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This is to certify that the
thesis entitled

ISOTOPOLOGUE FRACTIONATION DURING MICROBIAL
REDUCTION OF N20 IN SOIL

presented by

MALEE JINUNTUYA

has been accepted towards fulfillment
of the requirements for the

MS. degree in Geological Sciences

 

 

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ISOTOPOLOGUE FRACTIONATION DURING MICROBIAL REDUCTION OF
N20 IN SOIL

Malee Jinuntuya

A THESIS
Submitted to
Michigan State Univeristy
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Geological Sciences

2007

ABSTRACT

ISOTOPOLOGUE FRACTIONATION DURING MICROBIAL REDUCTION OF N20
IN SOIL

By
Malee J inuntuya
Reduction of N20 is a challenge to studies using isotope values to resolve global
budgets and microbial sources of this critical greenhouse gas. Prior research has
demonstrated that the difference in 5'5N between the central (or) and outer ([3) N atoms in
the N20 can be used to distinguish N20 derived from nitrification and denitrification
(Sutka et al., 2003; 2006; Toyoda et al., 2005). If intramolecular distribution of 15N,
however, is altered during reduction, apportionment of N20 to nitrification and
denitrification will be inaccurate. Isotopologue analyses of N20 within soil mesocosm
experiments were used to investigate fractionation during N20 reduction at four levels of
water filled pore space (WFPS) 60, 80, 100% (saturation) and 10% in excess. Soils were
obtained from the Kellogg Biological Station Long Term Ecological Research Site
(Michigan). Isotopic enrichment factors (8) for 8'5 N, 5'80, SUN“ and SISNB ranged from
-4.2 to -9.0, -l2.5 to -23.6, -6.4 to -10.0 and -2.0 to -7.9, respectively. With the exception
of site preference (SP), lower fractionation factors were observed at higher WFPS
demonstrating the importance of diffusion in limiting the expression of enzymatic
fractionation. Isotopic discrimination in SP was small and the a values varied between
-4.5 and 0 %o. Strong correlations were evident between 8'80 and 5'5 N and 5180 and
S'SN“, with slopes of 2.7 and 2.0, respectively. These relationships (1) provide a
definitive means for establishing that isotope effects during reduction are present and (2)

may provide a means to determine the source signatures even when reduction occurs.

DEDICATION

This work is dedicated to my parents, George K. Brown and Yaeko J inuntuya, for

believing that I am a scientist at heart.

iii

ACKNOWLEDGEMENTS

I would like to thank Dr. Nathaniel Ostrom for his support, guidance and patience
throughout the course of this work. I would also like to thank the rest of my committee
members, Drs. Grahame Larson and David Long for their invaluable advice and
suggestions throughout my graduate career here at MSU. Many thanks go out to Drs.
Robin Sutka, Hasand Gandhi and Peggy Ostrom, for their support and patience in
teaching me. Robin, I can never thank you enough. Thanks for pushing me beyond the
limit of what I thought I could do. You are truly instrumental to my success. Also, I
would like to thank Dr. Lina Patino for all the encouragement she has provided,
especially in times when I needed it most. Special thanks go out to my three partners in
crime, Karen Tefend, Colleen McLean (TP) and Meredith Benedict. Your sheer
enthusiasm and humor is what got me through this. I have earned more than colleagues;
you are my friends for life. In addition to this fantastic group of people, I would like to
acknowledge other graduate students in the department, for making life at MSU anything
but boring. To my family, Karo, Yaeko, Sukanya, Angela and the Nortmans, you have
been inspirational to me. This work is dedicated to you. Lastly, to my husband Dan,
thanks for not letting me take the easy way out. This feat would not have been

accomplished without your love and support.

iv

TABLE OF CONTENTS

LIST OF TABLES ................................................................................... vii
LIST OF FIGURES ................................................................................ viii
INTRODUCTION .................................................................................. 1
MATERIALS AND METHODS .................................................................. 7
RESULTS ........................................................................................... 10
Experiment 1 ............................................................................... 10
Rates of N20 reduction .......................................................... 10
N20 isotopologue enrichment factors
Experiment 2 ............................................................................... 17
Rates of N20 reduction .......................................................... 17
N20 isotopologue enrichment factors .......................................... 17
Relationships between 6'80 and SISN and 8180 and SN“ ........................... 21
DISCUSSION
Isotopic effects on N20 during N20 reduction ......................................... 24
N20 reduction and source apportionment based on SP ............................... 27
The relationship between 8180 and 515N, and 5180 and 8'5N" ....................... 28
CONCLUSIONS .................................................................................... 30

APPENDIX 1. Ion current ratios of N20 measured over time for 60 (A), 80 (B) and 100

(C) % WFPS, experiment 1. All treatments were conducted in triplicates
(R1,R2and R3... ................................................................. 32

APPENDIX 2. Isotopic compositions of N20 isotopologues for 60 (A), 80 (B) and 100
(C) % WFPS. The 5 values were calculated from the ion current ratios
with reference to VSMOW and air (Toyoda and Yoshida, 1999). All
treatments were conducted in triplicate (R1, R2 and R3) .................. 35

APPENDIX 3. Isotopic compositions (corrected évalues) of N20 isotopologues,
isotopomers and site preference (SP) for 60 (A), 80 (B) and 100 (C) %
WFPS, experiment 1. All treatments were conducted in triplicate (R1,
R2 and R3) ......................................................................... 38

APPENDIX 4. Headspace concentration data for 60, 80 and 100 % WFPS, experiment 1
over time. All treatments were conducted in triplicate (R1, R2 and
R3) ................................................................................. 41

APPENDIX 5.

APPENDIX 6.

APPENDIX 7.

APPENDIX 8.

APPENDIX 9.

Ion current ratios of N20 measured over time for 60 and 110 % WFPS,
experiment 2 ...................................................................... 42

Isotopic compositions of N20 isotopologues for 60 (A) and 110 (B) %
WFPS. The 5 values were calculated from the ion current ratios with
reference to VSMOW and air (Toyoda and Yoshida, 1999) ............... 44

Isotopic compositions (corrected d values) of N20 isotopologues, .
isotopomers and site preference (SP) for 60 (A) and 110 (B) % WFPS,
experiment 2 ..................................................................... 46

Headspace concentration data for 60 and 110 % WF PS, experiment 2
over time .......................................................................... 48

Rate of N20 reduction for 60, 80 and 100 % WFPS, experiment 1 for
replicate 1 (open square), replicate 2 (closed square) and replicate 3
(open triangle) ................................................ _ ................... 49

APPENDIX 10. Site preference as a function of ln(C/Co) for 60, 80 and 100 % WFPS,

experiment 1 for replicate 1 (open square), replicate 2 (closed square)
and replicate 3 (open triangle) ............................................... 50

APPENDIX 11. Rate of N20 reduction for 60 (open square) and 110 (closed triangle) %

WFPS over time, experiment 2 ............................................... 51

APPENDIX 12. Site preference (SP) as a function of ln(C/Co) for 60 (open square) and

110 (closed triangle) % WFPS over time, experiment 2 .................. 52

REFERENCES ...................................................................................... 53

vi

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

LIST OF TABLES

Headspace concentrations, microbial reduction rates of N20 and substrate
depletion expressed as percent for 60, 80 and 100 %WF PS. The results
represent data obtained from three replicates (R1, R2 and R3) during time series
experiment .............................................................................. 11

The a values for time series experiment 1 at 60, 80 and 100 % WFPS during
microbial reduction of N20 as determined from the Rayleigh equation (Eq. 1).
The value for sSP is determined as a difference between 8'5NCl and alsNl3
(Toyoda et al., 2005) ................................................................... 13

Concentration and reduction rate of N20 in soil mesocosms for 60 and 110
WFPS, during time series experiment 2. N20 depletion, expressed as percent,
reflects the amount of substrate reduced within the headspace samples in each
soil mesocosm treatment ............................................................... 17

The e values for 60 and 110 % WFPS during N20 reduction, experiment 2. The
a values represent values obtained from the slope of the natural log plot with
isotope values as a function of natural log of residual substrate concentration

(C) relative to initial substrate concentration (Co) .................................. l9

Slopes of the relationship between 8180 vs. 5'5 N and 8180 vs. SISNCl for 60, 80
and 100 % WFPS, experiment 1, and 60 and 110 % WFPS, experiment 2,
during N20 reduction. All treatments in experiment 1 were conducted in
triplicate. Experiment 2 was not replicated .......................................... 21

vii

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

LIST OF FIGURES

N20 production during nitrification and denitrification. The production of N20
can occur during oxidation of hydroxylarnine (a), reduction of nitrite (b) and
step-wise reduction of nitrate (c). Adapted from Wrage et al., 2001 .............. 2

N20 headspace concentration as a function of time for 60 (a), 80 (b) and 100 %
WFPS (c), experiment 1 for replicate mesocosms 1 (open square), 2 (close
square) and 3 (open triangle) ........................................................... 12

