently, studies of the NPSG
have revealed a significant contribution of nitrogen fixation to primary production (Karl
et al., 1997 and Capone er al., 1997). The ETNP is a major site for the loss of fixed
nitrogen via denitrification and a recent study suggests that nitrogen fixation has the
potential to balance the nitrogen losses (Brandes et al., 1998). However, our results
indicate that nitrification is an alternative explanation for isotopically light nitrate in the
euphotic zone of the NPSG and ETNP during the period of our study. In contrast to
nitrogen fixation, nitrification does not increase the pool size of available nitrogen.
Furthermore, the oxidation of ammonia to nitrate results in coupling of nitrification and
denitrification that contributes to a loss of fixed nitrogen to the system. If the loss of
nitrogen via coupled nitrification/denitrification in the euphotic zone is found to be
significant, it has the potential to impact estimates of productivity and organic matter
export.

The distribution of nitrification activity in the water column is inconsistent with
the new production paradigm of Dugdale and Goering (1967). A central component of
this paradigm is that increases in the productivity result from delivery of new sources of

nitrogen such as nitrate from deep water or nitrogen fixation (Dugdale and Goering,

19

1967). Thus, nitrate delivered to the euphotic zone via upwelling from deep waters and
nitrogen derived from fixation are termed “new nitrogen” (Dugdale and Goering, 1967;
Eppley and Peterson, 1979). In contrast, ammonia is termed “regenerated nitrogen” since
it is the product of Short-term recycling processes in the euphotic zone (Dugdale and
Goering, 1967). In order to maintain steady state levels in productivity, imports of new
nitrogen and exports from the system in the form of sinking particles and fisheries harvest
must be balanced (Eppley and Peterson, 1979). An important assumption is that
nitrification occurs solely in deep waters. It is now recognized that nitrification in the
euphotic zone is significant and contributes to the phytoplankton nitrate demand (Dore
and Karl, 1996; Ward, et al., 1989). Our results support the interpretation of these studies
(Dore and Karl, 1996; Ward, et al., 1989) and for the first time provide corroborating
stable nitrogen isotopic evidence for euphotic zone nitrification. It is clear that euphotic
zone nitrification must be accounted for in models of primary productivity and studies of
nitrogen isotope biogeochemistry.

The production of isotopically light nitrate in the euphotic zone has implications
for interpretation of nitrogen isotope signals in modern and ancient environments.
Variations in the 5'5 N of sedimentary organic matter are thought to be indicative of past
changes in nutrient inventories or utilization (Ganeshram et al.,1995 and Farrell et al., -
1995). Ganeshram et al., (1995) suggested that nitrate concentrations in oxygen deficient
waters are regulated by variations in the rate of denitrification (Ganeshram et al., 1995).
They assumed that the isotopic composition of organic matter produced in near surface
waters and deposited in sediments is a function of fractionation during phytoplankton

uptake and the isotopic composition of the source nitrate (Ganeshram et al., 1995). A

20

second assumption is that nutrients in near surface waters of the ETNP are completely
utilized by phytoplankton. Thus, the nitrogen isotopic composition of the organic matter
should be identical to that of its source (nitrate) and denitrification controls the 6'5 N of
nitrate (Ganeshram er al., 1995). Increases in denitrification would be reflected as an
increase in the nitrogen isotopic composition of nitrate and associated organic matter.
However, euphotic zone nitrification could obscure the isotopic signature of
denitrification by contributing MN enriched nitrate to the nutrient pool. Therefore, the
expectation that the nitrogen isotopic composition of organic matter is a function of the
extent of denitrification may not be entirely correct. An alternative explanation for
fluctuation in the nitrogen isotopic composition of sedimentary organic matter is
variation in the importance of euphotic zone nitrification. This could result from
increases in upwelling, which would stinrulate primary productivity and subsequently
increase the supply of regenerated NHa to support euphotic zone nitrification. At present
time, the relative importance of increases in the rate of denitrification versus euphotic
zone nitrification as mechanisms to influence the isotopic composition of nitrate during
glacial periods is unknown. However, our results definitively demonstrate that it is not
appropriate to conclude that changes in the rate of denitrification and fractionation during
assimilatory nitrate reduction are the only processes driving sedimentary nitrogen

isotopic signals.

21

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36

CHAPTER 2
NITROGEN ISOTOPOMER SITE PREFERENCE OF N20 PRODUCED BY
NITROSOMONAS E UROPAEA AND ME T H YLOCOCC US CAPSULA T US BATH
ABSTRACT
The relative importance of individual microbial pathways to nitrous oxide (N20)

