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Soil Organic Matter Composition Impacts its Degradability and Association with Soil Minerals
by
Joyce S. Clemente
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Graduate Department of Chemistry University of Toronto
Copyright by Joyce S. Clemente, 2012
ii
SOIL ORGANIC MATTER COMPOSITION IMPACTS ITS DEGRADABILITY AND ASSOCIATION WITH SOIL MINERALS
Doctor of Philosophy Degree, 2012
Joyce S. Clemente
Graduate Department of Chemistry, University of Toronto
ABSTRACT
Soil organic matter (OM) is a complex mixture of compounds, mainly derived from plants and
microbes at various states of decay. It is part of the global carbon cycle and is important for
maintaining soil quality. OM protection is mainly attributed to its association with minerals.
However, clay minerals preferentially sorb specific OM structures, and clay sorption sites
become saturated as OM concentrations increase. Therefore, it is important to examine how
OM structures influence their association with soil minerals, and to characterize other protection
mechanisms. Several techniques, which provide complementary information, were combined to
investigate OM composition: Biomarker (lignin phenol, cutin-OH acid, and lipid) analysis,
using gas chromatography/mass spectrometry; solid-state 13C nuclear magnetic resonance
(NMR) spectroscopy; and an emerging method, solution-state 1H NMR spectroscopy. OM
composition of sand-, silt-, clay-size, and light fractions of Canadian soils were compared. It
was found that microbial-derived and aliphatic structures accumulated in clay-size fractions, and
lignin phenols in silt-size fractions may be protected from further oxidation. Therefore, OM
protection through association with minerals may be structure-specific. OM in soils amended
with maize leaves, stems, and roots from a biodegradation study were also examined. Over
time, lignin phenol composition, and oxidation; and aliphatic structure contribution changed less
iii
in soils amended with leaves compared to soils amended with stems and roots. Compared to
soils amended with leaves and stems, amendment with roots may have promoted the more
efficient formation of microbial-derived OM. Therefore, plant chemistry influenced soil OM
turnover. Synthetic OM-clay complexes and soil mineral fractions were used to investigate
lignin protection from chemical oxidation. Coating with dodecanoic acid protected lignin from
chemical oxidation, and overlying vegetation determined the relative resistance of lignin
phenols in clay-size fractions from chemical oxidation. Therefore, additional protection from
chemical oxidation may be attributed to OM composition and interactions between OM
structures sorbed to clay minerals. Overall, these studies suggest that while association with
minerals is important, OM turnover is also influenced by vegetation, and protection through
association with clay minerals was modified by OM structure composition. As well, OM-OM
interaction is a potential mechanism that protects soil OM from degradation.
iv
ACKNOWLEDGEMENTS
First, this dissertation would not have been possible if not for my supervisor, Prof.
Myrna Simpson. Thank you for this and future opportunities that result from this work. Thank
you for your time, for being a mentor and guiding me through the many challenges throughout
the years. I appreciate that you were always available when I needed your help and advice.
I would also like to thank Prof. André Simpson for his enthusiasm and assistance with
the numerous NMR experiments. I am grateful to my committee member, Prof. Deborah
Zamble, her perspective and suggestions throughout the years have made this dissertation better.
I am additionally thankful to Prof. Scott Mabury for serving on my comprehensive exam
committee. I would also like to thank Prof. Kagan Kerman, my fourth committee member, and
Prof. Paul Voroney, my external examiner, for taking the time and effort to serve on my defence
committee. Finally, thank you to the chair of my examining committee, Prof. Nick Eyles.
I would like to acknowledge members of the M. Simpson and A. Simpson groups. It
was great talking and laughing with you. Dr. X. Feng and Dr. Y. Xu are thanked for training me
in my first year as a PhD student, and Dr. D. McNally for NMR training. Analysis of the
hundreds of samples in this thesis would not have been possible without help from David Wolfe,
Katherine Hills, and Magda Celejewski. I appreciate your dedication and wish you all the best.
I’d also like to express my thanks to Anna Liza Villavelez, for coordinating exams, meetings,
and answering a lot of questions.
Our project collaborators have provided samples and invaluable insight: thank you to
Dr. E. Gregorich, Dr. J. Whalen, and Dr. S. Yanni. I would also like to acknowledge funding
sources for the various projects: the Natural Sciences and Engineering Research Council Green
Crop Network (MJS); a University of Toronto Fellowship, a Teaching Reduction Fellowship, an
Ontario Graduate Scholarship, and a Doctoral Thesis Completion Grant (JSC).
To my friends, old and new: thank you for listening, for all the fun times, for being there
through the not-so-fun times, and for simply being there. Most of all, I am very grateful to my
family. They always remind me of the important things in life. I thank my sisters for exploring
the land of the Torontosaurus with me. You’ve grown up to be such wonderful people - I’m
proud of you too. To my parents: for letting me pursue what I think is best, I wouldn’t have had
the courage to make these choices if not for your unwavering love and support – thank you.
v
TABLE OF CONTENTS
ABSTRACT ................................................................................................................................... ii
ACKNOWLEDGEMENTS ............................................................................................................ iv
TABLE OF CONTENTS ................................................................................................................ v
LIST OF FIGURES ........................................................................................................................ ix
LIST OF TABLES .......................................................................................................................... xi
LIST OF APPENDICES .............................................................................................................. xii
ABBREVIATIONS ..................................................................................................................... xiii
CHAPTER 1 1
INTRODUCTION: SOIL ORGANIC MATTER COMPONENTS, MEASUREMENT, AND SEQUESTRATION ............................................................................................................... 1
1.1 General Overview ........................................................................................................... 2
1.2 Biologically-derived Compounds ................................................................................... 4
1.2.1 Carbohydrates ........................................................................................................... 4
1.2.2 Amino acids, Peptides, Proteins ............................................................................... 4
1.2.3 Lipids ......................................................................................................................... 5
1.2.4 Cutin and Suberin ..................................................................................................... 7
1.2.5 Lignin ........................................................................................................................ 9
1.3 Methods Used to Characterize Soil Organic Matter ..................................................... 11
1.3.1 Biomarkers ............................................................................................................... 13
1.3.1.1 Solvent extractable biomarkers ........................................................................ 13
1.3.1.2 Thermal and chemical cleavage of polymers in soil organic matter ................ 14
1.3.2 Nuclear Magnetic Resonance Spectroscopy ........................................................... 17
1.3.2.1 Solid-state 13C nuclear magnetic resonance spectroscopy ............................... 17
1.3.2.2 Solution-state 1H nuclear magnetic resonance spectroscopy ........................... 18
1.4 Mechanisms Responsible for Soil Organic Matter Accumulation ................................ 21
1.4.1 Inherent Chemical Recalcitrance ............................................................................. 22
1.4.2 Physical Protection of Soil Organic Matter ............................................................ 24
1.5 Objectives and Hypotheses ............................................................................................ 27
1.6 Thesis Summary ............................................................................................................. 29
vi
1.7 Statement of Authorship and Publication Status ........................................................... 31
CHAPTER 2 34
COMPARISON OF NUCLEAR MAGNETIC RESONANCE METHODS FOR THE ANALYSIS OF ORGANIC MATTER COMPOSITION FROM SOIL DENSITY AND PARTICLE FRACTIONS ............................................................................................................. 34
2.1 Abstract ......................................................................................................................... 35
2.2 Introduction ................................................................................................................... 35
2.3 Experimental Methods .................................................................................................. 39
2.3.1 Soil Description and Sample Isolation ................................................................... 39
2.3.2 Solid-State 13C Cross Polarization-Magic Angle Spinning (CP/MAS) NMR Spectroscopy ......................................................................................................... 40
2.3.3 Solution-State 1H NMR .......................................................................................... 41
2.4 Results and Discussion .................................................................................................. 41
2.4.1 Organic Matter Characterization by Solid-State 13C NMR .................................... 41
2.4.2 Organic Matter Characterization by Solution-state 1H NMR Spectroscopy .......... 46
2.4.3 Comparison of Solid-state 13C and Solution-state 1H NMR Methods .................... 48
2.5 Conclusions ................................................................................................................... 52
2.6 Acknowledgements ....................................................................................................... 53
CHAPTER 3 54
ASSOCIATION OF SPECIFIC ORGANIC MATTER COMPOUNDS IN SIZE FRACTIONS OF SOILS UNDER DIFFERENT ENVIRONMENTAL CONTROLS ................ 54
3.1 Abstract ......................................................................................................................... 55
3.2 Introduction .................................................................................................................. 55
3.3 Materials and Methods .................................................................................................. 57
3.3.1 Soil Sampling, Fractionation, and Carbon and Nitrogen Analysis .......................... 57
3.3.2 Solvent Extraction and Copper (II) Oxide Oxidation .............................................. 59
3.3.3 Solid-state 13C NMR ............................................................................................... 61
3.3.4 Solution-state 1H NMR and Diffusion Edited 1H NMR ......................................... 62
3.4. Results ........................................................................................................................... 63
3.4.1 Fractionation Yields and Carbon and Nitrogen analysis ........................................ 63
3.4.2 Solvent Extractable Lipids, Lignin-derived Phenols and Cutin-derived OH-acids ...................................................................................................................... 64
3.4.3 Organic Matter Patterns in Solid-state 13C NMR Spectra ....................................... 67
vii
3.4.4 Organic Matter Patterns in Solution-state 1H NMR Spectra ................................... 70
3.5 Discussion ...................................................................................................................... 76
3.5.1 Preservation of Organic Matter in Particle-size Fractions ....................................... 76
3.5.2 Environmental Controls on Organic Matter Preservation ....................................... 78
3.6 Conclusions .................................................................................................................... 80
3.7 Acknowledgements ....................................................................................................... 80
CHAPTER 4 81
COMPARISON OF SOIL ORGANIC MATTER COMPOSITION AFTER INCUBATION WITH MAIZE LEAVES, ROOTS, AND STEMS ............................................. 81
4.1 Abstract ......................................................................................................................... 82
4.2 Introduction ................................................................................................................... 82
4.3 Materials and Methods .................................................................................................. 84
4.3.1 Biodegradation of OM in Soils Amended with Maize Leaves, Stems, and Roots ..................................................................................................................... 84
4.3.2 Lignin-derived Phenol and Cutin-derived OH-acid Extraction and Analysis ......... 85
4.3.3 Solid-state 13C NMR ............................................................................................... 87
4.3.4 Solution-state 1H NMR ........................................................................................... 88
4.3.5 Changes in Organic Matter Chemistry and Statistical Analyses ............................ 89
4.4. Results .......................................................................................................................... 89
4.4.1 Lignin-derived Phenols and Cutin-derived OH-acids ............................................ 89
4.4.2 Organic Matter Characterization by Solid-state 13C NMR ...................................... 90
4.4.3 Organic Matter Characterization by Solution-state 1H NMR .................................. 95
4.5 Discussion ................................................................................................................... 100
4.6 Conclusions .................................................................................................................. 106
4.7 Acknowledgements ..................................................................................................... 107
CHAPTER 5 108
PHYSICAL PROTECTION OF LIGNIN BY ORGANIC MATTER AND CLAY MATERIALS FROM CHEMICAL OXIDATION ..................................................................... 108
5.1 Abstract ....................................................................................................................... 109
5.2 Introduction ................................................................................................................. 109
5.3 Materials and Methods ................................................................................................ 112
5.3.1 Preparation of Organic Matter-clay Complexes ................................................... 112
5.3.2 Soil Sampling, and Density Fractionation ............................................................ 113
viii
5.3.3 Chemical Oxidation of Soil Density Fractions and Organic Matter-clay Complexes .......................................................................................................... 113
5.3.4 Carbon Content, Solvent Extraction and Lignin-derived Phenol Analysis .......... 114
5.3.5 Data Analysis ........................................................................................................ 116
5.4 Results and Discussion ................................................................................................ 116
5.4.1 Composition of Organic Matter-clay Complexes Before Chemical Oxidation ............................................................................................................ 116
5.4.2 Chemical Oxidation of Lignin-clay and Lignin-clay-dodecanoic acid Complexes .......................................................................................................... 120
5.4.3 Chemical Oxidation of Humic Acid-clay Complexes .......................................... 122
5.4.4 Chemical Oxidation of Soil Density Fractions ..................................................... 127
5.5 Conclusions ................................................................................................................. 133
5.6 Acknowledgements ..................................................................................................... 134
CHAPTER 6 135
SUMMARY, CONCLUSIONS, AND FUTURE RESEARCH ................................................. 135
6.1 Summary and Conclusions .......................................................................................... 136
6.2 Future Research ........................................................................................................... 139
6.2.1 Microbial Derived Organic Matter ....................................................................... 139
6.2.2 Preservation of Organic Matter Associated with Clay ........................................ 140
6.2.2.1 Organic matter composition in soil fractions ................................................. 140
6.2.2.2 Protection of organic matter sorbed to clay minerals .................................... 141
REFERENCES .......................................................................................................................... 147
ix
LIST OF FIGURES
Figure 1.1 Possible fates of carbon in soil OM ............................................................................ 3
Figure 1.2 Sample lipid structures isolated with solvent extraction of soils, plants, microorganisms .......................................................................................................................... 5
Figure 1.3 Cutin and suberin structures. ...................................................................................... 8
Figure 1.4 Lignin composition. ................................................................................................. 10
Figure 1.5 Phenol monomer and dimers extracted after CuO oxidation ................................... 16
Figure 1.6 Solid-state 13C NMR spectrum of soil organic matter ............................................. 19
Figure 1.7 Detection of relatively large compounds using diffusion edited (DE) NMR. .......... 21
Figure 1.8 Solution-state diffusion edited (DE) 1H NMR can detect differences in signals from plant, soil and cultured microbe extracts............................................................. 21
Figure 2.1 Solid-state 13C cross polarization/magic angle spinning (CP/MAS), solution-state 1H nuclear magnetic resonance (NMR), and diffusion edited (DE) 1H NMR spectra of Agricultural (AM) soil, clay-size, and light fractions. ............................................ 42
Figure 2.2 Solid-state 13C cross polarization/magic angle spinning (CP/MAS), solution-state 1H nuclear magnetic resonance (NMR), and diffusion edited (DE) 1H NMR spectra of Northern Grassland (NG) soil, clay-size, and light fractions. ................................. 43
Figure 2.3 Solid-state 13C cross polarization/magic angle spinning (CP/MAS), solution-state 1H nuclear magnetic resonance (NMR), and diffusion edited (DE) 1H NMR spectra of Grassland-Forest Transition (GFT) soil, clay-size, and light fractions. .................. 44
Figure 2.4 Relative contribution of the various structures to A) the solid-state 13C cross polarization/magic angle spinning (CP/MAS) nuclear magnetic resonance (NMR), B) solution-state 1H NMR, and C) diffusion edited (DE) 1H NMR spectra of Agricultural (AM) Soil, Northern Grassland (NG), and Grassland-Forest Transition (GFT) light, clay-size fraction, and whole soil. ............................................................................................ 45
Figure 3.1 Solvent extractable lipids (µg/g C) from various soil fractions from Alberta. ........ 65
Figure 3.2 Lignin-derived phenol and cutin-derived OH-acid distribution. .............................. 66
Figure 3.3 Solid-state 13C CP/MAS NMR spectra of soils and their respective fractions. ....... 68
Figure 3.4 Solution state 1H-NMR and diffusion edited (DE) 1H-NMR of Southern Grassland soil fractions. ........................................................................................................... 71
Figure 3.5 Solution state 1H-NMR and diffusion edited (DE) 1H-NMR of Northern Grassland soil fractions. ........................................................................................................... 72
Figure 3.6 Solution state 1H-NMR and diffusion edited (DE) 1H-NMR of Grassland-Forest Transition soil fractions. ............................................................................................... 73
Figure 4.1 Change in lignin phenol monomer composition (%) with incubation. .................... 92
x
Figure 4.2 Change in oxidation (%) of vanillyl (Ad/Alv) and syringyl (Ad/Als) lignin phenol. Changes in lignin-derived phenol dimers (Dimers/Monomers) and cutin-derived OH-acids (OH-acids/Monomers) with incubation are also shown. ............................ 93
Figure 4.3 Solid-state 13C CP/MAS NMR spectra, of maize leaves, stems, roots, un-amended soil, and soils incubated with maize tissues at time = 1 and 36 weeks. ................... 94
Figure 4.4 Changes in the contribution (%) of alkyl, O-alkyl, anomeric, aromatic, and carboxyl or carbonyl chemical groups to total OM in soil amended with maize tissues, and incubated for 36 weeks. ..................................................................................................... 96
Figure 4.5 Solution-state 1H NMR spectra of maize tissues, un-amended soil, and soils incubated with maize tissues for 1 and 36 weeks. ................................................................... 97
Figure 4.6 Solution-state diffusion edited 1H NMR spectra of maize tissues, un-amended soil, and soils amended with maize tissues after 1 and 36 weeks. ........................... 98
Figure 4.7 Change in the contribution (%) of aliphatic methyl and methylene, aliphatic methyl and methylene near O and N, O-alkyl, and aromatic chemical groups to total NaOH-extractable OM from soil amended with maize tissues and incubated for 36 weeks. ..................................................................................................................................... 101
Figure 5.1 Lignin oxidation in lignin-clay and lignin-clay-dodecanoic acid complexes after chemical oxidation. ........................................................................................................ 121
Figure 5.2 Increase (%) in extractable dodecanoic acid after chemical oxidation of lignin-clay-dodecanoic acid complexes. ................................................................................ 123
Figure 5.3 Changes in lignin concentrations in humic acid-clay complexes (created at pH 4 and 7) and peat soil after chemical oxidation. .............................................................. 124
Figure 5.4 Changes in lignin phenol composition in humic acid-clay complexes (created at pH 4 and 7) and peat soil after chemical oxidation:........................................................... 126
Figure 5.5 Increase in extractable a) n-alkanol and b) organic acids after chemical oxidation of humic acid-clay complexes (created at pH 4 and pH 7), and peat soil.............. 128
Figure 5.6 Decreased lignin concentrations in Southern Grassland (SG) and Grassland-Forest Transition (GFT) soil mineral fractions after chemical oxidation. ............................. 129
Figure 5.7 Changes in lignin phenol monomer composition of Southern Grassland (SG) and Grassland-Forest Transition (GFT) soils after chemical oxidation. ................................ 130
Figure 6.1 Elemental composition measured using X-ray Photoelectron Spectroscopy (XPS) of the lignin-clay and lignin-dodecanoic acid (C12)-clay complexes. ......................... 143
Figure 6.2 Time of Flight-Secondary Ion Mass Spectrometry analysis of a) lignin-clay and b) lignin-clay-dodecanoic acid (C12)-clay complexes. .................................................... 144
Figure 6.3 Liquid chromatography of a) bovine serum albumin (BSA), b) BSA digested with trypsin in buffer, and the supernatant of c) trypsin added to montmorillonite clay, d) BSA-clay complex, and e) trypsin digest of BSA-clay complex. ..................................... 146
xi
LIST OF TABLES LIST OF TABLES
Table 1.1 Summary of methods used to analyze soil organic matter. ....................................... 12
Table 1.2 Characteristics of sand-, silt-, and clay-size fractions. ............................................... 25
Table 1.3 Common interactions between organic matter and clay minerals. ............................ 27
Table 2.1 Structural characteristics and organic matter sources detected using solid-state 13C nuclear magnetic resonance (NMR) and solution-state 1H NMR (in DMSO-d6). ............ 38
Table 2.2 Soil characteristics and alkyl/O-alkyl ratios from solid-state 13C nuclear magnetic resonance (NMR), solution-state 1H NMR, and diffusion edited (DE) 1H NMR of clay-size, light fraction, and soil from Agricultural (AM), Northern Grassland (NG), and Grassland-Forest Transition (GFT) soils. ............................................................... 51
Table 3.1 Soil fraction distribution, carbon and nitrogen contents, and C/N ratios of sand-, silt-, clay-size and light fractions. ................................................................................. 63
Table 3.2 Relative contribution (%) of alkyl (0-50 ppm), O-alkyl (50-95 ppm), anomeric (95-110 ppm), aromatic (110-160 ppm), and carboxyl + carbonyl (160-200 ppm) structures to the solid-state 13C CP/MAS NMR spectra after integration and resulting alkyl/O-alkyl ratios. ................................................................................................................. 69
Table 3.3 Contribution of lignin- and carbohydrate-derived structures (O-alkyl) relative
to: lipids, protein, waxes, cutin and suberin (alkyl/O-alkyl) or protein (O-alkyl/1H) in soil fraction extracts from solution-state 1H NMR and diffusion edited (DE) 1H NMR spectra. ..................................................................................................................................... 75
Table 4.1 C and N content, lignin-derived phenols, cutin-derived OH-acids, total organic matter (OM), and base-extractable OM composition of maize leaves, stems, and roots. .................................................................................................................................. 91
Table 5.1 Characteristics of lignin-clay complexes and lignin-clay complexes coated with dodecanoic acid prior to chemical oxidation. ................................................................ 117
Table 5.2 Carbon (C) content, composition of extractable lipids, lignin-derived phenols, and cutin-derived OH-acid contributions in humic acid-clay complexes before chemical oxidation. ................................................................................................................ 118
Table 5.3 Carbon (C) content, lignin-derived phenol and cutin-derived OH-acid composition in Southern Grassland and Grassland-Forest Transition sand-, silt-, and clay-size fractions prior to chemical oxidation. ..................................................................... 132
Table 6.1 Enzyme activity measured as change in trypsin substrate concentrations with time. ....................................................................................................................................... 145
xii
LIST OF APPENDICES LIST OF APPENDICES
APPENDIX A1: Supplementary Material for Chapter 3 (Association of Specific Organic Matter Compounds in Size Fractions of Soils under Different Environmental Controls) ................................................................................................................................ 167
APPENDIX A2: Supplementary Material for Chapter 4 (Comparison of Soil Organic Matter Composition after Incubation with Maize Leaves, Roots, and Stems) ........ 171
xiii
ABBREVIATIONS
Ad/Al Ratio of vanillic acid to vanillin and syringic acid to syringaldehyde
Ad/Als
Ratio of syringic acid to syringaldehyde
Ad/Alv
Ratio of vanillic acid to vanillin
AM
Agricultural soil
BSA
Bovine serum albumin
C
Carbon
C/N
Carbon to nitrogen ratio
CP
Cross-polarization
CP/MAS
Cross-Polarization/Magic Angle Spinning
CuO
Copper (II) Oxide
D2O
Deuterated H2O
DE
Diffusion edited
DMSO-d6
Perdeuterated dimethyl sulfoxide
FI/MS
Field Ionization/Mass Spectrometry
FTIR
Fourier Transform Infrared spectroscopy
GC/MS
Gas Chromatography/Mass Spectrometry
GFT
Grassland-Forest Transition
HF
Hydrofluoric acid
HPLC
High Performance Liquid Chromatography
xiv
LC
Liquid chromatography
MAS
Magic Angle Spinning
MS
Mass Spectrometry
NG
Northern Grassland
NMR
Nuclear Magnetic Resonance spectroscopy
OH-acids
Hydroxy-acids
OM
Organic matter
SG
Southern Grassland
ToF-SIMS
Time of Flight-Secondary Ion Mass Spectrometry
Tukey-HSD
Tukey-Honestly Significant Difference
XPS
X-ray Photoelectron Spectroscopy
1
CHAPTER 1
INTRODUCTION: SOIL ORGANIC MATTER COMPONENTS, MEASUREMENT, AND SEQUESTRATION
Authorship and Contributions: Written by Joyce S. Clemente, with critical comments from Myrna J. Simpson.
2
1.1 General Overview
Non-living soil organic matter (OM) constitutes the organic component of soil, and is
composed of compounds from various organisms at different stages of decay, including
abiotically altered OM, or humus; and char or black carbon (Kögel-Knabner, 2002; Schnitzer,
1991). Soil OM influences the physical properties (i.e. aggregation, water holding capacity),
chemistry (i.e. pH, interactions with contaminants, charge), biological activity, and productivity
of soils (Dinel et al., 1991a; Janzen et al., 1998; Schnitzer, 1991; Tiessen et al., 1994; Wicke and
Reemtsma, 2010). Soil OM stores about 2.5 times more carbon than total vegetation, and twice
the amount of atmospheric carbon (Batjes, 1998). For net soil OM formation to occur, OM
input must exceed its mineralization (Batjes, 1998; Janzen et al., 1998; Lorenz et al., 2007).
Soil OM can therefore be a source or sink of CO2, depending on whether net accumulation or
mineralization of OM occurs. Disturbance of the soil environment, resulting in net
mineralization and loss of soil OM may lead to increased atmospheric CO2 concentrations,
which may influence various aspects of the biosphere (Batjes, 1998; Lorenz et al., 2007). The
scientific interest in soil OM was best stated in a review by Hedges et al. (2000), in which the
authors considered the existence of OM (such as those in soil) as a “thermodynamic anomaly”
because so much energy can be gained by organisms if OM is mineralized to CO2. The stability
of soil OM against degradation and mineralization is therefore important in maintaining and
defining the overall environment.
Various studies have focused on the fate of plant-derived compounds, since these are
thought to control soil OM formation (Kögel-Knabner, 2002). Fig. 1.1 illustrates the sources
and fate of biologically-derived compounds, as discussed by Baldock et al. (2004): Products of
plant, animal or insect biomass degradation are used by microorganisms as carbon and energy
sources, which results in microbial biomass production. Degradation products may also be lost
from soils through migration into the water table, erosion, and mineralization to CO2.
Alternatively, the various degradation products may become sequestered in soil through the
formation of more stable compounds (humus formation), and physical protection through
association with soil minerals. It is important to determine how the chemistry of OM structures
are related to these protection mechanisms and the overall soil OM protection in order to
3
Figure 1.1 Possible fates of carbon in soil OM based on Baldock et al. (2004), where processes and compounds inside the dotted line occur in soil.
understand carbon turnover and storage in soils. This knowledge will facilitate predictions on
how soil OM will respond to changes in land management and climate.
Soil OM stability is believed to be influenced by several variables including
environmental factors such as temperature and pH; the inherent chemical characteristics of these
compounds and the ability of degrading organisms to process such compounds; and the
characteristics of the soil mineral fractions, such as the amount and type of clay minerals present
(Christensen, 2001; Mikutta et al., 2006b; Six, 2004; von Lutzow et al., 2006; Zech et al., 1997).
Unfortunately, studies investigating the stability of OM in soils are complicated by the complex
nature of soil OM itself, in addition to the varied interactions that occur in soil systems. To
date, there is still controversy regarding the structure and definition of stable or stabilized soil
Microbial biomass
CO2
Degradation products
Abioticdegradation
Microbial degradation
Dissolved Organic Matter
Physical protection
Humus Formation
Stable organic matter
PlantsAnimals,insects
Runoff, loss to water table
4
OM (Kleber, 2010; von Lutzow and Kögel-Knabner, 2010). Therefore, although progress has
been made in characterizing soil OM chemistry, and in defining factors that may lead to its
stability, further studies are needed to characterize the dynamics of specific soil OM
components, how this relates to soil mineralogy, and the specific mechanisms that stabilize OM
in soils. The remainder of this review will address, in more detail, the sources of soil OM; the
identity and characteristics of compounds that are major contributors to soil OM; methods used
to study soil OM composition; and the factors that influence soil OM preservation, and
degradation.
1.2 Biologically-derived Compounds
Biologically-derived compounds can be categorized into carbohydrates, peptides, lipids,
aliphatic polymers (cutin and suberin), and lignin, which vary in function, chemistry,
degradability, and contribution to soil OM. The discussion will focus on plant- and microbial-
derived compounds since plants and microbes are hypothesized to be the major contributors to
soil OM (Baldock et al., 1992; Kögel-Knabner, 2002; Simpson et al., 2007a).
1.2.1 Carbohydrates
Carbohydrates are found in all organisms, have diverse functions (energy source,
storage, cell structure and protection), are the most abundant biomolecules (Voet and Voet,
1995), and are estimated to make up ~25-50% of soil OM (Kaal et al., 2007; Oades, 1984).
Carbohydrates range in size, from monomers such as glucose, to polymers such as cellulose,
which contains >10,000 glucose units (Kögel-Knabner, 2002; Voet and Voet, 1995).
Carbohydrate polymers (starch, cellulose, and hemicellulose) are the most abundant plant-
derived compounds, and are estimated to contribute >50% to total plant biomass (Kögel-
Knabner, 2002; von Lutzow et al., 2006). In addition, polymers composed of N-acetyl hexoses
make up the cell walls of bacteria (N-acetylglucosamine, and N-acetylmuramic acid) and fungi
(N-acetylglucosamine;(White, 2000).
1.2.2 Amino acids, Peptides, Proteins
Peptides and proteins are important components of the cell machinery (Voet and Voet,
1995). Enzymatic proteins are responsible for catalyzing biological reactions, which includes
the degradation of soil OM and plant material (Fontaine et al., 2003; Hoppe et al., 1988;
5
Kandeler et al., 1999; Kleber, 2010; Kolattukudy, 1985; Tien and Kirk, 1983; Warren, 1996),
while collagen and peptides, such as that found in peptidoglycan, are also important in
maintaining cell structure (Voet and Voet, 1995; White, 2000). The contribution of peptides to
plant biomass is considered to be lower compared to its concentration in microbial and animal
biomass, which results in relatively lower nitrogen levels in plants (Wolf and Snyder, 2003). In
soils, proteins and peptides may be preserved through reaction with and encapsulation by other
OM (Hsu and Hatcher, 2006; Zang et al., 2000). Most of the nitrogen in soil OM may be found
in proteins, and nitrogen availability is essential in maintaining soil processes (Wolf and Snyder,
2003).
1.2.3 Lipids
Lipids are found in plants, animals and microorganisms. For example lipids are
components of phospholipids in membranes, lipoprotein in cells, and waxes that coat plant
surfaces (Bull et al., 2000b; Kögel-Knabner, 2002; Otto and Simpson, 2005; Schnitzer, 1991;
van Bergen et al., 1997; Voet and Voet, 1995; Volkman et al., 1980). These compounds are
biologically important in cell protection, and in maintaining cell function (Voet and Voet, 1995).
Lipid structures that are linear, branched, saturated, unsaturated, and cyclic, which may contain
alcohol, carboxylic acid, and ester functional groups all contribute to the lipids found in soil
OM. (Fig. 1.2).
Figure 1.2 Sample lipid structures isolated with solvent extraction of soils, plants, microorganisms and identified using gas chromatography/mass spectrometry (GC/MS) based on Bull et al. (2000b), Kögel-Knabner (2002), and Otto and Simpson (2005).
n-alkane
n-alkenen-alkanoic acid
n-alkanol
n
n
n
n
n
n n
Branched alkane
Ester
Steroid
6
Although some lipids, such as cholesterol, hexadecanoic acid, and octadecanoic acid are
found in many organisms, lipid structure and molecular weight has been used to differentiate
between plant- and microbial-derived lipids (Bull et al., 2000a; 2000b; Feng and Simpson, 2007;
2008; Feng et al., 2010; Otto and Simpson, 2005; Quenea, 2004; 2006; van Bergen et al., 1997;
Volkman et al., 1980; Wiesenberg et al., 2004; 2006; 2010). For example, phytosterols (-
sitosterol, stigmasterol, campesterol, stigmastanol) are plant-derived, while ergosterol is derived
from fungi (Feng et al., 2010; Otto and Simpson, 2005). Branched alkanes and alkanoic acids
(iso and ante-iso) are also thought to be microbial-derived (Feng and Simpson, 2007; Kaneda,
1991; Otto and Simpson, 2005; Wu and Palmquist, 1991). For straight-chain lipids, those with
>C20 are plant-derived, while those with <C20 are plant- and microbial-derived (Bull et al.,
2000a; Otto and Simpson, 2005; Quenea, 2004; van Bergen et al., 1997; Wiesenberg et al.,
2010). Although microorganisms that can synthesize lipids with up to C33 have been identified
(Ladygina et al., 2006), synthesis of such high molecular weight lipids in bacteria is generally
inhibited by cellular controls (White, 2000), and lipids with C14-C20 are more common (Voet
and Voet, 1995). On the other hand, lipids with >C20 from surface waxes are extracted from
plant tissues (Bull et al., 2000b; Otto and Simpson, 2005). It is also more common to observe n-
alkanols, and n-alkanoic acids with even carbon numbers because these are synthesized by
polymerization of C2 units (Voet and Voet, 1995). However, microorganisms are also known to
synthesize fatty acids with odd carbon numbers, by using compounds with odd number of
carbon, such as valeric acid, to intitiate synthesis (Fozo and Quivey, 2004; Kaneda, 1991; Wu
and Palmquist, 1991). Therefore, increased contributions from n-alkanols, and n-alkanoic acids
with odd number of carbon are thought to indicate contributions from microbial-derived lipids
(Feng and Simpson, 2007; Otto and Simpson, 2005; van Bergen et al., 1997; Volkman et al.,
1980; Wiesenberg et al., 2010). Plant-derived n-alkanes are dominated by compounds with odd
carbon number (Feng and Simpson, 2007; Otto and Simpson, 2005; Quenea et al., 2006;
Wiesenberg et al., 2010), because n-alkanes are synthesized by the removal of a carbonyl group
from fatty acids (Cheesbrough and Kolattukudy, 1984). N-alkanes in microorganisms on the
other hand, are a mixture of compounds with odd and even carbon number (Han and Calvin,
1969) because the fatty acid precursors are a mixture of these compounds (Fozo and Quivey,
2004; Kaneda, 1991; Wu and Palmquist, 1991), and n-alkane is produced through both carbonyl
group removal from fatty acids and reduction of alcohols (Park, 2005).
7
Lipid structure may also influence their relative degradability. For example, branching
has been observed to slow down biodegradation (Atlas and Bartha, 1998). The degradation of
carboxylic acids is also faster compared to alkanes, because alkane degradation additionally
requires oxidation (Atlas and Bartha, 1998; Wiesenberg et al., 2004). Although degradation of
compounds with up to C44 have been observed (Atlas, 1981), lipids with lower molecular weight
are degraded faster than those with higher molecular weight (Atlas, 1981; Atlas and Bartha,
1998; Whyte et al., 1998). Low molecular weight fatty acids can also be incorporated directly
into biomass by bacteria (Doumenq et al., 2001). The properties of lipids, such as structure and
degradability, are important in soils because these compounds are thought to stabilize soil
aggregates (Dinel et al., 1991a).
1.2.4 Cutin and Suberin
Cutin and suberin are plant-derived polymers that, together with waxes, repel water,
protect plant tissues from injury, prevent water loss, and help control passive diffusion through
plant cells (Arrieta-Baez and Stark, 2006; Jeffree, 2006; Kögel-Knabner, 2002; Kolattukudy,
1980, 1981; Koller et al., 1982; Perumalla and Peterson, 1986; Zeier et al., 1999). Cutin is
found in plant stems, leaves, and fruits; while suberin is found in bark and roots (Kögel-
Knabner, 2002; Kolattukudy, 1980, 1981; Perumalla and Peterson, 1986). These polymers are
on the surface of plant cells that are part of protective tissues (Buchanan et al., 2000; Jeffree,
2006).
Both cutin and suberin contain polymeric ethers or esters and phenolic components are
only found in suberin (Fig. 1.3). Cutin is composed of monomers that have lower molecular
weights, dominated by C16 and C18 units, compared to the C18-C30 units in suberin (Fig.
1.3a;(Kögel-Knabner, 2002; Kolattukudy, 1980, 1981). Cutin monomers are also more likely to
have alkene substitutions, and are more oxidized mid-chain through epoxide and hydroxyl
substitutions; unlike suberin, which are thought to not have mid-chain substitutions (Fig.
1.3a;(Kögel-Knabner, 2002; Kolattukudy, 1980). The aromatic structures in suberin (Fig. 1.3b)
are similar to lignin precursors (Fig. 1.4). Although there have been several studies on cutin and
suberin (Arrieta-Baez and Stark, 2006; Deshmukh et al., 2005; Fang et al., 2001; Kolattukudy,
1980), their exact covalent structures are still largely unknown (Deshmukh et al., 2005; Fang et
al., 2001).
8
Figure 1.3 Cutin and suberin structures with a) the aliphatic monomers constituting cutin and suberin are depicted with n = carbon number, and x = CH3, OH, or COOH [based on Bernards (2002), Franke et al. (2005), Kogel-Knabner (2002), and Kolattukudy (1980)], and b) models of cutin polymer, where n = number of repeating units [based on Franke et al. (2005), Kogel-Knabner (2002), and Kolattukudy (1980)], and suberin polymer attached to the cell wall where S = remainder of the suberin polymer (Bernards, 2002); 2008 Canadian Science Publishing, reproduced with permission).
n
n
n
n
n nn
Epoxide(cutin)
Alkene(cutin)
Unsubstitutedcarboxylic acid
(cutin & suberin)
Hydroxylatedmid-chain
(cutin)
a)
b)
n
Unsubstitutedn-alkanol(suberin)
n
cell
wal
l car
bohy
drat
es
Cutin polymer Suberin polymer
9
Cutin and suberin are degraded by Streptomyces sp., and various fungi (Kolattukudy,
1981, 1985). These polymers then become part of soil OM (Chefetz, 2007; Feng and Simpson,
2008; Otto et al., 2005), and abundance of cutin- or suberin-derived structures is used to
determine the contributions of shoot- and root-derived polymers to soil OM (Feng and Simpson,
2008; Mendez-Millan et al., 2010b; Otto et al., 2005). The stability of cutin has also allowed its
use in paleobotanical studies (Gupta et al., 2006). Cutan and suberan, which are thought to be
more condensed, less oxidized forms of cutin and suberin, may also be associated with plants
(Kögel-Knabner, 2002). However, there is some evidence that these compounds may not be
produced by the majority of plant species (Gupta et al., 2006; Kögel-Knabner, 2002), and will
not be discussed.
1.2.5 Lignin
Lignin is the third most abundant plant polymer after cellulose and hemicellulose; and is
important in maintaining plant structure, and in protecting plant tissues (Buchanan et al., 2000;
Kögel-Knabner, 2002). The polymer is a component of plant leaves, stems, and roots; and is
found in tissues responsible for nutrient transport and protection (Buchanan et al., 2000). As
such, lignin may be associated with hemicellulose, cutin and suberin (Buchanan et al., 2000;
Kögel-Knabner, 2002; Perumalla and Peterson, 1986; Zeier et al., 1999). White-rot fungi are
commonly responsible for lignin degradation, while brown-rot fungi and Streptomyces sp. are
also capable of degrading lignin but to a limited extent (Antai and Crawford, 1981; Atlas and
Bartha, 1998; Bergbauer and Eggert, 1994; Kirk et al., 1976; 1978; Sylvia et al., 1999).
To synthesize lignin, three types of precursors (Fig. 1.4a) are thought to be polymerized
to form an amorphous, poly-aromatic structure (Fig. 1.4c;(Buchanan et al., 2000; Koenig et al.,
2010; Kögel-Knabner, 2002). The three most common inter-aromatic bonds are depicted in Fig.
1.4b, with -O-4 linkages being the most common (Koenig et al., 2010; Kögel-Knabner, 2002).
Lignin has been characterized using: Time of Flight – Secondary Ion Mass Spectrometry (MS)
(Kleen, 2005; Saito et al., 2005; 2008), analysis of lignin monomer and dimers using gas
chromatography/ mass spectrometry (GC/MS) (Hatfield and Chaptman, 2009; Hedges and Ertel,
1982); nuclear magnetic resonance (NMR) spectroscopy of lignin extracts (Koenig et al., 2010;
Nimz et al., 1981; Obst and Landucci, 1986); and chemical modelling (Faulon et al., 1994).
However, the structure of lignin is still an area of investigation because of the heterogeneity of
10
Figure 1.4 Lignin composition a) Lignin precursors and monomers isolated after oxidation of the polymer [based on Koenig et al. (2010), and Hedges and Ertel (1982)], b) major inter-aromatic linkages between aromatic structures [based on Kogel-Knabner, (2002)], and c) model lignin polymer (Adler, 1977); Springer Sciences and Business Media, reproduced with permission).
