Soil Organic Matter

6 r Characterization of soil organic matter
Karolien Denef, Alain F. Plante and Johan Six
6.1 INTRODUCTION
referred to as the Walkley–Black method. Wet and dry
Soil organic matter (SOM) generally refers to the non- combustion methods are considered to yield absolute
living organic material within the soil matrix that was values of carbon, while other methods such as Walkley–
once part of, or produced by, a living organism. It is Black are calibrated against the wet and dry combustion
usually determined on soil that has passed through a 2- methods.
mm sieve, and therefore is free of coarse animal residues, Whole-soil SOM content is a dynamic property.
surface litter and large roots. Soil organic matter can be It represents the balance between plant, animal, micro-
of plant, animal or microbial origin, and consists of a bial and erosional inputs, and losses due to mineraliza-
continuum of materials in various stages of alteration tion, leaching and erosion. A posteriori measurements
due to both biotic and abiotic processes (Baldock and of changes in whole soil SOM contents after changes in
Skjemstad, 2000). Methods used in the past to estimate land-use or management practices provided the origi-
directly SOM content involved the destruction of the nal basis for estimates of SOM dynamics. However, the
organic matter by treatment with hydrogen peroxide bulk of whole soil SOM is stabilized and has turnover
(H2 O2 ) or by ignition of the soil at high temperature times measured in hundreds to thousands of years.
(Nelson and Sommers, 1996). Both of these techniques, Changes in whole soil SOC after changes in land-use or
however, are subject to significant error: oxidation of management practices are therefore difficult to detect
SOM by H2 O2 is incomplete, and some inorganic soil in the short term (< 5 years). High spatial variability
constituents decompose upon heating. poses an additional problem, and several studies have
While different elements such as C, N, P, S etc. reported the number of samples and time required to
are bound into organic compounds, we will concentrate demonstrate a minimum detectable difference in whole-
on soil organic carbon (SOC) for the purposes of this soil SOC (e.g. Garten and Wullschleger, 2000; Conen
chapter because it is the dominant element, and because et al., 2003; Smith, 2004).
of its role in the global carbon cycle. Organic carbon to The increased demand for better SOC stock assess-
SOM conversion factors for surface soils typically range ments and better predictions of the changes in SOC
from 1.72 to 2.0 g SOM g−1 C (Nelson and Sommers, stocks as a result of land use/land cover and climate
1996). Direct measurement of total soil carbon involves change has become a significant driver for the past few
the conversion of all forms of carbon to carbon diox- decades of SOM research. This has triggered large-scale
ide (CO2 ) by wet or dry combustion and subsequent and long-term measurements of SOC pools as well as
quantification of the evolved CO2 . Soil organic carbon more mechanistic process level studies (Sollins et al.,
may be determined by (1) analysis of soil samples for 2007). A large number of SOM fractionation and char-
total carbon and inorganic carbon, followed by sub- acterization techniques have been developed to gain
traction of the inorganic carbon portion from the total insight in the stabilization and destabilization mecha-
carbon, (2) determination of total carbon after destruc- nisms that underlie SOM dynamics in the short and
tion of inorganic carbon (e.g. by acid treatment) or (3) long term (Kögel-Knabner, 2000; Six et al., 2000a; von
oxidation of organic compounds by Cr2 O7 2− (Nelson Lützow et al., 2006; Kleber et al., 2007; Sollins et al.,
and Sommers, 1996). The last approach is generally 2007; von Lützow et al., 2007) and/or to identify SOM
Soil Carbon Dynamics: An Integrated Methodology, eds Werner L. Kutsch, Michael Bahn and Andreas Heinemeyer.
Published by Cambridge University Press. © Cambridge University Press 2009.
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92 K. DENEF et al.
fractions that respond more rapidly to changes in, for leaving the recalcitrant portion behind, resulting in
example, land use, management or climate than whole a relative accumulation of refractory compounds in
soil SOM and thus could serve as early indicators or carbon-depleted soils (Rühlmann, 1999; Kiem et al.,
verifiers for total SOM change (Six et al., 1998; Leifeld 2000). This approach, however, requires an undesirably
and Kögel-Knabner, 2005). long time to distinguish labile from recalcitrant SOC
The intention of this chapter is to give a general and is limited to systems with negligible organic inputs.
overview of existing SOM fractionation and characteri- One of the best means by which biological fractiona-
zation techniques, to discuss their shortcomings and to tion can be assessed is through the use of isotopes (13 C,
14
suggest directions for future research by giving some C, 15 N) in labelling experiments, 13 C natural abun-
examples of promising new techniques. dance or 14 C radiocarbon dating experiments. Carbon-
13 natural abundance studies utilize known and dated
vegetation changes (Cerri et al., 1985; Balesdent et al.,
6.2 OVERVIEW OF TECHNIQUES TO
1987; Balesdent and Mariotti, 1996). Examples of these
FRACTIONATE AND CHARACTERIZE
vegetation changes include shifts from C4 grasslands
SOIL ORGANIC MATTER
to C3 crops, or cultivation of C4 corn in temperate
Soil organic matter consists of many classes of com- areas dominated by C3 native vegetation. While dif-
pounds with different biochemical properties and vary- ferent plant parts and compounds have different 13 C to
12
ing degrees of association with the mineral matrix, C ratios, and while microbial decomposition of these
which differentially contribute to the overall dynam- materials results in some fractionation, SOC generally
ics of total SOM. Most models of SOM dynamics, retains the integrated signature of its parent vegeta-
however, do not explicitly take into account the bio- tion (Balesdent and Mariotti, 1996). The progressive
logical, physical or chemical mechanisms that act to change in the isotopic signature allows a quantitative
stabilize SOM. Instead, they divide SOM into sev- estimate of the turnover time and size of the SOC com-
eral kinetic compartments (e.g. active, slow and passive, ponents. Radiocarbon (14 C) measurements can provide
according to the CENTURY model) based on assumed estimates of much longer turnover times (Wang et al.,
turnover times, and define their dynamics in terms of 1996; Paul et al., 1997; Trumbore, 2000), but estimates
transfers from one compartment to another (e.g. Par- must take into account recent 14 C-bomb inputs. While
ton et al., 1987, 1988; Jenkinson, 1990). These models there is evidence from the radiocarbon dating of micro-
could greatly benefit from the integration of measurable bial respiration to suggest that microbial biomass will
and separable SOM fractions defined by different stabi- mineralize the labile portion first (e.g. Trumbore, 2000),
lization mechanisms and degrees of stabilization (Chris- other studies examining isotopic signatures of respired
tensen, 1996b; Elliott et al., 1996; Six and Jastrow, 2002; CO2 and phospholipid fatty acids (PLFA) suggest that
Smith et al., 2002). Numerous fractionation schemes a component of the microbial community is capable
have been developed with the purpose of separating of using substrates with older carbon (e.g. Waldrop
‘functional’ SOM fractions that are more homogeneous and Firestone, 2004; Rethemeyer et al., 2005; Kramer
in terms of their biochemical properties and turnover and Gleixner, 2006). The idea of active recycling of
times. Their relevance for clarifying SOM stabilization SOM carbon by soil microbes, as suggested by Gleixner
and destabilization mechanisms or for SOM modelling et al. (2002), could form a possible explanation for the
purposes has been recently reviewed by von Lützow older isotopic signature observed in biochemically labile
et al. (2007). SOM compounds (Gleixner et al., 1999, 2002; Derrien
et al., 2006) and respired CO2 and microbial PLFA (see
references above). Shifts from younger to older carbon
6.2.1 Soil organic matter fractionation
utilization could also be induced by changes in environ-
6.2.1.1 Biological fractionation mental conditions (e.g. temperature, N additions) and
Biological fractionation separates SOM into labile and have been ascribed to changes in microbial commu-
recalcitrant portions through microbial mineraliza- nity composition and accompanying changes in enzyme
tion. The underlying assumption is that the micro- activities (Dioumaeva et al., 2002; Waldrop and Fire-
bial biomass will mineralize the labile portion first, stone, 2004).