Isotopologue values as a function of ln(C/Co) for 60 % WFPS, experiment 1 for
replicate 1 (R1, open square), replicate 2 (R2, close square) and replicate 3 (R3,

open triangle). The slope of this relationship defines e (Eq. 1) ................... 14
Isotopologue values as a function of ln(C/Co) for 80 % WFPS. The slope of
these relationships defines 8 (Eq. 1) ................................................... 15
Isotopologue values as a function of ln(C/Co) for 100 % WFPS. The slope of
these relationships defines 8 (Eq. 1) ................................................... 16

Concentration of N20 in soil mesocosms for 60 (open square) and 110 % (close
diamond) WFPS in experiment 2 as a function of time expressed in umol. . . ...18

Isotopic compositions for SISN, 5180, SUN“, S'SNB and SP for 60 (open square)
and 110 % (close diamond) WF PS, experiment 2. Plots A through D represents
isotOpe values obtained from the natural leg plot. The slope of the line in the
natural log plot is the fractionation (enrichment) factor (a) by the Rayleigh
model. The SP values were calculated based on the difference between the
SUN0L and 8'5NB and the resulting values can be shown in Table 4 ................ 20

5180 as a function of 5'5N (A) and 5'5NOI (B) for experiment 1 at 60 (open
square), 80 (open triangle) and 100 % (open diamond) WFPS with. The initial
isotopologue values represent values from Tank A standard (open circle) of N20
that was added to the mesocosm headspace to initiate the experiment. Values
for r2 and p (t-test) in all cases are greater than 0.90 and less than 0.01 ,
respectively ............................................................................... 22

5180 as a function of SISN (A) and 5'5N“ (B) for experiment 2 square), 80 at 60
(open square) and 100 % (open diamond) WFPS. The initial isotopologue
values represent values from Tank C standard (open circle) of N20 that was
added to the mesocosm headspace to initiate the experiment. Values for r2 and
p (t-test) in all cases are equal to or greater than 0.90 and less than 0.01,
respectively ............................................................................... 23

viii

INTRODUCTION

Increasing concerns over changes in global climate have warranted a closer
examination of the microbial processes that produce the trace gas nitrous oxide (N20).
Nitrous oxide, a greenhouse gas, is emitted into the troposphere from terrestrial
ecosystems and plays an important role in altering stratospheric chemistry, including
depletion of the ozone layer (Prather et al., 2003). In response to anthropogenic activities
tropospheric N20 has been increasing at an average rate of 0.3 % per year since 1980
(IPCC, 2001; Prinn et al., 1990 and Rasmussen et al., 1986). Compared to the radiative
forcing of carbon dioxide, N20 traps radiant energy 296 times more efficiently (IPCC,
2001), thereby contributing about 6 % to the overall global warming (Dalal et al., 2003).
Even though N20 contributes only a fraction of the total warming effect, a small
percentage increase in emission can potentially lead to a large accumulation of N20 in the
troposphere as a result of its long residence time of approximately 120 years
(Minschwaner et al., 1998; Olsen et al. 2001). Therefore, accurate apportionment of
microbial sources is important in effectively mitigating N20 emissions.

Nitrous oxide is derived from both natural and anthropogenic sources. The major
source of N20 flux from terrestrial ecosystems are microbial processes stimulated by
agricultural activities, mainly application of nitrogen based fertilizers and tilling (Mosier
and Kroeze, 1998; Nevison and Holland, 1997), which is found to stimulate microbial
processes that produce this gas (Stein and Yung, 2003). Nitrification (aerobic) and
denitrification (anaerobic) are the two primary microbial processes that produce and

regulate N20 within the Earth’s troposphere (Figure 1). Three separate pathways are

 

Denitrification
, . - . ............................ 1

A l
2 ...... . ............. ( 92 ......... i
Nitrification I

 

 

 

I
I
I
I
I
I
I
I

—>
A
E
v
—->

 

 

Nitrifier Denitrification

 

r
I
I
I
I
I
I
I
r.
fx
'3
i
I
l
I
I
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' -

 

 

 

 

 

Figure 1. N20 production during nitrification and denitrification. The production of N20
can occur during oxidation of hydroxylamine (a), reduction of nitrite (b) and step-wise
reduction of nitrate (c). Adapted from Wrage et al., 2001.

responsible for the microbial formation of N20: (1) oxidation of hydroxylamine during
nitrification (Figure 1 a), (2) reduction of nitrite by nitrifier denitrification (Figure 1 b),
and (3) through stepwise reduction of nitrate to N2 during denitrification (Figure 1 c)
(Wrage et al., 2001; 2004a). Therefore, nitrous oxide emissions from soils can be
mitigated if the microbial process leading to its production is known.

Prior approaches to evaluate microbial production pathways have relied on the
use of inhibitors or natural abundance isotope data. For example, acetylene is commonly
used to block N20 reduction to N2 to evaluate rates of denitrication (Groffman et al.,
2006). The use of inhibitors, such as acetylene to evaluate N20 production, however,
results in alteration of microbial activity and may not accurately reflect production
pathways (Tilsner et al., 2003; Wrage et al., 2004b; 2004c). The natural abundance

isotope approach is based on the difference in B'SN between N20 and the substrates of

nitrification and denitrification (NH4+ or NO3', respectively). A difference of 60 %o

indicates production from nitrification, whereas a difference of 30%o reflects production
from denitrification (Perez et al., 2000). Limitation of the substrates may reduce
expression of fractionation. Thus, it is conceivable that nitrification can produce N20
that is depleted in 15N by 30 %o relative to the substrate. Because of this non conservative
behavior, bulk nitrogen isotope values (8'5 N) may not definitively distinguish production
pathway, and a conservative tracer is needed.

The distribution of 15 N within N20 molecule has been shown to be an effective
tracer of the origins of N20 (Toyoda and Yoshida, 1999). Within the asymmetrical linear
structure of the N20 molecule (N -N-O), the combination of five isotopes (MN, '5 N, '6O,
I70, I80) yields 12 possible combinations of isotopes (isotopologues). The five most
common isotopologues in order of abundance are: l4N'4N160, 15NMNHSO, l4N15 N160,
l4NMNWO, and 14NMNmO (Yung and Miller, 1997). The abundance ratios of 15NMNHSO
and 14N'5 N160 isotopomers with respect to l4NMNMO provide the basis to define the
isotopic composition of central and outer N atoms as SISN‘JLl and SISNB, respectively. The
difference between SISNO‘ and SUNB yields position-specific isotopic information, and is
commonly expressed as Site Preference (SP). Such information provides an additional
insight into the pathways of microbial reactions (Sutka et al., 2003; 2006). Furthermore,
the key advantages to the use of SP in contrast to bulk isotopes (515 N and 8180) are (1)

SP is independent of the isotopic composition of substrates, and therefore, is a

 

' Delta (5) expresses the isotopic composition of N and O in N20 with respect to Air (0 %o) and Vienna
Standard Mean Ocean Water (VSMOW) (0 %o), and is defined by the equation,

Rsam
5 = [(Rg—plc—J — I] x 1000 , where R refers to the ratio ofthe heavy ('SN, '80) to light (”N, '60)
tan dard

isotopes. Delta is expressed as per mil (%o).

conservative tracer, and (2) does not vary over the course of the reaction (Toyoda et al.,
2005; Sutka et al., 2006).

Sutka et al. (2006), and Toyoda et al. (2005), demonstrated the effectiveness of
ISN-SP as a tracer for the microbial sources during biogeochemical reactions in pure
cultures. The SP in N20 produced during hydroxylamine oxidation (nitrification) (Figure
1 a) and nitrate reduction (denitrification) (Figure 1 e) have markedly distinct values of
approximately 33 and 0%o, respectively. Reduction of nitrite by ammonia-oxidizing
bacteria during nitrifier denitrification (Figure 1 b) yields a SP that was not
distinguishable from N20 produced during nitrate reduction. Since N20 produced by
both nitrifier denitrification and denitrification are reductive processes, they are
combined and collectively termed “denitrification.” The distinctive SP values associated
with nitrification and denitrification allow microbial production of N20 to be
differentiated.

While SP values characteristic of production by nitrification and denitrification
have been identified, the affect of N20 reduction on SP remains uncertain. Recently
changes in SP during N20 reduction have been established within pure microbial cultures
(Ostrom et al., 2007). An isotopic enrichment factor of approximately 6960 was
observed. While small in magnitude, this value indicates that alteration of SP due to N20
reduction cannot be neglected in field studies using SP to evaluate microbial sources.
Thus, in the presence of N20 reduction SP is not truly a conservative tracer.