production is not well known. The intramolecular distribution of ‘5N in N20 provides a
basis for distinguishing biological pathways. Concentrated cell suspensions of M.
capsulatus Bath and N. europaea were used to investigate the site preference of N20 by
microbial processes during nitrification. The average site preference of N20 formed by
hydroxylamine oxidation by M. capsulatus Bath (5.5 +/- 3.1 ° 00) and N. europaea (-1.4
+/- 1.7 ° 00) and nitrite reduction by N. europaea (-7.7 +/- 3.1 ° 00) differed significantly
(AN OVA, fags): 247.9, p=0). These results demonstrate that the mechanisms for
hydroxylamine oxidation are distinct in M. capsulatus Bath and N. europaea. The
average filsO-NZO values of N20 formed during hydroxylamine oxidation for M.
capsulatus Bath (53.1 +/- 2.9 ° 00) and N. europaea (-23.4 +/- 7.2) and nitrite reduction by
N. europaea (4.6 +/- 1.4) were significantly different (ANOVA, 11235): 279.98, p=0) .
Although the nitrogen isotope value of the source hydroxylamine was similar, the A15 N
associated with hydroxylamine oxidation by M. capsulatus Bath and N. europaea (-2.3
and 26.0 °/oo for M. capsulatus Bath and N. europaea respectively) provided evidence
that differences in isotopic fractionation were associated with two mechanisms. The site
preferences in this study are the first measured values for isolated microbial processes.
The differences in site preference are significant and indicate that isotopomers provide a

basis for apportioning biological processes of N20.

37

INTRODUCTION
N20 is a trace gas that on a molecule for molecule basis is 200 times more

effective than carbon dioxide as a greenhouse gas (Lacis et al., 1981). In addition,
concentrations of N20 in the atmosphere have been increasing at a rate of 0.3% per year
(Prinn et al., 1990; Rasmussen and Khalil, 1986). The rise in atmospheric concentrations
of N20 is thought to result predominantly from microbial activity (J iang and Bakken,
1999) and not anthropogenic emissions. In certain environments, such as agricultural
fields, management practices could mitigate emissions of carbon dioxide and trace gases
by stimulating or inhibiting microbial processes (Robertson et al., 2000). Mitigation of
future emissions by management in such environments depends on knowledge of the
microbial processes responsible for trace gas production. To date, many studies have
focused on defining the relative importance of nitrification and denitrification, but have
not addressed the possibility of methanotrophic nitrification as a mechanism for the
production of N20 (Blackmer et al., 1980; Kim and Craig, 1990; Dore et al, 1998, Naqvi
et al., 1998; Perez et al, 2001; Ostrom et al., 2001). The possible contribution of
methane oxidizing organisms to N20 production was demonstrated in a culture study by
Yoshinari (1987). Defining the relative importance of methane oxidizers and autotrophic
nitrifiers will be an important step in managing N20 emissions from terrestrial
environments (Robertson et al., 2000).

The microbial processes in soil and water that produce N20 include nitrification,
denitrification, methanotrophic nitrification and heterotrophic nitrification (J iang and
Bakken, 1999). Recently, the importance of autotrophic nitrification as a source of N20

in oceanic environments has been emphasized (Dore etal., 1998; Naqvi et al., 1998).

38

During nitrification, N20 is formed by two mechanisms. In the first mechanism, N20 is a
by-product of the oxidation of hydroxylamine to nitrite by the enzyme hydroxylamine
oxidoreductase (Jiang and Bakken, 1999). In the second, nitrite is reduced to N20 by the
enzyme nitrite reductase (Ritchie and Nicholas, 1972; Poth and Focht, 1985). N20
production via nitrite reduction is stimulated by oxygen limitation (Ritchie and Nicholas,
1972; Poth and F ocht, 1985). This pathway was first documented in studies using the
organism, Nitrosomonas europaea and is referred to as nitrifier denitrification (Ritchie
and Nicolas, 1972; Poth and Focht, 1985). This term distinguishes it from denitrification
by heterotrophic denitrifiers, although the enzymes involved are similar (Ritchie and
Nicholas, 1974; Poth and Focht, 1985).

Like autotrophic nitrifiers, methanotrophic bacteria are capable of oxidizing
ammonia and hydroxylamine. An important consequence of ammonia oxidation by
methanotrophs is the production of N20 (Hanson and Hanson, 1996). While the
production of N20 by methane oxidizers has been evaluated in culture studies (Yoshinari,
1985), N20 production by methanotrophs in field-based studies has not been
unequivocally demonstrated.

Two studies provide important information on the role of methanotrophs in
nitrification (Bodelier and Frenzel, 1999; Mandemack et al., 2000). Using competitive
inhibition, Bodelier and Frenzel (1999) showed that methanotrophs were major
contributors to nitrification in the rice rhizosphere. The results of Mandemack et al.,
(2000) suggest the involvement of methane oxidizers in N20 production in landfill soils.
In incubations of these soils, the production of N20 was stimulated by pre-treatment with

1 % methane and rates were proportional to methane concentration (Mandemack et al.

39

2000). In addition, fatty acids isolated within the zone of maximum N20 flux were
indicative of type H methanotrophs (Mandemack et al., 2000). While these studies
provide evidence for a role of methane oxidizers as potential contributors to N20
production, they do not definitively demonstrate production of N20 by methanotrophs.