Characterization of soil organic matter 93
Bulk soil (intact)

1. Dispersion
2. Sieving
3. Sedimentation Density fractionation Sieving
Increasing density
Light fraction (LF) Organo-mineral SOM fractions
> 250 µm: macroaggregates

1. Density
Density free light fraction POM Microaggregate isolator
50 (or 53)–2000 µm: sand-sized
(POM) < 250 µm: microaggregates
sand occluded POM (in aggregates)

2–50 (or 53) µm: silt-sized 2. Dispersion, sieving
< 2 µm: clay-sized
and density
Primary organo-mineral Secondary organo-mineral

Uncomplexed SOM
complexes complexes
Figure 6.1 Overview of the most commonly used physical fractionation methods to isolate uncomplexed SOM based on size (POM)
or density (LF) from mineral-bound organic matter in primary particle and aggregate size fractions.
6.2.1.2 Physical fractionation particles. However, it is difficult to isolate either com-

The physical organization of a soil controls SOM acces- pletely mineral-free SOM or SOM-free minerals, par-
sibility to soil microbes and could as such determine ticularly in fine-textured soils. Assuming a density of
the biochemical characteristics and stability of SOM. 1.4 g cm−3 for the organic phase (Adams, 1973; Mayer
Most physical fractionation techniques therefore aim at et al., 2004) and 2.6 g cm−3 for the mineral phase,
isolating homogeneous fractions (in terms of proper- Chenu and Plante (2006) calculated the SOM contents
ties and turnover) that represent functional SOM pools of density fractions using a simple mixing model and
that are formed by a specific stabilization mechanism found that a density fraction of < 1.6 g cm−3 would
(e.g. recalcitrance, spatial inaccessibility and organo- contain > 73% SOM and a density fraction of > 2.2
mineral interactions (Sollins et al., 1996; Six and Jas- g cm−3 would contain < 21% SOM. The most pop-
trow, 2002; von Lützow et al., 2006). The distribution of ular density reagent is sodium polytungstate (SPT,
SOM among physical fractions in the soil is generally Na6 (H2 W12 O40 )), which is non-toxic, expensive but
assessed by applying various degrees of disruption to can be regenerated (Six et al., 1999b), but also solu-
the soil, followed by the separation of physical fractions bilizes a significant and functionally distinct portion
based on size or density (Elliott and Cambardella, 1991; of SOM (Crow et al., 2007). Microscopic investiga-
Christensen, 1992; Golchin et al., 1994; Six et al., 1998, tions and chemical characterization indicate a domi-
2000b; Christensen, 2001; Sollins et al., 2006) (Fig. 6.1). nant plant origin of uncomplexed SOM (Oades et al.,
This allows the separation of uncomplexed SOM and 1987; Golchin et al., 1994). In comparison to whole
of various primary (i.e. sand, silt, clay) or secondary soil, uncomplexed SOM is enriched in carbohydrates
(i.e. soil aggregates) organo-mineral fractions differ- (Dalal and Henry, 1988) and has a higher lignin con-
ing in size or density. Physical fractionation of SOM tent and C/N ratio (Gregorich et al., 2006). Uncom-
has revealed that soil size and density separates differ plexed SOM fractions often respond more rapidly to
in chemistry, origin, rates of turnover and dynamics land-use or soil-management changes than mineral-
(Puget et al., 2000; Liao et al., 2006a, 2006b; Sollins associated SOM fractions (e.g. Cambardella and Elliott,
et al., 2006). 1992; Gregorich and Janzen, 1996; Six et al., 1998;
Uncomplexed SOM has been defined by Gregorich Gerzabek et al., 2001; Chan et al., 2002). However,
et al. (2006) as SOM that is not bound to mineral several studies have suggested that more stabilized
94 K. DENEF et al.
mineral-associated SOM fractions are better indicators upon mechanical or chemical aggregate disruption and
for soil quality changes upon changes in land use or sieving (Fig. 6.1). Chemical and isotopic comparison
management despite their longer turnover times com- of the free LF and aggregate-occluded POM frac-
pared to uncomplexed organic materials (Jastrow, 1996; tions generally suggest that aggregate-occluded POM
Stemmer et al., 1999; Chenu et al., 2001; Denef et al., is more microbially processed and relatively more sta-
2004; Leifeld and Kögel-Knabner, 2005; Denef et al., bilized than the free LF (Golchin et al., 1994; Helfrich
2007). Related to this, controversy also exists about the et al., 2006; Liao et al., 2006b). However, the outcome of
turnover rate of uncomplexed SOM. Skjemstad et al. such compositional comparisons are greatly influenced
(1990) reported surprisingly long residence times for by the type of fractionation method used (Kölbl et al.,
density-separated uncomplexed SOM (i.e. light frac- 2005).
tion, LF). In soils converted to pasture, they found that Separation of organic matter in primary particle size
60% of the LF was derived from old rainforest veg- fractions is usually obtained by an assumed ‘complete’
etation 35 years after conversion to pasture, and 20% dispersion of the soil, followed by sieving and sedi-
of the LF was forest derived after 83 years. Different mentation (Christensen, 1996a) (Fig. 6.1). For the pur-
factors such as vegetation type, climate, soil texture, pose of primary particle fractionation, complete dis-
soil mineralogy, faunal activity, as well as methodol- persion is usually defined by or calibrated against soil
ogy (e.g. choice of separation solution density) can sig- texture, even though silt- and clay-sized microaggre-
nificantly influence the amount, quality and turnover gates may persist after the dispersion treatment (e.g.
of uncomplexed SOM (Skjemstad and Dalal, 1987). North, 1976; Balabane and Plante, 2004). Chemical
Future work should consider the composition and sta- characterization of SOM in primary particle fractions
bility of physical SOM fractions in different soil types has demonstrated an increased contribution of plant-
and environments following standardized fractionation (vs. microbial-) derived substances from clay- to silt-
procedures. to sand-sized organo-mineral fractions, with signifi-
There also exists confusion about the nomencla- cant enrichment of the silt-sized fraction in aromatic
ture of uncomplexed SOM. Light fraction and par- compounds (Baldock et al., 1992; Guggenberger et al.,
ticulate organic matter (POM) are the two most com- 1995; Christensen, 1996a). In addition, a decrease in
mon isolated fractions to assess uncomplexed SOM. lignin content (Amelung et al., 1999) and C/N ratio of
As Gregorich et al. (2006) suggested, LF is a fraction organo-mineral complexes (Guggenberger et al., 1995)
of uncomplexed SOM isolated by density alone (using with decreasing particle size have been observed, indi-
dense liquids typically in the range of 1.6–2.0 g cm−3 ), cating an enhanced degree of SOM humification or a
while POM is a fraction isolated by size alone (typ- higher microbial processing from sand- to silt- to clay-
ically > 50 or 53 μm). The uncomplexed nature of sized complexes. These observations suggest that, as
LF SOM obtained at densities < 1.6–2.0 g cm−3 was plant residues are decomposed while residing in the
supported by Sollins et al. (2006) who showed a wide sand-sized fraction, newly formed microbial products
range of C/N ratios between 10 and 40 for the < 2.0 become associated with clay-sized complexes, and more
g cm−3 fractions, whereas a more consistent C/N ratio recalcitrant substances end up in the silt-sized fraction.