Even though the isotopomer effect during N20 reduction has been identified in

pure culture, the affect on SP by a natural soil microbial community has not been

established. The Rayleigh model has been the convention for defining isotope

fractionation that occurs during many microbial reactions.
55 = 650 + sln(C/C0). (1)

This equation quantifies the isotopic composition of the residual substrate (5,) of a
particular microbial reaction in relation to the initial substrate, the isotopic enrichment
factor (a), and the ratio of the natural log of observed concentration to the initial
concentration (C/Co). The e, or the fractionation factor (or) describes the fractionation

during reactions that are restricted by masses of molecules and is defined as:
OL = —, (2)

where k2 and k1 are the reaction rate involving the heavy and light isotopically substituted
compounds and

s=(0t—1)1000 (3) .
(Marriotti et al., 1981; 1988; Ostrom et al., 2002). The model assumes, in a biological
process, fractionation is a single, unidirectional isotope reaction (Marriotti etal., 1981).
However, in biological processes this assumption is not entirely accurate since, for
instance reduction of N03' to N2 occurs via a series of individual reaction steps (Figure 1)
during denitrification (Firestone and Davidson, 1989). The magnitude of the expressed
fractionation during reduction of N03' to N2 is dependent upon which step in the reaction
sequence is rate limiting. The most common factors limiting reduction are diffusion and
the availability of enzymes to carry out each reduction step. The fractionation during
diffusion is small; however, enzymatic fractionation tends to be large (Ostrom et al.,

2002). For example, in water logged soil (100 % WFPS), N20 reduction is expected to

be limited by diffusion as the movement of the gas (N20) into the cell through water is a
slow process, therefore net fractionation is expected to be small. On the other hand, at 60
% WFPS, diffusion is less likely to limit the supply of N20 to the cells but rather the
enzymatic activity is the key rate limiting step. Under this condition the expressed
fractionation is large. The magnitude of fractionation during N20 reduction will vary
depending upon whether diffusion or enzymatic reduction is rate limiting. Thus, a low
level of fractionation is observed when diffusion is limiting, and a high level when
enzymatic reduction is significant (Ostrom et al., 2002). Consequently, the magnitude of
isotopic fractionation is expected to decrease with increasing WFPS and the use of SP to
evaluate sources of N20 production requires an understanding of the importance of this
alteration.

Isotopic fractionation during microbial reduction of N20 was evaluated in this
study at four levels of water soil-water content (60, 80, 100 and 110 % WFPS). By
varying the WFPS, the relative importance of diffusion and enzymatic reduction were
indirectly controlled. Soils used in this study w ere obtained from an uncultivated
successional agricultural field plots within the WK. Kellogg Biological Stations Long
Term Ecological Research Site (KBS LTER) (Hickory Comers, MI). Uncultivated soil
was used due to the rapid rate of N20 reduction previously observed (Ostrom et al.,
2007). Since fractionation factors during N20 reduction are expected to differ with water
content, fractionation at 60 % WFPS is expected to be the greatest. Knowledge of the
magnitude of fractionation may provide a basis for correcting isotope shifts that occur
during microbial denitrification and may allow for accurate apportionment of N20 fluxes

from soils even when SP is altered by reduction.

MATERIALS AND METHODS

Surface soil from an uncultivated successional field (treatment plot 7) was
collected from the K88 LTER to construct experimental mesocosms. This plot is
maintained as a native successional field following the abandonment of spring tillage in
1989. Soil was sieved through a screen with mesh size 2 mm, and air dried for 72 h and
stored dry at 24 °C until the construction of mesocosms.

Soil mesocosms were constructed of 250 mL glass serum bottles and filled with
approximately 40 g of dried soil. The amount of water added per gram of soil was
determined based on the soil properties to obtain target WFPS values 60, 80, 100
(saturation), and 10 % in excess of saturation using the Gravimetric Water Content

formula:

GWC =

 

(WFPS x TSP)
Pb ’

(4)

where TSP is the total soil porosity (%) and pb is the bulk density of the soil (g/cm3).
The pb of air dried sieved soil was calculated using:

_ weight of dry soil (5)

 

volume of core

Total soil porosity is defined as:

TSP = [I - [Fiji x 100 , (6)
95

where pS is the average particle density in most mineral soils and has the value of 2.65

g/cm3 (Robertson et al., 1999). Once GWC was determined, filtered distilled water was

added according to method followed by Bergsma et al. (2002) into each mesocosm, to
achieve to levels of 60, 80, 100 and 110 % WFPS. Each level of WFPS was prepared in
triplicate.

Anaerobic conditions were created by purging each mesocosm with ultra high
purity N2 gas (99.999%). An initial incubation period that varied from 2-4 weeks was
conducted to ensure the removal of any initial oxidized inorganic N by natural
denitrification. During this time, 2.5 mL of headspace gas was sampled once or twice
with a gas-tight syringe (Hamilton, Reno, NV) and the concentration of N20 was
measured until production was no longer evident. Prior to removal of headspace gas, an
equal amount of N2 was injected into the mesocosms to ensure maintenance of
atmospheric pressure. Sample gas was then stored in a 10 mL glass serum bottle purged
with N2, stoppered (using a rubber, butyl stopper), crimped, and analyzed on a
multicollector Isoprime mass spectrometer interfaced to a Trace Gas system (GV
Instruments, UK) (Sutka et al., 2003).

Once N20 production was no longer evident, a time series experiment was
implemented over the course of 10-50 h. Initially, 250 uL of isotopically characterized
N20 (substrate) was added with a gas-tight syringe to the mesocosm headspace. The
reduction of N20 was monitored every 4 h until at least 60% of the N20 was reduced. At
each time point, a gas sample of the headspace was taken and stored as described above.
Samples were analyzed immediately (or within 8 h of sampling) on the mass
spectrometer for concentration and isotopologue abundances (BUN, 5'80, SUN“ and
EISNB). The Isoprime mass spectrometer has 5 collectors capable of monitoring

simultaneously m/z 30, 31, 44, 45 and 46 that are required for measuring N20

isotopologues. The relative abundance of 15 N associated with N atom within the N20
molecule was obtained by analysis of the molecular (N 20) and fragment ions (NO+)

produced within the ion source of the mass spectrometer (Toyoda and Yoshida, 1999).

. . . 45/44 46/44 31/30 ‘
Isotope ratios were calculated from 1011 current ratios of 5 , 5 and 5 from

which 5‘5N, 5'30, a‘SN" and 5‘5NB are calculated as described by Toyoda and Yoshida

(1999). The 515 N, 5'5NCL and 5'80 values of the N20 added to the mesocosm headspace
were either 1.58, 14.90 and 41.70 %o (Tank A), respectively, or -0.93, 0.70 and 38.10 %o
(Tank C), respectively. Two sets of experiment were conducted where the second
experiment was employed two months subsequent to the first and the headspace was
injected using isotopically characterized N20 standard from Tank C. Finally, SP for each
treatment was determined as:

___15

SP Na— ”NB. (7)

RESULTS

Experiment 1
Rates of N20 reduction

Headspace concentrations of N20 in all WF PS treatments (60, 80 and 100 %)
manifested significant depletions as a function of time (Figure 2). The headspace
concentrations in the three replicates for each treatment (60, 80 and 100 %) WFPS
decreased from initial average concentrations for each treatment of 41.5, 40.4 and 42.7
uM/L to final concentrations of 18 (~60 % N20 depletion), 4 (~80 % depletion) and 15
uM/L (~70 % depletion), respectively (Figure 2 a,b,c and Table 1). Depletion of N20
within the 60 % WFPS treatment occurred in the span of 9 h for all replicates with the
exception of replicate I, where depletion of substrate N20 occurred almost
instantaneously (Figure 2a). Over time, the rate of N20 reduction decreased with
increasing WFPS in all treatments and depicted average rates of -73.3 :t 14.6, -44.9 :t 4.2

and -24.4 :t 3.5 mmol N20 g'l soil h for 60, 80 and 100 WFPS, respectively (Table 1).

N20 isotopologue enrichment factors

Microbial reduction of N20 during the time series experiment within mesocosm
treatments of 60, 80 and 100 % WFPS resulted in progressive enrichments of '5N and 18O
in N20 (Table 2). The 8 values for l5N, 18O, 15NO‘, l5NB, and SP were determined based
on the relationship between the isotopologue val ues and the natural log of the residual
concentration data for 60, 80 and 100, experiment 1 substrate concentrations relative to

the initial substrate concentrations (Eq. 1) (Figures 3, 4 and 5). The 8'5 N value for the

10

100 % WF PS mesocosm is -4.2i1.5 %o. AS WE PS decreases the 3 values become more
negative (-6.0d:0.3 %o and -7.8d:0.7 %o for 80 and 60 WFPS, respectively). The same
trend of decreasing fractionation with increasing WFPS is also evident in 8'80, s'SN‘Jl and

SISNB. Changes in sSP as a function of WFPS are not evident and are minimal, varying

between -1 and -2 %o (Table 2).

TABLE 1. Headspace concentrations, microbial reduction rates of N20 and substrate
depletion expressed as percent for 60, 80 and 100 %WFPS. The results represent data
obtained from three replicates (R1, R2 and R3) during time series experiment.

 

 

 

 

 

WFPS Total Elapsed Rates [N20]
(%) (h) (mmol N20 g" soil h") Depleted (%)
60Rl 7 -84.3 53
60R2 8 -56.0 54
60R3 9 -76.2 63
AVG ~73.3 57
STD 14.6 5

80R] 17 -40.1 67
80R2 18 -47.0 87
80R3 17 -47.6 80
AVG -44.9 78
STD 4.2 10
100R] 28 -28.4 71
100R2 ‘ 28 -21 .9 63
100R3 28 -22.8 63
AVG -24.4 66

STD

3.5

5

 

60 [A] -

 

 

 

 

0 l l 1

0 1O 20 30 40
Time elapsed (h)

60 [B] .