OC H3
OH
OCH3
O
H
p-coumaryl alcohol
coniferylalcohol
sinapylalcohol
a)
b)
c)
coumarylvanillyl syringyl
Precursors Products
X = H, OCH3 R = H, OH, CH3
4
1
1
4
1
5455
1
14
4
-O-4 -55-5
11
the polymer; and the difficulty in isolating the intact polymer because of its size and insolubility
(Buchanan et al., 2000). These challenges are similar to those faced in attempts to determine the
exact structure of cutin and suberin (Buchanan et al., 2000). Nevertheless, lignin aromatic
composition is known to change with plant maturity and tissue location (Guillaumie et al., 2007;
Meyer et al., 1998; Nimz et al., 1981). For example, concentrations of syringyl decrease as
Arabidopsis and maize mature, and are less abundant in maize leaves (Guillaumie et al., 2007;
Meyer et al., 1998). There is also evidence that lignin composition was controlled by vegetation
type, in that grasses (including corn) have high concentrations of coumaryl monomers, while
only vanillyl monomers are extracted from woody gymnosperm (Nimz et al., 1981; Tareq et al.,
2004). The observed environmental stability of lignin-derived phenols (detected by GC/MS
after CuO oxidation) decreases in the order vanillyl > syringyl > coumaryl; since vanillyl
monomers are enriched in samples at a more advanced stage of degradation (Bahri et al., 2006;
Goni et al., 1993). The composition of lignin may therefore influence the degradability and
contribution of various plants and plant tissues to soil OM upon plant decay.
1.3 Methods Used to Characterize Soil Organic Matter
Methods used to characterize soil OM can be grouped into four categories as
summarized in Table 1.1. Bulk elemental analysis provides carbon and nitrogen concentrations
after chemical or thermal reactions, and provides broad information on soil OM content
(Chatterjee et al., 2009; Schimel et al., 1994). Methods that determine structural composition
include Fourier transform infrared (FTIR) and NMR spectroscopy. The structural information
obtained using FTIR and NMR spectroscopy is diagnostic of contributions from groups of
compounds, which may have different reactivity in soils (Chefetz, 2007; Preston, 2001;
Schnitzer, 1991; Simpson et al., 2011). To identify specific compounds, GC/MS is often
utilized. This is a targeted approach, and may involve thermal (pyrolysis) or chemical
preparation of OM (Kögel-Knabner, 2000). The bulk elemental and GC/MS methods may also
be combined with isotope analysis to determine OM age and turnover (Cayet and Lichtfouse,
2001; Kögel-Knabner, 2000; von Lutzow et al., 2007; Wang et al., 1996). Since the methods
summarized in Table 1.1 provide different perspectives on OM, several methods are often
combined to gain a more complete picture of soil OM fate and composition.
12
Table 1.1 Summary of methods used to analyze soil organic matter.
Type of Method Examples Information obtained
Elemental composition (Chatterjee et al., 2009; Schimel et al., 1994)
C, N, P content, C/N ratio OM content, amount of limiting nutrients, and microbial- vs. plant-derived OM contribution
Structural Composition (Chefetz, 2007; Preston, 2001; Simpson et al., 2011)
Fourier Transform Infrared (FTIR) spectroscopy
13C solid-state nuclear magnetic resonance (NMR) spectroscopy
13C or 1H solution-state NMR
Structures found in OM (i.e. carboxyl, aromatic, polymethylene), 13C NMR and FTIR of soil OM may have broad overlapping peaks; solution-state 1H NMR is better resolved and multi-dimensional solution-state 1H NMR can give more specific structural information.
Identification of specific compounds using GC/MS (Kögel-Knabner, 2000; Otto and Simpson, 2005; Otto et al., 2005)
Extraction using solvents prior to gas chromatography/mass spec-trometry (GC/MS; lipids, water extractable OM)
Chemolysis (CuO oxidation, base hydrolysis, acid hydrolysis) or thermolysis prior to GC/MS
Some compounds are specific to certain organisms such as lignin in terrestrial plants, ergosterol from fungi. OM contributions from groups of organisms can therefore be derived.
Age and turnover of OM (Cayet and Lichtfouse, 2001; Kögel-Knabner, 2000; von Lutzow et al., 2007; Wang et al., 1996; Yanni et al., 2011)
Can analyze bulk carbon or specific compounds (when combined with biomarker methods) using high resolution MS
13C, 14C, 15N detection
13C turnover determined in soils grown with wheat (C3) and maize (C4) plants, which have different 13C abundance.
Isotopically labelled plants have an excess of 13C or 14C, which can be detected.
14C decay in fossil carbon
13
1.3.1 Biomarkers
Biomarkers are compounds whose structures can be traced to their biological source,
since some compounds can be specific to particular groups of organisms (Section 1.2;(Amelung
et al., 2008; Kögel-Knabner, 2000). For example, lignin, cutin, and suberin are derived from
plants, while peptidoglycan is derived from bacteria. Biomarker analysis is used to determine
the fate of biologically-derived compounds in soils and to characterize the structural
composition of soil OM (Amelung et al., 2008; Kögel-Knabner, 2000; Simpson et al., 2008).
GC/MS or liquid chromatography (LC)/MS are used to determine biomarker concentrations
(Amelung et al., 2008; Hedges et al., 2000; Kögel-Knabner, 2000; Poirier et al., 2005). As such,
the compounds of interest must have the appropriate molecular weight, and high molecular
weight polymers must be broken down to monomers or dimers (Kögel-Knabner, 2000; Simpson
et al., 2008). Biomarkers can therefore be divided into two general categories: compounds that
can be directly extracted from soils and organisms, and compounds that can be analyzed after
thermolysis or chemolysis.
1.3.1.1 Solvent extractable biomarkers
Directly extractable compounds are isolated using solvents that have different polarities
such as methanol, dichloromethane, or ether. These biomarkers are composed of decomposition
products, carbohydrates (Fischer et al., 2007), and lipids (Bull et al., 2000a; 2000b; Naafs et al.,
2004; Otto and Simpson, 2005; Quenea et al., 2006; Wiesenberg et al., 2010; Ziegler and Zech,
1989). Analysis of low molecular weight degradation products is important in determining the
composition of OM that may be lost through leaching to the water table (Fig. 1.1;(Fischer et al.,
2007). Besides degradation products, the source and dynamics of total lipids are commonly
evaluated because lipid extracts can also be diagnostic of OM source, as discussed in section
1.2.3. For example, lipids with >C20, and phytosterols are thought to be plant-derived;
ergosterol is derived from fungi; and branched-chain fatty acids and n-alkane with even carbon
number are likely derived from microorganisms (Bull et al., 2000a; 2000b; Naafs et al., 2004;
Otto and Simpson, 2005; Wiesenberg et al., 2010). By analyzing lipid concentrations,
Wiesenberg (2010) found increased concentrations of microbial-derived lipids, and decreased
contributions from plant-derived lipids upon converting grassland soil to wheat monoculture.
Otto and Simpson (2005) also found that microbial-derived lipids were minor contributors to
14
grassland soils in Alberta. Solvent extractable OM can therefore be diagnostic of soil OM
dynamics.
1.3.1.2 Thermal and chemical cleavage of polymers in soil organic matter
Plant-derived polymers such as cellulose, lignin, cutin, and suberin; and microbial
derived polymers such as peptidoglycan, and chitin must first be thermally or chemically
cleaved to their constituent monomers and dimers to facilitate analysis. Although thermal
cleavage (analytical pyrolysis) has been used in several studies (Kögel-Knabner, 2000;
Leinweber and Schulten, 1999; May et al., 1977; Nierop, 1998), the following discussion will
focus on chemolysis and CuO oxidation in particular, which is a key method used throughout
this thesis and in modern soil OM studies (Bahri et al., 2006; 2008; Feng and Simpson, 2007;
Feng et al., 2011; Heim and Schmidt, 2007b; Kiem and Kögel-Knabner, 2003; Kögel-Knabner,
2000; Otto and Simpson, 2006a; Wysocki et al., 2008).
Chemolysis involves cleavage of bonds through chemical reactions. Similar to
pyrolysis, products of chemolysis may be non-volatile and are reacted with methylating or
silylating reagents to make them amenable to GC. Afterwards, MS and flame ionization
detectors are used to detect the chemolytic products (Kögel-Knabner, 2000; Said-Pullicino et al.,
2007). Chemolytic methods include acid hydrolysis of carbohydrates and proteins
(Guggenberger et al., 1994; Kiem and Kögel-Knabner, 2003; Poirier et al., 2005; Said-Pullicino
et al., 2007; Schnitzer, 1991); base hydrolysis of cutin and suberin (Bull et al., 2000a; Otto et al.,
2005); CuO oxidation, tetramethyl ammonium hydroxide facilitated cleavage, and thioacidolysis
of lignin (Bahri et al., 2006; Hedges and Ertel, 1982; Otto and Simpson, 2006a; Wysocki et al.,
2008); and CuO oxidation of cutin (Goni and Hedges, 1990a; Mendez-Millan et al., 2010a).
Because specific structures are targeted by each chemolytic method, these have the advantage of
reducing soil OM complexity, and the investigator can focus on a specific group of compounds.
For example, after acid hydrolysis of soil samples, the relative contributions of microbial- and
plant-derived carbohydrates (mannose + galactose/arabinose + xylose) can be determined
(Guggenberger et al., 1994; Oades, 1984; Poirier et al., 2005; Said-Pullicino et al., 2007).
Increased contributions from microbial-derived carbohydrates have been observed during
decomposition of compost (Said-Pullicino et al., 2007). Higher concentrations of roots
15
compared to stems and leaves with soil depth, have also been observed by comparing
contributions of cutin- and suberin-derived biomarkers (Feng and Simpson, 2007).
Mild alkaline CuO oxidation is used to de-polymerize lignin, and is becoming widely
used. For example, a study by Hedges and Ertel (1982) alone has been cited 278 times in the
last twelve years. This method is estimated to be 30-90% efficient at extracting lignin phenols
(Ertel and Hedges, 1984), and is slightly more sensitive than other chemolytic methods such as
tetramethyl ammonium hydroxide extraction (Wysocki et al., 2008) and thioacidolysis (Zeier
and Schreiber, 1998). CuO oxidation releases p-hydroxyphenols, and lignin-specific phenols
from the lignin polymer (Fig. 1.5;(Goni et al., 1993; Hedges and Ertel, 1982), which can be
detected by GC/MS or GC/flame ionization detector. Of the eight lignin phenol monomers,
syringyl and vanillyl phenols are lignin-specific, while coumaryl phenols are also thought to
occur in suberin (Opsahl and Benner, 1995).
The relative concentrations of the various monomers is used in characterizing the
oxidation stage (Bahri et al., 2006; Goni et al., 1993; Guggenberger et al., 1994; Hedges et al.,
1988; Otto and Simpson, 2006a), and vegetation source (Goni and Hedges, 1990b; Goni et al.,
1993; Otto and Simpson, 2006a; Tareq et al., 2004) of lignin. That ratios of vanillic
acid/vanillin (Ad/Alv), and syringic acid/syringaldehyde (Ad/Als) increased upon lignin
degradation was established following incubation of birch with white rot fungi (Hedges et al.,
1988). Thereafter, Ad/Alv, and Ad/Als have been used to determine the degradation state of
lignin under different temperatures, soil management techniques, and plant biomass input, such
that factors that contribute to lignin preservation in soil OM may be characterized (Feng and
Simpson, 2007; 2011;(Feng et al., 2008; Heim and Schmidt, 2007a; Opsahl and Benner, 1995;
Otto et al., 2005; Yanni et al., 2011). Although less widely utilized, decreased concentrations
and increased oxidation (increased carboxy phenol dimer concentrations) of lignin phenol
dimers, is also diagnostic of the levels of lignin degradation (Goni and Hedges, 1992; Opsahl
and Benner, 1995; Otto and Simpson, 2006a). Ratios of syringyl/vanillyl phenols and
coumaryl/vanillyl phenols are diagnostic of contributions from woody flowering plants, non-
woody flowering plants, woody non-flowering plants, and non-woody non-flowering plants
(Goni and Hedges, 1990b; Otto and Simpson, 2006a; Tareq et al., 2004; Wysocki et al., 2008).
For example, only vanillyl phenols are extracted from woody non-flowering plants (i.e. pine),
while all three groups of lignin phenols are extracted from non-woody flowering plants (i.e.
16
Figure 1.5 Phenol monomer and dimers extracted after CuO oxidation based on Goni et al. (1993), and Hedges and Ertel (1982).
maize;(Goni and Hedges, 1990b; Tareq et al., 2004; Wysocki et al., 2008). As with other
biomarker methods, CuO oxidation coupled to GC/MS may be combined with 13C isotope
analysis to determine the turnover of lignin in soils (Bahri et al., 2006; Heim and Schmidt,
2007a). Besides lignin phenols, CuO oxidation also releases OH-acids from cutin (Goni and
5,5’ Dimer α-2-methyl
α-1-monoketone α-5-monoketone
β-1-diketone4-O-5’
Inter-aromatic Linkages in Lignin Phenol Dimers
CuO oxidation
OH
OHH
H
OH
HH
Vanillin
Acetovanillone
Vanillic acid Syringic acid
Syringaldehyde
Acetosyringone
Ferulic acid
Coumaric acid
Vanillyl Syringyl Coumaryl
Monomers
17
Hedges, 1990a, b; Mendez-Millan et al., 2010a). Although extraction of cutin-derived OH-acids
(described in Section 1.2) by CuO oxidation is not as efficient as base hydrolysis (Mendez-
Millan et al., 2010a), it is nevertheless useful in determining relative contributions of cutin to
soil OM (Goni and Hedges, 1990a, b).
1.3.2 Nuclear Magnetic Resonance Spectroscopy
NMR spectroscopy can detect NMR-active nuclei, such as 1H, 13C, 15N, and 31P
(Silverstein and Webster, 1998), and NMR spectroscopy methods that detect 1H, 13C, and 15N
nuclei are commonly used because of their abundance in soil OM (Kögel-Knabner, 2000;
Preston, 2001; Simpson et al., 2011). NMR spectroscopy methods, in contrast to biomarker-
GC/MS methods, are non-targeted approaches to analyzing soil OM. Since a large portion of
OM is uncharacterized (Hedges et al., 2000), soil OM components that may not be amenable to
biomarker approaches may be detected using NMR spectroscopy. Because the composition of
soil OM is very complex, NMR analysis results in signal overlap, which limits our ability to
determine the concentration of specific compounds (i.e. fructose may not be differentiated from
other carbohydrates). However, the relative abundance of structural groups in the resulting
spectra can be related to the relative concentration of their source compounds (Kögel-Knabner,
2000; Mao et al., 2000; Preston, 2001; Schnitzer, 1991; Simpson et al., 2011). For example,
polymethylene signals attributed to lipids, cutin and suberin can be differentiated from aromatic
signals attributed to lignin (Feng and Simpson, 2011; Simpson et al., 2008; Simpson et al.,
2011). These techniques have been used to analyze whole soil and NaOH extractable humic
and fulvic acids (Baldock et al., 1992; Golchin et al., 1996; Kelleher and Simpson, 2006;
Preston et al., 2000; Simpson et al., 2007a; 2007b). Simpson et al. (2011) divided
environmental NMR spectroscopy techniques into four categories: solid-state, semi-solid-state,
solution-state, and imaging. In this thesis, 1-dimensional solid-state 13C CP (cross-
polarization)/MAS (magic angle spinning) NMR, and solution-state 1H NMR spectroscopy are
used because these methods give complementary information on OM composition.
1.3.2.1 Solid-state 13C nuclear magnetic resonance spectroscopy
Solid-state 13C NMR spectroscopy is widely used to investigate the structural
composition of soil OM (Kögel-Knabner, 2000; Preston, 2001; Schnitzer, 1991; Simpson et al.,
2011). This method requires minimum sample preparation, and soils with high organic carbon
18
(>17%), and low paramagnetic compound concentrations (C/Fe > 1) can be directly analyzed
without pre-treatment (Baldock et al., 1992; Kinchesh et al., 1995; Preston, 2001; Schmidt et al.,
1997). However, most soils must be treated with chemicals such as hydrofluoric acid to remove
the majority of minerals thereby concentrating OM, and decreasing the concentration of
paramagnetic compounds (Rumpel et al., 2006; Schmidt et al., 1997). Sensitivity is further
improved by using cross-polarization, which results in a 4x signal enhancement and decreased
experiment time (Simpson et al., 2011). Because the orientation of structures in solids is more
rigid than in solutions, samples must also be spun at the magic angle (MAS at 54.7o) to the
applied magnetic field, to decrease the broadening of spectra caused by dipolar coupling
(Andrew, 2010). Fig. 1.6 illustrates the information derived from solid-state 13C NMR spectra.
A solid-state 13C NMR spectrum can be divided into general regions, which correspond to
structures found in lipids, cutin, suberin, and peptide side-chains (alkyl); carbohydrates (O-alkyl
and anomeric); lignin (O-alkyl, aromatic, and carboxyl); and degradation products (carboxyl)
(Fig. 1.6;(Baldock et al., 1992; Preston et al., 1997; 2000; Simpson et al., 2008).
Structural differences detected by solid-state 13C NMR spectroscopy have been used to
determine the relative degradation of soil OM components, such as carbohydrates, lignin, and
cutin (Almendros et al., 2000; Feng et al., 2008; 2011; Gregorich et al., 1996; Schnitzer, 1991),
and the synthesis of microbial-derived compounds (Golchin et al., 1996). In most cases, the
alkyl concentrations increased relative to O-alkyl concentrations as plant and litter were
degraded (Adani et al., 2006; Almendros et al., 2000; Chabbi and Rumpel, 2004; Feng et al.,
2008; 2011; Gregorich et al., 1996; Simpson et al., 2008), and increased alkyl/O-alkyl ratios
suggest increased soil OM degradation (Simpson et al., 2008). However, because the magnitude
of change in alkyl/O-alkyl ratios is sometimes minor, and appears to depend on the plant species
(Almendros et al., 2000), contributions from other structures (Fig. 1.6; carbohydrate- and lignin-
derived O-alkyl, aromatic and carboxyl) must still be considered. Upon degradation of plants,
the intensity of lignin-derived aromatic, O-alkyl signals and cutin, suberin and lipid-derived
alkyl signals increase (Adani et al., 2006; Almendros et al., 2000; Chabbi and Rumpel, 2004).
1.3.2.2 Solution-state 1H nuclear magnetic resonance spectroscopy
Solution-state NMR spectroscopy is used to determine the distribution of structures in
OM extracted from soils and dissolved in an appropriate solvent (usually D2O or DMSO-d6).
19
Figure 1.6 Solid-state 13C NMR spectrum of soil organic matter, with information on the general chemical shift regions based on Preston et al. (1997) and Quideau et al. (2001).
200 180 160 140 120 100 80 60 40 20 0Chemical Shi ft (ppm) 04080120160200
Chemical Shift (ppm)
1
2
2b2a
34a
4b
5
4
1a
1b
Structure
(chemical shift in ppm)
1. Alkyl (10-50)
2. O-alkyl (50-95)
3. Anomeric (95-110)
4. Aromatic (110-160)
5. Carbonyl (160-230)
Assignment and Potential Sources
Polymethylene in lipids, lignin, amino acids/peptides, cutin and suberin from plants.
The region at 10-20 ppm (1a) is assigned to terminal methyl groups, while the region between 20-50 ppm (1b) is assigned to mid-chain methylene groups.
Lignin and carbohydrates.
Lignin-derived signal observed at 56 ppm (2a), while carbohydrate-derived signal observed at 63-65 ppm (2b).
Carbohydrates
Lignin, proteins, and black C. The region observed at 110-140 ppm (4a) is assigned to other aromatic C and the region observed at 140-160 ppm (4b) is assigned to phenols.
Lignin, peptides, and degradation products.
The region between 160-200 ppm is assigned to carboxyl C, and carbonyl C is observed at 160-230 ppm.
20
13C NMR has greater dispersion than 1H NMR spectroscopy (Silverstein and Webster, 1998).
However, compared to signals in solid-state 13C NMR spectra of soil OM, solution-state 1H
NMR spectroscopy signals are usually more resolved because of the free movement of
molecules, which results in sharper signals (Andrew, 2010). Solution-state 1H NMR of soil OM
is therefore considered to provide more structure-specific information than solid-state 13C NMR
spectroscopy, but is a less targeted approach than biomarker-GC/MS methods (Feng and
Simpson, 2011). One type of solution-state method, diffusion edited (DE) 1H NMR
spectroscopy, enhances signals from relatively large molecules (Simpson et al., 2011). In DE 1H NMR spectroscopy, the sample tube is subjected to a magnetic gradient (Fig. 1.7; magnetic
strength 1-4). This gradient helps detect molecular motion because a compound that has
diffused to an area with a different gradient strength will not be observed (Fig. 1.7;(Lam and
Simpson, 2009; Simpson, 2002; Simpson et al., 2011). The likelihood of diffusion is
determined, in part, by molecular weight since according to the Stokes-Einstein equation (Fig.
1.7), the frictional coefficient is larger and thus the diffusion coefficient is smaller for molecules
that have larger radii (Laidler et al., 2003). The greater number of solvent molecules, which
must be disturbed by a larger molecule to allow its diffusion, results in greater resistance to
movement (a larger frictional coefficient), requiring more time to diffuse (Fig. 1.7 t2;(Laidler et
al., 2003). DE 1H NMR spectroscopy therefore emphasizes signals from relatively large, or
aggregated compounds (Simpson et al., 2011).
By comparing one-dimension 1H NMR and DE 1H NMR spectra to those from previous
studies, which further assigned these signals using multi-dimensional experiments and analyzing
purified compounds, changes in signal intensity can be related to changes in concentrations of a
group of compounds (Feng et al., 2008; 2011; Feng and Simpson, 2011; Simpson et al., 2003;
2007a; 2007b; 2011). Similar to solid-state 13C NMR signals, chemical shift ranges correspond
to structures derived from aliphatics (lipid, cutin, suberin, and protein side-chains),
carbohydrates, lignin, and proteins (Fig. 1.8). For example, peptide (aliphatic side chains, 1H,
and amide), and peptidoglycan (N-acetyl) signals were more intense in cultured microbes
(Simpson et al., 2007a), and increased intensities from these signals indicate increased
contributions from microbial-derived OM. The most effective application of solution-state 1H
NMR spectroscopy in soil OM research is in combination with biomarker methods, and solid-
state 13C NMR spectroscopy (Feng et al., 2008; 2011; Hertkorn et al., 2002; Pautler et al., 2010).
21
Figure 1.7 Detection of relatively large compounds using diffusion edited (DE) NMR, where time needed for particle 1 to diffuse is less than particle 2 (t1 < t2). Solvent is represented as triangles associated with the particles [based on Lam and Simpson (2009), Simpson et al. (2011), and Laidler et al. (2003)].
Figure 1.8 Solution-state diffusion edited (DE) 1H NMR can detect differences in signals from plant, soil and cultured microbe extracts, where vertical arrows in the soil spectrum indicate similar signals between cultured microbes and soils (adapted with permission from Simpson et al. (2007a); 2007 American Chemical Society).
Using all three methods, changes in OM composition during bio- and photo-degradation
experiments, and upon disturbance of Arctic environments were observed (Feng et al., 2008;
2011; Pautler et al., 2010).
1.4 Mechanisms Responsible for Soil Organic Matter Accumulation
The existence of soil OM indicates that at some point, the rate of input of organic
material from various organisms exceeded the degradation rate (Janzen et al., 1998).
Radiocarbon analyses suggest that a fraction of soil OM may be a few hundred to a few
Diffusion coefficient = (BT)/(6r)
where: B = Boltzmann constantT = temperaturer = particle radius
n = solvent viscosity6r = frictional coefficient
Mag
netic
grad
ient
t2
t1
Magnetic strength 1
Magnetic strength 2
Magnetic strength 3
Magnetic strength 4Lignin +
carbohydrate OCH3
Lignin + protein aromatic
Aliphatic CH2 and CH3
Protein amide
Lignin + Carbohydrate
Vegetation
Lignin
Soil
Cultured Microbes Side Chain Protons
22
thousand years old, depending on land management, and depth, with older OM in undisturbed
soil, associated with minerals, and at greater depth (Gupta et al., 2006; Krull et al., 2006;
Tiessen et al., 1994; von Lutzow et al., 2007; Wang et al., 1996). These data indicate that there
may be components of OM in soils that are resistant to degradation, and characterizing the
mechanisms responsible for this level of recalcitrance or stabilization is an active area of
research. There are currently two types of protection mechanisms that are hypothesized to be
responsible for inhibiting OM degradation in soils: the inherent recalcitrance of the compound,
and physical protection through interactions between OM and the soil mineral phase (Baldock
and Skjemstad, 2000; Christensen, 1987; Marschner et al., 2008; Mikutta et al., 2006b; Six et
al., 2002; von Lutzow et al., 2007; 2008; Zech et al., 1997).
1.4.1 Inherent Chemical Recalcitrance
Inherent recalcitrance refers to the resistance of OM to degradation, which can be
attributed to the structure, chemical, and physical properties of the molecule (Baldock and
Skjemstad, 2000; Lorenz et al., 2007; Six et al., 2002). In particular, the rate and extent of
degradation of a compound can be controlled by various kinetic and thermodynamic
considerations, such as bond dissociation energy and stability of products relative to reactants
(Kleber, 2010). However, living organisms (especially microorganisms) are thought to be
important in breaking down soil OM (Kleber, 2010; Powlson et al., 1987; von Lutzow et al.,
2008). Therefore, the costs of degrading a particular substrate (enzyme production, transport)
are balanced against the benefits of utilizing that substrate (amount of bond energy harvested,
and carbon available for biomass synthesis), as observed by several studies that considered the
degradation rates of pure compounds, such as sugar and fatty acids (Gommers et al., 1988;
McDermitt and Loomis, 1981). Furthermore, polymerization increases the resistance of a
compound to degradation, since inter-molecular linkages must first be broken to produce a
compound that is small enough to be transported into a cell (Atlas and Bartha, 1998; Baldock et
al., 2004; Warren, 1996). Another important consideration is the availability of the substrate to
organisms and degrading enzymes, as indicated by a positive correlation between water
solubility and degradability of various soil OM compounds (Kleber, 2010). Therefore, smaller,
water soluble compounds, and those that require less biological investment to degrade while
providing sufficient carbon and energy source are expected to be less recalcitrant.
23
The relative degradability of carbohydrates, proteins, lipids, lignin, cutin and suberin
from plants have been assessed using degradation studies; wherein compounds that are depleted
faster are more labile (Aerts and deCaluwe, 1997; Baldock and Skjemstad, 2000; Machinet et
al., 2011; Taylor et al., 1989; von Lutzow et al., 2007). According to these studies,
carbohydrates and proteins are labile; while lignin was relatively recalcitrant. Lignin was an
important determinant of plant biomass degradation rate (Ghidey and Alberts, 1993; Johnson et
al., 2007; Melillo et al., 1982; Moretto and Distel, 2003; Preston et al., 2000). Although lignin
is considered more recalcitrant than carbohydrates, polymethylene compounds (lipids, cutin and
suberin) are thought to be the most recalcitrant components of soil OM (Lorenz et al., 2007).
This hypothesis is derived from the enrichment of polymethylene signals in NMR spectra and
cutin- and suberin-derived biomarkers as plant material is degraded (Feng et al., 2008; Feng and
Simpson, 2011; Kelleher and Simpson, 2006). The polymethylene signal is also enriched in soil
fractions, such as clay, that contain older OM (Baldock et al., 1992; Guggenberger et al., 1995;
von Lutzow et al., 2007).
Given the relative resistance of carbohydrates, lignin, cutin, and suberin against
degradation, their abundance in plant tissues may also influence tissue degradation in soil.
Several studies found that plant roots have higher concentrations of lignin compared to leaves
and stems, which may be responsible for less mass loss, and CO2 production in soils amended
with roots compared to those amended with leaves and stems (Johnson et al., 2007; Moretto and
Distel, 2003; Yanni et al., 2011). Enrichment of root-derived suberin in soils and soil horizons
also suggest preferential stabilization of root-derived carbon in soil (Feng and Simpson, 2007;
Mendez-Millan et al., 2010b; Nierop, 1998; Otto and Simpson, 2006b; Rumpel et al., 2002). In
one study, grass degradation was monitored over time using NMR spectroscopy, which also
showed less alteration of soil OM structures in soils amended with grass roots, compared to soils
amended with grass blades (Kelleher and Simpson, 2006), further supporting the relative
recalcitrance of root-derived compounds. However, although polymethylene compounds are
recalcitrant relative to lignin and carbohydrates, incubation studies of cutin indicate that these
compounds are not immune to microbial degradation (Chefetz, 2007; Hauff et al., 2010;
Kolattukudy, 1980, 1985; Stimler et al., 2006). Furthermore, analysis of cutin- and suberin-
derived biomarkers suggest that polymethylene compounds found in soil are more degraded
than those in the parent plant material (Feng and Simpson, 2007; 2008; Otto and Simpson,
24
2006b). Protection of OM over hundreds to thousands of years may therefore involve other
factors, including interactions with the soil mineral fraction, and it is important to determine
whether this protection is specific to the structure of OM.
1.4.2 Physical Protection of Soil Organic Matter
Whole soil can be separated based on size and density to generate the sand-, silt-, clay-
size, and light (uncomplexed OM) fractions (Cambardella and Elliott, 1993; Christensen, 2001;
Gregorich et al., 1996; Schulten and Leinweber, 1999). Density fractionation is achieved by
suspending whole soil in either a salt solution (sodium iodide or sodium polytungstate), which
was adjusted to a specific density (1.2-2.0 g/mL), or water (Blackwood, 2003; Cambardella and
Elliott, 1993; Christensen, 2001; Gregorich et al., 1996; Marriott and Wander, 2006; Schulten
and Leinweber, 1999). The least dense fraction (light fraction), floats in these solutions, is
mainly composed of OM that is not associated with minerals (and therefore not physically
protected), and includes relatively recent plant-derived OM deposits (Gregorich et al., 1996;
Janzen et al., 1992; Leifeld and Kögel-Knabner, 2005; Marriott and Wander, 2006; Six et al.,
2002). The structures observed in this fraction are at earlier stages of decomposition compared
to those associated with mineral fractions, and therefore more closely resembles those of
overlying vegetation, (Cambardella and Elliott, 1993; Gregorich et al., 1996; Janzen et al., 1992;
von Lutzow et al., 2007; Zech et al., 1997). Compared to mineral fractions, carbon associated
with the light fraction is characterized by higher lignin biomarker concentrations (Gregorich et
al., 1996; Leifeld and Kögel-Knabner, 2005); nitrogen that is in a more stable form, likely
sequestered in undegraded biomass (Sollins et al., 1984); and microbial populations that are
more similar to populations closely associated with plants, such as those found in the
rhizosphere (Blackwood, 2003). OM associated with the light fraction are also younger (based
on isotope analysis), and have faster degradation rates compared to those associated with
mineral fractions (Gregorich et al., 1995; 1996; von Lutzow et al., 2007).
The characteristics of soil mineral fractions (sand-, silt-, and clay-size) are summarized
in Table 1.2. Because sand-size fractions have the largest particle size, smallest surface
area/volume ratio, and have the least reactive surface; plant-derived OM associated with this
fraction are degraded more quickly than those associated with silt- and clay-size fractions
(Christensen, 2001; Kögel-Knabner et al., 2008; Six et al., 2002; von Lutzow et al., 2007). In
25
Table 1.2 Characteristics of sand-, silt-, and clay-size fractions (Baldock et al., 1992; Bergaya and Lagaly, 2006; Christensen, 2001; Guggenberger et al., 1994; Heim and Schmidt, 2007a; Velde and Menunier, 2008).
Sand Silt Clay
Size (µm) 20-2000 2-20 <2
Surface area (N2-BETa) <10 m2g-1 10-50 m2g-1 25-100 m2g-1
Mineral 1o minerals More 1o minerals than clay, more weathered than sand
Inorganic precipitates and highly weathered (2o) minerals
C/N Large Medium Small
OM relative to totalb soil
<10% 20-40% 50-75%
OM properties Mainly from plants Mainly plant-derived aromatic
Microbial products, plant-derived OM depleted
aAdsorption isotherm is calculated based on the Brunauer-Emmett-Teller equation where clay is exposed to different concentrations of nitrogen, and surface area is calculated based on monolayer sorption of nitrogen on the clay surface (Laidler et al., 2003). bAssociated OM is influenced by contributions of each particle-size fraction to whole soil.
contrast, the silt- and clay-size fractions contains 70-90% of total soil OM, which are believed to
be older, and have slower turnover times (von Lutzow et al., 2007). These fine soil fractions are
therefore thought to be responsible for stabilizing soil OM (Christensen, 2001; Guggenberger et
al., 1994; 1995; Kaiser and Guggenberger, 2000, 2007; Six et al., 2002; von Lutzow et al., 2007;
Zech et al., 1997). This stability is attributed to protection of OM in silt-, and clay- size
aggregates; and physico-chemical interactions between OM and clay, which limit access of
degrading enzymes to OM (Chenu and Plante, 2006; Feng et al., 2005; Kaiser and
Guggenberger, 2007; Mikutta et al., 2007; Mikutta and Kaiser, 2011; Six, 2004; Virto et al.,
2008; 2010; von Lutzow et al., 2007).
Silt-size fractions were found to preferentially sequester lignin-derived phenols, which
were also less oxidized (Heim and Schmidt, 2007a). Although the mechanism responsible for
26
lignin preservation in silt-size fractions has not been elucidated (Heim and Schmidt, 2007a),
protection of lignin in microaggregates observed in silt-size fractions is likely (Chenu and
Plante, 2006). The importance of soil OM protection within aggregates is responsible for
decreased carbon content upon conversion of forest or grassland to agriculture, since agriculture
disturbs aggregates, which releases the sequestered carbon (Guggenberger et al., 1994; Janzen et
al., 1998). Using solid-state 13C NMR spectroscopy, it was found that in contrast to the silt-size
fraction, organic carbon associated with clay-size fractions are dominated by polymethylene
structures, likely derived from plant-derived cutin, and suberin; as well as lipids originating
from plants and microbes (Baldock et al., 1992; Baldock and Skjemstad, 2000; Guggenberger et
al., 1995). 13C isotope abundance in lipid biomarkers was consistent with the hypothesis that
clay-associated polymethylene structures were derived from both plant and microbial biomass
(Quenea et al., 2006). These studies suggest that various polymethylene structures, which are
considered to be chemically recalcitrant (Kögel-Knabner, 2002; von Lutzow et al., 2007), were
also stabilized in the clay-size fraction. Microbial-derived OM were sequestered in clay-size
fractions, as hexoses are enriched relative to pentoses in this fraction (Guggenberger et al.,
1994). There may be contributions from both living and dead microorganisms since sorption to
clay minerals is also hypothesized to enhance microbial survival in soils (Amato and Ladd,
1992; Marshall, 1975). From the differences in the relative abundance of the various OM
structures associated with soil mineral fractions, it appears that organo-mineral interactions may
be structure specific.
OM composition was influenced by the type of clay (Feng et al., 2005; Wattel-Koekkoek
et al., 2001). For example, smectites (i.e. montmorillonite) were observed to sorb greater
concentrations of peptides and aromatic structures compared to kaolinite (Feng et al., 2005;
Wattel-Koekkoek et al., 2001), while goethite sorbed greater concentrations of carboxylic acids
compared to kaolinite and montmorillonite (Ghosh et al., 2009). The most important primary
interactions between clay minerals and OM include ligand exchange, van der Waals, cation
bridging, and hydrophobic interactions (Feng et al., 2005; Mikutta et al., 2007), as described in
Table 1.3. The preferential sorption of specific OM structures by clay minerals may be
attributed to dominant OM-clay interactions, which are controlled by the type of clay mineral
(Feng et al., 2005; Mikutta et al., 2007; Wattel-Koekkoek et al., 2001). For example, Mikutta et
al. (2007) contrasted OM sorption to clay with a hydroxylated amphoteric surface (goethite),
27
Table 1.3 Common interactions between organic matter and clay minerals (Baron et al., 1999; Feng et al., 2005; Laidler et al., 2003; Mikutta et al., 2007).
Interaction Description
Ligand exchange Displacement of surface hydroxyl and water groups by OM.
van der Waals Attraction due to induced polarity in non-ionic compound.
Cation bridging Attraction between a negative OM compound and the permanent negative clay surface charge, mediated by a divalent cation (i.e. Ca2+).
Hydrophobic interaction Attraction due to increased entropy of water molecules as hydrophobic surfaces interact.
Electrostatic Attraction due to differences in surface charge.
neutral siloxane surface (pyrophillite), and a surface with a permanent negative charge
(vermiculite); and found that ligand exchange dominated sorption to goethite, cation bridging
and van der Waals forces dominated sorption to pyrophillite, and cation bridging dominated
sorption to vermiculite (Mikutta et al., 2007). These studies further support the hypothesis that
interactions between OM and clays responsible for OM preservation are both OM and clay
specific.
1.5 Objectives and Hypotheses
The overall objective of this thesis is to investigate factors that contribute to soil OM
accumulation, with emphasis on how these relate to OM chemistry. Various NMR spectroscopy
and biomarker methods have been developed to characterize soil OM composition, and
combining these methods allows us to obtain a better picture of soil OM dynamics (Feng et al.
2010; 2011;(Feng and Simpson, 2011; Pautler et al., 2010; Simpson et al., 2007b; 2011);
Simpson et al. 2008). Furthermore, recent developments in solution-state 1H NMR
spectroscopy methods have provided novel perspectives on the chemistry and nature of soil OM,
and these methods may help us understand OM sequestration in soils. There have been various
studies that characterized the influence of factors such as temperature, precipitation, plant tissue
chemistry, and OM-clay interactions on OM sequestration in soils. However, despite these
efforts, the mechanisms responsible for OM sequestration in soils and soil fractions are still not
28
fully characterized. Although chemical recalcitrance is not as important as OM-mineral
associations in sequestering OM, it is hypothesized that specific structures, such as high
molecular-weight polymethylene compounds, are preferentially preserved over time. This may
be due to specific interactions between polymethylene structures and clay, as well as resistance
of these compounds to degradation. Therefore, to further our knowledge, this research focuses
on examining how structures found in the starting material influences the composition of
sequestered and protected OM, with reference to environment, plant tissue, and OM-mineral
association. The specific objectives of this thesis are as follows:
Objective 1: To compare the structures observed through solid-state 13C NMR, solution-state 1H NMR, and DE 1H NMR spectroscopy, as they relate to soil physical fractions; to further
define how information from these methods can be combined to enhance our understanding of
OM sequestration mechanisms.
Objective 2: To determine the extent of interaction between various factors that influence OM
sequestration mechanisms by comparing the composition of OM in the sand-, silt-, clay-size,
and light fractions of three Canadian Prairie soils that have similar mineralogy, but different
temperature and vegetation.
Objective 3: To compare the relative influence of maize leaf-, stem- or root-tissue amendment
on the changes in lignin phenol and OM composition in soils during a biodegradation study.
Objective 4: To characterize the influence of OM structures sorbed to clay minerals on the
protection of lignin phenols from chemical degradation.
Through these research objectives, the following hypotheses are tested:
1) Polymethylene compounds are enriched in clay-size fractions, while lignin phenols in silt-
size fractions are protected from degradation. It therefore appears that associations between OM
and soil minerals are structure specific. However, temperature and overlying vegetation also
influence OM stability. It is hypothesized that although environment and vegetation influence
carbon sequestration in soils, OM structure will also exert a control on sequestration in fine soil
fractions. This is expected to result in similar enrichment patterns, across different types of soils
29
when the structural composition of relatively fresh OM in light fraction is compared to the OM
composition in fine soil fractions.
2) Contributions from suberin and higher concentrations of lignin are thought to result in
greater contributions from plant roots compared to leaves and stems to soil OM. These studies
are usually carried out by measuring rate of mineralization (i.e. CO2 production) and mass loss.
However, the degradation process is complex in that plant tissue derived OM may be
sequestered in soil, lost through mineralization, or transformed into microbial-derived OM (Fig.
1.1.). We wanted to detect changes in soil OM composition as plant tissues are degraded, since
there are a limited number of studies that address this, and none combine biomarker, solid-state,
and solution-state NMR spectroscopy methods. It is hypothesized that greater concentrations of
more recalcitrant structures in plant tissues may result in less change in OM composition of soils
amended with those tissues during biodegradation.