of 10 was altered for fractions with a density of > 2.0 g It is generally believed that carbon is more stabilized
cm−3 . Depending on the fractionation method, LF and in finer particle size fractions, due to adsorption and
POM fractions can greatly differ in their biochemical chemical binding on clay mineral surfaces as well as
properties (Gregorich et al., 2006). When LF or POM is aggregate formation (Baldock and Skjemstad, 2000).
separated after chemical or mechanical disruption of the Evidence for this comes from carbon isotope analyses
soil, it includes uncomplexed SOM that was occluded in of particle-size fractions generally showing increasing
aggregates as well as uncomplexed SOM located outside mean residence time with decreasing particle size (e.g.
aggregates. Light fraction obtained by density fraction- Anderson and Paul, 1984; Balesdent, 1996). However,
ation on intact or minimal disrupted soil consists only when comparing mean residence times of soil organic
of non-aggregate-occluded light fraction (i.e. free LF) carbon across particle size fractions from several stud-
(Christensen, 2001). After removal of the free LF, the ies, von Lützow et al. (2007) found lower turnover times
aggregate-occluded POM fraction is usually obtained for the fine (< 0.2 μm) vs. coarse (0.2–2 μm) clay
fraction, which could be attributed to an incomplete greatest carbon enrichment in the fractions of interme-
disaggregation of coarse clay-sized microaggregates diate magnetic susceptibility, whereas both the highly
during dispersion (Guggenberger and Zech, 1999; magnetic fraction of predominantly well crystallized
Chenu and Plante, 2006). Another plausible explana- Fe and the non-magnetic fraction were carbon poor
tion, however, could be found in differences in miner- (Shang and Tiessen, 1998). The authors attributed this
alogical properties. Soil mineral properties (e.g. mineral to the fact that highly crystalline Fe oxides have a much
assemblage, mineral charge, effective cation exchange lower sorption capacity to bind with SOM compared
capacity (ECEC), specific or reactive surface area, poly- to poor-crystalline Fe- and Al-oxides (Turchenek and
valent cations, organometal complexes, crystallinity and Oades, 1979; Duiker et al., 2003). Mikutta et al. (2005)
type of oxides) have been found to have a signifi- attempted to chemically characterize poorly crystalline
cant impact on the amount, chemical composition and minerals by selective dissolution with acid oxalate and
turnover of SOC in clay fractions due to different dithionite–citrate, and physically characterized them by
SOM–mineral binding mechanisms for different clay surface area determinations (BET–N2 ). Their results
minerals (Wattel-Koekkoek et al., 2003; Kleber et al., supported HGMS findings that the capacity of the
2004; Schöning et al., 2005). In many soils, the type of mineral matrix to protect SOM against decomposi-
soil mineral assemblage appears to be even more impor- tion is largely controlled by the content of poorly crys-
tant for SOC storage than total clay content (Saggar talline minerals. Chemical and isotopic characterization
et al., 1996; Torn et al., 1997; Percival et al., 2000). of HGMS fractions revealed a greater C/N ratio and a
Residence times of organo-mineral associations not vegetation-resembling 13 C signature of the most mag-
only depend on mineralogy, but also greatly on soil type, netic (well crystallized) clay fraction, suggesting that the
vegetation, land use and environmental conditions as SOM in this fraction is derived mostly from plant mate-
well as method of age determination (13 C versus 14 C) rials and is relatively untransformed by microbial pro-
and SOM fractionation. Carbon turnover times esti- cesses (Shang and Tiessen, 2000). The more enriched
mated by 13 C natural abundance are generally an order δ 13 C values and lower C/N ratio, characterizing the
of magnitude smaller than those estimated by radiocar- non-magnetic clay fraction, suggested a more decom-
bon dating because of the different time scales mea- posed fraction of mainly microbial origin.
sured by the two different methods (Paul et al., 2001; Soil organic matter associated with secondary
Six and Jastrow, 2002). Comparing 13 C and 14 C mean organo-mineral complexes or soil aggregate fractions
residence times of particle size fractions across different encompass primary particle-associated SOM, uncom-
ecosystems, von Lützow et al. (2007) found wide and plexed SOM, micro-organisms (bacteria, fungal hyphae
overlapping ranges of turnover times of SOM in differ- and micro-fauna) and fine roots. Aggregate size sep-
ent particle size fractions. Therefore, it seems absolute aration methods have been utilized in many stud-
values for turnover times of SOM fractions cannot be ies to identify mechanisms controlling SOM stor-
compared among studies. age as well as changes in C and N pools associated
High gradient magnetic separation (HGMS) has with land-use/land-cover changes (Elliott, 1986; Beare
been used as a tool to separate Fe oxides and Fe-rich et al., 1994b; Cambardella and Elliott, 1994; Six et al.,
clay minerals from soil clays (Schulze and Dixon, 1979; 1998, 2000b, 2002; Liao et al., 2006a), following the
Jaynes and Bigham, 1986). This technique has been concept of aggregate hierarchy as proposed by Tis-
applied in tropical SOM turnover studies to investi- dall and Oades (1982). This concept assumes a hier-
gate why SOM appears to be less stable (i.e. faster archical order of aggregates with different types of
turnover rates) under tropical than under temperate organic binding agents acting at the different stages:
conditions (Jenkinson and Ayanaba, 1977; Trumbore, primary organo-mineral fractions are bound together
1993; Tiessen et al., 1994). In tropical soils, SOM is into microaggregates (< 250 μm) by persistent materi-
mainly stabilized through complexation with Fe and Al als of mainly microbial origin. Microaggregates are held
oxyhydroxides (Parfitt et al., 1997; Schwertmann et al., together within macroaggregates (> 250 μm) through
2005) and interactions of clay particles with Fe and Al the binding action of roots and fungal hyphae (tempo-
ions (Theng, 1976). Results from studies using HGMS rary) and readily decomposable plant- and microbial-
(Hughes, 1982; Shang and Tiessen, 1998) revealed the derived polysaccharides (transient). According to this
96 K. DENEF et al.
concept, macroaggregates are more enriched in car- isolated by an isolator device (Six et al., 2000b) that
bon but at the same time more transient and sensitive allows complete break-up of macroaggregates while
to physical disturbance compared to microaggregates minimizing the breakdown of the released microaggre-
(Tisdall and Oades, 1982). Support for aggregate hier- gates (Fig. 6.1). Research should be further directed at
archy comes from studies reporting greater concentra- the chemical (Simpson et al., 2004), microbial (Mum-
tions of organic carbon and biomass carbon (Elliott, mey et al., 2006a, 2006b), and isotopic characterization
1986; Singh and Singh, 1995; Jastrow et al., 1996; Six (Six et al., 2000b; Denef et al., 2001b) of this microag-
et al., 2000a; Ashman et al., 2003), as well as POM gregate fraction among other potential diagnostic SOM
contents (Cambardella and Elliott, 1993; Beare et al., fractions to better understand their dynamics as well
1994b; Puget et al., 1996) in macroaggregates compared as their fundamental importance to soil structure and
to microaggregates. Moreover, relative to SOM asso- function.
ciated with smaller sized aggregates, macroaggregate-
associated SOM has been generally found to be (1) more 6.2.1.3 Chemical fractionation
labile, as supported by direct relationships between Similar to physical fractionation, the goal of chemical
C/N ratios of aggregate-SOM and aggregate size and fractionation of SOC is to isolate pools of carbon with
mineralization studies (Elliott, 1986; Beare et al., 1994a; similar properties and dynamics, and sometimes to iso-
Aoyama et al., 1999; Gregorich et al., 2003); (2) less late an organic fraction from the background mineral
decomposed, as demonstrated by decreasing plant- soil material. Depending on the carbon fraction of inter-
derived and increasing microbial-derived components est, a chemical treatment will be selected that removes
with decreasing aggregate size (Monreal et al., 1997) more or less SOC. Conversely, chemical treatments
and (3) younger, as demonstrated by 13 C natural abun- such as hydrofluoric acid (HF) have been designed
dance (e.g. Skjemstad et al., 1990; Puget et al., 1995; to dissolve the mineral portion of the sample, leaving
Angers and Giroux, 1996; Jastrow et al., 1996; Six et al., behind the SOC (Skjemstad et al., 1994; Schmidt et al.,
1999a) as well as 13 C tracer studies (Angers et al., 1997; 1997). These latter treatments are generally used to
Denef et al., 2001a). While aggregate hierarchy is gen- improve spectroscopic analyses of whole-soil samples
erally expressed in fine textured soils dominated by 2:1 rather than a means to fractionate SOM.