 

N20(HM)
or A
o o
l:

I >

 

 

 

0 10 20 30 40

60 [C] 1

 

 

 

 

0 1O 20 3O 40
Time elapsed (h)

FIGURE 2. N20 headspace concentration as a function of time for 60

(a), 80 (b) and 100 % WFPS (c), experiment 1 for replicate mesocosms
1 (open square) , 2 (close square) and 3 (open triangle).

12

TABLE 2. The a values for time series experiment 1 at 60, 80 and 100 % WFPS during
microbial reduction of N20 as determined from the Rayleigh equation (Eq. 1). The value
for eSP is determined as a difference between 8'5NCl and 8'le3 (Toyoda et al., 2005).

 

 

 

 

 

WFPS (%) a 'SN-bulk e '80 e ”N“ e ISNB e SP
60R] .79 -19.8 -9.0 -6.9 -22
60R2 -8.5 -19.8 -11.5 -55 -6.0
60R3 -7.0 -l7.6 -8.5 -5.5 81

AVG -7.8 -19.1 -9.7 —5.9 -3.8
STD 0.7 1.3 1.6 0.8 2.0
80R] -6.2 -15.8 -7.3 -52 -2.1
80R2 -6.0 -15.7 -7.5 -4.5 -30
80R3 -5.6 -14.0 -7.5 -3.8 -3.7
AVG -6.0 -152 -74 -45 -29
STD 0.3 1.0 0.1 0.7 0.8
100R] -5.9 -16.3 -7.4 -4.3 -3.1
100R2 -3.7 -125 -6.8 -07 -6.1
100R3 -3.0 -8.8 .51 -0.9 -42
AVG -4.2 -125 -6.4 -2.0 -4.5
STD 1.5 '3.8 1.2 2.1 1.5

 

13

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16

Experiment 2
Rates of N20 reduction

A second N20 reduction experiment was conducted two months subsequent to the
first experiment but using the same soil sample and identical procedures as the first
experiment. Two treatment conditions were conducted for the second experiment; 60 and
110 % WF PS. A saturation level of 60% was repeated in experiment 2 because the rapid
rate of reduction in the first experiment resulted in a limited number of data points. A
saturation condition of 110 % WF PS was conducted to study isotopologue fractionation
during reduction under a supersaturated condition. The concentration of N20 in the 60
and 110 % WFPS mesocosms declined as a function of time, however, the rates of
reduction (-16 and -21 mmol N20 g"l soil h for 60 and 110 %, respectively) was much

lower than those observed in experiment 1 (Figure 6, Tables 3).

N20 isotopologue enrichment factors

Strong correlations were evident between the natural log of the initial substrate
concentration divided by to the residual concentration and all isotopologue values during
the 60 and 110 % WFPS experiments (Figures 7). Such strong linear correlations
TABLE 3. Concentration and reduction rate of N20 in soil mesocosms for 60 and 110
WP PS, during time series experiment 2. N20 depletion, expressed as percent, reflects the

amount of substrate reduced within the headspace samples in each soil mesocosm
treatment.

 

 

WFPS Total Elapsed Rates N20 depleted
(%) hour (mmol N20 g" soil h") (%)
60 59 - 1 5.7 86
1 l 0 53 -2 1 .0 91

 

17

 

 

 

 

60
1:101:l .
A40 ~ [1330 ..
E D O
E; D DD 0
0 El
N U
z D ’
20 ~ ”D
a
QC]
. “u
0 El
0
O I 1 I
0 20 40 60 80

Elapsed time (h)

Figure 6. Concentration of N20 in soil mesocosms for 60 (open
square) and 110 % (closed diamond) WFPS in experiment 2 as a
function of time expressed in 11M.

18

TABLE 4. The 8 values for 60 and 110 % WFPS during N20 reduction, experiment 2.

The a values represent values obtained from the slope of the natural log plot with isotope
values as a function of natural log of residual substrate concentration (C) relative to initial

substrate concentration (C0).

 

 

WFPS (%) e I5N e '80 e ”N“ e l5NB e SP
60 -90 -23.6 -10.0 -7.9 -21
110 -5.3 -15.9 -53 -5.3 0.0

 

corroborate the use of the Rayleigh model to determine the isotopic enrichment factors
(Eq. 1) (Table 4). The a values for 60 WFPS were -9.0, -23.6, -10.0, -7.9, and -2.1 %o
and -5.3, -15.9, -5.3, -5.3 and 0.0 %o for 110 WFPS (Table 4). Similar to the initial
experiment conducted two months prior, the 8 values for all isotopologues in the second
experiment decrease with increasing WF PS with the exception of SP. The magnitude of
the 8 values during N20 reduction for the 60 and 110 % WFPS mesocosms in experiment
2 were Slightly less than those observed in experiment 1 (Table 2). Additionally, in
contrast to the first experiment, sSP for 60 % WF PS mesocosm exhibited a smaller shift
(-3.8 vs -2.1 %o) while the sSP value for the 110 % WFPS mesocosm was 0 %o. The lack
of variation in sSP under super saturated condition indicates that no observable
fractionation had occurred and there was no change in the relative isotopic compositions

of the or and B positions during reduction.

19

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20

Relationships between 8180 and 8'5N and 5'80 and 815N°L

While a values are clearly variable as function of WFPS and between experiments
1 and 2, there are consistent relationships between isotopologue values. For example, in
all treatments there are strong correlations between 5180 and 815 N values with an average

slope of 2.7 :t 0.2 (Table 5, Figure 8a, 9a). Similarly, consistent relationships are evident

between 6180 and S'SNO‘ with an average slope of 2.0 i 0.3 (Table 5, Figure 8b, 9b).

TABLE 5. Slopes of the relationship between 5'80 vs. 6'5 N and 5180 vs. SISN" for 60,
80 and 100 % WFPS, experiment 1, and 60 and 110 % WFPS, experiment 2, during N20
reduction. All treatments in experiment 1 were conducted in triplicate. Experiment 2
was not replicated.

 

 

 

WFPS (%) 5'80 vs 5'5N 5‘80 vs 5'5N“

Experiment 1 60R] 2.5 2.2
60R2 2.3 1.7

60R3 2.5 2.0

80R] 2.5 2.2

80R2 2.6 2.1

80R3 2.4 1.8

100R1 2.8 2.2

100R2 3.2 1.71
100R3 2.9 1.64

Experiment 2 60 2.6 2.2
110 2.9 2.7

AVG 2.7 2.0

STD 0.2 0.3

 

21

90 [A] -

 

 

 

 

 

 

 

 

60 ~
63
,9 <— Tank A
To standard

30 .

0 l l T
0 1O 20 30 40
5‘5N (%o)
90 [B] ~
A
A

60 ~
83
.9
To

30 4

Tank A
standard
0 - - -
0 10 20 3O 40
615Na (%0)

Figure 8. 5180 as a function of 5'5N (A) and SISNOL (B) for experiment 1
at 60 (open square), 80 (open triangle) and 100 % (open diamond) WFPS.
The initial isotopologue values represent values from Tank A standard
(open circle) of N20 that was added to the mesocosm headspace to initiate
the experiment. Values for r2 and p (t-test) in all cases are greater than
0.90 and less than 0.01, respectively.

22

90 [A] «

 

 

 

 

 

60 — Tank 0
a? standard
.9
Tao

30 .

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EN (%o)
90 [B] -

 

 

 

 

 

a8
.9
'70
Tank C
standard
0 - r
O 1 0 20 3O

5‘5N“ (%o)

Figure 9. 5180 as a function of SUN (A) and SUN“ (B) for experiment 2
at 60 (open square) and 100 % (open diamond) WFPS. The initial
isotopologue values represent values from Tank C standard (open circle)
of N20 that was added to the mesocosm headspace to initiate the
experiment. Values for r2 and p(t-test) in all cases are equal to or greater
than 0.90 and less than 0.01, respectively.

23

DISCUSSION

Isotopic effects on N20 during N20 reduction

Owing to complexity of natural microbial communities, published isotopologue
enrichment factors for N20 reduction in soils has not been chronicled as extensively as in
pure culture (Yoshida et al., 1984; Wahlen and Yoshinari, 1985; Yamazaki et al., 1987;
Barford et al., 1999; Cavagelli and Robertson, 2001; Sutka et al., 2005, Ostrom et al.,
2007). A challenge to isotope studies is that a values for many microbial processes,
notably N20 reduction tend to be variable (Mandemack et al., 2000; Ostrom et al., 2007).
Microbial processes are commonly multi-step reactions, which violates the fundamental
requirements of the Raleigh model (Eq. 1) that is designed for a single step uni-
directional reaction. The net fractionation expressed during a microbial process tends to
be the step that is rate limiting (Marrioti et al., 1988; 1981, Ostrom et al., 2007). For N20
reduction, the key rate limiting steps are diffusion (little fractionation) and enzymatic
reduction (large fractionation). By varying the WFPS, this study was able to determine
the importance of diffusion in controlling isotopic fractionation during N20 reduction.