3) Interactions with clay minerals protect OM from biological, chemical, and physical
degradation, and less OM degradation occurs when interactions between OM and clay minerals
are stronger. It has also been suggested that clay minerals have a finite capacity for protecting
OM. It is hypothesized that interactions between OM may also be a factor in inhibiting
degradation. Therefore, lignin in OM-clay complexes, and mineral fractions from Prairie soils
should be protected from chemical (NaClO2) degradation, and the level of protection should
depend on lignin association with compounds (OM-OM interactions) that have low reactivity to
NaClO2.
1.6 Thesis Summary
Chapter 1: Introduction: Soil Organic Matter Components, Measurement, and Sequestration
Chapter 2: Comparison of NMR Methods for the Analysis of Organic Matter Composition
from Soil Density and Particle Fractions
This chapter was published in Environmental Chemistry and addresses objective 1.
Agricultural, Northern Grassland, and Grassland-Forest Transition soils were separated into
particle and density fractions. The OM composition of soils, clay-size and light fractions were
then analyzed using solid-state 13C CP/MAS NMR, solution-state 1H NMR, and solution-state
30
DE 1H NMR spectroscopy to compare the OM composition and degradation level detected by
these methods. Using the abundance of polymethylene compared to carbohydrate and lignin
signals (alkyl/O-alkyl ratios), these methods consistently found more advanced OM degradation
in clay-size compared to light fractions. Solution-state 1H NMR spectroscopy methods were
also able to detect that peptide, peptidoglycan or chitin signals, likely from microbial-derived
OM, were enriched in clay-size fractions. Lignin-derived aromatic signals were found in the
samples using these NMR spectroscopy methods. By combining solid-state 13C and solution-
state 1H NMR spectroscopy methods, which provided complementary information, we were
able to gain a better understanding of the fate and preservation of soil OM.
Chapter 3: Association of Specific Organic Matter Compounds in Size Fractions of Soils
Under Different Environmental Controls
This chapter was published in Organic Geochemistry, and addresses objective 2. The
sand-, silt-, clay-size and light fractions from three soils, which differed in vegetation and
temperature (Northern Grassland, Southern Grassland, Grassland-Forest Transition), were
isolated. Solvent extractable lipids and CuO oxidized lignin phenols were analyzed using
GC/MS, solid-state 13C NMR was used to analyze total OM, while solution-state 1H NMR, and
solution-state DE 1H NMR spectroscopy were used to analyze base-extractable OM (humic
substances). Despite differences in environmental variables, long chain aliphatic compounds
accumulated in the fine fractions of these soils. Microbial-derived OM was also more abundant
in finer soil fractions. The chemical patterns in the soil fractions, suggest that specific
associations between soil minerals and OM may be more important than environmental controls
in sequestering these compounds.
Chapter 4: Comparison of Soil Organic Matter Composition after Incubation with Maize
Leaves, Roots, and Stems
This chapter was submitted to Geoderma, and addresses objective 3. Changes in OM
composition of soils amended with maize leaves, stems, and roots as these tissues degraded over
36 weeks were monitored using GC/MS of CuO oxidation products; solid-state 13C NMR,
solution-state 1H NMR, and solution-state DE 1H NMR spectroscopy. Changes in OM
composition of soils amended with leaves, stems, and roots were compared. During incubation,
there were greater changes in OM composition of soils incubated with stems, likely due to high
31
carbohydrate concentrations in maize stems. Soil amended with leaves had greater
concentrations of aliphatic compounds; therefore leaves are potential sources of stable aliphatic
compounds in soil. Over the 36-week incubation, there were greater contributions from
microbial-derived OM to soils amended with roots. The OM composition of the starting plant
material therefore influenced the relative changes in soil OM composition during
biodegradation. This study demonstrates a link between plant tissue composition and soil OM
turnover.
Chapter 5: Physical Protection of Lignin by Organic Matter and Clay Minerals from Chemical
Oxidation
This chapter is in prep for submission to Organic Geochemistry, and addresses objective
4. Organo-mineral complexes composed of lignin and montmorillonite; lignin, dodecanoic acid
and montmorillonite; and humic acid and montmorillonite (sorbed at pH 4 and 7) were created
using different OM loadings. These samples, together with sand-, silt-, and clay-size fractions
of Southern Grassland, and Grassland-Forest Transition soils were reacted with an acidic
NaClO2 solution. Samples before and after reactions were then extracted after CuO oxidation,
and analyzed using GC/MS to determine changes in lignin phenol oxidation. This study
evaluated whether protection of lignin from chemical degradation through association with
montmorillonite was modified by the presence of other OM structures. For the synthetic
complexes, the results suggest that coating of lignin-clay complexes with dodecanoic acid
protected lignin from degradation, and that the content and possible OM conformation on the
clay influenced the level of protection. It was also found that Grassland-Forest Transition
fractions, which had higher carbon content, and whose OM input was derived from grass and
woody vegetation, contained lignin phenols that were more resistant to chemical oxidation.
Chapter 6: Summary, Conclusions, and Future Research
1.7 Statement of Authorship and Publication Status
Chapter 1: Introduction: Soil Organic Matter Components, Measurement, and Sequestration
Authorship and Contributions: Written by Joyce S. Clemente, with critical comments from
Myrna J. Simpson.
32
Chapter 2: Comparison of NMR Methods for the Analysis of Organic Matter Composition
from Soil Density and Particle Fractions
Authors: Joyce S. Clemente, Edward G. Gregorich, André J. Simpson, Rajeev Kumar, Denis
Courtier-Murias, Myrna J. Simpson
Contributions: JSC and MJS framed the research questions. MJS and EGG provided the soil
samples. JSC, Aleksandra Spasojevic, and Magda Celejewski isolated and extracted the soil
fractions. EGG provided carbon and nitrogen data for the agricultural soil. AJS designed the
NMR acquisition parameters. JSC, RK and DC-M acquired the solid-state 13C NMR data, while
JSC and Dr. Andrew Baer acquired the solution-state 1H NMR, and DE 1H NMR spectroscopy
data. JSC, MJS, and AJS analyzed the data. The paper was written by JSC, MJS and AJS, with
critical input from the co-authors.
Status: Published in Environmental Chemistry 9, 97-107 (2012).
Chapter 3: Association of Specific Organic Matter Compounds in Size Fractions of Soils
under Different Environmental Controls
Authors: Joyce S. Clemente, André J. Simpson, Myrna J. Simpson
Contributions: The study was designed by JSC and MJS. Soil samples were provided by MJS.
Soil samples were fractionated and extracted by JSC with assistance from Katherine Hills and
David Wolfe. NMR acquisition parameters were designed by AJS. Biomarker analyses were
performed by JSC, and NMR analyses were performed by JSC with assistance from Dr. David
McNally. Data analyses were performed by JSC, with guidance from MJS and AJS. The
manuscript was written by JSC and MJS, with critical comments from AJS.
Status: Published in Organic Geochemistry 42, 1169-1180 (2011).
Chapter 4: Comparison of Soil Organic Matter Composition after Incubation with Maize
Leaves, Roots, and Stems
Authors: Joyce S. Clemente, Myrna J. Simpson, André J. Simpson, Sandra F. Yanni, Joann K.
Whalen
33
Contributions: Soils amended with maize tissues and biodegraded were provided by SFY and
JKW. The research questions were framed by JSC, with input from MJS. Sample extractions
were performed by JSC, with assistance from David Wolfe. Biomarker analyses were
performed by JSC. NMR acquisition parameters were designed by AJS. NMR data acquisition
was performed by JSC with assistance from Dr. David McNally. Data analyses were performed
by JSC, with input from MJS. The manuscript was written by JSC and MJS, with critical
comments from the co-authors.
Status: Submitted to Geoderma (in revision).
Chapter 5: Physical Protection of Lignin from Chemical Oxidation by Organic Matter and
Clay Minerals
Authors: Joyce S. Clemente and Myrna J. Simpson
Contributions: JSC and MJS designed the study. Soil samples and humic acid extracted from
peat soil were provided by MJS. Samples were prepared, extracted, and analyzed by JSC with
assistance from Katherine Hills and David Wolfe. Data analysis was performed by JSC with
input from MJS. The chapter was written by JSC and MJS.
Status: In-prep for submission to Organic Geochemistry
Chapter 6: Summary, Conclusions and Future Research
Author: Joyce S. Clemente
Contributions: Dr. R. Sodhi at Surface Interface Ontario, obtained the X-ray Photoelectron
Spectroscopy (XPS) and Time of Flight-Secondary ion Mass Spectrometry (ToF-SIMS) data,
and processed the XPS data. JSC analyzed the ToF-SIMS data with guidance from Dr. Sodhi.
JSC performed the protein-clay interaction experiments, and analyzed the data. The chapter was
written by JSC with critical comments from Myrna J. Simpson.
34
CHAPTER 2
COMPARISON OF NUCLEAR MAGNETIC RESONANCE METHODS FOR THE ANALYSIS OF ORGANIC MATTER COMPOSITION FROM
SOIL DENSITY AND PARTICLE FRACTIONS
Authors: Joyce S. Clemente, Edward G. Gregorich, André J. Simpson, Rajeev Kumar, Denis
Courtier-Murias, Myrna J. Simpson
Contributions: JSC and MJS framed the research questions. MJS and EGG provided the soil
samples. JSC, Aleksandra Spasojevic, and Magda Celejewski isolated and extracted the soil
fractions. EGG provided carbon and nitrogen data for the agricultural soil. AJS designed the
NMR acquisition parameters. JSC, RK and DC-M acquired the solid-state 13C NMR data, while
JSC and Dr. Andrew Baer acquired the solution-state 1H NMR, and DE 1H NMR data. JSC,
MJS, and AJS analyzed the data. The paper was written by JSC, MJS and AJS, with critical
input from the co-authors.
Status: Published in Environmental Chemistry 9, 97-107 (2012). Copyright 2012, reprinted
with permission from CSIRO Publishing.
35
2.1 Abstract
Organic matter (OM) associated with fine soil fractions are hypothesized to be protected from
complete biodegradation by soil microbes. It is therefore important to understand the structure
and stage of decomposition of OM associated with various soil mineral fractions. Solid-state 13C nuclear magnetic resonance (NMR) has been used extensively to investigate the OM
composition of soils and soil fractions. Solution-state 1H NMR is not used as commonly to
study OM associated with mineral fractions but is an emerging tool for analyzing soil OM
because 1H NMR spectra can be better resolved and the information gained complements
structural information obtained from solid-state 13C NMR. This study compares solution-state 1H NMR and solid-state 13C NMR methods for assessing OM degradation and composition in
three different soils, and their light and clay-size fractions. The alkyl/O-alkyl degradation
parameter was consistent across all the NMR methods in that OM associated with clay-size
fractions were at more advanced stages of degradation as compared to that in light density soil
fractions. Solution-state 1H and diffusion edited (DE) 1H NMR results showed the presence of
high concentrations of microbial-derived peptidoglycan and peptide side-chains in clay-sized
fractions. Lignin was also identified in clay-sized fractions using solid-state 13C and solution-
state 1H NMR techniques. The combination of solid-state 13C and solution-state 1H NMR
methods provides a more detailed analysis of OM composition and thereby facilitates a better
understanding of the fate and preservation of OM in soil.
2.2 Introduction
The fate of organic matter (OM) in soil is controlled by a combination of various
biological, chemical and physical processes regulated by soil structure, the availability and
chemical nature of the substrate, as well as interactions with soil mineral components
(Christensen, 2001; Kiem and Kögel-Knabner, 2003; Kleber et al., 2011; Six et al., 2002; von
Lutzow et al., 2008). OM associated with the sand-size and light density fractions has been
observed to turnover more rapidly than that associated with silt- and clay-size fractions because
it is hypothesized that OM in these fine fractions is physically protected from biodegradation
(Christensen, 1987; Gregorich et al., 1995; Post and Kwon, 2000). A compilation of 14C data by
von Lutzow et al. (2007) indicated that OM associated with silt- and clay-size fractions are older
and more recalcitrant because of protection from biodegradation through sorption to mineral
36
surfaces. Several studies have evaluated the chemical composition of clay-associated OM to
further elucidate the mechanisms that control the turnover and persistence of OM in soil (Feng
et al., 2005; Ghosh et al., 2009; Simpson et al., 2006). Higher concentrations of microbial-
derived carbohydrates in clay-size fractions and decreased C/N ratios with decreasing particle
size (Guggenberger et al., 1994; Kiem and Kögel-Knabner, 2003) also suggest greater
contributions from microbial-derived OM to finer soil fractions. Physically protected OM can
be disturbed through agricultural management (e.g. tillage) and these soils usually have a lower
carbon content compared to undisturbed land (Gregorich et al., 1995; Guggenberger et al., 1994;
Janzen et al., 1998; Wilhelm et al., 2004). It is hypothesized that uncomplexed OM is readily
decomposed as a consequence of cultivation (Christensen, 2001; Janzen et al., 1998).
Insights into the relationships between OM composition and their preservation in various
soil fractions have been studied using solid-state 13C nuclear magnetic resonance (NMR)
methods (Baldock et al., 1992; Blackwood, 2003; Chenu and Plante, 2006; Gregorich et al.,
1996; Guggenberger et al., 1994; 1995; Heim and Schmidt, 2007a; Kaiser and Zech, 2000;
Leifeld and Kögel-Knabner, 2005; Mikutta et al., 2007; Quenea et al., 2006; Quideau et al.,
2001; Virto et al., 2008), which is used to characterize the total OM associated with physical
fractions of a variety of soils (Baldock et al., 1992; Clemente et al., 2011; Guggenberger et al.,
1995; Quideau et al., 2001). Solid-state 13C NMR spectroscopy is widely used because it is
non-destructive and provides semi-quantitative information about the composition of soil OM
with little pre-treatment (Rumpel et al., 2006; Schmidt et al., 1997). Previous studies using
solid-state 13C NMR spectroscopy have shown enrichment of alkyl structures in finer soil
fractions (Baldock et al., 1992; Guggenberger et al., 1995; Leifeld and Kögel-Knabner, 2005;
Rumpel et al., 2004). In addition, the ratio of alkyl to O-alkyl components (alkyl/O-alkyl) has
been observed to increase with decreasing particle size, suggesting that OM in these fine
fractions is at a more advanced state of degradation (Baldock et al., 1992; Gregorich et al., 1996;
Guggenberger et al., 1995; Kiem and Kögel-Knabner, 2003; Rumpel et al., 2004). Solid-state
NMR spectroscopy is an excellent tool for such studies as it provides an overview of the types
of carbon structures present and does not require sample extractions. The main disadvantage of
solid-state 13C NMR stems from the broad resonances which results from the multitude of OM
components as well as strong dipolar coupling interactions in the solid-state. This can result in
low resolution spectra, thus making it difficult to definitively determine the precise identity and
37
source of OM structures that resonate within broadly defined regions, namely the akyl and
aromatic regions which have been linked to recalcitrant OM components. As such, there is a
need to further confirm the generic information obtained from solid-state 13C NMR experiments.
For example, some studies that also used OM biomarker methods observed lower concentrations
of saponifiable and extractable lipids associated with fine fractions, which suggests that the low
resolution from solid-state 13C NMR may not provide a detailed picture of OM composition and
source (e.g. plant- versus microbial-derived;(Quenea et al., 2006; Wiesenberg and Schwark,
2006). The use of complementary NMR methods may offer additional information about the
presence or absence of specific OM components obtained by solid-state 13C NMR analysis
alone.
Over the past decade, several solution-state 1H NMR methods have been employed to
advance the fundamental knowledge of OM composition (Clemente et al., 2011; Feng et al.,
2008; Simpson et al., 2007a; 2007b). This research also entailed the use of several multi-
dimensional NMR experiments, which facilitated the detailed structural elucidation of
components typically observed in a one-dimensional 1H NMR spectrum for soil-derived OM
(Simpson et al., 2007b; 2011). Furthermore, solution-state 1H NMR has been more sensitive to
subtle changes in OM chemistry and can be more resolved than solid-state 13C spectra of soil
OM (Feng et al., 2011; Feng and Simpson, 2011). However, solution-state 1H NMR can only be
used to analyze soluble components of soil OM (i.e. NaOH extractable humic substances),
which represents 50% to 80% of soil organic carbon (Simpson et al., 2007a). Thus, although it
offers improved resolution and detail, solution-state 1H NMR may not provide a complete
picture of all soil OM components. Consequently, when used in tandem with solid-state 13C
NMR, which can measure total OM components, solution-state 1H NMR methods may provide
a higher level of detail and specificity for the structural elucidation and source apportionment of
soil OM than solid-state 13C NMR alone.
Despite the advantages of combining its use with the more commonly used solid-state 13C NMR, solution-state 1H NMR remains underused to identify components associated with
soil density and particle size fractions (Clemente et al., 2011). Therefore, the focus of this study
is to compare OM structural information obtained from two solution-state 1H NMR methods (1H
and diffusion edited (DE) 1H NMR) with that obtained from solid-state 13C NMR. In Table 2.1,
we summarize the structures identified using solid-state 13C and solution-state 1H NMR methods
38
Table 2.1 Structural characteristics and organic matter sources detected using solid-state 13C nuclear magnetic resonance (NMR) and solution-state 1H NMR (in DMSO-d6) compiled from the literature (Mao et al., 2000; Preston et al., 2000; Simpson et al., 2007a; 2007b). Structural units are highlighted using bold font where appropriate.
Structural Unit Solid-state 13C NMR Solution-state 1H NMR Potential Sources R-CH3
(terminal methyl groups) R = branched or un-branched aliphatic C
Observed at 10-20 ppm. Observed at 0.8 ppm. Lipids, lignin, amino acids in peptides
R-(CH2)n – CH3
Mid-chain methylene groups
Observed between 20-50 ppm, polymethylene has a distinct apex at ~30-33 ppm.
The polymethylene CH2 is observed at 1.2 ppm, a lower CH2/CH3 signal ratio suggests greater contributions from amino acid side chains.
Cutin and suberin from plants, lipids, and amino acid side chains
-O-(R)n<3-CH2- -HN-(R)n<3-CH2- R = branched or un-branched aliphatic C
X = sugar ring in peptidoglycan or chitin
Not resolvable – main region resonates from 29-50 ppm.
Detected between 1.2-3.9 ppm with N-acetyl at ~1.8-2.0 ppm.
Lipids, degradation products, amino acids N-acetyl in peptidoglycan and chitin
R-O-CH2- R-O-CH3
R = aromatic, aliphatic, or other sugar C
O-alkyl observed at 50-95 ppm. Methoxy observed at 56 ppm. Hexose ring carbons observed between 62-76 ppm. Anomeric C observed between 95-105 ppm.
Methoxy and ethoxy are observed between 2.9-4.1 ppm. The anomeric region is observed at 4.8-5.2 ppm.
Lignin, carbohydrates and peptides
R = amino acid side chain
-C resonates at around 46 ppm (not resolvable within the O-alkyl region).
-H observed at 4.1-4.8 ppm. Peptides
R = amino acid side chain
Not observed in 13C NMR. Amide observed at 7.8-8.4 ppm (in DMSO-d6 only).
Peptides
Observed at 140-160 ppm when R is O or N, and at 110-140 ppm when R is C.
Detected between 6.2-7.8 ppm; aromatic amino acids typically resonate at 7.2, 7.0, and 6.6 ppm; aromatic structures in lignin are centered at 6.7 ppm.
Lignin, peptides, and black C
Carboxyl C observed at 170 ppm and carbonyl C observed at 190 ppm.
Variable chemical shift, sharp signals or a broad hump are usually observed at >9.0 ppm.
Lignin, peptides, and other degradation products
39
and the most likely OM source that corresponds to these components. We also use DE 1H NMR
spectroscopy, which emphasizes the large or aggregated OM, and when combined with solution-
state 1H NMR spectroscopy, provides additional information on the relative size and
degradation stage of extractable compounds (Feng et al., 2011; Feng and Simpson, 2011;
Kelleher et al., 2006) associated with the various fractions. We analyzed the clay-size and light
fractions of three different soil types: a maize cropped agricultural (AM) soil, a Northern
Grassland (NG) soil, and a Grassland-Forest Transition (GFT) soil. These soils were developed
on glacial till but vary in texture, mineralogy and OM content. The overall objective is to
examine the chemistry of OM associated with fine soil fractions from soils that vary in OM
source and concentration using a variety of NMR methods.
2.3 Experimental Methods
2.3.1 Soil Description and Sample Isolation
Three soils were used in this study: an agricultural soil cropped with maize for 14 years
(AM; collected from the field experiment at Agriculture and Agri-Food Canada’s Central
Experimental Farm in Ottawa, Ontario); Northern Grassland (NG; collected near Edmonton,
Alberta); and a Grassland-Forest Transition soil (GFT; collected near Tofield, Alberta). Soils
were collected from the 0-15 cm depth, air-dried, and ground to pass through a 2 mm sieve. The
AM soil site has a mean annual precipitation of 880 mm and a mean annual temperature of 5.8
˚C. The NG and GFT soil sites have a mean annual precipitation of 452-413 mm and mean
annual temperature of 1.7-3.3 oC (Janzen et al., 1998; Otto et al., 2005). The AM soil (classified
as an Orthic Melanic Brunisol) developed on glacial till, has a sandy clay loam texture and the
clay fraction contains feldspar, chlorite, vermiculite, illite and small amounts of interstratified
minerals (MacLean and Brydon, 1963). The NG and GFT soils (classified as an Orthic Black
Chernozem and an Orthic Dark Grey Chernozem, respectively) developed on calcareous till, had
a loam to clay loam texture and high amounts of montmorillonite and illite clays as well as, to a
lesser extent, chlorite and kaolinite (Bentley, 1979). Previous studies in our laboratory
investigated the NG and GFT soils to characterize the OM composition using several techniques
(Clemente et al., 2011; Feng and Simpson, 2007; Otto et al., 2005; Otto and Simpson, 2005;
2006b; 2006a; 2007; Simpson et al., 2007a; Simpson et al., 2008).
40
Clay-size, and light fractions were isolated using density and size fractionation
techniques (Gregorich and Beare, 2008). Air-dried soil was mixed with a NaI solution (Fisher
Scientific, Fairlawn, NJ), which had been adjusted to a density of 1.7 g/mL, the ratio between
soil and NaI was kept at 1 g soil for every 2 mL of NaI. The resulting suspension was shaken
overnight. The free-floating light fractions were isolated by vacuum filtration and washed with
de-ionized water. The residual heavy fraction was washed through a 53 µm sieve, the material
that passed through the sieve was collected quantitatively, and then subjected to sedimentation
for the required period of time to isolate the suspended clay (<2 µm). The clay-size fractions
were then collected, freeze-dried, and ground to a powder. Soil, light density and clay-size
fractions were analyzed for total carbon, inorganic carbon and nitrogen. Inorganic carbon was
not detected in these soils; therefore, total carbon represents organic carbon in each sample.
2.3.2 Solid-State 13C Cross Polarization-Magic Angle Spinning (CP/MAS) NMR Spectroscopy
Soil and the clay-size fraction were repeatedly treated with 10% hydrofluoric acid (HF),
then washed with deionized water, and then freeze-dried prior to NMR analysis. Treatment with
HF improved the solid-state NMR spectra by concentrating the OM and significantly decreasing
the concentration of paramagnetic material (Schmidt et al., 1997), without significantly
changing the 13C CP/MAS detectable OM distribution (Rumpel et al., 2004). HF treated
samples and light fraction were packed into 4 mm zirconium rotors, which were closed with
Kel-F caps. Solid-state 13C NMR spectra were acquired on a 500 MHz Bruker BioSpin Avance
III spectrometer (Bruker BioSpin, Rheinstetten, Germany) with a 4 mm broad-band CP/MAS
probe head, using a 13 kHz spinning speed and a ramp-CP contact time of 1 ms and a recycle
delay of 1 s. NMR spectra were processed using a zero-filling factor of 2 and 75 Hz line
broadening. Chemical shift ranges were integrated using AMIX v.3.7.10 (Bruker BioSpin).
Chemical shifts were assigned according to published studies (Arigoni et al., 1997; Baldock et
al., 1992; Deshmukh et al., 2005; Hu et al., 2000; Preston et al., 2000) and integrated based on 5
general regions (Table 2.1): alkyl carbon found in lipids, waxes, cutin, and suberin (0-50 ppm);
O-alkyl carbon found in methoxy and ethoxy carbons in lignin, and methoxy carbons found in
carbohydrates (50-95 ppm); anomeric carbons found in carbohydrates (95-110); aromatic
carbons found in lignin with possible contributions from carbons found in protein side-chains
and black carbon (110-160 ppm); and carboxyl carbons (160-200 ppm;(Mao et al., 2000;
41
Preston et al., 2000). Alkyl/O-alkyl ratios were calculated by dividing the area of the alkyl
region (0-50 ppm) with the area of the O-alkyl region (50-95 ppm;(Guggenberger et al., 1995).
2.3.3 Solution-State 1H NMR
Humic substances (humic and fulvic acids) were exhaustively extracted with 0.1 M
NaOH, passed through a 0.2 µm PVDF filter (Millipore, Billerica MA), cation-exchanged using
Amberjet 1200(H) (Aldrich, St. Louis, MO), and then freeze-dried. NaOH extracts have
previously been found to represent 50% to 80% of soil organic carbon (Simpson et al., 2007a).
These were then further dried over P2O5, re-dissolved in DMSO-d6 (Cambridge Isotopes,
Andover MA), and then transferred into 5 mm NMR tubes. Samples were analyzed using a
Bruker BioSpin Avance 500 MHz spectrometer using a 5 mm Quadrupley tuned inverse (QXI)
probe fitted with an actively shielded z-gradient. Solution-state 1H NMR spectra were acquired
using 256 scans and 32 K time domain points. DE 1H NMR spectra, which emphasize relatively
large or aggregated molecules, were acquired using a Bipolar-gradient-Pulse Pair Longitudinal
Eddy current Delay (BBPLED) sequence, with 1024 scans, 16384 time domain points, a
diffusion time of 0.2 s, and 2.5 ms encoding/decoding gradients at 50 gauss/cm. All solution-
state 1H NMR data were processed using a zero-filling factor of 2. A line broadening of 1 Hz
was used for processing solution-state 1H NMR spectra, whereas a line broadening of 10 Hz was
used for processing DE 1H NMR experiments. Solution-state 1H NMR assignments (Table 2.1)
are based on previous work by Simpson et al. (2007a; 2007b), in which one-dimensional
structural assignments have been confirmed through a range of multi-dimensional NMR
experiments.
2.4 Results and Discussion
2.4.1 Organic Matter Characterization by Solid-State 13C NMR
Soil and corresponding fractions have similar structural components but the 13C NMR
signal intensities indicate varying abundances within each fraction (Figs. 2.1-2.4). Alkyl signals
include CH, CH2, and CH3 constituents found in proteins, lipids, waxes, as well as those found
in cutin and suberin (Deshmukh et al., 2005; Hu et al., 2000; Preston et al., 2000). Within this
region, signals that resonate between 11-27 ppm may be attributed to CH, CH3 in acetyl, and
CH2 which are to carbonyl, to external methane or to an alcohol; these may be derived
42
Figure 2.1 Solid-state 13C cross polarization/magic angle spinning (CP/MAS), solution-state 1H nuclear magnetic resonance (NMR), and diffusion edited (DE) 1H NMR spectra of Agricultural (AM) soil, clay-size, and light fractions. Highlighted regions in the solid-state 13C spectra are attributed to (1) alkyl (0-50 ppm); (2) O-alkyl (50-95 ppm); (3) anomeric (from carbohydrates; 95-110 ppm); (4) aromatic (C-linked and phenol; 110-160 ppm); (5) carboxyl + carbonyl (160-200 ppm). Highlighted regions in solution-state 1H and DE 1H NMR spectra are attributed to (1) aliphatic methyl and methylene (0.3-1.3 ppm); (2) aliphatic methyl and methylene near O and N (1.3-2.9 ppm); (3) O-alkyl, mainly from carbohydrates and lignin (2.9-4.1 ppm); (4) 1H from proteins (4.1-4.8 ppm); (5) aromatic, from lignin and proteins (6.2-7.8 ppm); (6) amide from proteins (7.8-8.4 ppm). Other signals, which may be protein derived are labelled as P1-P6 as described in the text. Asterisks are used to mark the N-acetyl signal from peptidoglycan or chitin.
1.0 02.03.04.06.07.08.09.0 5.0
ammonia
1.0 02.03.04.06.07.08.09.0 5.0
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DMSO-d6
CH2
CH3
AlkylO-Alkyl
P6 P5
P2 CH3 (P1)
B) Solution-state 1H NMR C) Solution-state DE 1H NMR
12345
A) Solid-state 13C NMR
Light
Clay
Soil
Chemical Shift (ppm)
*
*
*
*
*
*
0200 40160 80120
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AlkylO-Alkyl
43
Figure 2.2 Solid-state 13C cross polarization/magic angle spinning (CP/MAS), solution-state 1H nuclear magnetic resonance (NMR), and diffusion edited (DE) 1H NMR spectra of Northern Grassland (NG) soil, clay-size, and light fractions. Highlighted regions in the solid-state 13C spectra are attributed to (1) alkyl (0-50 ppm); (2) O-alkyl (50-95 ppm); (3) anomeric (from carbohydrates; 95-110 ppm); (4) aromatic (C-linked and phenol; 110-160 ppm); (5) carboxyl + carbonyl (160-200 ppm). Highlighted regions in solution-state 1H and DE 1H NMR spectra are attributed to (1) aliphatic methyl and methylene (0.3-1.3 ppm); (2) aliphatic methyl and methylene near O and N (1.3-2.9 ppm); (3) O-alkyl, mainly from carbohydrates and lignin (2.9-4.1 ppm); (4) 1H from proteins (4.1-4.8 ppm); (5) aromatic, from lignin and proteins (6.2-7.8 ppm); (6) amide from proteins (7.8-8.4 ppm). Other signals, which may be protein derived are labelled as P1-P6 as described in the text. Asterisks are used to mark the N-acetyl signal from peptidoglycan or chitin.
01.02.03.04.05.06.07.08.09.0 01.02.03.04.05.06.07.08.09.0
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DMSO-d6
CH2
CH3Light
Clay
AlkylO-Alkyl
P6P5
P2
CH3 (P1)
B) Solution-state 1H NMR C) Solution-state DE 1H NMR
Soil
04080120160200
Chemical Shift (ppm)
12345
A) Solid-state 13C NMR
P4 P3*
*
*
*
*
*
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O-AlkylAlkyl
44
Figure 2.3 Solid-state 13C cross polarization/magic angle spinning (CP/MAS), solution-state 1H nuclear magnetic resonance (NMR), and diffusion edited (DE) 1H NMR spectra of Grassland-Forest Transition (GFT) soil, clay-size, and light fractions. Highlighted regions in the solid-state 13C spectra are attributed to (1) alkyl (0-50 ppm); (2) O-alkyl (50-95 ppm); (3) anomeric (from carbohydrates; 95-110 ppm); (4) aromatic (C-linked and phenol; 110-160 ppm); (5) carboxyl + carbonyl (160-200 ppm). Highlighted regions in solution-state 1H and DE 1H NMR spectra are attributed to (1) aliphatic methyl and methylene (0.3-1.3 ppm); (2) aliphatic methyl and methylene near O and N (1.3-2.9 ppm); (3) O-alkyl, mainly from carbohydrates and lignin (2.9-4.1 ppm); (4) 1H from proteins (4.1-4.8 ppm); (5) aromatic, from lignin and proteins (6.2-7.8 ppm); (6) amide from proteins (7.8-8.4 ppm). Other signals, which may be protein derived are labelled as P1-P6 as described in the text. Asterisks are used to mark the N-acetyl signal from peptidoglycan or chitin.
01.02.03.04.05.06.07.08.09.0 01.02.03.04.05.06.07.08.09.0
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DMSO-d6
CH2
CH3Light
Clay
AlkylO-Alkyl
P5
P2
CH3 (P1)
P6
B) Solution-state 1H NMR C) Solution-state DE 1H NMR
Soil
04080120160200
12345
A) Solid-state 13C NMR
Chemical Shift (ppm)
P4 P3
*
*
*
*
*
*
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AlkylO-Alkyl
45
Figure 2.4 Relative contribution of the various structures to A) the solid-state 13C cross polarization/magic angle spinning (CP/MAS) nuclear magnetic resonance (NMR), B) solution-state 1H NMR, and C) diffusion edited (DE) 1H NMR spectra of Agricultural (AM) Soil, Northern Grassland (NG), and Grassland-Forest Transition (GFT) light, clay-size fraction, and whole soil. The region between 4.8 and 6.2 ppm in the solution-state 1H NMR, and DE 1H NMR spectra were omitted as structures that resonate in this region were not major contributors to the signals in the spectra. For solution-state 1H NMR spectra, the sum of the aliphatic region (1.3-2.9 ppm) and the O- and N-substituted aliphatic region (1.3-2.9 ppm) were used to determine the total contribution of alkyl structures to the solution-state NMR spectra.
from lipids and proteins with some contributions from phytols (Arigoni et al., 1997; Deshmukh
et al., 2005; Preston et al., 2000). Signals observed at 27-36 ppm are likely derived from
branched or unbranched aliphatic carbons, such as those found in cutin, suberin, and other
waxes, as well as contributions from CH2 groups found in microbial- and plant-derived lipids
(Deshmukh et al., 2005; Hu et al., 2000; Preston et al., 2000). Signals from polymethylene
0
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40
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Clay-size
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Light
Clay-size
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Agricultural (AM)
Northern Grassland (NG)
Grassland-Forest Transition (GFT)
A) Solid-state13C B) Solution-state 1H C) Solution-state DE 1H
Rel
ativ
e C
on
trib
uti
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(%
)
Structural Unit
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46
structures were more intense in clay-size fractions (Figs. 2.1-2.3) for all soils, suggesting that
there is more cutin-derived polymethylene carbon in these fractions and potentially microbial-
derived lipids associated with clay surfaces.
The O-alkyl region of the solid-state 13C NMR spectra was most prominent in the light
fractions (Figs. 2.1-2.3), and corresponds to structures found in carbohydrates and lignin with
minor contributions from proteins (Preston et al., 2000; Spaccini and Piccolo, 2009). The
observed enrichment of these signals in the light fraction (Fig. 2.4) is consistent with the OM
containing fresh plant material. Within the O-alkyl region, the signal centered at 56 ppm is
typically attributed to methoxy structures found in lignin and amino acids in peptides, whereas
signals from 63-76 ppm are attributed to hexose ring carbon such as those found in
carbohydrates (Preston et al., 2000; Quideau et al., 2001; Spaccini and Piccolo, 2009). The
intensity of the methoxy signal (56 ppm) increased, whereas the intensity of signals that arise
from carbohydrates decreased in the clay-sized as compared to the light fractions (Figs. 2.1-2.3).
These results suggest an accumulation of lignin or peptide in the fine fractions. For AM soil
fractions, the intensity of the aromatic and phenolic signals (110-160 ppm) were greater in the
light fraction compared to the clay-size fraction, whereas the intensity of these signals was
higher in the clay-size compared to the light fraction of NG and GFT soils (Figs. 2.1-2.3).
Aromatic signals arise from lignin but may also have contributions from amino acids, suberin,
and black carbon (Preston et al., 2000; Quideau et al., 2001). Overall, the solid-state 13C NMR
results suggest that cutin-derived and lignin-derived OM are concentrated in the fine fractions of
soil in the NG and GFT soils and are consistent with other studies (Baldock et al., 1992;
Guggenberger et al., 1995; Quideau et al., 2001). However, it is difficult to ascertain the
contribution of microbial-derived peptides in soil fine fractions, which has also been observed
by other studies using biomarker methods (Guggenberger et al., 1994; Kaiser and Zech, 2000).
Consequently, additional information from solution-state 1H NMR spectra will aid in assigning
the observed signals and their variation with soil fractions.
2.4.2 Organic Matter Characterization by Solution-state 1H NMR Spectroscopy
Several structural components are represented by signals in the solution-state 1H NMR
spectra (Figs. 2.1-2.3) and include: aliphatic structures found in lipids, waxes, cutin, and suberin
(0.6-1.3 ppm); O- and N-substituted aliphatic structures found in peptides, lipids, and
47
degradation products (1.3-2.9 ppm; such as those in peptides and β or γ to COOH in lipids); O-
alkyl structures found in lignin and carbohydrate, with some contributions from protein and
hydroxyalkanoic acids (2.9-4.1 ppm); 1H on α C from peptides, with some overlap from 1H in
anomeric structures from carbohydrates (4.1-4.8 ppm, anomeric protons are mainly detected at
4.8-5.2 ppm); as well as aromatic structures mainly attributed to lignin and peptide side-chains
(6.2-7.7 ppm); and amide structures from peptides (7.8-8.4 ppm). As with solid-state 13C NMR
spectra, solution-state 1H NMR spectra show an enrichment of polymethylene components in
the clay-size fraction of all three soils and O-alkyl carbon in the light fraction of the NG and
GFT soil (Fig. 2.4). The differentiation between aromatic and amide containing structures can
be used with solution-state DE 1H NMR results (as discussed in the next paragraph) to confirm
the exact source of OM.
Solution-state DE 1H NMR spectra show the presence of mostly large or rigid
components (Simpson et al., 2007a; 2007b; 2011); Figs. 2.1-2.3). Some peaks in the solution-
state 1H NMR spectra were no longer observed in the DE 1H NMR spectra, indicating that these
signals are from small compounds (Simpson et al., 2007a; 2007b), such as OM degradation
products. These sharp signals were most prominent in the AM clay-size fraction and soil (Fig.
2.1), but were less prominent in the NG clay-size fraction and soil extracts (Fig. 2.2). Signals in
the O-alkyl region (centered at ~3.8 ppm) and in the aromatic region (centered at ~6.7 ppm)
observed in solution-state DE 1H NMR spectra were more prominent in the light fractions (Figs.
2.1, 2.2), which is typical of structures found in lignin (Simpson et al., 2007b). A signal at ~1.8
ppm arises from N-acetyl groups, such as those found in peptidoglycan (Simpson et al., 2007b;
White, 2000) or fungal chitin (White, 2000). This signal was observed in all samples, but was
most intense in the DE 1H NMR spectrum for the AM soil and especially its clay-size fraction
(Fig. 2.1). Signals from CH2 were also attenuated by diffusion editing (Fig. 2.1-2.3), suggesting
that many of the CH2-containing compounds in the samples are mobile; this may be attributed to
small molecules such as short-chain lipids, found in microbial membranes (Simpson et al.,
2007a). In the AM clay-size fraction and soil samples, the CH2 signal was more intense than the
CH3 signal in the DE 1H NMR spectrum (Fig. 2.3), indicating larger contributions from
polymethylene compounds in the clay-size compared to the light fraction. A notable peak in the
aromatic region of all DE 1H NMR spectra at 7.2 ppm (P5; Figures 1-3) is attributed to
phenylalanine in proteins (Simpson et al., 2007a; 2007b). Additional aromatic peaks were
48
observed at 6.6 (P3) and 7.0 (P4) ppm in the DE 1H NMR spectra of NG and GFT clay-size
fraction and soils (Figs. 2.2-2.3), which are attributed to tyrosine in proteins. These amino acid-
derived aromatic signals, along with a higher abundance of CH3 relative to CH2 signals in DE 1H NMR spectra (P1; which arises from methyl rich amino acids), and amide signals between
7.8 and 8.4 ppm (P6; Figs. 2.1-2.3), denotes the presence of proteins, as this pattern was
observed in the 1H NMR spectra of bovine serum albumin, previously analyzed in our
laboratory (Simpson et al., 2007a). The pattern of signals in these spectra (P1-P6) also shows
similarities to soil microbial biomass previously isolated and studied by solution-state 1H and
DE 1H NMR (Simpson et al., 2007a). Other peaks (6.7-6.8 ppm) that were also observed in the
DE 1H NMR spectra of light fractions have been reported for lignin and tyrosine found in
proteins (Simpson et al., 2007a; 2007b). These, as well as signals corresponding to
phenylalanine, were also observed in clay-size fractions and soil extracts. Overall, these results
indicate that the clay-size fraction contains high concentrations of microbial-derived peptides
(for all three soils) and lignin (in the NG and GFT soils).