The intramolecular distribution (isotopomers) of 15 N in N20 is emerging as a new
tool for defining the relative importance of microbial sources of N20 (Toyoda and
Yoshida, 2000; Perez et al, 2001; Popp et al., in press; Toyoda et al., in press). In the
N20 molecule, the central nitrogen atom is preferred to as the alpha (or) and the terminal or
end nitrogen atom is referred to as beta (B) (Toyoda and Yoshida, 1999). Quantifying the
distribution of 15N in N20 is possible due to the fragmentation of N20+ to NO+ in the ion
source of the mass spectrometer. The isotopic composition of 15N in the a position is
determined from the fragment ion, NO+, after correction for rearrangement that occurs in
the ion source (Toyoda and Yoshida, 1999). The relative abundance of 15 N in the
molecule N20 is commonly expressed in 5 notation.

6 = [(Rsamplc/Rsmdard) — 1] * 1000 (Equation 8)

Where:

Rsamplc = 15’N/ 14N and ‘SO/wO for sample
Rsmdard = 15N/MN and 18O/mO for standard

The standards for nitrogen and oxygen isotope measurements are atmospheric N2 and
VSMOW, respectively. The site preference is equal to the difference between S'SN“ and
5‘5NB.

Site preference = 515N“ — S'SNB (Equation 9)

Where:

4O

5'5N“ = (‘5R“(,,m,I.,/‘5R“(..d, — 1) *1000
B'SNB = (‘5R5(,,mpI.I/'5R”IIIII) — 1) *1000
15Ror = [l4NlSNl60/14N14NIOO]

ISRB =[15N14N16O/MNMNI6O]

Studies by Toyoda and Yoshida (2000) and Breninkenmeijer and Rockman
(2000) were a critical step in suggesting that isotopomers could provide a basis to
apportion sources of N20. Toyoda and Yoshida (2000) demonstrated that the site
preference of N20 varied throughout the atmosphere, but the general trend was a lower
site preference in the troposphere samples and a greater site preference in the
stratospheric air samples. The low site preference for the troposphere samples is likely a
result of local soil emissions and fossil fuel combustion sources (Toyoda and Yoshida,
2000). An important research goal since the Toyoda and Yoshida (2000) study is source
apportionment. Thus far, efforts have been directed at determining the isotopomer
compositions of N20 in environments that have been implicated as sources, such as
oceans and soils, to determine which source is most significant (Perez et al., 2001; Popp
et al., in press; Toyoda et al., in press). While this work has increased our understanding
of the role of biological mechanisms in determining the isotopomeric composition of '
N20, the isotopomer signature of distinct microbial pathways is still undefined. In order
to determine the isotopomer signatures associated with individual microbial pathways, it
is important to utilize experimental conditions that isolate distinct pathways.

This study examines the isotopomer site preference of N20 produced via

hydroxylamine oxidation by cultures of a methane oxidizer and autotrophic nitrifier and

41

reduction of nitrite via nitrifier denitrification. Nitrosomonas europaea was selected as a
candidate autotrophic nitrifier because the physiology and biochemistry of ammonia
oxidation is best understood in this species (Poth and Focht, 1985; Bothe et al., 2000).
Among the methane oxidizing organisms, we chose Methylococcus capsulatus Bath
owing to its ability to oxidize ammonia (Dalton, 1977). The objective of this study is to
define the site preference distribution for the N20 produced during methanotrophic
nitrification, autotrophic nitrification and nitrifier denitrification. This is an important
first step in the use of isotopomers as a tool for apportioning microbial sources and in
particular, evaluating the importance of methanotrophic nitrification as a source of N20
in certain environments.

MATERIALS AND NIETHODS

Culture and Media

A culture of Methylococcus capsulatus Bath grown on solidified nitrate mineral
salts (N MS) medium was provided by A. DiSpirito from Iowa State University. The
cultures were maintained in modified nitrate mineral salts medium (NMS)-Whittenbury
medium amended with 5 uM CuSO4 in 25 mL test tubes with a headspace of 3:7 (vzv)
methane:air (Whittenbury and Dalton, 1981). Prior to inoculation, 5 mL of medium
containing all components except the phosphate and vitamin solution were dispensed and
autoclaved. The medium was cooled and amended with the phosphate and vitamin
solution. The final pH was adjusted to 6.9-7.1 using 1 M Na2C03. The complete NMS
medium was inoculated with 0.1 mL of stock culture and incubated horizontally at 40 °C.
Stock cultures were stored in medium containing 5 % v/v DMSO held in 2.5 mL

cryogenic vials (N algene) and frozen at -70°C.