In this study, 3'5 N and 8'80 values for reduction of N20 in soil mesocosms at all
levels of WFPS were -9.0 to -4.2 %o and -23.6 to -12.5 %o, respectively. These isotope
effects are generally small relative to other processes in the nitrogen cycle. For
examples, N20 production by nitrification and denitrification has reported 8 values of -68
and -28.6 %o, respectively (Yoshida, 1988; Barford et al., 1999). Such differences reflect
the unique behavior of isotopes during contrasting enzymatic reactions (Schmidt et al.,

2004). Similarly, enzymatic reduction of N20 in pure cultures has been known to

24

produce a values as high as -39 %o (Yamazaki et al., 1987) for '5 N and -42 %o (Wahlen
and Yoshinari, 1985) for 18O. Yamagishi et al. (in press) also noted that fractionation
during reduction of N20 in the eastern tropical North Pacific and the Gulf of California
was expressed to a large degree and demonstrated slightly higher 2: values than those
collected from soils (-11.6 :1: 1.0 for SISN and -30.5 :t 3.2 %o for 8'80). At the low end of
the of the spectrum, incubation of landfill cover soils produced 8'5 N and 3'80 of -2.4 and
-4.9 %o (Mandemack et al., 2000), respectively. Isotopic discrimination for 8'5N and
8180 in other soil studies is as large as —9.2 and -25.1 %o (in soil mesocosms at a single
saturation level) (Ostrom et al., 2007), -9.8 and -24.9 %o (forest soils) (Menyailo and
Hungate, 2006), respectively. These results suggest that isotopic discrimination in soils
is expressed to a lesser degree than in pure cultures and oceans.

In microbial processes that consist of more than one reaction step, it is the rate
limiting step that controls the observed fractionation for the entire process. Diffusion in
general is associated with little to no fractionation (Brandes and Devol, 1997).
Enzymatic fractionation tends to be quite large, and is likely the predominant factor
controlling the large degree of discrimination observed for N20 reduction in pure culture
(Wahlen and Yoshinari, 1985; Yamazaki et al., 1987). If the supply of substrate to the
enzyme is limiting, then all of the substrate is converted to the product and no
fractionation is observed. If the substrate is not limiting, however, the enzymatic
fractionation tends to be fully expressed. Diffusion is the predominant factor in
controlling the supply of substrate to the enzyme where N20 reduction occurs. The
relative importance of the rate of diffusion and enzymatic reduction ultimately controls

the observed or net isotopic fractionation. Thus, when diffusion across the cell boundary

25

is the rate limiting step, the observed fractionation for N20 reduction tends to be small.
This is in contrast to when diffusion is not rate limiting, the overall fractionation tends to
be large. In this study, the importance of diffusion was indirectly controlled by adjusting
the WFPS. The trend of decreasing a values with increasing WF PS is consistent with the
importance of diffusion as outlined above (Tables 2 and 4). Diffusion is not the only
factor controlling the net fractionation. The enzyme activity can vary depending on the
supply of electron donor (organic carbon) or enzyme abundance. Thus, net fractionation
varies depending upon the relative rates of diffusion versus enzyme activity.

With the exception of SP, all mesocosm treatments demonstrated consistent
declines in the 8 values for SUN, 5'80, BISNG, SISNB (Tables 2 and 4) with increases in

WFPS (60, 80, 100 and 110 %). This is consistent with reduction of enzyme activity due
to diffusion limiting the transport of N20 into the cell. In contrast, there appeared to be
no apparent relationship between SP and WFPS. The resulting a values for SP for
experiments 1 and 2 appear to be independent of the saturation conditions; -3.8 %o (60 %,
experiment 1), -2.9 %o (80 %, experiment 1), -4.5 %o (100 %, experiment 1), —2.1 %o (60
%, experiment 2) and O %o (110 %, experiment 2). However, the 8 values of SP during
N20 reduction were small and only ranged between approximately 0 and -5 %o. A trend
may have been present but likely could not be resolved because the small a values
observed approach the experimental uncertainty (i 0.8 to i 2.0, tables 2 and 4).
Comparison of experiments 1 and 2 yielded some disagreement in the a values for
each level of WFPS. The a values in the second 60 % WFPS experiment were larger

than in experiment 1; -9.0 vs -7.8 %o for 15N, -23.6 vs 19.1 %o for '80 , -10.0 vs -9.7 %o

for 15N“ and -7.9 vs -5.9 %o for 15NB. Experiment 2 was repeated two months subsequent

26 ‘

to the initial experiment with the intention of obtaining supplementary isotope data.
However, the rate of reduction was significantly slower in experiment 2, approximately
-20 mmol g'l soil h'l as opposed to -73 mmol g'l soil h]. A slower rate of reduction
possibly allowed greater expression of enzymatic fractionation as evidenced by the
higher a values in the 60% WFPS of experiment 2. A key plausible explanation for the
disparity between the first and second 60 % WFPS treatments could be attributed to the
degradation of the enzymes during storage (2 months). Thus, the rate of diffusion was
likely constant between experiments 1 and 2 but the lower enzyme activity in experiment

2 allowed expression of a greater degree of fractionation.

N20 reduction and source apportionment based on SP

In this study, isotopic discrimination during reduction of N20 was observed in SP
and the a values ranged from -4.5 to 0 %o (Table 2 and 4). This is in contrast to studies of
N20 production, which showed no discrimination in SP (Sutka et al, 2006; Toyoda et al.,
2005). With the exception of experiment 2, the sSP value of -4.5 %o in experiment 1 is
comparable to the values of -5.0 and -6.8 %o for pure cultures of Pseudomonas stutzeri
and Pseudomonas denitrificans (Ostrom et al., 2007), and -6.4 %o from ocean
environments (Yamagishi et al., in press). Fractionation in SP during N20 reduction is
problematic in source apportionment studies that rely on constant SP values. For
example, N20 reduction would cause an increase in SP from the value of 0 %o associated
with production of N20 from denitrication towards that of 33 %o associated with
production from nitrification (Sutka et a1, 2006; Ostrom et al, 2007). Consequently, the

effect on SP, 8'5N and 5'80 due to N20 reduction will ultimately affect source

27

apportionment and could become critical in regions or times with high rates of N20
reduction. The importance of this process remains unresolved as direct measures of N20

reduction in field studies are rare (Ostrom et al., 2007).

The relationship between 8'80 and 6'5N, and 8'80 and SEN“

Nitrous oxide reduction is a challenge to studies using stable isotopes to resolve
the origins of this gas and, therefore, it is critical to establish a mean for recognizing
when reduction is important. A potential means for recognizing N20 reduction resides in
the relationship among N20 isotopologues. This study found a consistent relationship
between 6180 and SISN during reduction defined by a slope of 2.7 across all levels of
WFPS (Figure 7). This relationship was present in experiments 1 and 2 despite I
differences in initial isotopologue values of the standards (tank A vs tank C) used to
initiate N20 reduction. With respect to other N20 reduction studies in soils, the slope of
2.7 for 5'80 vs 5'5 N agrees remarkably well with the slopes of 2.5 reported by Ostrom et
al. (2007), 2.5 reported by Menyailo and Hungate (2006) and 2.0 reported by
Mandemack et al. (2000).

This study, however, extends beyond previous work on N20 reduction by
establishing a relationship between 5'80 and BISNG for the first time in soils. In response
to the reduction of N20, 5'80 vs. 615Na is linearly correlated and displays a slope of 2.0
for all experiments. In N20 reduction in pure culture a slope of 1.7 for 5'80 vs.

SUN0L (Ostrom et al., 2007) was observed , which is comparable to the slope of 2.2 in this

study. Thus, a definitive means of identifying when N20 reduction is predominant are

28

relationships between 6'80 and SN and 5'80 and 5'5NCl of 2.7 and 2.0, respectively.
These relationships differ markedly from the slope of less than 1 common in flux

chamber studies (for both 5'80 and 5'5N and 5180 and 5‘5N“) (Ostrom et al., 2007).

29

CONCLUSIONS

The isotopic composition of N20 has been used to define microbial sources of
N20 and resolve global budgets of this important greenhouse gas (Kim and Craig, 1990;
Perez et al., 2001; Sutka et al, 2006). N20 reduction is a challenge to the studies because
the isotope signal of the various sources is altered during this reaction. Furthermore the
variable nature of a values indicates that the magnitude of isotope shifts during reduction
is also variable, which undermines corrections for this reaction. For example, the a
values for all N20 isotopologues consistently decline with increasing WFPS during N20
reduction. Site preference has been proposed as a conservative tracer of the production
of N20 from nitrification and denitrification (Sutka et al., 2006), however, this study
demonstrated that N20 reduction alters SP (8 S -4.5%o) and potentially source signals.
With the exception of SP, all mesocosm treatments demonstrated consistent declines in
the 8 values for N20 isotopolgues with increasing WFPS. A pragmatic method for
recognizing that isotope signatures are altered during reduction is the relationships
between 6180 and BUN, and 5'80 and ESISNOI that approach 2.7 and 2.2, respectively.
These relationships do not vary with WFPS and may provide a means for correcting for

reduction if and when it occurs.