minerals where SOM is the primary aggregate bind- The simplest chemical fractionation of SOM uses
ing agent, this is not the case in oxide-dominated soils cold distilled or deionized water to isolate dissolved
(Oades and Waters, 1991; Feller et al., 1996; Six et al., organic matter (DOM) as a pool of readily decompos-
2000a; Zotarelli et al., 2005; de Azevedo and Schulze, able carbon (Chantigny, 2003). Hot-water extraction
2007). was first proposed by Keeney and Bremner (1966) to
Many methods have combined different physical determine easily available soil N, and has since been
fractionation methods in an attempt to target spatially applied to isolate labile C (Haynes, 2005). Hot-water
explicit SOM fractions where carbon stabilization is extractable SOC consists largely of labile carbohydrates
most pronounced or land-use, management or envi- and amino acids (Leinweber et al., 1995), and is primar-
ronmental forces have the greatest impact (Six et al., ily of microbial origin (Sparling et al., 1998). A number
2000b; Del Galdo et al., 2003; Denef et al., 2004; of other chemical fractionations have been devised to
Leifeld and Kögel-Knabner, 2005; Del Galdo et al., isolate labile pools of SOC. Blair et al. (1995) suggested
2006). For example, the use of complex fractiona- that a fraction of organic carbon oxidizable with potas-
tion schemes by Six and colleagues have demonstrated sium permanganate (KMnO4 ) was a useful index of
across widely varying ecosystems, soil types and envi- labile SOC. However, Tirol-Padre and Ladha (2004)
ronments the importance of macroaggregate-occluded found better correlations of permanganate oxidizable
microaggregates (53–250 μm) as long-term stabiliza- carbon with total SOC than with water-soluble carbon
tion sites for SOM (Six et al., 2000b; Del Galdo et al., and microbial biomass carbon. Skjemstad et al. (2006)
2003; Denef et al., 2004; Kong et al., 2005) and the also compared the method to POM and found that
potential of this aggregate fraction to serve as an early permanganate oxidizable carbon was relatively insen-
indicator for total SOC stock changes upon land-use sitive to rapid SOC gains, which is contrary to the
change (Denef et al., 2007). These microaggregates were definition of a labile carbon pool. Both studies found
SOM (humus)
extract with alkali
(insoluble) (soluble)
Humin
treat with acid
Hymatomelanic extract with (precipitated) (not precipitated)

acid alcohol
Humic acid Fulvic acid
redissolve in base
add electrolyte XAD-8
(pigments adsorbed)
elute base
desalt
(precipitated) (not precipitated)
Grey humic acid Brown humic acid Generic fulvic acid
Figure 6.2 Chemical fractionation scheme of soil organic matter based on solubility characteristics (from Stevenson, 1994).
that the method is sensitive to the presence of lignin, humin, and other fractions including hymatomelanic
and concluded that caution should be exercised when acid and grey and brown humic acids (Stevenson, 1994;
attempting to relate permanganate oxidizable carbon to Fig. 6.2). An enormous literature exists on the fraction-
labile SOC. ation and analysis of humic substances from soils, and
For much of the history of its study, SOM has the reader is directed to several in-depth reviews (Aiken
been frequently synonymous with ‘humic substances’. et al., 1985; Hayes et al., 1989; Stevenson, 1994). The
Humic substances conventionally defined as ‘a series operational definition of SOM fractions based on sol-
of relatively high-molecular weight, brown to black ubility characteristics has led to significant criticism,
coloured substances formed by secondary synthesis and many have abandoned the methods and termi-
reactions’ (Stevenson, 1994). They are essentially a cat- nology altogether. Stevenson (1994) identified several
egory of organic substance that cannot be classified into problems in the fractionation procedure (including the
the normal categories such as proteins, lipids, polysac- dissolution of fresh organic debris). The fractionation
charides etc. This vague definition has often led to is generally not quantitative and it is nearly impossi-
defining these materials operationally in terms of the ble to devise a scheme that separates humic from non-
methods used to extract them from soils (MacCarthy, humic substances because both share some of the same
2001), and decades of research has shown much of this chemical functional groups, and because it is difficult
characterization to be impractical or incorrect. Humic to identify the point in the decay process at which the
substances have traditionally been isolated using aque- transition from non-humic to humic substance occurs
ous base or neutral salt solutions, and combinations (MacCarthy, 2001). Moreover, the usefulness of the
of aqueous base (e.g. 0.5N NaOH) and pyrophosphate obtained fractions as kinetic fractions also appears to
(e.g. 0.1M Na4 P2 O7 ) (see review by Hayes, 2006). Soil be rather limited. While several researchers have found
organic matter fractionation operationally based on sol- that the age or mean residence times for each of the
ubility in acid and base was first introduced by Spren- fractions based on 14 C-dating were significantly dif-
gel (1837) and evolved through the mid-nineteenth ferent (e.g. Campbell et al., 1967; Anderson and Paul,
century through the work of Berzelius (1839) and 1984), Balesdent (1996) found similar kinetics of enrich-
Mulder (1861). The classic fractionation scheme con- ment in new 13 C and concluded that the fractionation
sists of humic acid, fulvic acid, the non-extractable scheme was not useful in investigating carbon turnover.
98 K. DENEF et al.
Lastly, difficulties in characterizing humic substances decreasing total carbon. Generally, these chemical treat-
(described later) have also demonstrated that the ments remove a greater proportion of SOM than acid
approach has limited value for predicting SOM hydrolysis, and SOC in the resistant residues is smaller
behaviour. than what is generally assumed to exist based on
Chemical fractionation of SOM to isolate stable turnover times calculated using common models. Zim-
or recalcitrant SOC is frequently performed using merman et al. (2007a) compared NaOCl treatment to
acid hydrolysis. A common acid hydrolysis technique acid hydrolysis and found that NaOCl removed more
involves refluxing whole-soil or isolated physical frac- SOC (63 to 91%) than did acid hydrolysis (35 to 66%).
tions in 6M HCl at 116 ◦ C for 18 hours, though other They also found that 14 C activities of NaOCl-resistant
acids and temperatures have been used. The hydrolysis SOC were lower than those of hydrolysis-resistant
reaction, through the process of protonation and hydra- SOC, suggesting that SOM isolated by NaOCl treat-
tion, solubilizes compounds with O- and N-containing ment is older than that obtained by acid hydrolysis. The
functional side-groups (Barriuso et al., 1987). Fatty authors concluded that oxidation with NaOCl is better
acids, proteins and polysaccharides are susceptible to than acid hydrolysis with HCl to obtain an operationally
acid hydrolysis treatment, whereas long-chain alkyls, defined resistant fraction of SOM.
