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METHANE OXIDATION IN TERRESTRIAL ECOSYSTEMS:
PATTERNS AND EFFECTS OF DISTURBANCE

By

Pongthep Suwanwaree

A DISSERTATION
Submitted to
Michigan State University

in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Crop and Soil Sciences

2003

ABSTRACT

METHANE OXIDATION IN TERRESTRIAL ECOSYSTEMS:
PATTERNS AND EFFECTS OF DISTURBANCE

By

Pongthep Suwanwaree

Methane oxidation in aerobic upland soils is an important sink for annually
increasing atmospheric methane. The methane oxidation capacity of soil may be
declining due to the conversion of natural forest to other uses. I investigate methane
oxidation along a gradient of soil disturbance from mature old-growth forest to
agricultural ﬁelds. I found that soils in the mature deciduous forest oxidize substantially
more CH4 than mid-successional forest and no-till agricultural ﬁelds.

A single application of 100 kg N fertilizer per hectare signiﬁcantly decreased CH4
oxidation rates in both mature deciduous forest and mid-successional communities but
had no effects in agricultural ﬁelds, which already had low oxidation rates. In contrast, a
10 cm depth plowing did not show a signiﬁcant detectable effect on soil CH4
consumption in any soils. Additionally, lower levels of N fertilization (30 kg N ha'l)
signiﬁcantly affected CH4 oxidation in coniferous forest, but not in deciduous forest nor
in mid-successional communities.

Methane oxidation signiﬁcantly differed among soil depths. In deciduous forest
soil cores incubated in the laboratory, CH4 uptake was highest at 5-10 cm depth whereas
there were no signiﬁcant differences with depth (0-5, 5-10, 10-20, and 20-30 cm) in

coniferous forest, in a mid-successional community, nor in no-till agricultural soils.

Organic and conventional com-com-soybean—wheat management systems also
had similar average soil CH4 uptake rates. Nevertheless, CH4 oxidation signiﬁcantly
differed among crops, such that organic com had the highest rates of oxidation while
organic soybean and wheat had the lowest rates. Rates in conventionally managed corn,
soybean, and wheat crops were similar and intermediate.

Soil methane oxidation was not signiﬁcantly correlated with C02 emission. Soil
nitrate and ammonium were weak indicators of changes of CH; oxidation among land use
types and soil depths. Nitriﬁcation potential was also a good indicator of CH4 oxidation
rates.

Results suggest a major effect of agricultural management on CH4 oxidation in
terrestrial ecosystems, with reductions in oxidation capacity related more to changes in
nitrogen availability than physical soil disturbance itself. Low-level nitrogen additions
(10 kg N ha"), similar to rates of N-deposition, however, were not sufﬁcient to reduce

oxidation in undisturbed forests or mid-successional communities.

ACKNOWLEDGMENTS

I consider the four years at KBS and six years in the US as one of the most
extraordinary journeys in my life. I value this time so dearly and mainly attribute this to
all the support I received and to people I had the great fortune of meeting. My ﬁrst
deepest appreciation goes to my advisor — Prof. G.P. Robertson for his “hands off ’
mentoring, advice, encouragement, and untiring editing of my dissertation. I am heartily
thankful for his untiring support in this endeavor to push my abilities to the test in order
to pursue my degree.

I would also like to extend my deepest appreciation to my committee members —
Prof. Alvin J. Smucker, Prof. Michael J. Klug, and Prof. Bernard Knezek for their
support and guidance in my research.

This dissertation could not be successfully operated without the assistance of
Claire McSwiney for the backbreaking gas and soil sampling in the hot and humid ﬁelds
and forests, Star Shelton and Christine Easley for gas and soil analysis and lab assistance,
Barb Fox for administrative work, Andrew Corbin, Joe Simmons, Greg Parker for ﬁeld
maintenances, and Brady West from Center for Statistical Consultation and Research,
University of Michigan for Statistics help.

I also greatly appreciate the campus and KBS faculty members, staff, and friends
who have remained helpful and supportive as always. Special thanks for my fellow
students — Tim Bergsma, Skip Vanbloem, Richard Smith, Natalie Dubious, Laura
Broughton, Ann-Marie F ortuna, Stuart Grandy, Terry Loecke, and Sven Bohm for their

unfading friendships and support.

iv

In pursuing my educational goals, I am genuinely thankful to the Michigan State
University, KBS, NSF as well as to the Royal Thai Government for providing me with
support and opportunities.

Last, but not the least, I am sincerely grateful to my parents for their love,

support, and patience for my being far away for years. This dissertation is dedicated to

them.

TABLE OF CONTENT

LIST OF TABLES ........................................................................................................... viii

LIST OF FIGURES .......................................................................................................... xii

CHAPTER 1

METHANE OXIDATION IN ARABLE SOILS ................................................................ 1
Introduction .............................................................................................................. 1
Methanotrophic bacteria .......................................................................................... 2
Environmental factors inﬂuencing soil methane oxidation ..................................... 7
Other methane oxidizing organisms ...................................................................... l3
Methane oxidation in aerobic soils ........................................................................ 14
Agricultural effects ................................................................................................ 1 5
Summary ................................................................................................................ 20
References .............................................................................................................. 21

CHAPTER 2

METHANE OXIDATION IN SUCCESSIONAL AND AGRICULTURAL

ECOSYSTEMS: EFFECTS OF NITROGEN AND SOIL DISTURBANCE ................... 49
Introduction ............................................................................................................ 49
Methodology .......................................................................................................... 50
Results .................................................................................................................... 53
Discussion .............................................................................................................. 57
Conclusion ............................................................................................................. 60
References .............................................................................................................. 61

CHAPTER 3

SOIL METHANE OXIDATION IN ORGANIC VS. CONVENTIONAL

AGRICULTURE ............................................................................................................... 84

vi

Introduction ............................................................................................................ 84

Methodology .......................................................................................................... 85
Results .................................................................................................................... 88
Discussion .............................................................................................................. 96
Conclusion ........................................................................................................... 100
References ............................................................................................................ l O 1

CHAPTER 4

METHANE OXIDATION IN SUCCESSIONAL FOREST: EFFECTS OF NITROGEN

DEPOSITION .................................................................................................................. 130
Introduction .......................................................................................................... 1 3O
Methodology ........................................................................................................ 13 1
Results .................................................................................................................. 135
Discussion ............................................................................................................ 140
Conclusion ........................................................................................................... 144
References ............................................................................................................ 145

CHAPTER 5

EFFECTS OF DIFFERENT LEVELS OF NITROGEN FERTILIZER ON SOIL CH4

OXIDATION AND C02 FLUX 1N CONTINUOUS CORN .......................................... 171
Introduction .......................................................................................................... 1 7 1
Methodology ........................................................................................................ l 72
Results .................................................................................................................. 174
Discussion ............................................................................................................ 176
Conclusion ........................................................................................................... 179
References ............................................................................................................ 179

vii

LIST OF TABLES

CHAPTER 1

Table 1.1 Estimated sources and sinks of methane ............................................................ 38
Table 1.2 General characteristics of methanotrophic bacteria ........................................... 39
Table 1.3 Characterization of methanotrophs .................................................................... 39
Table 1.4. Annual methane uptake in different global ecosystems ................................... 40

Table 1.5 Methane oxidation of aerobic soils under ﬁeld condition in various
locations of the world ......................................................................................... 41

Table 1.6 Average methane oxidation in different ecosystems; summary data from
Table 1.5 ............................................................................................................. 42

CHAPTER 2

Table 2.1 Soil properties for the mature deciduous forest, coniferous forest, mid-
successional forest, and no-till agricultural ﬁeld at the KBS LTER site. ........... 66

Table 2.2 The effects of tillage and N fertilizer on average methane oxidation,
carbon dioxide ﬂux, and soil properties in mature deciduous forest, mid-
successional forest, and no-till agriculture at the KBS LTER site ..................... 67

Table 2.3 The analysis of variance to determine effects of sites on methane
oxidation rate, carbon dioxide emission, and soil properties before the
experiment from mature deciduous forest, mid-successional community,
and no-till agricultural ﬁeld at the KBS LTER site. ........................................... 68

Table 2.4 The analysis of covariance to determine effects of site, and soil
properties on methane oxidation rate before the experiment from mature
deciduous forest, mid-successional community, and no-till agricultural
ﬁeld at the KBS LTER site. ................................................................................ 69

Table 2.5 The analysis of variance to determine effects of site, treatment, and soil
properties on methane oxidation rate after the experiment from mature
deciduous forest, mid-successional community, and no-till agricultural
ﬁeld at the KBS LTER site ................................................................................. 70

Table 2.6 The analysis of covariance to determine effects of site, and soil
properties on carbon dioxide emission before the experiment from

viii

mature deciduous forest, mid-successional community, and no-till
agricultural ﬁeld at the KBS LTER site .............................................................. 71

Table 2.7 The analysis of covariance to determine effects of site, treatment, and
soil properties on carbon dioxide emission after the experiment from
mature deciduous forest, mid-successional community, and no-till
agricultural ﬁeld at the KBS LTER site .............................................................. 72

Table 2.8 Analysis of variance to determine effects of site, treatment, and day on
soil properties from mature deciduous forest, mid-successional
community, and no-till agricultural ﬁeld at the KBS LTER site ........................ 73

Table 2.9 Relationships among methane oxidation, carbon dioxide, and other soil
properties before and after experimental treatment in mature deciduous

forest, mid-successional community, and no-till agricultural sites .................... 74
CHAPTER 3
Table 3.1 Agronomic ﬁeld activities for the sites studied ............................................... 104

Table 3.2 Soil properties of conventional and organic management systems at the
KBS LF L site .................................................................................................... 105

Table 3.3 Net average methane oxidation and carbon dioxide emission rates and
other soil properties in conventional and organic systems at the KBS
LF L site ............................................................................................................. 106

Table 3.4 Analysis of covariance to determine effects of treatment, crop, day, and
soil properties on methane oxidation rates from conventional and
organic agriculture at the KBS LF L site ........................................................... 107

Table 3.5 Average soil methane oxidation and carbon dioxide ﬂuxes, and soil
properties among crops in conventional and organic agriculture systems ....... 108

Table 3.6 Analysis of covariance to determine effects of treatment, crop, day, and
soil properties on carbon dioxide emission rates from conventional and
organic agriculture at the KBS LF L site ........................................................... 109

Table 3.7 Analysis of variance to determine effects of site, treatment, and day on
soil properties from conventional and organic agriculture at the KBS
LFL site ............................................................................................................. 110

Table 3.8 Relationships among methane oxidation, carbon dioxide and other soil
properties in conventional and organic treatments at the KBS LFL site .......... 111

ix

Table 3.9 Differences in average soil methane oxidation rates and other properties
between soil cores from conventional and organic systems at the KBS
LFL site ............................................................................................................. 112

Table 3.10 Average methane oxidation, soil properties, and nitriﬁcation rates

among crops in conventional and organic agriculture along soil proﬁle at
the KBS LFL site .............................................................................................. 113

Table 3.11 Analysis of covariance for methane oxidation in laboratory soil cores
as affected by management, crops, soil depth, and soil properties at the
KBS LF L site .................................................................................................... 114

Table 3.12 Analysis of variance to determine effects of crop, treatment, and depth
on properties of soil cores from conventional and organic agriculture at

the KBS LFL site .............................................................................................. 115
Table 3.13 The relationships among methane oxidation and other soil properties

in soil cores from different depths at the KBS LFL site ................................... 116
CHAPTER 4

Table 4.1 Soil properties for the mature deciduous forest, coniferous forest, mid-
successional forest, and no-till agricultural ﬁeld at the KBS LTER site. ......... 148

Table 4.2 Analysis of covariance to determine effects of site, treatment, and soil
properties on methane oxidation rates in mature deciduous forests,
coniferous plantations, and mid-successional communities at the KBS
LTER site .......................................................................................................... 149

Table 4.3 Effects of N fertilizer on methane oxidation, carbon dioxide emission,
and soil properties in coniferous forest, deciduous forest, and mid-
successional forest at the KBS LTER site ........................................................ 150

Table 4.4 Analysis of covariance to determine effects of site, treatment (added
nitrogen), and soil properties on carbon dioxide emissions in mature
deciduous forests, coniferous plantations, and mid-successional
communities at the KBS LTER site .................................................................. 151

Table 4.5 Analysis of variance to determine effects of site, treatment, and day on
soil pr0perties from coniferous forest, mature deciduous forest, and mid-
successional forest at the KBS LTER site ........................................................ 152

Table 4.6 The relationships among methane oxidation, carbon dioxide and other
soil properties in coniferous forest, mature deciduous forest, and mid-
successional forest at the KBS LTER site ........................................................ 153

Table 4.7 Fertilization effects on average methane oxidation, soil properties, and
nitriﬁcation rates among crops in coniferous forest, deciduous forest,
mid-successional forest, and no-till agricultural ﬁeld at the KBS LTER
site .................................................................................................................. 154

Table 4.8 Analysis of covariance for methane oxidation as affected by site,
fertilization, and soil depth, on soil properties from coniferous forest,
mature deciduous forest, mid-successional forest, and no-till agriculture
at KBS LTER site ............................................................................................. 155

Table 4.9 Analysis of variance to determine effects of site, treatment, and depth
on soil properties from coniferous forest, mature deciduous forest, mid-
successional forest, and no-till agriculture at the KBS LTER site ................... 156

Table 4.10 The relationship among methane oxidation and other soil properties
from the soil depth study in coniferous forest, mature deciduous forest,
mid-successional forest, and no-till agriculture at KBS LTER site .................. 157

CHAPTER 5

Table 5.1 Net methane oxidation, carbon dioxide emission, and soil properties at
various N fertilizer levels in continuous corn plots after fertilization
across all sample dates ...................................................................................... 182

Table 5.2 Analysis of covariance to determine effects of N fertilizer and soil
properties on methane oxidation rates before fertilization ............................... 183

Table 5.3 Analysis of covariance to determine effects of N fertilizer and soil
properties on methane oxidation rates aﬁer fertilization .................................. 184

Table 5.4 Analysis of variance to determine effects of N fertilizer and soil
properties on carbon dioxide emission rates before fertilization ...................... 185

Table 5.5 Analysis of covariance to determine effects of N fertilizer and soil
properties on carbon dioxide emission rates after fertilization ......................... 186

Table 5.6 Analysis of covariance to determine effects of N fertilizer on soil
properties aﬁer fertilization .............................................................................. 187

Table 5.7 The relationships among methane oxidation, carbon dioxide and other

soil properties before and after fertilization at the N rate continuous
cornﬁeld study at the KBS site ......................................................................... 188

xi

LIST OF FIGURES

CHAPTER 1

Figure 1.1 Electron micrographs showing type I membrane systems of
Methylomonas methanica ................................................................................ 43

Figure 1.2 Electron micrographs showing type 11 membrane systems of
Methylocystis parvus ........................................................................................ 44

Figure 1.3 Methane oxidation in methanotrophs ............................................................... 45

Figure 1.4 Electron transfer pathway between the three protein components of the

sMMO .............................................................................................................. 46
Figure 1.5 Ribulose monophosphate (RMP) pathway for formaldehyde ﬁxation ............ 47
Figure 1.6 Serine pathway for formaldehyde ﬁxation ....................................................... 48
CHAPTER 2

Figure 2.1 The reduction of methane oxidation due to soil disturbance, and
ammonium nitrate fertilizer (100 kg N ha") in mature forests, mid-

successional communities, and no-till comﬁelds at KBS LTER site .............. 75
Figure 2.2 Average daily methane oxidation in the KBS LTER site ................................. 76
Figure 2.3 Average daily carbon dioxide ﬂuxes in the KBS LTER site ............................ 77

Figure 2.4 Net carbon dioxide ﬂuxes at KBS LTER site due to soil disturbance,
and ammonium nitrate fertilizer (100 kg N ha") in mature forests, mid-

successional communities, and no-till comﬁelds ............................................ 78
Figure 2.5 Average daily soil moisture in the KBS LTER site ......................................... 79
Figure 2.6 Average daily soil temperature in the KBS LTER site .................................... 80
Figure 2.7 Average daily soil nitrate in the KBS LTER site ............................................. 81
Figure 2.8 Average daily soil ammonium in the KBS LTER site ..................................... 82

Figure 2.9 Relationships between methane oxidation and carbon dioxide ﬂux
from mature deciduous forest, mid-successional communities, and no-
till agricultural ﬁeld at the KBS LTER site ..................................................... 83

xii

CHAPTER 3

Figure 3.1 Net methane oxidation of different crops in conventional and organic
agriculture at KBS LF L site ........................................................................... 117

Figure 3.2 Net soil carbon dioxide ﬂuxes of different crops in conventional and
organic agriculture at KBS LF L site .............................................................. 117

Figure 3.3 Average daily soil methane oxidation under different crops from
conventional (top) and organic management systems (bottom) at the
KBS LFL site in 2001-2002 ........................................................................... 118

Figure 3.4 Average daily soil carbon dioxide emissions under different crops from
conventional (top) and organic management systems (bottom) at the
KBS LFL site in 2001-2002 ........................................................................... 119

Figure 3.5 Average daily soil moisture in different crops under conventional (top)
and organic management systems (bottom) at the KBS LFL site in
2001-2002 ...................................................................................................... 120

Figure 3.6 Average daily soil temperature under different crops in conventional
(top) and organic management systems (bottom) at the KBS LFL site
in 2001-2002 .................................................................................................. 121

Figure 3.7 Average daily soil nitrate levels under different crops in conventional
(top) and organic management systems (bottom) at the KBS LFL site
in 2001-2002 .................................................................................................. 122

Figure 3.8 Average daily soil ammonium levels in different crops under
conventional (top) and organic management systems (bottom) at the
KBS LFL site in 2001-2002 ........................................................................... 123

Figure 3.9 Relationships between methane oxidation and carbon dioxide ﬂuxes in
conventional and organic agriculture at the KBS LFL site ............................ 124

Figure 3.10 Soil methane oxidation with depth in soil cores from corn, soybean,
and wheat rotations under conventional and organic management at
the KBS LFL site ........................................................................................... 125

Figure 3.11 Soil moisture with depth in soil cores from corn, soybean, and wheat
rotations under conventional and organic management at the KBS LFL
she .................................................................................................................. 126

Figure 3.12 Soil nitrate with depth in soil cores from corn, soybean, and wheat
rotations under conventional and organic management at the KBS LF L
site .................................................................................................................. 127

xiii

Figure 3.13 Soil ammonium with depth in soil cores from corn, soybean, and
wheat rotations under conventional and organic management at the
KBS LFL site ................................................................................................. 128

Figure 3.14 Soil nitriﬁcation with depth in soil cores from corn, soybean, and
wheat rotations under conventional and organic management at the
KBS LFL site. ................................................................................................ 129

CHAPTER 4

Figure 4.1The effects of different N fertilizer levels on net methane oxidation in

coniferous forest, deciduous forest, and mid-successional communities
at the KBS LTER site .................................................................................... 158

Figure 4.2 The effects of different N fertilizer levels on net daily methane
oxidation in coniferous forest (top), deciduous forest (middle), and
mid-successional communities (bottom) at the KBS LTER site ................... 159

Figure 4.3 The effects of different N fertilizer levels on net carbon dioxide
emission in coniferous forest, deciduous forest, and mid-successional
communities at the KBS LTER site ............................................................... 160

Figure 4.4 The effects of different N fertilizer levels on net daily carbon dioxide
emission in coniferous forest (top), deciduous forest (middle), and
mid-successional communities (bottom) at the KBS LTER site ................... 161

Figure 4.5 The effects of different N fertilizer levels on daily soil moisture in
coniferous forest (top), deciduous forest (middle), and mid-
successional communities (bottom) at the KBS LTER site ........................... 162

Figure 4.6 The effects of different N fertilizer levels on daily soil temperature in
coniferous forest (top), deciduous forest (middle), and mid-
successional communities (bottom) at the KBS LTER site ........................... 163

Figure 4.7 The effects of different N fertilizer levels on daily soil nitrate in
coniferous forest (top), deciduous forest (middle), and mid-
successional communities (bottom) at the KBS LTER site ........................... 164

Figure 4.8 The effects of different N fertilizer levels on daily soil ammonium in
coniferous forest (top), deciduous forest (middle), and mid-
successional communities (bottom) at the KBS LTER site ........................... 165

Figure 4.9 The effects of N fertilizer on methane oxidation along soil depth from
soil cores taken from deciduous, coniferous forest, mid-successional
communities, and no-till cornﬁeld at the KBS LTER site, after
incubation in laboratory ................................................................................. 166

xiv

Figure 4.10 The effects of N fertilizer on soil moisture along soil depth from soil
cores taken from deciduous, coniferous forest, mid-successional
communities, and no-till cornﬁeld at the KBS LTER site, after
incubation in laboratory ................................................................................. 167

Figure 4.11 The effects of N fertilizer on soil nitrate along soil depth from soil
cores taken from deciduous, coniferous forest, mid-successional
communities, and no-till cornﬁeld at the KBS LTER site, after
incubation in laboratory ................................................................................. 168

Figure 4.12 The effects of N fertilizer on soil ammonium along soil depth from
soil cores taken from deciduous, coniferous forest, mid-successional
communities, and no-till cornﬁeld at the KBS LTER site, after
incubation in laboratory ................................................................................. 169

Figure 4.13 The effects of N fertilizer on soil nitriﬁcation along soil depth from
soil cores taken from deciduous, coniferous forest, mid-successional
communities, and no-till cornﬁeld at the KBS LTER site, after

incubation in laboratory ................................................................................. 170
CHAPTER 5
Figure 5.1 Layout of the N-fertilizer rates study at the KBS LTER site ......................... 189

Figure 5.2 Net methane oxidation at various N fertilizer levels in continuous corn
plots at the KBS LTER site ............................................................................ 190

Figure 5.3 Net daily methane oxidation at various N fertilizer levels in continuous
corn plots at the KBS LTER site .................................................................... 190

Figure 5.4 Net carbon dioxide emission at various N fertilizer levels in continuous
corn plots at the KBS LTER site .................................................................... 191

Figure 5.5 Net daily carbon dioxide ﬂux at various N fertilizer levels in
continuous corn plots at the KBS LTER site ................................................. 191

Figure 5.6 Net soil temperature at various N fertilizer levels in continuous corn
plots at the KBS LTER site ............................................................................ 192

Figure 5.7 Net daily soil temperature at various N fertilizer levels in continuous
corn plots at the KBS LTER site .................................................................... 192

Figure 5.8 Net soil moisture at various N fertilizer levels in continuous corn plots
at the KBS LTER site .................................................................................... 193

XV

Figure 5.9 Net daily soil moisture at various N fertilizer levels in continuous corn
plots at the KBS LTER site ............................................................................ 193

Figure 5.10 Net soil nitrate at various N fertilizer levels in continuous corn plots
at the KBS LTER site .................................................................................... 194

Figure 5.11 Net daily soil nitrate at various N fertilizer levels in continuous corn
plots at the KBS LTER site ............................................................................ 194

Figure 5.12 Net soil ammonium at various N fertilizer levels in continuous corn
plots at the KBS LTER site ............................................................................ 195

Figure 5.13 Net daily soil ammonium at various N fertilizer levels in continuous
corn plots at the KBS LTER site .................................................................... 195

xvi

Chapter 1

Methane Oxidation in Arable Soils

Introduction

Methane (CH4) is the most abundant greenhouse gas in the atmosphere, after
water vapor and carbon dioxide (CO2). It has an average atmospheric lifetime of 12 years.
Methane today contributes around 20% of radiative forcing by greenhouse gases since
1750 (IPCC 2001) and has a global warming potential 62 times that of C02 over a 20
year time horizon. The current (1998) concentration of CH4 in the atmosphere is 1,745
ppb, approximately 2.5 fold higher than the 700 ppb concentration of preindustrial times
based on ice-core studies (Blunier et al. 1993). This increase is due principally to the
increased use of fossil fuels and the expansion of animal and lowland rice agriculture.
The current annual rate of increase is 7.0 ppb (IPCC 2001), which is slightly lower than a

decade ago (Dlugokencky et al. 1994) for still unknown reasons.

Saturated soils in wetlands are the primary natural sources of CH. emission,
responsible for approximately 21% of global emissions, while over half of global
emissions are from anthropogenic sources such as agriculture, natural gas activities, and
landﬁlls (Table 1.1). More than 80% of CH4 produced is removed from the atmosphere
by reaction with hydroxyl radicals (0H). Of the remainder, about half remains in the
atmosphere to contribute to the 7.0 ppb annual increase, and the rest appears to be
removed by biological methane oxidation in soils. Microbial oxidation in aerobic soils is

the only biological sink for atmospheric CH4, Globally some 30 Tg are oxidized

annually, close to the 37 Tg annual CH4 atmospheric increase. Though small relative to
total global emissions of 598 Tg y'l, this sink could signiﬁcantly affect atmospheric CH4
concentrations and thus global warming. Understanding methane oxidation in soil,
carried out by methanotrophic bacteria, is therefore important for understanding CH4

mitigation potentials.

Methanotrophic Bacteria

Methane-oxidizing bacteria (methanotrophs) are obligately aerobic, Gram-
negative bacteria that utilize methane as their sole source of carbon and energy. Some can
also use methanol but none grow on multicarbon compounds. The ﬁrst methanotroph to
be isolated, by Sohngen in 1906, was named Bacillus methanicus (Sohngen 1906), later
renamed Methylomonas methanica. Prior to 1970 only a few more had been isolated.

In 1970, after studying more than 100 methanotroph isolates, Whittenbury and
colleagues proposed ﬁve genera of methanotrophs based on cell morphology, form of the
resting state, intracytoplasmic membrane structure, and other physiological
characteristics (Whittenbury et a1. 1970). They are Methylomonas, Methylobacter,
Methylococcus, Methylocystis, and Methylosinus; four new genera have been added
since: Methylomicrobium, Methylocaldum, Methylosphaera and Methylocella (Murrell
and Radajewski 2000).