30

APPENDICES

31

APPENDIX 1. Ion current ratios of N20 measured over time for 60 (a), 80 (b) and 100 (c) %
WFPS, experiment 1. All treatments were conducted in triplicates (R1, R2 and R3).

(A)

 

 

 

 

 

 

 

 

 

WFPS Time elapsed Peak height Peak height 645/44 546/44 831/30
(%) (h) of N20 of NO
60R1 t0 0 12.62 3.45 0.46 0.14 2.43
60R1 t1 3 12.86 3.51 1.82 3.67 3.69
60R1 t2 7 8.56 2.33 6.37 14.2 8.99
60R2 t0 0 8.93 2.44 0.69 0.72 1.87
60R2 t1 4 7.57 2.07 3.33 6.17 5.27
60R2 t2 8 5.07 1.39 7.75 16.22 10.86
60R3 t0 0 10.84 2.96 0.41 -0.04 1.62
60R3 t1 4 8.25 2.25 3.35 5.83 5.66
60R3 t2 9 5.19 1.43 7.58 16.72 9.96

 

32

 

APPENDIX 1. Continued

(3)

 

 

 

WFPS Time elapsed Peak height Peak height 645/44 646/44 631/30
(%) (h) of N20 of NO
80R1 t0 0 10.9 2.98 0.44 0.12 1.87
80R! t1 3 11.52 3.14 1.36 1.51 2.82
80R1 t3 11 8.89 2.42 3.99 8.19 5.75
80R1 t4 14 11.94 3.26 5.81 12.9 7.5
80R1 t5 17 12.65 3.46 7.41 17.24 10.92
80R1 t6 19 8.98 2.46 10.76 23.72 12.48
80R1 t7 20 9.58 2.62 12.04 27.13 14.58
80R1 t8 21 14.23 3.91 12.68 30.04 16.8
80R2 t0 0 9.81 2.68 0.65 -0.24 1.27
80R2 t1 4 8.24 2.25 1.99 3.2 4.54
80R2 t2 8 6.05 1.65 3.67 7.67 5.72
80R2 t3 12 6.28 1.72 7.04 13.63 9.09
80R2 t4 16 6.65 1.82 9.89 22.47 12.97
80R2 t5 18 5.88 1.61 13.35 30.41 15.34
80R3 t0 0 10.86 2.97 0.53 -0.18 1.89
80R3 t1 4 10.17 2.78 1.61 2.05 3.74
80R3 t2 8 7.31 2 3.33 5.64 3.55
80R3 t3 12 7.12 1.95 5.49 10.63 6.3
80R3 t4 15 8.81 2.41 6.87 14.99 10.19
80R3 t5 17 9.46 2.59 8.78 19.48 11.04
80R3 t7 21 6.69 1.84 14.86 33.51 18.35
80R3 t8 22 8.22 2.26 16.74 36.93 21.39

 

 

 

 

 

 

 

33

 

APPENDIX 1. Continued

(C)

 

 

 

 

. Time
WFPS elapsed Peak height Peak height 645/44 646/44 631/30
(%) (h) of N20 of NO
100R1 t0 0.000 11.36 3.1 0.44 -0.06 1.74
100R1 t1 4.133 16.08 4.45 0.6 1.25 2.59
100R1 t2 8.317 13.5 3.69 1.34 2.47 3.39
100R1 t3 12.317 11.55 3.15 2 3.63 4.18
100R1 t4 16.133 18.82 5.33 2.36 6.12 4.56
100R1 t5 18.717 18.38 5.17 2.9 7.73 5.25
100R1 t6 21.200 14.3 3.93 3.81 9.26 5.29
100R1 18 24.467 19.08 5.38 4.65 12.76 7.71
100R1 t9 26.367 16.05 4.42 5.8 15.48 8.82
100R1 t10 28.400 11.78 2.78 1.61 2.05 3.74
100R1 t11 29.650 13.97 3.84 8.85 22.04 3.84
100R2 to 0.000 10.48 2.86 0.59 0 1.48
100R2 t1 4.050 9.95 2.72 0.64 0.52 1.64
100R2 t2 8.183 9.32 2.55 1.24 1.29 2.52
100R2 t3 11.717 11.62 3.18 1.78 2.4 1.95
100R2 t4 15.367 17.9 5 1.65 3.72 3.82
100R2 t5 17.833 16.85 4.65 1.88 4.75 3.85
100R2 t6 20.350 14.23 3.9 2.5 5.46 4.35
100R2 t8 24.650 14.75 4.12 3.32 7.49 4.45
100R2 t10 28.167 16.87 4.79 4.6 11.27 7.14
100R2 t11 29.500 18.98 5.45 4.91 13.9 8.53
100R3 t0 0.000 11.37 3.11 0.51 -0.33 2.13
100R3 t1 4.100 9.26 2.53 1.04 0.48 1.34
100R3 t2 8.267 9.55 2.61 1.06 1.67 2.82
100R3 t3 11.983 11.13 3.04 1.84 2.92 3.54
100R3 t4 15.100 16.7 4.62 1.85 3.74 3.53
100R3 t5 17.567 17.07 4.73 1.91 4.42 3.9
100R3 t6 20.133 14.73 4.04 2.72 5.28 5.09
100R3 t8 23.950 11.51 3.17 3.65 6.84 5.23
100R3 t9 26.283 19.68 5.65 3.49 8.81 6.11
100R3 t10 27.933 18.38 5.26 3.95 9.61 6.82

 

 

 

 

 

 

34

 

APPENDIX 2. Isotopic compositions of N20 isotopologues for 60 (a), 80 (b) and 100 (c)
% WFPS. The 6 values were calculated from the ion current ratios with reference to

VSMOW and air (Toyoda and Yoshida, 1999). All treatments were conducted in triplicate

(R1, R2 and R3)

(A)

 

 

 

 

 

 

 

 

WFPS Time elapsed 6‘5N 81511“ 6‘5N‘3 8‘80
(%) (h)
60R1 10 0 2.06 17.61 -13.49 41.81
60R1 11 3 3.4 18.84 —12.03 45.49
60R1 12 7 7.92 24.24 -8.39 56.47
60R2 10 0 2.29 16.94 ~12.36 42.41
60R2 11 4 4.93 20.48 -10.62 48.09
60R2 12 8 9.33 26.24 -7.59 58.57
60R3 10 0 2.01 16.7 -12.67 41.62
60R3 11 4 4.96 20.94 -1102 47.74
60R3 12 9 9.13 25.19 -6.92 59.10

 

35

 

APPENDIX 2. Continued

(3)

 

 

 

 

 

 

 

 

WFPS Time elapsed 6‘5N 615M“ 8151:" 8‘80
(%) (h) 3‘

80R1 10 0 2.04 16.97 -12.89 41.79
80Rl 11 3 2.97 17.97 -1202 43.23
80R1 13 11 5.57 20.9 -9.76 50.2
80R1 14 14 7.37 22.62 -7.88 55.12
80R1 15 17 8.94 26.26 -8.38 59.65
80R1 16 19 12.3 27.63 -302 66.39
80R1 17 20 13.56 29.38 -27 69.95
80R1 18 21 14.16 32.2 -3.87 73

80R2 10 0 2.27 16.3 -11.76 41.41
80R2 11 4 3.59 19.83 -12.64 45

80R2 12 8 5.25 20.9 -1041 49.66
80R2 13 12 8.64 24.37 -7.08 55.86
80R2 14 16 11.42 28.28 -5.44 65.1

80R2 15 18 14.86 30.49 -077 73.37
80R3 10 0 2.14 17.01 -1272 41.47
80R3 11 4 3.22 18.98 -1254 43.8
80R3 12 8 4.94 18.54 -8.65 47.54
80R3 13 12 7.09 21.37 -7.19 52.74
80R3 14 15 8.43 25.56 -8.7 57.29
80R3 15 17 10.33 26.25 -5.6 61.98
80R3 17 21 16.37 33.74 -1 76.6
80R3 18 22 18.27 37 -047 80.16

 

36

 

APPENDIX 2. Continued

(C)

 

 

 

 

 

 

 

 

WFPS Time elapsed 615N 8‘5N“ 6‘5NB 6‘80
(%L (h)