waxes, lignin and other aromatics are resistant to hydrol-
ysis (Martel and Paul, 1974; Schnitzer and Preston, 6.2.1.4 Black carbon fractionation and
1983; Paul et al., 2000). Studies have found that the quantification
non-hydrolyzable fraction from a wide range of samples One SOC fraction that has received an increasing
represents 35–65% of the total soil carbon (Paul et al., amount of attention is generally referred to as ‘black
2006), and is generally 1300–1800 years older than total carbon’ (BC). Black carbon exists in soil as the prod-
soil carbon (Leavitt et al., 1996; Paul et al., 1997, 2001). uct of vegetation fires and the incomplete combustion
The acid hydrolysis method has been broadly adopted in of fossil fuels. This pool of SOM is particularly dif-
studies of SOM dynamics and coupled with 14 C-dating ficult to isolate because it consists of a continuum of
to estimate the size and turnover rate of the stable SOM combustion products ranging from slightly charred and
pool in models (Leavitt et al., 1996; Falloon et al., 1998; degradable biomass to highly condensed and refractory
Paul et al., 2006). Recent criticism of the technique has soot (Hedges et al., 2000), which results in physical and
argued that under land-use change, non-hydrolyzable chemical heterogeneity. This heterogeneity results in
carbon in soils may be lost or gained during relatively the occurrence of BC in both coarse and fine size frac-
short periods of time relative to the age of the resis- tions, as well as in both light and heavy fractions. Black
tant carbon pool, which is inconsistent with its assumed carbon is distinguished from the rest of SOM by its
recalcitrant chemical nature (Paul et al., 2006; Plante presumed biochemical recalcitrance and long turnover
et al., 2006). times, although several studies have demonstrated that
Other chemicals have been used to separate stable BC is less recalcitrant than commonly assumed (see
or recalcitrant SOM from labile fractions. These chem- Knicker, 2007 for a recent review). Techniques for
ical treatments include hydrogen peroxide (H2 O2 ), the identification and quantification of BC fall into six
sodium hypochlorite (NaOCl) and disodium perox- general classes: microscopic, optical, thermal, chemi-
odisulphate (Na2 S2 O8 ). Some of these treatments were cal, spectroscopic and molecular marker (Schmidt and
originally designed to remove as much SOC as possible Noack, 2000; Masiello, 2004). Additionally, techniques
to improve the characterization of the mineral fraction exist that blend these six measurement types. Micro-
(Mikutta et al., 2005), though none are 100% effec- scopic techniques measure the number of charcoal
tive. The SOM remaining in treated residues has been pieces identifiable under an optical microscope, while
regarded as a resistant fraction. Theng et al. (1992) optical techniques measure the ‘blackness’ of a sample.
and Righi et al. (1995) proposed that H2 O2 -resistant Thermal methods measure BC remaining after oxida-
SOM represented a resistant fraction protected in inter- tion upon heating, while chemical techniques measure
clay layers. However, Plante et al. (2004) found that, BC remaining after chemical oxidation. Spectroscopic
similar to acid hydrolysis, the proportion of H2 O2 - techniques target NMR regions characteristic of com-
resistant clay-associated carbon did not increase with bustion products and estimate total BC concentration
based on the strength of these bands after removal of its molecular composition and complexity, lignin is
non-BC SOM; while molecular marker techniques mea- often considered to be a recalcitrant biomolecule, rela-
sure the concentration of a particular compound or class tively resistant to decomposition. However, its contri-
of compounds associated with BC. Clearly, each of these bution to the stable (passive) SOM pool is under debate
techniques measures a different region within the com- (Heim and Schmidt, 2007). The most widely used
bustion continuum, causing significant over- or under- technique for soil lignin analysis is the alkaline cupric
estimations. In a study comparing six BC quantification oxide (CuO) oxidation method (Hedges and Ertel,
methods on eight soils, Schmidt et al. (2001) found val- 1982; Kögel, 1986), which releases several lignin phenol
ues ranging over two orders of magnitude, differences monomers with benzoyl- (H), vanillyl- (V), syringyl-
between methods varying by factors of 14 to 571 and (S) and cinnamyl- (C) structures and acid, aldehyde
no systematic methodological biases. Further evidence and ketone side-chains (Kögel and Bochter, 1985). The
of a continuum of BC in terms of thermal stability was sum of V-, S- and C- phenolic CuO oxidation products
provided by Leifeld (2007) using oxidative differential (VSC) provides a quantitative measure of the lignin
scanning calorimetry, a dynamic thermal method that content in the soil, while the mass ratios of acids to
scans the reactivity (thermal stability) of a sample over aldehydes of the V- and S-units indicate the state of
a wide temperature range. Spectroscopic and molecular oxidative degradation of the lignin phenols and allows
marker techniques (e.g. Brodowski et al., 2005b) hold an assessment of the humification state of the SOM
great potential to quantify the widest range of BC mate- (Hedges and Ertel, 1982; Kögel, 1986; Hedges et al.,
rials because they focus on the chemical signature of 1988; Hatcher et al., 1995). Another commonly used
burning. Currently, the most frequently used means of technique for the analysis of lignin involves the ther-
fractionating and quantifying BC is using a preparatory mochemolysis with tetramethylammonium hydroxide
oxidation step followed by quantification of the aromatic (TMAH) (Hatcher et al., 1995; Nierop et al., 2001).
region by 13 C-NMR (e.g. Simpson and Hatcher, 2004). With TMAH thermochemolysis, lignin monomers are
obtained analogous to those obtained with the CuO pro-
cedure, revealing similar information about the content
6.2.2 Soil organic matter characterization
and state of degradation of lignin. However, TMAH
In addition to attempts to separate SOM into functional thermochemolysis is more sensitive in detecting lignin
fractions by physical or chemical fractionation, a great degradation than the CuO method. It also is less time
deal of effort has been put into characterizing the chem- consuming and thus more suitable for routine analy-
ical composition of SOM, either following extraction ses (Hatcher et al., 1995). A major drawback of the
from the soil matrix or in situ. TMAH thermochemolysis technique, however, is its
inability to determine if an aromatic methoxyl group
6.2.2.1 Compound-specific characterization produced by the procedure was originally present as a
Within a chemical SOM extract, molecular compounds hydroxyl or a methoxyl functionality, because the pro-
can be characterized and quantified by online coupling cedure involves methylation of phenols while leaving
of gas (GC) or liquid (LC) chromatographs to con- the original methoxyl groups unaltered (Filley, 2003).
ventional GC or LC detectors (e.g. flame ionization Using 13 C-labelled TMAH and structural mass spec-
(FID), refractive index) or mass-spectrometers (MS). trometry analysis, Filley et al. (1999) were able to dis-
Such compound-specific analyses can provide informa- tinguish between original methoxyl groups on non-
tion about the origin (e.g. plant vs. microbial derived, degraded lignin and those analytically added by TMAH
or fungal vs. bacterial) or biochemical composition (e.g. (i.e. 13 C-labelled methyl group) on lignin residues that
carbohydrates, lignin, lipids, proteins) of the SOM frac- had undergone demethylation during microbial decay.
tion, and could help us better understand the underly- It therefore provides a rapid and sensitive tool for track-
ing mechanisms of carbon stabilization (Lorenz et al., ing microbial modifications of lignin in different ter-
2007). restrial environments (Filley et al., 2000). Moreover,
13
Lignin, for example, is a structurally complex C-TMAH thermochemolysis can distinguish methy-
biopolymer exclusively produced by vascular plants, lated compounds from non-lignin sources (such as tan-
and used as tracer for plant origin of SOM. Due to nins or other phenolics), as opposed to the conventional
100 K. DENEF et al.
(unlabelled) TMAH method, where these compounds and plant-derived sugars to the total extracted carbohy-
are usually ascribed to a lignin source (Filley et al., 2006; drate fraction varies depending on the specific extrac-
Nierop and Filley, 2007). tion method adopted, in particular the type of extrac-
Carbohydrates form another important and widely tion solvent used (Cheshire, 1979; Haynes and Francis,
studied class of SOM compounds of both plant and 1993; Ball et al., 1996; Puget et al., 1999).