2.4.3 Comparison of Solid-state 13C and Solution-state 1H NMR Methods
A major advantage of using solution-state 1H NMR spectroscopy is the additional
information obtained through improved resolution, especially within the aliphatic and aromatic
regions (Table 2.1). For example, methyl and methylene structures were successfully resolved
from the methyl and methylene near O and N structures using solution-state 1H and DE 1H
NMR experiments. Furthermore, aromatic resonances related to peptide- and lignin-derived
signals can also be resolved using solution-state 1H NMR methods. In solid-state 13C NMR
spectra, these structures are not resolved because of signal overlap (Table 2.1). The integration
results from all three NMR methods used are displayed in Fig. 2.4. There is general consistency
in the relative abundance of the various components among all three NMR methods, even
though solution-state 1H NMR methods focus on only the soluble OM components. This
agreement suggests that solution-state 1H NMR methods are indeed representative of the overall
OM characteristics, in addition to being more sensitive to small changes in OM chemistry that
are not always detectable by solid-state 13C NMR methods (Feng et al., 2011; Feng and
Simpson, 2011).
49
Clay-associated OM had a high concentration of polymethylene structures, which
indicates that plant-derived waxes and cuticles may selectively accumulate in this fraction (Figs.
2.1-2.3). Lipids, waxes, cutin, and suberin are believed to increase relative to carbohydrates and
lignin components as plant biomass degradation progresses (Baldock et al., 1992; Deshmukh et
al., 2005; Guggenberger et al., 1995; Kelleher et al., 2006). Alkyl OM may also be derived
from microbial lipids (Baldock et al., 1992; Blackwood, 2003; Golchin et al., 1996; Simpson et
al., 2007a). Thus, the observed enrichment of alkyl carbon in solid-state 13C NMR spectra of
clay-size fractions may be from cutin-derived compounds, which have been preferentially
stabilized and preserved due to their association with clay mineral surfaces. This enrichment
can also be attributed to contributions from microbial-derived lipids generated in situ (also
suggested by the solution-state 1H NMR results). Additional information on the identity of
structures that contribute to the alkyl region of solid-state 13C NMR may help elucidate whether
plant- or microbial-derived compounds are enriched in this fraction.
The general alkyl region of solid-state 13C NMR spectra can be differentiated by
improved resolution in solution-state 1H NMR, and DE 1H NMR spectra. For example, CH3,
polymethylene CH2, and methyl and methylene signals near O and N (Table 2.1; Figs. 2.1-2.3)
can be distinguished. Signals observed using solution-state 1H NMR and DE 1H NMR spectra
suggest that the humic fraction of soil OM contains compounds which have different relative
sizes and states of oxidation (Figs. 2.1-2.3). In particular, comparisons between solution-state 1H NMR, and DE 1H NMR signals suggest higher concentrations of polymethylene structures to
total humic extracts, which is consistent with the solid-state 13C NMR observations (Figs. 2.1-
2.3). Contributions from peptides (P1-P6) and microbial-derived peptidoglycan or chitin signals,
which were observed in the methyl near O and N region were also enhanced in the DE 1H NMR
spectra of clay-size fractions (Figs. 2.1-2.3), which suggests greater microbial-derived peptides
in fine fractions. Similarly, Simpson et al. (2007a) observed that the protein signature in 1H
NMR and DE 1H NMR spectra increases with microbial-derived contributions to soil OM.
Guggenberger et al. (1994) also found higher concentrations of microbial-derived (amino)
sugars in clay-size fractions which corroborates the findings reported here. Microbial-derived
peptides and peptidoglycan or chitin were greatest in the clay-size fraction of the agricultural
(AM) soil, and is consistent with a study which suggests that a greater proportion of soil carbon
in agricultural soils may be microbial-derived (Wardle, 1992). Therefore, information obtained
50
using solution-state 1H NMR and DE 1H NMR spectroscopy suggest that microbial-derived
peptides and possibly lipids may contribute to the alkyl carbon signal observed using solid-state 13C NMR.
The O-alkyl and aromatic containing compounds detected using solid-state 13C NMR
spectra suggest increased contributions from lignin in clay-size fractions of NG and GFT soils
but not the AM soil. Baldock et al. (1992) similarly found that contributions from aromatic
compounds to solid-state 13C NMR spectra did not always increase with decreasing particle size.
Solid-state 13C NMR spectra display contributions from both carbon-linked and phenolic
aromatic compounds within the aromatic region of light fractions, whereas only carbon-linked
signals were observed in the clay-size fractions (Fig. 2.1-2.3). According to a study by Chabbi
and Rumpel (2004), a decrease in the phenolic signal in the 13C NMR spectra corresponded to a
decrease in extractable lignin phenol biomarkers, which indicates that the majority of phenolic-
derived compounds arise from lignin. We hypothesize that select lignin-derived structures may
be preserved in clay-size fractions. The DE 1H NMR spectra also suggest greater concentrations
of large lignin compounds in humic extracts from light compared to clay-size fractions (Figs.
2.1-2.3). Together, these observations suggest that the various structures that compose lignin
may not be uniformly degraded, and this suggests that various lignin types have different
stabilities against degradation (Bahri et al., 2006; Ertel and Hedges, 1984; Hedges et al., 1988).
The plant inputs for the soils studied here vary (grassland versus maize versus grassland-forest),
therefore the type of lignin and its stability will also differ as it has been reported that some
lignin units are more susceptible to degradation than others (Hedges et al., 1988; Otto et al.,
2005). Similarly, these soils have varying textures, mineralogy and OM content. The AM soil
has the coarsest texture and this may result in less available mineral surface area for OM
sorption. The NG and GFT soils both have greater clay content than the AM soil and their fine
textures are dominated by montmorillonite and illite, which may assist with the preservation of
lignin and other OM components. Furthermore, the high organic carbon content of the NG and
GFT soils as compared to the AM soil suggests that OM-OM interactions may also play a role
in the sorptive preservation of OM components.
The alkyl/O-alkyl ratio has been used as an indicator of OM degradation stage because
this ratio has been observed to increase with progressive stages of degradation. In addition,
other studies have also shown good agreement between C/N ratios and degradation parameters
51
Table 2.2 Soil characteristics from combustion analysis (C and N concentrations) and alkyl/O-alkyl ratios from solid-state 13C nuclear magnetic resonance (NMR), solution-state 1H NMR, and diffusion edited (DE) 1H NMR of clay-size, light fraction, and soil from Agricultural (AM), Northern Grassland (NG), and Grassland-Forest Transition (GFT) soils.
Soil
Fraction Total C (mg/g) Total N
(mg/g) C/N Alkyl/O-alkyl
(13C NMR) Alkyl/O-alkyl
(1H NMR) Alkyl/O-alkyl (DE 1H NMR)
Agricultural (AM)
Light 200.3 10.6 22.1 0.3 0.7 0.6
Clay 27.5 2.7 12.1 1.0 1.0 0.8
Soil 16.6 1.2 17.5 0.8 0.7 0.7
Northern Grassland (NG)
Light 302.0 15.8 22.3 0.6 1.1 1.0
Clay 57.6 6.4 10.5 1.0 1.2 1.8
Soil 44.0 4.0 13.0 1.0 1.3 1.7
Grassland-Forest Transition (GFT)
Light 328.0 14.6 26.2 0.5 1.1 0.8
Clay 54.5 5.9 10.8 0.8 1.6 1.7
Soil 50.0 3.0 19.0 0.7 1.4 1.2
52
(i.e. alkyl/O-alkyl ratios) derived from solid-state 13C NMR spectra (Baldock et al., 1992;
Guggenberger et al., 1995; Quideau et al., 2001). Here, we also note consistency with C/N
ratios, solid-state 13C NMR and solution-state 1H NMR alkyl/O-alkyl ratios (Table 2.2). For all
samples analyzed, the clay-size fraction contains more degraded OM compared to light fraction
and for AM and GFT, the OM in the clay-size fraction is more degraded than OM in the whole
soil. Although, the solution-state 1H NMR alkyl/O-alkyl ratios show similar trends, the analysis
of the DE 1H NMR data provides additional information. For example, for both the NG and
GFT soils, the alkyl/O-alkyl ratio for the clay-size fraction increases with diffusion editing,
suggesting that larger, more rigid molecules at an advanced stage of degradation are prominent
in these fractions. This is not the case for the agricultural soil (AM), which indicates that the
mechanisms of soil OM turnover and preservation vary with environmental factors, such as soil
management as well as OM content.
2.5 Conclusions
This study highlights the importance of using complementary and advanced methods for
characterizing the composition of soil OM in fine fractions. The increased resolution afforded
by solution-state 1H NMR spectroscopy differentiated between peptide- and lignin-derived
aromatic structures; detected N-containing amides and N-acetyl in peptidoglycan or chitin that
may be responsible for nitrogen enrichment in finer fractions; and confirmed contributions from
long-chain alkyl constituents in clay-size fractions. Signals from a variety of methyl and
methylene containing compounds, and O-alkyl-containing compounds were the major
contributors to the solid- and solution-state NMR spectra of the various soil samples. Lignin-
and carbohydrate-derived structures decreased in the NG and GFT clay-size fractions, consistent
with their hypothesized fate in soil (Guggenberger et al., 1995). These compounds are the most
abundant biopolymers found in plants (Kögel-Knabner, 2002), and clay-size fractions are
hypothesized to contain more degraded plant material as compared to light density fractions
(Christensen, 1987; Gregorich et al., 1995). The enrichment of relatively large structures in
extractable OM in the clay-size fraction compared to light density fractions also corroborates
studies that found enrichment of microbial-derived OM in fine fractions (Guggenberger et al.,
1994; 1995). The concentration of small structures attributed to peptide side-chains and
degradation products in NG and GFT light density fractions may indicate slower microbial
53
utilization of such compounds in these soils, whereas OM in agricultural soils may have faster
turnover causing these structures to quickly disappear (Guggenberger et al., 1994; 1995).
Results from all three NMR methods indicate that the overall state of OM decomposition in the
clay-size fraction was consistently at a more advanced stage than compared to the light density
fraction. However, additional information on the structures that contributed to the humic
fraction of OM associated with light density and clay-size fractions, and whole soil were found
using solution-state 1H NMR and DE 1H NMR spectroscopy. In particular, contributions from a
variety of compounds, including small polymethylene structures and microbial-derived OM
were observed using solution-state 1H and DE 1H NMR spectroscopy.
2.6 Acknowledgements
The authors thank Aleksandra Spasojevic and Magda Celejewski for assistance with sample
preparation, as well as Dr. Andrew Baer for assistance with liquid-state NMR analyses. Funding
for this project was provided by the NSERC Green Crop Network.
54
CHAPTER 3
ASSOCIATION OF SPECIFIC ORGANIC MATTER COMPOUNDS IN SIZE FRACTIONS OF SOILS UNDER DIFFERENT ENVIRONMENTAL
CONTROLS
Authors: Joyce S. Clemente, André J. Simpson, Myrna J. Simpson
Contributions: The study was designed by JSC and MJS. Soil samples were provided by MJS.
Soil samples were fractionated and extracted by JSC with assistance from Katherine Hills and
David Wolfe. NMR acquisition parameters were designed by AJS. Biomarker analyses were
performed by JSC, and NMR analyses were performed by JSC with assistance from Dr. David
McNally. Data analyses were performed by JSC, with guidance from MJS and AJS. The
manuscript was written by JSC and MJS, with critical comments from AJS.
Status: Published in Organic Geochemistry 42, 1169-1180 (2011). Copyright (2011), reprinted
with permission from Elsevier.
55
3.1 Abstract
Inherent chemical recalcitrance and association of organic matter (OM) with minerals are
mechanisms responsible for the long-term preservation of OM in soils. The structural
characteristics of OM are also believed to control specific interactions between OM and soil
minerals. However, the extent of the relationship between recalcitrance and mineral protection,
and the specificity of these chemically-driven interactions are not clearly understood at the
molecular-level. To measure chemical patterns of OM sequestration in sand-, silt-, clay-size and
light fractions, we analyzed three soils, which mainly differed in carbon content and overlying
vegetation, but have similar clay mineralogy, using biomarker analysis and nuclear magnetic
resonance (NMR). Despite differences in environmental controls, long chain aliphatic
compounds generally accumulated in the fine fractions of all soils. This accumulation is likely
due to the strong interaction between recalcitrant forms of OM and soil minerals. For example,
polymethylene and >C20 organic acids accumulated in fine fractions, while lignin-derived
phenols were protected from oxidation in silt-size fractions. Diffusion edited solution-state 1H
NMR suggested that contributions from microbial-derived OM was greater in finer fractions,
which is likely due to the accumulation of microbial-derived compounds or higher microbial
activity in clay micro-sites. Our data suggest that, for these Prairie soils, the specific structure
of OM and not environmental factors is responsible for long-term preservation of OM in mineral
fractions. Further research is necessary to understand the interplay between these preservation
mechanisms such that the long-term fate of OM can be further elucidated.
3.2 Introduction
The preservation of organic matter (OM) in soils is an important factor in soil quality,
turnover, and productivity (Janzen et al., 1998). Two main mechanisms that preserve soil OM
are inherent recalcitrance and mineral protection (Christensen, 1987; Marschner et al., 2008;
Mikutta et al., 2006b; Six et al., 2002; von Lutzow et al., 2008; Zech et al., 1997). The first
mechanism, inherent recalcitrance, is the resistance of specific OM compounds to degradation
because of their structural properties (Lorenz et al., 2007; Six et al., 2002). For example,
measurements of OM turnover and residence times suggest that aliphatic structures (lipids,
waxes, cutin, cutan, suberin, and suberan) may be retained in soils longer than labile
compounds, such carbohydrates and lignin (Lorenz et al., 2007; Mikutta et al., 2006b). The
56
second mechanism, mineral protection, may occur through formation of OM-mineral
complexes, as well as physical and chemical sorption to mineral (especially clay) surfaces
(Mikutta et al., 2006b; Six et al., 2002; von Lutzow et al., 2007). Physical protection
mechanisms limit degradation by making OM inaccessible to microbes and degrading enzymes
(Christensen, 2001; Six et al., 2002). While chemical recalcitrance is believed to be important
in short-term OM preservation, long-term preservation has been attributed to physical protection
(Christensen, 2001; Mikutta et al., 2006b; Six et al., 2002). Consequently, it is important to
understand how these two mechanisms are inter-dependent and related.
Interactions between OM compounds and soil minerals have been investigated mainly
by sorption of OM or model OM compounds to clay minerals, or analysis of OM composition in
soil particle size fractions. Sorption studies with OM compounds or isolates and clay minerals
have shown that specific compounds interact with clay minerals, and that the composition of
sorbed compounds was influenced by the type of clay mineral (Asselman and Garnier, 2000;
Feng et al., 2005; Ghosh et al., 2009; Simpson et al., 2006). Several studies observed that
polymethylene structures were preferentially sorbed to kaolinite and montmorillonite (Feng et
al., 2005; Ghosh et al., 2009; Simpson et al., 2006), while lignin had low affinity for these clays
(Asselman and Garnier, 2000) suggesting that aliphatic compounds will be selectively preserved
over time. Proteins were also observed to sorb to some clay surfaces, such as montmorillonite
(Feng et al., 2005; Ghosh et al., 2009). The type of clay also influenced sorption of OM since
carboxyl groups preferentially interacted with goethite (Ghosh et al., 2009). Alternatively,
detailed analysis of soil density and particle-size fractions has identified specific groups of
compounds that accumulate in the sand-, silt-, and clay-size fractions (Aoyama et al., 1999;
Baldock et al., 1992; Guggenberger et al., 1994; Quideau et al., 2001; Wattel-Koekkoek et al.,
2001). For example, clay-size fractions are enriched with aliphatic compounds, while sand-size
fractions contain higher concentrations of carbohydrates (Baldock et al., 1992; Guggenberger et
al., 1995; Quideau et al., 2001). Older lignin-derived phenols (Heim and Schmidt, 2007a) and
high concentrations of aromatic OM (Baldock et al., 1992; Guggenberger et al., 1995) have been
observed in silt-size fractions.
While sorption studies provide detailed information on specific interactions between OM
compounds and clay minerals, it is equally important to investigate soil samples by
fractionation, as this approach may provide information on the relationship between compound
57
structure and long-term OM preservation through physical protection. Furthermore, when
analyzing soil samples, it is also necessary to determine the role of environmental factors, such
as vegetation, climate and moisture, on OM preservation. In addition to clay content and
mineralogy, vegetation and climate are believed to determine soil OM compound distribution
(Baldock et al., 1992; Guggenberger et al., 1994; 1995; Mikutta et al., 2006b; 2007; Quideau et
al., 2001; Zech et al., 1997). The overlying vegetation was found to influence the amount of
microbial-derived OM, oxidation level of lignin-derived phenols (Guggenberger et al., 1994), as
well as 13C nuclear magnetic resonance (NMR) profiles of soil mineral fractions (Guggenberger
et al., 1995; Quideau et al., 2001).
The main objectives of this study were to provide a more detailed understanding of OM
associated with various soil fractions and to determine whether preservation of compounds
through association with minerals is influenced by chemical structure or by environmental
conditions, such as climate and vegetation. To achieve these objectives, the overall composition
of OM associated with sand-, silt-, clay-size and light fractions was analyzed using both solid-
and solution-state NMR spectroscopy, and biomarker analysis by gas chromatography/mass
spectrometry (GC/MS). We applied this molecular-level approach to Canadian Prairie soil
samples that are of similar age, have similar clay mineralogy (Dudas and Pawluk, 1969), and
have developed on similar parent materials but developed under different climatic conditions
and/or vegetation (Otto and Simpson, 2005). Thus, further study of these soil samples allows
the investigation of the interplay between OM content, vegetation, chemical structure, and OM-
mineral associations on the preservation of OM in the various density and particle-size fractions.
While environmental factors and the two preservation mechanisms are recognized to be
interdependent (Mikutta et al., 2006b; Six et al., 2002) the extent of the interactions between
these factors and their relative importance to long-term OM preservation, is largely unknown.
3.3 Materials and Methods
3.3.1 Soil Sampling, Fractionation, and Carbon and Nitrogen Analysis
Three soil samples from the Alberta Prairie Ecozone were collected: Southern Grassland
(SG; collected near Lethbridge, Alberta), Northern Grassland (NG; collected near Edmonton,
Alberta), and Grassland-Forest Transition (GFT; collected near Tofield, Alberta). These soils
vary in vegetation, carbon content, and climate but developed from similar parent materials and
58
have similar clay mineralogy (Dudas and Pawluk, 1969). Previous studies in our laboratory
investigated these soils to characterize the relationships between climate and the composition of
OM at the molecular-level (Feng and Simpson, 2007; Otto et al., 2005; Otto and Simpson, 2005;
2006b; 2006a; 2007; Simpson et al., 2007a; Simpson et al., 2008). Our previous studies
indicated that these soils contain different amounts of lignin-derived phenols and cutin-derived
OH-acids, and other compounds which implied varying levels of OM degradation (Otto et al.,
2005; 2006b; Otto and Simpson, 2006a). Climate in these areas range from semi-arid (for SG)
to sub-humid (for GFT), and mean annual temperatures range between 1.7 oC and 5.0 oC
(Janzen et al., 1998). These soils are part of the Dark Brown (SG), Black (NG), and Dark Gray
(GFT) Chernozemic groups (Soil Classification WorkingsoGroup, 1998). Overlying vegetation
in the SG and NG soil sites are dominated by Western Wheatgrass, while the GFT soil site is
dominated by both grasses and stands of Quaking Aspen. Detailed descriptions of the soils and
sampling areas can be found in Dudas and Pawluk (1969) and Janzen et al. (1998). After
collection, these soils were air-dried, passed through a 2 mm mesh sieve, and then stored at
room temperature in glass containers.
Sand-, silt-, clay-size and light fractions were isolated using density and size
fractionation techniques (Gregorich and Beare, 2008). Visible roots and other plant material
were manually removed prior to fractionation. Fifty grams of air-dried soil were mixed with
100 mL of a NaI solution (Fisher Scientific, Fairlawn, NJ), which had been adjusted to a density
of 1.7 g/mL. The resulting suspension was shaken overnight and then centrifuged at 1450 rpm
for 50 minutes. The free-floating light fraction was removed by aspiration, filtered using a GF/F
glass microfibre filter (0.7 µm cut off; Whatman, Kent, UK), then washed with de-ionized
water. The residual heavy fraction was washed through a 53 µm sieve, and the material that
passed through the sieve (silt- and clay-size particles) was collected quantitatively. The sieved
material was then suspended in distilled H2O, and the differences in sedimentation time
(calculated using Stokes’ law), was used to isolate the suspended clay- (<2 µm) from silt-size
(53 to 2 µm) particles. After it was collected by aspiration, a calcium chloride solution was
added to the clay-size fraction to promote flocculation, and avoid loss of ultrafine particles.
Both the silt- and clay-size fractions were then isolated by centrifugation at 5000 rpm for 40
minutes. The sand-size particles remaining on the sieve were thoroughly washed with water and
quantitatively collected. All fractions were freeze-dried, then ground, and stored at room
59
temperature prior to analysis. Sample yields revealed that recovery was ~95% for each sample
(Table 3.1) but this does not seem to reflect any selective OM compound loss because we
observed the same constituents but in varying distribution as previous studies, which studied
these same soil samples (Otto et al., 2005; 2006b; Otto and Simpson, 2006a; Simpson et al.,
2007a). Total carbon and nitrogen were analyzed using the LECO combustion method at the
University of Guelph (Ontario, Canada). Using the method of Bundy and Bremner (1972), Otto
and Simpson (2006b) did not detect inorganic carbon in the surface horizons of these soils:
therefore, total carbon represents organic carbon.
3.3.2 Solvent Extraction and Copper (II) Oxide Oxidation
Three sub-samples from each soil fraction were sequentially extracted with methanol
(Fisher Scientific), dichloromethane:methanol (Fisher Scientific; 1:1 v/v), and dichloromethane
to extract free lipids (Otto and Simpson, 2005). The combined solvent extracts were then
filtered through GF/A and GF/F glass microfibre filters, concentrated by rotary evaporation, and
dried under a stream of nitrogen gas (Praxair, Toronto, ON), in 2 mL vials. Lignin-derived
phenols were extracted using CuO oxidation following the method of Hedges and Ertel (1982),
as modified by Otto and Simpson (2006a). Cutin-derived hydroxy-acids (OH-acids) can also be
extracted with CuO oxidation (Filley et al., 2008; Goni and Hedges, 1990a; Mendez-Millan et
al., 2010a). After solvent extraction, the soil residues were air-dried, and then extracted with
copper (II) oxide and ammonium iron (II) sulfate hexahydrate [Fe(NH4)2(SO4)26H2O] with 2
M NaOH in Teflon-lined bombs. The bombs were flushed with nitrogen and incubated at 170 oC for 2.5 h. The supernatants were acidified to pH ~1 using 6 M HCl (Caledon Laboratories,
ON) and kept for 1 h at room temperature, in the dark to prevent cinnamic acid polymerization.
After centrifugation at 2700 rpm for 30 min, the supernatant was extracted with diethyl ether
(Fisher Scientific), the extracts were concentrated, transferred to 2 mL glass vials, and then
dried under a stream of nitrogen gas.
The CuO oxidation residues were re-dissolved in dichloromethane prior to
derivatization. Both solvent extracts and CuO extracts were derivatized by adding N,O-
bis(trimethylsilyl)-trifluoroacetamide (BSTFA; Sigma-Aldrich, Columbus, GA) and pyridine,
followed by heating at 70 oC for 1 hr. After cooling, the samples were analyzed using GC/MS.
To do this, 3 µL of sample was injected using an Agilent 7683 autosampler (Agilent
60
Technologies, Santa Clara, CA) in splitless mode. The sample was then eluted from the Agilent
6890N GC, which had an HP-5MS fused silica capillary column (25 m x 0.201 mm inner
diameter x 0.33 µm film thickness) by using the following temperature gradient: hold at 65 oC
for 2 min, followed by a temperature increase from 65 oC to 300 oC at a rate of 6 oC/min, and a
final isothermal hold at 300 oC for 20 min. The Agilent 5973N mass spectrometer was operated
at 70 eV in the electron impact mode. Data were acquired and processed using the Agilent
Chemstation G1701DA v. D software. Compounds were identified by comparison with mass
spectra from commercial libraries (Wiley, NIST) and authentic standards. Tetracosane (Sigma-
Aldrich), ergosterol (Sigma-Aldrich), and lauric acid (Sigma-Aldrich) were used as external
standards to estimate the relative amounts of n-alkanes, steroids, n-alkanols, and organic acids in
the solvent extracts. Relative amounts of extractable lignin phenols were estimated by using
vanillic acid (Sigma-Aldrich) as an external standard, while relative amounts of OH-acids were
estimated using lauric acid as an external standard.
Eight main lignin-derived phenol monomers were identified and quantified according to
Hedges and Ertel (1982) and Otto and Simpson (2006a) which included: vanillyl (vanillin,
acetovanillone, vanillic acid), syringyl (syringaldehyde, acetosyringone, syringic acid), and
coumaryl (coumaric acid, ferulic acid) groups. The relative contributions (%) of these three
lignin monomer groups were calculated to determine changes in their distribution across the
various fractions. The acid to aldehyde ratios of vanillyl (Ad/Alv) and syringyl (Ad/Als) groups
were calculated as these reflect the oxidation state of lignin-derived phenols, where higher
values indicate that lignin is more oxidized (Hedges et al., 1988). Lignin-derived phenol dimers
were also identified according to Goni and Hedges (1992) and Otto and Simpson (2006a): these
comprised of 2-syringylsyringic acid, 2-syringylsyringaldehyde, 2-vanillylsyringic acid,
dehydrodivanillic acid, dehydrovanillinvanillic acid, dehydrovanillinacetovanillone,
dehydrodivanillin, and dehydroacetovanillonevanillic acid. The concentrations of lignin-derived
phenol dimers were divided by the concentrations of lignin-derived phenol monomers
(dimers/monomers). Cutin-derived OH-acids were also observed in the CuO oxidation extracts,
and these were identified according to Filley et al. (2008), Goni and Hedges (1990a), and
Mendez-Millan et al. (2010a): these comprised of 16-hydroxyhexadecanoic acid, 12-
hydroxyoctadecanedioic acid, 9,10-dihydroxyhexadecanoic acid, and 9,10,18-
trihydroxyoctadecanoic acid. Similar to lignin phenol dimers, the relative amounts of OH-acids
61
compared to lignin phenol monomers were calculated (OH-acids/monomers). The
dimers/monomers and OH-acids/monomers ratios were used to determine the sequestration of
lignin phenol monomers relative to lignin phenol dimers and cutin-derived OH-acids, in the
various fractions.
Multivariate comparisons were performed on chemical groups identified in solvent
extracts and CuO oxidation extracts (n = 3) using SPSS v. 18.0. Analysis of variance followed
by Tukey Honestly Significant Difference (HSD) were used to determine whether differences
between means were significant ( = 0.05).
3.3.3 Solid-state 13C NMR
Sand-, silt-, clay-size, and soil samples for solid-state 13C cross polarization magic angle
spinning (CP/MAS) NMR analysis were repeatedly treated with 10% HF acid (Fisher
Scientific), then washed with deionized water to remove excess salts, as described by Schmidt et
al. (1997). HF acid treatment has been shown to improve the signal to noise of solid-state 13C
NMR by concentrating OM and significantly decreasing the concentrations of paramagnetic
material (Schmidt et al., 1997), without necessarily changing the distribution of 13C CP/MAS
detectable OM (Rumpel et al., 2006). All the HF-treated samples were freeze-dried and ground
into a powder. Samples were packed in 4 mm zirconium rotors, which were then closed with
Kel-F caps. Solid-state 13C NMR spectra were acquired on a 500 MHz Bruker BioSpin Avance
III spectrometer (Bruker BioSpin, Rheinstetten, Germany) with a 4 mm probe, using a 13 kHz
spinning speed, a ramp-CP contact time of 1 ms, a recycle delay of 1 s, and 4096 scans. Glycine
was used to calibrate chemical shifts. NMR spectra were processed using a zero-filling factor of
2 and 75 Hz line broadening. Chemical shifts were assigned according to published studies
(Baldock et al., 1992; Hu et al., 2000; Preston et al., 2000). The spectra were integrated, using
AMIX software (v. 3.7.10; Bruker BioSpin), based on five regions corresponding to: alkyl
carbon derived from waxes, cutin, suberin, lipids, and waxes (0-50 ppm); O-alkyl derived from
methoxy and ethoxy carbons in lignin and methoxy carbons in carbohydrates (50-95 ppm);
anomeric carbon in carbohydrate (95-110); aromatic carbons found in lignin, with possible
contributions from protein and black carbon (110-160 ppm); and carboxyl or carbonyl carbon
(160-200 ppm;(Mao et al., 2000; Preston et al., 2000). Alkyl/O-alkyl ratios were calculated by
dividing the area of the alkyl region (0-50 ppm) with the area of the O-alkyl (50-110 ppm)
62
region (Baldock et al., 1992; Guggenberger et al., 1995; Quideau et al., 2001; Simpson et al.,
2008).
3.3.4 Solution-state 1H NMR and Diffusion Edited 1H NMR
Light fractions and HF-treated sand-, silt-, and clay-size fractions were exhaustively
extracted with 0.1 M NaOH in order to isolate humic substances. Solution-state 1H NMR and
diffusion edited (DE) 1H NMR spectra of humic substances extracted from the SG, NG, and
GFT soils have been previously published in Simpson et al. (2007a). Combined extracts were
passed through a 0.2 µm PVDF filter (Millipore, Billerica MA), cation-exchanged using
Amberjet 1200(H) (Aldrich, St. Louis MO), and then freeze-dried. The resulting solid was
further dried over phosphorous pentoxide, then re-dissolved in DMSO-d6 (Cambridge Isotopes,
Andover MA), and transferred into 5 mm NMR tubes. Samples were analyzed using a Bruker
BioSpin Avance 500 MHz spectrometer equipped with a 5 mm QXI probe. Solution-state 1H
NMR spectra were acquired using 128 scans and 16384 time domain points. DE 1H NMR
spectra, which emphasize relatively large or aggregated molecules, were acquired using 1024
scans, 16384 time domain points, 0.2 s diffusion time, as well as 2.5 ms encoding/decoding
gradients at 50 gauss/cm. Both solution-state 1H and DE 1H NMR were processed using a zero-
filling factor of 2 and a line broadening of 1 Hz. Structural assignments in 1H NMR spectra for
humic substances are based on published data that were collected for commercially available
compounds, purified natural compounds, and multi-dimensional NMR experiments (Simpson et
al., 2007a; 2007b; 2011; Song et al., 2008). Resonance assignments were made as follows:
alkyl CH3 and CH2 (0.6-1.3 ppm); alkyl near O or N, for example peptide or protein side chains
and alkyl β or γ to the COOH in lipids (1.3-2.9 ppm); O-alkyl signals mainly from lignin and
carbohydrate, with some contributions from protein and hydroxyalkanoic acids (2.9-4.1 ppm); 1H on peptides, with minor contributions from carbohydrate anomeric protons (4.1-4.8 ppm;
majority of anomeric protons are detected at 4.8-5.2 ppm); aromatic (6.2-7.8 ppm); and amide
signals (7.8-8.4 ppm). Alkyl/O-alkyl, and O-alkyl/α1H ratios were calculated after the major
regions were integrated using AMIX (v. 3.7.10; Bruker BioSpin).
63
Table 3.1 Soil fraction distribution, carbon (C) and nitrogen (N) contents, and C/N ratios of sand-, silt-, clay-size and light fractions. Lignin phenol values are the sum of vanillyl + syringyl + coumaryl monomers with standard errors of the mean (n = 3).
Light Sand Silt Clay Soila Southern Grassland (SG) Fraction Yield (%)b 1.9 46.3 30.5 16.1 Total C (mg g-1) 260.0 9.0 22.2 40.5 28.0 Total N (mg g-1) 18.0 0.8 2.3 4.7 3.0 C/N 16.9 13.1 11.3 10.1 11.0 Lignin Phenol (mg g-1 C)
4.9 0.5 2.2 0.1 1.5 0.05 0.6 0.05 4.2
Northern Grassland (NG) Fraction Yield (%)b 1.2 32.8 40.1 21.3 Total C (mg g-1) 302.0 9.8 36.6 57.6 44.0 Total N (mg g-1) 15.8 0.7 3.0 6.4 4.0 C/N 22.3 16.3 14.2 10.5 13.0 Lignin Phenol (mg g-1 C)
2.7 0.1 1.7 0.06 0.8 0.01 0.4 0.02 3.2
Grassland-Forest Transition (GFT) Fraction Yield (%)b 4.0 37.1 36.1 17.8
Total C (mg g-1) 328.0 13.9 27.2 54.5 50.0 Total N (mg g-1) 14.6 0.9 1.8 5.9 3.0 C/N 26.2 18.0 17.6 10.8 19.0 Lignin Phenol (mg g-1 C)
2.2 0.1 2.0 0.03 1.1 0.06 0.5 0.03 1.1
a Soil C/N data from Otto and Simpson (2006a). b Fraction yields are based on total recovery as discussed in the materials and methods.
3.4. Results
3.4.1 Fractionation Yields and Carbon and Nitrogen analysis
Fraction yields for each soil, as well as the carbon and nitrogen contents of each fraction
are listed in Table 3.1. For all soil samples, the C/N ratios decreased with decreasing particle-
size and were highest for the light fraction (Table 3.1). C/N ratios were also lower in SG
compared to NG sand-, silt-size, and light fractions, while GFT had higher ratios compared to
NG fractions. The light fraction accounted for ~26% of GFT total soil carbon as compared to
64
~18% for the SG and 8% of NG soil carbon. This indicates that contributions of light fraction,
as well as differences in C/N ratios of sand-, silt-size and light fractions may be influenced by
carbon content (climate and vegetation). In contrast, clay-size fractions from all soils had
similar C/N ratios (Table 3.1). For each soil, the majority of carbon (40% to 60%) and nitrogen
(50% to 65%) were associated with silt- and clay-size fractions.
3.4.2 Solvent Extractable Lipids, Lignin-derived Phenols and Cutin-derived OH-acids
Solvent-extractable lipid concentrations differed in the various fractions (Fig. 3.1).
Significantly greater concentrations of relatively labile monounsaturated organic acids (C16:1,
C18:1) and plant-derived phytosterols were detected in all three light fractions (Fig. 3.1). On the
other hand, high molecular weight (>C20) organic acids, which are likely more difficult to
degrade, were accumulated in silt- and clay-size fractions. The majority of linear n-alkanes and
n-alkanols extracted from each fraction consisted of high molecular weight (>C20) compounds
(Fig. 3.1), which are also more persistent than low molecular weight (<C21) linear lipids. In
addition, only n-alkanols with even carbon numbers were extracted from all fractions, which
suggests that these n-alkanols are predominantly plant-derived (Otto et al., 2005). Alternatively,
the low preference for n-alkanes with odd carbon numbers over those with even carbon numbers
suggests contributions from microbes. Linear lipids associated with the silt- and clay-size
fractions have been transformed by microorganisms, as suggested by greater similarities in the
distribution of linear lipids in these fractions (Appendix A1, Figs. A1.1-A1.3).
Relative concentrations of cutin-derived OH-acids, as well as lignin-derived phenol
monomers and dimers are shown in Fig. 3.2, and lignin phenol yields are listed in Table 3.1.
Variations in the relative abundance of these compounds indicate differences in the degradation
state of lignin-derived phenols and cutin-derived OH-acids (Bahri et al., 2006; Ertel and Hedges,
1984; Feng and Simpson, 2008; Goni and Hedges, 1992; Hedges et al., 1988). Lignin-derived
phenols from clay-size fractions had the highest Ad/Alv and Ad/Als ratios (Fig. 3.2), which
denotes that lignin-derived phenol monomers in these fractions were more oxidized (Hedges et
al., 1988). Alternatively, contributions of acidic forms of lignin dimers (dehydrodivanillic acid,
dehydrovanillinvanillic acid, 2-syringylsyringic acid, 2-vanillylsyringic acid) to clay-size
fractions increased with soil carbon content as there was 1.7 times more acid dimers in SG, 3.5
times more acid dimers in NG, and only acid dimers detected in GFT clay-size fractions.
65
Figure 3.1 Solvent extractable lipids (µg/g C) from various soil fractions from Alberta, which includes phytosterols (sitosterol + campesterol + stigmasterol), n-alkane (C24-C33), n-alkanol (<C21 = C16-C20; >C20 = C21-C32), organic acid (C16:1, C18:1 = sum of C16 and C18 monoun-saturated acid; <C21 = C10-C20; >C20 = C21-C28). Error bars represent standard error of the mean (n = 3). Means with different letters are significantly different (Tukey-HSD; = 0.05). Different upper-case letters indicate significant differences for the total extractable phytosterol, n-alkane, n-alkanol, or organic acid, while lower-case letters indicate significant differences in the contribution of the compounds in each fraction.
0
200
400
600
A
BC BC
0
200
400
600A
BC C
0
200
400
600A
B
B B
0
100
200
EvenOddA
AA
A
0
100
200
300 >C20 <C21
AA A A
c a b b
j l k k
0
400
800
1200
Light Sand Silt Clay
C16:1, C18:1 >C20 <C21
A
C CB
a
ky
bkx x xy
j jc c
0
100
200EvenOdd
A
B B
AB
0
100
200
300>C20 <C21
A
BABAB
a ba
a
k k kj
0
400
800
1200
Light Sand Silt Clay
C16:1, C18:1 >C20 <C21
A A
BABa
y yx xyjk k
j j
bb b
Org
anic
Aci
d (µ
g/g
C)
0
100
200Even
OddA
BBB
0
100
200
300 >C20 <C21
BB
B
A
ba a
a
j k k k
0
400
800
1200
Light Sand Silt Clay
C16:1, C18:1 >C20 <C21A
BB
B
y x x xy
kk
jkj
a
bb
b
Soil Fraction
Southern Grassland
Northern Grassland
Grassland-Forest Transition
N-a
lkan
ol (
µg/
g C
)N
-alk
ane
(µg/
g C
)P
hyto
ster
ol
(µg/
g C
)
>C20 <C21 >C20>C20<C21
<C21
C16:1, C18:1 C16:1, C18:1
C16:1, C18:1>C20 >C20
>C20<C21 <C21
<C21
66
Figure 3.2 Lignin-derived phenol and cutin-derived OH-acid distribution. The relative contribution of the three groups of lignin-derived phenol monomers are defined as vanillyl = vanillin + acetovanillone + vanillic acid; syringyl = syringaldehyde + acetosyringone + syringic acid; coumaryl = ferulic acid + coumaric acid; and the relative oxidation (Ad/Al) of lignin-derived phenols are defined as vanillyl = vanillic acid/vanillin, syringyl = syringic acid/syringaldehyde. The contribution of lignin phenol dimers relative to monomers is expressed as dimers/monomers ratios and contribution of cutin-derived OH-acid relative to lignin phenol monomers is expressed as OH-acids/monomers ratios. Error bars represent standard error of the mean (n = 3). Means with different letters are significantly different (Tukey-HSD; = 0.05).