100R1 10 0 2.05 16.83 -12.74 41.6
100R1 11 4 2.18 17.73 -13.38 42.97
100R1 12 8 2.93 18.57 -12.71 44.24
100R1 13 12 3.59 19.4 -12.21 45.45
100R1 14 16 3.91 19.69 -11.88 48.06
100R1 15 19 4.43 20.38 -11.51 49.74
100R1 16 21 5.35 20.33 -9.62 51.33
100R1 18 24 6.15 22.89 -10.60 54.99
100R1 19 26 7.29 23.99 -941 57.83
100R1 110 28 9.19 26.17 -7.79 61.41
100R1 111 30 10.33 27.86 -7.19 64.66
100R2 10 0 2.2 16.53 -1213 41.66
100R2 11 4 2.24 16.68 -12.20 42.21
100R2 12 8 2.85 17.64 -11.93 43
100R2 13 12 3.39 16.91 -10.13 44.16
100R2 14 15 3.22 18.99 -12.54 45.55
100R2 15 18 3.44 18.96 -1209 46.63
100R2 16 20 4.07 19.48 -1134 47.36
100R2 18 25 4.88 19.47 -9.70 49.48
100R2 110 28 6.13 22.32 -10.05 53.43
100R2 111 30 6.39 23.76 -10.98 56.18
100R3 10 o 2.13 17.29 -13.04 41.32
100R3 11 4 2.66 16.33 -11.o1 42.16
100R3 12 8 2.65 17.96 -12.66 43.41
100R3 13 12 3.44 18.7 -11.82 44.71
100R3 14 15 3.43 18.65 -11.79 45.57
100R3 15 18 3.48 19.04 -12.08 46.28
100R3 16 20 4.31 20.33 -11.72 47.17
100R3 18 24 5.25 20.39 -9.89 48.79
100R3 19 26 5.03 21.3 -11.24 50.86
100R3 110 28 5.49 22.06 -11.07 51.69

 

37

 

APPENDIX 3. Isotopic compositions (corrected 6 values) of N20 isotopologues,
isotopomers and site preference (SP) for 60 (a), 80 (b) and 100 (c) °/o WFPS,
experiment 1). All treatments were conducted in triplicate (R1, R2 and R3)

 

 

 

 

 

 

 

 

(A) WFPS Time elapsed 615N 81511“ 6‘5N" SP
(%) (h)

60R1 10 0 1.5 15.32 -1232 27.64
60R1 11 3 2.84 16.55 -10.87 27.42
60R1 12 7 7.36 21.95 -7.23 29.18
60R2 10 0 1.73 14.65 -1 1.19 25.84
60R2 11 4 4.37 18.92 -10.18 29.1
60R2 12 8 8.66 24.15 —6.83 30.98
60R3 10 0 1.45 14.41 -11.51 25.92
60R3 11 4 4.4 19.38 -10.58 29.96
60R3 12 9 8.46 23.1 -6.18 29.28

 

38

 

APPENDIX 3. Continued

(3)

 

 

 

 

 

 

 

 

WFPS Time elapsed 615N 615N0L 615NB SP
(%) 10)

80R1 t0 0 1.48 14.68 -11.72 26.40
80R1 t1 3 2.41 15.68 -10.86 26.54
80R1 13 11 5.01 18.61 -8.59 27.2
80R1 t4 14 6.81 20.33 -6.71 27.04
80R1 t5 17 8.38 23.97 -7.21 31.18
80R1 16 19 11.74 25.34 -1.86 27.20
80R1 17 20 13.00 27.09 -1.09 28.18
80R1 t8 21 13.6 29.91 -2.71 32.62
80R2 t0 0 1.71 14.01 -10.59 24.6
80R2 t1 4 3.03 17.54 -11.48 29.02
80R2 112 8 4.69 19.34 -9.96 29.3
80R2 13 12 7.97 22.28 -6.34 28.62
80R2 t4 16 10.425 27.24 -6.39 33.63
80R2 t5 18 13.865 29.45 -1.72 31.17
80R3 t0 0 1.58 14.72 -11.56 26.28
80R3 t1 4 2.66 16.69 -11.37 28.06
80R3 t2 8 4.38 16.98 -8.22 25.20
80R3 t3 12 6.42 19.28 . -6.44 25.72
80R3 t4 15 7.435 24.52 -9.65 34.17
80R3 t5 17 9.335 25.21 ~6.54 31.75
80R3 t7 21 15.68 32.65 -1.29 33.94
80R3 t8 22 17.58 ' 35.91 -0.75 36.66

 

39

 

APPENDIX 3. Continued

(C)

 

 

 

 

 

 

 

 

WFPS Time elapsed 615N 615M“ 6‘5NB SP
(%) (h)
100R1 10 0 1.49 14.54 -11.56 26.10
100R1 11 4 1.62 15.44 -1220 27.64
100R1 12 8 2.37 16.28 -11.54 27.82
100R1 13 12 3.03 17.11 -11.05 28.16
100R1 14 16 3.35 17.40 -1070 28.10
100R1 15 19 3.87 18.09 -1035 28.44
100R1 16 21 4.79 18.04 -8.46 26.50
100R1 18 24 5.59 20.60 942 30.02
100R1 19 26 6.73 21.70 -8.24 29.94
100R1 110 28 8.63 23.88 -6.62 30.50
100R1 111 30 9.77 25.57 -6.03 31.60
100R2 10 o 1.64 14.24 -10.96 25.20
100R2 11 4 1.68 15.12 -11.76 26.88
100R2 12 8 2.29 16.08 -11.50 27.58
100R2 13 12 2.72 14.82 -9.38 24.20
100R2 14 15 2.23 17.95 -13.50 31.45
100R2 15 18 2.45 17.92 -13.03 30.95
100R2 16 20 3.40 17.39 -1o.59 27.98
100R2 18 25 4.19 18.38 -10.00 28.38
100R2 110 28 5.44 21.23 -10.35 31.58
100R2 111 30 5.70 22.67 -11.27 33.94
100R3 10 o 1.57 15.00 -11.86 26.86
100R3 11 4 2.10 14.77 -1o.57 25.34
100R3 12 8 2.09 16.40 -12.22 28.62
100R3 13 12 2.77 16.61 -1107 27.68
100R3 14 15 2.76 16.56 -11.04 27.60
100R3 15 18 2.49 18.00 -1303 31.03
100R3 16 20 3.64 18.24 -10.96 29.20
100R3 18 24 4.56 19.30 -10.18 29.48
100R3 19 26 4.34 20.21 -11.53 31.74
100R3 110 28 4.80 20.97 -1137 32.34

 

40

 

APPENDIX 4. Headspace concentration data for 60, 80 and 100 % WFPS, experiment 1
over time. All treatments were conducted in triplicate (R1, R2 and R3).

 

 

 

 

 

 

WFPS Total Elapsed Concentration (pmol)
(%) (h) [Initial] LFinal]
60R1 7 49.02 23.11
60R2 8 33.86 14.95
60R3 9 41.71 15.35
Avg 41.53 17.80
STD 7.59 4.60
80R1 17 41.95 13.69
80R2 18 37.47 4.92
80R3 17 41 .79 8.23
Avg 40.41 8.95
STD 2.54 4.43
100R1 28 43.84 12.69
100R2 28 40.23 14.92
100R3 28 43.89 16.25
Avg 42.65 14.62
STD 2.10 1.80

 

41

 

APPENDIX 5. Ion current ratios of N20 measured over time for 60 (A) and 110 (B) %
WFPS, experiment 2.

 

 

 

 

 

 

 

 

 

(A)
WFPS Time elapsed Peak height Peak height 645/44 646/44 631/30
(%) (h) of N20 of NO

60 t0 0 11.57 3.30 1.42 0.09 -3.68
60 t2 4 14.05 4.15 1.81 1.29 0.97
60 t3 5 11.00 3.14 1.75 1.52 -1.24
60 t4 6 12.31 3.57 2.01 1.48 -2.08
60 t5 8 13.42 3.95 2.54 2.97 —0.17
60 t6 10 13.28 3.90 2.85 3.31 0.05
60 t7 11 11.69 3.38 2.34 3.81 -2.31
60 t8 13 12.63 3.70 3.16 4.72 -1.14
60 t9 15 12.29 3.60 3.24 5.33 -0.89
60t10 18 12.83 3.76 4.52 6.11 -0.92
60 t11 22 12.70 3.72 4.77 8.43 -0.55
60 t12 25 12.17 3.53 5.53 9.59 -0.60
60 t13 29 11.65 3.36 5.87 11.30 —1.23
60 U4 33 11.75 3.39 7.23 13.68 1.14
60 t15 35 12.12 3.55 6.91 14.61 1.17
60 t16 38 14.80 4.43 8.43 16.48 5.66
60 H7 40 14.08 4.20 9.50 18.14 5.28
60 t19 49 14.45 4.35 12.29 25.98 11.55
60 t20 52 12.58 3.74 13.57 29.35 9.02
60 t21 55 11.93 3.53 14.90 33.93 13.50
60 t22 57 11.70 3.46 16.23 36.74 12.14
60 t23 59 11.84 3.53 16.60 40.37 15.47

 

42

 

APPENDIX 5. Continued.