microbial origin. Although carbohydrates are consid- Amino sugars comprise only about 1–5% of the
ered to be among the most easily biodegradable sub- total SOC pool, but are reliable molecular markers
strates (Cheshire, 1979), their concentration in soils for total (living and decomposing) microbial biomass
is substantial, suggesting physical or chemical stabi- as they are synthesized by micro-organisms but not
lization or active recycling of this carbon pool by soil by higher plants (Benzing-Purdie, 1984; Zhang and
microbes (Gleixner et al., 1999, 2002; Derrien et al., Amelung, 1996; Chantigny et al., 1997). The concen-
2006). The determination of carbohydrates is com- tration of amino sugars is therefore routinely applied to
monly carried out in three steps: (1) extraction of car- indicate microbial contributions to SOM (Zhang et al.,
bohydrates from samples by hot-water extraction (e.g. 1998; Amelung, 2001; Solomon et al., 2001; Turrion
Ball et al., 1996) or acid hydrolysis with sulphuric acid et al., 2002; Glaser et al., 2004). Amino sugars are
(Cheshire and Mundie, 1966; Oades et al., 1970; Larre- usually determined according to the method of Zhang
Larrouy and Feller, 2001) or 4M trifluoroacetic acid and Amelung (1996): (1) extraction by acid hydroly-
(TFA) (Amelung et al., 1996; Rumpel and Dignac, sis with 6N hydrochloric acid, followed by purification
2006) followed by purification, (2) derivatization by and neutralization, (2) derivatization by the aldonon-
either the trimethylsilyl, alditol acetate, aldononitrile itrile (Guerrant and Moss, 1984) or methyl boronic
acetate, trifluoroacetate or O-methyloxime acetate pro- acid (Gross and Glaser, 2004) procedure and (3) GC
cedure (see reviews by Guerrant and Moss, 1984; Black analysis of the monomers. Comparisons of the concen-
and Fox, 1996) or by methyl boronic acid derivatization trations of glucosamine, galactosamine, mannosamine
(Gross and Glaser, 2004; Bock et al., 2007), and sub- and muramic acid, in the soil with those in the soil
sequent (3) GC analysis of the monomers. Compound- microbial biomass showed that amino sugars are mainly
specific characterization of carbohydrate SOM differ- derived from microbial necromass and become stabi-
entiates between microbial-derived sugars (dominated lized in the soil (Glaser et al., 2004). Due to the pre-
by hexoses) and plant-derived sugars (dominated by dominant fungal origin of glucosamine compared to
pentoses) (Oades, 1984; Moers et al., 1990). Different the predominant and even exclusive bacterial origin of
ratios of hexoses to pentoses have been used to indi- galactosamine and muramic acid, respectively (Parsons,
cate the origin of soil carbohydrates (Oades, 1984; Hu 1981; Amelung, 2001), the ratios of glucosamine over
et al., 1995; Glaser et al., 2000). In particular, ratios of muramic acid or galactosamine (or the sum of these
(mannose + galactose)/(xylose + arabinose) and (rham- two) have been used to differentiate between the rela-
nose + fucose)/(xylose + arabinose) < 0.5 indicate a tive contribution of fungi and bacteria to SOM turnover
dominance of plant polysaccharides while ratios > 2.0 and accumulation in soil (Zhang et al., 1998; Guggen-
indicate a high microbial sugar synthesis (Oades, 1984; berger et al., 1999a; Amelung, 2001). Amino sugar anal-
Guggenberger and Zech, 1994; Rumpel and Dignac, ysis has been combined with physical fractionation to
2006). Carbohydrate analyses on physical SOM frac- clarify microbial-derived SOM dynamics and SOM
tions have shown predominantly plant origin of par- (de)stabilization mechanisms. For example, by ana-
ticulate organic matter >50 μm (POM) and increasing lyzing amino sugar concentrations in different aggre-
proportions of microbial-derived sugars with decreas- gate size fractions, fungal-derived SOM accumulation
ing particle size (Cheshire, 1979; Angers and Mehuys, under no-till was attributed to the preferential stabiliza-
1990; Guggenberger et al., 1994, 1995; Guggenberger tion of fungal-derived amino sugars in macroaggregate-
and Zech, 1999; Puget et al., 1999; Larre-Larrouy and occluded microaggregates (Simpson et al., 2004). Zhang
Feller, 2001; Larre-Larrouy et al., 2003; Derrien et al., et al. (1998) proposed sorption onto clay particles as a
2006; Bock et al., 2007), suggesting an increasing micro- major stabilization mechanism of amino sugars based
bial conversion of saccharides with decreasing particle on observations of progressive accumulation of amino
size. However, the relative contribution of microbial- sugars in SOM with decreasing particle size. However,
a lack of correlation between clay or TOC content and (e.g. Wershaw, 1999; Piccolo, 2001; Sutton and Sposito,
individual amino sugar concentrations did not support 2005).
sorption as a major stabilization mechanism (Glaser Recent advances in analytical instrumentation have
et al., 2004). The possibility of microbial recycling (cf. allowed the chemical characterization of SOM in situ,
Gleixner et al., 2002) of amino sugars as preservation that is, without extraction. Two approaches represent
mechanism remains to be further investigated. the current state of the art for the characterization of
Extractable lipids form another important group of SOM in situ: pyrolysis mass spectrometry (Py-MS)
organic substances in the soil and have been of par- and nuclear magnetic resonance (NMR). Nuclear mag-
ticular interest in SOM research due to their role in netic resonance spectroscopy has been applied to humic
SOM accumulation and SOM stability (Naafs et al., substances in the liquid state for decades (e.g. Barton
2004; Rumpel et al., 2004b), their use as vegetation and Schnitzer, 1963). Since then, solid-state 13 C-NMR
tracers (VanBergen et al., 1997; Marseille et al., 1999; spectroscopy has become an important tool in the char-
Bull et al., 2000b; Wiesenberg et al., 2004a) as well acterization of SOM (Wilson, 1987). Solid-state 13 C-
as microbial tracers (Zelles, 1999), and their diagnos- NMR is generally used to determine the concentrations
tic potential to infer SOM transformation and stabi- of the main functional groups of SOM in whole-soil
lization processes (Bull et al., 2000a; Quenea et al., samples and isolated physical fractions. The most com-
2004; Otto and Simpson, 2005). Soil lipids consist of mon type of 13 C-NMR used in SOM studies is cross-
various components such as alkanoic acids, alkanols, polarization, magic angle spinning NMR (CPMAS 13 C-
alkenes, alkanes, ketones, resins, terpenoids and steroids NMR) (Preston, 1996; Kögel-Knabner, 1997). The
present both as free compounds or combined as waxes, MAS technique compensates for the chemical-shift
triglycerides and/or phospholipids (Dinel et al., 1990; anisotropy of solid samples, while cross-polarization
Kögel-Knabner, 2002). A wide range of procedures has between 1 H and 13 C spins leads to improved signal
been applied for soil lipid extraction, including Soxhlet enhancement, which allows investigation of solid sam-
extraction (Naafs et al., 2004; Nierop et al., 2005), soni- ples at natural 13 C abundance. The principle of 13 C-
cation followed by solvent extraction (Otto et al., 2005), NMR involves the excitation of nuclei by a magnetic
simple shaking with solvent (Quenea et al., 2004) and field. Solid-state 13 C-NMR spectra are recorded as a
accelerated solvent extraction (Rumpel et al., 2004b; difference, or chemical shift (δ, ppm), between the res-
Wiesenberg et al., 2004b; Jansen et al., 2006). How- onance frequency of the sample and a reference (typ-
ever, 100% recoveries are not always guaranteed as sig- ically tetramethylsilane, TMS, (CH3 )4 Si) (Fig. 6.3).