Coumaryl monomers are believed to be less persistent compared to syringyl and vanillyl lignin
phenol monomers (Bahri et al., 2006; Ertel and Hedges, 1984; Kiem and Kögel-Knabner, 2003),
and its greater abundance in light fractions signifies that lignin in these fractions were at a
relatively early stage of degradation (Fig. 3.2). The concentration of lignin-derived phenols
decreased from light to mineral fractions (Table 3.1). However, lignin-derived phenol dimers
and cutin-derived OH-acids were significantly enriched relative to monomers, in mineral
0
0.5
1
1.5
2
2.5
3
VanillylSyringyl
a
bbb
j
kkl
l
0
0.5
1
1.5
2
2.5
3
VanillylSyringyl
a
b
bb
j
kkl
l
0
0.5
1
1.5
2
2.5
3
VanillylSyringyl
a
bc
bj
kl
mLign
in O
xida
tion
(Ad/
Al)
Soil Fraction
0
0.05
0.1
0.15
0.2
0.25
Light Sand Silt Clay
Dimers/Monomers
OH-acids/Monomersj
k
l
m
aa
bb
0
0.05
0.1
0.15
0.2
0.25
Light Sand Silt Clay
Dimers/Monomers
OH-acids/Monomers
jj
jkk
aab
bc
c
0
0.05
0.1
0.15
0.2
0.25
Light Sand Silt Clay
Dimers/Monomers
OH-acids/Monomers j
k
l
m
aa
ab
b
0
10
20
30
40
50
60 Vanillyl Syringyl Coumaryl
b l
ya j
z
a ak jk
z z
0
10
20
30
40
50
60 Vanillyl Syringyl Coumaryl
j
a a a
b kj jy
zz z
Lign
in P
heno
ls (
%)
Dim
er a
nd O
H-a
cid
Enr
ichm
ent
Southern Grassland
Northern Grassland
Grassland-Forest Transition
abb
cj j
kl
y
z z z
0
10
20
30
40
50
60 Vanillyl Syringyl Coumaryl
67
fractions compared to light fractions (Fig. 3.2). This observation may be attributed to the
relative preservation of these compounds as lignin-derived phenol dimers and cutin-derived OH-
acids may be more stable against degradation compared to lignin-derived phenol monomers.
The relative concentrations of lignin-derived phenols were similar to whole soils (Otto and
Simpson, 2006a) in that there was relatively more extractable lignin-derived phenol monomers
detected in SG fractions compared to NG and GFT fractions (Table 3.1).
3.4.3 Organic Matter Patterns in Solid-state 13C NMR Spectra
The differences in intensity of NMR signals is related to differences in abundance of the
corresponding chemical structure, which may be attributed to soil characteristics (climate,
vegetation) or particle size (Baldock et al., 1992; Guggenberger et al., 1995; Quideau et al.,
2001). 13C CP/MAS NMR-detected total OM from soils and their fractions were composed of
similar chemical structures but at varying abundances (Fig. 3.3). Integrated values of the alkyl,
O-alkyl, anomeric, aromatic, and carboxyl regions (labeled a-e respectively in Fig. 3.3) are
listed in Table 3.2. Alkyl signals (Fig. 3.3, region a) may be derived from CH, CH2, and CH3 in
proteins, lipids, waxes, or cutin and suberin (Deshmukh et al., 2005; Hu et al., 2000; Preston et
al., 2000). Signals in the 27-36 ppm region are likely from polymethylene compounds derived
from plant cuticles (Deshmukh et al., 2005; Hu et al., 2000). Contributions from these relatively
stable structures were also greater in SG compared to NG and GFT fractions (Fig. 3.3). Signals
in the O-alkyl region (Fig. 3.3, region b, Table 3.2) may be attributed to carbon bonds and
methoxy groups that correspond to carbohydrates and lignin, with minor contributions from
proteins and ethers (Preston et al., 2000). For all soils, these signals decreased from light
fraction and with decreasing particle size (Table 3.2). There were also lower contributions from
O-alkyl signals to OM in NG sand- and silt-size fractions compared to the same fractions from
SG and GFT fractions (Table 3.2). The most intense signal observed in the O-alkyl region,
which was centered at 74 ppm, has been attributed to carbohydrates (Preston et al., 2000;
Quideau et al., 2001). Other signals, which are also likely carbohydrate-derived, are observed at
95-110 ppm (anomeric; Fig. 3.3, region c) and 63 ppm (i.e. C6 of glucose; Fig. 3.3;(Preston et
al., 2000). The signal at 56 ppm is typically attributed to methoxy or ethoxy groups attached to
aromatic units (typical of lignin, with possible minor contributions from amino acids;(Preston et
al., 2000; Quideau et al., 2001). The decrease in intensity of the 63 ppm signal relative to the 56
ppm signal from light to silt-size fraction was associated with a decrease in the intensity of the
68
Figure 3.3 Solid-state 13C CP/MAS NMR spectra of soils and their respective fractions. Highlighted regions are attributed to: a) alkyl (0-50 ppm) b) O-alkyl (50-95 ppm) c) anomeric (from carbohydrates; 95-110 ppm) d) aromatic (C-linked and phenol; 110-160 ppm) e) carboxyl + carbonyl (160-200 ppm).
04080120160200 04080120160200 04080120160200
Chemical Shift (ppm)
Southern Grassland Northern Grassland Grassland-Forest Transition
Light
Sand
Silt
Clay
Soil
abcde abcde abcde
69
Table 3.2 Relative contribution (%) of alkyl (0-50 ppm), O-alkyl (50-95 ppm), anomeric (95-110 ppm), aromatic (110-160 ppm), and carboxyl + carbonyl (160-200 ppm) structures to the solid-state 13C CP/MAS NMR spectra after integration and resulting alkyl/O-alkyl ratios.
Structural Group Light Sand Silt Clay Soil Southern Grassland Alkyl 22 31 32 35 32 O-alkyl 47 34 35 36 35 Anomeric 8 5 5 5 5 Aromatic 16 21 19 14 19 Carboxyl + carbonyl 7 9 9 10 9 Alkyl/O-alkyl 0.5 0.9 0.9
1.0 0.9
Northern Grassland Alkyl 26 28 28 32 29 O-alkyl 43 26 27 32 29 Anomeric 8 4 5 5 5 Aromatic 17 31 30 19 26 Carboxyl + carbonyl 6 11 10 12 11 Alkyl/O-alkyl 0.6 1.1 1.1
1.0 1.0
Grassland-Forest Transition Alkyl 25 24 24 29 26 O-alkyl 50 38 30 37 36 Anomeric 8 7 6 7 7 Aromatic 14 22 32 17 23 Carboxyl + carbonyl 3 9 8 10 8 Alkyl/O-alkyl 0.5 0.6 0.8 0.8 0.7
anomeric signal relative to the aromatic signal (Fig. 3.3; Table 3.2). This suggests decreased
carbohydrate contributions in silt-size fractions. In contrast, contributions from carbohydrate-
derived signals (anomeric and 63 ppm) were more intense relative to lignin-derived (aromatic
and 56 ppm) signals in clay-size fractions.
Contributions of lignin-derived structures in sand- and silt-size compared to clay-size
and light fractions (Fig. 3.2) are consistent with integration of aromatic signals from solid-state 13C NMR spectra (Table 3.2). The aromatic region (128 ppm) was less intense in SG soil,
especially in sand- and silt-size fractions (Fig. 3.3, Table 3.2). In addition, the intensity of
signals associated with phenolic (150 ppm) structures also became less intense from light
70
fraction and with decreasing particle size (Fig. 3.3), which indicates a decreased contribution
from lignin–derived compounds. Other aromatic structures, which include suberin (Preston et
al., 2000), amino acids (Preston et al., 2000), and black carbon (Simpson and Hatcher, 2004),
may have also contributed to aromatic signal intensity but given the detection of lignin-derived
phenols by CuO oxidation, it is likely that the majority of signals in this region are from lignin.
The ratio of alkyl/O-alkyl decreases with progressive OM degradation and is used to
assess the relative degradation of OM (Baldock et al., 1992; Guggenberger et al., 1995; Quideau
et al., 2001). In this study, higher alkyl/O-alkyl ratios in mineral fractions were observed
suggesting an enrichment of alkyl carbon and a greater extent of OM degradation in mineral
fractions as compared to their respective light fractions. This observation is consistent with high
Ad/Al ratios for lignin phenols also observed in clay fractions (Fig. 3.2) which also imply that
lignin in this fraction is at an advanced stage of degradation.
3.4.4 Organic Matter Patterns in Solution-state 1H NMR Spectra
Solid-state 13C CP/MAS NMR detects signals from all OM components whereas
Simpson et al. (2007a) reported that 56-79% of soil OM was extractable for 1H NMR analysis.
Solution-state 1H NMR offers increased resolution and sensitivity which provides additional
information and detail about OM structural composition that cannot be obtained from solid-state 13C NMR alone (Feng and Simpson, 2011). Solution-state 1H NMR and DE 1H NMR spectra of
humic substances isolated from sand-, silt-, clay-size and light fractions are shown in Figs. 3.4-
3.6. Sharper signals in 1H NMR spectra, including one that resonates at 3.9 ppm (Figs. 3.4-3.6),
were not observed in the DE 1H NMR spectra (Figs. 3.4-3.6) indicating that these peaks arise
from relatively small compounds (Simpson et al., 2007a) such as degradation products. These
smaller compounds were found in all mineral fractions, but were especially noticeable in the
GFT mineral fractions (Fig. 3.6), which suggests that GFT fractions contained higher
concentrations of degradation products and altered OM.
Signals mainly attributed to polymethylene, carbohydrates, and lignin were also detected
in the solution-state 1H NMR spectra of all samples (Figs. 3.4-3.6). Intensity of signals
attributed to CH2 in polymethylene structures was greater in mineral compared to light fractions
(Figs. 3.4-3.6). This signifies greater contributions from relatively small plant- or microbial-
derived lipids and waxes, since the relative intensity of these signals was also attenuated after
71
Figure 3.4 Solution state 1H-NMR and diffusion edited (DE) 1H-NMR of Southern Grassland soil fractions. The highlighted chemical shift regions are attributed to: a) aliphatic methyl and methylene (0.6-1.3 ppm); b) aliphatic methyl and methylene near O and N (1.3-2.9 ppm); c) O-alkyl, mainly from carbohydrate and lignin (2.9-4.1 ppm); d) α 1H from proteins (4.1-4.8 ppm e) aromatic, from lignin and proteins (6.2-7.8 ppm); f) amide from proteins (7.8-8.4 ppm). Other signals, which may be protein-derived are labeled as P1-P6 as described in the text.
01.02.03.04.05.06.07.08.09.001.02.03.04.05.06.07.08.09.0
abcdef
DMSO-d6
CH2
CH3Light
Sand
Silt
Clay
Chemical Shift (ppm)
AlkylO-Alkyl
3.9
P6P5
P4P3
P2
CH3 (P1)
1H NMR DE 1H NMR
72
Figure 3.5 Solution state 1H-NMR and diffusion edited (DE) 1H-NMR of Northern Grassland soil fractions. The highlighted chemical shift regions are attributed to: a) aliphatic methyl and methylene (0.6-1.3 ppm); b) aliphatic methyl and methylene near O and N (1.3-2.9 ppm); c) O-alkyl, mainly from carbohydrate and lignin (2.9-4.1 ppm); d) α 1H from proteins (4.1-4.8 ppm); e) aromatic, from lignin and proteins (6.2-7.8 ppm); f) amide from proteins (7.8-8.4 ppm). Other signals, which may be protein-derived are labeled as P1-P6 as described in the text.
01.02.03.04.05.06.07.08.09.0 01.02.03.04.05.06.07.08.09.0
abcdef
DMSO-d6
CH2
CH3Light
Sand
Silt
Clay
Chemical Shift (ppm)
AlkylO-Alkyl
3.9
P6P5
P4P3
P2
CH3 (P1)
1H NMR DE 1H NMR
73
Figure 3.6 Solution state 1H-NMR and diffusion edited (DE) 1H-NMR of Grassland-Forest Transition soil fractions. The highlighted chemical shift regions are attributed to: a) aliphatic methyl and methylene (0.6-1.3 ppm); b) aliphatic methyl and methylene near O and N (1.3-2.9 ppm); c) O-alkyl, mainly from carbohydrate and lignin (2.9-4.1 ppm); d) α 1H from proteins (4.1-4.8 ppm); e) aromatic, from lignin and proteins (6.2-7.8 ppm); f) amide from proteins (7.8-8.4 ppm). Other signals, which may be protein-derived are labeled as P1-P6 as described in the text.
01.02.03.04.05.06.07.08.09.0 01.02.03.04.05.06.07.08.09.0
abcdef
DMSO-d6
CH2
CH3
Light
Sand
Silt
Clay
Chemical Shift (ppm)
AlkylO-Alkyl
3.9
P5
P4P3
P2
CH3 (P1)
P6
1H NMR DE 1H NMR
74
diffusion editing. On the other hand, contributions from O-alkyl signals attributed to
carbohydrates and lignin were greater in light compared to mineral fractions (Figs. 3.4-3.6).
Because of the greater sensitivity of solution-state 1H NMR as compared to solid-state 13C
NMR, as well as the selectivity of DE 1H NMR for relatively large compounds, differences in
alkyl/O-alkyl ratios between the mineral fractions were enhanced after diffusion editing (Table
3.3). For example, the alkyl/O-alkyl ratio from the DE 1H NMR spectrum of SG clay-size
fraction spectra was higher than ratios in the NG and GFT clay-size fraction spectra (Table 3.3)
implying that lignin or carbohydrates were depleted in the SG clay-size fraction. Compared to
the light fraction, there were greater decreases in contribution from O-alkyl signals in clay-size
fractions after diffusion editing (Figs. 3.4-3.6; Table 3.3). This indicates that relatively small O-
alkyl structures (carbohydrates and lignin) contributed to OM associated with clay-size fractions
of these soils. An apex in the aromatic region (region e; 6.7-6.8 ppm) was previously observed
in SG vegetation and commercial lignin (Simpson et al., 2007a). In this study, except for
signals attributed to peptides, the majority of signals in the aromatic region (region e; Figs. 3.4-
3.6) were likely lignin-derived. Similar to lignin-derived phenols, aromatic signal contribution
was greater in light fraction and decreased with mineral size (Figs. 3.4-3.6).
Previous solution-state 1H NMR studies identified microbial-derived peptides in OM
extracts (Simpson et al., 2007a; 2007b). In this study, the same series of signals (P1-P6) were
observed (Figs. 3.4-3.6) and collectively suggest the presence of peptides in the various
fractions. First, signals attributed to α1H and amides, which are characteristic of peptides, were
observed at 4.1-4.8 ppm (P2) and 7.8-8.4 ppm (P6) respectively (Simpson et al., 2007a). Second,
although signals in the aromatic region (e) are commonly attributed to lignin, the relative
heights and distance between signals P3 (6.6 ppm) and P4 (7.0 ppm) are consistent with the
presence of tyrosine, while P5 (7.2 ppm) may be attributed to phenylalanine (Simpson et al.,
2007a). Both tyrosine and phenylalanine are aromatic amino acids found in peptides. Finally,
because the CH3 (P1) signals had similar intensities as the CH2 signals, these CH3 groups cannot
be accounted for by polymethylene-containing compounds such as cutin and suberin alone, and
a large proportion of these CH3 groups may be derived from methyl-rich amino acids in peptides
(Simpson et al., 2007a; 2007b). Diffusion editing also revealed signals from relatively large or
rigid components in region b. These signals, when combined with those previously attributed to
75
Table 3.3 Contribution of lignin- and carbohydrate-derived structures (O-alkyl) relative to: lipids, protein, waxes, cutin and suberin (alkyl/O-alkyl) or protein (O-alkyl/1H) in soil fraction extracts from solution-state 1H NMR and diffusion edited (DE) 1H NMR spectra.
Light Sand Silt Clay
Structural Group
Southern Grassland
Alkyl/O-alkyl 1H 0.8 1.5 1.5 1.6
DE 1H 0.7 1.5 1.7 2.1
O-alkyl/1H 1H 6.4 5.5 4.2 3.9
DE 1H 5.3 4.2 3.7 3.0
Northern Grassland
Alkyl/O-alkyl 1H 1.1 1.3 1.3 1.2
DE 1H 1.0 1.5 1.6 1.8
O-alkyl/1H 1H 5.2 5.5 4.6 5.0
DE 1H 5.0 4.4 3.9 3.5
Grassland-Forest Transition
Alkyl/O-alkyl 1H 1.1 1.1 1.5 1.6
DE 1H 0.8 1.0 1.3 1.7
O-alkyl/1H 1H 5.6 5.2 5.2 4.2
DE 1H 5.6 4.9 4.5 3.7
peptides, denote contributions from microbial-derived OM in soil fractions (Simpson et al.,
2007a).
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3.5 Discussion
3.5.1 Preservation of Organic Matter in Particle-size Fractions
In this study, enrichment of specific OM signatures, namely those from peptides,
carbohydrates, lignin, phytosterols, and aliphatics, in the particle-size fractions was related to
their possible sources, structure and relative recalcitrance. Microbial-derived OM is enriched in
proteins (polypeptides) and N compared to plant-derived OM (Kögel-Knabner, 2002). In this
study, the enrichment of peptide-derived signals in 1H and DE 1H NMR spectra (Figs. 3.4-3.6,
Table 3.3), as well as lower C/N ratios (Table 3.1) suggests greater concentrations of microbial-
derived OM in finer fractions. Although peptides are considered relatively labile, these
compounds may be preserved in finer fractions by forming stable complexes within soil OM
(Hsu and Hatcher, 2005; Zang et al., 2000). The greater contributions of relatively labile
carbohydrates to clay- compared to silt-size fractions (Fig. 3.3) also implies that these
compounds may be microbial-derived as other studies found that microbial-derived
carbohydrates increased with decreasing particle size (Guggenberger et al., 1994; Kiem and
Kögel-Knabner, 2003). Although it is likely that peptides and carbohydrates accumulated in
finer fractions through specific interactions with clay minerals, it is also possible that microbes
(whether active or dormant) may be associated with clay-rich microenvironments in soils since
survival of microorganisms may be enhanced through encapsulation with clay (Marshall, 1975;
Mikutta et al., 2007). The precise nature of the interactions between microbial-derived OM and
clay minerals deserves further study, as this has important implications on current models of
OM turnover.
Lignin is a plant-derived, heterogeneous phenolic polymer, which must first be
depolymerized such that it can be used as a substrate by microbes (Kögel-Knabner, 2002). In
this study, relatively small lignin components (likely depolymerization products) may have
contributed to the aromatic region since a variety of smaller structures detected using solution-
state 1H NMR, were no longer detected by DE 1H NMR (Figs. 3.4-3.6; aromatic and O-alkyl
regions). The relative stability of specific lignin-derived phenols may facilitate their enrichment
in finer fractions (Fig. 3.2). For example, lignin-derived phenol dimers, which are hypothesized
to be more stable than lignin-derived phenol monomers (Goni and Hedges, 1992; Goni et al.,
1993), accumulated in the mineral fractions of all three soils and especially in the finer fractions
77
of SG and NG soils (Fig. 3.2). Syringyl and vanillyl monomers, which are more stable than
coumaryl monomers (Bahri et al., 2006; Ertel and Hedges, 1984; Hedges et al., 1988; Kiem and
Kögel-Knabner, 2003), also accumulated in SG, NG, and GFT mineral fractions (Table 3.1; Fig.
3.2), further supporting previous observations where more stable lignin-derived phenols
accumulated through association with mineral fractions. Our data also supports the hypothesis
that protection of lignin-derived structures may be attributed to the formation of silt-size
microaggregates (Heim and Schmidt, 2007b), which are stabilized by various OM compounds
(Chenu and Plante, 2006; Dinel et al., 1991b; Virto et al., 2008). For example, radiocarbon 14C
dating indicate that OM associated with finer fractions had long residence times, which could
range between 800-1660 y in silt- and 75-4409 y in clay-size fractions (von Lutzow et al.,
2007). In particular, measurements of 13C abundance after C3/C4 plant turnover indicate
enrichment of older lignin in silt-size fraction, which further suggests that lignin preservation
may rely on particle-size-specific mechanisms (Heim and Schmidt, 2007a).
Aliphatic compounds, derived from cutin, suberin, waxes, and lipids are believed to be
the most stable components of soil OM (Baldock et al., 1992; Kelleher et al., 2006; Kögel-
Knabner, 2002; Lorenz et al., 2007; Mikutta et al., 2006b), and accumulate in finer particle-size
fractions (Baldock et al., 1992; Lorenz et al., 2007). This study found that increased alkyl/O-
alkyl ratios from solution-state DE 1H NMR (Table 3.3), which indicates enrichment of aliphatic
compounds relative to lignin and carbohydrates, can be directly related to the enrichment of
cutin-derived OH-acids compared to lignin-derived phenol monomers (Fig. 3.2). Unlike
solution-state DE 1H NMR data, solid-state 13C NMR was not as sensitive to shifts in alkyl/O-
alkyl ratios along mineral fractions (Table 3.2), perhaps due to the abundance of relatively small
lignin or carbohydrates. This explanation is consistent with the differences in the alkyl/O-alkyl
ratios between light and clay-size fractions, which was smaller in 1H NMR and greater in DE 1H
NMR spectra (Table 3.3), since relatively small structures (mainly in the O-alkyl region) were
not detected after diffusion editing.
Although aliphatic compounds have been shown to sorb to clay (Feng et al., 2005), this
study found that the specific structure of aliphatic compounds may control their distribution in
finer fractions. While differences in the concentrations of n-alkanes and n-alkanols associated
with the various fractions seemed to be influenced by soil type (Fig. 3.1), more stable and higher
molecular-weight aliphatic structures were preserved in finer particles as indicated by the
78
distribution of organic acids (Fig. 3.1) and other aliphatic compounds (Fig. 3.3). Specifically,
relative contributions of labile <C21, and mono-unsaturated organic acids decreased with particle
size, while contributions of relatively stable >C20 organic acids were significantly greater in silt-
and clay-size fractions (Fig. 3.1). These data are consistent with the study by Wiesenberg et al.
(2010), who found greater concentrations of >C20 organic acids in mineral fractions. In our
study, we observed the enrichment of >C20 organic acids specifically in silt- and clay-size
fractions. While previous studies only reported alkyl contents in soil fractions (Baldock et al.,
1992; Guggenberger et al., 1995), the 13C NMR spectra in this study were resolved enough to
discern polymethylene structures (30 and 33 ppm). Polymethylene and aliphatic compounds
were accumulated, relative to other alkyl structures, in finer fractions (Fig. 3.3). Although
microbial lipids may have contributed to the structures in the aliphatic region (Baldock et al.,
1992; Guggenberger et al., 1995), the polymethylene structures and >C20 organic acids together
imply that cutin- and suberin-derived compounds were accumulated in finer fractions. These
plant-derived materials are degraded in soils (Otto and Simpson, 2006b), but at a slower rate
compared to other compounds (Baldock et al., 1992; Feng and Simpson, 2008; Kelleher et al.,
2006), which leads to their enrichment. Other studies have also shown that aliphatic compounds
are selectively sorbed to kaolinite and montmorillonite surfaces, which may also enhance their
long-term preservation in soil (Feng et al., 2005; Ghosh et al., 2009; Simpson et al., 2006).
3.5.2 Environmental Controls on Organic Matter Preservation
Both climate and vegetation, which result in variations in carbon content, are also
believed to influence the distribution of OM compounds in soils and their particle-size fractions
(Baldock et al., 1992; Guggenberger et al., 1994; 1995; Quideau et al., 2001; Zech et al., 1997).
OM in GFT soils was less degraded, as suggested by greater contribution of light fraction to
GFT soil compared to Grassland soils. The greater contribution of this fraction may be
attributed to the higher stability of wood-derived OM in GFT soil, relative to grass-derived OM
in NG and SG soils (McCulley et al., 2004). This is consistent with higher light fraction
concentrations observed by Liao et al. (2006) after woody plants replaced grasses. OM
associated with sand-, silt-, and clay-size fractions of GFT was also less degraded (suggested by
higher C/N ratios; Table 3.1), in contrast to the similar degradation state of OM in clay-size
fractions of all three soils (suggested by similar C/N ratios; Table 3.1). This finding was also
consistent with greater contributions from small degradation products in the 1H NMR spectra of
79
GFT soil mineral fractions (Fig. 3.6), which may represent compounds that were more slowly
mineralized.
All clay fractions had similar C/N ratios but there was enrichment of n-alkanes and n-
alkanols with >C20 in the GFT clay-size fraction (Figs. 3.1; Appendix A1, Figs. A1.1-A1.3).
This enrichment may also be related to the greater recalcitrance of OM in GFT compared to
Grassland (NG and SG) soils because there were also greater concentrations of lipids extracted
from the GFT soil (Otto and Simpson, 2005). Preservation of these compounds in clay-size
fraction may be attributed to their relative recalcitrance, as suggested by Otto and Simpson
(2005). However, unlike whole soils, where only plant-derived n-alkanes (which had odd
carbon number) were observed, the greater contributions from n-alkanes with even carbon
number suggests microbial-derived n-alkanes were also extracted from the various fractions.
Previous studies have shown that n-alkanes may be trapped within the OM matrix (Lichtfouse et
al., 1998), or aggregates (Dinel et al., 1991b; Gobe et al., 2000), and disruptions of OM and
OM-mineral associations during the fractionation process may have released these compounds
from the soil matrix. However, enrichment of these linear lipids in clay-size fraction may not be
attributed solely to the higher carbon content in GFT soils since studies indicate that phytosterol
preservation was also related to carbon content (Gobe et al., 2000), but unlike n-alkanes and n-
alkanols, our study shows that phytosterol concentrations decreased with particle size (Fig. 3.1).
This suggests that besides the inherent recalcitrance of n-alkanes and n-alkanols with >C20, the
preservation of these compounds may have resulted from their linear structure, which enhanced
their interactions with other soil OM. Despite the enrichment of n-alkanes and n-alkanols in the
GFT clay-size fraction (Fig. 3.1), overall contributions of polymethylene structures were greater
in SG fractions (Fig. 3.3). This is consistent with higher concentrations of hydrolysable lipids
detected in SG soils by Otto and Simpson (2006b). This difference may again be attributed to
variations in the biochemistry of the overlying vegetation. Plants produce more waxes and cutin
in order to survive drier environments (Lorenz et al., 2007), and the SG sampling site receives
less precipitation (Janzen et al., 1998). In addition, the lower C/N ratios in the sand-, silt-, clay-
size, and light fractions (Table 3.1) imply that OM from this soil is more degraded, and the more
recalcitrant cutin- and suberin-derived polymethylene compounds may have accumulated in this
soil. Further studies are required to understand the chemical and physical processes behind
these interactions.
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3.6 Conclusions
As this study highlights, mechanisms responsible for OM protection are interdependent.
While relatively labile OM that accumulated in finer fractions may be attributed to in situ
microbial synthesis, relatively recalcitrant OM structures were preferentially stabilized through
OM-mineral interactions and accumulated in silt- and clay-size fractions. These interactions
appear to be controlled by OM structure, because differences in the degradation state of OM in
the various soils had little control on the enrichment of specific OM components in finer
fractions. For example, aromatic structures were preferentially preserved in silt-size fractions,
while stable linear aliphatic compounds were preferentially preserved in clay-size fractions.
There were also indications that the enrichment of n-alkanes, and n-alkanols are controlled by
carbon content, which means that OM-OM interactions is also a possible protection mechanism
that has not yet been fully explored. Determining the relative roles of chemical recalcitrance, as
opposed to physical and chemical interactions with minerals, on the protection of soil OM
remains a challenge, and further studies are necessary to determine if the patterns we observed
apply to other soil systems.
3.7 Acknowledgements
The authors thank Katherine Hills and David Wolfe for assistance with sample extractions. We
would also like to thank Dr. David McNally for assistance with NMR experiments. Funding for
this project was provided by the NSERC Green Crop Network. J.S.C. also thanks the Ontario
Government for support via an Ontario Graduate Scholarship.
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CHAPTER 4
COMPARISON OF SOIL ORGANIC MATTER COMPOSITION AFTER INCUBATION WITH MAIZE LEAVES, ROOTS, AND STEMS
Authors: Joyce S. Clemente, Myrna J. Simpson, Andre J. Simpson, Sandra F. Yanni, Joann K.
Whalen
Contributions: Soils amended with maize tissues and biodegraded were provided by SFY and
JKW. The research questions were framed by JSC, with input from MJS. Sample extractions
were performed by JSC, with assistance from David Wolfe. Biomarker analyses were
performed by JSC. NMR acquisition parameters were designed by AJS. NMR data acquisition
was performed by JSC with assistance from Dr. David McNally. Data analyses were performed
by JSC, with input from MJS. The manuscript was written by JSC and MJS, with critical
comments from the co-authors.
Status: Submitted to Geoderma (in revision).
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4.1 Abstract
As plants are major contributors to soil organic matter (OM), the chemical composition of plant
tissues entering the soil should be related to the soil OM composition. We examined the OM
composition of maize leaves, stems, and roots, to determine if differences in plant tissue
composition altered the soil OM composition during a 36-week degradation experiment. Lignin
phenols were measured by gas chromatography/mass spectrometry (GC/MS), and soil OM and
humic substances were characterized using solid-state 13C and solution-state 1H nuclear
magnetic resonance (NMR) spectroscopy, respectively. Lignin phenol composition, oxidation,
and depletion relative to cutin-derived OH-acids changed less in leaf- compared to stem- and
root-amended soils over time, and may be due to greater vanillyl concentrations in leaves. Soil
amended with stems had higher concentrations of carbohydrates in soil OM. Humic substances
from leaf-amended soils had higher concentrations of aliphatic components, likely due to higher
concentrations of aliphatic compounds in leaf tissues, which suggests that compounds derived
from leaves are potential contributors to the stable pool of soil OM. After 36-weeks of
incubation, the contribution of microbial-derived OM was greatest in humic extracts from root-
amended soils, and increased contribution from these compounds was detected earlier in these
soils than stem- and leaf-amended soils. This indicates that root amendment may enhance
contributions from microbial-derived OM. Our study suggests that changes in soil OM
composition over time was related to the chemical composition of the plant tissue, and
demonstrates the important link between plant chemistry and soil OM turnover.
4.2 Introduction
Maize (Zea mays L.) is a major cereal crop grown on more than one-fifth of the
agricultural land worldwide (Amos and Walters, 2006), and production is expected to increase
to meet food and biofuel demands (FAO-UN, June 2011). Thus, understanding the fate of
maize residues in agricultural soils is critical to the future management and sustainability of
agricultural ecosystems (Patzek, 2008). It is hypothesized that most plant-derived C is
eventually mineralized to CO2, and approximately 10% - 20% is preserved in soil (Lorenz et al.,
2007). Plant-derived C that remains in soil may be preserved as intact plant material, or
transformed into degradation products and microbial-derived organic matter (OM) (Baldock and
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Skjemstad, 2000). However, how the chemical composition of plant residue influences the
relative pools of soil carbon is yet to be analyzed at the molecular-level.
The long-term (between several months to several years) preservation of some plant-
derived compounds has been partly attributed to their biochemical recalcitrance (Baldock and
Skjemstad, 2000; Feng et al., 2008; Mikutta et al., 2006b; von Lutzow et al., 2007). For
example, carbohydrates and proteins are more easily degraded than lignin, while long-chain
aliphatic compounds found in cuticular waxes, cutin, and suberin are more slowly degraded
(Lorenz et al., 2007; Mikutta et al., 2006b). A number of studies suggest that higher lignin/N
ratios (Johnson et al., 2007; Melillo et al., 1982; Moretto and Distel, 2003; Preston et al., 2000;
Yanni et al., 2011) or cutin/N ratios (Gallardo and Merino, 1993) results in slower rates of plant
residue degradation. Roots have higher concentrations of lignin compared to leaves and stems,
and contain suberin, which are not always found in plant biomass above-ground (Johnson et al.,
2007; Mendez-Millan et al., 2010b; Nierop, 1998). Therefore, the chemical composition of
roots is believed to result in their preservation in litter bag (Moretto and Distel, 2003) and
mineralization studies (Johnson et al., 2007; Yanni et al., 2011). Moreover, high resolution-
magic angle spinning 13C nuclear magnetic resonance (NMR) monitoring of grass degradation
over time showed less chemical alteration of OM in soil amended with grass roots, compared to
soil amended with grass blades (Kelleher et al., 2006), further suggesting that root-derived
compounds may be more recalcitrant. Higher concentrations of root-derived suberin were also
detected in surface and subsurface soil horizons, which collectively suggests the preferential
incorporation and preservation of root-derived C is related to its chemical composition,
specifically the lignin, cutin and suberin concentrations (Feng and Simpson, 2007; Mendez-
Millan et al., 2010b; Nierop, 1998; Otto and Simpson, 2006b; Rumpel et al., 2002). However,
other studies suggest that roots are not always more recalcitrant than leaves and stems (Riederer
et al., 1993; Wang et al., 2004). Consequently, additional studies are needed to better
understand how the chemical composition of maize roots, compared to leaves and stems,
governs their degradation and preservation in soil, and how each of these tissues contribute to
the composition of soil OM.
The influence of maize tissue chemistry on soil OM composition can be better
understood by combining information from solid-state 13C and solution-state 1H NMR, as well
as biomarker methods (Feng and Simpson, 2011). Solid-state 13C NMR is able to characterize
84
structures in whole soil OM and can be related to various plant-derived compounds in soil.
However, the resolution in solid-state 13C NMR spectra is limited because the chemical
heterogeneity of soil OM results in signal overlap, which may prevent identification of specific
compounds (Chabbi and Rumpel, 2004; Feng and Simpson, 2011; Preston et al., 2000; Simpson
et al., 2008). Biomarker methods, such as CuO oxidation, on the other hand are able to identify
and quantify specific compounds, such as lignin-derived phenols (Chabbi and Rumpel, 2004;
Ertel and Hedges, 1984; Goni and Hedges, 1990b, 1992; Hedges and Ertel, 1982; Otto and
Simpson, 2006a; Simpson et al., 2008). Solution-state 1H NMR methods are used as a bridge
between solid-state 13C NMR and biomarker methods, since solution-state 1H NMR spectra of
humic substances are better resolved (Feng and Simpson, 2011), and allows the identification of
specific components such as lipids, lignin, carbohydrates, peptides, and peptidoglycan
(Simpson et al., 2007a; 2007b). Solution-state 1H NMR has also been found to be more
sensitive to changes in OM composition in comparison with solid-state 13C NMR (Feng et al.,
2008; Simpson et al., 2011).
We investigated the composition of OM in leaves, roots, stems, and soil samples
amended with these plant materials before, during, and after 36 weeks of incubation. Analyses
were done using gas chromatography-mass spectrometry (GC/MS) after CuO oxidation. We
also employed solid-state 13C NMR, solution-state 1H, and diffusion edited (DE) 1H NMR to
examine structural changes to total (soil) OM and NaOH-extractable (soil humic substances)
organic compounds. In soils, roots are generally believed to be degraded to a lesser extent than
leaves and stems, because roots are mineralized more slowly. Our objectives are to test whether
degradation of roots also correspond to less extensive changes in soil OM composition
compared to degradation of leaves and stems; and whether changes in contributions of plant
material to plant-derived OM, degradation products, and microbial-derived OM, can be
observed using advanced molecular-level methods.
4.3 Materials and Methods
4.3.1 Biodegradation of OM in Soils Amended with Maize Leaves, Stems, and Roots
The samples analyzed in this study were a subset from a larger investigation on aerobic
biodegradation of maize tissues that examined the influences of genetic modification and added
lignin (Yanni et al., 2011). The soil used in this study was sampled from a long-term maize
85
experiment at the Macdonald Research Farm, Ste. Anne de Bellevue, Quebec, Canada (45o
30’N, 73o 35’W) and is classified as a Dystric Gleysol (815 g sand kg-1, 96 g clay kg-1, with a
pH of 6.0, 17.6 g organic C kg-1 and 1.6 g N kg-1; Yanni et al., 2011). The clay minerals were
composed of vermiculite, illite, smectites, and chlorite (Simard et al., 1990). The soil was air-
dried and passed through a 2 mm sieve. Maize plants (DKC 38-33, MON810 Bt insertion
event) were grown in the greenhouse and harvested at the V9-V10 stage, separated into leaves,
stems, and roots and then dried at 50 oC for 24 h, and ground to pass through a 1mm sieve. The
soil (50 g) and tissues (0.5 g), resulting in a 1% residue addition, were placed in 120 cm3 plastic
vials, mixed thoroughly, and moistened to 40% water-filled pore space. In comparison, it is
estimated that soil OM increases by 1-3% over 10 years, when above-ground biomass is
returned to agricultural soils (Janzen et al., 1998; Wilhelm et al., 2004). These vials were then
placed in 1-L glass jars, and 10 mL distilled water added to the jars to maintain soil humidity.
The jars (n = 4) were capped with an air-tight lid, incubated in the dark at 20 oC, and lids were
removed to aerate the jar for 15 min every week. Based on respiration results from Yanni et al.
(2011), we selected soils amended with tissue and biodegraded for 1, 2, 4, 16, and 36 weeks,
which corresponded to time-points in the exponential (weeks 1, 2, 4) and stationary (weeks 16,
36) respiration phases. An un-amended soil served as a control and was also analyzed. These
samples were freeze dried and ground to a powder for subsequent chemical analyses.
4.3.2 Lignin-derived Phenol and Cutin-derived OH-acid Extraction and Analysis
Lignin-derived phenols were isolated using CuO oxidation following the method of
Hedges and Ertel (1982), as modified by Otto and Simpson (2006a). Cutin-derived hydroxy-
acids (OH-acids) were also isolated by CuO oxidation (Filley et al., 2008; Goni and Hedges,
1990a; Mendez-Millan et al., 2010a). Three sub-samples from each soil were extracted with
organic solvents (dichloromethane and methanol) to remove free compounds and then extracted
with Copper (II) oxide (Sigma-Aldrich, Columbus, GA) and ammonium iron (II) sulfate
hexahydrate [Fe(NH4)2(SO4)26H2O] (Sigma-Aldrich), which were suspended in 2 M NaOH,
and placed in Teflon-lined bombs. These samples were then flushed with N2 gas and incubated
at 170 oC for 2.5 h. After cooling, the resulting supernatants were acidified to pH ~1 using 6 M
HCl (Caledon Laboratories, ON), then kept for 1 h at room temperature, in the dark to prevent
cinnamic acid polymerization. Colloidal particles that formed after acidification were removed
by centrifugation at 2700 rpm (1450 rcf) for 30 min, and the supernatant was extracted with
86
diethyl ether (Fisher Scientific), concentrated, transferred to 2 mL glass vials, and dried under
nitrogen.
CuO oxidation residues were re-dissolved in dichloromethane prior to derivatization.
Derivatization was performed by adding N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA;
Sigma-Aldrich) and pyridine to the samples, followed by heating at 70 oC for 1 hr. After
cooling, the samples were diluted with dichloromethane and analyzed using gas
chromatography/mass spectrometry (GC/MS). One microlitre of sample was injected using an
Agilent 7683 autosampler (Agilent Technologies, Santa Clara, CA) in splitless mode. The
sample was eluted from the Agilent 6890N GC, which had an HP-5MS fused silica capillary
column (30 m x 0.25 mm inner diameter x 0.25 µm film thickness) with the following thermal
gradient: temperature hold at 65 oC for 2 min, temperature increase from 65 oC to 300 oC at a
rate of 6 oC per min, and an isothermal hold at 300 oC for 20 min. The mass spectrometer
(Agilent 5973N) was operated at 70 eV in the electron impact mode. Data were acquired and
processed using Agilent Chemstation G1701DA (v. D) software. Vanillic acid (Sigma-Aldrich)
and lauric acid (Sigma-Aldrich) were used as external standards. Compounds were identified by
comparison with mass spectra from the Wiley MS library, NIST library, and authentic
standards.
Eight main lignin-derived phenol monomers were identified according to Hedges and
Ertel (1982) and Otto and Simpson (2006a). These monomers can be divided into three major
groups: vanillyl (vanillin, acetovanillone, vanillic acid), syringyl (syringaldehyde,
acetosyringone, syringic acid), and coumaryl (coumaric acid, ferulic acid) monomers. The acid
to aldehyde ratios of vanillyl (Ad/Alv) and syringyl (Ad/Als) monomers were calculated, as
these reflect the oxidation state of lignin-derived phenols, where higher values indicate that
lignin is more oxidized (Hedges et al., 1988). The following lignin-derived phenolic dimers
were also identified (Goni and Hedges, 1992; Otto and Simpson, 2006a): 2-syringylsyringic
acid, 2-syringylsyringaldehyde, dehydrovanillinvanillic acid, dehydrovanillinacetovanillone,
dehydrodivanillin, and dehydroacetovanillonevanillic acid. The ratio of lignin phenol
dimers/monomers was calculated to determine the relative enrichment of lignin-derived phenol
dimers in soils amended with different maize tissues. Cutin-derived OH-acids (16-
hydroxyhexadecanoic acid, 12-hydroxyoctadecanedioic acid, 9,10-dihydroxyhexadecanoic acid,
and 9,10,18-trihydroxyoctadecanoic acid were also identified according to Filley et al. (2008),
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Goni and Hedges (1990a), and Mendez-Millan et al. (2010a). Cutin-derived OH-acids were
divided by the total lignin-derived phenol monomers (OH-acids/Monomers) to determine the
relative enrichment of cutin-derived OH-acids as biodegradation progressed, using all eight
lignin phenol monomers, as opposed to the six used by Opsahl and Benner (1995). Since CuO
oxidation extracts only a sub-set of lignin-derived compounds (Ertel and Hedges, 1984), relative
contributions rather than absolute concentrations of lignin phenols in maize leaves, stems, roots,
and amended soils were compared.