Methanotrophs have been isolated from a wide variety of environments including
soils, sediments, freshwater, seawater and even more extreme environments such as hot
springs, Antarctic tundra, acid peat bogs, and soda lakes (Hanson and Hanson 1996).

There are two distinct groups of methanotrophs. The low afﬁnity methanotrophs

can oxidize methane only in concentrations of methane higher than 40 ppm (Le Mer and

Roger 2001). This is the “known” group, which we can culture and study in laboratories.
The other group is the high afﬁnity methanotrophs, which can oxidize methane at much
lower concentrations (<12 ppm) as in the atmosphere (1.7 ppm). Members of this group
have not yet been cultivated. Most of what we know about methanotrophs is from the
bacteria we can culture, which may not represent the more prevalent methanotrophs

found in natural soils.

Characteristics of Methanotrophs

Methanotrophs have been classiﬁed into three types (I, II, and X) based on their
cell morphology, membrane type, resting stage, pathways of carbon assimilation,
physiology, and phospholipid fatty acid content (Table 1.2).

Type I methanotrophs belong to the 7—subclass of the Proteobacteria. They
contain bundles of membranes (Fig. 1.1), assimilate formaldehyde produced from the
oxidation of methane via the ribulose monophosphate (RMP) pathway, and contain
predominantly 16-carbon (C16) fatty acids. They include four genera: Methylomonas,
Methylomicrobium, Methylobacter, and Methylosphaera (Table 1.3).

Type II methanotrophs belong to the a—subclass of the Proteobacteria. They
generally have membranes around the periphery of the cell (Fig. 1.2), assimilate
formaldehyde via the serine pathway, and contain predominately 18-carbon (C13) fatty
acids. They include three genera: Methylosinus, Methylocystis, and Methylocella.

Type X was added to accommodate methanotrophs similar to Methylococcus
capsulatus. They belong to the 7—subclass of the Proteobacteria and assimilate

formaldehyde primarily via the RMP pathway, like type I methanotrophs. However, they

also possess low levels of serine pathway enzymes, and they can ﬁx nitrogen and carbon
dioxide. Type X methanotrophs can also grow at higher temperatures than Type I and
Type II methanotrophs. Two genera of Type X methanotrophs are Methylococcus and

Methylocaldum.

Physiology and Biochemistry
Methane is ﬁrst catalysed by methane monooxygenase (MMO) to produce
methanol. The methanol is further oxidized to formaldehyde by the enzyme methanol
dehydrogenase (MDH) and the formaldehyde can be assimilated into cell carbon by the
RMP or serine pathway. Formaldehyde can also be further oxidized to fonnate by
formaldehyde dehydrogenase (FADH) and then ﬁnally CO2 by formate dehydrogenase
(FDH) to provide reducing power for the initial oxidation of methane and for biosynthetic

reactions (Fig. 1.3).

Methane Monooxygenase

There are two forms of methane monooxygenase; a soluble, cytoplasmic enzyme
complex (sMMO), which is found in only some methanotrophs, and a membrane-bound,
particulate enzyme (pMMO), which is present in all known methanotrophs.

The most extensively characterized enzyme is the sMMO, which is a non-heme
iron-containing enzyme complex consisting of three components (Fig. 1.4). Protein A is a
hydroxylase containing three subunits of approximately 60, 45 and 20 kDa, arranged in a
a2 52 72 complex. The 0 subunits contain a diiron centre believed to be the site of the

monooxygenation reaction. Protein B, a coupling protein of 16 kDa, facilitates electron

ﬂow and/or the interactions between Proteins A and C. Protein C (38 kDa) is the
reductase component of the sMMO and catalyses the transfer of reducing equivalents
from NADH to the hydroxylase (Murrell et al. 2000).

The pMMO enzyme probably consists of three membrane-associated polypeptides
of around 47, 27 and 25 kDa. The ﬁrst two probably contain the active site. The active
enzyme contains two iron and approximately 15 copper atoms per mol. The pMMO also
associates with small copper-binding polypeptides of 1,218 and 779 Da (Murrell et al.
2000)

The sMMO is not present in all methanotrophs and is found only in Type II
genera Methylosinus, Methylocystis, and Methylocella. It is also found in some species of
Methylococcus, Methylomonas, Methylomicrobium, and Methylocaldum (Table 1.3). In
contrast to pMMO, sMMO has extremely broad substrate speciﬁcity and can oxidize a
wide range of non-growth compounds such as alkanes, alkenes, and aromatic compounds
(see more examples in Knowles, 1993 . This enzyme is expressed only under copper-
deﬁcient conditions for methanotrophs and apparently suppressed by pMMO.

The pMMO is found in all cultured methanotrophs during copper-sufﬁcient
growth conditions and appears to require copper ions for both expression and activity. It
has a relatively narrow substrate speciﬁcity but will, in addition to oxidising methane,

also co-oxidise a number of short chain alkanes, alkenes, and ammonia.

Ribulose Monophosphate Pathway

The ribulose monophosphate (RMP) pathway cycle occurs in Type I and X

methanotrophs. This pathway consists of three parts: ﬁxation, cleavage, and

rearrangement (Fig. 1.5). First, ﬁxation is a result of an aldol condensation of three
formaldehyde molecules and three ribulose 5-phosphate (ribulose monophosphate)
molecules, forming three molecules of fructose 6-phosphate. Second, during cleavage,
one molecule of fructose 6-phosphate is converted to 2-keto 3-deoxy 6-
phosphogluconate, which is cleaved to yield pyruvate and glyceraldehyde 3-phosphate.
Finally, the remaining two molecules of fructose 6-phosphate and glyceraldehyde 3-
phosphate undergo a series of rearrangement reactions, which regenerate three molecules

of ribulose 5- phosphate.

Serine Pathway

The serine pathway cycle occurs in Type II and X methanotrophic bacteria. First,
formaldehyde reacts with glycerine to form serine, catalyzed by serine hydroxymethyl
transferase (Fig 1.6). The serine then undergoes a series of reactions to form two
molecules of 2-phosphoglycerate, one of which forms a 3-phosphoglycerate molecule.
The 3-phosphog1y-cerate becomes cellular material by another metabolic pathway. The
remaining 2-phosphogly—cerate molecule forms Phosphoenolpyruvate, which then
combines with carbon dioxide to form oxaloacetate. The oxaloacetate then proceeds
through a series of reactions to ﬁnally form glycerine.

The ribulose monophosphate pathway is more efﬁcient than the serine pathway
since all carbon atoms for cell material construction are derived from formaldehyde.
Additionally, the formaldehyde is at the same oxidation level as cellular biomass, so there

is no need for more reducing power.

Environmental Factors Inﬂuencing Soil Methane Oxidation
The factors affecting CH4 oxidation in soil generally divide into two categories:

chemical and biological factors, which will be discussed below.

Chemical Factors
Methane oxidation is affected by chemical factors such as methane

concentrations, oxygen, water, temperature, pH, nitrogen, and other carbon compounds.

Methane Availability

Methane is the primary source of carbon and energy for methanotrophs, so any
factors that affect the availability of methane will affect the growth and function of
methanotrophic bacteria. The minimum threshold for CH4 oxidation (<0.1 to 0.4 ppm) in
upland soil is much lower than that in sediments (2—3 ppm) (Born et al. 1990, Whalen
and Reeburgh 1990, Whalen et al. 1990, Yavitt et al. 1990). This means that aerobic soils
can consume CH4 at lower concentrations, as in atmosphere, than sediment soil in water,
which get CH4 from anaerobic fermentation at lower depths. Because CH4 is the sole
source of energy for methanotrophs, when soil is exposed to CH4 at higher concentrations
than atmospheric, the numbers of methanotrophs and CH4 oxidation tends to (Whalen et
al. 1990, Bender and Conrad 1992, 1994a, Schnell and King 1995). This also happens in
soil sediment under water or soil saturated by water after precipitation.

In contrast, periodically low CH4 concentrations do not always decrease soil CH4
consumption capacity. For example, some taiga soils lose their CH4 uptake capacity when

incubated with low CH4 concentration (<0.03 ppm) for a period of time, whereas

temperate forest soils do not lose the ability when incubated with similar CH4
concentration (Schnell and King 1995, Benstead and King 1997, Gulledge et al. 1998).
This might be because methanotrophic bacteria in temperate forest soil can persist in low
CH4 by either staying in a dormant state or by using carbon from other sources than CH4,
whereas the bacteria population in taiga soil perishes from a lack of energy source.
Methane availability is primarily limited by gas diffusion (Whalen et al. 1990,
Nesbit and Breitenbeck 1992, Dorr et al. 1993, Koschorreck and Conrad 1993), which is
usually governed by soil moisture, texture, and composition. Therefore, these factors

should be considered when we try to predict CH4 oxidation in soil.

Oxygen Availability

Oxygen is required for methane oxidation. To oxidize one mole of CH4 to CO2
requires two moles of 02 (Fig. 1.3). The importance of 02 to methane oxidation was ﬁrst
demonstrated in the 19705 (Smimova 1971a, b, Nesterov et al. 1977). Methanotrophic
bacteria are sensitive to 02 concentration. Low 02 concentration (0.2 and 2%) partially
inhibits methane uptake in incubated temperate forest soil (Schnell and King 1995) and
CH4 oxidation ceases when soils are incubated under anoxic conditions (Bender and
Conrad 1995). In contrast, anaerobic conditions promote methane production in soil by

methanogens.

Water
The optimum water content for soil methane uptake varies depending on soil type

and texture, but generally is 10-35% w/w (Bender and Conrad 1995, Schnell and King

1996, van den Pol-van Dasselaar et al. 1998), 50% water-holding capacity (Tom and
Harte 1996), 40-60% water-ﬁlled pore space (Castro et al. 1995), or —0.2 MPa water
potential (Schnell and King 1996). This optimum soil water content shows that CH4
uptake is low at both low and high water content. Rates of CH4 uptake generally decrease
with increasing soil water content (Steudler et al. 1989, Crill 1991, Adamsen and King
1993) because CH4 and oxygen diffusion and transport to methanotrophs are reduced.
Water also increases CH4 production in soil by increasing the number of anaerobic
microsites in soil crumbs and aggregates, thus reducing the net uptake of CH4 by soil
(Yavitt et al. 1990). Conversely, rates of CH4 uptake decrease at lower water contents
because of the physiological response of methanotrophs to water stress (Czepiel et al.
1995, Dobbie and Smith 1996). Therefore in dry sites such as deserts, some studies have
demonstrated increased rates of CH4 consumption associated with precipitation because

rain alleviated water stress for CH4 oxidizing bacteria (Striegl et al. 1992).

Temperature

Most of the known methanotrophic bacteria are mesophilic except for
Methylomonas vinelandii, Methylococcus thermophilus, and Methylococcus capsulatus,
which can grow at 45 °C or above (Anthony 1982, Green 1992, Bowman et a1. 1995).
The optimum temperature for CH4 oxidation is generally between 25-35 0C both in the
laboratory (Whittenbury et al. 1970, Whalen et al. 1990, Bender and Conrad 1995) and in
the ﬁeld (Nesbit and Breitenbeck 1992, Dunﬁeld et al. 1993). However, some CH4
uptake can occur even at —2 °C in soil core experiments (Prieme and Christensen 1997)

and -5 °C in winter soils (Castro et al. 1995) but rates are very low . Different soils

exhibit different CH4 oxidation responses to temperature, indicating that different CH4
oxidizing bacteria populations adapt to different temperatures in nature. However, the 010
values of CH4 consumption range from 1.0-2.9 (Born et al. 1990, Crill 1991, King and
Adamsen 1992, Lessard et al. 1994, Prieme and Christensen 1997) indicating only a
modest direct sensitivity of soil CH4 oxidation toward seasonal temperature changes
(King 1993). This may be because methanotrophs are located in subsoil layers, in which
temperature is more constant and has more soil moisture than surface soil. This may also
explain why atmospheric temperature changes in the ﬁeld have a small effect on CH4 ﬂux

(King and Adamsen 1992, Peterjohn et a1. 1993, Crill et a1. 1994).

pH

Soil pH plays a very important role in methane consumption since pH directly
affects the physiology of methanotrophs and can determine the availability of toxic
elements and nutrients (e. g. ammonium, aluminum, iron). Methane uptake can occur in
environments with pH ranging from <4 to 9 (Born et al. 1990, King 1992, Dorr et al.
1993). But the optimum pH for CH4 consumption and growth of methanotrophs in vitro
is 6.0 to 7.0 for most known methanotrophic bacteria (Whittenbury et al. 1970, Dunﬁeld
et al. 1993, Bender and Conrad 1995, Amara] et a1. 1998). However, reported responses
of soil methane consumption to pH have varied among sites and studies (Hiitsch et al.
1994, Sitaula et al. 1995, Amara] et a1. 1998, Hiitsch 1998a). Methane uptake was lower
in low-pH (pH 4.8-5. 1) grassland soils (Hiitsch et al. 1994). In contrast, hardwood and
pine plantations still show high CH4 oxidation rates even at low pH 3.3 (Steudler et al.

1989) and pH 2.3 (Conrad 1996). Generally, acidiﬁcation decreases methane oxidation in

10

forest soils (Benstead and King 2001, Sitaula et al. 2001). Different types of acid
compounds also have different effects on soil CH4 uptake. For instance, nitric acid
inhibits CH4 oxidation more than sulfuric acid (Benstead and King 2001, Bradford et al.

2001) and nitric acid affects agricultural soils more than forest soils.

Nitrogen Compounds

Although nitrogen is an essential nutrient for methanotrophs, different kinds of
nitrogen compounds have different effects on CH4 consumption. Methanotrophic bacteria
prefer nitrate as a nitrogen source rather than ammonium (Whittenbury et a1. 1970,
Mancinelli 1995) because ammonium appears to inhibit CH4 oxidation (Bender and
Conrad 1994b, King and Schnell 1994, Hﬁtsch 1996, Gulledge et al. 1997, King and
Schnell 1998). However, in some cases, no immediate effect was observed after NH4+
addition (Delgado and Mosier 1996, Dobbie and Smith 1996, Gulledge et al. 1997). The
mechanism of N114+ inhibition is complex and thought to include both a competitive
inhibition of the CH4 monooxygenase as well as a toxic inhibition by hydroxylamine or
nitrite produced via NH4+ oxidation (King and Schnell 1994). Additionally, the inhibition
from a salt effect has also been demonstrated (MacDonald et a1. 1997, Gulledge and
Schimel 1998). The amount of exchangeable NH4+ could be another important factor for
CH4 uptake inhibition by NH4+(De Visscher et al. 1998).

The effect of nitrate, another product of NI—I4+ oxidation, on CH4 oxidation is
ambiguous. Some authors did not ﬁnd an effect of N03' (Boeckx and Van Cleemput
1996), while others did (Whalen 2000). A recent report shows a greater effect of NO3'

than NH4+ on the inhibition of CH4 oxidation in coniferous forest soil (Wang and Ineson

11

2003). It has also been suggested that N-tumover rates (mineralisation and gross
nitriﬁcation) inﬂuence CH4 uptake rather than the actual soil mineral N (N Hf, N03' or

NO2') content (Mosier et al. 1991, Steudler et al. 1996a).

Other Carbon Compounds
It is often assumed that atmospheric CH4 oxidizers use CH4 as their sole carbon
and energy source, but recent studies have indicated that methanotrophs can also utilize
non- CH4 substrates, speciﬁcally methanol, forrnate (Roslev et a1. 1997, Benstead et al.
1998, Jensen et al. 1998), and acetate (West and Schmidt 1999). Moreover, C02 is also
required for the growth and function of methanotrophs in bioreactors (Acha et al. 2002),

although likely to never be limiting in aerobic soils.

Biological Factors
The interaction of methanotrophic bacteria with other organisms in soil can, by

effects on methanotroph population size, affect soil CH4 uptake capacity.

Competition
Although no direct competition studies have been reported for methanotrophs in
soil, methanotrophs compete with other soil inhabitants for some nutrients. For example,
nitrogen, phosphate, and oxygen are in ﬁnite supply, and various soil organisms compete
for these resources. An active population of methanotrophs has been shown to inhibit the
ammonium oxidation activity of nitriﬁers due to competition for 02 (Megraw and

Knowles 1987). The plant-growth promoting rhizopseudomonad strains, Pseudomonas

12

aeruginosa 7NSK2 and P. ﬂuorescens ANP15, transiently reduced CH4 oxidation by 20-
30% during a in vitro 5 day incubation (Arif et al. 1996). Among the same
methanotrophs, Methylomonas trichosporium OB3b (a Type II methanotroph) outgrows
Methylobactor albus BG8 (a Type I methanotroph) in bioreactors under copper and
nitrate limiting conditions, due to the M. trichosporium’s ability to express sMMO under

copper limitation and nitrogenase under nitrate deﬁciency (Graham et al. 1993).

Predation

Nearly all bacteria in soil are susceptible to predation by other soil
microorganisms. The populations of predator and prey are dependent and ﬂuctuate but
normally are in balance. Protozoa are the major predators of bacteria, consuming
approximately 150-900 g bacteria m'2 y'1 in arable soil (Clarholm 1981). Soil bacteria are
also consumed by other soil bacteria (McInemey 1986, Casida 1988). However, no direct
studies of predation on methanotrophic bacteria have been reported to date yet. Predation
nonetheless may potentially limit the consumption of available methane in soil by

methanotrophs (Mancinelli 1995).

Other Methane Oxidizing Organisms
Methane oxidation also occurs in anoxic environments such as ocean sediments,
soda lakes, and peat by a consortium of CH4 oxidizing archaea and sulfate reducing
bacteria (Hanson and Hanson 1996, Valentine and Reeburgh 2000, Kotelnikova 2002).
Some yeasts such as Rhodotorula glutinis, Rhodotorula rubra, Sporobolomyces

gracilis, and Sporobolomyces roseus can grow very slowly on methane (Wolf 1981).

13

Methanotrophic bacteria can also form symbioses with some marine invertebrates such as

deep-sea mytilid mussels and the tubeworrn Siboglinum posez’doni (Cavanaugh 1993).

Methane Oxidation in Aerobic Soils

The uptake of methane by soils was ﬁrst demonstrated in swamp soils in Virginia,
USA, when the water table dropped below the sediment surface (Harriss et al. 1982).
Two years later it was observed in tropical soils (Seiler et al. 1984). Since then, soil
methane oxidation has been measured in many ecosystems in both tropical and temperate
zones, including temperate forests (Steudler et al. 1989, Adamsen and King 1993,
Ishizuka et al. 2000, Robertson et al. 2000), tropical forests (Keller et al. 1986, Keller and
Reiners 1994, Prieme and Christensen 1999), grasslands (Whalen and Reeburgh 1990,
Mosier et al. 1993, Tate and Striegl 1993, Mosier et al. 1997b), and deserts (Striegl et al.

1992).

Oxidation Rates in Different Ecosystems

Methane uptake rates in different ecosystems in various parts of the world are
presented in Table 1.4. From these data, published from 1986 until 2000, it is clear that
CH4 oxidation is higher in forest soils than grassland and arable soils. The average of the
minimum and maximum values of CH4 uptake are in Table 1.5

These summary data show a reduction of CH4 consumption in arable soils of
59% from grassland soils and 77% from forest soils, consistent with 71% reduction in
European studies (Smith et a1. 2000) and a 63% decrease in Ghana studies (Prieme and

Christensen 1999).

14

It is apparent that, irrespective of fertilization type or level, tillage system, soil
texture and structure, and climate conditions, conversion of forests or grasslands to

agriculture results in a reduced capacity for soil CH4 uptake.

Global Methane Sinks Related to Soil and Land Use

Table 1.6 shows the soils and land use related CH4 sink calculated from CH4
uptake rates in various land uses (Reeburgh et a1. 1993) and land use data (Bouwman
1990). Methane oxidation rates vary from 0.023 mg CH4 rn'2 h'1 in taiga soils (Whalen et
al. 1991) to 0.028 mg CH4 m'2 h" in desert soils (Striegl et al. 1992) to 0.16 mg CH4 m'2
h'1 in temperate forest soils (Steudler et al. 1989). Temperate forest soils have the highest
CH4 consumption rates, approximately 28% of total global uptake, surprisingly followed
by desert and semidesert soils at 19%, and tropical rain forest soils at 12%. The cultivated
lands consume only 6% of global CH4 uptake while constituting 10% of global land area.

From Table 1.6, the total global annual CH4 sink is 43.7 Tg CH4, higher than the
IPCC calculation of 30 Tg CH4 (IPCC 1995), 29 Tg CH4 (Smith et al. 2000), 38 Tg CH4

(Ridgwell et al. 1999), and a model value of 17 Tg CH4 (Potter et al. 1996).

Agricultural Effects
Agricultural practices reduce soil CH4 oxidation capacity compared with
undisturbed soils in forests and grasslands. Fertilization, tillage, and the application of
pesticides and herbicides, and lime can potentially affect CH4 oxidation (Mosier et al.
1991, Clymo et al. 1995, Goulding et al. 1995, Arif et al. 1996, Mosier et al. 1997a,

Powlson et al. 1997, Robertson et al. 2000, Hi’rtsch 2001); each of these is discussed

15

brieﬂy below.

Nitrogen Fertilizer

Nitrogen fertilization appears to be the number one factor inhibiting CH4
oxidation in agriculture. Numerous studies have conﬁrmed the reduction of soil CH4
uptake after N fertilization in forest, grassland, arable, and landﬁll soils (Steudler et al.
1989, Mosier et al. 1991, Hansen et a1. 1993, Hiitsch et al. 1993, Bronson and Mosier
1994, Crill et a1. 1994, Hiitsch et a1. 1994, Castro et a1. 1995, Willison et al. 1995b,
Hiitsch 1996, Tlustos et al. 1998, Hilger et al. 2000). However, N fertilizer had no effect
on CH4 oxidation in a regularly fertilized wheat ﬁeld (Mosier and Schimel 1991), in
irrigated crops in Colorado (Bronson and Mosier 1993), and in acid oxisol sites in Puerto
Rico (Mosier et al. 1998). Different forms of nitrogen have different effects on soil CH4
consumption (see section 3.1.5). Nitrogen fertilizer can immediately interfere with
methane monooxygenase or on a longer-term basis can cause changes in microbial

populations (Adamsen and King 1993).

Tillage
Tillage alone reduced CH4 oxidation in a continuous wheat plot compared with
woodland over 150 years (Hﬁtsch et a1. 1994, Willison et al. 1995b). In grassland, tillage
reduced CH4 consumption by 35% immediately and the effect persisted for more than 3
years (Mosier et al. 1997b). Disking after plowing further reduced CH4 consumption
another 60-70% (Kessavalou et al. 1998a). However, plowing had no apparent effect on

CH4 uptake in tropical savanna (Sanhueza et al. 1994), Piedmont comﬁelds (Burke et al.

16

1999), nor in an acid oxisol in Puerto Rico (Mosier et a1. 1998). On the other hand,
plowing a plot in Denmark heathland increased CH4 consumption to seven times that of
native heathland (Kruse and Iversen 1995). In general, it might be expected that reduced
tillage is beneﬁcial for CH4 consumption in agriculture since methanotrophs are sensitive
to soil disturbance. Tillage reduction can restore CH4 oxidation in some soils. For
instance, no-till management showed higher CH4 oxidation rates than the plowed
treatments of wheat-fallow rotation in Nebraska (Kessavalou et al. 1998b), and also in
continuous spring barley in Alaska (Cochran et al. 1997). Fiﬁeen years of direct-drilling
resulted in 4.5-11 times the CH4 oxidation rates of continuous plowed treatments (Hiltsch
1998b). On the other hand, Robertson et a1. (2000) found no difference in oxidation
between tilled and no till in a 10-year Michigan study. The vertical proﬁle of soil CH4
oxidation also reveals different zonation in plowed, direct-drilled, set aside treatments,
and forest soil. Methane oxidation showed the highest activity below 25 cm in the plowed
and set aside treatments, while under direct-drilling maximum rates were at 5-15 cm and

under forest soil at 5- 10 cm (Hiitsch 1998b).

Pesticides
Few studies have investigated the inhibitory effects of pesticides, herbicides, and
insecticides on soil CH4 oxidation. For example, the herbicide dichlorophenoxy acetic
acid (2,4-D) had a strong negative effect on CH4 oxidation in sandy Belgium soils in vitro
(Arif et al. 1996). The herbicides Lenacil and Mikado and the fungicide oxadixyl
signiﬁcantly reduced CH4 uptake in sandy soils, while atrazine, Mikado and

dimethenamid decreased CH4 consumption in clayey soils in Germany (Boeckx et al.

17

1998). Among 30 agrochemicals tested, only the herbicide bromoxynil and the
insecticide methomyl effectively decreased CH4 oxidation in Canadian soils (Topp et al.
1999). The insecticide Dimethoat 40 BC, the herbicide Tolkan and the fungicides Tilt 250
EC, Tilt Top, and Corbel drastically reduced atmospheric CH4 uptake in slurries from a
Danish forest soil (Prieme and Ekelund 2001). In the tropics, the insecticides HCH and
Carbuforan inhibited CH4 oxidation in ﬂooded rice soils (Kumaraswamy et al. 1997,
Kumaraswamy et al. 1998). Pesticide utilization in agriculture is thus also a potential

inhibitor for soil CH4 oxidation.