nificant amounts of lipids can be trapped in complex Different chemical shift regions are assigned to differ-
organic polymers (e.g. humin) that cannot be directly ent chemical functional groups (Table 6.1). A review of
13
extracted with solvents (Grasset and Ambles, 1998). C-NMR on more than 300 bulk soil samples showed
The composition of total lipid extracts from soils is a remarkable similarity between all soils with respect
usually determined by gas chromatography–mass spec- to the distribution of different forms of carbon, despite
trometry (GC/MS). the wide range of land use, climate, cropping practice,
fertilizer or manure application, and different spec-
6.2.2.2 Whole-soil SOM characterization trometer characteristics and experimental conditions
For a time during the mid to late 1900s, the chemi- used (Mahieu et al., 1999). The analysis found that
cal structure of the majority of SOM (i.e. humic sub- functional groups in whole soils were always in the
stances) was constructed from the products of chemical same order of abundance: O-alkyl C (mean of 45%
and pyrolytic degradations followed by ‘an imagina- of the spectrum, increasing with soil carbon content),
tive synthesis of all the information and the postula- alkyl C (25%), aromatic C (20%), and carbonyl and
tion of a grand structure’ (Burdon, 2001). However, amide C (10%, decreasing with soil carbon content).
more recent innovations in spectroscopic, microscopic, These results suggest that the application of NMR for
pyrolysis and soft ionization techniques suggest a new differentiating SOM quality due to land-use or cli-
concept of humic substances consisting of a supramolec- mate differences may be limited. A significant prob-
ular association of many relatively small and chemically lem with 13 C-NMR is that the low carbon concentra-
diverse organic molecules of plant and microbial origin tion in some soil and fraction samples results in long
102 K. DENEF et al.
Table 6.1 Chemical shift assignments in CPMAS
e ic
ph xyl
om c
ar ol i
ac at
-a l
al kyl
N lky
13
O tal
o
C-NMR spectra (from Swift, 1996).
en
l
rb
l
ky
-a
ca
Shift
range (ppm) Possible assignments
(a)
0–50 Unsubstituted saturated aliphatic C
atoms
10–20 Terminal methyl groups
15–50 Methylene groups in alkyl chains
25–50 Methine groups in alkyl chains
(b) 29–33 Methylene C α, β, δ, ε from terminal
methyl groups
35–50 Methylene C atoms of branched alkyl
chains
41–42 α-C in aliphatic chains
(c)
45–46 R2 NCH3
50–95 Aliphatic C singly bonded to one O or
N atom
51–61 Aliphatic esters and ethers; methoxy,
200 100 0 ethoxy
Chemical shift (ppm)
57–65 C in CH2 OH groups; C6 in
polysaccharides
Figure 6.3 Solid-state 13 C-NMR spectra of (a) free POM, (b) 65–85 C in CH(OH) groups; ring C atoms of
aggregate-occluded POM and (c) heavy, mineral-associated
polysaccharides; ether-bonded
SOM fractions (from Poirier et al., 2005).
aliphatic C
90–110 C singly bonded to two O atoms;
acquisition times, low resolution and low signal-to-noise anomeric in polysaccharides, acetal or
ratios of the spectra (Kögel-Knabner, 1997). Another ketal
problem is the potential presence of paramagnetic ele- 110–160 Aromatic and unsaturated C
ments, which broaden NMR signals and lead to spectra 110–120 Protonated aromatic C, aryl H
with overlapping resonance lines. Some of the organic 118–122 Aromatic C ortho to O-substituted
carbon bound to paramagnetic nuclei may therefore be aromatic C
invisible to 13 C-NMR. These problems can be over- 120–140 Unsubstituted and alkyl-substituted
come by concentrating the organic matter in the sam- aromatic C
ple through dissolution of the mineral material with 140–160 Aromatic C substituted by O and N;
HF. While some SOM is solubilized and lost during aromatic ether, phenol, aromatic
HF treatment, no significant alteration of the struc- amines
tural composition of SOM occurs (Schmidt et al., 1997; 160–230 Carbonyl, carboxyl, amide, ester C atoms
Goncalves et al., 2003). 160–190 Largely carboxyl C atoms
While 13 C-NMR spectroscopy can generate infor- 190–230 Carbonyl C atoms
mation on the gross chemical composition of SOM, spe-
cific compounds are not identified. An alternative tech-
nique is analytical pyrolysis, which generates data at the The thermal energy imparted by heating the samples
molecular level (Schnitzer and Schulten, 1992). Analy- in an inert atmosphere causes the breakdown of chem-
tical pyrolysis has been widely used to study the chemical bonds within the SOM macromolecules, yielding
ical composition of plant materials, extracted humic pyrolysis products characteristic of the original struc-
substances and SOM (Leinweber and Schulten, 1999). ture. The thermolytic degradation of SOM is followed
Figure 6.4 Integrated spectrum from the Py-MBMS analysis of a 0–5 cm layer of a forest soil sample. m/z values of compounds of
interest are marked above the peaks (from Magrini et al., 2002).
by analytical characterization of the pyrolysis products. and phenolic acids that represent an important part of
The most common techniques are pyrolysis gas chro- the macromolecule, as well as by the production of arte-
matography mass spectrometry, Py-GC/MS (Saiz- facts from fatty acids (Saiz-Jimenez, 1994).
Jimenez, 1994), pyrolysis field ionization mass spec- The big advantage of 13 C-NMR over analytical
trometry, Py-FIMS (Schnitzer and Schulten, 1995) pyrolysis is that it is non-destructive and avoids the
and more recently pyrolysis molecular beam mass spec- potential for secondary reactions. However, 13 C-NMR
trometry, Py-MBMS (Magrini et al., 2002). In each is less sensitive than analytical pyrolysis, and is subject to
case, the resulting mass spectra can be used as a fin- interferences by paramagnetic metal ions. While NMR
gerprint of that sample (Fig. 6.4). Interpretation of the and analytical pyrolysis are widely differing experimen-
pyrolysis data requires detailed knowledge of the pyrol- tal methods that generate data at different chemical
ysis behaviour of the compounds under study. Similar ‘scales’, the two methods can identify essentially the
to other mass spectrometry approaches, databases con- same types of compounds in SOM (Table 6.3). Results
tinue to grow as more users apply the technique. Table from both techniques have significantly contributed to
6.2 summarizes the identification of major signals in typ- the new paradigm that humic substances consist of
ical Py-FIMS spectra. The difficulty, however, is that a highly diverse, aliphatic admixture of biomolecules
many pyrolysis products can originate from chemically rather than the old model that they are dominated by
diverse SOM precursors (Saiz-Jimenez, 1994). One distinct, highly aromatic chemical compounds (Schul-
solution that is proposed is simultaneous pyrolysis and ten and Leinweber, 2000).
derivatization with tetramethylammonium hydroxide
(TMAH) (Chefetz et al., 2000). Pyrolysis/methylation
6.3 SHORTCOMINGS
results in the hydrolysis and methylation of polar com-
ponents, yielding methyl esters of carboxylic acids and The reductionist approach of separating heterogeneous
of hydroxyl groups. The result is that many polar com- SOM into more homogeneous and diagnostic frac-
pounds become volatile, rendering them more amenable tions has generated an enormous amount of information
for GC analysis. Comparisons of pyrolysis/methylation about the physical and chemical characteristics of SOM
to conventional pyrolysis demonstrate that pyrolysis, as (see above and Sollins et al., 1996; Six et al., 2002; Krull
traditionally performed, is biased by the thermal degra- et al., 2003; von Lützow et al., 2006). Yet, a good under-
dation of functional groups mainly in benzenecarboxylic standing of the feedback and interactions between these
104 K. DENEF et al.
Table 6.2 Identification of major signals in Py-FIMS spectra (from Schnitzer and Schulten, 1992).