42

A frozen stock of Nitrosomonas europaea ATCC 19718 was purchased and
shipped on dry ice from the American Type Culture Collection, Manassas, VA. The
recipe for the medium used to grow N. europaea was provided by A. Hooper, University
of Minnesota. The medium consisted of 7.91 g NH4804, 0.76 g KzHPO4' 3HzO, 1 mL
0.5 % w/v Phenol red solution, 1 mL of trace element solution A and 3 mL of trace
element solution B per 1.0 L of E-Pure water (A. Hooper, pers.comm). Trace element
solution A is an autoclaved mixture containing 7.91 g CuSO4, FeSO4, and 5.89 g EDTA
in 1.0 L of E-Pure water. Trace element solution B is an autoclaved mixture containing
68 g MgClz ' 6H20 and 3.08 g CaClz ' 2HzO per 1.0 L of E-Pure water. Approximately
100 mL of sterile media was dispensed into 300 mL tissue culture flasks and the final pH
was adjusted to 7.5-7.7 with 5 % (w/v) K2CO3. At least 10 mL of culture was added to
inoculate fresh media in tissue culture flasks. The cultures were incubated horizontally at
27 °C. The cultures were checked for purity by inoculating 10 mL of culture in a 20 mL
volume of tryptic soy broth (TSB) in a 60 mL serum bottle. Growth was not observed in
these tests after 30 days. Stock cultures were stored in a 5 % v/v DMSO at -70 °C.

Concentrated cell suspensions

Concentrated cell suspensions were used to obtain sufficient concentrations of
N20 for subsequent mass spectrometric analysis. Approximately 1 mL of Methylococcus
capsulatus Bath culture was used to inoculate each of ten separate 160 mL serum bottles
with approximately 30 mL of NMS media and 30 % methane in the headspace. After
40 hours of incubation at 40 0C, the ten cultures were combined in two 250 mL centrifuge
bottles (Nal gene). Cells were concentrated by centrifugation (8,000G for 10 minutes at

5 ° C). After the supernatant was decanted, the concentrated cells were combined in one

43

centrifuge cup and resuspended with fresh NMS medium to an approximate volume of
20 mL. For each experiment, four 25 mL butyl stoppered serum test tubes (Bellco) were
prepared with 3 mL of concentrated cells and 300 11L of a 0.01 M hydroxylamine. The
tubes were stoppered with air in the headspace. The tubes were incubated horizontally at
40 0C.

Prior to concentration, Nitrosomonas europaea was maintained in 300 mL
tissue culture flasks that contained 100 mL of media in each bottle. The tissue culture
flasks were incubated horizontally at 27 0C for three days. Prior to concentration, a
subsample was used to inoculate 20 mL of TSB in a 60 mL serum bottle to test for
heterotrophic contamination. In all cases, there was no growth in the bottles with TSB
following 30 days of incubation. For sufficient cell densities, ten flasks were
concentrated via centrifugation (8,000G for 10 minutes at 5 ° C). The cell pellets were
washed once and resuspended with 20 mL of 0.1 M phosphate buffer (pH 7.5) containing
171 5.1M CaClz and 78.7 uM MgClz (Poth and F ocht, 1985). For experiments involving
hydroxylamine oxidation, four tubes with 2 mL of cell suspension and 300 uL of 0.01 M
hydroxylamine solution were stoppered with a headspace of air. To test the isotopomer
composition of N20 produced during the nitrifier denitrification pathway, experiments
were conducted with nitrite. For each nitrifier-denitrification experiment, at least four
serum test tubes were prepared with 2 mL of cell suspension and 300 pL of 0.01 M
NaNOz solution. The headspace of the nitrite reduction experiments was purged with N2

and stoppered.

44

Mass spectrometric analysis of N 20 and isotope units

Nitrous oxide was sampled from the headspace of the concentrated cell
suspensions using a 500 11L or 5 mL gas tight syringe. The gas sample was injected
immediately into a Trace Gas system (Micromass) interfaced to an Isoprime (Micromass)
mass spectrometer. Using helium as the carrier gas, NzO was purified and concentrated
by cryogenic trapping followed by chromatographic separation on a Porapak Q column.
Prior to introduction into the mass spectrometer, carbon dioxide and water were removed
from the headspace sample in the Trace Gas system with Carbosorb. The nitrogen and
oxygen isotopic composition of N20 and the fragment ion NO+ is determined in a single
analysis. Triplicate analyses of a N20 sample demonstrate reproducibility of +/- 0.9 for
SlsNa-NZO, +/- 0.3 for s‘SN-Nzo and 5'80-Nzo. Calculations of s'SNa-Nzo and
BISNB-NZO were done by equations in Toyoda and Yoshida (1999). We observed a
rearrangement factor of 18 % versus previous literature estimates of 8 % (Toyoda and
Yoshida, 1999; Breninkenmeijer and Rockman, 2000). This difference suggests that the
rearrangement factor must be evaluated for each instrument. The correction for 17O for
the 5'5N-NZO value was made according to method described by Brand (1995). The
concentration of N20 was determined from the area under the mass 44 trace.