Liming

Soil acidiﬁcation tends to decrease CH4 oxidation in soil, and therefore liming
might be expected to increase soil CH4 uptake by alleviating soil acidity. However,
results are mixed. Liming had an insigniﬁcant effect on CH4 oxidation in hardwood forest
in the Adirondack region of New York after 2 years of application, but it signiﬁcantly
reduced CH4 uptake in incubated forest soils (Yavitt et al. 1993) and in German spruce
forest soils four years after application (Butterbach-Bahl and Papen 2002). In contrast,
dolomite slightly increased CH4 oxidation in Sweden spruce forests aﬁer seven years of
liming (Klemedtsson and Klemedtsson 1997) and in an acid oxisol in western Puerto
Rico after several years (Mosier et a1. 1998). Liming also signiﬁcantly increased CH4
uptake rates in four out of ﬁve deciduous and spruce forest plots in Germany (Borken and
Brumme 1997), in Dutch forests (Saari et al. 1997), and in landﬁll soils (Hilger et al.

2000). Therefore, the effect of liming is varied depending on certain soil conditions.

18

Vegetation and Crop Management

Until recently, the effects of plants on CH4 oxidation have been studied
exclusively in wetlands and paddy soils. Only limited data are available for aerobic arable
soils. Arif et al. (1996) found no differences in CH4 oxidation between corn and an
unplanted control in sandy soil incubation studies. Additionally, in a wheat-fallow
rotation, CH4 oxidation rates were not different between wheat and the fallow period
(Kessavalou et al. 1998b). The plant itself thus does not appears to be the primary factor
affecting CH4 oxidation; rather CH4 oxidation seems more affected by secondary direct
factors such as pesticide treatments, pH changes, and tillage. An exception appears to be
the rice in India dryland soils (Singh et al. 1999), where differences appear related to
methanotrophs being more abundant in the rice rhizosphere than in either bulk cropped
soil or bare fallow soil (Dubey and Singh 2001).

The removal of crop surface residue decreased CH4 uptake in spring barley due to
the reduction of soil moisture (Cochran et a1. 1997). However, the application of crop
residues after harvest has different effects. Fresh sugar beet leaves mixed with a loamy
soil reduced CH4 oxidation by 20%, while wheat straw had no effect (Hﬁtsch 1998a).

In ley and legume crop rotations in Norway, the tractor trafﬁc alone reduced the
accumulated CH4 uptake by 52% while its combination with fertilizer decreased CH4
consumption by 78% (Hansen et al. 1993, Sitaula et al. 2000); the effect persists even

aﬁer soils are sieved before incubation (Sitaula et al. 2000).

19

Soil Methane Oxidation Recovery
Soils in the temperate zone generally take more than 100 years to recover a CH4
consumption capacity as high as the undisturbed soils nearby after abandonment to
grasslands or forests (Smith et al. 2000). It takes more than 200 years for European soils
(Prieme et al. 1997) and longer than this for soils in North America (Ojima et al. 1993,
Hudgens and Yavitt 1997, Mosier et al. 1997b, Robertson et a1. 2000). However, it is
possibly much faster in the tropics as; perhaps as little as 50 years (Keller and Reiners

1994)

Summary

Methane oxidation by upland soils is an important process for alleviating global
warming potentials. Although the net annual oxidation rate is small it rivals the annual
increase of atmospheric CH4. Therefore, if we can promote this soil sink we can help to
keep atmospheric CH4 stable.

However, the expansion of deforestation and urbanization decrease the capacity
of soil to consume CH4. Agricultural practice, especially nitrogen addition, is the major
soil CH4 oxidation inhibitor. Many studies have been conducted to try to understand the
mechanisms of this inhibition but much is still unknown. Because agriculture is important
to us, it is necessary to ﬁnd ways to improve agricultural productivity and reduce
greenhouse effects simultaneously. Reducing soil disturbances and N application or

ﬁnding more appropriate N fertilizers might be key for developing mitigation strategies.

20

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35

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36

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37

Table 1.] Estimated sources and sinks of methane (IPCC 1995, 2001)

 

 

 

Annual release Range
(Tg CH4) (Tg CH4)
Natural sources
Wetlands 115 55-150
Termites 20 10-50
Oceans 10 5-50
Others 15 10-40
Anthropogenic sources
Enteric fermentation and animal waste 110 85-130
Energy production and use 100 70-120
Riceﬁelds 60 20-100
Biomass burning 40 20-80
Landﬁlls 40 20-70
Domestic sewage 25 15-80
Total sources 5 3 5 598 * 410-660
Sinks
Consumption in atmosphere 485 546* 420-520
Oxidation in upland soils 30 15-45
Total sinks 5 15 576* 430-600
Atmospheric increase 37 22* 35-40

 

*IPCC (2001) adjustment
Tg=10”g

38

Table 1.2 General characteristics of methanotrophic bacteria (modiﬁed after King 1992

and Hanson and Hanson 1996).

 

 

Character Type 1 Type II Type X
Cell morphology Rods Rods, vibroid Coccoid
Membrane structure
Bundles of vesicles Yes No Yes
Peripheral paired membranes No Yes No
Resting stages Cysts Exospores or cysts Cysts
Rosettes No Yes No
Carbon assimilation pathway RMPa Serineb RMP/serine
Tricarboxylic acid cycle Incomplete Complete Incomplete
Nitrogen ﬁxation No Yes Yes
Diagnostic fatty acid carbon 16 18 16
length
Carbon dioxide ﬁxation No No Yes

 

a Incorporation of formaldehyde via the ribulose monophosphate pathway
b Incorporation of formaldehyde via the serine pathway

Table 1.3 Characterization of methanotrophs (modiﬁed after Murrell et al. 1998)

 

 

Genus Type Formaldehyde Enzyme type Mol% G+C
assimilation DNA content
pathway

Methylomonas Type I RMP pMMO 51-59

Methylobacter Type I RMP pMMO 49-54

Methylomicrobium Type I RMP pMMO 50-60

Methylosphaera Type I RMP pMMO 43-46

Methylosinus Type II Serine pMMO/sMMO 62-63

Methylocystis Type II Serine pMMO/SMMO 62-63

Methylocella Type II Serine pMMO/sMMO na

Methylococcus Type X RMP/serine pMMO 59-66

Methylocaldum Type X RMP/serine pMMO/sMMO 56-58

 

39

Table 1.4. Annual methane uptake in different global ecosystems (adapted from

Bouwman 1990 and Reeburgh et al. 1993).

 

 

Land cover type Area Uptake rate System uptake
(10” m2) (mg CH4 m'2 y“) (Tg CH4 y")

Tropical rain forest 17.0 0.3 5.]
Tropical seasonal forest 7.5 0.3 2.3
Temperate forest 12.0 1.1 12.1
Boreal forest 12.0 0.2 2.4
Woodland-schrubland 8.5 0.3 2.6
Temperate grassland 9.0 0.3 2.7
Savanna 15.0 0.3 4.5
Tundra and alpine 8.0 0.1 0.8
Desert-semidesert 42.0 0.2 8.4
Cultivate land 14.0 0.2 2.8
Total 145 - 43.7

 

40

Table 1.5 Methane oxidation of aerobic soils under ﬁeld condition in various locations of

the world (modiﬁed after Boeckx and van Cleemput 2001; Hiitsch 2001; Prieme and

Christensen 1999; Smith et a1. 2000).

 

 

 

Location Average CH4 oxidation rate Literature
(mg CH4 m'2 d")
Arable Grassland Forest
Canada
Ottawa 0-0.13 0.04-1.10 Lessard et a1. (1994)
1.0-3.0 Adamsen and King (1993)
Denmark 0.04-0.08 0.03-0.25 0.14-0.33 Ambus and Christensen (1995)
0.2 0.7 Dobbie et al. (1996)
Germany 0. 12096 Butterbach-Bahl et a1. (1998)
0.04-0.05 Ruser et al. (1998)
Gottingen 0.11 Flessa et al. (1996)
0.03-0.68 Brumme and Borken (1999)

Heidelberg 0.005-0.55 0.25-3.56 Born et a]. (1990)

Konstanz 0-0.62 0-1.79 Koschorreck and Conrad (1993)
Ireland 1.34 Butterbach-Bahl et al. (1998)
Japan 0.6 2.1 Minami et al. (1993)

1.8-7.6 Ishizuka et a]. (2000)
New Zealand 0.04-0.05 vander Weerden et a1. (1999)
Norway 0.8-1.4 Sitaula et al. (1995)
Poland 0.2 1.0 Dobbie et al. (1996)
Scotland 0.02-0. 12 0.85 0.86-1.06 MacDonald et a]. (1996)
0.7 1.4 Dobbie et a]. (1996)
0.82 2.19-5.97 Dobbie and Smith ( 1996)
0.19-0.36 MacDonald et a1. (1997)
Sweden 0.38 Klemedtsson and Klemedtsson
(1997)
0.48 MacDonald et al. (1997)
UK
0.18 0.48 1.05 Willison et al. (1995a)
0-0. 13 0-0. 19 0024 Goulding et a]. (1996)
USA
Alaska 0.67-0.87 Goulding et al. (1997)
Colorado 0.17 0.35 Mosier et a1. (1991)
0.03-0.20 0.35-0.84 Mosier and Schimel (1991)
0.09 Delgado and Mosier (1996)
0.18 0.82 Ojima et a]. (1993)
Durham 4.9 Crill (1991)
Massachusetts 3.20-4.16 Steudler et a]. (1989)
3.84-5.44 Castro et a]. (1994)
Michigan 0.24 1.92 Paustian et al. (1995)

Table 1.5 (cont’d)

0.24 0.73 1.22
Nebraska 0.91-1.03 1.16
New York 0.60
2.1-6.9
0.25 0.75
Piedmont 0.3 1.4
Tropic
Brazil 1.71
0.96
-0.72
0.38
0.35 1.15
Cameroon 0.41 1 .8
Congo 0.46 0.62
1.0
Costa Rica -0.9-(-0.2) 1.20-1.26
Ecuador -0.07 -0.62-0.82
Ghana 0.16 0.89 0.94
India 11.52 864-1368
6.0
Malaysia 1.34
Mali 0.072
Panama 0.032 0.58
Puerto Rico 0.5
0.19
South Africa -1.63-0.96
1.23
Venezuela -0.82 1.15
-1.13

Robertson et al. (2000)
Kessavalou et al. (1998b)
Yavitt et al. (1993)
Goldman et al. (1995)
Hudgens and Yavitt (1997)
Burke et a]. (1999)

Steudler et al. (1996b)
Andersen and Poth (1998)
Poth et al. (1995)

Keller et a1. (1986)
Goreau and de Mello (1988)
MacDonald et a1. (1998)
Delmas et al. (1991)
Delmas et al. (1992)
Keller and Reiners (1994)
Keller et al. (1986)
Prieme and Christensen (1999)
Singh et a]. (1997)

Singh et al. (1999)
MacDonald et al. (1999)
Delmas et al. (1991)
Keller et al. (1990)
Steudler et al. (1991)
Mosier et al. (1998)

Zepp et al. (1996)

Seiler et al. (1984)
Scharffe et al. (1990)
Sanhueza et al. (1994)

 

Note: a negative number means net CH4 production

Table 1.6 Average methane oxidation in different ecosystems; a summary of data from

 

 

Table 1.5

Ecosystems Average Minimum Maximum
Forest 1.66 1.28 2.05
Grassland 0.89 0.85 0.88
Agriculture 0.37 0.33 0.41

 

Unit = mg CH4 m'2 d‘I

42

 

 

 

Figure 1.1 Electron micrographs showing type I membrane systems of Methylomonas

methanica (Green 1992).

43

 

Figure 1.2 Electron micrographs showing type 11 membrane systems of Methylocystis

parvus (Green 1992).

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48

Chapter 2

Methane Oxidation in Successional and Agricultural Ecosystems:

Effects of Nitrogen and Soil Disturbance

Introduction

Upland soil is an important global sink for the greenhouse gas methane,
consuming about 30 Tg CH4 y", slightly more than the annual atmospheric loading rate
of 22 Tg CH4 y'l(IPCC 2001). Increased soil methane uptake could thus help to mitigate
increasing concentrations of methane in the atmosphere, now at 1,745 ppb CH4 (IPCC
2001).

Land use and in particular agriculture has a big impact on rates of soil CH4
oxidation: a number of studies have shown that undisturbed forest and grassland soils
consume more methane than similar soils converted to agriculture (e. g. Ambus and
Christensen 1995, Willison et al. 1995a, Goulding et a1. 1996, MacDonald eta]. 1996,
Prieme and Christensen 1999, Robertson et al. 2000). Various agricultural practices
including fertilization, tillage, and the use of insecticides and herbicides have been
demonstrated to inhibit soil methane uptake to different degrees in a variety of different
studies (Mosier and Schimel 1991, Goulding et al. 1995, Arif et a1. 1996, Mosier et al.
1997a, Powlson et al. 1997, Topp et al. 1999, Hiitsch 2001).

Nitrogen fertilizer has been shown most often to reduce methane oxidation in
forest, grassland, arable, and landﬁll soils, especially when applied in the ammonium
form (Steudler et a1. 1989, Mosier et al. 1991, Hansen et al. 1993, Hﬁtsch et a1. 1993,

Bronson and Mosier 1994, Crill et a1. 1994, Hiitsch et al. 1994, Castro et al. 1995,

49

Willison et al. 1995b, Hiitsch 1996, Tlustos et al. 1998, Hilger et al. 2000). Tillage also
has been shown to decrease methane oxidation in both natural and agricultural soils
(Hutsch et al. 1994, Willison et al. 1995a, Cochran et a1. 1997, Mosier et al. 1997b),
however, it had no effect (Sanhueza et al. 1994, Mosier et a]. 1998, Burke et a1. 1999)
and even increased (Kruse and Iversen 1995) methane uptake in some soils.

These studies of N fertilizer and tillage effects on soil methane consumption have
investigated each factor alone and in a particular ecosystem. In this study I attempt to
study both factors simultaneously by simulating soil disturbance (as tillage) and applying
N fertilizer separately and in combination in different type of ecosystems along a 3-point
disturbance gradient that includes old growth deciduous forest, mid-successional old
ﬁeld, and no-til] agricultural sites. In this way I am able to separate the effects of tillage

on N availability separate from its effects on other soil properties.

Methodology
Site Description

The study was conducted at the Long-term Ecological Research (LTER) site at
the WK. Kellogg Biological Station (KBS), Hickory Corners, Michigan (42° 24’N, 85°
24’W, elevation 288 m). Annual rainfall at KBS averages 890 mm y'1 with about half
falling as snow; potential evapotranspiration (PET) exceeds precipitation for about 4
months of the year. Mean annual temperature is 9.7 °C. I measured methane ﬂuxes at
three replicated sites along a management intensity gradient: mature deciduous forests

(DF), mid-successional ﬁelds (also called mid-successional communities, SF) abandoned

50

from agriculture 40-60 years ago, and a no-till com-soybean-wheat rotation (T2)
established in 1988 (see details at http://lter.kbs.msu.edu).

The major plants in the deciduous forest sites are red maple (A cer rubrum L.),
sugar maple (Acer saccharum Marsh), white oak (Quercus alba L.), northern red oak
(Quercus rubra L.), ﬂowering dogwood (Camus florida L.), and sassafras (Sassafras
albidum (Nutt.) Nees). The dominant plants in the mid-successional communities are
Canada goldenrod (Solidago canadensis L.), quackgrass (Elytrigia repens (L.) Nevski),
timothy (Phleum pratense L.), white hearth aster (Aster pilosus Willd.), Kentucky
bluegrass (Poa pratensis L.), common yarrow (Achillea millefolium L.), Canada
bluegrass (Poa compressa L.), autumn olive (Elaeagnus umbellata Thunb.), sassafras
(Sassafras albidum (Nutt.) Nees), gray goldenrod (Solidago nemoralis Ait.), smooth
brome (Bromus inermis Leyss.), gerrnander Speedwell (Veronica chamaedrys L.),
orchardgrass (Dactylis glomerata L.), ﬂowering spurge (Euphorbia corollata L.), and
honeysuckle (Lonicera spp.).

The no-till system was planted to corn (Zea mays L.) during 2002, the year that
this study was conducted.

All sites were replicated within the larger landscape (n=3 locations) and were on
the same Kalamazoo/Oshtemo soil series (Austin 1979). The soils at these sites are Typic
Hapludalfs (ﬁne or coarse-loamy, mixed, mesic soils) derived from glacial till about
12,000 years ago (Crum and Collins 2003).

Four 0.5 x 0.5 m plots were established in each replicate site and a 2 x 2 factorial
design was imposed with N fertilizer and tillage as factors. To one plot was added 100 kg

N ha'1 ammonium nitrate (N H4NO3), another plot was physically disturbed by hand

51

shoveling to simulate soil tillage to 10 cm depth, another plot was both fertilized and

tilled, and a fourth plot served as control.

In Situ Gas Sampling

1 measured in situ methane oxidation rates using a static chamber technique
(Hutchinson and Livingston 1993, Robertson et al. 2000). Static chambers were
fashioned from 25 cm diameter PVC pipe: bases (25 cm diameter x 10 cm high x 3 cm
depth) were installed in each plot and left in place except during agronomic operations.
Immediately prior to sampling, a 4.5 cm high cap was placed on each base and sealed
with a latex skirt wrapped with an elastic band. At 10-minute intervals, four 10 mL
headspace samples were removed through rubber septa in each cap using a syringe and
put into 3 mL glass sample vials pre-ﬂushed with headspace air. Within 3 days via]
contents were measured for CH4 using a gas chromatograph (GC 5890 Series II, Hewlett
Packard) equipped with a ﬂame ionization detector (F ID), and for CO2 using an infrared
gas absorption (IRGA) analyzer (EGA CO2 Analyzer, Analytical Development CO. LTD.
Hoddesdon, England).

Chambers were sampled one day before treatment and l, 6, 16, 23, 52, 73 and 101

days after treatment.

Soil Analyses
Soil temperature was measured at 0-5 cm depth using a temperature probe. Soil
samples for other analyses were taken from the top 10 cm of soil using a 2.5 cm diameter

soil probe. Fresh soils were passed through a 4 mm sieve and mixed by hand, and then

52

sub samples were taken for moisture content and mineral N analysis. Prior to analysis,
soils were stored in a refrigerator at 4°C. Soil moisture content was measured
gravimetrically by drying the soil samples at 65°C for 3 days or until dry; further drying
these soils at 105 °C typically removes only 0.8 g H20 100 g 8011'1 more moisture (data
not shown). Mineral N measurements were obtained by extracting 20 g of dry soil with
100 mL 1 M KC1 for 24 h then ﬁltering through 2-um pore size glass ﬁber ﬁlters. The
ﬁltrates were frozen prior to analysis for NH4+ and N03' using an Alpkem continuous
ﬂow analyzer (Alpkem 3550, 01 Analytical, College Station, TX) (Bundy and Meisinger

1994).

Statistical Analyses

The data were divided into two parts: before fertilization (day 0) and after
fertilization (day 1, 6, 16, 23, 52, 73, and 101). For analysis of the ﬁrst part, I used SPSS
version 10.0.] (SPSS Inc. 2001) for the analysis of variance (ANOVA), analysis of
covariance (ANCOVA), and correlation analysis. I used Proc Mixed of SAS program
version 8.0 (SAS Institute 1999) for the ANOVA and ANCOVA for the post-fertilization
data, for which I treated site and treatment as ﬁxed effects and day and site x treatment x
day as random effects. Methane and CO2 data were natural log transformed before
ANOVA and AN COVA to homogenize variances. I used untransformed data for

correlation analysis.

Results

Methane Oxidation in the Field

53

Methane oxidation rates were highest in the mature deciduous forest (Table 2.2),
where average rates in the Control treatment were 32 ($3.2, n=3 sites x 7 sample dates)
11g CH4-C rn'2 h'1 and daily rates over all treatments ranged from 0 to 73 ug CH4-C m'2
h". Added nitrogen reduced methane oxidation substantially (Figure 2.1), with the effect
appearing to increase over a period of time until day 52 (Figure 2.2). In contrast, plowing
had no signiﬁcant effect on methane oxidation in mature forests and no-till comﬁelds,
nor in mid-successional communities. There was also no signiﬁcant fertilizer x plowing
interaction (Table 2.5). Methane oxidation in both fertilized and plow x fertilized plots in
the mature forests dropped sharply after treatment and started to increase after day 52,
when CH4 uptake in the plow x fertilized plots began to increase faster than in the
fertilizer only plots (Figure 2.2a). In contrast, CH4 oxidation in plowed plots was similar
to control plots throughout the experiment.

Methane oxidation rates in the mid-successional sites were intermediate between
rates in the forest and cropped systems (Table 2.2). Average oxidation in the control
plots was 24 (i1 .6) ug CH4-C m“2 h'1 and rates over all plots ranged from 2.4 to 50 pg
CH4—C m'2 h'l. Following treatment, N fertilizer and fertilizer x plowing reduced
methane oxidation rates in these plots signiﬁcantly, from 24 to 15 pg CH4-C m'2 h’l on
average. In contrast, plowing alone had no detectable effect on oxidation. Similar to
ﬂuxes in mature forests, CH4 oxidation in fertilized and in plow x fertilized plots
exponentially decreased after treatment but started to increase at day 23 (Figure 2.2b).

In the No-till corn sites, CH4 oxidation rates were variable and low; the average
control plot rate was 4.0 (i0.7) ug CH4-C m'2 h". Rates across all treatments ranged

from -8 to 17 pg CH4-C rn'2 h'l (negative oxidation rates indicate methane production).

54

Neither added nitrogen nor plowing signiﬁcantly affected soil methane uptake in this site

(Figure 2.2c).

Carbon dioxide ﬂuxes in the ﬁelds

In situ carbon dioxide production ranged from 20 to 335 mg CO2-C m'2 h’1 among
the different sites and treatments. Before treatment, there were signiﬁcantly different
(P<0.0001) CO2 ﬂuxes among sites (Table 2.3 and 2.4); CO2 ﬂux was highest in mature
forest followed closely by mid-successional communities then no-till corn ﬁeld (Figure
2.4). However, after treatment CO2 ﬂuxes were highest in mid-successional communities
(124.9 i131 mg CO2-C rn'2 h") followed closely by mature deciduous forest (108.9 d: 7.7
mg CO2-C m'2 0‘) and no-till cornﬁeld (104.9 i 9.2 mg CO2-C rn'2 h'l) in control plots
(Table 2.2 and Figure 2.3).

Although treatment had overall no signiﬁcant effect on CO2 ﬂuxes (Table 2.7), N
fertilizer signiﬁcantly increased rates of CO2 production in both mid-successional
communities and mature forests but not in no-til] cornﬁeld (Figure 2.3 and Table 2.2).
Fertilizer x plowing also had the same effect on CO2 production as fertilizer alone. In
contrast, plowing alone did not show any signiﬁcant effect on soil CO2 ﬂuxes in any site
(Table 2.2 and Figure 2.3).

Early in the experiment, CO2 ﬂuxes in the mid-successional and mature forests
were greater than in the no-till cornﬁeld, but these differences were not persistent (Figure
2.4). Carbon dioxide ﬂuxes likely differed among dates of sampling (P=0.059; Table
2.7), however CO2 ﬂuxes in mature and mid-successional communities decreased

modestly after treatment and increased signiﬁcantly at day 52 then declined drastically,

55

whereas in the no-till com site CO2 ﬂuxes varied very little among the sampling dates

(Figure 2.4).

Soil physical and chemical factors

Average soil moisture (0-10 cm depth) was highest in mature forests (18 g H20-
100 g soil'l) followed by mid-successional communities (0.9 g H2O-100 g soil") and no-
till comﬁelds (1.0 g H20- 100 g soil") respectively (Table 2.2). Soil moisture did not vary
signiﬁcantly among treatments and likely differed (P=0.053) across sample dates (Table
2.8 and Figure 2.5). It was highest after rainfall at day 16 and 52.

Soil temperature was, on average, 2-4 oC cooler in the mature forests (16°C
average) than the mid-successional communities (18 °C) and no-till cornﬁeld (20°C;
Table 2.2). Temperature dropped over the course of the experiment, but the relative
ranking of the sites remained unchanged (Figure 2.6). There were no treatment effects on
soil temperature and it signiﬁcantly varies among sample dates (Table 2.8).

Soil nitrate in control plots was signiﬁcantly different among sites (Table 2.8). It
was highest in no-till comﬁelds (30.4 i 7.4 pg NO3'-N g soil'l), followed by mature
forests (4.1 :1: 0.7 ug N03'-N g soil"), and mid-successional communities (0.5 :t 0.1 ug
NO3'-N g soil’l) (Table 2.2). Fertilizer also signiﬁcantly increased soil nitrate (Table
2.7). Nitrate was highest in fertilized x plow plots followed by fertilized plots and control
plots. Levels of soil nitrate dramatically increased after fertilizer application in every site
and then declined rapidly until reaching pre-fertilization levels at day 73 for mature
forests and day 52 for mid-successional communities (Figure 2.7). However, nitrate in

no-till cornﬁeld soils remained high during the sampling period.

56

Unlike nitrate, soil ammonium did not vary by site (Table 2.8). Average soil
ammonium levels in control plots were similar for all sites (ca. 22.2 pg NH4+-N g soil";
Table 2.2). Treatment effects were signiﬁcant, similar to those for nitrate (Table 2.8),
with fertilized treatments having an order of magnitude more N than control and plowed
treatments, which did not differ. The temporal patterns of soil ammonium were also

similar to those for nitrate (Figure 2.8).

Controls on Methane and Carbon Dioxide Fluxes

Methane had a very strong and signiﬁcant correlation (r=0.70, P<0.0], n=3 forest
sites x 3 replications x 4 plots) with CO2 initially but it disappeared after treatment (Table
2.9 and Figure 2.9).

Before treatment, temperature seemed to be a signiﬁcant factor affecting the rate
of soil CH4 uptake (Table 2.4). However, aﬁer treatment no soil factor showed a
signiﬁcant effect on CH4 ﬂux (Table 2.5).