 

 

 

(B)
WFPS Time elapsed Peak height Peak height 645/44 646/44 631/30
(%) (h) of N20 of NO
110 to 0 11.62 3.32 1.14 0.26 -3.43
110 t2 12 14.00 4.13 1.93 1.31 -1.47
110 t3 16 10.72 3.05 1.72 1.28 -2.16
110 14 21 13.48 3.95 2.62 2.50 -1.04
110 t5 24 12.93 3.80 2.19 2.74 -0.44
110 t6 28 12.23 3.58 2.47 3.47 -3.24
110 t7 35 12.29 3.59 3.15 5.50 -2.31
110 t8 42 11.22 3.21 4.51 8.76 -2.13
110 t11 48 12.70 3.75 7.04 16.04 3.47
110 t12 49 12.23 3.58 8.32 18.40 4.38
110113 51 10.85 3.11 10.44 25.18 5.92
110 t14 52 9.57 2.73 11.96 29.50 6.21
110 t15 53 8.51 2.42 15.25 37.00 11.51

 

 

 

 

 

 

 

43

 

APPENDIX 6. Isotopic compositions of N20 isotopologues for 60 (A) and 110 (B) %
WFPS. The 6 values were calculated from the ion current ratios with reference to
VSMOW and air (Toyoda and Yoshida, 1999).

 

 

 

 

 

 

 

 

(A)
WFPS Time elapsed 51511 8‘5N“ 8‘5NB 6180
(%) (“L
60 10 0 -1.47 -3.46 4.57 37.92
60 12 4 -1.12 1.72 0.14 38.88
60 13 5 -1.56 -0.78 2.51 39.48
60 14 6 -1.37 -174 4.02 39.29
60 15 8 -1.21 0.33 2.99 40.71
60 16 10 -123 0.55 3.40 41.07
60 17 11 -1.46 -2.13 4.98 41 .79
60 18 13 -1.32 -0.87 5.40 42.62
60 19 15 -137 -0.63 5.29 43.29
60110 18 -1.29 -0.73 8.05 44.03
60 111 22 -1.31 -044 8.16 46.46
60 112 25 -139 -0.58 9.84 47.72
60 114 33 -1.45 1.13 11.49 52.02
60 115 35 -140 1.13 10.77 52.96
60116 38 -101 6.07 8.93 54.58
60117 40 -1.12 5.58 11.64 56.38
60 119 49 -1.06 12.14 10.49 64.50
60 120 52 -133 9.07 16.07 68.21
60 121 55 -142 13.87 13.84 73.06
60 122 57 -1.46 12.15 18.20 76.01
60 123 59 -1.44 15.72 15.23 79.78

 

44

 

APPENDIX 6. Continued.

 

 

 

 

(B)
WFPS Time elapsed 5‘5N 6‘5N“ 8‘5N‘3 6‘80
(%) (0)
11010 0 -1.47 -3.18 3.69 38.10
11012 12 -1.13 -1.04 3.16 38.91
11013 16 -1.60 -1.81 3.49 39.26
11014 21 -1.20 -0.63 4.14 40.20
11015 24 -1.28 0.04 2.55 40.52
110 16 28 -1.38 -3.17 6.31 41.36
11017 35 -137 -224 6.70 43.47
110 18 42 -1.52 -2.24 9.40 46.98
110111 48 -1.31 3.65 8.45 54.39
110112 49 -1.38 4.52 10.14 56.88
110113 51 -1.58 5.86 12.92 64.10
110114 52 -1.76 5.93 15.82 68.75
110 115 53 -1.91 11.45 16.83 76.66

 

 

 

 

 

45

 

APPENDIX 7. Isotopic compositions (corrected 6 values) of N20 isotopologues,

isotopomers and site preference (SP) for 60 (A) and 110 (B) % WFPS, experiment 2.

 

 

 

 

 

 

 

 

(A)
WFPS Time elapsed 615M 6‘5N“ 615NB SP
(%) (h)
60 10 o -091 1.38 -321 4.59
60 12 4 -0.18 6.56 -6.92 13.48
60 13 5 -0.69 4.06 -543 9.50
60 14 6 -023 3.10 -3.56 6.66
60 15 8 0.45 5.17 .427 9.44
60 16 10 0.75 5.39 -3.89 9.28
60 17 11 -003 2.71 -277 5.48
60 18 13 0.95 3.97 -2.08 6.05
60 19 15 0.97 4.21 -227 6.49
60t10 18 2.37 4.11 0.62 3.49
60 111 22 2.55 4.40 0.69 3.71
60 112 25 3.24 4.26 2.22 2.04
50 114 33 4.86 5.97 3.75 2.22
60 115 35 4.55 5.97 3.14 2.84
60 t16 38 6.49 10.91 2.06 8.85
60 117 40 7.47 10.37 4.58 5.80
60 119 49 10.25 16.98 3.53 13.45
60 120 52 11.25 13.91 8.59 5.32
60 121 55 12.44 18.71 6.16 12.55
60 122 57 13.72 16.99 10.46 6.54
60 123 59 14.04 20.56 7.53 13.04

 

46

 

APPENDIX 7. Continued.

03)

 

 

 

 

 

 

 

 

WFPS Time elapsed 615N 81514“ 8151113 SP
(96) (0)

11010 0 -121 1.66 -4.08 5.74
110 12 12 -007 3.80 394 7.74
11013 16 -0.76 3.03 -4.54 7.58
11014 21 0.55 4.21 -309 7.31
110 15 24 0.02 4.88 —4.84 9.72
11016 28 0.20 1.67 -127 2.94
110 17 35 0.87 2.60 -0.86 3.47
110 18 42 2.06 2.60 1.51 1.09
110111 48 4.74 8.49 0.98 7.51
110112 49 5.96 9.36 2.56 6.80
110113 51 7.81 10.70 4.92 5.78
110114 52 9.12 10.77 7.47 3.30
110115 53 12.24 16.29 8.19 8.11

 

47

 

APPENDIX 8. Headspace concentration data for 60 and 110 °/0 WFPS,
experiment 2 over time.

 

 

 

WFPS Total Elapsed Concentrations (mM)
(%) (h) [initial] [final]
60 59 46.80 6.70
1 10 53 47.10 4.30

 

48

 

 

   
   
  

 

 

 

1.4 60 %
.. R1:y=-0.08x+1.18
FA 1'2 R2 = 0.99
'm 1.0 ‘ , __
«_ R2. y - -0.06x + 0.84
g 0.8 R2 = 1.00
E 0.6 4
g- 0.4 -
-— R3: y = -0.08x + 1.03
0-2 ‘ R2 =1.00
0.0 1 1
0 5 10 15
Elapsed time (h)
1.2 80 %

 

    
  

R1: y = -0.04x + 1.00
A R2 = 0.98

R2: y = -0.05x + 0.93
R2 = 0.99

  
 
 
 
    

[~20] (um0|*9")
O
O)

 

 

 

 

 

 

 

0.4 1
0.2 -
0.0 - r -
0 5 10 15 20 25
Elapsed time (h)
1.4 e 100 %
1 R1:y=-0.03x+ 1.18
A 1.2 R2=0.96
to 1.0 R2:y=-0.02x+0.95
L 2:
E 0.8 - R 0.98
2‘ 0.6 ~
0
~ .4 —
E O R3:y=-0.02x+0.96 A
0.2 * R2=0.89
0.0 I I I
0 10 20 , 30 40
Elapsed time (h)

APPENDIX 9. Rate of N20 reduction for 60, 80 and 100 % WFPS,
experiment 1 for replicate 1 (open square), replicate 2 (closed square) and
replicate 3 (open triangle)

49

 

 

 

 

 

 

 

 

 

 

 

 

4O -— 60 %
35 -
a? .
0- 30 - A D A I
(D a El
25 -
20 -
-2 -1 O
ln(C/Co)
40 1— 80 %
A
35 ~ A D I A
g 30 ' A D
Q I I I
‘0 D :1 1:: n n A
25 - A A
20 1 -
-3 -2 -1
ln(ClCo)
35 '— 100 % I
A
an i of
A 30 -1 A 1:] D
83 IA
v 'AA 6.1::
% El
25 - A
I
20 1 - -
-3 -2 -1
ln(C/Co)

APPENDIX 10. Site preference as a function of ln(C/Co) for 60, 80 and 100 %

WFPS, experiment 1 for replicate 1 (open square), replicate 2 (closed
square) and replicate 3 (open triangle).

50

 

 
     
  

1-0 ‘ y=-0.016x+ 1.143

R2 = 0.992

0

[N20] oimoi*g")
p o
O) 00

y= -0.021x+ 1.356
R2 = 0.916

 

 

 

.0 .0 .0
ON-h-
1 L
.0
D

O

20 40 60 80
Time elapsed (h)

APPENDIX 11. Rate of N20 reduction for 60 (open square) and 110 (closed triangle) °/0
WFPS over time, experiment 2.

51

 

 

 

 

20
15 --
D D Cl
C]
a?
0. 1O - D b D
(D . .
D O O .00
5 ~ ° ° 0 Ch” ii
0 a «E.
0 c1
0
0 i -
-3 -2 -1 O
ln(C/Co)

APPENDIX 12. Site preference (SP) as a function of ln(C/Co) for 60 (open square) and
110 (closed triangle) % WFPS over time, experiment 2.

52

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53

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