Standard characterization

A Keeling plot approach was used to determine the BISN“ and site preference of
an in-house pure (>99.9%) N20 tank standard (Flanagan and Ehleringer, 1998). A series
mixtures of 98 % + Na-N20 (Cambridge Isotope Laboratories, MA) with the tank
standard NzO (SlsN-NZO = 0.83 °/oo, SIBO-NZO = 37.68 ° 00) were performed to provide

an expected range in SUN“ of 10 to 170 °/oo. Mixtures were performed by injecting

45

known volumes of the Cambridge Isotope standard using a gas-tight syringe and injecting
into a glass 150 mL vessel (exact volume known) containing the pure N20 tank standard
(both the syringe and glass vessel were at atmospheric pressure). To obtain appropriate
quantity of N20 for analysis on the Trace Gas system 500 11L of the resulting mixture
was injected into a second glass vessel (500 mL) containing pure N2 at atmospheric
pressure. A volume of 200 uL from each of the N20-N2 mixtures was injected into the
Trace Gas system. Each isotopic analysis was completed in triplicate. On an xy plot
Qa/Qm vs. SlsNa-NZO of the mixture, the y—intercept of the best fit line for the 8 mixtures
yielded the S‘SNa-NZO of the laboratory N20 tank standard (Q. = moles of 9s % +
N°-Nzo and Qm = total moles of N20). Determinations of the ”N‘-N20 of the tank
standard were based on the intercept of the plot of Qa/Qm vs. 515Na-NZO based on the
following mass balance relationships.

5QO = 5aQa + 5oQo (Equation 10)

5m = Qa(5a — 5o)/Qm + 5o

Where:

Qm = Qa + Qb

Qa = moles of N20 (Cambridge Isotope Lab standard)

Qm = total number of moles of N20 (mixture)

Q, = moles of N20 (tank standard)

53 = 515Na-N20 (Cambridge Isotope Lab standard)
on, = 5‘5N°-Nzo (mixture)

5b = SISNQ-NZO (tank standard)

Controls

46

Control experiments were necessary to verify that the stimulated microbial
process was responsible for N20 rather than undesired abiological or biological
mechanisms. The first control tested whether hydroxylamine can produce N20
abiologically during the time course of a typical incubation (8 hours). In this experiment,
2 mL of phosphate buffer and 300 uL of hydroxylamine were incubated with a headspace
of 100 % air at 27 °C. After 8 hours, a 1 mL of a headspace gas was introduced in the
mass spectrometer. A second control experiment with 2 mL of concentrated N. europaea
cells, 300 11L sodium nitrite and a headspace of air was conducted to test whether N20
was formed via nitrifier denitrification. After 8 hours of incubation, a 1 mL headspace
sample was analyzed for N20 concentration. To verify that nitrite reduction does not
occur in concentrated cell cultures of M. capsulatus Bath, a third control experiment was
conducted. A volume of 2 mL of cell suspension, 300 1.1L of 0.01 M hydroxylamine and
100 uL of 99 % If’N-sodium nitrite were incubated in a test tube with air in the
headspace. A 1 mL headspace sample was analyzed to determine N20 concentration

RESULTS AND DISCUSSION
Standard Characterization

The 515 Nat of the standard N20 was obtained from a plot of QalQm vs. 515 Na-NZO

of the mixture (Figure 10). The y-intercept of a best-fit line (y = l38520x + 3.61; R2 =

0.9862) yields the 5'5N° oflaboratory N20 tank standard of 3.61 °/,, (Figure 10).

Controls
Abiological or anoxic microsite N20 production was not detected in the control
experiments. In a control experiment to test abiological production of N20 from

hydroxylamine, MO was not detected after 8 hours of incubation. In a second control

47

experiment with a concentrated cell suspension of N. europaea, a headspace of air (21 0/o
02) and nitrite as the substrate, the possibility of denitlification within anoxic microsites
was examined. The results of the experiment demonstrated N2O was not produced after
8 hours. Since anoxic conditions stimulate the nitrifier denitrification pathway for N20,
the result of this experiment suggests that anoxic microsites, if present, were not a site of
active denitrification in the concentrated N. europaea cell cultures. To evaluate whether
nitrite reduction was active in M. capsulatus Bath, 3 third control was established using
concentrated cells, nitrite as the substrate and a headspace of N2 (0 % 02). Using an
isotopic mass balance equation, the fraction of N20 calculated to be a product of 15NO2’
in the third control experiment was less than 1 %. Therefore, in the studies of
hydroxylamine oxidation by M capsulatus Bath, further reduction of nitrite is not of
concern.
Bulk nitrogen and oxygen isotopic composition of N 20

The amount of N20 in the headspace increased in the time course experiments
indicating production of N20 (Tables 1, 2 and 3). The average 8‘5N values of N20
formed by hydroxylamine oxidation by M. capsulatus Bath (0.0 +/- 1.2 °/oo),
hydroxylamine oxidation by N. europaea (-28.3 +/- 6.7) and nitrifier denitrification by
N. europaea (-34.8 +/- 2.7) were significantly different (ANOVA, f(2,35)= 247.9, p=0). In
addition, the average 5'80 values of N20 formed by hydroxylamine oxidation for M.
capsulatus Bath (53.1 +/- 2.9 °/oo), hydroxylamine oxidation by N. europaea (-23.4 +/-
7.2) and nitrifier denitrification by N. europaea (4.6 +/- 1.4) were significantly different
(ANOVA, f(2,35)= 279.98, p=0). The average stable oxygen and nitrogen isotopic values

of N20 varied in the hydroxylamine oxidation experiments despite the fact that a single