Only soil temperature had a signiﬁcant effect on CO2 ﬂuxes before treatment
(Table 2.6) but only soil moisture signiﬁcantly affected CO2 after treatment (Table 2.7).
Moisture was also strongly correlated with CO2 (Table 2.9). Even though soil
temperature, nitrate, and ammonium were signiﬁcantly correlated with CO2 emission
before treatment, only soil ammonium remained signiﬁcantly but weakly correlated after

treatment (Table 2.9).

Discussion

Methane Oxidation

57

Soil CH4 oxidation was highest in mature forest, followed by mid-successional
communities and no-till agriculture, respectively (Table 2.2 and Figure 2.1). Agricultural
ﬂuxes were less than 25% of those in the forests and successional sites. The rate decline
along the successional gradient is similar to that observed in previous agriculture and
natural vegetation comparisons (e. g. Mosier et al. 1991, Goulding et a]. 1995, Arif et al.
1996, Powlson et al. 1997, Robertson et a1. 2000, Hiitsch 2001).

Before treatment, soil temperature appeared to be the most signiﬁcant factor
controlling differences in CH4 oxidation among sites. Mature forest soils have
signiﬁcantly higher levels of total carbon, total nitrogen, and ammonium as compared to
the no-till com ﬁeld soils, which had higher soil nitrate levels and greater bulk density
(Table 2.1). The lower soil bulk density in the forest implies more gas diffusion, which
has been shown to increase soil CH4 uptake by methanotrophs in soil crumbs and soil
aggregates (Ball eta]. 1997a, Ball et al. 1997b). Mature deciduous forest soils were also
acidic, which suggests acid-adapted CH4 oxidizing bacteria in this forest.

However, after treatment, no physical or chemical factor had a signiﬁcant effect
on CH4 uptake (Table 2.5). Across all treatments, mature forest soil still had more CH4
oxidation than the no-till cornﬁeld soils, while mid-successional communities soils were
intermediate (Figure 2.1) and also had higher soil moisture, lower soil temperature, and
less soil nitrate than the mid-successional communities and no-till cornﬁeld (Table 2.2).

Fertilizer (100 kg N ha") markedly inhibited soil CH4 consumption up to 60% in
mature forest soils and to 40% in mid-successional communities soils (Table 2.2). These
results were similar to most previous studies (Bender and Conrad 1994, King and Schnell

1994, Hiitsch 1996, Gulledge et al. 1997, King and Schnell 1998), indicating the

58

detrimental effect of inorganic N on CH4 oxidation. However, added nitrogen did not
further suppress CH4 oxidation in no-till cornﬁeld as found in some studies (Mosier and
Schimel 1991, Bronson and Mosier 1993, Tate and Striegl 1993, Mosier et a1. 1998),
probably due to the already high soil nitrate and ammonium levels in these soils.

Soil tillage alone did not show any signiﬁcant effect on soil CH4 uptake in any of
these sites, but faintly increased CH4 oxidation in mature forest and comﬁelds and faintly
decreased CH4 uptake in mid-successional communities (Figure 2.1). The depth of tillage
in this study was about 10 cm less than the 20 cm depth of normal tillage, and thus has a
less destructive effect on soil structure than normal agricultural tillage, but nevertheless
represents a strong disturbance my results were similar to those from other studies in
tropical savanna (Sanhueza et al. 1994), Piedmont comﬁelds (Burke et al. 1999), and an
acid oxisol site in Puerto Rico (Mosier eta]. 1998).

Interestingly, although not statistically signiﬁcant, soil tillage slightly alleviated
the inhibition effect of N fertilizer in mature and mid-successional communities,
especially after day 52 (Figure 2.2) when soil nitrate and ammonium had already declined
markedly (Figure 2.7 and 2.8). The tillage might have increased soil aeration in these
plots allowing a greater ﬂow of CH4 into soil micro sites and providing CH4 oxidizing
bacteria more access to the gas.

Soil CH4 consumption in this study showed some seasonal changes (P=0.065)
over the course of this 101-day investigation (Table 2.5). This suggests that fertilizer has
a stronger effect on soil CH4 consumption than other seasonal physical factors such as

soil temperature and moisture in this study.

59

That CH4 oxidation was strongly and positively related to C02 production before
fertilization (Table 2.9) indicates that conditions were favorable for both soil respiration

and CH4 oxidation.

Carbon dioxide

Carbon dioxide ﬂuxes differed among sites only after treatment (Table 2.6 and
2.7). Even though treatments did not signiﬁcantly affect CO2 ﬂuxes (Table 2.7), the
fertilizer and fertilizer x plow combination plots in mid-successional and mature forests
had signiﬁcantly higher CO2 production, approximately 25% more than control and
plow-alone plots (Table 2.2 and Figure 2.3). In no-till cornﬁeld plow and N fertilizer
treatments had no noteworthy effect on soil CO2 emission.

Soil moisture was the most important positive factor affecting soil CO2 emission

(Table 2.7 and 2.9).

Conclusion

The mature forest soils had the highest overall methane oxidation rates, followed
by mid-successional and agricultural systems, respectively. Nitrogen added to the forest
and mid-successional sites substantially reduced methane oxidation within 2 weeks of
nitrogen addition. In the no-till site, where oxidation rates were already low and soil
nitrogen levels already high, additional nitrogen had no effect on soil CH4 uptake.
Plowing had no detectable effect on methane oxidation in any of the three sites. N
fertilizer also signiﬁcantly increased CO2 emission in mature forest and mid—successional

communities. The impact of agriculture on methane oxidation is likely due primarily to

60

greater nitrogen availability via N fertilization rather than to the disruption of soil
structure or other effects of plowing. Increasing nitrogen inputs to mid-successional and

mature ecosystems reduces rates of oxidation that would otherwise be relatively high.

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64

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65

Table 2.] Soil properties for the mature deciduous forest, coniferous forest, mid-

successional communities, and no-till agricultural ﬁeld at the KBS LTER site.

 

 

Soil property Deciduous forest Mid-successional No-till cornﬁeld
communities
Texture Sandy loam Sandy loam Sandy loam
pH‘ 5.20 i 0.12 a 5.66 i 0.04 b 6.45 3: 0.09 c
Bulk density (g om'3)2 1.22 i 0.08 a 1.40 e 0.04 b 1.57 i 0.06 bc
Total carbon (mg c g soil")3 1.52 i 0.12 a 0.97 :t 0.03 ab 0.73 i 0.09 b
Total nitrogen (mg N g soil“)3 0.123 i 0.09 a 0.093 t 0.003 a 0.087 4: 0.009 b
Nitrate (11g N g soir‘)4 1.93 x 0.21 a 0.38 i 0.05 b 2.79 i 0.30 c
Ammonium (pg N g soir‘)4 3.50 i 0.37 ab 4.46 i 0.58 a 2.08 i 0.73 b

 

Note: Values are means (is.e., n=varied). Data are from the KBS LTER website htgpzllkbs.msu.edu/lter/.

 

I sampled 8 March 2000 to a depth of 10 cm.
2 sampled 10 April 1996 to a depth of 15 cm.
3 sampled 7 April 1999 to a depth of 25 cm.

4 average values from 2000 season to a depth of 25 cm.

66

Table 2.2 The effects of tillage and N fertilizer on average methane oxidation, carbon dioxide ﬂux, and soil
properties in mature deciduous forest, mid-successional communities, and no-till agriculture at the KBS

 

 

LTER site.
Control Tillage Fertilizer Fertilizer x
Tillage
CH. (pg cat-c m‘2 h")
Mature forest 31.6i3.2 Aa 33.7:t3.6 Aa 12.7i1.8 ABb 14.8i2.1 Ab
Mid successional communities 23.7i1.6 Aa 22.0i2.3 Ba 14.8i1.8 Ab 16.1i1.7 Ab
No-till cornﬁeld 4.03:0.7 B 5.0i1.1 C 4.311.] B 2.313 B
co2 (mg CO2-C m'2 h")
Mature forest 108.9i7.7a 118.8i9.6a 135.9i13.l Ab 132.7112] Ab
Mid successional communities 124.9il3.la 118.2i14.8a 153.83c13.6 Ab 156.3i15.5 Ab
No-till cornﬁeld 104.9i9.2 95.6i9.5 83.1i7.4 B 91.5i9.8 B
Moisture (g 1120' 100g soil")
Mature forest 17.6il.5 A 17.9i1.8 A 20.1i1.6 A 18.5:t1.6 A
Mid successional communities 9.1i0.9 B 9.4:t1.0 B 8.4i0.8 B 7.9:t0.8 B
No-till cornﬁeld 9.7:t0.6 B 10.1i0.5 B 10.8i0.5 B 10.6i0.6 B
Temperature (°C)
Mature forest 16.3i0.6 AB 16.3:t0.6 AB 16.3i0.6 AB 16.3:t0.6 AB
Mid successional communities 18.6:t0.5 AB 18.6105 AB 18.6i0.5 AB 18.6i0.5 AB
No-till cornﬁeld 20.4i0.5 BC 20.4ds0.5 BC 20.4i0.5 BC 20.4i0.5 BC
Nitrate (ng No3 '-N g soil")
Mature forest 4.1:t0.7 a 6.1:tl.1 a 105.2i19.9ABb 154.0i33.7 b
Mid successional communities 0.55:0.1 a 1.2i0.3 a 77.3:t22.9 Aab 137.7i41.6 b
No-till cornﬁeld 30.4i7.4 a 19.73c8.2 a 176.2i25.6 Bb 181.5:I:]7.9 b
Ammonium (11g NHai-N g soil")
Mature forest 21.3i2.8 a 22.0i3.5 a 101.8i20.1 ab 181.2i38.4 b
Mid successional communities 24.9:t5.4 a 26.9:t4.3 a 98.2i19.6 ab 18031427 b
No-till cornﬁeld 20.3i4.7 ab 27.2i8.2 abc 124.4i25.0 bcd 161.8i28.0 cd

 

Note: Values are means i standard error of mean for four replicates.

Values followed by different higher case letters are signiﬁcantly different (P<0.05) among sites.
Values followed by different lower case letters are signiﬁcantly different (P<0.05) among treatments within

a site.

67

Table 2.3 Analysis of variance to determine effects of sites on methane oxidation rate,
carbon dioxide emission, and soil properties before treatment in mature deciduous forest,

the mid-successional community, and the no-till agricultural ﬁeld at the KBS LTER site.

 

 

SS df MS F P
CH4 Between Groups 8444 2 4222 36 <0.0001
Within Groups 3812 33 115
Total 12257 35
CO2 Between Groups 87304 2 43652 43 <0.0001
Within Groups 33162 33 1005
Total 120466 35
Nitrate Between Groups 790 2 395 59 <0.0001
Within Groups 219 33 7
Total 1010 35
Ammonium Between Groups 3637 2 1819 ll <0.0001
Within Groups 5235 33 159
Total 8873 35
Moisture Between Groups 1830 2 915 35 <0.0001
Within Groups 866 33 26
Total 2697 35
Temperature Between Groups 640 2 320 124 <0.0001
Within Groups 85 33 2
Total 726 35

 

68

Table 2.4 Analysis of covariance to determine effects of site and soil properties on

methane oxidation rate before treatment in mature deciduous forest, the mid-successional

community, and the no-till agricultural ﬁeld at the KBS LTER site.

 

 

Source SS df MS F P
Site 1.8 2 0.88 6.2 0.008
Treatment 0.] 3 0.05 0.3 0.808
Site x treatment 0.6 6 0.09 0.7 0.676
Nitrate 0.4 1 0.36 2.5 0.127
Ammonium 0.008 1 0.008 0.06 0.810
Moisture 0.3 1 0.3 1.9 0.180
Temperature 0.8 l 0.8 5.6 0.028
Error 2.8 20 0.14

Total 442 36

Corrected Total 16.2 35

 

69

Table 2.5 Analysis of variance to determine effects of site, treatment, and soil properties
on methane oxidation rate after treatment in mature deciduous forest, the mid-

successional community, and the no-till agricultural ﬁeld at the KBS LTER site.

 

 

Variable Num. (if a Den. df a F P
Site 2 230 105 <0.0001
Treatment 3 230 14.9 <0.0001
Site x Treatment 6 230 3.8 0.00]
Day 6 230 1.5b 0.065
Site x Treatment x Day 36 230 -01" 0.916
Nitrate 1 230 0.2 0.629
Ammonium l 230 0.1 0.808
Moisture 1 230 2.4 0.123
Temperature 1 230 0. 1 0.777

 

a Num. df and Den. df refer to numerator and denominator degrees of freedom,
respectively

b Z value

70

Table 2.6 Analysis of covariance to determine effects of site, and soil properties on

carbon dioxide emission before treatment in mature deciduous forest, the mid-

successional community, and the no-till agricultural ﬁeld at the KBS LTER site.

 

 

Source SS df MS F P
Site 0.2 2 0.1 1.6 0.220
Treatment 0.] 3 0.04 0.6 0.623
Site x treatment 0.08 6 0.01 0.2 0.970
Nitrate 0.002 1 0.002 0.04 0.843
Ammonium 0.001 1 0.001 0.01 0.898
Moisture 0.03 1 0.03 0.46 0.504
Temperature 0.27 l 0.27 4.3 0.050
Error 1.26 20 0.06

Total 852 36

Corrected Total 9.2 35

 

71

Table 2.7 Analysis of covariance to determine effects of site, treatment, and soil

properties on carbon dioxide emission after treatment in mature deciduous forest, the

mid-successional community, and the no-til] agricultural ﬁeld at the KBS LTER site.

 

 

Variable Num. (if 8‘ Den. df a F P
Site 2 230 17.6 <0.0001
Treatment 3 230 0.7 0.573
Site x Treatment 6 230 2.2 0.045
Day 6 230 1.6b 0.059
Site x Treatment x Day 36 230 1.9b 0.058
Nitrate 1 230 2.6 0.109
Ammonium l 230 3.3 0.071
Moisture l 230 24.0 <0.0001
Temperature 1 230 1 . 1 0.295

 

a Num. df and Den. df refer to numerator and denominator degrees of freedom,

respectively

b Z value

72

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73

Table 2.9 Relationships among methane oxidation, carbon dioxide, and other soil
properties before and after experimental treatment in mature deciduous forest, the mid-
successiona] community, and the no-till agricultural sites. Values are Pearson correlation
coefﬁcients (r). a n = 3 sites x 3 replications x 4 sample plots. b n = 3 sites x 3 replications

x 4 treatments x 7 sample dates.

 

 

CO2 Moisture Temperature Nitrate Ammonium
Before treatmenta
CH4 0.701** -0.678** -0.168 -0.401* 0.179
C02 1.00 0.536* -0.831** -0.336* 0.512**
After treatmentb
CH4 0.054 0.094 -0.339** -0.279** 0216‘”
C02 1.000 O.306** 0.009 0.055 0.170**

 

* Correlation is signiﬁcant at the 0.05 level (2-tailed).

** Correlation is signiﬁcant at the 0.01 level (2-tailed).

74

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Aa
A3 I
35 -|- DControl
*‘ IPlow
30 “ IFertilizer
.- Aa Ba IPlow+Fer
"a: 25 -
E
9’ 20 i
Q
I
0
g 15 -
10 ~
5 T ,1;
0 - .
Mature Forests Mid-successional No-till Comﬁelds
Communities

Figure 2.1 The reduction of methane oxidation due to soil disturbance and ammonium
nitrate fertilizer (100 kg N ha!) in mature forests, mid-successional communities, and no-
til] comﬁelds at the KBS LTER site. Vertical bars are standard errors of mean (s.e., n = 3
sites x 7 sample dates). Different higher and lower case letters represent signiﬁcant

differences (P<0.05) of treatments among sites and within site respectively.

75

70 2 -~~ ~- - ~ - ‘
a. Mature forest 4

 

604
50'
40| “ i
30 2' l

H" ¢ ...... o
.3. Q a A

pg CH4-C in2 h"

 

b. Mid-successional communities

60*

 

 

 

pg CH4-C rn'2 I'i'1

 

 

 

 

70
60 , c. No-till cornﬁeld +COWO.

- - A- - ~P|ow
50 — -X — Fertilizer
40 _ — O— -PIow+Fer
30 i

 

 

 

 

1 6 16 23 52 73 101

Day after treatment

Figure 2.2 Average daily methane oxidation in the KBS LTER site: a) mature deciduous
forests, b) mid-successional communities, c) no-till comﬁelds, with standard error bars
(n=3 sites).

76

200

 

 

 

   

 

 

 

 

 

 

 

 

 

180 Ab Ab DControl
EIPlow
160 Ab Ab IFertilizer
r: 140 " lPlow+Fer
I: Aa
‘r 120 ,
E Aa
B
L-,’, 100 . ‘1" Ba a
o '.
U 80
a) .....
E 60 4
40
20
0 r ..
Mature Forests Mid-successional No-till Comﬁelds
Communities

Figure 2.3 Net carbon dioxide ﬂuxes due to soil disturbance, and ammonium nitrate
fertilizer (100 kg N ha'l) in mature forests, mid-successional communities, and no-till
comﬁelds at the KBS LTER site. Vertical bars are standard errors of mean (s.e., n = 3
sites x 7 sample dates). Different higher and lower case letters represent signiﬁcant

differences (P<0.05) of the treatment among sites and among treatments within the same

site, respectively.

77

 

300 ;

a. Mature forest

 

 

 

 

 

 

 

 

 

.. 250 1
'3: 200 I If .
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0
300 ~
c. No-till cornﬁeld —I—Control
250 — . . A. . .plow
.7 — -x — Fertilizer
f 200 -_ - O- -Plow+Fer
’E
‘3‘ 150 - °‘
3 .I. .. -------------- ‘ l
a 100 'i ' "I /§- —' . L- _—_':;—_--x’ w.:.(- A \
E \ ‘ I/ J- ~ . - ‘w . - 7.? N. l
s . ~ ‘ \- - u . ‘
50 5 ' ‘ .= ..
0 ~ .
l 6 16 23 52 73 101
Day after treatment

Figure 2.4 Average daily carbon dioxide ﬂuxes in the KBS LTER site: a) mature
deciduous forests, b) mid-successional communities, c) no-till comﬁelds, with standard
error bars (n = 3 sites).

78

30‘

 

25

 

20

I I

 

\ ' ~
‘ o \ -~ ....
15 4 I 'O Q ‘0’
\ o ‘ ‘
D
a

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30

 

b. Mid-successional communities
25

20

 

 

g H201009 soil"

 

 

 

 

 

 

 

 

 

30
‘ +Control
25 c. No-till cornﬁeld _ _ a- - Plow
v; — -X — Fertilizer
’3 20 — O —P|ow+Fer
m
or
c
2
O __ 1
f .—=-—-"*—"
or
O .
1 6 16 23 52 73 101

Day after treatment

Figure 2.5 Average daily soil moisture in the KBS LTER site: a) mature deciduous
forests, b) mid-successional communities, c) no-till comﬁelds, with standard error bars
(n =3 sites).

79

35

 

30

 

 

 

—O—Mature Forests
5 - - I- - Mid-successional Communities
- ‘A— - No-till comﬁelds

 

 

1 6 16 23 52 73 101

Day after treatment

Figure 2.6 Average daily soil temperature in the KBS LTER site: a) mature deciduous
forests, b) mid-successional communities, 0) no-till comﬁelds, with standard error bars
(n =3 sites).

80

700

600 ‘1

300

pg N03'N 9 soil"

100 ,

700

600 .

500

pg NO3-N 9 soil"

200

100

pg NO3-N 9 soil“1

400 ‘

200 i

500 ,1

 

 

400 .

300+:

 

b. Mid-successional communities

 

 

 

 

 

 

 

 

+Control
- - A- - ~Plow
— -x — Fertilizer

— o — Plow+Fer

 

 

 

52
Day after treatment

Figure 2.7 Average daily soil nitrate in the KBS LTER site: a) mature deciduous forests,
b) mid-successional communities, c) no-till comﬁelds, with standard error bars (11 = 3

sites).

81

700~-— _ - _. .. _____._.___
600 a. Mature forest

500 4

400 i ‘ 1
300 l. I i
\ r ix
200 (Ting—A
.,,~ EX
100 1':

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pg NHf-N 9 soil‘1

 

700

 

500 . b. Mid-successional communities

01
O
0

pg NHf-N g soil-1
(.0 -h-
8

 

 

 

 

+Control
- - A- - -Plow
500 l — -x — Fertilizer

600 c. No-till cornﬁeld

— o — Plow+Fer

00-h
O
O

200 .

pg NHf-N 9 soil'1

9

o
1:49:4—4
—§o—i

100

 

 

 

 

Day after treatment

Figure 2.8 Average daily soil ammonium in the KBS LTER site: a) mature deciduous

forests, b) mid-successional communities, c) no-till comﬁelds, with standard error bars
(n=3 sites).

82

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83

Chapter 3

Soil Methane Oxidation in Organic vs. Conventional Agriculture

Introduction

The oxidation of methane by methanotrophs in soil is a small but signiﬁcant sink
of atmospheric methane. Previous studies show higher methane consumption rates in
forest and prairie than in agricultural soils (Ambus and Christensen 1995, Willison et al.
1995a, Goulding et a]. 1996, MacDonald eta]. 1996, Prieme and Christensen 1999),
suggesting a loss of soil oxidation capacity upon conversion to agriculture (Keller and
Reiners 1994). However, oxidation rates have been reported only for row crops under
conventional management (e. g.Wi]lison et al. 1995b, Boeckx and van Cleemput 2001,
Hiitsch 2001). Fertilizer, especially nitrogen, has been demonstrated to signiﬁcantly
reduce soil CH4 uptake (Hiitsch 1996, Jambert et al. 1997, Hiitsch 1998a, Tlustos et al.
1998). Many pesticides and herbicides also inhibit soil CH4 oxidation to some degree
(Topp 1993, Arif et a1. 1996, Boeckx et al. 1998, Prieme and Ekelund 2001).

It is possible, then, that different management systems, in particular those that
rely more on organic than synthetic sources for nutrients and pest control, may have a
higher oxidation capacity. If so, then agricultural management for soil methane oxidation
may be a practical means for helping to mitigate the buildup of atmospheric methane.

Robertson et a]. (2000) found no differences between an organically managed
com-soybean-wheat system and a conventionally managed counterpart in Michigan, but

they did not include compost or manure application, common in organic farming.

84

The aim of this study is to compare the effects of conventional vs. organic
agricultural practices on methane oxidation, and within each system to examine

differences across seasons and between different crops within the rotation cycle.

Methodology
Site Description

The study was conducted at the WK. Kellogg Biological Station's (KBS) Living
Field Lab (LFL), which is a set of ﬁeld plots under a variety of organic and conventional
crop management systems established 10 years prior to this study. It is located on
Kalamazoo loam and Oshtemo sandy loam soils (Typic Hapludalfs; Table 1). I measured
methane oxidation in two com-com (Zea mays L.) - soybean (Glycine max L.) - wheat
(T riticum aestivum L.) cropping systems, one managed organically with compost and
cover crops vs. one managed conventionally with pesticides and inorganic nitrogen
applied as per best management practice (BMP).

Compost material contained ~50% dairy manure and ~50% oak leaves (Quercus
rubra) on a dry weight basis. Materials were composted for a minimum of one year. The
total amount of N applied to corn in the form of compost was 117 kg N ha'1 yr'l or an
average dry compost mass of 4480 kg ha'1 yr". Compost was not applied to the soybean
crop. Nitrogen was applied as urea to treatments in com under N fertilizer management at
a rate of 110 kg N ha'1 y'l. Cumulatively, compost applied plus previous compost
applications provided 40- 53 kg N ha’1 yr'l inorganic N (Willson 1998).

The cover crops in the organic system varied with crop; crimson clover (T rifolium

incarnatum L.) was sown into standing corn in late June and red clover (T rr'folium

85

pratense L.) was frost seeded into wheat in late March (Table 3.1). Both systems were
replicated four times in a split-split—plot randomized complete block design. Each plot is
4.5 x 15 m (15 x 50 ft). Soil properties and yield of these experiment plots are presented

in Table 3.2.

In Situ Gas Sampling

1 measured in situ methane oxidation rates using the static chamber technique
(Hutchinson and Livingston 1993). Static chambers were fashioned from 25 cm diameter
PVC pipe: bases (25 cm diameter x 10 cm high) were installed in each plot and left in
place except during agronomic Operations. Immediately prior to sampling, a 4.5 cm high
cap was placed on each base and sealed with a latex skirt wrapped with an elastic band.
At 15-20 minute intervals, four 10 mL headspace samples were removed through rubber
septa in each cap using a syringe, and put into 3 mL glass sample vials pre-ﬂushed with
headspace air. Within 24 hours via] contents were measured for CH4 using a gas
chromatograph (GC 5890 Series II, Hewlett Packard) equipped with a ﬂame ionization
detector (FID), and measured for CO2 using an infrared gas absorption (IRGA) analyzer
(EGA C02 Analyzer, Analytical Development CO. LTD. Hoddesdon, England).

Chambers were sampled twice per month during the growing season and once per

month during other parts of the year.

Soil Analyses
Soil temperature was measured at 0-5 cm depth using a temperature probe. Soil

samples for other analysis were taken from the top 10 cm of soil using a 2.5 cm diameter

86

soil probe. Fresh soils were passed through a 4 mm sieve and mixed by hand, and then
sub samples were taken for moisture content and mineral N analysis. Prior to analysis,
soils were stored in a reﬁigerator at 4°C for up to 4 weeks. Soil moisture content was
measured gravimetrically by drying the soil samples at 65°C for 3 days or until dry.
Further drying at 105°C removes an additional 0.8 g H2O°100g soil'1 moisture in these
soils. Mineral N measurements were obtained by extracting 20 g of dry soil with 100 mL
1 M KC1 for 24 h then ﬁltering through 2-pm pore size ﬁberglass ﬁlters. The ﬁltrates
were frozen prior to analysis for NI-I4+ and N03' using a continuous ﬂow analyzer

(Alpkem 3550, 01 Analytical, College Station, TX) (Bundy and Meisinger, 1994).