4.3.3 Solid-state 13C NMR
Soil samples for solid-state 13C cross polarization/magic angle spinning (CP/MAS) NMR
analysis were repeatedly treated with 10% HF acid (Fisher Scientific), washed with deionized
water as described by Schmidt et al. (1997), and freeze-dried. HF acid treatment improves
solid-state NMR spectra by concentrating OM and decreasing paramagnetic material
concentrations (Schmidt et al., 1997), without significantly changing 13C CP/MAS detectable
OM (Rumpel et al., 2006). Freeze-dried tissues (leaves, stems, and roots) from maize were
ground to pass through a 105 µm mesh sieve prior to analysis. Samples (maize tissues or HF
treated soil) were packed in 4 mm zirconium rotors and closed with Kel-F caps. Solid-state 13C
CP/MAS NMR spectra were acquired on a 500 MHz Bruker BioSpin Avance III spectrometer
(Bruker BioSpin, Rheinstetten, Germany) using 24000 scans, while NMR spectra of maize
tissues were acquired using 4096 scans. Spectra for all samples were acquired using a 13 kHz
spinning speed, a ramp-CP contact time of 1 ms, and a recycle delay of 1s. Glycine was used as
an external standard to calibrate the chemical shifts. NMR spectra were processed using a zero-
filling factor of 2 and 75 Hz line broadening. Chemical shift ranges were integrated using
AMIX v. 3.7.10 (Bruker BioSpin) and assigned based on published studies (Baldock et al.,
1992; Hu et al., 2000; Mao et al., 2000; Preston et al., 2000). Five structural regions were
integrated: alkyl (0-50 ppm), such as those found in high and low molecular weight lipids, cutin
or suberin; O-alkyl (50-95 ppm), such as those found in carbohydrates or lignin, with some
contributions from peptides; anomeric (95-110), such as those found in carbohydrates; aromatic
(110-160 ppm), such as those found in lignin, with some contributions from suberin, peptides, or
possibly black C; and carboxyl + carbonyl (160-200 ppm), such as those found in lipids or
degradation products, with some contributions from lignin. Alkyl/O-alkyl ratios, which increase
88
with progressive degradation, were calculated by dividing the area of the alkyl region (0-50
ppm) by the area of the O-alkyl (50-95 ppm) region (Baldock et al., 1992; Simpson et al., 2008).
4.3.4 Solution-state 1H NMR
Samples were exhaustively extracted with 0.1 M NaOH to isolate humic substances from
soil and precursors from plant tissues for solution-state 1H NMR analysis. To minimize
hydrolysis and side-reactions of compounds during extraction, samples were extracted using
milliQ water, flushed with N2, and stored at -20 oC. The resulting solutions were passed through
a 0.2 µm PVDF filter (Millipore, Billerica, MA), cation-exchanged using Amberjet 1200(H)
(Aldrich, St. Louis, MO), freeze-dried, and then dried further over phosphorous pentoxide. In
this study, 0.03 g of leaves, 0.02 g of stems, and 0.11 g of roots material were extracted per g of
biomass; and approximately 50% - 80% of total soil OM may be extracted using this method
(Simpson et al., 2007a). Samples were re-dissolved in D2O (pH adjusted to 10 using 10 mM
NaOD to assist with dissolution) and then transferred into 5 mm NMR tubes for analysis using a
Bruker BioSpin Avance III 500 MHz spectrometer. Solution-state 1H NMR spectra were
acquired using 1024 scans and 16384 time domain points. Diffusion edited (DE) 1H NMR
spectra, which emphasize relatively large or aggregated molecules, were acquired using 1024
scans, 16384 time domain points, diffusion time of 0.2 s, and 2.5 ms encoding/decoding
gradients at 50 gauss/cm. At this gradient strength, signals from structures in compounds,
which have similar diffusion as 21 kDa maltodextrin or a 70 kDa protein are enhanced (Lam
and Simpson, 2009). Solution-state 1H NMR spectra were processed using a zero-filling factor
of 2, and 1 Hz of line broadening. DE 1H NMR spectra were processed using the same
parameters, except line broadening of 10 Hz was used. Structural groups were identified based
on previous work by Simpson et al. (2007a; 2007b) where one-dimensional structural
assignments have been confirmed through a range of multi-dimensional NMR experiments.
Resonances were integrated into the following four general regions: alkyl, such as those from
polymethylene, or proteins (0.6-1.4 ppm); O- or N-substituted aliphatic compounds, such as
those in peptides or alkyl β or γ to COOH in lipids (1.4-2.9 ppm); O-alkyl mainly from lignin or
carbohydrate, with some contributions from protein or hydroxyalkanoic acids (2.9-4.1 ppm);
and aromatic, mainly from lignin, suberin or peptides (6.2-7.7 ppm).
89
4.3.5 Changes in Organic Matter Chemistry and Statistical Analyses
Overall changes in OM chemistry were monitored by examining contributions of lignin-
derived phenols (vanillyl, syringyl, and coumaryl); alkyl, O-alkyl, aromatic, and carboxyl from
solid-state 13C NMR; and aliphatic, O-alkyl, and aromatic from solutions state 1H NMR and DE 1H NMR of plant material. Changes (loss or enrichment) during the course of the experiment
were calculated as biodegradation progressed (change = % week n - % week 1) for soils
amended with the different tissues (week 1 was used as the reference point to calculate the
relative extent of change during biodegradation). Differences in the oxidation state of lignin-
derived phenols (Ad/Alv, Ad/Als), lignin-derived phenolic dimer relative to monomer
contributions (Dimers/Monomers), and cutin-derived OH-acids relative to monomer
contributions (OH-acids/Monomers) in the tissue-amended soils were also calculated (change =
([week n – week 1] / week 1)*100) to examine trends in OM chemistry as biodegradation
progressed. Independent sample t-test and multivariate comparisons were also performed on
chemical groups identified in CuO oxidation extracts (n = 3) using SPSS v. 19.0. For
multivariate comparisons, analysis of variance followed by Tukey Honestly Significant
Difference (HSD) was used to determine whether differences between means were significant.
4.4. Results
4.4.1 Lignin-derived Phenols and Cutin-derived OH-acids
Prior to incubation, the elemental composition, lignin phenol monomer concentrations,
distribution of lignin phenol dimers relative to lignin monomers, and cutin-derived OH-acids
relative to lignin monomers from maize varied for each tissue (Table 4.1). Although the
concentrations of lignin phenol monomers in maize biomass (Table 4.1) were lower than lignin
concentrations measured using the Klason method (Yanni et al. 2011), the relative amounts of
lignin phenol monomers and lignin residue in maize leaves, stems, and roots were consistent
with both methods. The C/N and lignin phenol/ N ratios in maize roots were higher than those in
leaves and stems (Table 4.1). The relative contribution (%) of vanillyl monomers, dimers, and
cutin-derived OH-acids were significantly greater in leaf tissues than other tissues, and root
tissues contained more coumaryl monomers than the other tissues (Table 4.1). Lignin phenols in
leaf tissues had higher Ad/Als ratios compared to lignin phenols extracted from stem- and root-
tissues (Table 4.1).
90
Relative changes in lignin phenol and OH-acid distribution in soils amended with maize
leaves, stems, and roots during biodegradation (Figs. 4.1-4.3) show varying trends over the
course of the experiment. For example, vanillyl monomers increased whereas coumaryl
monomers decreased (Fig. 4.1). The extent of change in vanillyl and coumaryl monomer
distribution during biodegradation was lowest in leaf-amended soils (Fig. 4.1). Stem- and root-
amended soils showed significant changes in the oxidation state of syringyl and vanillyl phenols
during biodegradation (Fig. 4.2). We observed enrichment of lignin phenol dimers and cutin-
derived OH-acids relative to lignin phenol monomers (Dimers/Monomers and OH-
acids/Monomers) during biodegradation (Fig. 4.2), but changes in the ratio of OH-
acids/monomers were significant only in soils amended with stems and roots (Fig. 4.2).
4.4.2 Organic Matter Characterization by Solid-state 13C NMR
Alkyl, O-alkyl, anomeric, aromatic, and carboxyl signals were observed and the
integration values for these regions (Fig. 4.3, region a-e) for maize tissues are listed in Table 1.
Alkyl signals (Fig. 4.3, region a; Table 4.1) may be derived from CH, CH2, and CH3 in proteins,
high and low molecular weight lipids, as well as cutin and suberin (Deshmukh et al., 2005; Hu
et al., 2000; Preston et al., 2000). Within the alkyl region, signals observed at 27-36 ppm are
likely from long-chain aliphatic (i.e. polymethylene) structures found in plant cuticles
(Deshmukh et al., 2005; Golchin et al., 1996; Hu et al., 2000; Preston et al., 2000), and these
signals were more intense in leaf tissues compared to stem- and root-tissues (Fig. 4.3, Table
4.1). Structures that resonate in the O-alkyl region contributed over 50% (Table 4.1) to the total
OM in maize tissues, and were least intense in leaves (Fig. 4.3, Table 4.1). These structures are
found mainly in carbohydrates and lignin, with minor contributions from peptides (Fig. 4.3,
region b; Table 4.1). The differences in the alkyl/O-alkyl ratios of un-amended soil, and soils
amended with maize tissues were small (data not shown).
The solid-state 13C NMR spectra of week 1 soils amended with maize tissues had more
intense O-alkyl signals compared to the un-amended soil (Fig. 4.3). The signal at 63 ppm in the
O-alkyl region and the anomeric signal (Fig. 4.3, region c) at 95-110 ppm, are characteristic of
sugars and carbohydrates (Preston et al., 2000; Quideau et al., 2001; Spaccini and Piccolo,
2009). The signal at 56 ppm in the O-alkyl region is typically attributed to methoxy and ethoxy
compounds, such as those found in lignin, with possible contributions from structures found in
91
Table 4.1 C and N content, lignin-derived phenols, cutin-derived OH-acids, total organic matter (OM), and base-extractable OM composition of maize leaves, stems, and roots. Total lignin monomers (vanillyl + syringyl + coumaryl monomers); oxidation state of vanillyl (Ad/Alv) and syringyl (Ad/Als) monomers; relative contributions (%) of vanillyl, syringyl, and coumaryl monomers; relative contributions of lignin phenol dimers and monomers (Dimers/Monomers); and contributions of cutin-derived OH-acids relative to lignin-derived monomers (OH-acids/Monomers) are listed with standard errors of the mean (n = 3). Values within a row with different letters are significantly different (Tukey-HSD, = 0.05). OM in total tissues was observed using solid-state 13C NMR. OM extracted using NaOH was observed using 1H NMR, and DE 1H NMR. For the solution-state spectra: integration values between 4.1-6.2 ppm were not included because there was no difference in the contribution from this region to the different tissues; alkyl values used to determine Alkyl/O-alkyl ratios for solution-state 1H NMR were calculated as the sum of signals attributed to CH3, CH2 and CH3 + CH2 near O and N. NMR values are expressed as (%) of the total spectra.
Leaves Stems Roots Elemental Analysis1 Carbon (mg g-1 residue) 452 401 245 Nitrogen (mg g-1 residue) 35.5 30.8 14.8 C/N 12.7 13.0 16.6 Lignin Phenols and Cutin OH-acids Total lignin monomers (mg g-1 C) 8.2 0.5b 14.4 1.0b 28.2 2.5a Vanillyl (%) 14 0.2a 10 0.2b 8 0.8c Syringyl (%) 23 0.1 26 0.3 24 1.4 Coumaryl (%) 63 0.2b 64 0.4ab 69 2.2a Ad/Alv 0.4 0.0 0.4 0.0 0.4 0.1 Ad/Als 0.9 0.0a 0.6 0.03b 0.2 0.03c Dimers/Monomers 0.03 0.002a 0.02 0.001b 0.003 0.002c OH-acids/Monomers 0.01 0.002a 0.002 6x10-4b not detected Solid-state 13C NMR Alkyl 21 13 13 O-alkyl 55 64 62 Anomeric 10 13 12 Aromatic 7 6 9 Carboxyl and carbonyl 7 4 4 Alkyl/O-alkyl 0.4 0.2 0.2 Solution-state 1H NMR CH3 and CH2 13 6 10 CH3 and CH2 near O and N 21 16 13 O-alkyl 53 63 58 Aromatic 8 10 14 Alkyl/O-alkyl 0.6 0.3 0.4 Solution-state DE 1H NMR CH3 and CH2 14 9 11 CH3 and CH2 near O and N 14 10 12 O-alkyl 60 67 62 Aromatic 6 9 11 Alkyl/O-alkyl 0.5 0.3 0.4
1 C and N content (mg g-1 plant biomass) from Yanni et al. (2011).
92
Figure 4.1 Change in lignin phenol monomer composition (%) with incubation. Error bars represent standard error of the mean (n = 3). Letters represent significant differences (Tukey-HSD, = 0.05) at week 36 between different tissue amendments. (#) indicate that there was a significant difference (t-test, α = 0.05) after 36 weeks compared to week 1 for each tissue amendment.
-4
0
4
8
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0 10 20 30 40
-14
-10
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-4
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Leaves
Stems
Roots
Vanillyl
Syringyl
Coumaryl
Incubation Time (weeks)
Ch
an
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in L
ign
in P
he
no
l C
om
po
sit
ion
(%
) a#ab#
b
a#ab#
b#
aab
b
###
93
Figure 4.2 Change in oxidation (%) of vanillyl (Ad/Alv) and syringyl (Ad/Als) lignin phenol. Changes in lignin-derived phenol dimers (Dimers/Monomers) and cutin-derived OH-acids (OH-acids/Monomers) with incubation are also shown. Error bars represent standard error of the mean (n = 3). Letters represent significant differences (Tukey-HSD = 0.05) at week 36 between different tissue amendments. (#) indicate that there was a significant difference (t-test, α = 0.05) after 36 weeks compared to week 1 for each tissue amendment.
-20
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40
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LeavesStemsRoots
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20
40
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Ad/Alv
Ad/Als
Incubation Time (weeks)
Ch
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)
a# ab
b
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-40
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40
80
120
160
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Leaves
Stems
Roots
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40
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Dimers/Monomers
OH-acids/Monomers
a#
b#b#
Incubation Time (weeks)C
ha
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e in
Lig
nin
Ph
en
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Co
ntr
ibu
tio
n (
%)
##
94
Figure 4.3 Solid-state 13C CP/MAS NMR spectra, of maize leaves, stems, roots, un-amended soil, and soils incubated with maize tissues at time = 1 and 36 weeks. The spectra are labelled to reflect the major chemical groups: a) alkyl found in high and low molecular weight lipids, waxes, cutin, or suberin, b) O-alkyl found in sugars or lignin, with some contributions from peptides, c) anomeric found in carbohydrates, d) aromatic found in lignin, protein, or black carbon, and e) carboxyl or carbonyl signals.
220 200 180 160 140 120 100 80 60 40 20 0Chemical Shift (ppm)
220 200 180 160 140 120 100 80 60 40 20 0Chemical Shift (ppm)
220 200 180 160 140 120 100 80 60 40 20 0Chemical Shift (ppm)4080120160200 0 4080120160200 0 4080120160200 0
220 200 180 160 140 120 100 80 60 40 20 0Chemical Shift (ppm)
4080120160200 0
Leaves Stems Roots
Tissue before incubation
Amended soil after 1 week incubation
Amended soil after 36 weeks incubation Un-amended soil
abcde
27-3611-27
Chemical Shift (ppm)
abcde abcde
abcde
95
amino acids. Aromatic signals (region d) can be from structures found in lignin, suberin,
proteins, and/or black carbon; and lignin-derived structures are believed to be the main
contributors to signals in this region (Preston et al., 2000; Quideau et al., 2001; Spaccini and
Piccolo, 2009). Accordingly, the intensities of the 63 ppm and anomeric (105 ppm) signals
relative to the 56 ppm and aromatic (130 ppm) signals may be used to determine the relative
distribution of carbohydrates versus lignin. When the spectral profiles of amended soils
incubated for 1 week were compared to spectral profiles of amended soils incubated for 36
weeks, decreased intensity of signals attributed to structures found in carbohydrates (63 and 105
ppm) compared to signals attributed to structures found in lignin (56 and 130 ppm) was
observed for soil amended with plant tissues (Fig. 4.3). This trend was especially distinct in soil
amended with maize stems (Fig. 4.3). Changes in the distribution of the five major structural
groups (alkyl, O-alkyl, anomeric, aromatic, carboxyl and carbonyl) in the OM of soils amended
with maize leaves, stems, and roots during biodegradation is shown in Fig. 4.4. Signals from
alkyl structures and aromatic structures increased during incubation, whereas signals from O-
alkyl structures decreased (Fig. 4.4).
4.4.3 Organic Matter Characterization by Solution-state 1H NMR
Solution-state 1H NMR and DE 1H NMR spectra of NaOH extracts isolated from maize
leaves, stems, and roots; un-amended soil; and soil amended with maize tissues are shown in
Figs. 4.5 and 4.6 respectively, and corresponding integration values for maize tissues are listed
in Table 4.1. Concentrations of methyl (CH3) and methylene (CH2) structures were more
enriched in solution-state NMR spectra of leaves (Figs. 4.5 and 4.6, region a; Table 4.1), and
contributions from polymethylene structures is suggested by the higher intensity of the CH2
relative to the CH3 signal (Fig. 4.5). These structures are typically found in high and low
molecular weight lipids, cutin, and suberin (Simpson et al., 2007a; 2007b). The 1H NMR
spectrum for the leaf extract also showed greater contributions from methyl and methylene near
O and N in relatively small compounds (Fig. 4.5, region b; Table 4.1), likely from structures
found in peptides, with some contributions from lipids. The O-alkyl region of the stems and
roots spectra (Fig. 4.5, region c; Table 4.1) was more intense, which suggests greater
contributions from structures found in carbohydrates and lignin. The more intense aromatic
96
Figure 4.4 Changes in the contribution (%) of alkyl, O-alkyl, anomeric, aromatic, and carboxyl or carbonyl chemical groups to total OM in soil amended with maize tissues, and incubated for 36 weeks.
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Alkyl
O-Alkyl
Aromatic
Carboxyl and Carbonyl
Incubation Time (weeks)
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Stems
Roots
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Anomeric
Incubation Time (weeks)
97
Figure 4.5 Solution-state 1H NMR spectra of maize tissues, un-amended soil, and soils incubated with maize tissues for 1 and 36 weeks. Spectra are labelled to reflect chemical shift regions representing: a) alkyl from high and low molecular weight lipids, cutin, suberin; b) alkyl closer to O and N found in lipids, or peptides; c) O-alkyl from sugars or lignin, with minor contributions from peptides; and d) aromatic signals from lignin or proteins. Signals from N-acetyl, which may be attributed to peptidoglycan or chitin from microorganisms are also labelled (*).
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0Chemical Shift (ppm)
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0Chemical Shift (ppm)
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0Chemical Shift (ppm)01.02.03.04.05.06.07.08.09.0 01.02.03.04.05.06.07.08.09.0 01.02.03.04.05.06.07.08.09.0
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0Chemical Shift (ppm) 01.02.03.04.05.06.07.08.09.0
Leaves Stems Roots
Tissue before incubation
Amended soil after 1 week incubation
Amended soil after 36 weeks incubation Un-amended
Soil
abcd
CH3
CH2
Chemical Shift (ppm)
*
* *
*
*
* *
abcd abcd
abcd
98
Figure 4.6 Solution-state diffusion edited 1H NMR spectra of maize tissues, un-amended soil, and soils amended with maize tissues after 1 and 36 weeks. Spectra are divided to reflect the following major chemical groups: a) aliphatic methyl and methylene from high and low molecular weight lipids, cutin, or suberin; b) aliphatic methyl and methylene near O and N, lipids, or peptides; c) O-alkyl from sugars or lignin; and d) aromatic signals from lignin or proteins. Signals from N-acetyl, which may be attributed to peptidoglycan or chitin from microorganisms are also labelled (*), and peptide-derived signals are labelled P1 and P2.
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0Chemical Shift (ppm)
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0Chemical Shift (ppm)
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0Chemical Shift (ppm)
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0Chemical Shift (ppm) 01.02.03.04.05.06.07.08.09.001.02.03.04.05.06.07.08.09.0 01.02.03.04.05.06.07.08.09.001.02.03.04.05.06.07.08.09.0
Tissue before incubation
abcd
CH3 (P1)
CH2
Leaves Stems Roots
Amended soil after 1 week incubation
Amended soil after 36 weeks incubaton Un-amended
Soil
Chemical Shift (ppm)
*
*
*
*
*
* *
abcd abcd
abcd
P2 P2P2
LigninLigninLignin
CH3 (P1)CH3 (P1)
CH2CH2
99
signals in the 1H NMR spectrum of root extract (Fig. 4.5, region d; Table 4.1) also suggest
greater contributions from aromatic protons, such as those found in lignin and suberin.
Plant tissue extracts were dominated by low molecular weight compounds that were no
longer observed after diffusion editing (Figs. 4.5 and 4.6). In the DE 1H NMR spectra of all
tissues, an apex at 6.7 ppm (Fig. 4.6), which was previously assigned to aromatic structures
found in lignin (Simpson et al., 2007a), was observed. This signal is most intense in the DE 1H
NMR spectra of roots (Fig. 4.6), which suggests greater contributions from relatively large
lignin-derived structures. Signals from lignin-derived aromatic structures were least intense in
the spectra of leaf-tissue extracts (Fig. 4.6). Previous studies found that a combination of
signals (P1-P2) in solution-state DE 1H NMR spectra suggest the presence of peptides, which
coincided to signals in the spectrum of bovine serum albumin (Simpson et al., 2007a; 2007b).
The higher intensity of the CH3 signal compared to the CH2 signal after diffusion editing (P1),
highlights the presence of methyl-rich amino acids (Simpson et al., 2007b), and the signal at 7.2
ppm can be attributed to phenylalanine (P2) found in peptides. These signals were observed in
the DE 1H NMR spectra of all tissues, but were most intense in extracts from leaves (Fig. 4.6).
Contributions from peptides was also confirmed by the detection of amide signals in the
solution-state DE 1H NMR analysis of tissue extracts dissolved in DMSO-d6 (see Appendix 2,
Fig. A2.1).
The spectra of amended soils incubated for 1 week also differed from the spectra of
amended soils incubated for 36 weeks (Figs. 4.5 and 4.6). For example, sharp signals from
aromatic structures (Fig. 4.5, region d) in the 1H NMR spectra, which are consistent with the
presence of lignin-derived compounds, were most intense in soil amended with stems (week 1,
Fig. 4.5). Sharp peaks observed in the O-alkyl region of the DE 1H NMR spectra of soils
amended with tissues were no longer observed after 36 weeks of incubation (Fig. 4.6) and an
apex at 3.7 ppm, previously observed in commercial lignin (Simpson et al., 2007b), became
more discernible. The intensity of the CH2 signal was greater relative to CH3 signals in the 1H
NMR (Fig. 4.5) and DE 1H NMR (Fig. 4.6) spectra of all soils compared to maize tissues,
indicating an enrichment of polymethylene structures (likely from cutin or suberin) in soil. The
CH2 signal in the 1H NMR spectrum of soil amended with leaves was less intense after 36
weeks compared to the CH2 signal after 1 week (Fig. 4.5), which suggests depletion of relatively
100
small aliphatic compounds as leaves were degraded. The attenuation of the CH2 signal in all the
soil spectra after diffusion editing suggests contributions from peptides to all soils, and the
intensity of the CH2 relative to the CH3 signal was less in soil amended with roots after 36
weeks (Fig. 4.6). In addition, a signal likely derived from N-acetyl structures in microbial chitin
or peptidoglycan, the major contributor to signals in region b (methyl and methylene near O and
N), increased after 36 weeks (Figs. 4.5 and 4.6).
Changes over time for the five major regions identified from solution-state 1H NMR and
DE 1H NMR spectra of soil amended with leaves, stems, and roots are shown in Fig. 4.7.
Aliphatic methyl and methylene structures and aliphatic methyl and methylene near O and N
generally increased, while a decrease in O-alkyl structures was observed over time (Fig. 4.7).
Aromatic compounds did not change significantly with time (Fig. 4.7). The contribution of
relatively small compounds to the signals observed in the 1H NMR spectra, resulted in different
patterns from that observed in the DE 1H NMR spectra (Fig. 4.7). A comparison of 1H and DE 1H NMR results revealed that relatively large aliphatic components in leaves did not change
through time, which suggest the resistance of leaf waxes and cutins to biodegradation. Some
changes in the contribution of the various structures were specific to the tissue amendment (Fig.
4.7). For example, O-alkyl structures decreased for stem- and root-amended soil but did not
change significantly for leaf amended soils. There was also a greater decrease in contributions
from O-alkyl signals to the DE 1H NMR spectra of stem-amended soils with time (Fig. 4.7). In
the DE 1H NMR spectra, the extent of increase in the relative contribution of aliphatic methyl
and methylene near O and N plateaued after 4 weeks in soils amended with roots, which was
earlier than soils amended with stems and leaves (Fig. 4.7).
4.5 Discussion
The relative contributions of carbohydrates, lignin, cutin, and suberin over time were
similar for all soil amendments at the end of the 36 week incubation, but some distinct trends
were discernible. Decreased contributions from anomeric structures (Fig. 4.3) confirms the
susceptibility of carbohydrate compounds to degradation and is consistent with the rapid
degradation of labile constituents when fresh plant material is added to soil (Kelleher et al.,
2006; Machinet et al., 2009), likely due to preferential utilization of more easily degradable
substrates, such as carbohydrates and small fatty acids by microbes (White, 2000). Lignin
101
Figure 4.7 Change in the contribution (%) of aliphatic methyl and methylene, aliphatic methyl and methylene near O and N, O-alkyl, and aromatic chemical groups to total NaOH-extractable OM from soil amended with maize tissues and incubated for 36 weeks.
-5
-3
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1
3
5
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Stems
Roots
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-3
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Incubation Time (weeks)
1H NMR DE 1H NMRC
ha
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Re
lati
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Ab
un
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nc
e (
%)
Aliphatic methyl and methylene
O-Alkyl
Aromatic
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Aliphatic methyl and methylene near O and N
-5
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Stems
Roots
102
degrades more slowly in soil and is hypothesized to be part of the stable OM component
(Guggenberger et al., 1994; Kiem and Kögel-Knabner, 2003; Kögel-Knabner, 2002). Structures
from both carbohydrates and lignin contribute to the O-alkyl region in solid-state 13C NMR, and
the degradation of these compounds resulted in the enrichment of compounds that contained
alkyl structures (mostly cutin and suberin; Figs. 4.2, 4.4, 4.7), which are thought to be more
resistant to biodegradation (Kögel-Knabner, 2002). Enrichment of alkyl structures is consistent
with processes that occur during degradation of fresh plant material when compounds that
contain O-alkyl structures are depleted (Baldock et al., 1992; Kelleher et al., 2006; Kögel-
Knabner, 2002). Despite these similarities in overall changes in soil OM as tissues degraded
over time, the contributions and composition of these groups of compounds to the various
tissues differed (Table 4.1; Figs. 4.3, 4.5, 4.6). Over the course of the study, there were also
notable variations that occurred in leaf-, stem-, and root-amended soils (Figs. 4.1-4.7), which
may have important implications on OM stabilization in soils. For example, more intense
carbohydrate-derived signals were observed in stem tissues (Table 4.1; Figs. 4.3, 4.5, 4.6;
Appendix A2, Fig. A2.1), which indicates higher carbohydrate concentrations, resulted in the
greatest decline of signals derived from carbohydrates in soils amended with stems (Figs. 4.3,
4.4 and 4.7). The type of carbohydrates found in stems may also be important in influencing
their relative degradation. In particular, the concentrations of water-soluble carbohydrates
(starch, glucose, and fructose) are reported to be four times higher in stems compared to leaves
and roots (Johnson et al., 2007). These carbohydrates are likely more easily degraded than
structural carbohydrates, such as cellulose, because they contain less complex linkages between
sugar monomers (Warren, 1996), thereby making carbohydrates found in stems more
susceptible to degradation compared to those found in leaves and roots.
The composition of lignin phenols in the various tissues also differed in terms of lignin
monomer composition, as well as the concentration of lignin dimers. Leaf tissues had
significantly greater concentrations of vanillyl monomers (Table 4.1) as compared to stems and
roots, which suggest that some leaf-derived lignin may be more recalcitrant. This is consistent
with previous studies that observed greater vanillyl contributions in maize leaves (Guillaumie et
al., 2007), and greater coumaryl monomer contribution in maize roots (Hatfield and Chaptman,
2009). Because of higher concentrations of relatively recalcitrant vanillyl monomers in leaf
tissues, lignin-derived compounds from leaves were degraded to a lesser extent than lignin from
103
roots and stems (Figs. 4.1, 4.2 and 4.5), similar to other studies where coumaryl and syringyl
were degraded preferentially over vanillyl type phenols (Bahri et al., 2006; Ertel and Hedges,
1984; Goni and Hedges, 1992; Goni et al., 1993; Hedges et al., 1988; Kiem and Kögel-Knabner,
2003). Lignin phenol dimers in soils amended with leaves, stems, and roots were enriched
relative to lignin monomers over the 36-week incubation, but to varying extents (Table 4.1; Fig.
4.2), and this relationship was more distinct in the leaf-amended soil (Table 4.1). The
significant enrichment of lignin phenol dimers in leaf-amended soil (Fig. 4.3) may be attributed
to the relative stability of these compounds once tissues are partially degraded. Because lignin
phenol monomers in soil amended with leaves were less oxidized (Figs. 4.1 and 4.2), differences
in the enrichment of lignin phenol dimers in soils amended with various tissues suggests that the
stability of these dimers may depend on other tissue-specific characteristics of lignin (coumaryl,
syringyl and vanillyl composition, inter-aromatic linkages, tissue anatomy), which may be more
important in the early stages of lignin degradation.
Leaf tissues had higher concentrations of aliphatic structures (Table 4.1). The
polymethylene structures observed in leaf tissues were composed of two groups (Table 4.1): low
molecular weight lipids, which are easily decomposed; and aliphatic polymers (such as cutin),
which are more resistant to degradation. Depletion of low molecular-weight lipids over time
(Fig. 4.7, 1H NMR), is consistent with observations that solvent-extractable fatty acids were
degraded faster compared to cutin, suberin (Almendros et al., 2000; Feng and Simpson, 2008;
Sun et al., 1997), and lignin phenol monomers (Feng and Simpson, 2008). Since some
organisms preferentially degrade short-chain fatty acids over glucose (White, 2000), low-
molecular weight lipids may have been an important pool of labile compounds in soils amended
with leaves. Compared to other tissues, such as roots, leaves are known to contain greater
concentrations of lipids (Stefanov et al., 1993). Higher input of lipids from leaves is especially
relevant given the lower contributions from relatively labile O-alkyl structures, found in sugars
and carbohydrates, in leaf tissues (Table 4.1). The increase in polymethylene signals (Figs. 4.4
and 4.7) and cutin-derived OH-acids (Fig. 4.2) through time were less distinct in soils amended
with leaves. This trend may be attributed to greater amounts of cutin and waxes on the leaf
surface (Table 4.1), which limited microbial access to some of the underlying leaf polymers
(Fig. 4.7). Cutin, suberin, and other epicuticular waxes protect plant material from microbial
damage (Kolattukudy, 1985; Koller et al., 1982), and large concentrations of cutin-derived
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structures have been observed in degraded leaf litter (Almendros et al., 2000). Previous
biodegradation studies have also shown less alteration in the composition of pine needles; which
have more cutin and epicuticular waxes; compared to corn (Feng et al., 2011), and grass
(Kelleher et al., 2006). The overall alkyl carbon contribution to leaf-amended soils after 36
weeks of incubation (Figure 6), suggests that leaf tissues may be an important source of both
labile (low molecular-weight aliphatic compounds), and recalcitrant (cutin) aliphatic
compounds. Leaf-derived cutin may enhance soil OM stability, since aliphatic compounds have
been shown to stabilize soil aggregates (Dinel et al., 1991a), which is thought to be one of the
mechanisms responsible for soil OM preservation (Six et al., 2002; Six, 2004).
The biodegradation of OM constituents also coincided with an increase in N-acetyl
groups found in microbial chitin or peptidoglycan (Fig. 4.7), which indicates increases of
microbial-derived OM compounds over time. These data are consistent with increased
contribution from microbial-derived OM after addition of fresh plant material to soils (Powlson
et al., 1987), and the hypothesis that microbial-derived OM may be major contributors to soil
OM (Simpson et al., 2007a). Increases in microbial-derived structures was observed as early as
4 weeks (Fig. 4.7, DE 1H NMR) and was most pronounced in soils amended with roots. Further
support for greater contributions of microbial-derived OM to root-amended soils was the lower
intensity of the CH2 relative to the CH3 signal after 36 weeks (Fig. 4.6). Increased intensity of
the N-acetyl signal combined with lower intensity of the CH2 relative to the CH3 signal, was
previously observed in cultured microbes, and suggests increased contributions from peptide
side chains (Simpson et al., 2007a). In contrast, increases in microbial-derived structures was
observed later (36 weeks) in soils amended with leaves (Fig. 4.7, DE 1H NMR). Differences in
contributions from microbial-derived OM extracted at the various sampling times, from soils
amended with the various tissues, may be attributed to differences in tissue chemistry. In
particular, greater contributions from lignin-derived aromatic structures, and lesser contribution
from N-containing peptides were observed in roots compared to other tissues (Figs. 4.5, 4.7, See
Appendix A2 Fig. A2.1), and may have resulted in higher lignin/N ratios in roots. Higher
lignin/N ratios was previously found to result in greater N immobilization (Melillo et al., 1982).
In our study, it is likely that increases in microbial-derived OM (which have higher N-
containing peptide concentrations) as plant material was degraded may have contributed to
carbon and nitrogen immobilization. We hypothesize that greater microbial-derived OM
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contribution to soils amended with roots may be possible through more efficient conversion
rates of root-derived carbon (compared to stem- and leaf-derived substrates) to microbial
biomass. It has been suggested that assimilation of root-carbon into fungal-carbon may mediate
root-carbon contributions to soil OM (Godbold et al., 2006), and there is some evidence that
root-carbon was more efficiently converted to microbial-carbon compared to shoot-carbon
(Kramer et al., 2010; Puget and Drinkwater, 2001). However, the underlying mechanisms
responsible for these differences in substrate transformation efficiencies in soils remain unclear.
Yanni et al. (2011) calculated contributions of 13C from maize leaves, stems, and roots at
the end of 36 weeks of incubation, and found that soils amended with leaves and stems had
relatively low maize-derived carbon retention compared to soils amended with roots.
Approximately 90-95% of the carbon dioxide was evolved over the first 4 weeks of the
degradation study (Yanni, personal communication). Similarly, we observed that for the most
part, changes in lignin phenol and total OM and humic substance composition occurred during
the first 4 weeks (Figs. 4.1, 4.4, 4.7). Our results complement this study and show that with
only 1% w/w maize tissues added to soils, variations in plant tissue chemistry did influence the
extent of degradation over the 36 week period as well as the overall soil OM composition. Our
results show that labile constituents, such as carbohydrates, were degraded first followed by low
molecular weight aliphatic compounds and lignin. The abundance of low molecular weight
aliphatic compounds and carbohydrates in the leaves and stems resulted in higher rates of
mineralization, followed by slower degradation of lignin compounds. The notable differences in
OM composition of soils amended with maize stems over time, suggest that stem-derived OM
input may not be a major contributor to soil OM. In contrast, the more subtle changes in OM
composition of soil amended with maize leaves with time suggest that leaf-derived OM input
may be important in sequestering plant compounds in soil. In particular, greater concentrations
of persistent aliphatic structures found in cutin-derived OH-acids were detected in leaves, and
also suggests the potential preservation of these leaf-derived components in soils. Finally,
lignin/N ratio is hypothesized to be a better predictor of degradation rates than C/N ratios
(Johnson et al., 2007; Melillo et al., 1982; Moretto and Distel, 2003; Preston et al., 2000; Yanni
et al., 2011), and higher lignin phenol/N ratio of roots suggest that these should be mineralized
slower than leaves and stems. The high concentration of lignin in roots likely resulted in slower
mineralization rates (as observed by Yanni et al, 2011), which may be related to C sequestration
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in microbial-derived OM in soils since we observed microbial-derived OM enrichment in root-
amended soils.
Overall, this study when combined with the mineralization data reported by Yanni et al.
(2011) emphasizes the need to evaluate the influence of structural composition of soil OM in
detail. Although measuring carbon mineralization to CO2 is important in determining carbon
fluxes in soil, it is equally important to characterize the source, formation, and turnover of OM
compounds that remain and may become stabilized in soils. This hypothesis is consistent with a
study, which found that the degradation of various lipids, cutin, suberin, and lignin were not
always reflected in the overall soil OM mineralization rates (Feng et al., 2008) since carbon
dioxide evolution does not reflect the degradation of individual structures. More recently, a
review by Schmidt et al. (2011) recommended an ecosystem approach in characterizing soil OM
sequestration and stabilization because current models, such as those that use lignin/N ratios
may not accurately describe soil OM turnover. Consistent with this, we believe that structures
comprising OM should be characterized at the molecular-level to acquire a more accurate
picture of mechanisms responsible for OM sequestration and stability. For example, in this
study, lignin composition (i.e. vanillyl, syringyl, coumaryl) of maize leaves, stems and roots was
important for assessing the extent of change in lignin phenol composition as these tissues were
biodegraded over time.
4.6 Conclusions
Our results further highlight the role of tissue chemistry in biodegradation processes.
Based on our study, it appears that although maize stems may be degraded faster because of
their high carbohydrate concentrations, corn leaves contain greater contributions from
compounds that are hypothesized to be responsible for long-term soil OM stability. Although
roots are known to be more slowly mineralized than leaves and stems, results from this study
also suggest that root tissues are nevertheless degraded and likely transformed to microbial-
derived OM. Future research should monitor microbial community structure (such as
phospholipid fatty acid analysis) with plant biodegradation to confirm this hypothesis. The
variation in substrate utilization may result in shifts in microbial community, activity, and
structure. The degradation products and microbial-derived OM may also have different
stabilities in soils, and greater sequestration of carbon in soils is possible through transformation
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products that are more stable than the starting plant-derived compounds. These compounds
should be characterized to fully ascertain the impact of the various tissues to soil OM. Since the
conversion of plant-derived OM to CO2 is also thought to be controlled by nitrogen availability,
future research may also examine the role of nitrogen limitation.
4.7 Acknowledgements
We thank Dr. David McNally for assistance with NMR experiments, and David Wolfe for
assistance with biomarker extractions. Funding for this project was provided by the NSERC
Green Crop Network. J.S.C. also thanks the Ontario Government for support via an Ontario
Graduate Scholarship.
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CHAPTER 5
PHYSICAL PROTECTION OF LIGNIN BY ORGANIC MATTER AND CLAY MATERIALS FROM CHEMICAL OXIDATION
Authors: Joyce S. Clemente and Myrna J. Simpson
Contributions: JSC and MJS designed the study. Soil samples and humic acid extracted from
peat soil were provided by MJS. Samples were prepared, extracted, and analyzed by JSC with
assistance from Katherine Hills and David Wolfe. Data analysis was performed by JSC with
input from MJS. The chapter was written by JSC and MJS.
Status: Submitted to Organic Geochemistry.