48

substrate solution was used (Table 1 and 2). The isotopic disparities among the
hydroxylamine experiments likely result from a difference in the enzymes responsible for
hydroxylamine oxidation between M. capsulatus and N. europaea. Whereas, cytochrome
P-460 is the enzyme responsible for hydroxylamine oxidation in M. capsulatus Bath
(Zahn et al., 1994), the primary enzyme used for hydroxylamine oxidation in N.
europaea is hydroxylamine oxidoreductase. Furthermore, the cytochrome P-460 enzyme
purified from M. capsulatus Bath differs in terms of activity from that of N. europaea
(Zahn etal., 1994). Thus, the enzymes used for oxidation of hydroxylamine in the two
organisms are distinct. This could invoke a difference in the fractionation factor
associated with the nitrification reaction and, thus, account for the disparities in the bulk
nitrogen and oxygen isotope values of N20 produced by the two organisms studied here.
The Al5 N for the cultures demonstrates that the two hydroxylamine experiments differ in
the degree of fractionation (-2.3 and 26.0 °/oo for M. capsulatus Bath and N. europaea
respectively).
A15 N = 815N(subsImIe) — BlstroducI) (Equation 11)

Where:

8'5 N of hydroxylamine = -2.3 °/oo

515Nofnitrite= 1.1 0/00 1
The A15 N for the nitrifier-denitrification experiment is 35.9 °/oo. This is similar to an
estimate of the fractionation factor during N20 production by nitrifiers in a laboratory
study (8 = 30 0Am) and suggests that hydroxylamine oxidation may be the rate limiting
step in nitrification(Yoshida, 1988). The study by Yoshida (1988) and the current study

demonstrate that depletions in 15 N of N20 could result from hydroxylamine oxidation and

49

nitrite reduction by nitrifiers. While bulk stable nitrogen and oxygen isotopic values
provide insight into isotopic fractionation, a basis for distinguishing N2O formed by
hydroxylamine oxidation and nitrite reduction by nitrifiers still remains.

Nitrogen isotopomeric site preference of N 20

Average site preferences of N20 formed by hydroxylamine oxidation by M.
capsulatus Bath (5.5 +/- 3.1 °/oo), hydroxylamine oxidation by N. europaea (-1.4 +/- 1.7
0Am) and nitrite reduction by N. europaea (-7.7 +/- 3.1 04,0) are significantly different
(ANOVA, F234 = 72.09, p=0). This result indicates that the site preferences of the N20
formed by the three pathways in this study are distinct. This preliminary data set
provides the first reported values of the isotopomeric site preference of individual
microbial pathways.

A negative site preference resulted from the processes of hydroxylamine
oxidation and nitrite reduction by N. europaea (Tables 2 and 3). Equilibrium conditions
were shown to impose a positive site preference (45 °/oo) (Y ung and Miller, 1997).
During kinetic isotope effects, such as the three pathways in this study, the formation of
N20 involving a hyponitrite intermediate can result in enrichment in 15N at N“ (Popp et
al., in press; Toyoda et al., in press). However, measurements with a negative site
preference are reported by Yamulki et al. (2001) and the current study. In the proposed
mechanism for the production of N20 via dissimilatory nitrite reduction by Weeg-
Aerssens et al., (1988), the N-N bond of N20 is formed by nucleophilic attack of a
second nitrite on a Fe-coordinated nitrosyl species (Figure 11). In terms of kinetic
fi'actionation, the key reactions in the sequential mechanism that could produce a site

preference are steps 1 and 3. This is the case because nitrite is abundant in culture and,

50

therefore, not rate limiting. The first N added in step 1 ultimately occupies the B position
and fractionation could result in the process of nitrite binding to the ferrous heme (Figure
11). Nucleophilic attack of nitrite on the coordinated nitrosyl species (step 3) resulting in
the formation of the N-N bond can also lead to isotopic discrimination (Figure 11). The
relative difference in the magnitude of the kinetic isotope effect (degree of discrimination
against 'SN) resulting from steps 1 and 3 will determine the site preference. In order for a
negative site preference to result, the NB of N20 must be more enriched in 15N than N“.
We propose that the isotopic fractionation involved in nucleophilic attack on the
coordinated nitrosyl group is greater than the isotope effect associated with binding of
nitrite to ferrous heme. Alternatively, step 3 may be rate limited to a greater extent than
step 1 thereby favoring depletion of lsN in N“ (Figure 11). This occurs because as a
reaction approaches completion, the isotopic composition of the product approaches that
of the initial substrate. Thus, the observed discrimination against 15 N will be greater for
rate-limited reactions. While the mechanism associated with N20 formation by
hydroxylamine oxidation is unknown, it is possible that a negative site preference could
result from a similar difference in fractionation factors or rate limitation associated with
yet undescribed pathways in the formation of the N-N bond of N20.