Soil Depth Study

Soil cores (5 cm diameter x 30 cm deep) were taken from the ﬁeld and stored
intact at 4°C for one week, after which they were separated into four different depth
increments (0-5, 5-10, 10-20, and 20-30 cm). Each soil increment was transferred to a
473 mL Mason jar for 0-5 cm and 5-10 cm increments and to a 946 mL Mason jar for 10-
20 cm and 20-30 cm increments; jars were left open and allowed to pre-incubate with
atmospheric methane at room temperature for one week in order to reduce soil moisture
levels to ca. 15% w/w. At the end of this period, methane oxidation was measured by
sealing the jar with a cap and removing 0.5 mL of headspace at four 10-minute intervals.
The headspace aliquot was immediately injected into the gas chromatograph for methane
analysis. After the CH4 oxidation study, soil samples were sieved and separated for soil

moisture, soil inorganic N content, and nitriﬁcation assays.

87

Nitriﬁcation Assay
A sieved soil sample of 10 g was placed in a 100 mL Erlenmeyer ﬂask and
combined with 50 mL of the phosphate buffer solution and ammonium sulfate (Hart et al.
1994) and sealed with Paraﬁlm. The ﬂasks were placed on a shaker table and shaken for
24 h. Soil slurry samples of 10 mL were taken before and another after 24 h incubation,
centrifuged for 30 min and ﬁltered through 0.2 pm ﬁberglass ﬁlter. The aliquots were
frozen until analysis of nitrate as above. Rates of nitrate production were calculated on

the basis of the difference between the initial and ﬁnal nitrate concentrations.

Statistical Analyses

I used Proc Mixed of SAS program version 8.0 (SAS Institute 1999) for the
analysis of variance (ANOVA) and analysis of covariance (ANCOVA) for ﬁeld data, in
which I treated crop nested in management as ﬁxed effects and day and crop x
management x day as random effects. I also used SPSS program version 10.0.] (SPSS
Inc. 1999) for ANOVA and AN COVA for the soil depth study and for the correlation
analysis for both ﬁeld and soil depth data. Methane and CO2 data were natural log
transformed before ANOVA and ANCOVA to homogenize variances. I used

untransformed data for correlation analysis.

Results
Conventional Crops vs. Organic Crops

Overall, average seasonal soil methane consumption in the conventional rotation

was 3.8 (:120.3) pg CH4-C m—2 h-l, slightly but not signiﬁcantly higher than rates in the

88

organic treatment at 3.3 (:l: 0.4) pg CH4-C m2 h-1 (Table 3.3 and 3.4). Mean seasonal CH4

oxidation rates in soils under conventional corn, soybean, and wheat were similar at ca.
3.8 pg CH4-C m2 h-1 (Table 3.5). In contrast, analysis of variance shows signiﬁcant
differences in CH4 uptake rates among crops in the organic rotation (Table 3.5 and Figure
3.1). Methane oxidation under organic corn was the highest at 4.8 (106 s.e., n= 4
replicate plots x 15 sampling dates) pg CH4-C m.2 h], and oxidation rates under organic

soybean and wheat were about 50% lower, at 2.5 (i 0.7) and 3.4 (i 0.5) pg CH4-C m.2

h], respectively (Table 3.5). Only dates of sampling were signiﬁcant for CH4 uptake rates
(Table 3.4).

In contrast to CH4, the average seasonal CO2 ﬂux from organic treatments (94.0
i 6.3 mg C02-C m.2 hi1) was 22% higher than ﬂuxes in conventional treatments (73.5 i

4.6 mg C02-C m-2 hJ; Table 3.3 and 3.6). Crops and sample. dates also signiﬁcantly

affected soil CO2 emission. Soils under organic wheat produced the most C02 (130 i 15
mg C02-C m-2 h.1 on average) followed by conventional wheat (93.7 i107 mg C02-C
m.2 hJ; Table 3.5 and Figure 3.2). The organic corn and soybean soils produced 42%
less co; than organic wheat soils (ca. 75 mg C02-C m’2 h") but 18% more CO2 than

their conventional counterparts (ca. 63 mg C02-C m.2 hi).

Seasonal Trends in Conventional Rotations
Methane oxidation in the conventional systems was generally highest in late
summer and early fall, and lowest in early spring and summer (Figure 3.3a). Oxidation

rates in conventional treatments ranged from —5 to 15 pg CH4-C rn'2 h" (a negative

89

oxidation rate indicates methane production). For com the highest methane uptake rates
were in late September (9.4 d: 1.0 pg CH4-C m'2 h], n = 4 replicates) and the lowest rates
were in December and March (around 1.0 pg CH4-C In2 0‘). For soybeans, rates were
also highest in late September (8.3 i: 2.1 pg CH4-C m‘2 h'l); lowest rates occurred in July
(-0.1 :t 0.9 pg CH4-C m'2 h'l). For winter wheat, oxidation rates were also highest after
harvest in early August (6.6 :t 3.2 pg CH4-C m'2 hi) and lowest in November and June
(around 1.9 pg CH4-C in2 b").

As for CH4 uptake, CO2 ﬂuxes in the conventional systems were highest in late
summer and early fall, and lowest in winter and early spring (Figure 3.3a). Fluxes ranged
from 3.4 to 345 mg C02-C m'2 h'1 in conventional treatments. For com, the highest CO2
production rates were in October (109 i 11 mg C02-C rn'2 h'l, n=4 replicates) and the
lowest rates were in January (11.5 i 1.7 mg C02-C m’2 h'l). For soybeans, rates were also
highest in August (140 :t 19 mg C02-C m'2 h"); lowest rates occurred in January (7.6 d:
0.9 mg C02-C m'2 h"). For wheat, ﬂux was highest in August (293 d: 36 mg C02-C m'2

h") and lowest in January (6.8 i 1.1 mg C02-C m'2 h").

Seasonal Trends in Organic Rotations
Methane oxidation in the organic rotations also exhibited seasonal trends, with
high rates of oxidation in the late growing season and lower rates in early spring except
for wheat (Figure 3.3b). Oxidation rates ranged from —20 to 17 pg CH4-C m'2 h". For
com, the highest methane uptake rates were in the July — October period (9.2 :i: 1.6 pg
CH4-C m2 h", n = 4 replicates, in late September) and the lowest were during winter to

early spring (-4.1 :1: 4.3 pg CH4-C m‘2 h'1 in November). For soybeans, rates were also

90

highest in late September (10.6 :12 pg CH4-C m’2 h!) and low in the spring, although
the lowest rate followed a rain event in early September (-2.5 i 6.0 pg CH4-C rn'2 h'l). In
winter wheat, which is harvested in late August, oxidation was highest in September -
October (6.0 i: 1.1 pg CH4-C rn'2 h'1 for late September) and lowest in early September
(-3.7 :t 5.0 pg CH4-C m'2 h'l) following crop harvest and rain. As for conventional
wheat, seasonal variation in organic wheat was lower than in other crops; in particular,
oxidation was moderately high in spring when for other crops oxidation rates were
lowest.

Carbon dioxide production in the organic systems was similar to that in
conventional systems, but generally of higher magnitude especially during the wheat part
of the rotation. The ﬂux was highest in August and lowest in January (Figure 3.4b). Daily
ﬂuxes ranged from 1.8 to 544 mg C02-C rrl'2 h'l. For com the highest C02 production
rates were in October (162 d: 21 mg C02-C rn'2 h], n = 4 replicates) and the lowest rates
were in January and March (around 22 mg C02-C rn'2 h'l). For soybean, rates were also
highest in August (197 :l: 39 mg C02-C m“2 0'); lowest rates occurred in January (19.1 3:
3.3 mg C02-C rn’2 h'l). For wheat, ﬂuxes were highest in August (432 i 47 mg C02-C

m'2 h") and lowest in January (6.5 i 2.5 mg C02-C m'2 h“).

Yield
Overall, conventional rotations produced more yield than organic rotations (Table
3.3). Conventional corn and soybean yielded 21% and 43% than organic counterparts

while organic and conventional wheat had similar yields.

91

Soil Moisture

Soil moisture was signiﬁcantly different among crops, management, and sample
dates (Table 3.7). Organic rotations had signiﬁcantly higher average soil moisture (14.9 i
0.4 g H20-100g soil") than conventional rotations (12.5 i 0.3 g H2O'100g soil", n = 4
replicates x 15 sample dates; Table 3.3). Moisture ranged from 1 to 23 g H20-100g soil'1
and the highest average soil moisture was in organic soybean and corn (around 15 g H2O-
100g 5011']; Table 3.5). Soil moisture remained constant from early October 2001 (at
around 17 g H20 100g soil'1 in conventional treatments and 20 g H20-100g soil'1 in
organic treatments; Figure 3.3) to March 2002, then slightly decreased until June before
exponentially decreasing to 3-8 g H20-100g soil'l. Soi] moisture increased to around 18 g
H20-100g soil'l after rainfall in early August then fell again in early September before

increasing at the end of the month (Figure 3.5).

Soil Temperature

Organic rotations also had higher average soil temperature (14.8 i 0.4 0C) than
those of conventional rotations (12.5 i 0.3 °C; Table 3.3). Within rotations, soil
temperatures were not signiﬁcantly different among crops (Table 3.4); nor was there a
signiﬁcant crop x treatment x day interaction, but temperature did differ signiﬁcantly
among dates of sampling (Table 3.7). Soil temperature was highest in summer around
late June (to around 30 °C in both rotations), and another peak occurred in late August
(around 25 °C) (Figure 3.6). Soil temperatures were lowest in winter (from December to

March), but were above freezing during the sample dates.

92

Soil Inorganic Nitrogen

Soil nitrate contents were similar between conventional and organic management
treatments (Table 3.3). However, there were signiﬁcant differences among crops (Table
3.5 and 3.7), crop x management x sampling date interactions, and among sampling
dates. Average soil nitrate in organic rotations (8.88 i 0.73 pg N g soil", it = 4 replicates
x 3 crops x 15 sample dates) was slightly but not signiﬁcantly higher than those in
conventional rotations (7.88 i 1.15 pg N g soil"; Table 3.3). Soil nitrate was highest in
conventional corn (12.2 i 3.2 pg N g soil") followed by organic soybean (11.2 i 1.4 pg
N g soil") and organic corn (9.2 i 1.3 pg N g soil"; Table 3.5). In conventional rotations,
soil nitrate reached its ﬁrst peak at mid January (around 16 pg N g soil") and its second
peak in conventional corn at mid July (44.7 i 41.3 pg N g soil'l; Figure 3.7a), just three
weeks after fertilization and two weeks after tillage (Table 3.1). In organic rotations, soil
nitrate began to increase from December 2001 to mid January 2002 then remained
constant until mid June (Figure 3.7b). Nitrate levels in organic rotations were higher
(approximately 20 pg N g soil") than in conventional rotations (10 pg N g soil'l) during
this period. Then nitrate levels declined until they reached their lowest levels in early
August. Under conventional management, soil nitrate began to decline ﬁrst in wheat,
followed by soybean and corn respectively, but under organic management, soil nitrate
ﬁrst decreased in wheat and then in corn and soybean correspondingly.

Soil ammonium differed signiﬁcantly by sample date (Table 3.7) but not by
management (Table 3.3) or crops (Table 3.4 and 3.7). Conventional corn had the highest
average soil ammonium (21.4 i 5.4 pg N g soil") followed by the other crops at

approximately 15 pg N g soil'l (Table 3.5). The seasonal trends for soil ammonium were

93

similar to those of soil nitrate. Ammonium levels reached their highest point in mid-July
after fertilization and tillage and reached a second peak in mid-August (Figure 3.8a).
Whereas in organic management, soil ammonium was highest in late May and mid-

August, levels much lower than in conventional corn (Figure 3.8b).

Controls on Methane and Carbon Dioxide Flux in the Field

Analysis of covariance shows that CH4 oxidation was not affected by any
measured soil factor (Table 3.4). Correlation analysis also shows a weak but signiﬁcant
negative correlation between CH4 oxidation and moisture (r=-0.152, p<0.01, n = 2
managements x 3 crops x 4 replicate plots x 15 sample dates; Table 3.8). In contrast, CO2
ﬂuxes were signiﬁcantly affected by soil nitrate, ammonium, and temperature but not soil
moisture (Table 3.6). Carbon dioxide ﬂuxes were signiﬁcantly correlated with soil
temperature and nitrate (Table 3.8). Fluxes of CO2 and CH4 were not correlated (Table

3.8 and Figure 3.9).

Soil Depth Study
CH4 oxidation in soil cores did not differ between conventional and organic
systems (Table 3.9). Oxidation rates were similar at approximately 0.05 pg CH4-C kg
soil'l h'l. Rates also did not vary among crops but did signiﬁcantly vary by soil depth
(Table 3.10 and 3.11), and generally uptake was highest at 10-30 cm depth (Table 3.10
and Figure 3.10). Soil CH4 uptake rates ranged from —0.05 to 0.21 pg CH4-C kg 5011'1 h".
Some soil cores from organic rotations showed net CH4 production in the incubation

(Table 3.10).

94

Soil moisture in collected cores differed signiﬁcantly between management
systems and among crops, but not among soil depths (Table 3.12 and Figure 3.11). Soil
cores from the conventional systems had less soil moisture (10.5 i 0.3 g H2Ot100g soil'l)
than those from the organic systems (11.5 i: 0.4 g H20-100g soil"; Table 3.9).

Soil nitrate was signiﬁcantly different among crops and soil depths but not
between management systems (Table 3.9 and 3.12). Nitrate was highest at the soil surface
then declined along the soil proﬁle (Figure 3.12). Organic soybean had the highest levels
of soil nitrate (Table 3.10).

Like nitrate, ammonium varied signiﬁcantly among crops and soil depths (Table
3.12) but not management systems (Table 3.9). There also was more ammonium at the
soil surface vs. lower soil levels (Table 3.10 and Figure 3.13). Conventional and organic
corn had the highest soil ammonium content among other crops (Table 10).

Soil nitriﬁcation signiﬁcantly differed between management systems and among
soil depths (Tables 3.9 and 3.12). Soil from organic systems had higher nitriﬁcation rates
(0.3] i 0.03 pg N kg 5011'1 h") than did soils from the conventional systems (0.20 i 0.02
pg N kg soil‘l h"; Table 3.9). Nitriﬁcation was also highest in surface soil rather than
deeper soils (Table 3.10 and Figure 3.14). Organic wheat had the highest nitriﬁcation rate

followed by organic com and soybean.
Controls on Methane Oxidation in Soil Depth

Soil moisture was the only factor almost signiﬁcantly (P=0.069) affecting CH4

uptake in incubated soil cores (Table 3.11); soil nitrate and ammonium did not

95

signiﬁcantly affect oxidation. Nevertheless, all three factors were negatively and

signiﬁcantly correlated with CH4 oxidation (r=-0.23 to —0.5 1; Table 3.13).

Discussion
Field Study
Methane Oxidation
Soil methane oxidation in organic cropping systems was overall 15% lower than
in the conventional systems but this difference was not statistically signiﬁcant. Mean

CH4 oxidation rates were similar among crops in the conventional system (ca. 3.8 pg
-2 -l . . . .
CH4-C m h ). In the organic system ox1datlon rates were 50% lower 1n the wheat and

soybean rotations (ca. 2.4 pg CH4-C rn'2 h'l) than in the corn, which exhibited rates
similar to those in the conventional counterparts. The lower CH4 consumption rate in
organic soybean and wheat was mainly due to high net CH4 emission in November and
early September while there was lower CH4 production in conventional systems on the
same dates. The November CH4 emission in the organic soybean and wheat might be a
result of high soil moisture at that time, combined with compost application from late
October in organic wheat plots. However, CH4 production occurred in early September
even in low soil moisture at 0-10 cm depth. Therefore, the source of CH4 production is
likely in a deeper soil horizon, perhaps due to a higher soil water table caused by high
rainfall two weeks before.

Most crops showed a similar seasonal pattern of methane oxidation. In general,

methane oxidation was highest in late summer and fall, and lowest in early spring.

96

Oxidation in the winter wheat rotations was less seasonally variable than in the other
crops, with lowest rates immediately following harvest in July.
Net CH4 production occurred on several sample dates and more frequently in

organic systems.

Carbon dioxide

Unlike CH4, overall CO2 ﬂuxes in the organic rotation were 22% higher than in
the conventional rotation; largely because of higher soil CO2 emission in organic wheat.
Corn and soybean had similar soil CO2 production rates while wheat had the highest CO2
ﬂux in both management systems, from 33% higher in conventional to 43% higher in
organic rotations. This might be in part from the compost application into organic wheat,
which increased available carbon in soil. However, organic corn, which also received
compost, did not show any increase in CO2 emissions. Another factor might be the clover
crops in the organic wheat, but organic corn, which also had a cover crop, had CO2 ﬂuxes
no different from non-cover crop organic soybeans. Additionally, the organic system had
more total carbon, total nitrate, and higher soil pH as compared to the conventional
treatment (Table 3.2).

Although soil moisture was not a statistically signiﬁcant predictor of CO2 ﬂux,
CO2 emission during summer sampling dates were correlated with soil moisture
especially at the peak of both CO2 production and soil moisture content in late August.
Overall soil moisture content in the organic treatment was also higher than in the

conventional treatment for every crop.

97

Both soil nitrate and ammonium reached their peak approximately three weeks
after fertilization in conventional com, the highest among other treatments. However,
conventional corn had overall soil nitrate no different than organic corn and soybean.
Although not statistically different, soil ammonium in conventional corn was

approximately 30% higher than in other plots.

Soil Depth Study

In the laboratory soil core study, CH4 consumption (either overall or at various
depths) did not differ between conventional and organic systems, nor among crops within
systems.

Only soil depth signiﬁcantly affected soil CH4 uptake in the laboratory study, as
occurred in with previous studies (Adamsen and King 1993, Koschorreck and Conrad
1993, Czepiel et al. 1995). However, the highest oxidation region was 20-30 cm depth,
deeper than in other studies; others have found highest oxidation at 3-6 cm in temperate
soil cores (Czepiel et a1. 1995), 10-12 cm in German forest soils (Koschorreck and
Conrad 1993), 9-12 cm in subarctic spruce-lichen woodland in Canada, and 4-8 cm in
temperate mixed forest in Maine (Adamsen and King 1993), and 5-15 cm in no-till
agricultural soils in UK (Hiitsch 1998b).

One possible reason for higher oxidation at this depth is that CH4 might be
produced at an adjacent level, providing CH4 for a larger oxidizing population at 20-30
cm as the CH4 diffuses upward or downward. Methane production still occurred at 5-10
cm depths even after a week of drying. Further experiments to verify this possibility are

needed.

98

Soil moisture was a signiﬁcant predictor of CH4 oxidation in the ﬁeld but it was
not a good predictor for differences in CH4 oxidation among soil depths because moisture
did not differ among depths. In contrast, soil nitrate and ammonium were not signiﬁcant
predictors for CH4 uptake in both ﬁeld and laboratory studies even though both declined
with soil depth, supporting the hypothesis that higher soil N leads to less soil CH4
oxidation as has been found in other studies. Soil nitriﬁcation also declined with depth,
suggesting that nitrifying bacteria were not responsible for the CH4 oxidation trends in
these soils. In fact, soil CH4 consumption was strongly negative correlated with soil
nitriﬁcation, ammonium, nitrate, and moisture.

From this study, organic and conventional systems are too simplistic a division to
deﬁne differences in soil CH4 oxidation. Each had similar net average soil CH4
consumption. The organic corn had the highest CH4 consumption, perhaps because it had
less inorganic nitrogen than those of the conventional plots but had higher soil moisture
and soil carbon from compost. The organic soybean and wheat had the lowest CH4
oxidation, perhaps from the decomposition and N mineralization of the remaining
compost in organic wheat and N ﬁxation from soybean. Soil from organic management
tends to produce CH4 easily when wet. The high soil carbon and organic matter might
contribute to this phenomenon.

It appears to be very difﬁcult to increase CH4 oxidation in agricultural soils. First,
the inorganic nitrogen is very high. Second, tillage destroys soil structure and disrupts the
colonization of soil microorganisms. Third, some pesticides are CH4 oxidation inhibitors.
Forth, soil becomes compacted from trafﬁc in the ﬁeld. Nevertheless, the best available

ways to increase CH4 oxidation appear to be related to reducing tillage and N

99

fertilization. Organic N should slowly release N to soil microbes thus decrease its toxicity
to methanotroph. But organic matter addition might increase CH4 production when soil

WCI.

Conclusion

Soil methane oxidation in organic cropping systems was overall 12% lower than
in the conventional system primarily due to low oxidation rates in organic wheat and
soybean crops. Mean annual oxidation rates were similar among crops in the
conventional system. In the organic system oxidation rates were lower in the wheat and
soybean rotations than in the corn, which exhibited rates higher than those in the
conventional system. Most crops showed a similar seasonal pattern of methane oxidation.
In general, methane oxidation was highest in late summer and fall, and lowest in early
spring. Oxidation in the winter wheat rotations was less seasonally variable than in the
other crops, with lowest rates following harvest in July. Net methane production occurred

on several sample dates and more frequently in organic systems.

100

References

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arctic forest soils - rates, vertical zonation, and responses to water and nitrogen.
Applied and Environmental Microbiology 59:485-490.

Ambus, P., and S. Christensen. 1995. Spatial and seasonal nitrous oxide and methane
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Arif, M. A. S., F. Houwen, and W. Verstraete. 1996. Agricultural factors affecting
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Boeckx, P., and 0. van Cleemput. 2001. Estimates of N20 and CH4 ﬂuxes from
agricultural lands in various regions in Europe. Nutrient Cycling in
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Boeckx, P., 0. Van Cleemput, and T. Meyer. 1998. The inﬂuence of land use and
pesticides on methane oxidation in some Belgian soils. Biology and Fertility of
Soils 27:293-298.

Bundy, L. G., and J. J. Meisinger. 1994. Nitrogen availability indices. Pages 951-984 in
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Part 2. Microbiological and biochemical properties. SSSA Book Series, no. 5.,
Madison, WI.

Czepiel, P. M., P. M. Crill, and R. C. Harriss. 1995. Environmental-factors inﬂuencing
the variability of methane oxidation in temperate zone soils. Journal of
Geophysical Research-Atmospheres 100:9359-9364.

Goulding, K., T. Willison, C. Webster, and D. Powlson. 1996. Methane ﬂuxes in aerobic
soils. Environment Monitoring Assessment 42:175-187.

Hart, S. C., J. M. Stark, E. A. Davidson, and M. K. Firestone. 1994. Nitrogen
mineralization, immobilization, and nitriﬁcation. Pages 985-1018 in R. W.
Weaver, J. S. Angle, and B. S. Bottomley, editors. Methods of soil analysis. Part
2. Microbiological and biochemical properties. SSSA Book Series, no. 5.,
Madison, WI.

Hutchinson, G. L., and G. P. Livingston. 1993. Use of chamber systems to measure trace
gas ﬂuxes. Pages 63-78 in L. A. Harper, A. R. Mosier, J. M. Duxbury, and D. E.
Rolston, editors. Agricultural Ecosystem Effects on Trace Gases and Global
Climate Change. American Society of Agronomy, Madison, Wisconsin.

101

Hiltsch, B. W. 1996. Methane oxidation in soils of two long-term fertilization
experiments in Germany. Soil Biology & Biochemistry 28:773-782.

Hﬁtsch, B. W. 1998a. Methane oxidation in arable soil as inhibited by ammonium, nitrite,
and organic manure with respect to soil pH. Biology and Fertility of Soils 28:27-
35.

Hﬁtsch, B. W. 1998b. Tillage and land use effects on methane oxidation rates and their
vertical proﬁles in soil. Biology and Fertility of Soils 27:284-292.

Hﬁtsch, B. W. 2001. Methane oxidation in non-ﬂooded soils as affected by crop
production. European Journal of Agronomy 14:237-260.

Jambert, C., R. Delmas, D. Serca, L. Thouron, L. Labroue, and L. Delprat. 1997. N20
and CH4 emissions from fertilized agricultural soils in southwest France. Nutrient
Cycling in Agroecosystems 48:105-114.

Keller, M., and W. A. Reiners. 1994. Soil atmosphere exchange of nitrous oxide, nitric
oxide, and methane under secondary succession of pasture to forest in the Atlantic
lowlands of Costa Rica. Global Biogeochemical Cycles 8:399-409.

Koschorreck, M., and R. Conrad. 1993. Oxidation of atmospheric methane in soil -
Measurements in the ﬁeld, in soil cores and in soil samples. Global
Biogeochemical Cycles 7: 109-121.

MacDonald, J. A., U. Skiba, L. J. Sheppard, K. J. Hargreaves, K. A. Smith, and D.
Fowler. 1996. Soil environmental variables affecting the ﬂux of methane from a
range of forest, moorland and agricultural soils. Biogeochemistry 34:113-132.

Prieme, A., and S. Christensen. 1999. Methane uptake by a selection of soils in Ghana
with different land use. Journal of Geophysical Research-Atmospheres
104:23,617-623,622.

Prieme, A., and F. Ekelund. 2001. Five pesticides decreased oxidation of atmospheric
methane in a forest soil. Soil Biology & Biochemistry 33:831-835.

Robertson, G. P., E. A. Paul, and R. R. Harwood. 2000. Greenhouse gases in intensive
agriculture: Contributions of individual gases to the radiative forcing of the
atmosphere. Science 289: 1922- 1 925.

SAS Institute. 1999. SAS/STAT User's Guide, Version 8. SAS Institute Inc., Cary, NC.