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5.1 Abstract
The role of organic matter (OM) concentration, structure, and composition and how these relate
to mineral protection is important for the understanding of long-term soil OM dynamics.
Various OM-clay complexes were constructed by sequential sorption of lignin and dodecanoic
acid to montmorillonite. Humic acid-montmorillonite complexes were prepared at pH 4 and 7 to
vary OM conformation prior to sorption. Results obtained with constructed OM-clay complexes
were tested with isolated mineral fractions from two soils. Oxidation with an acidic NaClO2
solution was used to chemically oxidize lignin in the OM-clay complexes, sand-, silt-, and clay-
size soil fractions to test whether or not it can be protected from chemical attack. Gas
chromatography/mass spectrometry was used to analyze lignin-derived phenols, cutin OH-acid
(after CuO oxidation), fatty acid, and n-alkanol concentrations and composition. We found that
carbon content was not solely responsible for lignin stability against chemical oxidation. Lignin
was protected from chemical oxidation through coating with dodecanoic acid, and sorption of
humic acid to clay minerals in a stretched conformation at pH 7. Therefore, interactions
between OM constituents as well as OM conformation are important factors that protect lignin
from chemical oxidation. Lignin-derived phenol dimers in the Grassland-Forest Transition soil
fractions were protected from chemical oxidation to a greater extent compared to those in
Grassland soil fractions. Therefore, although lignin was protected from degradation through
mineral association, the extent of this protection was also related to OM content and the specific
stability of lignin components.
5.2 Introduction
The preservation of soil organic matter (OM) is important for maintaining soil quality
and productivity (Janzen et al., 1998). The level of OM decomposition is higher in sand-size
fractions which has led to the hypothesis that OM in fine-sized soil fractions is protected from
biodegradation through associations with mineral surfaces (Baldock et al., 1992; Christensen,
2001; Guggenberger et al., 1995; Quideau et al., 2001; Six et al., 2002). Furthermore,
radiocarbon data (14C) used to estimate OM age coupled with stable isotope (δ13C) turnover
studies, suggest that silt- and clay-size fractions contain older, and more slowly degraded OM
(von Lutzow et al., 2007) and long term preservation of OM is attributed to its association with
fine soil fractions (Christensen, 2001; Kaiser and Guggenberger, 2003; Mikutta et al., 2007; Six
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et al., 2002). Of particular interest is the stabilization of OM in clay-size fractions, because this
fraction may contain as much as 50-75% of total soil OM (Christensen, 2001). However, clay
surfaces have a finite amount of interactions sites, which can be saturated at high organic carbon
concentrations (Six et al., 2002). Mikutta et al. (2006b) reported that that OM was protected
from degradation, through association with clay minerals. Sorption studies also suggest that
clay mineralogy, structure, and composition influenced the structural composition of OM sorbed
to clay surfaces (Asselman and Garnier, 2000; Feng et al., 2005; Ghosh et al., 2009; Simpson et
al., 2006). It is therefore important to understand factors that may contribute to the stability of
OM associated with clay-size fractions to better manage OM input, and organic carbon
sequestration in soils (Christensen, 2001; Six et al., 2002).
OM associated with clay-size fractions may be stabilized through a number of physical
and chemical interactions (Christensen, 2001; Kögel-Knabner et al., 2008; Six et al., 2002).
One of which is the formation of microaggregates, where clay particles are coated with OM,
which then subsequently limits access of degrading enzymes resulting in OM protection (Chenu
and Plante, 2006; Six, 2004). Sorptive interactions between OM and clay mineral surfaces;
which are governed by van der Waals interactions, ligand exchange, divalent cation bridging,
electrostatic or hydrophobic bonding (Feng et al., 2005; Mikutta et al., 2007); also contribute to
OM stabilization by mineral surfaces. The dominant mode of bonding is determined by solution
properties (pH, ionic strength, presence of cations); valence of cations in solution; presence of
competing ions (such as polyphosphate); properties of minerals; and composition of the OM
sorbate (Asselman and Garnier, 2000; Chi and Amy, 2004; Feng et al., 2005; Ghosh et al., 2009;
Mikutta et al., 2007). For example, ligand exchange was found to contribute to the sorption of
peat humic acid to montmorillonite clay at weakly acidic pH values (Feng et al., 2005). OM
was also found to sorb to goethite clay mainly through ligand exchange, while Ca2+- mediated
cation bridging was found to be the dominant mechanism in OM sorption to vermiculite clay
(Ghosh et al., 2009; Mikutta et al., 2007). The differences in strength and relative contribution
of these sorption mechanisms were found to also play a role in the desorption of OM from clay,
and subsequent biodegradability (Mikutta et al., 2007).
The fate of lignin in soil environments is also hypothesized to be regulated by
interactions with mineral surfaces (Heim and Schmidt, 2007a, b). Model sorption experiments
have observed higher concentrations of aromatic structures sorbed to montmorillonite as
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compared to kaolinite (Feng et al., 2005) and suggests that lignin stabilization in soil may also
be tied to interactions with clay minerals. Recent studies have reported that lignin-derived
phenols are at a more advanced oxidation stage in clay-sized fractions as compared to those in
sand- and silt-size fractions (Clemente et al., 2011; Kiem and Kögel-Knabner, 2003; Thevenot
et al., 2010). Recent studies also suggest that lignin phenols may be sequestered in silt-size
fractions and may be older and less oxidized compared to those in clay-size fractions (Heim and
Schmidt, 2007a). In contrast, Feng et al. (2008) reported accelerated lignin oxidation with 14
months of soil warming. The authors hypothesized that the soil type (sandy loam) provided
little physical protection from enhanced microbial activity that was observed using phospholipid
fatty acid concentrations. Therefore, it is important to ascertain whether lignin in clay-size
fractions are preferentially degraded by microbes in sites with high clay content, or if oxidized
lignin become sorbed to clay minerals and subsequently leads to longer environmental
persistence. It is also necessary to determine whether lignin associated with clay is protected
from degradation, and to characterize the factors that influence this protection because lignin
associated with clay minerals represents an important part of stabilized OM (Thevenot et al.,
2010).
In this study, the factors that contribute to the stabilization of lignin on clay mineral
surfaces were investigated using clay-size fractions isolated from soil and model OM
compounds sorbed to montmorillonite. The influence of OM conformation was tested by
creating humic acid-clay complexes at pH 4 and 7, since humic substances form coiled and
compact aggregates at acidic pH, and become stretched and disaggregated as pH increases
(Avena and Wilkinson, 2002; Chien and Bleam, 1998). These OM-mineral compounds were
subjected to chemical oxidation to test whether or not lignin is physically protected from
enhanced chemical oxidation. We also examine the role of other OM compounds in the
protection of lignin, as this was identified in our previous study as an important consideration of
OM stabilization in soils (Clemente et al., 2011). The objectives of this study are to: 1)
determine whether the presence of other OM compounds can protect lignin from chemical
oxidation; 2) determine the role of OM concentration in protection of lignin from chemical
oxidation; 3) determine whether the sorption mechanism and OM conformation influences
lignin protection at pH 4 and pH 7; and 4) to test findings with model OM-clay complexes by
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examining lignin protection from chemical oxidation in the sand-, silt-, and clay-size fractions
of two soils, which have similar clay mineralogy.
5.3 Materials and Methods
5.3.1 Preparation of Organic Matter-clay Complexes
Sodium-rich montmorillonite (SWy-2) was purchased from the Source Clay Repository
(Clay Minerals Society; Purdue University). The clay was suspended in a 3 mM NaCl + 2 mM
CaCl2 solution at a 1:500 weight ratio to maintain the concentrations of Na+ and Ca2+, which are
the major exchangeable cations associated with this clay mineral. Dissolved lignin (Alkali
Lignin; Sigma-Aldrich) was added to the suspension (pH = 7), using the following
concentrations: 0.4, 4, 20, and 100 g lignin/100 g clay. Preliminary experiments found that
vanillyl monomers were the main lignin phenols extracted using CuO oxidation from alkali
lignin (data not shown). Peat humic acid from Pahokee Peat (International Humic Substances
Society) was isolated and characterized as described in Salloum et al. (2001). Three humic
acid-clay complexes were prepared in a similar manner as those with alkali lignin, with the
following concentrations: 0.4, 4, and 20 g humic acid/100 g clay. The pH was adjusted to either
7.0 or 4.0 using HCl and NaOH, which changes the conformation of humic substances, coiled at
pH 4 and stretched at pH 7 (Avena and Wilkinson, 2002; Chien and Bleam, 1998). The
suspensions were placed on a shaker for 20 h, and then centrifuged at 4500 rpm for 1 h to isolate
the OM-clay complexes. Complexes were then washed 10 times with the 3 mM NaCl + 2 mM
CaCl2 salt solution to isolate the OM-clay complexes and avoid disturbing cation-mediated
associations. The complexes were then freeze-dried and ground to pass through a 106 µm sieve
to reduce the contributions of any large aggregates. To create the lignin-clay-dodecanoic acid
complexes, 1 g of the lignin-clay complexes (described as loadings 2, 3, and 4, which
correspond to 4, 20, and 100 g lignin/g clay respectively) were mixed in acetone with 200 mg
dodecanoic acid overnight on a shaker. Preliminary tests showed that the alkali lignin chosen
for this study was soluble in water, but not in acetone. Therefore, extraction of lignin from the
lignin-clay complexes was minimized by using acetone to dissolve the dodecanoic acid. This
was verified by CuO oxidation followed by gas chromatography/mass spectrometry (GC/MS)
analysis of the acetone supernatant which did not detect any lignin-derived phenols (data not
shown). The OM-clay complexes were collected by centrifugation at 9500 rpm, washed twice
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with 30 mL acetone, then 5 times with the 3 mM NaCl + 2 mM CaCl2 salt solution. This was
dried in the fume hood to remove residual acetone, then freeze dried, and ground to pass through
a 106 µm sieve.
5.3.2 Soil Sampling, and Density Fractionation
Two samples from the Alberta Prairie Ecozone were collected: Southern Grassland (SG;
collected near Lethbridge, Alberta), and Grassland-Forest Transition (GFT; collected near
Tofield, Alberta) soils. Detailed descriptions of the soils and sampling areas can be found in
Dudas and Pawluk (1969) and Janzen et al. (1998), and characteristics of the sand-, silt-, and
clay-size fractions are described by Clemente et al. (2011). These soils contain high amounts of
montmorillonite and illite clays as well as chlorite and kaolinite but to a lesser extent (Bentley,
1979). Overlying vegetation in the SG and NG soil sites are dominated by Western Wheatgrass,
while the GFT soil site is dominated by both grasses and stands of Quaking Aspen (Otto and
Simpson, 2006a). Previous studies in our laboratory investigated these soils to characterize the
relationships between climate and the composition of OM at the molecular-level (Clemente et
al., 2011; Feng and Simpson, 2007; Otto and Simpson, 2005; 2006a; 2006b; 2007). Our
previous studies indicated that these soils contain different amounts of lignin-derived phenols
and cutin-derived OH-acids, and other compounds which implied varying levels of OM
degradation (Otto et al., 2005; 2006a; 2006b). After collection, these soils were air-dried,
passed through a 2 mm mesh sieve, and then stored at room temperature in glass containers.
Sand-, silt- and clay-size fractions were isolated using density and size fractionation
techniques (Clemente et al., 2011; Gregorich and Beare, 2008). All fractions were freeze-dried,
then ground, and stored at room temperature prior to analysis. Sample yields (which included
light fraction, not used in this study) revealed that recovery was ~95% for each sample but this
does not seem to reflect any selective OM compound loss because we observed the same
constituents but in varying distribution as previous studies, which analyzed these same soil
samples (Otto and Simpson, 2005; 2006a; 2006b).
5.3.3 Chemical Oxidation of Soil Density Fractions and Organic Matter-clay Complexes
Approximately 0.5 g of OM-clay complex, peat soil, silt-, or clay-size fraction, and 1 g
of sand-size fraction (n = 3) were suspended in a 20 mL salt solution containing 0.5 M
phosphate buffer, which maintains the pH at 4.5, 3 mM NaCl, and 2 mM CaCl2. OM-clay
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complexes and soil physical fractions (n = 3) were oxidized by dissolving 1 g of NaClO2 in the
salt solution (resulting in a 5% weight concentration). NaClO2 concentrations were determined
using preliminary experiments with high lignin concentrations to ensure that enough chemical
oxidant was added for the complete chemical oxidation of lignin. Samples that were suspended
in salt solution without NaClO2 were compared to chemically oxidized samples, so that changes
in lignin oxidation and OM composition due to chemical oxidation can be determined. Both
chemically oxidized and non-oxidized samples were then centrifuged for 30 min at 9500 rpm.
The samples were suspended and washed with the 3 mM NaCl + 2 mM CaCl2 solution five
times, freeze dried and ground prior to solvent extraction and CuO oxidation.
5.3.4 Carbon Content, Solvent Extraction and Lignin-derived Phenol Analysis
OM-clay complexes and soil fractions were analyzed for total carbon using the LECO
combustion method at the University of Guelph (Ontario, Canada). Using the method of Bundy
and Bremner (1972), Otto and Simpson (2006a) did not detect inorganic carbon in the surface
horizons of the soils: therefore, total carbon represents organic carbon content. Montmorillonite
clay was also analyzed for carbon, and the value (0.14% carbon by weight) was subtracted from
the OM-clay complexes to determine sorbed organic carbon concentrations. Samples that were
chemically oxidized (n = 3) and samples that did not receive NaClO2 were sequentially
extracted with methanol (Fisher Scientific), dichloromethane:methanol (Fisher Scientific; 1:1
v/v), and dichloromethane to extract free lipids (Otto and Simpson, 2005). Only one sample that
did not receive NaClO2, for each complex and soil fraction, was used because preliminary
experiments (using lignin-clay complexes and soil fractions) indicated that the standard error in
oxidized samples were greater compared to samples without NaClO2. The combined solvent
extracts were then filtered through GF/A and GF/F glass microfibre filters, concentrated by
rotary evaporation, and dried under a stream of nitrogen gas (Praxair, Toronto, ON) in 2 mL
vials. Lignin-derived phenols were extracted using CuO oxidation following the method of
Hedges and Ertel (1982), as modified by Otto and Simpson (2006a). Cutin-derived hydroxy-
acids (OH-acids) can also be extracted with CuO oxidation (Filley et al., 2008; Goni and
Hedges, 1990a; Mendez-Millan et al., 2010a). After solvent extraction, the OM-clay
complexes, and soil residues were air-dried, and then extracted with Copper (II) oxide and
ammonium iron (II) sulfate hexahydrate [Fe(NH4)2(SO4)26H2O] with 2 M NaOH in Teflon-
lined bombs. The bombs were flushed with nitrogen and incubated at 170 oC for 2.5 h. The
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supernatants were acidified to pH ~1 using 6 M HCl (Caledon Laboratories, ON) and kept for 1
h at room temperature, in the dark to prevent cinnamic acid polymerization. After
centrifugation at 2,700 rpm for 30 min, the supernatant was extracted with diethyl ether (Fisher
Scientific); the extracts were concentrated, transferred to 2 mL glass vials, and then dried under
a stream of nitrogen gas.
The CuO oxidation residues were re-dissolved in dichloromethane prior to
derivatization. Both solvent extracts and CuO extracts were derivatized by adding N,O-
bis(trimethylsilyl)-trifluoroacetamide (BSTFA; Sigma-Aldrich, Columbus, GA) and pyridine,
followed by heating at 70 oC for 1 hr. After cooling, the samples were analyzed using GC/MS.
To do this, three microlitres of sample was injected using an Agilent 7683 autosampler (Agilent
Technologies, Santa Clara, CA) in splitless mode. The sample was then eluted from the Agilent
6890N GC, which had an HP-5MS fused silica capillary column (30 m x 0.25 mm inner
diameter x 0.25 µm film thickness) by using the following temperature gradient: hold at 65 oC
for 2 min, followed by a temperature increase from 65 oC to 300 oC at a rate of 6 oC per min,
and a final isothermal hold at 300 oC for 20 min. The Agilent 5973N mass spectrometer was
operated at 70 eV in the electron impact mode. Data were acquired and processed using the
Agilent Chemstation G1701DA v. D software. Compounds were identified by comparison with
mass spectra from commercial libraries (Wiley, NIST) and authentic standards. Lauric acid
(Sigma-Aldrich) was used as an external standard to estimate the relative amounts of organic
acids and n-alkanol in the solvent extracts. Relative amounts of extractable lignin phenols were
estimated by using vanillic acid (Sigma-Aldrich) as an external standard, while relative amounts
of OH-acids were estimated using lauric acid as an external standard.
Eight main lignin-derived phenol monomers were identified and quantified according to
Hedges and Ertel (1982) and Otto and Simpson (2006a) which included: vanillyl (vanillin,
acetovanillone, vanillic acid), syringyl (syringaldehyde, acetosyringone, syringic acid), and
coumaryl (coumaric acid, ferulic acid) groups. The relative contributions (%) of these three
lignin monomer groups were calculated to determine changes in their distribution in the various
samples. The acid to aldehyde ratios of vanillyl (Ad/Alv) and syringyl (Ad/Als) groups were
calculated as these reflect the oxidation state of lignin-derived phenols, where higher values
indicate that lignin is more oxidized (Hedges et al., 1988). Lignin-derived phenol dimers were
also identified according to Goni and Hedges (1992) and Otto and Simpson (2006a): these
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comprised of 2-syringylsyringic acid, 2-syringylsyringaldehyde, 2-vanillylsyringic acid,
dehydrodivanillic acid, dehydrovanillinvanillic acid, dehydrovanillinacetovanillone, dehydrodi-
vanillin, and dehydroacetovanillonevanillic acid. The concentrations of lignin-derived phenol
dimers were divided by the concentrations of lignin-derived phenol monomers
(dimers/monomers). Cutin-derived OH-acids were also observed in the CuO oxidation extracts,
and these were identified according to Filley et al. (2008), Goni and Hedges (1990a), and
Mendez-Millan et al. (2010a): these comprised of 16-hydroxyhexadecanoic acid, 12-hydroxy-
octadecanedioic acid, 9,10-dihydroxyhexadecanoic acid, and 9,10,18-trihydroxyoctadecanoic
acid. Similar to lignin phenol dimers, the relative amounts of OH-acids compared to lignin
phenol monomers were calculated (OH-acids/monomers). The dimers/monomers and OH-
acids/monomers ratios were used to determine the stability of lignin phenol monomers relative
to lignin phenol dimers and cutin-derived OH-acids against oxidation. Characteristics of lignin-
clay, lignin-clay coated with dodecanoic acid; humic acid-clay and peat soil; and soil size
fractions before chemical oxidation are listed in Tables 5.1, 5.2 and 5.3, respectively.
5.3.5 Data Analysis
Changes in the various parameters, which resulted from NaClO2 oxidation, were
expressed as percentages of the difference between oxidized samples and samples that did not
receive NaClO2 (controls): change = ([oxidized sample-control]/control)*100. Here, a positive
change indicates an increase after oxidation, and a negative value indicates a decrease after
oxidation. Multivariate comparisons were performed on chemical groups identified in solvent
extracts and CuO oxidation extracts (n = 3) using SPSS v. 19.0. Analysis of variance followed
by Tukey Honestly Significant Difference (HSD) were used to determine whether differences
between mean changes were significant ( = 0.05). A 2-tailed, independent sample t-test was
used to determine significant differences ( = 0.05) between lignin-clay and lignin-clay-
dodecanoic acid complex, complexes prepared by humic acid sorption to clay at pH 4 and
sorption at pH 7, and SG and GFT soils.
5.4 Results and Discussion
5.4.1 Composition of Organic Matter-clay Complexes Before Chemical Oxidation
Higher concentrations of organic carbon and extractable lignin phenols in OM-clay
complexes were observed with higher lignin loadings (Table 5.1). Similarly, organic carbon
117
Table 5.1 Characteristics of lignin-clay complexes and lignin-clay complexes coated with dodecanoic acid prior to chemical oxidation. Lignin loadings (1 through 4) correspond to added lignin concentrations of 1) 0.4, 2) 4, 3) 20, 4) 100 g lignin/ 100 g clay.
Lignin Loading
1 2 3 4
Lignin-clay complexes
C content (%) 0.2 1.0 2.5 4.1
Lignin phenols (µg/gsample) 529 811 1989 2551
Ad/Alv 1.0 1.3 0.6 0.5
Lignin-clay complexes coated with dodecanoic acid
C content (%) 1.6 3.1 4.4
Lignin phenols (µg/gsample) 727 1408 1483
Ad/Alv 0.8 0.6 0.5
Dodecanoic acid (µg/gsample) 260 485 367
content, and lignin phenol concentrations in humic acid-clay complexes increased with humic
acid loadings at both pH 4 and 7 (Table 5.2). This trend is consistent with previous studies
where the amount of material sorbed to clay increased with higher starting concentrations of
OM in solution (Asselman and Garnier, 2000; Bayrak, 2006; Feng et al., 2005; Ghosh et al.,
2009). Only vanillyl phenol monomers (vanillin, acetovanillone, vanillic acid) were detected in
the CuO oxidation extracts of lignin-clay and lignin-clay-dodecanoic acid complexes (Table 5.1)
and is consistent with the lignin phenol monomer composition of alkali lignin measured in
preliminary studies (data not shown). For lignin-clay complexes, the higher Ad/Alv ratios at
lower lignin loadings (Table 5.1) suggest preferential association of more oxidized lignin
phenols on clay surfaces. There was also less lignin phenol extracted from lignin-dodecanoic
acid-clay complexes 3 and 4, compared to the corresponding lignin-clay complexes, which may
be attributed to displacement of sorbed lignin by dodecanoic acid. This is consistent with
preferential sorption of aliphatic structures observed by Feng et al. (2005). However, more
detailed sorption studies are necessary to confirm this hypothesis. Vanillyl monomers were also
118
Table 5.2 Carbon content, composition of extractable lipids, lignin-derived phenols, and cutin-derived OH-acid contributions in humic acid-clay complexes before chemical oxidation. Humic acid loadings (1 through 3) correspond to added humic acid concentrations of: 1) 0.4, 2) 4, and 3) 20 g peat humic acid/ 100 g clay at pH 4 and pH 7.
Humic Acid Loading Peat Soil
pH 4 pH 7
1 2 3 1 2 3
C content (%) 0.1 1.1 3.9 0.1 1.1 3.5 45.7
C9-C28 Fatty acid (µg/gsample)
9 12 11 9 31 22 408
n-alkanols (µg/gsample) 3 4 2 4 2 2 36
Lignin phenols (µg/gsample) 10 135 456 8 175 370 2130
Ad/Alv 1.2 1.3 2.1 1.3 1.7 1.7 2.8
Ad/Als bdl 0.6 1.4 bdl 0.3 0.9 2.1
% Vanillyl 100 37 57 100 64 59 55
% Syringyl bdl 48 35 bdl 27 34 33
% Coumaryl bdl 16 9 bdl 9 7 13
Dimers/monomers 2.3E-7 0.3 0.04 3.5E-7 0.1 0.01 0.02
OH-acid/monomers 7.3 0.2 0.01 5.5 0.1 0.01 0.06
bdl = below detection limit
119
preferentially sorbed to montmorillonite in humic acid-clay complexes (Table 5.2). In both SG
and GFT soils, higher Ad/Al ratios in the clay- compared to sand- and silt-size fractions suggest
greater oxidation of lignin in clay-size fractions. These data are consistent with previous
studies, which concluded that lignin in fine particle-size fractions are more oxidized (Clemente
et al., 2011; Guggenberger et al., 1994; Heim and Schmidt, 2007a; Quideau et al., 2001). Lignin
phenols, which consisted mainly of vanillyl monomers, were also isolated from peat soil (Table
5.2) and enrichment of vanillyl monomers in soils is attributed to the greater chemical
recalcitrance of these lignin phenol structures (Bahri et al., 2006; Hedges et al., 1988).
However, the affinity between oxidized lignin phenols, vanillyl monomers and clay mineral
surfaces observed in the OM-clay complexes (Tables 5.1 and 5.2), and higher Ad/Al ratios in
soil clay-size fractions also collectively suggest that there is preferential association between
clay minerals and specific lignin phenol structures.
Aliphatic compounds may have been associated with lignin-clay-dodecanoic acid and
humic acid-clay complexes through different modes. Similar concentrations of dodecanoic acid
were extracted from lignin-clay-dodecanoic acid complexes regardless of lignin concentrations
(Table 5.1), which suggests that dodecanoic acid coated lignin-clay complexes to a similar
extent regardless of the lignin loading used. These results indicate additional protection of OM
through interactions between OM compounds such as lignin and dodecanoic acid, in addition to
protection through OM-clay interactions. Similarly, proteins and n-alkanes are hypothesized to
be protected from degradation through association with other soil OM structures (Lichtfouse et
al., 1998; Simonart et al., 1967). On the other hand, both cutin-derived OH-acids and plant-
derived organic acids are found in humic acid extracts from peat soil (Table 5.2). These
compounds, along with other structures such as lignin phenols, are likely to simultaneously sorb
to clay minerals during humic acid-clay complex formation. In the humic acid-clay complexes,
the ratio of OH-acid to monomers decreased with increased humic acid loadings (Table 5.2) and
indicates that OM sorbed to clay at higher humic acid concentrations consisted of a smaller
proportion of cutin OH-acids. Peat soil, which has a high carbon content (Table 5.2) also had
lower OH-acid to lignin phenol monomer ratios (Table 5.2), which is attributed to the slow rate
of plant material degradation (Johnston et al., 1997). Previous studies, also found that aliphatic
compounds (such as cutin) preferentially associate with clay mineral surfaces (Chi and Amy,
2004; Feng et al., 2005). These studies, together with our observations indicate that the smaller
120
proportion of cutin OH-acids at higher humic acid loadings may present another mechanism for
OM interaction with clay minerals. For example, at higher humic acid concentrations, aliphatic
compounds on clay surfaces may enhance indirect association of other compounds (such as
lignin and carbohydrates) with clay minerals through hydrophobic interactions between OM and
previously sorbed aliphatic compounds. These interactions may be more favorable than direct
interactions between compounds, such as lignin and carbohydrates, and clay mineral surfaces.
Fatty acids with C9-C28, with a preference for even carbon number, as well as C18, C24, and C26
n-alkanol were also detected in the solvent extracts of the humic acid-clay complexes (Table
5.2). This even carbon preference is typical of plant-derived cuticular waxes, and is consistent
with the plant origin of peat humic acid, while contributions from lipids with <C20 also indicate
contributions from microbial lipids (Bull et al., 2000a; Bull et al., 2000b; Otto and Simpson,
2005). Humic acid-clay complexes (notably loadings 2 and 3) constructed at pH 7, had greater
organic acid concentrations as compared to humic acid-clay complexes constructed at pH 4
(Table 5.2), which may be due to a higher proportion of fatty acids that ionize at pH 7 (Kanicky
et al., 2000) and subsequently sorbed to clay through Ca2+- mediated cation bridges (Mikutta et
al., 2007). At higher pH values, humic acids adopt a more stretched conformation (Avena and
Wilkinson, 2002; Chien and Bleam, 1998), which may also enhance interactions between fatty
acids and the clay mineral surface.
5.4.2 Chemical Oxidation of Lignin-clay and Lignin-clay-dodecanoic acid Complexes
Coating lignin-clay complexes with dodecanoic acid, a compound that is not altered by
the chemical oxidant (Hoigne and Bader, 1994), resulted in further protection of vanillyl phenols
from chemical oxidation (Fig. 5.1). Extractable lignin phenols from lignin-clay-dodecanoic acid
complex increased slightly at the highest loading (Fig. 5.1, loading 4). An increase in
extractable lignin phenols is believed to occur when lignin is depolymerized, but only slightly
oxidized (Said-Pullicino et al., 2007). Vanillyl concentrations decreased to a greater extent in
lignin-clay compared to lignin-clay-dodecanoic acid complexes at similar lignin loadings,
suggesting that lignin protection from oxidation through OM-OM interactions between lignin
and dodecanoic acid may also have occurred (Fig. 5.1). Protection of lignin by dodecanoic acid
may have limited access of water (and the oxidizing agent). In soils, hydrophobicity is
hypothesized to limit the degradation of OM (Kleber, 2010). Long chain aliphatic compounds,
121
Figure 5.1 Lignin oxidation in lignin-clay and lignin-clay-dodecanoic acid complexes after chemical oxidation a) changes in vanillyl monomer concentrations and b) changes in vanillyl oxidation (Ad/Alv). Values plotted are changes (%) after chemical oxidation. Loading numbers (1-4) indicate higher lignin concentrations respectively prior to oxidation (Table 5.1). Positive values suggest an increase, while negative values suggest a decrease due to chemical oxidation. Error bars represent the standard error of the mean (n = 3 digests). Letters indicate significant difference between samples with different lignin loadings (Tukey HSD, α = 0.05), and (*) indicate significant difference between samples with and without dodecanoic acid (t-test, = 0.05).
such as dodecanoic acid, are also known to stabilize microaggregates (Dinel et al., 1991a;
1991b), and protection of lignin within these aggregates may have also inhibited its chemical
oxidation.
Lignin loading
a.
b.
-60
-40
-20
0
20
40
60
80
100
120
Lignin
Lignin and Dodecanoic acid
Ch
ang
e in
van
illy
l oxi
dat
ion
(% A
d/A
l v)
2*1 3 4
a
b
c
c
-100
-80
-60
-40
-20
0
20Lignin Lignin and dodecanoic acid
Ch
ang
e in
to
tal v
anil
lyl c
on
cen
trat
ion
(%
µg
/g s
amp
le)
1 2* 3 4*
a
bbc
cx
xy
y
122
At the highest lignin concentration (Fig. 5.1, loading 4), lignin phenol oxidation resulted
in higher Ad/Alv ratios but less lignin was removed at this loading since vanillyl yields
decreased to a lesser extent (Fig. 5.1, loading 4). However, at the lowest lignin concentration
(Fig. 5.1, loading 1), there was a smaller increase in Ad/Alv ratios after chemical oxidation
likely because the chemical oxidation products were no longer identified as distinct lignin
phenols. These data suggest that greater concentrations of lignin on clay resulted in OM-OM
interactions, which protected lignin from chemical oxidation. The concentration of dodecanoic
acid that was extracted after chemical oxidation was also higher in lignin-clay-dodecanoic acid
complexes with lower lignin concentrations (Fig. 5.2, loading 2). This pattern indicates that the
stability of interactions between dodecanoic acid and the lignin-clay complex was also
dependent on the amount of lignin sorbed to montmorillonite, which is disrupted as lignin is
chemically oxidized (Fig. 5.2). This observation is consistent with the hypothesis that when
OM sufficiently coats the active sites on clay minerals, the OM that is subsequently added sorbs
to the previously formed OM layer through hydrophobic interactions (Chi and Amy, 2004;
Kaiser and Guggenberger, 2000). Therefore, both the lower degradation of lignin phenols and
lower concentrations of extractable dodecanoic acid at higher lignin loadings indicate that
increased hydrophobic interactions and enhanced OM-OM interactions may further protect clay-
associated OM.
5.4.3 Chemical Oxidation of Humic Acid-clay Complexes
To investigate whether the composition and conformation of OM sorbed to
montmorillonite plays a role in lignin protection from chemical oxidation; changes in lignin
concentration, composition and extractable n-alkanol and fatty acids after chemical oxidation
were measured for humic acid-montmorillonite complexes prepared at pH 4 and 7 (Figs. 5.3-
5.5). Despite differences in the conformation of humic acid sorbed to clay minerals at pH 4 and
7, there were similarities in the oxidation of lignin phenols in both sets of humic acid-clay
complexes. With the exception of the pH 7 complex with the lowest humic acid concentration
(Fig. 5.3, loading 1), lignin phenol concentrations decreased with chemical oxidation, which
suggests lignin removal in the majority of humic acid-clay complexes (Fig. 5.3a). Increased
OH-acid/monomer ratios (Fig. 5.3b), is also consistent with lignin removal and illustrates the
resulting enrichment of cutin-derived OH-acids relative to lignin phenols with chemical
oxidation. This enrichment is due to the 106-1011 times greater reactivity of phenols to chlorine
123
Figure 5.2 Increase (%) in extractable dodecanoic acid after chemical oxidation of lignin-clay-dodecanoic acid complexes. Values plotted are differences in % extracted before and after oxidation, and larger loading numbers (2-4) indicate higher lignin concentrations prior to oxidation (Table 5.1). Positive values suggest an increase in extracted dodecanoic acid as a consequence of lignin oxidation. Error bars represent the standard error of the mean (n = 3 digests). Different letters indicate significant difference in extractable dodecanoic acid among complexes with different lignin loadings (Tukey-HSD, = 0.05).
dioxide, the main oxidizer produced by acidifying NaClO2 (Svenson et al., 2006), as compared
to alcohols and fatty acids (Hoigne and Bader, 1994). Humic acid-clay complexes were created
by simultaneous, competitive sorption of a complex mixture of OM compounds within the
humic acid, which is in contrast to the layering of lignin-clay with dodecanoic acid. The
simultaneous mode of sorption may have led to less efficient protection of lignin in the resulting
humic acid-clay complexes (Fig. 5.3) compared to that in lignin-clay-dodecanoic acid
complexes (Fig. 5.1). The number of sorption sites on clay surfaces are believed to be finite
(Kaiser and Guggenberger, 2003; Mikutta et al., 2007; Six et al., 2002), and aliphatic
compounds that preferentially sorb to montmorillonite (Feng et al., 2005; Ghosh et al., 2009)
may out compete lignin. Therefore, at high humic acid loadings, some lignin may have
associated with clay mineral surfaces through interactions with OM, as suggested by lower OH-
acid/lignin ratios at higher humic acid concentrations (Table 5.2). However, aromatic structures
may not always interact with aliphatic structures within humic acids (Chien and Bleam, 1998),
which may also limit lignin protection. This hypothesis is further supported by similar levels of
chemical oxidation of lignin phenols in peat soil and humic-acid clay complexes with higher
humic acid concentrations (Fig. 5.3, loadings 2 and 3).
For humic acid-clay complexes created at pH 4 and 7, preferential oxidation of syringyl
and coumaryl monomers resulted in higher concentrations from vanillyl monomers after
-0.5
0.5
1.5
2.5
3.5
Lignin loading
2 43
Incr
ease
in
ext
ract
able
do
dec
ano
icac
id(%
mg
/g s
amp
le)
a
bb
124
Figure 5.3 Changes in lignin concentrations in humic acid-clay complexes (created at pH 4 and 7) and peat soil after chemical oxidation: a) decreased concentrations of lignin phenol monomers, and b) increased OH-acid/monomer ratios. Values plotted are changes (%) after chemical oxidation, and larger loading numbers (1-3) indicate greater humic acid concentrations prior to oxidation (Table 5.2). Positive values suggest an increase in lignin phenol concentrations, while negative values suggest a decrease due to oxidation. Error bars represent the standard error of the mean (n = 3 digests). Different letters indicate significant differences between complexes with different humic acid loadings (Tukey-HSD, α = 0.05), and (*) indicates significant difference between complexes created at pH 7 and pH 4 (t-test, α = 0.05).
chemical oxidation (Fig. 5.4a). This trend is consistent with the reported stability of vanillyl
compared to syringyl and coumaryl monomers (Bahri et al., 2006; Ertel and Hedges, 1984). As
well, Ad/Alv ratios increased (Fig. 5.4d), while Ad/Als ratios decreased (Fig. 5.4e) after
chemical oxidation. A previous study concluded that syringic acid and coumaric acid were
more labile than ferulic acid, acetosyringone, syringaldehyde, and vanillyl monomers (Bahri et
a.
b.
Humic acid loading
-130
-90
-50
-10
30
70
110
150
pH 7 pH 4
Ch
ang
e in
lig
nin
ph
eno
l mo
no
mer
co
nce
ntr
atio
ns
(% µ
g/g
sam
ple
)
PeatSoil
1* 2 3*
y
xyx
b
a a
ay
-500
500
1500
2500
3500
4500
pH 7
pH 4
PeatSoil
1 2 3
Ch
ang
e in
OH
-aci
ds/
mo
no
mer
s (%
)
y
xy
x
xy
ab
ab
a
b
125
al., 2006). Therefore, syringic acid may not always accumulate relative to syringaldehyde,
which leads to decreased Ad/Als. In contrast, vanillin is thought to be more labile than vanillic
acid (Bahri et al., 2006; Hedges et al., 1988). The relative changes in lignin phenol composition
(Fig. 5.4) therefore suggest that the relative recalcitrance of OM structures may also contribute
to OM protection in OM-clay complexes.
Despite the similarities in overall lignin oxidation for the complexes created at pH 4 and
7, pH controlled composition and conformation of OM sorbed to clay minerals, which resulted
in noticeable differences in lignin protection. Lignin was removed to a lesser extent at the
lowest humic acid concentration at pH 7 (Fig. 5.3, loading 1), which also had the lowest lignin
phenol concentrations prior to oxidation (Tables 5.1, 5.2). At these concentrations, lignin may
have sorbed to high affinity sites, which inhibited OM degradation (Mikutta et al., 2007).
Enhanced monolayer sorption on clay due to the stretched conformation of humic acids at pH 7
(Avena and Wilkinson, 2002; Chien and Bleam, 1998) may have also resulted in stronger OM-
clay interactions, and lignin protection. Therefore, although aromatic structures (such as those
in lignin phenols) may not be preferentially sorbed by montmorillonite (Asselman and Garnier,
2000), availability of high affinity sites on clay and monolayer sorption, are possible
mechanisms that protected lignin phenols at the lowest humic acid concentrations from chemical
oxidation (Fig. 5.3).
In complexes with the highest carbon concentrations prior to oxidation (Table 5.2,
loading 3), chemical oxidation resulted in increased Ad/Alv ratios, which was greatest in the
humic-clay complex prepared at pH 4 compared to that prepared at pH 7 (Fig. 5.4d). This
difference is likely because vanillin was less oxidized in the pH 7 complex, such that ~30% of
the vanillin was detected after oxidation, while only ~2% of vanillin was detected in the pH 4
complex after chemical oxidation (not shown). Since vanillic acid is a product of vanillin
oxidation (Hedges et al., 1988), and changes in lignin phenol concentrations were similar for
both pH values, these data suggest that vanillin in the pH 4 complex was more susceptible to
oxidation. This hypothesis is further supported by the higher dimers/monomers ratio in the pH 7
compared to the pH 4 complex (Fig. 5.4f, humic acid loading 3), which resulted from higher
residual dimer concentrations in the pH 7 complex. Although dimers/monomers ratios in soils
increase through enrichment of more stable lignin dimers as lignin monomers are degraded
(Goni and Hedges, 1992; Goni et al., 1993; Opsahl and Benner, 1995; Thevenot et al., 2010),
126
Figure 5.4 Changes in lignin phenol composition in humic acid-clay complexes (created at pH 4 and 7) and peat soil after chemical oxidation: a) changes in vanillyl contribution (vanillin + acetovanillone + vanillic acid) b) decreased syringyl contribution (syringaldehyde + acetosyringone + syringic acid), c) decreased coumaryl contribution (coumaric acid + ferulic acid), d) changes in vanillyl oxidation (Ad/Alv), e) changes in syringyl oxidation (Ad/Als), and f) changes in dimers/monomers. Values plotted are changes (%) after chemical oxidation, and larger loading numbers (1-3) indicate greater humic acid concentrations prior to oxidation (Table 5.2). Positive values suggest an increase in lignin phenol concentrations, while negative values suggest a decrease due to oxidation. Error bars represent the standard error of the mean (n = 3 digests). Different letters indicate significant difference in samples with different humic acid loadings (Tukey-HSD, α = 0.05), and (*) indicate significant difference between complexes created at pH 7 and pH 4.
Humic acid loading
d.
e.
f.
-120
-100
-80
-60
-40
-20
0
pH 7
pH 4
PeatSoil
1 2 3
De
cre
as
ed
co
um
ary
l c
on
trib
uti
on
(%
)
x
y
by
a
a
a.
b.
c.