Field studies have indicated that N20 formed via biological mechanisms is
characterized by low isotopomeric site preferences (Table 4). The average site
preference of N20 in tropospheric samples is approximately 19 ° 00 (Y oshida and Toyoda,
2000). The 19 °/(,0 site preference is thought to result from mixing of biologically and
combustion derived N20 of low site preference with N20 of higher site preferences such

as that produced by photolysis in the stratosphere (Yoshida and Toyoda, 2000). Using an

51

isotope mass balance equation, Yoshida and Toyoda (2000) estimated that the range of
site preferences from soil and oceanic sources was -O.5 to +15 °/oo. This is consistent
with other field studies that demonstrate low site preferences in areas of the water column
with active nitrification or soils with nitrogen additions (Table 4). In a study of N20
production in the North Pacific, Popp et al.(in press) estimated that the site preference of
the in situ source of N20 was likely between 0 and 8 °/oo, Although the measured value of
the site preference of N20 formed during hydroxylamine oxidation by autotrophic
nitrifiers (-1.4 +/- 1.7 °/00) was slightly lower than the estimated range in the field study
(0 and 8 0Am), given the possible errors in estimation and analytical measurement they
could be considered similar. This provides further evidence that autotrophic nitrification
was the dominant in situ source of N20 at ALOHA (Popp et al., in press). The site .
preference we observed for the nitrifier-denitrification pathway (-7.7 +/- 3.1 oloo) is much
lower than that estimated for oceanic and soil sources (05 to +15 °/oo; Yoshida and
Toyoda, 2000). This may suggest that the nitrifier-denitrification pathway is not a
dominant mechanism for N20 production in soil and oceanic sources, however, the
number of site preference measurements in the literature are few. This is consistent with
Ostrom et al. (2001) who proposed that oxidation of hydroxylamine was the predominant
pathway of N20 production in the central North Pacific, but also proposed that nitrifier
denitrification was significant within a narrow depth interval. The isotopomeric
composition of N20 formed via hydroxylamine oxidation by M. capsulatus Bath (5.5 +/-
3.1 °/oo) is similar to the range of estimated values for soil and oceanic sources. This is
suggestive of an underappreciated role of methanotrophic nitrification to N20 production

in soil and oceanic environments.

52

CONCLUSIONS

The A15 N of hydroxylamine oxidation and nitrifier denitrification by N. europaea
were, 26.0 and 35.9 °/oo, respectively, and similar to the fractionation previously reported
of 8 = 30 °/oo (Y oshida, 1988). However, discrimination against ”N during N20
production by hydroxylamine oxidation by M capsulatus Bath was small (-2.3 oAm). This
suggests that B'SN-N2O could be used to distinguish N2O production by autotrophic
nitrification and methanotrophic nitrification. Isotopic fractionation of oxygen isotopes
during hydroxylamine oxidation by M. capsulatus Bath and N. europaea were
significantly different. This is consistent with a study demonstrating that different
enzymes are involved in hydroxylamine oxidation in M. capsulatus Bath and
N. europaea. These results suggest that, in addition to nitrogen isotope values, oxygen
isotope values can be used to distinguish N20 produced by methanotrophic nitrification
and autotrophic nitrification.

The site preference of N20 formed by hydroxylamine oxidation by M. capsulatus
Bath and N europaea and nitrite reduction by N. europaea were significantly different.
Therefore, site preference can distinguish N20 formed by hydroxylamine oxidation by
M. capsulatus Bath and N. europaea. In addition to differentiating hydroxylamine
oxidation mechanisms, site preference can discern N2O produced by nitrifier
denitrification from hydroxylamine oxidation. The disparity in site preference is likely a
consequence of differences in the enzymes responsible for N20 production between the
two organisms. In sequential mechanisms for the production of N20, the degree of
discrimination or rate limitation in individual steps of the reaction pathway could impose

a distinct site preference. The results of this study indicate that site preference provides a

53

basis for resolving specific microbial pathways of N20 production. This information
could be critical for predicting and managing N2O emissions from some ecosystems, such
as agricultural systems, where activities to mitigate carbon dioxide emissions may

enhance N2O production.

54

 

 

 

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N
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O
(amrxgw) aplxo snomu-mmg

 

 

 

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~83 u
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5 m + xommmfl u >
o?

 

 

 

 

 

 

.025 98:: mo 838 :38 n so . 02x0 msobEIoZ e\e ma .«o 83:. U ad 80:3 @3983
02x0 355:. on“ .«o 85.5.“ng 87. on. wcfitouofiano 2 5889? BE wczoov— .3 «Burn

55

Table 1. Summary of nitrous oxide data including concentration, site preference, S‘SN-
N20 and 6'80-N2O from concentrated Methylococcus capsulatus Bath cell cultures with
hydroxylamine as the substrate. Each letter (A, B, C and D) corresponds to a separate
experiment and number indicates the time course sample. Nitrous oxide formation via
hydroxylamine oxidation is stimulated in this set of experiments. 'Anomalous value
(37.7 °/oo) omitted in the calculation of average and standard deviation of the 5'80-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