SPSS Inc. 1999. SPSS. SPSS Inc.

102

Tlustos, P., T. W. Willison, J. C. Baker, D. V. Murphy, D. Pavlikova, K. W. T. Goulding,
and D. S. Powlson. 1998. Short-terrn effects of nitrogen on methane oxidation in
soils. Biology and Fertility of Soils 28:64-70.

Topp, E. 1993. Effects of selected agrochemicals on methane oxidation by an organic
agricultural soil. Canadian Journal of Soil Science 73:287-291.

Willison, T. W., K. Goulding, and D. Powlson. 1995a. Effect of land use change and
methane mixing ratio on methane uptake from United Kingdom soil. Global
Change Biology 1:209-212.

Willison, T. W., C. P. Webster, K. W. T. Goulding, and D. S. Powlson. 1995b. Methane
oxidation in temperate soils - Effects of land-use and the chemical form of
nitrogen fertilizer. Chemosphere 30:539-546.

Willson, T. C. 1998. Managing nitrogen mineralization and biologically active organic

matter fractions in agricultural soil. Ph.D. Michigan State University, East
Lansing, MI.

103

Table 3.1 Agronomic ﬁeld activities for the sites studied. Data are from the KBS LTER

website httn://kbs.msu.edu/lter/.

 

Date

Field activity

 

10/3 0/2001
10/ 3 1/2001
3/ 19/2002
4/25/2002
4/25/2002
5/2/2002
5/20/2002
5/22/2002
5/22/2002
5/24/2002
5/28/2002
5/28/2002
6/7/2002
6/8/2002
6/13/2002
6/ 17/2002
6/27/2002
6/28/2002
7/8/2002
7/8/2002
7/15/2002
10/8/2002
10/8/2002
10/ 17/2002
10/23/2002
10/23/2002
10/23/2002

Apply compost 409 kg plot'1 in organic wheat

Plant wheat Pioneer 25R49 2.5 bu/a

Plant red clover 7 kg acre'1 in organic wheat

Apply compost 350 kg plot'1 in organic corn

F ertilize 46-0-0 N 18 kg acre'1 in conventional wheat
Chisel in conventional corn and soybean

Field cultivation in conventional corn and soybean
Plant corn Pioneer 37M34 28,000 seeds/a

Fertilize 28% N 1.3 kg acre'1 in conventional corn
Plant soybean 150,000 seeds/a

Broadcast herbicide mix 7 in conventional corn
Broadcast herbicide Dual] 11 in conventional soybean
Rotary hoe in organic corn and soybean -

Rotary hoe in organic corn and soybean

Rotary hoe in organic corn and soybean

Row cultivation in organic corn and soybean
Fertilize 28% N 50 kg acre'1 in conventional corn
Row cultivation in organic corn and soybean

Row cultivation in both conventional and organic corn and soybean
Plant crimson clover 5.4 kg acre'1 in organic corn
Harvest wheat

Chisel in both conventional and organic soybean
Harvest soybean

Harvest corn

Field cultivation both conventional and organic soybean
Plant wheat Pioneer 25R26 red winter 2.5 bu/a
Apply compost 300 kg plot'1 in organic wheat

 

104

ea 00 0o 8.8 a o. 88 804 00 Bass a

as 02o 860 e 9 800 E4 40 Bass _

 

 

 

2.0 a :0 00.0 4 08 03.0 n 00.0 8.0 .4 ~50 2.0 H 40.0 42.0 n 00.0 9-2 :0 2o;
00.0 a 00.0 08 0 e: 02 0 S0 04 40.0 a 0.0 na Rd 0 00.0 a 2.0 n 00.0 No-08 0 z 03 2802
000404.: 00.0.4 00.: m: 404.: 08 004.: 0: a 0:: 00: 040.2 N20.828
80.0 n 08.0 45.0 at. 08.0 8.0 n 08.0 5.0 a 000.0 8.0 a 000.0 80.0 0 040.0 $-08 0 z 0:0 5088 .860
8.0 n 3.0 2.0 0 0:0 :0 4. 00.0 8.0 n 00.0 3.0 a 8.0 00.0 n 00.0 ~88 0 0 0:0 .508 060

m 1.0 a 00.0 00.0 0 08 8.0 a $0 4 2.0 a 3.0 8.0 a Re :0 n 3.0 in
000:3 :00nxom :80 00055 :0008m :80 b00008 00m

8080008 08080

8080008 0:283:00

 

 

08080008 8805 0:08 8080 Amodvmv 8008.00 428008880 0.8 000:0— 0000 830— 888.00 .3 003028 00=_0> 080800.: 803000 :08

0800 05 .80 Amodvmv 8008.00 38008880 08 000:0— 0000 8an: 8000.80 43 0032—8 0020> .\:00_\:00.=08.0€<\8:: 000003 89: mmM 05 808 0:0 800

00:00:00: .88 :8 :008 00 880 0:00:80 H 8008 08 0030> .080 4.2 mg 0.0 00 080000 8080w0:08 080w8 0:0 0880800 00 000888 00m N.m 030:.

105

Table 3.3 Net average methane oxidation and carbon dioxide emission rates and other
soil properties in conventional and organic systems at the KBS LF L site. Values are

means i 1 SE for 4 replications x 15 sampling dates; signiﬁcance values are based on

 

 

ttCStS.

Factor Conventional Agriculture Organic Agriculture t-testa
CH4 (pg CH4—C m'2 if) 3.80 0 0.28 3.25 d: 0.37 ns
C02 (mg C02-C m‘2 h") 73.5 :t 4.6 94.0 :1: 6.4 *
Moisture (g H20-100g soil’l) 12.5 i 0.3 14.9 0 0.4 ***
Temperature (°C) 12.5 :t 0.3 14.8 d: 0.4 **
Nitrate (11g N g soil") 7.88 d: 1.15 8.88 0 0.73 ns
Ammonium (11g N g soil") 16.7 d: 2.0 15.2 i 0.9 ns
Yield (MT ha") 4.01 t. 0.8 3.18 i 0.7 ns

 

3 ns, not signiﬁcant, P>O.l; * P<0.05; **P<0.0l; ***P<0.0001

106

Table 3.4 Analysis of covariance to determine effects of treatment, crop, day, and soil

properties on methane oxidation rates from conventional and organic agriculture at the

 

 

KBS LF L site.

Variable Num. Df a Den. Df a F P
Management 1 84 2. l3 0. 149
Crop 2 84 2.56 0.083
Crop x Management 2 84 2.08 0.131
Crop x Management x Day 28 266 0.06b 0.477
Day 14 266 1.79b 0.037
Nitrate 1 266 0.00 0.969
Ammonium l 266 0. 15 0.696
Moisture l 266 0.1 1 0.742
Temperature 1 266 0.09 0.760

 

a Num. df and Den. df refer to numerator and denominator degrees of freedom,

respectively.

b Z value

107

 

4040.2 240.2 00400: 4._40.0_ 0040.: 4.044.: 9-0802000203828884

108

 

 

0.: 4 0.0 4._ 4 0.: 0._ 4 0.0 0 0.0 4 0.4 00 0.0 4 0.0 4 0.0 4 0.2 9.03. 0 2-002 0.0 88:2

_._4_.0_ _._4_.0_ 240.2 0040.0. 0040.2 0040.: 6002388800

0.0 40.: 0.0 4 0.2 0 0.0 4 :2 0.0 4 0.2 0.0 4 0.0_ 4 0.0 4 0.2 9.08 0000040 00 23202

00 4.2 4 0.02 4 0.0 4 0.40 4 4.0 4 0.00 4 00_ 4 0.00 0.0 4 0.00 0.0 4 0.00 9-0 we 0-400 0:0 N00

0 00.0 4 04.0 0 00.0 4 04.0 0 000 4 00.4 44.0 4 00.0 00.0 4 00.0 04.0 4 00.0 :0 4.: 04:0 03 £0
00055 :000400m 500 00055 :000000m 0.80

003300034 0_:0w00 050000.594N 0000000280 0E0t0>

 

06000400 5805 000:0 w:0E0 Amodvmv 00008400
3:005:90 000 00002 0000 0030. 0:88.00 43 0032.8 0020> ”08000400 0:0E0w0008 0003000 Amcdvmv 00008.00 30:02.2:w0
000 0000— 0000 0000: E00880 03 0032—8 0030> 00:00:00: 38 08 000:: 80 00:00 0:00:30 H 0:008 000 0020> 08000400 0033030

0_:0w:0 0:0 .0:00:0>:00 E 00000 w:0E0 00800000 000 0:0 00008 000080 :0800 0:0 :000008 0:082: :00 0w80>< m.m 030,—.

Table 3.6 Analysis of covariance to determine effects of treatment, crop, day, and soil

properties on carbon dioxide emission rates from conventional and organic agriculture at

 

 

the KBS LFL site.

Variable Num. (if a Den. df a F P
Management 1 84 3.9 0.05 1
Crop 2 84 4.3 0.017
Crop x Management 2 84 0.0 0.992
Crop x Management x Day 28 266 4.5b <0.0001
Day 14 266 2.2b 0.013
Nitrate 1 266 7.2 0.008
Ammonium l 266 6.3 0.013
Moisture 1 266 0.3 0.554
Temperature 1 266 1 5 .5 0.0001

 

a Num. df and Den. df refer to numerator and denominator degrees of freedom,

respectively.

b Z value

109

020> N 0

303000000: .8000000 00 0000w00 :8058050 0:00 08000005: 9 00000 :0 :0Q 0:0 .20 .832 4

 

 

 

00.0 00.0 :00 a .0 400.0 00.0 000.0 00.0 000 4_ 00:

000.0 00.0 040.0 30._ 000.0 00.0 03.0 a .0 000 00 000 0 088000002 0 080

0000 0.: 000.0 4.0 000.0 0.0 :40 0.0 40 0 088000002 0 080

_00.0 0: 000.0 0.0 80.0 0._ 000.0 00 40 0 080

004.0 0.0 004.0 0.0 400.0 0.0 0000.0v 0.40 40 _ 058000002
0 0 0 0 0 0 0 0

82:08:30 0000: Z 0030009000. 00305: 0 00 .:0Q 0 :0 :52 0.000.002

 

.20 0”: 0000 20 a 23.8000

2:030 0:0 _0:0_0:0>:00 88,0 000000000: :00 :o 0000 0:0 0E05000 .020 .00 0000000 05800000 8 00:0_._0> .00 00000—050 0am 030,—.

110

Table 3.8 Relationships among methane oxidation, carbon dioxide and other soil

properties in conventional and organic treatments at the KBS LFL site. Values are

Pearson correlation coefﬁcients (r). n = 2 management systems x 3 crops x 4 replicates x

15 sample dates.

 

 

C02 Moisture Temperature Nitrate Ammonium
CH4 0.053 -0. 152* 0.083 -0.034 0.011
C02 1.000 -0.094 0.41 1* -0.201* 0.090

 

* Correlation is signiﬁcant at the 0.01 level (2-tailed)

111

Table 3.9 Differences in average soil methane oxidation rates and other properties among
soil cores from conventional and organic systems at the KBS LFL site. Methane ﬂuxes
were measured in the laboratory; values are means i 1 SE for 4 replications x 3 crops x 4

depths; signiﬁcance values are based on t tests.

 

 

Factor Conventional Agriculture Organic Agriculture t-testa
CH4 (11g CH4-C kg soil'] h") 0.049 a: 0.007 0.051 0 0.008 ns
Moisture (g H20-100g soil") 10.5 a: 0.3 11.5 0 0.4 *
Nitrate (pg N g soil") 5.07 0 0.59 5.50 0 0.69 ns
Ammonium (11g N g soil") 3.85 0 0.23 4.21 0 0.22 ns
Nitriﬁcation (ug N kg soil" h") 0.20 0 0.02 0.31 0 0.03 **

 

3 ns not signiﬁcant (P>0.1); * P<0.05; **P<0.01.

112

 

 

 

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113

Table 3.11 Analysis of covariance for methane oxidation in laboratory soil cores as

affected by management, crops, soil depth, and soil properties at the KBS LFL site.

 

 

Source SS df MS F P
Crop 1.0x10r 2 5.3x10'6 0.5 0.600
Management 9.8x10'6 1 9.8x10'6 1.0 0.331
Depth 4.5x10“‘ 3 1.5x10“ 14.5 0.000
Crop x Management 5.6x10’5 2 2.010“5 2.7 0.072
Crop x Depth 1.1x10'4 6 1.9rt10'5 1.8 0.111
Management x Depth 2.1x10'5 3 7.0xlO'6 0.7 0.564
Crop x Management x Depth 1.0x104 6 1.7)(10'S 1.7 0.139
Nitrate 2.0x10'5 1 2.0x10'5 2.0 0.163
Ammonium 1.4x10'5 1 1.4x10'S 1.4 0.243
Moisture 3.2x10'5 1 3.2x10'5 3.1 0.082
Nitriﬁcation 1.1x10’5 1 1.1x10'5 1.1 0.301
Error 7.011104 68 1.0x10'5

Total 51] 96

Corrected Total 2.4x10'3 95

 

114

 

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115

Table 3.13 The relationships among methane oxidation and other soil properties in soil
cores from different depths at the KBS LFL site. Values are Pearson correlation

coefﬁcients (r). n = 2 management systems x 3 crops x 4 replicates.

 

Moisture Nitrate Ammonium Nitriﬁcation

 

CH4 -O.230* —0.351** -0.454** -O.505**

 

* Correlation is signiﬁcant at the 0.05 level (2-tailed)

** Correlation is signiﬁcant at the 0.01 level (2-tailed)

116

 

 

 

 

 

 

 

 

   

 

 

 

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Conventional

Organic

Figure 3.1 Net methane oxidation of different crops in conventional and organic
agriculture at the KBS LFL site. Vertical bars represent standard errors (n = 4 replicate
plots x 13 sample dates). Different higher and lower case letters represent signiﬁcant

differences (P<0.05) of crops within management systems and between management
systems respectively.

160

 

D corn Aa
140 ' Elsoybean

I wheat
120 —

100 a

80*

 

 

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mg C02-C m'2 h‘1
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Conventional Organic

Figure 3.2 Net soil carbon dioxide ﬂuxes of different crops in conventional and organic
agriculture at the KBS LFL site. Vertical bars represent standard error (n = 4 replicate
plots x 13 sample dates). Different higher and lower case letters represent signiﬁcant

differences (P<0.05) of crops within management systems and between management
systems respectively.

117

 

 

 

 

 

 

 

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Figure 3.3 Average daily soil methane oxidation under different crops from conventional
(top) and organic management systems (bottom) at the KBS LFL site in 2001-2002.
Vertical bars represent standard errors (n = 4 replicate plots).

118

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+-' > o r: h h > c —
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Figure 3.4 Average daily soil carbon dioxide emissions under different crops from

conventional (top) and organic management systems (bottom) at the KBS LF L site in
2001-2002. Vertical bars represent standard errors (n = 4 replicate plots).

119

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Figure 3.5 Average daily soil moisture in different crops under conventional (top) and

organic management systems (bottom) at the KBS LF L site in 2001-2002. Vertical bars
represent standard errors (n = 4 replicate plots).

120

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Figure 3.6 Average daily soil temperature under different crops in conventional (top) and

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121

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122

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123

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129

Chapter 4
Methane Oxidation in Mature and Successional Forests:

Effects of Nitrogen Deposition

Introduction

Atmospheric nitrogen deposition from air pollution has increased substantially
with the industrial revolution and urbanization (Holland et al. 1999). This extra input of
N into soil can alter both natural terrestrial and aquatic ecosystems (Holland et al. 1997).
Results from several studies in which high rates of added N depresses CH4 oxidation in
soil suggest that anthropogenic N inputs might decrease the soil sink for atmospheric CH4
in forests (Castro et al. 1992, Klemedtsson and Klemedtsson 1997, Saari et al. 1997,
Butterbach-Bahl et al. 1998, Sitaula et al. 2001, Butterbach-Bahl et al. 2002, Wang and
Ineson 2003). Circumstantial evidence also suggests a link. For example, a high latitude
coniferous forest in the U.K. receives more N deposition and has lower rates of soil CH4
oxidation than a lower latitude forest (MacDonald et al. 1997). Low N deposition
coniferous soils in Finland have CH4 uptake rates three times higher than high N
deposition Dutch forest soils (Saari et al. 1997).

However, not all studies support this linkage. Steinkamp et al. (2001) found
identical CH4 oxidation rates between fertilized and unfertilized German Black forest
soils, and Bradford et al. (2001) found that the chronic deposition of nitric acid and
ammonium sulphate had no signiﬁcant effect on soil CH4 uptake in deciduous woodland

in southwest England. Additionally, Borjesson and Nohrstedt (2000) found only transient

130

inhibition of CH; oxidation after N fertilization in Swedish forest soils during two years
of investigation.

There are also conﬂicting results for the effects of ammonium vs. nitrate on forest
soil CH4 consumption (Schnell and King 1994). The different patterns of reduction can
be explained partly by differences in the associated counter-ions of the ammonium or
nitrate salts (King and Schnell 1998).

A problem with many of the studies that have directly tested the effect of added N
on CH4 oxidation is that N-levels have been much higher than the levels added in acid
deposition, ca. 5-10 kg N ha'1 y", and usually have been added for a much shorter period
(ca. 1-3 years). The present study was designed to assess the impact of N deposition on
soil CH4 consumption in different types of temperate forest with N added at rates close to

those added by deposition, and for longer periods.

Methodology
Site Description

The study was conducted at the Long-Term Ecological Research (LTER) sites at
the WK. Kellogg Biological Station (KBS), Hickory Comers, Michigan (42° 24’N, 85°
24’W, elevation 288 m). Annual rainfall at KBS averages 890 mm y'1 with about half
falling as snow; potential evapotranspiration (PET) exceeds precipitation for about 4
months of the year. Mean annual temperature is 9.7 0C.

I measured methane ﬂuxes at three sites along a management intensity gradient:
mature deciduous forests (DF), 40-60 years old conifer plantations (CF), and mid-

successional communities (SF) abandoned from agriculture 40-60 years ago (see details

131

at http://lter.kbs.msu.edu). All sites were replicated within the larger landscape (n=3
locations) and were on the same Kalamazoo/Oshtemo soil series (Austin 1979). The soils
at these sites are Typic Hapludalfs (ﬁne or coarse-loamy, mixed, mesic soils) derived
from glacial till about 12,000 years ago (Crum and Collins 2003).

The major plants in the deciduous forest sites are red maple (Acer rubrum L.),
sugar maple (Acer saccharum Marsh), white oak (Quercus alba L.), northern red oak
(Quercus rubra L.), ﬂowering dogwood (Cornusﬂorida L.), and sassafras (Sassafras
albidum (Nutt.) Nees). The prevailing plants in coniferous forests are red pine (Pinus
resinosa Aiton), white pine (Pinus strobus L.), pi gnut hickory (Carya glabra (Miller)
Sweet), Norway spruce (Picea abies (L.) Karsten), wild black cherry (Prunus serotina
Ehrh.), and big-toothed aspen (Populus grandidentata Michx.).

The dominant plants in the mid-successional communities are Canada goldenrod
(Solidago canadensis L.), quackgrass (Elytrigia repens (L.) Nevski), timothy (Phleum
pratense L.), white hearth aster (Aster pilosus Willd.), Kentucky bluegrass (Poa pratensis
L.), common yarrow (Achillea millefolium L.), Canada bluegrass (Poa compressa L.),
autumn olive (Elaeagnus umbellata Thunb.), sassafras (Sassafras albidum (Nutt.) Nees),
gray goldenrod (Solidago nemoralis Ait.), smooth brome (Bromus inermis Leyss.),
germander speedwell (Veronica chamaedrys L.), orchardgrass (Dactylis glomerata L.),
ﬂowering spurge (Euphorbia corollata L.), and honeysuckle (Lonicera spp.).

The fertilized plots were establish in 1993, nine years prior to the start of this
study. Two levels of ammonium nitrate (NH4NO3) are applied annually in three equal
aliquots per year during April to November. At each replicated site, a 10 x 10 m fertilized

(F) plot receives 10 kg N ha'1 y'l and a 2 x 2 m highly fertilized (HF) plot receives 30 kg

132

N ha‘1 y". Average N deposition in this area is ca. 10 kg N ha'1 y"(National Atmospheric
Deposition Program website http://nadp.sws.uiuc.edu/). During the 2002 study year, the

fertilized dates were March 28, June 27, and September 17. From a 20 x 20 cm microplot
in the highly fertilized plot, forest litter was regularly remove from soil surface beginning

in May.

In Situ Gas Sampling

I measured in situ methane oxidation rates using the static chamber technique
(Hutchinson and Livingston 1993). Static chambers were fashioned from 25 cm diameter
PVC pipe: bases (25 cm diameter x 10 cm high) were installed in each plot and left in
place except during agronomic operations. Immediately prior to sampling, a 4.5 cm high
cap was placed on each base and sealed with a latex skirt wrapped with an elastic band.
At lO-minute intervals, four 10 mL headspace samples were removed through a rubber
septa in each cap using a syringe and put into 3 mL glass sample vials pre-ﬂushed with
headspace air. Within 3 days vial contents were measured for CH4 using a gas
chromatograph (GC 5890 Series II, Hewlett Packard) equipped with a ﬂame ionization
detector (FID), and for C02 using an infrared gas absorption (IRGA) analyzer (EGA C02
Analyzer, Analytical Development CO. LTD. Hoddesdon, England).

Chambers were sampled twice per month during the growing season from late

May to late September. All chambers were sampled on the same day.

Soil Analyses

Soil temperature was measured at 0-5 cm depth using a temperature probe. Soil

133

samples for other analysis were taken from the top 10 cm of soil using a 2.5 cm diameter
soil probe. Fresh soils were passed through a 4 mm sieve and mixed by hand, and then
subsamples were taken for moisture content and mineral N analyses. Prior to analysis,
soils were stored in a refrigerator at 4°C for up to 4 weeks. Soil moisture content was
measured gravimetrically by drying the soil samples at 65°C for 3 days or until dry.
Further drying at 105°C removes an additional 0.8 g H20 100g soil"1 moisture in these
soils. Mineral N measurements were obtained by extracting 20 g of dry soil with 100 mL
1 M KCl for 24 h and then ﬁltering the extract through 2-um pore size ﬁberglass ﬁlters.
The ﬁltrates were frozen prior to analysis for NIH“ and N03', which was performed using
an Alpkem continuous ﬂow analyzer (Alpkem 3550, 01 Analytical, College Station, TX)

(Bundy and Meisinger 1994).

Soil Depth Study

Soil cores (5 cm diameter x 30 cm deep) were taken from the coniferous forest,
mature deciduous forest, mid-successional communities, and no-tilled agricultural ﬁelds
and stored intact at 4°C for one week, after which they were then separated into four
different depth increments (0-5, 5-10, 10-20, and 20—30 cm). Each soil increment was
transferred to a 473 mL Mason jar for 0-5 cm and 5-10 cm increments and to a 946 mL
Mason jar for 10-20 cm and 20-30 increments; jars were left open and allowed to pre-
incubate with atmospheric methane at room temperature for one week in order to reduce
soil moisture levels to ca. 15% w/w. At the end of this period, methane oxidation was
measured by sealing each jar with a cap and removing 0.5 mL of headspace at four 10-

minute intervals. The headspace aliquot was immediately injected into the gas

134

chromatograph for methane analysis. After the CH4 oxidation study, soil samples were

sieved and separated for soil moisture, soil inorganic N content, and nitriﬁcation assays.

Nitriﬁcation Assay
A sieved soil sample of 10 g was placed in a 100 mL Erlenmeyer ﬂask and
combined with 50 mL of the phosphate buffer solution and ammonium sulfate (Hart et al.
1994), and sealed with Paraﬁlm. The ﬂasks were placed on a shaker table and shaken for
24 h. Soil slurry samples of 10 mL were taken before and another after 24 h incubation,
centrifuged for 30 min and ﬁltered through 0.2 pm ﬁlter paper. The aliquots were frozen
until analysis of nitrate as above. Rates of nitrate production were calculated on the basis

of the difference between the initial and ﬁnal nitrate concentrations.

Statistic Analyses
I used Proc Mixed of SAS version 8.0 (SAS Institute 1999) for the analysis of
variance (ANOVA) and analysis of covariance (ANCOVA) for ﬁeld data, in which I
treated site and treatment as ﬁxed effects and day and site x treatment x day as random
effects. I also used SPSS version l0.0.l (SPSS Inc. 2001) for ANOVA and ANCOVA for
the soil depth data, and also for correlation analyses for both ﬁeld and laboratory
samples. Methane and C02 data were natural log transformed before ANOVA and

ANCOVA to homogenize variances. I used untransformed data for correlation analysis.

Results

Field study

135

Methane Oxidation

Soil CH4 consumption was signiﬁcantly different among sites (Table 4.2). Among
control plots, CH4 uptake was highest in deciduous forest at 44.1 i 3.9 ug CH4-C m'2 h'1
(is.e, n=3 replicate sites x 9 sample dates) and lowest in mid-successional communities
at 23.1 i1.5 pg CH4-C m'2 h", while the coniferous forest was intermediate (Table 4.3).

Added nitrogen had a signiﬁcant effect on soil CH4 oxidation (Table 4.2); the site
x treatment and the site x treatment x day interactions were also signiﬁcant: nitrogen
additions signiﬁcantly reduced CH4 oxidation rates only in the coniferous forest sites,
where average rates declined from 28.7 i 3.6 to 16.4 i 2.3 ug CH4-C m'2 h'1 N with low
fertilizer addition and to 11.1 i 2.6 ug CH4-C m’2 h'1 with high N addition (Table 4.3 and
Figure 4.1). Removing the forest ﬂoor of the high N coniferous forest plots further
lowered oxidation rates to 6.2 i 1.8 ug CH4-C rn'2 h". In contrast, only forest ﬂoor
removal signiﬁcantly affected CH4 oxidation rates in the deciduous forest sites, where
rates declined from 44.1 :1: 3.9 to 23.7 i 5.8 pg CH4-C m’2 h'1 following the removal. In
the mid-successional communities oxidation rates were likewise unaffected by N—
fertilizer addition.