-20
0
20
40
60
80
100
120
pH 7
pH 4
PeatSoil
1 2* 3Ch
an
ge
in v
an
illy
l co
ntr
ibu
tio
n (
%)
x
yay
z
a a
b
-120
-100
-80
-60
-40
-20
0
pH 7
pH 4
De
cre
as
ed
syr
ing
yl c
on
trib
uti
on
(%
)
PeatSoil
1 2 3
-100
200
500
800
1100
pH 7pH 4
PeatSoil
1* 2 3*
Ch
an
ge
in
dim
ers
/mo
no
me
rs (
%)
y
y
y
a ax
bb
-240
-200
-160
-120
-80
-40
0
pH 7
pH 4
Ch
an
ge
in
syr
ing
yl o
xid
ati
on
(%
Ad
/Al s
)
PeatSoil
1 2 3
-100
-60
-20
20
60
100
140pH 7
pH 4
PeatSoil
1 2 3*
Ch
an
ge
in
va
nil
lyl o
xid
ati
on
(%
Ad
/Al v
)
xy
x
xy
y
127
the higher ratios in the pH 7 complex after oxidation may be attributed to better protection of
lignin in the pH 7 humic acid-clay complex.
After chemical oxidation, there was also a greater proportion of n-alkanol and organic
acids extracted from complexes created at pH 4 and peat soil compared to complexes created at
pH 7 (Fig. 5.5). These data further support the hypothesis that under the conditions used in this
study, OM-clay complexes created at pH 7 were less susceptible to oxidation than those created
at pH 4. The solution pH during the creation of OM-clay complexes may have influenced both
OM conformation and the mechanisms responsible for OM-clay interactions (Avena and
Wilkinson, 2002; Baalousha et al., 2006; Chien and Bleam, 1998; Feng et al., 2005; Mikutta et
al., 2007; Piccolo et al., 1996). Solution pH is believed to influence the conformation of humic
substances in that OM aggregation increases under acidic pH; while OM disaggregates under
basic pH. These pH-induced OM conformations may also influence the structures exposed to
solution and therefore the structures available to complex with clay, since a portion of the
structures are protected within the OM aggregate. For example, associations between carboxyl
groups, which are more likely to occur under acidic pH (Doan et al., 1997) may need to be
disrupted before complexation between these structures and clay can occur. Solution pH may
also influence the mechanisms responsible for OM-clay interactions because ligand exchange
was observed only in clay-OM complexes created at pH 4 (Feng et al., 2005), and Ca2+-
mediated cation bridges were more important at neutral pH values (Mikutta et al., 2007). In this
study, a PO43- buffer was used during oxidation, which may have weakened OM-clay sorption
through ligand exchange (Kaiser and Guggenberger, 2000; Mikutta et al., 2006a), thereby
increasing OM susceptibility to chemical oxidation (Mikutta et al., 2007). The stability of lignin
against degradation in the pH 7 complex compared to the pH 4 complex may therefore be
attributed to OM composition, and OM-clay interactions (such as ligand exchange) that are
favoured at pH 4.
5.4.4 Chemical Oxidation of Soil Density Fractions
Using Southern Grassland (SG), and Grassland-Forest Transition (GFT) soils, protection
of lignin from oxidation through association with sand-, silt-, and clay-size fractions in natural
systems was investigated. Similar to OM-clay complexes, cutin-derived OH-acids were
enriched as a result of lignin monomer chemical oxidation (Fig. 5.6). The depletion of syringyl
128
Figure 5.5 Increase in extractable a) n-alkanol and b) organic acids after chemical oxidation of humic acid-clay complexes (created at pH 4 and pH 7), and peat soil. Values plotted are changes (%) after chemical oxidation, and larger loading numbers (1-3) indicate greater humic acid concentrations prior to oxidation (Table 5.2). Error bars represent the standard error of the mean (n = 3 digests). Different letters indicate significant differences between samples with different peat humic acid loadings (Tukey-HSD, α = 0.05), and (*) indicate significant difference between complexes created at pH 7 and pH 4.
and coumaryl monomers, which resulted in enrichment of vanillyl monomers (Fig. 5.7) is
consistent with other observations that report enhanced stability of vanillyl monomers in the
environment (Bahri et al., 2006; Ertel and Hedges, 1984; Kiem and Kögel-Knabner, 2003).
Lignin removal and changes in lignin composition were also compared across the sand-, silt-,
and clay-size fractions. A previous study suggests that lignin phenols were preferentially
preserved in silt-size fractions (Heim and Schmidt, 2007a), likely through association with
aggregates (Six, 2004). However, destruction of aggregates in the SG and GFT silt-size
Humic acid loading
a.
b.
-200
200
600
1000
1400
pH 7
pH 4
1 2* 3 PeatSoilIn
crea
sed
ext
ract
able
n-a
lkan
ol
(% µ
g/g
sam
ple
)
-200
200
600
1000
1400
pH 7
pH 4
1 2 3 PeatSoilIn
crea
sed
ext
ract
able
org
anic
aci
ds
(% µ
g/g
sam
ple
)
a
ab
b
b
129
Figure 5.6 Decreased lignin concentrations in Southern Grassland (SG) and Grassland-Forest Transition (GFT) soil mineral fractions after chemical oxidation: a) decrease in lignin phenol monomers, and b) increase in OH-acid/monomer ratios. Values plotted are differences (%) before and after chemical oxidation. Error bars represent the standard error of the mean (n = 3 digests). Different letters indicate significant differences between fractions (Tukey-HSD, α = 0.05), and (*) indicates significant difference between SG and GFT fractions (t-test α = 0.05).
fractions (through grinding) may have resulted in less protection for OM as compared to lignin
in clay-size fractions (Figs. 5.6, 5.7). Decrease in lignin concentration and changes in lignin
phenol composition were smaller in clay-size compared to sand-, and silt-size fractions (Figs.
5.6, 5.7), which suggests that OM associated with clay was protected from chemical oxidation.
Such protection is consistent with the hypothesis that OM in finer fractions is older because they
are physically protected from degradation (Baldock and Skjemstad, 2000; von Lutzow et al.,
2007). Protection of OM in clay-size fractions observed in this study may be attributed to OM
protection within smaller microaggregates, and primary physical or chemical associations
between OM and clay. Microaggregates found in clay-sized fractions are believed to be stable
a.
b.
-100
-80
-60
-40
-20
0
SG GFT
Sand* Silt* ClayD
ecre
ase
in li
gn
in p
hen
ol m
on
om
ers
(% µ
g/g
sam
ple
)
xxy
y
aa
b
0
100
200
300
400SG GFT
Sand Silt Clay
Soil mineral fraction
Incr
ease
d O
H-a
cid
/mo
no
mer
s (%
) a
b
c
130
Figure 5.7 Changes in lignin phenol monomer composition of Southern Grassland (SG) and Grassland-Forest Transition (GFT) soils after chemical oxidation: a) increased vanillyl contribution (vanillyl + acetovanillone + vanillic acid), b) decreased syringyl contribution (syringaldehyde + acetosyringone + syringic acid), c) decreased coumaryl contribution (coumaric acid + ferulic acid), d) increased vanillyl oxidation, e) increased syringyl oxidation, and f) changes in dimers/monomers. Values plotted are changes (%) after chemical oxidation. Positive values suggest an increase in Ad/Al and dimers/monomers ratios, while negative values suggest decreases in values due to oxidation. Error bars represent the standard error of the mean (n = 3 digests). Different letters indicate significant difference between different fractions (Tukey-HSD, α = 0.05), and (*) indicate significant difference between SG and GFT soil fractions (t-test α = 0.05).
against physical manipulations (Chenu and Plante, 2006; Six, 2004). Sorption of OM to clay
also limits access of chemical reagents and enzymes to the reactive structures in OM
(Christensen, 2001; Mikutta et al., 2006b; Six et al., 2002; von Lutzow et al., 2007). In contrast,
chemical recalcitrance may be more important in protecting OM in sand-size fractions from
Soil mineral fraction
d.
e.
f.
-50
0
50
100
150
200
250SGGFT
Sand Silt* Clay
Inc
rea
se
d v
an
illy
l ox
ida
tio
n
(%A
d/A
l v)
a
ab
b
-50
0
50
100
150
200
250SGGFT
Sand* Silt Clay
Incr
ease
d s
yrin
gyl
ox
idat
ion
(%
Ad
/Al s
)
-80
-40
0
40
80SG GFT
Sand* Silt* Clay
Ch
an
ge
in
dim
ers
/mo
no
mer
s (
%)
a
b
c
a.
b.
c.
0
25
50
75
100SGGFT
Sand* Silt Clay
Incr
eas
ed v
an
illy
l co
ntr
ibu
tio
n (
%) a
b
c
-120
-95
-70
-45
-20
SGGFT
Sand Silt* Clay*
Dec
reas
ed s
yrin
gyl
co
ntr
ibu
tio
n (%
)
a
b
c
-120
-95
-70
-45
-20
SGGFT
Sand Silt ClayDec
reas
ed c
ou
mar
yl
co
ntr
ibu
tio
n (%
)
a
ab
b
131
oxidation because of the lower surface area and limited interactions between sand-size minerals
and OM (Christensen, 2001). Lignin concentrations in the sand-size fractions decreased to a
greater extent indicating that chemical recalcitrance may not be as effective as mineral
association (Figs. 5.6, 5.7) in protecting OM. This hypothesis is consistent with previously
reported faster turnover of lignin in larger particle-size fractions (Heim and Schmidt, 2007a; von
Lutzow et al., 2007).
The extent of lignin oxidation in the SG particle-size fractions was also compared to
those of GFT size fractions (Figs. 5.6, 5.7) to determine whether differences in organic carbon
content, and vegetation influenced lignin protection. The decrease in lignin phenol
concentrations, enrichment of cutin-derived OH-acids, enrichment of vanillyl monomers, and
increases in Ad/Alv ratios after digestion (Figs. 5.6, 5.7), in the sand- and silt-size fractions of
SG was greater than those of GFT fractions, all of which suggest that lignin in the SG fractions
were less protected. Because of the limited interactions between minerals and OM in sand-size
fractions (Christensen, 2001), and disturbance of silt-size aggregates, differences in the
oxidation of lignin in these fractions may be attributed to lignin phenol composition. This
hypothesis is consistent with higher vanillyl monomer concentrations (Table 5.3), which are
more stable than syringyl and coumaryl monomers (Bahri et al., 2006; Ertel and Hedges, 1984;
Kiem and Kögel-Knabner, 2003); and higher vanillyl dimer concentrations (not shown), which
are more stable monomers (Goni and Hedges, 1992; Goni et al., 1993; Otto and Simpson,
2006a) in GFT fractions. Because OM in sand-size fractions are at an earlier stage of
degradation, differences in lignin structures extracted from this fraction may be attributed to the
overlying vegetation. SG soils are dominated by Western Wheatgrass, while GFT soil is
dominated by Quaking Aspen as well as grasses (Otto and Simpson, 2006a). The replacement
of grass by trees has been observed to result in more recalcitrant soil OM (Liao et al., 2006).
Susceptibility of grass-derived lignin to degradation is also consistent with its structure: it was
observed to have greater concentrations of more labile coumaryl monomers (Nimz et al., 1981;
Opsahl and Benner, 1995; Otto and Simpson, 2006a); and more ester linkages, which are easily
decomposed (Nimz et al., 1981). These structural considerations are consistent with a previous
study, in which Klason lignin extracted from grass was also more easily degraded by
microorganisms, compared to those extracted from softwood and hardwood (Antai and
Crawford, 1981).
132
Table 5.3 Carbon content, lignin-derived phenol and cutin-derived OH-acid composition in Southern Grassland and Grassland-Forest Transition sand-, silt-, and clay-size fractions prior to chemical oxidation.
Soil Mineral Fraction
Sand Silt Clay
Sand Silt Clay
Southern Grassland (SG) Grassland-Forest Transition (GFT)
C content (%) 0.9 2.2 4.1 1.4 2.7 5.5
Lignin phenols (µg/gsample)
26 18 10 29 19 8
Ad/Alv 0.9 1.3 2.6 0.8 1.0 1.9
Ad/Als 0.7 1.0 1.5 0.6 0.8 1.4
Vanillyl (%) 40 41 41 48 50 49
Syringyl (%) 38 34 35 33 32 28
Coumaryl (%) 21 25 24 19 18 23
Dimers/monomers 0.2 0.2 0.1 0.2 0.1 0.1
OH-acids/monomers 0.1 0.2 0.3 0.1 0.20 0.4
The oxidation levels of lignin in the clay-size fractions in both soils were similar, as
were the increase in vanillyl, decrease in coumaryl monomer contributions, and increases in
Ad/Al ratios (Figs. 5.6, 5.7). These data suggest that overall, lignin monomers in the clay-size
fractions of both soils were protected to the same extent. The clay mineral composition of both
soils were dominated by montmorillonite and illite (Bentley, 1979). Therefore, the interactions
between clay and OM that were responsible for protecting lignin from chemical oxidation may
have been similar as well. However, syringyl monomer contributions decreased to a greater
extent, while dimer contributions decreased to a lesser extent in the GFT compared to the SG
clay-size fraction (Fig. 5.7b and 5.7f). These differences in the degradation levels of specific
structures in SG and GFT, suggest that OM composition prior to oxidation may have also
influenced lignin protection in the clay-size fractions, and is consistent with that observed in the
humic acid-clay complex (Fig. 5.4) as well. For example, the greater resistance of lignin dimers
in the GFT fractions (concentrations decreased by 16% in GFT, and ~50% in SG clay-size
fraction) may be attributed to the greater concentrations of dimers where aromatic rings are
133
directly linked (i.e. 5-5’ dimers) in GFT fractions, which is consistent with total soil dimer
concentrations found by Otto et al. (2006a). These structures are thought to be more recalcitrant
compared to when carbonyl or methyl groups link the aromatic structures (i.e. β,1-diketone
dimers; α,1-monoketone dimers; α ,5-monoketone dimers; and α,2-methyl dimers;(Goni and
Hedges, 1992). The mechanism responsible for greater decrease of syringyl monomers in GFT
clay-size fraction after NaClO2 oxidation on the other hand, is less clear, but may also be related
to the structure of lignin in GFT vegetation. Therefore, although the overall degradation of
lignin phenols in clay-size fractions was mainly limited by protection of OM through its
interactions with clay minerals, inherent recalcitrance of lignin structures may have also
influenced the relative degradability of specific structures. This is consistent with the
hypothesis that OM structure influences its preservation in soil minerals (Kaiser and
Guggenberger, 2000). It is also possible that lignin phenol dimers were unable to form strong
interactions with SG clay, which made them more susceptible to oxidation. The total OM in this
clay-size fraction contains higher concentrations of aliphatic structures (Clemente et al., 2011),
which may compete with lignin for sorption sites, since montmorillonite sorbs high
concentrations of aliphatic compounds (Feng et al., 2005).
5.5 Conclusions
By examining OM-montmorillonite clay complexes and mineral fractions from two
soils, we found that OM concentration and composition governed the protection of lignin from
NaClO2 oxidation. Coating lignin-clay complexes with dodecanoic acid, which was resistant to
NaClO2 oxidation, protected lignin from chemical oxidation. The stretched conformation of
humic acids in humic acid-clay complexes created at pH 7, which may have promoted
monolayer sorption of humic acids to montmorillonite, also enhanced protection of lignin from
chemical oxidation. Accordingly, interactions between OM structures may be another
protection mechanism that should be considered. Current models emphasize the importance of
interactions between OM and clay minerals and protection within microaggregates, as the main
mechanisms responsible for OM protection in soils. Our results are consistent with these
models in that overall, lignin in clay-size fractions of SG and GFT soils were protected against
chemical oxidation to the same extent. However, OM composition also appears to play a role in
lignin protection from chemical attack. This hypothesis is emphasized by the more advanced
oxidation of lignin dimers in SG fractions, compared to GFT fractions; and the greater
134
differences in the oxidation of lignin in sand- and clay-size fractions of SG soils compared to
those of GFT soils. The role of OM structure and composition in its preservation should be
considered because it has been hypothesized that clay sorption sites may become saturated as
soil carbon content increases. Therefore, as carbon concentrations increase, interactions
between OM may become more important in determining preservation. It can also be
envisioned that OM sorption to clay may become more stable with time, because of the
additional protection provided by overlying OM. These OM-clay interactions may depend on
the structures of both OM and clay minerals. Therefore, understanding how OM structures
control their association with soil minerals and subsequent preservation, may help predict the
resilience of protected OM against disturbance caused by changes in environmental conditions
and land management.
5.6 Acknowledgements
The authors thank Katherine Hills (funded through the Natural Sciences and Engineering
Research Council Undergraduate Student Research Awards Program) for assistance with sample
extractions. Funding for this project was provided by the Natural Sciences and Engineering
Research Council Green Crop Network. J.S.C. also thanks the Ontario Government for support
via an Ontario Graduate Scholarship.
135
CHAPTER 6
SUMMARY, CONCLUSIONS, AND FUTURE RESEARCH
Author: Joyce S. Clemente
Contributions: Dr. R. Sodhi at Surface Interface Ontario, obtained the X-ray Photoelectron
Spectroscopy (XPS) and Time of Flight-Secondary ion Mass Spectrometry (ToF-SIMS) data,
and processed the XPS dat. JSC analyzed the ToF-SIMS data with guidance from Dr. Sodhi.
JSC performed the protein-clay interaction experiments, and analyzed the data. The chapter was
written by JSC with critical comments from Myrna J. Simpson.
136
6.1 Summary and Conclusions
Soil OM is important because it is involved in the global carbon cycle, is essential in
supporting terrestrial life and agricultural productivity, and can mitigate the impact of
environmental contaminants (Batjes, 1998; Janzen et al., 1998; Stimler et al., 2006; Tiessen et
al., 1994; Wicke and Reemtsma, 2010; Wolf and Snyder, 2003). Soil OM also maintains
biological activity in that it promotes retention and biological availability of essential nutrients
such as N, S and P (McGill and Cole, 1981; Tiessen et al., 1994; Wolf and Snyder, 2003). Soils
with greater OM content are also better at retaining water and preventing erosion (Campbell et
al., 1989; Wallace, 1994). Finally, soil OM interacts with contaminants that may be harmful,
and may limit their interactions with the various organisms that dwell, or otherwise interact with
soils (Stimler et al., 2006; Wicke and Reemtsma, 2010). Plant biomass, which is mainly derived
from atmospheric carbon dioxide, is one of the main contributors to soil OM (Batjes, 1998;
Kögel-Knabner, 2002). Therefore, incomplete mineralization of plant material results in carbon
sequestration in soils (Batjes, 1998; Lorenz et al., 2007). On the other hand, disruption of
mechanisms responsible for OM sequestration may result in mineralization of plant material and
carbon dioxide release from soils (Janzen et al., 1998; Six, 2004). It is therefore important to
determine how OM is preserved in soils.
Characterizing the mechanisms responsible for soil organic matter (OM) preservation is
a broad topic that is further challenged by the heterogeneity of the OM structures. Interactions
between OM and clay also vary, and are thought to involve ligand exchange, van der Waals,
cation bridging, hydrophobic interactions, and electrostatic forces (Table 1.3; (Baron et al.,
1999; Feng et al., 2005; Mikutta et al., 2007). The work presented in this thesis focused on
characterizing the role of OM chemistry on its preservation and interactions with clay minerals
for the following reasons: OM structures that enter soils can be anthropogenically influenced by
vegetation shifts (Lorenz et al., 2007); and aliphatic structures, which are considered to be
chemically recalcitrant, are also enriched in clay-size fractions, which are hypothesized to
sequester older OM (Baldock et al., 1992; von Lutzow et al., 2007). The latter observation
suggests that associations of OM with clay minerals may be influenced by OM chemistry. It
should be noted that the studies presented within this thesis focused on montmorillonite clays,
with emphasis on Canadian temperate soils. Characterizing soil OM structural composition was
137
achieved by combining biomarker-gas chromatography/mass spectrometry (GC/MS) after
solvent extraction and CuO oxidation, and nuclear magnetic resonance (NMR) spectroscopy
methods. Combining information gathered using biomarker and NMR methods, has been used
by our group to acquire a better description of processes involved in soil OM dynamics (Feng et
al., 2008; 2010; 2011; Feng and Simpson, 2011; Pautler et al., 2010; Simpson et al., 2008).
Chapter 2 presents how information from solid-state 13C NMR, solution-state 1H NMR, and
diffusion edited (DE) 1H NMR can be combined to determine the structural composition of OM
associated with soil fractions. By combining these methods, three hypotheses were tested and
confirmed:
1) It was thought that aliphatic structures were sequestered in clay-size fractions, while
lignin phenols were protected through association with silt-size fractions (Baldock et al., 1992;
Christensen, 2001; Guggenberger et al., 1995; Heim and Schmidt, 2007a; von Lutzow et al.,
2007). Enrichment of >C20 lipids, aliphatic structures (likely cutin and suberin derived), and
microbial-derived peptidoglycan or chitin in clay-size fractions of three Canadian Prairie soils,
despite differences in temperature, precipitation, overlying vegetation, and carbon content
among the soils (Chapter 3), was consistent with this hypothesis. Furthermore, enrichment of
vanillyl monomers and lignin phenol dimers, which are considered to be relatively stable lignin
structures (Goni et al., 1993), indicates protection of specific lignin structures in fine fractions of
these soils. In previous studies, it was hypothesized that microbial-derived OM may be
important contributors to OM in clay-size fractions (Baldock et al., 1992; Guggenberger et al.,
1994; Kiem and Kögel-Knabner, 2003; Marshall, 1975; Mikutta et al., 2007). In addition, the
nature of aliphatic structures enriched in clay-size fractions, previously hypothesized to be
mainly derived from microbial lipids (Baldock et al., 1992), is still a topic of debate (Quenea,
2004; Wiesenberg et al., 2006; Wiesenberg et al., 2010). Using both solid- and solution-state
NMR methods; it was confirmed that the aliphatic structures, known to be enriched in clay-size
fractions, were likely a mixture of microbial- and plant-derived compounds (Chapter 3).
Therefore, this study demonstrated that interactions between specific OM structures and soil
minerals exerted a stronger control on the sequestration of specific OM structures in fine soil
fractions, regardless of overlying vegetation (Chapter 3).
2) Root tissues are thought to be a major contributor to plant-derived carbon in soils
(Johnson et al., 2007; Moretto and Distel, 2003; Rasse et al., 2005; Yanni et al., 2011), and root
138
tissues may be less degraded than leaves and stems in soils (Johnson et al., 2007; Kelleher et al.,
2006). However, changes in soil OM composition that result from amendment with different
plant tissues have not been fully explored. One study that utilized NMR suggested that the
magnitude of change in soil OM composition may depend on the plant tissue added to soil
(Kelleher et al., 2006). Therefore, a study was conducted to determine whether the
concentrations of various structures in plant tissues controlled the degree to which soil OM
composition changed as these tissues were degraded (Chapter 4). GC/MS analysis after CuO
oxidation, solid-state 13C NMR, and solution-state 1H NMR spectroscopy methods were used to
monitor changes in OM composition as maize tissues were degraded. It was found that
carbohydrate- and lignin- derived O-alkyl signals decreased to a greater extent, while aliphatic
signals increased to a greater extent in soil amended with maize stems compared to soils
amended with leaves and roots. Leaf tissues were also observed to contain greater
concentrations of aliphatic structures. Furthermore, while lignin phenol monomers and O-alkyl
structures (lignin and carbohydrates) derived from roots were degraded to a greater extent than
those from leaves; more root-derived carbon may have been converted to microbial-derived
carbon. This study suggests that degradation products from plant tissues may be important
additions to soil OM, and that it is necessary to further determine the relative contributions from
these products compared to structures from parent plant materials to soil.
3) It is believed that interactions of OM with clay minerals is a major mechanism
responsible for OM protection (Chapter 3;(Baldock et al., 1992; Christensen, 2001; Mikutta et
al., 2007; von Lutzow et al., 2007). Several studies have also suggested that specific
interactions between OM and clay minerals can be identified (Feng et al., 2005; Ghosh et al.,
2009; Mikutta et al., 2007; Wattel-Koekkoek et al., 2001). Because of the specific nature of
OM-clay mineral interactions, and observations that specific OM structures were sequestered
with clay-size fractions in soils (Chapter 3); it was necessary to determine whether OM
structural composition and conformation was also important in inhibiting the oxidation of OM
(Chapter 5). This hypothesis was tested with regards to the influence of aliphatic structures and
carbon content, on the preservation of lignin phenol structures associated with clay from
chemical (NaClO2) oxidation. OM-clay mineral complexes that were constructed in the
laboratory and mineral fractions isolated from Southern Grassland (SG) and Grassland-Forest
Transition (GFT) soils, which had different overlying vegetation, were used in this study. The
139
extent of change in lignin phenol concentration and oxidation was determined using CuO
oxidation-GC/MS analysis, while aliphatic structure concentrations were determined by
analyzing CuO oxidation and solvent extracts using GC/MS. Protection of lignin phenols
sorbed to clay minerals were attributed to coating with dodecanoic acid, and stretched
conformation of humic acid when sorbed to clay minerals. The composition of overlying
vegetation also resulted in different levels of protection of lignin phenols associated with clay-
size fractions of SG and GFT soils. Therefore, in the case of OM associated with clay minerals
and clay-size fractions, an additional level of protection from oxidation can be attributed to OM
composition and conformation, and OM-OM interactions.
The current discussion on soil OM stabilization mechanisms is centered on the role of
clay minerals and microaggregates on OM preservation, and the importance of this interaction is
supported by data on 13C stable isotope abundance and 14C isotopic decay (von Lutzow et al.,
2007). This thesis does not dispute the hypothesis that interactions of OM with soil minerals are
central to soil OM preservation (Christensen, 2001; Six et al., 2002; von Lutzow et al., 2007).
However, the studies presented in this thesis highlight the importance of considering OM
chemistry in evaluating the mechanisms responsible for soil OM preservation, and
transformation of plant- to microbial-derived OM. Overall, OM composition and interactions
between various OM structures further protect OM structures that are associated with clay-
minerals.
6.2 Future Research
6.2.1 Microbial Derived Organic Matter
The observed increase in N-acetyl signals occurred earlier and contributions of peptide
side-chains may be greater after biodegradation for 36 weeks in root-amended compared to leaf-
and stem-amended soils, which suggests that roots were transformed to microbial-derived OM
to a greater extent compared to stems and leaves (Chapter 4). Contributions of microbial-
derived OM can be verified by determining the concentration of microbial biomarkers, such as
phospholipid fatty acid methyl esters (Feng and Simpson, 2008; Feng et al., 2010; Otto et al.,
2005; Pautler et al., 2010), muramic acid (Ogawa et al., 2001), and D-amino acids (Jørgensen et
al., 2003; Jørgensen and Middelboe, 2006) to soil OM, as tissues are degraded. Biomarker
methods, such as phospholipid fatty acid methyl ester analysis, can also be combined with
140
compound-specific stable isotope analysis (Cifuentes and Salata, 2001). If the plant material
were enriched in 13C, plant carbon could be traced to their end products by calculating 13C
enrichment relative to 13C natural abundance in these compounds. With sample replicates, the
rate of mineralization and transformation of plant tissues could then be compared. This study
would help determine the relative contributions of compounds derived from plant tissues versus
compounds derived from microbial transformation products and microbial-derived OM to soil.
Such information will improve our understanding of the flow of carbon in soils (Fig. 1.1). The
efficient transformation of plant-derived carbon to microbial carbon, as opposed to
mineralization to CO2 is a potential mechanism for carbon sequestration in soils. It can be
imagined that plant-derived carbon may remain in the actively transformed soil carbon pool, if
mineralization to CO2 were kept to a minimum.
6.2.2 Preservation of Organic Matter Associated with Clay
6.2.2.1 Organic matter composition in soil fractions
In Chapters 2 and 3, changes in soil OM composition with particle size were determined
using Canadian soils, whose clay minerals were composed mainly of chlorite, vermiculite, and
illite (AM), or montmorillonite and illite (SG, Northern Grassland (NG), GFT;(Bentley, 1979).
The N-acetyl signal derived from microbial peptidoglycan or chitin was more intense in AM
compared to SG, NG, and GFT clay-size fractions (Chapters 2 and 3). This difference may be
attributed to variations in clay mineralogy or soil management and vegetation. Interactions
between OM and clay minerals were found to be influenced by the type of clay (Eusterhues et
al., 2003; Feng et al., 2005; Ghosh et al., 2009; Mikutta et al., 2007), the research described in
Chapters 2 and 3 could therefore be expanded to include soils with different clay mineralogy
(i.e. goethite contributions). In particular, using solution-state 1H NMR spectroscopy to analyze
physical fractions of various soils could provide additional insight into OM structure
sequestration in the clay-size fractions from various soils. Soils with different mineral
composition may preferentially sequester different OM structures. Such a study will determine
to what extent aliphatic and microbial-derived structures are enriched in clay-size fractions of
soils, which vary in clay mineralogy but have similar vegetation. As well, the OM composition
in the clay-size fraction of soils with similar mineralogy, but are managed differently (i.e.
grassland versus agricultural soil) may also be compared. This study will determine to what
141
extent the composition of OM associated with soil mineral fractions change upon conversion to
agriculture.
6.2.2.2 Protection of organic matter sorbed to clay minerals
The research described in Chapter 5, in which OM interactions modify the protection
afforded by clay, can be further explored by expanding the study to include clays other than
montmorillonite, and coating lignin with other compounds. Clay mineralogy influences the
sorbed OM structures, for example more aromatic and peptide structures are sorbed to
montmorillonite than kaolinite (Feng et al., 2005). Aliphatic compounds are also preferentially
sorbed to kaolinite and montmorillonite (Feng et al., 2005; Ghosh et al., 2009; Simpson et al.,
2006), while carboxylic acids are preferentially sorbed to goethite (Ghosh et al., 2009).
Therefore, sorption of OM structure followed by chemical degradation can be expanded to
include other types of clay minerals, such as goethite, kaolinite, and chlorite. These studies will
determine whether the same protection patterns are observed in lignin sorbed to clay minerals
other than montmorillonite. This research could also be expanded by coating lignin with a water
soluble compound, such as starch, instead of the hydrophobic compound (dodecanoic acid) used
here. In line with a review by Kleber et al. (2010), dodecanoic acid may have inhibited access
of the water-soluble NaClO2 oxidant to the underlying lignin. If lignin coated with starch were
more oxidized compared to lignin coated with dodecanoic acid, then hydrophobicity of the OM
coating is a pre-requisite for effective protection of OM from chemical oxidation.
Preferential association between specific OM structures, such as aliphatic compounds
(cutin, suberin, and lipids) and oxidized lignin phenols, with clay-size fractions of various soils
was observed (Chapters 2 and 3), which was consistent with other reports (Baldock et al., 1992;
Guggenberger et al., 1994; Guggenberger et al., 1995). In the OM-clay complexes prior to
chemical oxidation, there were greater concentrations of vanillyl phenols at lower OM
concentrations (Chapter 5), which further suggests specific interactions between OM structures
and clay minerals. The reasons behind this preference can be further investigated by measuring
sorption isotherms of model compounds to determine whether specific lignin structures are
preferentially sorbed to clay minerals. Preferential association between OM structures and clay
surfaces may also be verified by calculating the energy associated with OM sorption to clay
surfaces (Heinz et al., 2007; Tournassat et al., 2009).
142
The composition of OM sorbed to clay minerals, was also investigated in Chapter 5. To
further validate these results, OM composition on the clay surface can also be analyzed using
other methods. High resolution-magic angle spinning NMR spectroscopy has been used in
previous studies (Feng et al., 2005; Simpson et al., 2006), which can also be applied to organo-
clay samples such as those prepared to test hypotheses within this thesis (Chapter 5). The
lignin-clay and lignin-clay-dodecanoic acid complexes can also be analyzed using X-ray
photoelectron spectroscopy (XPS) and Time of Flight-Secondary Ion Mass Spectrometry (ToF-
SIMS). Preliminary XPS experiments indicate that for the highest lignin loading (Chapter 5,
Table 5.1, loading 4), carbon signal was less abundant than signals from the clay mineral, and
deconvolution of the carbon signal suggests contributions from a variety of carbon compounds.
The intensity of organic carbon was observed to increase, while intensity of signals from clay
(oxygen, silicon, aluminum) decreased, with increased lignin loading (Fig. 6.1). ToF-SIMS was
also used to analyze the lignin-clay, lignin-clay-dodecanoic acid complexes (Fig. 6.2). Signals
typical of lignin (m/z = 137, 151, 165) were identified based on previous studies where wood
was analyzed using ToF-SIMS (Kleen, 2005; Saito et al., 2005; Tokareva et al., 2007). The
carbon ion (m/z = 12) was more intense at higher lignin loadings (Fig. 6.2a, loadings 3 and 4),
which is consistent with higher carbon concentrations observed using elemental analysis
(Chapter 5), and XPS (Fig. 6.1). Signals attributed to lignin-derived vanillyl monomers was
more intense at higher lignin loadings in the lignin-clay-dodecanoic acid complexes (Fig. 6.2b),
but not in the lignin-clay complexes (Fig. 6.2a). The reason for differences in these observed
patterns need to be further investigated. For example, the major ions that can be attributed to
lignin used in this study should be analyzed to verify the major ions expected from this sample.
Nevertheless, the study by Feng et al. (2005), and the preliminary studies using XPS and ToF-
SIMS suggest that it is possible to expand our investigation on OM-clay mineral associations
using other sophisticated methods.
The OM-clay complexes and soil mineral fractions were reacted with a strong oxidizing
agent, NaClO2 (Chapter 5). However, in the environment, proteins catalyze OM degradation,
and proteins responsible for degradation of high molecular weight compounds, such as lignin
and cellulose, are released into the soil by microorganisms (Sylvia et al., 1999). It is therefore
important to determine how OM-clay associations modify enzyme activity. According to a
preliminary experiment using trypsin, enzyme activity decreased when this trypsin was added to
143
Figure 6.1 Elemental composition measured using X-ray Photoelectron Spectroscopy (XPS) of the lignin-clay and lignin-dodecanoic acid (C12)-clay complexes described in Chapter 5. Lignin loading increases with increasing loading number (x-axis). The relative contributions of a) oxygen, b) silicon, and c) aluminum from the clay mineral, and d) organic carbon from the sorbed OM are shown.
0
4
8
12
16
cla
y
C1
2 1 2
2 +
C1
2 3
3 +
C1
2 4
4 +
C1
2
52
54
56
58
60
cla
y
C1
2 1 2
2 +
C1
2 3
3 +
C1
2 4
4 +
C1
2
8
8.5
9
9.5
cla
y
C1
2 1 2
2 +
C1
2 3
3 +
C1
2 4
4 +
C1
2
19
21
23
25
cla
y
C1
2 1 2
2 +
C1
2 3
3 +
C1
2 4
4 +
C1
2
Lignin loading
Con
trib
utio
n to
tota
l ion
(%
)
Oxygen
Silicon
Aluminum
Organic C
a.
b.
c.
d.
144
Figure 6.2 Time of Flight-Secondary Ion Mass Spectrometry analysis (ToF-SIMS) of a) lignin-clay and b) lignin-clay-dodecanoic acid (C12)-clay complexes described in Chapter 5. Lignin loading increases with loading number (y-axis). Contributions of the ions indicated on top of the diagram are more intense with brighter colours. The relative contributions of silicon, carbon, and lignin-derived vanillyl ions (m/z = 137, 151, 165) to the total ion are also indicated.
C12
2
3
4
100 µm
0 5 10 15 20 25
0 0.2 0.4 0.6 0.8
C
Vanillyl
Si
Vanillyl
Vanillyl, C (% of Total ion)
Si (% of Total ion)
Lig
nin
lo
adin
g
1 00 µm
1
Clay
2
3
4
Vanillyl
0 5 10 15 20 25
0 0.2 0.4 0.6 0.8
C
Vanillyl
Si
Vanillyl, C (% of Total ion)
Si (% of Total ion)
Lig
nin
lo
adin
g
a.
b.
Greater ion contribution
145
clay or bovine serum albumin (BSA)-clay complexes (Table 6.1). This result was consistent
with a previous study, which found that protease activity decreased upon sorption to clay
(Kelleher et al., 2003). It also appears that enzyme activity decreased to a lesser extent when
trypsin was added to clay if another protein (BSA) was previously sorbed (Table 6.1). These
data further support the hypothesis that previously sorbed OM may block clay sorption sites
(Chapter 5). BSA in solution, and BSA complexed with clay were digested with trypsin for 24
h, and the supernatant was analyzed using HPLC coupled to a diode array detector (Fig. 6.3).
Although there seems to be more protein degraded in the absence of clay, peptides were also
released from the clay surface after digestion with trypsin (Fig. 6.3). Signals from the BSA-
clay complex were also less intense, which suggests that clay protected the protein from
degradation (Fig. 6.3). Further experiments are required to validate these results. However, this
approach may help characterize the behaviour of degrading enzymes in the environment.
Table 6.1 Enzyme activity measured as change in trypsin substrate concentrations with time (change in substrate concentration/min). Trypsin activity was measured using the supernatant, after incubating trypsin with the following mixtures: trypsin with buffer and BSA, buffer and Clay, or BSA-clay complexes for 0 or 15 minutes. Values show standard error (n = 3).
Time before measurement (min)
Sample Change in substrate concentration/min
0 Buffer + BSA (3.8 0.2) *10-2
0 Buffer + Clay (2.8 0.1) *10-4
0 BSA-clay (5.8 0.2) *10-4
15 Buffer + BSA (2.6 0.02) *10-2
15 Buffer + Clay (4.8 1.6) *10-5
15 BSA-clay (7.1 0.0) *10-5
146
Figure 6.3 Liquid chromatography of a) bovine serum albumin (BSA), b) BSA digested with trypsin in buffer, and the supernatant of c) trypsin added to montmorillonite clay, d) BSA-clay complex, and e) trypsin digest of BSA-clay complex. The arrow in each chromatogram indicates a background signal detected in all the samples.
147
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Zeier, J., Schreiber, L., 1998. Comparative investigation of primary and tertiary endodermal cell walls isolated from the roots of five monocotyledoneous species: chemical composition in relation to fine structure. Planta 206, 349-361.
Ziegler, F., Zech, W., 1989. Distribution pattern of total lipids and lipid fractions in forest humus. Zeitschrift Fur Pflanzenernahrung Und Bodenkunde 152, 287-290.
167
APPENDIX A1
SUPPLEMENTARY MATERIAL FOR CHAPTER 3
Association of Specific Organic Matter Compounds in Size Fractions of Soils under Different Environmental Controls
168
Figure A1.1 Relative contribution (%) of n-alkanes extracted from sand-, silt-, clay-size, and light fractions. Error bars represent standard error of the mean (n = 3).
0
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Light
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lativ
e C
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169
Figure A1.2 Relative contribution (%) of n-alkanol extracted from sand-, silt-, clay-size, and light fractions. Error bars represent standard error of the mean (n = 3).
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16 18 20 22 24 26 28 30 32
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170
Figure A1.3 Relative contribution (%) of organic acids extracted from sand-, silt-, clay-size and light fractions, values after the colon is
the unsaturation number. Error bars represent standard error of the mean (n = 3).
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e C
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171
APPENDIX A2
SUPPLEMENTARY MATERIAL FOR CHAPTER 4
Comparison of Soil Organic Matter Composition after Incubation with Maize Leaves, Roots, and Stems
172
Figure A2.1 Solution-state 1H NMR and diffusion edited (DE) 1H NMR spectra of maize tissue extracts in DMSO-d6. Spectra are divided to reflect the following major chemical groups: a) alkyl from waxes, cutin, suberin, or lipids; b) alkyl close to O and N, lipids, or peptides; c) O-alkyl from sugars or lignin; d) aromatic signals from lignin or proteins; and e) amide from peptides. Peptide-derived signals are labeled P1-P3.
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0Chemical Shift (ppm)
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0Chemical Shift (ppm)
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0Chemical Shift (ppm)
Leaves Stems Roots
1H NMR
DE 1H NMR
abcde
CH2
CH3
P2
01.02.03.04.05.06.07.08.09.001.02.03.04.05.06.07.08.09.001.02.03.04.05.06.07.08.09.0Chemical Shift (ppm)
abcde abcde
P1
P3 P3P3
P1 P1P2 P2
DMSO-d6 DMSO-d6 DMSO-d6
CH2CH2
CH3
CH3