NZOsmow~
Time site 6‘5N-Nzo... 5‘80-
preference (°Ioo) N2Ovsuow
Culture N20 (nM) Cloo) (°Ioo)
A-M.cap 1 20:09 282 7.2 -0.3 50.4
A-M.cap 2 20:28 451 8.5 -0.2 53.4
A-M.cap 3 21 :24 563 8.1 -3.3 56.2
B.M.cap 1 17:40 305 1 .5 1.7 37.7
B-M.cap 2 18:35 644 6.2 0.4 55.8
B-M.cap 3 19:18 676 7.5 0.7 57.1
C-M.cap 1 20:28 191 0.0 -0.2 49.8
C-M.cap 2 21 :05 260 10.0 -0.2 50.4
C-M.cap 3 21 :47 323 3.8 -0.3 51.3
D-M.cap 1 18:45 317 3.1 1.0 50.8
D-M.cap 2 19:20 387 3.2 0.2 51.9
D-M.cap 3 23:11 576 6.9 0.7 57.1
Average 5.5 0.0 53.1:
Standard deviation 3.1 1.2 2.9

56

Table 2. Summary of nitrous oxide data including concentration, site preference and,
6180-N2O and 8'5N-N2O from concentrated Nitrosomonas europaea cell cultures with
hydroxylamine as the substrate. Each letter (A, B, C and D) corresponds to a separate
experiment and number indicates the time course sample. Nitrous oxide formation by
hydroxylamine oxidation is stimulated in this set of experiments.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Site 8‘5N- 51°0-
preference N20... Nzovsuow
Culture Time N 20 (nM (°Ioo) (°Ioo) (°loo)
A-N.eur 1 15:28 89 -0.7 -20.4 33.5
A-N.eur 2 15:47 143 1.2 -21.4 31.9
A-N.eur 3 16:05 216 1.2 -24.7 28.1
B-N.eur 1 17:46 284 -1.4 -35.5 16.5
B-N.eur 2 18:29 417 -2.4 -37.5 15.3
B-N.eur 3 19:08 466 -1.8 -38.1 15.1
C-N.eur 1 19:58 158 -2.6 -30.2 20.5
C-N.eur 2 20:38 179 -1.5 -30.6 20.3
C-N.eur 3 21 :18 246 -0.9 -30.3 20.2
D-N.eur 1 14:03 246 -4.6 -27.2 21.4
D-N.eur 2 15:41 249 -2.7 -26.3 22.3
Average -1 .4 -28.3 23.4
Standard deviation 1.7 6.7 7.2

57

Table 3. Summary of nitrous oxide data including concentration, site preference, 5180-
N20 and SlSN-N2O from concentrated Nitrosomonas europaea cell cultures with nitrite
as the substrate. Each letter (A, B, C and D) corresponds to a separate experiment and
number indicates the time course sample. Nitrous oxide formation via nitrite reduction is
stimulated in this experiment.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Site 5‘5N- 51°0-
preference N203" Nzovsmow

Culture Time N20 (nM) (°loo) (°Ioo) (°Ioo)
A-Nit-denit 1 14:08 89 -10.1 -34.0 4.3
A-Nit-denit 2 14:58 119 -11.1 -33.1 4.1
A-Nit-denit 3 17:38 189 -6.4 -33.3 4.1
A-Nit-denit 4 19:00 204 -6.9 -33.1 4.2
A-Nit-denit 5 19:41 218 -10.8 -33.2 4.1
B-Nit-denit 1 20:18 51 -5.4 -36.0 3.4
B-Nit-denit 2 21 :36 74 -6.0 -36.7 3.5
B-Nit-denit 3 22:16 73 -7.5 -36.8 3.1
C-Nit-denit 1 19:47 98 -5.9 -38.2 8.0
C-Nit-denit 2 20:27 116 -6.0 -38.7 6.5
C-Nit-denit 3 20:47 122 -2.4 -39.1 6.5
D-Nit-denit 1 20:18 214 -11.8 -31.0 5.1
D-Nit-denit 2 21 :36 291 -11.4 -31.5 4.4
D-Nit-denit 3 22:16 559 -3.2 -33.1 3.8
Average -7.7 -34.8 4.6
Standard deviation 3.1 2.7 1.4

58

Table 4. Summary of isotopomeric compositions of nitrous oxide in the literature with an

emphasis on biological pathways.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Site

preference

(°Ioo) Study site Comments Authors
Tropospheric average trophospheric Yoshida and

19 sample value Toyoda, 2000
Hydroxylamine Methylococcus capsulatus

5.5 ’I. 3.1 oxidation Bath — Laboratory study Current study
Hydroxylamine Nitrosomonas europaea -

-1.4 *l. 1.7 oxidation Laboratory study Current study

Nitrosomonas europaea -

-7.7 +I. 3.1 Nitrite reduction Laboratory study Current study
North Pacific minimum between 100 Popp et al., in

~8 waters and 300 m press
Western Pacific isotopomeric minimum in Toyoda et al.,

~11 waters water column in press
agricultural Range of isotopomeric Perez et al.,

4.9 to 14.2 fields composition in study 2001
urine amended Range of measured Yamulki et al.,

-2 to +9 grassland values 2001

Yoshida and
-0.5 to +15 estimated value isotope mass balance Toyoda, 2000

 

59

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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