Sampling date also had no effect on CH4 oxidation rates (Table 4.2). Uptake rates
varied very little among sample dates in the mid-successional communities; rates in
control plots were between 20 and 35 ug CH4-C rn'2 h’1 for all 9 dates (Figure 4.2c). In
deciduous forest control plots, CH4 oxidation rates ranged from 17 to 59 pg CH4-C m'2
h'l, with the higher rates between June and September (Figure 4.2b). In the coniferous
forest, CH4 uptake was high only in August (to 52 ug CH4—C m'2 h'l) (Figure 4.2a);

otherwise control plot rates were similar to those in the mid-successional sites.

136

Carbon Dioxide Fluxes

C02 production rates were also signiﬁcantly different among sites and the site x
treatment interaction was signiﬁcant (Table 4.4). Among control plots, C02 ﬂux was
highest in mid-successional communities at 127 iIO mg C02—C m'2 h'lfollowed by
coniferous forest at 109 i 11 mg C02-C m'2 h"and then deciduous forest (70 i 7 mg
C02-C m‘2 h" (Table 4.3).

Added nitrogen had a signiﬁcant effect on C0; emissions (Table 4.4). The high N
additions signiﬁcantly decreased C02 ﬂuxes from 109 i 11 to 80 i 8 mg C02-C m'2 h" in
coniferous forest. In contrast, high N-additions signiﬁcantly increased C02 emissions
from 70 i 7 to 101 i 8 mg C02-C rn'2 h'1 in deciduous forest (Table 4.3 and Figure 4.3).
Forest ﬂoor removal signiﬁcantly decreased C02 ﬂuxes only in the coniferous forest, by
40% (Table 4.2).

Carbon dioxide ﬂuxes also varied signiﬁcantly bysample dates (Table 4.4). The
emission rates in coniferous forests and mid-successional communities reached their
highest levels (approximately 180 mg C02-C rn’2 h") in early August. The peak occurred
two weeks earlier in deciduous forest — in late July emissions were around 188 mg C02-C

m'2 h'1 (Figure 4.4).

Soil Properties
Soil moisture signiﬁcantly differed only among sites (Table 4.5). For example, in

control plots, average soil moisture was highest in mid-successional communities (18.6 i

0.7 g H20-100 g soil"), followed by deciduous forest (9.4 i 0.9 g H20- 100 g soil") and

137

coniferous forests (8.9 i 0.7 g H20- 100 g soil") (Table 4.3). Treatments did not affect
soil moisture, which varied (P=0.069) by sample dates (Table 4.3, 4.5 and Figure 4.5).

Soil temperature was slightly different among sites (P=0.044) (Table 4.5). Mid-
successional control soils were on average about a degree warmer (18.6 0C) than the
others (18.3 vs. 17.3 and 17.1 °C; Table 4.3) (Table 4.2). N addition did not signiﬁcantly
affect soil temperature, but soil temperature signiﬁcantly varied with sample dates (Table
4.5). Temperatures were highest in mid June (24.0 i 3.1 °C, n=3 replications) and started
to decline at the end of August (Figure 4.6).

Soil nitrate signiﬁcantly differed by site (Table 4.5). Among control plots, soil
nitrate was highest in coniferous forest (5.4 i 0.6 pg N g soil") followed by deciduous
forest (3.4 i 0.6 pg N g soil'l), and mid-successional communities (0.7 i: 0.2 pg N g
soil'l), respectively (Table 4.2). Although nitrate did not signiﬁcantly differ by sampling
dates (Table 4.5) it increased substantially after fertilization (Figure 4.7). The increases
following fertilization were highest in deciduous forest soils, intermediate in mid
successional communities, and lowest in coniferous forest. It was highest in mid-July and
climbing up at the mid-September (Figure 4.7b).

Soil ammonium was also signiﬁcantly different among sites (Table 4.5). Both
deciduous (15.7 i 2.8 pg N g soil") and mid-successional communities (15.4 i 2.8 pg N
g soil") had higher soil ammonium levels than those of coniferous forest (12.1 :t 1.6 pg
N g soil"; Table 4.2 and Figure 4.9). N addition had no signiﬁcant effect on soil
ammonium (Table 4.5). However, ammonium levels changed signiﬁcantly changed
seasonally (Table 4.5). Soil ammonium in all treatments, including control, began to

increase from the end of May until reaching the highest point at the end of June after the

138

second fertilization, and then declined steadily for the rest of the sampling period even

after the third fertilization (Figure 4.8).

Soil Depth Proﬁles

As for in situ ﬂuxes, overall soil CH4 oxidation rates in laboratory-incubated
cores were signiﬁcantly different among sites (Table 4.8). Deciduous forest soils had the
highest rates of oxidation at 0.49 :t 0.039 (s.e., n=6 soil cores x 4 depth x 2 treatments) pg
CH4-C kg dry soil"1 h'1 followed by mid-successional communities (0.20 i 0.018 pg CH4—
C kg dry soil" h'l), and coniferous forest (0. 17 i 0.024 pg CH4-C kg dry soil'1 h'l).
Methane uptake did not much differ among soil depths except in deciduous forest soils,
where consumption at the 5-10 cm depth was 28-5 8% higher than consumption at 0-5
cm, 10-20 cm, and 20-30 cm depth (Table 4.7 and Figure 4.9).

Fertilizer application (10 kg N ha“) in the forested sites also did not much affect
soil CH4 consumption within each site (Tables 4.7 and 4.8).

Soil moisture in cores was signiﬁcantly different among sites, treatments, and soil
depths (Table 4.9). Surface soils had the highest soil moisture when compared with lower
level soil proﬁles (Table 4.7 and Figure 4.10). Moisture decreased up to 53% at 20-30 cm
depth comparing to surface soils in deciduous fertilized soils.

Nitrate signiﬁcantly differed by site and treatment but not by depth (Table 4.9).
Although not signiﬁcant, soil nitrate was generally highest in surface soils rather than
deeper soils (Figure 4.11). For example, nitrate was 87% lower in surface soils than

deeper layers in coniferous control plots (Table 4.7).

139

In contrast, soil ammonium signiﬁcantly differed only among soil depths and not
among sites or treatments (Table 4.9). Ammonium was highest in surface soils then
signiﬁcantly decreased with depths up to 88% in soils from deciduous control plots
(Table 4.7 and Figure 4.12).

Soil nitriﬁcation also varied signiﬁcantly among sites, depth and nearly differed
among treatments (P=0.067; Table 4.9). Nitriﬁcation was lowest in mid-successional
communities soils (Table 4.7). Nitriﬁcation also signiﬁcantly decreased with soil depth,

up to 95% in cores from coniferous control plots (Figure 4.13).

Controls on Methane and Carbon Dioxide Fluxes

From analysis of covariance of ﬁeld data, none of the physical and chemical soil
properties measured signiﬁcantly affected CH4 uptake in either in situ or in soil core
incubation (Table 4.2 and 4.8). There were also no signiﬁcant correlations between CH4
ﬂux and soil properties in the ﬁeld (Tables 4.6). Methane uptake was weakly correlated
with nitrate (r = -0.l62) and ammonium (r = 0.152; Table 4.10). Methane and C02 ﬂuxes
were not signiﬁcantly correlated (Table 4.6).

Soil moisture nearly affected (P=0.063) in situ C02 ﬂuxes (Table 4.4) although
moisture and C02 ﬂux were not signiﬁcantly correlated (Table 4.6). Only nitrate was

signiﬁcantly but weakly correlated with C02 (r = -0.144) (Table 4.6).

Discussion
Field study

Methane Oxidation

140

As in an earlier study (Chapter 2), soil CH4 oxidation was signiﬁcantly different
among study sites. In control plots, CH4 uptake was highest in mature deciduous forest
followed by rates in coniferous forests and mid-successional communities, which were
similar (Table 4.3 and Figure 4.1) Deciduous forest soils had lower soil bulk density than
both coniferous and mid-successional communities (Table 4.1), which may have allowed
faster CH4 diffusion into the soil. Deciduous forest soils also had more total carbon, total
nitrogen, and higher ammonium levels than coniferous forest soils. The higher soil
nitrate in coniferous forests and higher soil ammonium in mid-successional communities
might explain their lower CH4 uptake. In contrast, deciduous forest soil had more acidity
than coniferous forests and mid-successional communities. Acidity is thought to suppress
soil CH4 consumption but this did not appear to be true in this study.

N fertilizer inhibited soil CH4 oxidation only in coniferous forest sites, where
rates declined from 28.7 i 3.6 to 16.4 i 2.3 pg CH4-C rn'2 h"1 with low fertilizer addition
and to 11.1 i 2.6 pg CH4-C m"2 h'1 with high N addition (Table 4.3). This suggests that
soil CH4 oxidation in mid-successional and mature native communities is unaffected by
up to 3 times the current levels of N deposition in this area. CH4 oxidation in planted
coniferous stands, on the other hand, appears suppressible by small N additions.

Soil nitrate slightly increased following N—fertilization in all sites (Figure 4.7),
but relatively low levels, especially in the coniferous forest and mid-successional
communities during the growing season, suggests strong biological sinks for nitrogen.
Levels added appeared not to create excess soil N, except in the deciduous forest where

fertilized plots had more N than control plots for most of the year.

141

Forest ﬂoor removal signiﬁcantly reduced CH4 uptake in both coniferous and
deciduous forests. This suggests that the litter layer is either a habitat for CH4 oxidation
or affects soil conditions in underlying layers. Perhaps the high C:N forest ﬂoor material
immobilizes inorganic N and its removal allows N to increase in the mineral soil, thereby
suppressing oxidation. Yavitt et al. (1993) also found a slight reduction of CH4
consumption aﬁer removing roots and leaf litter from the soil surface in a northern
hardwood forest in the Adirondack region of New York. These results also indicate that
leaf litter in these sites does not contain a CH4 oxidation inhibitor as found in some soil

surface extracts from boreal and temperate forest (Amaral and Knowles 1997).

Carbon dioxide

As for CH4 oxidation, C02 emissions were also signiﬁcantly different among
sites. Overall C02 production was highest in mid-successional communities, somewhat
lower in coniferous forests, and much lower in deciduous forests. This might be caused
by higher soil moisture in the mid-successional communities, which would have favored
microbial activity. Although soil ammonium was a fairly good predictor of C02 emission,
it was not different among sites.

Nitrogen fertilizer addition signiﬁcantly affected C02 emissions in coniferous and
deciduous forests. Fertilizer signiﬁcantly increased soil microbial activity. Forest ﬂoor
removal only signiﬁcantly decreased C02 emission in the high N fertilization coniferous
forest plots. This suggests that the forest ﬂoor was providing carbon and nutrients to soil

microorganisms in lower horizons in these plots.

142

CH4 oxidation with Soil Depth

In laboratory incubations, deciduous forest soil cores had the highest methane
oxidation rates, followed by mid-successional communities, and coniferous forests.
Although statistical analysis showed no detectable impact of soil physical and chemical
properties on CH4 uptake in soil cores, mid-successional communities had lower soil
nitrate and nitriﬁcation rates and less CH4 consumption than deciduous forest soil.

As in the ﬁeld study, fertilizer treatment (10 kg N ha") did not affect soil CH4
consumption in soil cores. Fertilizer increased soil nitrate in coniferous and deciduous
forests but not in the mid-successional communities. Plants and bacteria in mid-
successional communities soil appear to have a high ability to deplete nitrate after the
fertilization (see Figure 4.8). Soil ammonium increased slightly in fertilized plots of
coniferous and deciduous forests but, again, not in mid-successional communities. The
fertilized plots also had a higher nitriﬁcation rate than control plots, especially in
deciduous forest.

Soil CH4 consumption also did not signiﬁcantly differ among soil depths, except
in deciduous forest. There was a slight subsurface maximum at 5- 10 cm depth in
deciduous forest soil cores. CH4 oxidation in soil below 5 cm depth was slightly higher
than in 0-5 cm for mid-successional communities. Soil moisture also slightly decreased
with soil depth, especially in deciduous forest soil cores. Soil nitrate was highest at 5-20
cm depth of coniferous forest, deciduous forest, and no-till cornﬁeld but rarely found at
depths lower than 5 cm in mid-successional communities. Only soil ammonium showed a
signiﬁcant decrease along soil proﬁles of all study sites. Soil nitriﬁcation was also higher

near the surface.

143

Nitriﬁcation seems to be a gOOd indicator for soil CH4 uptake inhibition. Hﬁtsch
(2001) found a very strong negative correlation (r = -O.92) between CH4 oxidation and
nitriﬁcation from long-term fertilization treatments of the ﬁeld experiment "Ewiger
Roggenbau" at Halle, Germany. The use of nitriﬁcation inhibitors has also increased CH4
oxidation by 28% (W eiske et al. 2001). Kravchenko (2002) recently showed that
ammonium inhibition in peat soils was not a competitive mechanism but rather caused by
toxic substances from nitriﬁers. This suggests that nitriﬁers are not responsible for
methane oxidation as has been suggested in the past (see Hﬁtsch 2001), but rather than
inhibit methanotrophs by producing nitrite. This notion is consistent with our results,

which showed no relationships between nitriﬁcation and CH4 oxidation.

Conclusion

Low N deposition (10 kg N ha'l) had no detectable effect on CH4 ﬂuxes in all
sites while high N deposition (30 kg N ha!) signiﬁcantly reduced CH4 oxidation only in
coniferous forest. N deposition also signiﬁcantly increased C02 emission in deciduous
forest while in contrast, only high N deposition signiﬁcantly reduced C02 ﬂuxes in
coniferous forest. Forest ﬂoor removal also signiﬁcantly decreased CH4 oxidation in both
coniferous and deciduous forests while C02 emission was signiﬁcantly reduced only in
coniferous forest by litter removal. Methane oxidation signiﬁcantly differed among soil
depths only in deciduous forest. The highest oxidation rate was in 5-10 cm soil depth.
Soil factors had no signiﬁcant effect on CH4 uptake. Only soil moisture signiﬁcantly

associated with C02 ﬂuxes.

144

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147

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148

Table 4.2 Analysis of covariance to determine effects of site, treatment, and soil

properties on methane oxidation rates in mature deciduous forest, coniferous forest, and

mid-successional communities at the KBS LTER site.

 

 

Variable Num. df a Den. (if a F P
Site 2 276 32.5 <0.0001
Treatment 3 276 19.6 <0.0001
Site x Treatment 5 276 2.6 0.024
Day 8 276 0.7b 0.235
Site x Treatment x Day 39 276 -3.3b 0.001
Moisture 1 276 0.9 0.334
Temperature 1 276 0. 1 0.762
Nitrate l 222 0.6 0.424
Ammonium 1 222 1.6 0.205

 

a Num. (if and Den. df refer to numerator and denominator degrees of freedom,

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b Z value

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Table 4.4 Analysis of covariance to determine effects of site, treatment, and soil
properties on carbon dioxide emissions in mature deciduous forests, coniferous forests,

and mid-successional communities at the KBS LTER site.

 

 

Variable Num. df a Den. df a F P
Site 2 276 13.3 <0.0001
Treatment 3 276 3.4 0.018
Site x Treatment 5 276 4.5 0.001
Day 8 276 1.8b 0.036
Site x Treatment x Day 39 276 0.9b 0.386
Moisture 1 276 3.5 0.063
Temperature 1 276 l .4 0.233
Nitrate l 222 0.1 0.698
Ammonium 1 222 3.0 0.083

 

a Num. df and Den. df refer to numerator and denominator degrees of freedom,
respectively

b Z value

151

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152

Table 4.6 The relationships among methane oxidation, carbon dioxide and other soil

properties in coniferous forest, mature deciduous forest, and mid-successional

communities at the KBS LTER site. Values are Pearson correlation coefﬁcients (r); n= 3

replicate plots x 9 sampling dates x 4 treatments.

 

 

C02 Moisture Temperature Nitrate Ammonium
CH4 0.027 0.005 -0.089 -0.042 0.062
C02 1.000 0.095 0.113 -0. 144* 0.002

 

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154

Table 4.8 Analysis of covariance for methane oxidation as affected by site, fertilization,
and soil depth on soil properties from coniferous forest, mature deciduous forest, mid-

successional communities, and no-till agricultural ﬁelds at the KBS LTER site.

 

 

Source SS df MS F P

Site 3.2x10'2 3 1.0x10'2 37.3 0.000
Treatment 1.2x10‘1 1 1.2-<10“4 0.43 0.513
Depth 1.8x10'3 3 6.2-t10'4 2.18 0.092
Site x Treatment 7.0x10“4 2 3.5)(104 1.25 0.291
Site x Depth 7.5x10'3 9 8.4-r10“4 2.95 0.003
Treatment x Depth 5.3x10'5 3 1.8x10'5 0.06 0.979

Site x Treatment x Depth 1.3x10'3 6 2.2x10“1 0.77 0.593

Nitrate 2.3x10‘5 1 2.3x10'5 0.08 0.777
Ammonium 2.7x10“‘ 1 2.7x10‘4 0.96 0.329
Moisture 5.5x10'6 1 5.5-(10" 0.02 0.889
Nitrification 2.1x10‘4 1 2.1x10’4 0.74 0.390
Error 3.9x10'2 136 2.8x10‘4

Tota1 910 168

Corrected Total 8.9)(10'2 167

 

155

 

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Table 4.10 The relationship among methane oxidation and other soil properties from the
soil depth study in coniferous forest, mature deciduous forest, mid-successional
communities, and no-till agricultural ﬁelds at the KBS LTER site. Values are Pearson

correlation coefﬁcients (r); n = 6 soil cores x 4 sites x 2 treatments.

 

Moisture Nitrate Ammonium Nitriﬁcation

 

CH4 -0.044 -0.162* 0.152* -0.032

 

* Correlation is signiﬁcant at the 0.05 level (2-tailed)

** Correlation is signiﬁcant at the 0.01 level (2-tailed)

157

60

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A El Control
a
50 ~ Aa Aa ClLow N
I High N
,. I Hi h N w/o Floor
-; 40 9
‘1'
E Aa
o Aa Aa
'e 30 J Aa
I
Q 1'
g: 20 ~
10 ~
0 a .
Coniferous Forest Deciduous Forest Mid-successional
Communities

Figure 4.1 The effects of different N fertilizer levels on net methane oxidation in
coniferous forest, deciduous forest, and mid-successional communities at the KBS LTER
site. Bars are mean seasonal rates; vertical lines indicate standard errors (n= 3 replicate
plots x 9 sampling dates). The different higher and lower case letters indicate signiﬁcant

differences (P>0.05) of the same treatment among sites, and between control and the

treatment within the site, respectively.

158

160

140 a. Coniferous Forest —+-Control
- - C} - -Low N
120 r — -A — High N
100 — ->( — High N w/o Floor

 

 

pg CH4-C m'2 h'1

 

 

 

 

160
140 . b. Deciduous Forest
'7; 120 «
N
'E 100 .

 

 

 

 

340

   

 

160
140 -
120 .
100 .
80 ~
60
40 4
20 1

O . r
31 -May-02 30-Jun-02 31 -Jul-02 31 -Aug-02

 

c. Mid-successional Communities

pg CH4-C rn‘2 h'1

 

 

 

 

Figure 4.2 The effects of different N fertilizer levels on net daily methane oxidation in
coniferous forest (top), deciduous forest (middle), and mid-successional communities

(bottom) at the KBS LTER site. Vertical lines indicate standard errors (n= 3 replicate
plots).

159

160

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

EIControl
140 g D LOW N
IHigh N
120 , Ba IHigh N w/o Floor
3. L Aa
f 100 -
E
9 80 « Ba
9: T
a, 60 T
E
40 -
20 ~
0 r a
Coniferous Forest Deciduous Forest Mid-successional
Communities

Figure 4.3 The effects of different N fertilizer levels on net carbon dioxide emission in
coniferous forest, deciduous forest, and mid-successional communities at the KBS LTER
site. Bars are mean seasonal rates; vertical lines indicate standard errors (n= 3 replicate
plots x 9 sampling dates). The different higher and lower case letters indicate signiﬁcant
differences (P<0.05) of the same treatment among sites, and between control and the

treatment within the site, respectively.

160

300

 

 

 

 

 

 

 

 

 

 

 

 

a. Coniferous Forest
,_ 250m
'5 +Control
‘7‘ 200 --D--LowN
E . '.
o —-A-nghN ,.
| 150 *1 - a"
(5' —-x-—ngth/o Floor 2 ’ , n
0 100 ‘l ’, ."':” -\\‘.'§- -
U) . _,‘a—",_ ‘.o"’ v \
E 50 i ”33‘ ."'" \N. ..,..<,’ ______ x'/ \" ‘\.'
0
300 .
b. Dec1duous Forest
250 -
it:
“3 200
E
(a; 150 « ,z’ ‘\
.I~ ’ \
8 100 L- - ”I 1K .-
a- --.. , -’ e. 4’. ............ . a: .. ,
E - ' """"""" '- a
50 ‘ 9.3 ‘
O
300
250 c. Mid-successional Communities ‘
1t:
., 200~ . -—-"'.'..
E ’z’.' . ‘\
0 150 x \X
l . / o ‘-
6 .\\ ‘ E’,’ ' \ '-
0 -I’T'. ‘ ‘A ’
O) 100 g? "_.- , - .7 \
E :1" . ' =-
50 '1'
O . . .
31 -May-02 30-Jun-02 31 -Jul-02 31 -Aug-02

Figure 4.4 The effects of different N fertilizer levels on net daily carbon dioxide emission
in coniferous forest (top), deciduous forest (middle), and mid-successional communities
(bottom) at the KBS LTER site. Vertical lines indicate standard errors (n= 3 replicate

plots).

161

 

45
40 -

9 H20 1009 soir1

 

a. Coniferous Forest

 

+Control
- - D- - -Low N
-¢—W®N

 

 

45
40
35
30
25
20
15
10

9 H20 1009 soir1

45
40
35

25
20
15
10
5
O

9 H20 1009 soir1

31-May-02

30 ~<

 

b. Deciduous Forest

 

 

 

.4

 

c. Mid-successional Communities

 

 

l

30-Jun-02

l

31-Jul-02 31-Aug-02

Figure 4.5 The effects of different N fertilizer levels on daily soil moisture in coniferous
forest (top), deciduous forest (middle), and mid-successional communities (bottom) at the
KBS LTER site. Vertical lines indicate standard errors (n= 3 replicate plots).

162

 

 

10 —O—- Control

. - - D - -Low N
5 a. Coniferous Forest _ .A _ High N

 

 

 

 

 

5 b. Deciduous Forest

 

 

 

30
254
20 .
15 . '
10

 

°C

 

5 ‘ c. Mid-successional Communities
0 T
31 -May-02 30-Jun-02 31 ~Jul-02 31 -Aug-02

 

 

 

Figure 4.6 The effects of different N fertilizer levels on daily soil temperature in
coniferous forest (top), deciduous forest (middle), and mid-successional communities
(bottom) at the KBS LTER site. Vertical lines indicate standard errors (n= 3 replicate
plots).

163

 

3O , a. Coniferous Forest +Control
- - D - -Low N
25 — ~A — High N

 

 

 

 

 

 

 

30 , b. Deciduous Forest

7.6 25 11
m 20 "
a l
.2 5’“
5’15

2

U)

1

 

 

 

 

 

35

 

c. Mid-successional Communities
30 ~

25
20

15

pg NO3‘N 9 soil'1

10

/
5 , \

 

 

 

0 I .
31-May-02 30-Jun-02 31-Jul-O2 31-Aug-02

 

 

Figure 4.7 The effects of different N fertilizer levels on daily soil nitrate in coniferous

forest (top), deciduous forest (middle), and mid-successional communities (bottom) at the
KBS LTER site. Vertical lines indicate standard errors (n= 3 replicate plots).

164

\l
O

 

60 J a. Coniferous Forest +Control
- - c1- - 'Low N
50 1 — o — High N

 

ug NHf-N 9 soil’1
‘6’

 

 

 

 

b. Deciduous Forest

 

 

 

 

 

 

 

 

70 ,
6O ‘ c. Mid-successional Communities
73 50 -
a:
2°) 40 . , __ ‘
+V’ , / I -------- I \
g 30 ‘~.\
O) 20'/" . \\\‘\
1 v: ' -------- :6
1O ‘ - K“ 1.: ‘
0 . .
31-May-02 30—Jun-02 31001-02 31-Aug-02

Figure 4.8 The effects of different N fertilizer levels on daily soil ammonium in
coniferous forest (t0p), deciduous forest (middle), and mid-successional communities
(bottom) at the KBS LTER site. Vertical lines indicate standard errors (n= 3 replicate

plots).

165

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170

Chapter 5
Effects of Different Levels of Nitrogen Fertilization on

Soil CH4 Oxidation and C02 Flux in Continuous Corn

Introduction

Methane oxidation rates in agricultural soils tend to be substantially lower than
rates in forest and grassland soils (Mosier et al. 1991 , Goulding et al. 1995, Arif et al.
1996, Mosier et al. 1997, Powlson et al. 1997, Robertson et al. 2000, Hﬁtsch 2001).
Various studies have identiﬁed N fertilizer as a potentially important inhibitor of soil CH4
uptake (Steudler eta]. 1989, Topp 1993, Willison et al. 1995, Hiitsch 1996, Prieme and
Ekelund 2001).

Because the reduction of CH4 oxidation may depend on the amount of N added to
soil, reduced amounts of N fertilizer may allow increased CH4 oxidation in agricultural
soils. However, few investigators have studied the effects of different N fertilizer levels
on CH4 oxidation in agriculture. For example, N fertilizer had no effect on CH4 oxidation
in a regularly fertilized wheat ﬁeld (Mosier and Schimel 1991), on irrigated crops in
Colorado (Bronson and Mosier 1993), or on an acid oxisol in Puerto Rico (Mosier et al.
1998). Although Robertson et al. (2000) documented no increases in CH4 oxidation in a
reduced-fertilizer corn-soybean-wheat rotation, the sensitivity of CH4 oxidation in
agricultural soils to different levels of N fertilizer remains unknown.

The aim of this study is to evaluate whether different rates of N fertilizer will

have correspondingly different effects on soil CH4 consumption in continuous com.

171

Methodology
Site Description

The study was conducted at the Long-term Ecological Research (LTER) sites at
the W.K. Kellogg Biological Station (KBS), Hickory Comers, Michigan (420 24’N, 85°
24’W, elevation 288 m). Annual rainfall at KBS averages 890 m y'1 with about half
falling as snow; potential evapotranspiration (PET) exceeds precipitation for about 4
months of the year. Mean annual temperature is 9.7 °C.

The study site is on the Kalamazoo/Oshtemo soil series (Austin 1979). The soil is
Typic Hapludalfs (ﬁne or coarse-loamy, mixed, mesic soils) derived from glacial till
about 12,000 years ago (Crum and Collins 2003).

We measured methane ﬂuxes at the N rate study plots established in 1999 to
document the effect of different N-fertilizer levels on the growth and yield of corn (Zea
mays L.). The experiment has 9 levels of N fertilizer from O to 291 kg N ha'1 applied to
continuous corn rotation in a randomized complete block design with 4 replications
(Figure 1). In 2002, the year of this study, N-fertilizer was applied as 28% urea
ammonium nitrate (UAN). The ﬁrst 34 kg ha'1 N fertilizer was injected between rows at
planting and the remainder was injected at the 6-leaf stage of corn development on June

27‘“.

In Situ Gas Sampling
We measured in situ methane oxidation rates using the static chamber technique
(Hutchinson and Livingston 1993). Static chambers were fashioned from 25 cm diameter

PVC pipe: bases (25 cm diameter x 10 cm high) were installed in each plot and leﬁ in

172

place except during agronomic operations. Immediately prior to sampling, a 4.5 cm high
cap was placed on each base and sealed with a latex skirt wrapped with an elastic band.
At 30-40 minute intervals, four 10 mL headspace samples were removed through rubber
septa in each cap using a syringe, and put into 3 mL glass sample vials pre-ﬂushed with
headspace air. Within 3 days vial contents were measured for CH4 using a gas
chromatograph (GC. 5890 Series II, Hewlett Packard) equipped with a ﬂame ionization
detector (F ID), and measured for C02 using an infrared gas absorption (IRGA) analyzer
(EGA-15996 C02 Analyzer, Analytical Development CO. LTD., Hoddesdon, England).
Chambers were sampled one day before fertilization and l, 4, 12, 17, 33, 54, and

104 days after fertilization.

Soil Analyses

Soil temperature was measured at 0-5 cm depth using a soil temperature probe.
Soil samples for other analysis were taken from the top 10 cm of soil using a 2.5 cm
diameter soil probe. Fresh soils were passed through a 4 mm sieve and mixed by hand,
and then sub samples were taken for moisture content and mineral N analysis. Prior to
analysis, soils were stored for a week in a refrigerator at 4°C. Soil moisture content was
measured gravimetrically by drying the soil samples at 65°C for 3 days or until dry.
Further drying at 105°C removes an additional 0.8 g H20-100g soil'l moisture in these
soils. Mineral N measurements were obtained by extracting 20 g of dry soil with 100 mL
1 M KCl for 24 h then ﬁltering through 2-pm pore size ﬁberglass ﬁlters. The ﬁltrates

were frozen prior to analysis for NH4+ and N03' using an Alpkem continuous ﬂow

173

analyzer (Alpkem 3550, 01 Analytical, College Station, TX) (Bundy and Meisinger
1994)
Statistical Analyses

The data were divided into two parts: before fertilization (day 0; n= 1 date) and
after fertilization (days 1, 4, 12, 17, 33, 54, and 104; n = 7 dates). I used SPSS version
10.0.1 (SPSS Inc. 2001) for the analysis of covariance (ANCOVA) for the ﬁrst part and
also for the correlation analyses. I used Proc Mixed of SAS version 8.0 (SAS Institute
1999) for the analysis of covariance of the second data set. Methane and C02 data were
natural log transformed before ANOVA and ANCOVA to homogenize variances. I used

untransformed data for correlation analysis.

Results
Methane Oxidation

Methane oxidation averaged 3.34 i 0.32 (s.e., n= 4 replicate blocks x 8 sampling
dates x 9 N-fertilizer levels) pg CH4-C m’2 h’l across all sampling dates and fertilizer
levels. There were no treatment effects and CH4 consumption was not signiﬁcantly
affected by N addition (Tables 5.1, 5.2, 5.3 and Figure 5.2) except for the 34 kg N level
(P=0.013), which had the average highest rate (5.53 $1.51 pg CH4-C m'2 h") (Table 5 .1).
Oxidation rates ranged from —9 to 43 pg CH4-C m‘2 h'l (negative number indicates net
CH4 production).

Methane consumption did not signiﬁcantly change by sample dates (Table 5.3 and
Figure 5.3); average daily rates were low, between 1 and 5 pg CH4-C rn'2 h'l for all

treatments except for one sample date. The exception was on July 30th following a July

174

29th rainfall, for which the average daily rate increased to 10 pg CH4-C m'2 h", with the

34 kg N ha”1 fertilization level as high as 19 pg CH4-C m"2 h".

Carbon Dioxide Fluxes
Fertilizer signiﬁcantly affected C02 ﬂuxes (Tables 5.5) but only at a fertilizer
level of 291 kg N ha", in which co2 ﬂuxes (102.9 i 11.4 mg C02-C m'2 h") were
slightly higher than control and other treatments (Table 5.1 and Figure 5.4). C02
production rates ranged from 4 to 249 mg C02-C rn'2 h". Carbon dioxide ﬂuxes were
strongly and signiﬁcantly (p=0.043) different by sampling date (Table 5.5), and rates

were also highest on July 30th (Figure 5.5).

Soil Properties

Soil moisture signiﬁcantly differed among both fertilizer levels and sample dates
(Table 5.6 and Figure 5.6). Moisture ranged from 5.20 g HZO'lOOg soil'l (£0.18) on July
14th to 14.11 g H20°100g soil" (i048) on July 30th (Figure 5.7).

Soil nitrate and ammonium also signiﬁcantly varied by fertilizer levels and
sample dates (Table 5.6). Nitrate and ammonium signiﬁcantly increased with levels of N
addition (Figure 5.8 and 5.10). Ammonium and nitrate levels also exponentially increased
immediately after N application but declined rapidly until August 20th (Figure 5.9 and
5.11).

Soil temperature was not signiﬁcant different among N levels but changed

signiﬁcantly with dates of sampling (Table 5.6 and Figure 5.12). Soil temperature was

175

 

highest in early July around 35°C then steadily declined to 10 0C in early October (Figure

5.13).

Controls of Methane Oxidation and Carbon Dioxide Fluxes

Analysis of covariance reveals a signiﬁcant association of nitrate and ammonium
levels with CH4 oxidation (Table 5.3). In both cases correlations were negative,
indicating that higher levels of soil N were associated with lower CH4 oxidation (r=-0.22
for nitrate and —0. 19 for ammonium; Table 5.7) although the correlations were not
signiﬁcant (P>0.05). On the other hand, CH4 oxidation was positively correlated with soil
moisture both before (r=0.35) and after fertilization (r=0.18). Carbon dioxide ﬂux was the
strongest predictor of CH4 oxidation (1:035, P<0.01) for post fertilization ﬂuxes.

In contrast, nitrate and ammonium were poor predictors of C02 ﬂux (Tables 5.5

and 5.7), which was best predicted by soil temperature (r=0.30 and P<0.01).

Discussion
Methane oxidation rates in this study were low, on the order of 1-5 pg CH4-C m"2
h‘1 for most sample dates. With one exception, CH4 oxidation rates were not signiﬁcantly
affected by N-fertilizer levels, where oxidation was signiﬁcantly greater than at other
levels (Figure 5.2). There were also no signiﬁcant differences in seasonal oxidation rates
among the nine fertilizer levels. The exceptional response of the 34 kg N level appears
due to a single post-rain sample date, where oxidation in this treatment was as high as 20

pg CH4-C m'2 b" (Figure 5.3).

176

Despite a lack of response to fertilizer level, CH4 oxidation was related to soil
inorganic N contents; higher nitrate and ammonium levels were weakly associated with
lower C114 oxidation (Tables 5.3 and 5.7). This may indicate low population of CH4
oxidizing bacteria or fewer activities due to the competitive effect of ammonium to
methane monooxygenase enzyme.

The best prediction of CH; oxidation across all N levels was C02 ﬂux and soil
moisture. Higher oxidation rates were associated with higher soil respiration rates and
soil moisture. Faster soil carbon cycling — as indicated by higher microbial activity and
root respiration — appears to accelerate CH4 oxidation as well.

Soil C02 ﬂuxes were largely related to soil temperature differences (Tables 5.5
and 5.7). Rates of C02 emission varied over an order of magnitude over the course of
this study (from < 20 to > 200 mg C02-C rn'2 h'l), and were highest following rainfall
events in mid-summer (Figure 5.5). C02 ﬂuxes were likewise unaffected by N-fertilizer
levels (Figure 5.4).

The low rates of CH4 oxidation in the highly and moderately fertilized plots are
consistent with a number of other studies that have also found low oxidation rates in
fertilized wheat (Mosier and Schimel 1991, Sitaula et al. 2000).

I was surprised, however, that oxidation rates were equally low or (even lower) in
the low-fertilizer-N plots. The only other multiple-level N-fertilizer experiment in the
literature (Hﬁtsch et al. 1993) shows a progressive decline of soil CH4 oxidation
according to the amount of N-fertilization (O, 48, 96, and 144 kg N ha'l) in the
Rothamsted “Broadbalk Wheat Experiment.” This, together with two available fertilizer

experiments in wheat (Mosier and Schimel 1991), and timothy and clover (Sitaula et al.

177

2000), in which oxidation was also low at low fertilizer levels, suggests that some factors
other than (but affected by) fertilizers may affect CH4 oxidation.

It is also possible that a legacy of high fertilization at our site is still suppressing
oxidation. That is, in the Broadbalk experiment plots have received low rates of fertilizer
N for more than 140 years; the methanotroph community may take more than the 3 years
of low fertilizer-N in our study to oxidize at higher rates. In another study, near our sites,
soil in successional communities did not oxidize CH4 at rates higher than agricultural
sites for several years after the initiation of succession (Robertson et al. 2000), with rates
50% of nearby forest rates only after 50 years of succession. Thus, higher oxidation rates
at lower fertilization rates may take additional years to develop.

Surprisingly, fertilizer at 34 kg N ha'1 stimulated soil CH4 consumption in this
study. The low level of N added might be adequate to stimulate the growth of CH4
oxidizing bacteria as found in one nitrate fertilizer study (Hiltsch et al. 1994) but not too
high to suppress soil CH4 oxidation.

Carbon dioxide production was also not affected by different amounts of N
fertilizer except at the highest fertilizer level of 291 kg N ha’l. Carbon dioxide ﬂuxes
were mainly related to soil temperature (Tables 5.5 and 5.7).

The substantial increase in both CH4 oxidation and C02 production on July 30th
was likely caused by the sharp increase in soil moisture ﬁ'om 5% on July 14th to 15% on
July 30th due to heavy rain on July 29‘“. On the other hand, an early June rainfall
resulted in a signiﬁcant C02 response (Figure 5.5) but no CH4 response (Figure 5 .4).

This may be due to the high soil N levels still present in early June (Figure 5.9 and 5.11).

178

Conclusion
Nitrogen fertilizer added to these continuous corn plots did not affect soil CH4
oxidation nor C02 emission at any level of fertilizer addition but the lowest (CH4) or
highest (C02). Both C114 uptake rates and C02 ﬂuxes varied seasonally. Soil nitrate and
ammonium signiﬁcantly affected soil CH4 consumption but they did not inﬂuence C02

production. Only soil temperature was a signiﬁcant predictor for C02 ﬂuxes in this soil.

References

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Austin, F. R. 1979. Soil Survey of Kalamazoo County, Michigan. United States
Department of Agriculture, Soil Conservation Service, in cooperation with
Michigan Agricultural Experiment Station. The Soil Conservation Service,
Washington.

Bronson, K., and A. Mosier. 1993. Nitrous oxide emissions and methane consumption in
wheat and com-cropped systems in northeastern Colorado. Pages 133-144 in L.
A. Harper, A. Mosier, J. Duxbury, and D. E. Rolston, editors. Agricultural
Ecosystem Effects on Trace Gases and Global Climate Change. American Society
of Agronomy, Madison.

Bundy, L. G., and J. J. Meisinger. 1994. Nitrogen availability indices. Pages 951-984 in
R. W. Weaver, J. S. Angle, and B. S. Bottomley, editors. Methods of soil analysis.
Part 2. Microbiological and biochemical properties. SSSA Book Series, no. 5.,
Madison, WI.

Crum, J. R., and H. P. Collins. 2003. KBS soils in
http://lter.kbs.msu.edu/Soil/characterization/.

Goulding, K. W. T., B. W. Hiitsch, C. P. Webster, T. W. Willison, and D. S. Powlson.
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Hutchinson, G. L., and G. P. Livingston. 1993. Use of chamber systems to measure trace
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Rolston, editors. Agricultural Ecosystem Effects on Trace Gases and Global
Climate Change. American Society of Agronomy, Madison, Wisconsin.

Hﬁtsch, B. W. 1996. Methane oxidation in soils of two long-term fertilization
experiments in Germany. Soil Biology & Biochemistry 28:773-782.

Hﬁtsch, B. W. 2001. Methane oxidation in non-ﬂooded soils as affected by crop
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Hutsch, B. W., C. P. Webster, and D. S. Powlson. 1993. Long-term effects of nitrogen-
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Soil Biology & Biochemistry 25:1307-1315.

Hﬁtsch, B. W., C. P. Webster, and D. S. Powlson. 1994. Methane oxidation in soil as
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26:1613-1622.

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acid oxisol in western Puerto Rico; effects of tillage, liming, and fertilization. Soil
Biology & Biochemistry 30:2087-2098.

Mosier, A., and D. Schimel. 1991. Inﬂuence of agricultural nitrogen on atmospheric
methane and nitrous oxide. Chemical Industry 24:334—336.

Mosier, A., D. Schimel, D. Valentine, K. Bronson, and W. Parton. 1991. Methane and
nitrous oxide ﬂuxes in native, fertilized and cultivated grasslands. Nature
350:330-332.

Mosier, A. R., J. A. Delgado, V. L. Cochran, D. W. Valentine, and W. J. Parton. 1997.
Impact of agriculture on soil consumption of atmospheric CH4 and a comparison
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Cycling in Agroecosystems 49:71-83.

Powlson, D. S., K. W. T. Goulding, T. W. Willison, C. P. Webster, and B. W. Hﬁtsch.
1997. The effect of agriculture on methane oxidation in soil. Nutrient Cycling in
Agroecosystems 49:59-70.

Prieme, A., and F. Ekelund. 2001. Five pesticides decreased oxidation of atmospheric
methane in a forest soil. Soil Biology & Biochemistry 33:831-835.

Robertson, G. P., E. A. Paul, and R. R. Harwood. 2000. Greenhouse gases in intensive
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SAS Institute. 1999. SAS/STAT User's Guide, Version 8. SAS Institute Inc., Cary, NC.

180

Sitaula, B. K., S. Hansen, J. I. B. Sitaula, and L. R. Bakken. 2000. Methane oxidation
potentials and ﬂuxes in agricultural soil: Effects of fertilisation and soil
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SPSS Inc. 2001. SPSS Base 11.0 for Windows User's Guide. SPSS Inc.

Steudler, P. A., R. D. Bowden, J. M. Melillo, and J. D. Aber. 1989. Inﬂuence of nitrogen
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Topp, E. 1993. Effects of selected agrochemicals on methane oxidation by an organic
agricultural soil. Canadian Journal of Soil Science 73:287-291.

Willison, T. W., C. P. Webster, K. W. T. Goulding, and D. S. Powlson. 1995. Methane
oxidation in temperate soils - Effects of land-use and the chemical form of
nitrogen fertilizer. Chemosphere 30:539-546.

181

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182

Table 5.2 Analysis of covariance to determine effects of N fertilizer and soil properties

on methane oxidation rates before fertilization.

 

 

Source SS df MS F P
Fertilizer 0.27 8 0.03 0.42 0.867
Moisture 0.24 1 0.24 2.93 0.148
Temperature 1.5 x 10“ 1 1.5 x 10“ 0.002 0.968
Nitrate 0.12 1 0.12 1.52 0.273
Ammonium 0.12 l 0.12 1.49 0.277
Error 0.41 5 0.09

Total 64.8 18

Corrected Total 0.92 17

 

183

Table 5.3 Analysis of covariance to determine effects of N fertilizer and soil properties

on methane oxidation rates after fertilization.

 

 

Variable Num. df a Den. (if a F P

Fertilizer 8 236 0.9 0.558
Date 6 236 1.3b 0.106
Fertilizer x Date 48 236 0.6b 0.543
Moisture l 107 2.0 0.159
Temperature 1 236 0.3 0.619
Nitrate l 107 4.7 0.033
Ammonium 1 l 07 5 .5 0.021

 

a Num. df and Den. df refer to numerator and denominator degrees of freedom,
respectively

b Z value

184

Table 5.4 Analysis of variance to determine effects of N fertilizer and soil properties on

carbon dioxide emission rates before fertilization.

 

 

Source SS df MS F P
Fertilizer 1.76 8 0.22 2.4 0.175
Moisture 0.18 1 0.18 2.00 0.216
Temperature 0.01 1 0.01 0.15 0.718
Nitrate 0.05 l 0.05 0.61 0.470
Ammonium 0.07 1 0.07 0.80 0.413
Error 0.46 5 0.09

Total 245 18

Corrected Total 2.41 17

 

185

Table 5.5 Analysis of covariance to determine effects of N fertilizer and soil properties

on carbon dioxide emission rates aﬁer fertilization.

 

 

Variable Num. df a Den. df a F P

Fertilizer 8 236 2.67 0.008
Date 6 236 1.72b 0.043
Fertilizer x Date 48 236 4.9” 0.059
Moisture l 107 2.24 0.137
Temperature 1 236 15.5 0.0001
Nitrate l 107 0.7 0.416
Ammonium l 107 1.4 0.237

 

a Num. (if and Den. df refer to numerator and denominator degrees of freedom,
respectively.

b Z value

186

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187

Table 5.7 The relationships among methane oxidation, carbon dioxide and other soil

properties before and after fertilization across nine different N rates in continuous corn at

the KBS LTER site. Values are Pearson correlation coefﬁcients (r); a n = 9 fertilizer

levels x 4 replicates x 1 sample date, b n = 9 fertilizer levels x 4 replicates x 7 sample

 

 

dates.
C02 Moisture Temperature Nitrate Ammonium

Before F ertilizationa

CH4 -0.l40 0.346 -O.168 -0.223 -0.186

C02 1.000 -0.022 0.100 -0.003 -0.007
After Fertilizationb

CH4 0.345" 0.176* -0.067 -0.095 -0. 174

C02 1.000 0.115 0.295" 0.059 0.038

 

* Correlation is signiﬁcant at the 0.05 level (2-tailed)

** Correlation is signiﬁcant at the 0.01 level (2-tailed)

188

 

 

Border
Rows

 

 

 

 

 

 

27m Border 101
Rows F1

 

 

 

 

4.5 m

Figure 5.] Layout of the N-fertilizer rate study at the KBS LTER site. Unit = kg N ha".

189

 

 

 

 

0 34 67 101 134 168 202 246
N-Fertilizer Level (kg N ha")

291

Figure 5.2 Net methane oxidation at various N fertilizer levels in continuous corn plots at
KBS. Bars represent average seasonal ﬂuxes from ﬁeld plots fertilized at the rates
indicated. Vertical lines are standard errors of means (n= 4 replicate blocks x 8 sampling
dates). * means a signiﬁcant difference at 0.05 level from control (0 kg N ha"').

30

 

pg curc m'2 it"

 

 

 

 

- 1 0
26-Jun-02 26-J ul-OZ 26-Aug-02

 

26-Sep-02

Figure 5.3 Net daily methane oxidation at various N fertilizer levels in continuous corn

plots. Vertical lines are standard errors of means (n= 4 replicate plots). Fertilization
occurred on June 27'“.

190

140

 

120

100

mg corc m‘2 h"

 

 

 

 

101 134 168 202
N-Fertlllzer Level (kg N ha")

 

Figure 5.4 Net carbon dioxide emission at various N fertilizer levels in continuous corn
plots. Bars represent average seasonal ﬂuxes from ﬁeld plots fertilized at the rates

indicated. Vertical lines are standard errors of means (n= 4 replicate blocks x 8 sampling
dates).

 

  
 

 

 

 

 

250
200 - —o—o
_ . _ a l
"'s —a—-67 ‘
”E 150 ‘ - <>— -1o1
Li — -x— -134
8 — o -168.
m 100 - .—e—2o2
E - - a - -246 .
__—x—291J
50 '
0 t . .
26—Jun—02 26—Jul-02 26—Aug-02 26—Sep-02

Figure 5.5 Net daily carbon dioxide ﬂux at various N fertilizer levels in continuous com

plots. Vertical lines are standard errors of means (n= 4 replicate plots). Fertilization
occurred on June 27‘“.

191

28

 

 

 

 

0 34 67 101 134 168 202 246 291
N-Fertillzor Level (kg N ha")

Figure 5.6 Net soil temperature at various N fertilizer levels in continuous corn plots at
KBS. Bars represent average seasonal ﬂuxes from ﬁeld plots fertilized at the rates
indicated. Vertical lines are standard errors of means (n= 4 replicate blocks x 8 sampling
dates).

 

 

 

 

 

45
4o
' ’u 1
35 —o—o
—I—M
30 —A—67 l
25 - <>— -101 i
9 ~ _ *— '134 ‘
2o . l- o —1681
1—9—202 l
15 ‘. . D- . -246 l
10 l L+.ﬁ_29_U
5
o .
26-Jun—02 26-Jui-02 26—Aug-02 26-Sep—02

Figure 5.7 Net daily soil temperature at various N fertilizer levels in continuous corn
plots at the KBS LTER site. Vertical lines are standard errors of means (n= 4 replicate
plots).

192

 

9 H20 1009 solr‘

 

 

 

0 34 67 101 134 168 202 246 291
N-Fertillzer Level (kg N ha“)

Figure 5.8 Net soil moisture at various N fertilizer levels in continuous corn plots at the
KBS LTER site. Bars represent average seasonal ﬂuxes from ﬁeld plots fertilized at the
rates indicated. Vertical lines are standard errors of means (n= 4 replicate blocks x 8

sampling dates). * ,** and *** are signiﬁcant differences at P<0.05, 0.01 and 0.0001
from control (0 kg N ha'l).

 

 

9 H10 1009 soir‘

 

 

26-Jun-02 26-Jul-02 26-Aug-02 26—Sep—02

Figure 5.9 Net daily soil moisture at various N fertilizer levels in continuous corn plots at
the KBS LTER site. Vertical lines are standard errors of means (n= 4 replicate plots).

193

 

pg NO,'-N g 30“.,
8 8 8 8

._L
o

 

 

   

 

 

0 34 67 101 134 168
N-Fertlllzer Level (kg N ha")

Figure 5.10 Net soil nitrate at various N fertilizer levels in continuous corn plots at KBS.
Bars represent average seasonal ﬂuxes from ﬁeld plots fertilized at the rates indicated.
Vertical lines are standard errors of means (n= 4 replicate blocks x 8 sampling dates). *

,**land *** are signiﬁcant differences at P<0.05, 0.01 and 0.0001 from control (0 kg N
ha' ).

250

 

pg N03'-N 9 soil'1
8

 

 

50

   
 

 

 

0
26-Jun-02 26-Jul-02 26-Aug-02 26—Sep—02

Figure 5.11 Net daily soil nitrate at various N fertilizer levels in continuous corn plots at
the KBS LTER site. Vertical lines are standard errors of means (n= 4 replicate plots).
Fertilization occurred on June 27 .

194

200
180 - Hal:

 

 

 

 

 

 

0 34 67
N-Fertmzer Level (kg N ha")

Figure 5.12 Net soil ammonium at various N fertilizer levels in continuous corn plots at

KBS. Bars represent average seasonal ﬂuxes from ﬁeld plots fertilized at the rates

indicated. Vertical lines are standard errors of means (n= 4 replicate blocks x 8 sampling

dates). * ** and *** are signiﬁcant differences at P<0.05, 0.01 and 0.0001 from control
(0 kg N ha'l).

 

 

 

 

 

 

700
600 1'
'——o—0 l
500 — g — 34
1.5 —A—67 i
g 400 - <>- -101
2. — -x— -134
+ — o — 168
:2? 300 +202 1
g - - D - -246
200 - ;_——x—____391
100
o '7'"""““—I---—I"
26-Jun~02 26-Jul-02 26-Aug-02 26-Sep-02

Figure 5.13 Net daily soil ammonium at various N fertilizer levels in continuous com
plots at the KBS LTER site. Vertical lines are standard errors of means (n= 4 replicate
plots). Fertilization occurred on June 27th.

195

    

 
 

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