m/z Identification
60, 72, 82, 84, 96, 98, 110, 112, 114, 126, 132, 144, 162 Carbohydrates with pentose and hexose subunits
94, 108, 110, 122, 124, 126, 138, 140, 154 Phenols
124, 138, 140, 150, 152, 154, 164, 166, 168, 178, 180, Monomeric lignins
182, 194, 196, 208, 210, 212
246, 260, 270, 272, 274, 284, 286, 296, 298, 300, 310, Dimeric lignins
312, 314, 316, 326, 328, 330, 340, 342, 356
256, 270, 284, 298, 312, 326, 340, 354, 368, 382, 396, n-C16 to n-C34 fatty acids
410, 424, 438, 452, 466, 480, 494, 508
380, 394, 408, 422 n-C27 to n-C30 alkanes
92, 106, 120, 134, 148 Methyl-, dimethyl-, trimethyl-, tetramethyl- and
pentamethyl-benzene
106, 120, 134, 148, 162, 176, 190, 204, 218, 232, 246, Ethyl- to docosyl-benzene
260, 274, 288, 302, 316, 330, 344, 358, 372, 386
142, 156, 170, 184, 198 Methyl-, dimethyl-, trimethyl-, tetramethyl- and
pentamethyl-naphthalene
192, 206, 220, 234 Methyl-, dimethyl-, trimethyl-, tetramethyl- and
pentamethyl-phenanthrene
59 Acetamide
67 Pyrrole
79 Pyridine
81 Methyl pyrrole
95 Hydroxypyridine or formyl-pyrrole or dimethyl
pyrrole
103 Benzonitrile
109 Trimethyl pyrrole
123 Tetramethyl pyrrole
648, 662, 676, 704, 732 n-C44 to n-C50 alkyl monoesters
202, 216, 230, 244, 258, 272, 286, 300, 314, 328, 342 n-C10 to n-C20 alkyl diesters
characteristics of SOM and the biological activity medi- soil. Consequently, most characterization approaches
ated by SOM is still limited. The linkage between the are not sensitive enough to detect small differences in
bio-physico-chemical quality of SOM and the turnover SOM quality between different SOM pools. For exam-
of SOM remains especially poorly understood, or even ple, Six et al. (2001) detected no biochemical differences
elusive. This elusiveness is probably a result of poten- between free versus occluded POM fractions with solu-
tial shortcomings or traps inherently associated with the tion 13 C-NMR analyses. In contrast, isotope analyses
reductionist approach. Some of these inherent short- revealed clear differences in carbon dynamics between
comings are as follows. the two POM fractions, and compound-specific analy-
ses of CuO-oxidation products showed a more decom-
posed stage of the free POM than the occluded POM.
6.3.1 The remaining gap between SOM Helfrich et al. (2006) did not observe any trend in alkyl-
fractionation and characterization C/O-alkyl-C or aryl-C/O-alkyl-C across four different
Most SOM characterization techniques have been aggregate size classes by NMR analyses. However, iso-
developed and most often been applied to the whole tope analyses on the same aggregate size classes from
Table 6.3 Compounds identified in whole soils by whereas the heavy fraction is recalcitrant (Swanston
solid-state 13 C-NMR and by Py-FIMS (from Schnitzer, et al., 2002; Crow et al., 2006, 2007). A comparison
2001). of the 13 C abundance among different physical frac-
tions obtained from a combination of magnetic, particle
13 size and density fractionations by Shang and Tiessen
C-NMR Py-FIMS
(2000) showed that losses of old forest (C3)-derived car-
Carbohydrate C Carbohydrates bon and accrual of new sorghum (C4)-derived Carbon
Phenolic C Phenols occurred in different organo-mineral size fractions with
Aromatic C Lignin monomers different magnetic susceptibility. Another 14 C-labelling
Aromatic C Lignin dimers study comparing different physical fractionation meth-
CH3 , (CH2 )n , ester groups n-fatty acids ods (Magid et al., 1996) indicated that the active frac-
CH3 , (CH2 )n n-alkanes tions of SOM were distributed among several different
CH3 , (CH2 )n , ester groups n-alkylmonoesters size and density fractions. Bosatta and Ågren (1985)
CH3 , (CH2 )n , ester groups n-alkyldiesters would argue that it is impossible to meet uniformity in
CH3 , (CH2 )n , aromatic C n-alkylbenzenes SOM pools because the heterogeneity of SOM is best
CH3 , aromatic C methylnaphthalenes represented by a continuous quality function. There-
CH3 , aromatic C methylphenanthrenes fore any fractionation methodology would have to iso-
C in N-containing compounds N-compounds late an infinite number of fractions in order to have uni-
form fractions. Recently, however, many efforts have
been put towards modelling measured fractions (Chris-
the same soil indicated that these aggregates represent tensen, 1996b; Elliott et al., 1996; Arah, 2000; Six and
different SOM pools (John et al., 2005). Numerous Jastrow, 2002) and defining a reasonable number of dif-
other studies have also revealed a relationship between ferent fractions that are both measurable and modellable
aggregate size and carbon dynamics (e.g. Elliott, 1986; (Smith et al., 2002). The most promising methods iso-
Jastrow et al., 1996; Six et al., 1998). Nevertheless, Hel- late fractions physically, and then chemically character-
frich et al. (2006) did observe a greater alkyl-C/O-alkyl- ize labile vs. more recalcitrant moieties (e.g. Balesdent
C ratio in the free POM compared to the occluded and Mariotti, 1996; Six et al., 2002; Sohi et al., 2005;
POM by NMR, which indicates, in contrast to Six Olk and Gregorich, 2006; Zimmermann et al., 2007b).
et al. (2001), that occluded POM is more decomposed
than free POM. The same was found in several other
studies (e.g. Golchin et al., 1994; Sohi et al., 2001;
Kölbl and Kögel-Knabner, 2004; Kölbl et al., 2005), but 6.3.3 Biochemical characteristics of SOM have
the differences in proportions of the organic moieties seldom been directly linked to microbial
were not always very pronounced, and were sometimes functioning and resulting SOM dynamics
contradictory. As described above, many different approaches have
been employed to biochemically characterize SOM,
but this is based on the inherent complexity of the
6.3.2 The current fractionation methodologies
organic compounds without any link to microbial func-
frequently isolate non-uniform SOM pools with
tioning and with only a relationship to decompos-
different turnover times
ability through inference. Nevertheless, several stud-
The wealth of different fractionation methods that have ies do exist where physically isolated SOM fractions
been employed indicate that all the different method- were incubated to evaluate their potential decompos-
ologies are a step in the direction of defining more ability as a biological indication of stability (e.g. Cam-
uniform SOM pools, but none of them have totally bardella and Elliott, 1994; Schutter and Dick, 2002;
resolved the issue of non-uniformity (von Lützow et al., Crow et al., 2006; Oorts et al., 2006). Other studies have
2007). For example, laboratory incubations following attempted to clarify SOM stabilization mechanisms
density fractionations are not always in support of the through biological means by measuring extracellular
concept that the light fraction is readily decomposable enzyme activities in physically isolated SOM fractions
106 K. DENEF et al.
(Kandeler et al., 1999; Allison and Jastrow, 2006). How- land-use change (Six et al., 2000b; Del Galdo et al.,
ever, the decomposability of physically isolated frac- 2003; Denef et al., 2004; Kong et al., 2005), to account
tions can vary depending on the type of density reagent for much of the stock change in total SOC upon man-
used (Magid et al., 1996), and could be different in iso- agement change (Denef et al., 2004) and to be an early
lated state than in situ. Direct investigations of in situ indicator for future total SOC changes upon land-use
mineralization of physical SOM fractions are limited or management change (Denef et al., 2004, 2007).
to tracer (14 C, 13 C) incubations following changes in
labelled plant-residue carbon concentrations in differ-
ent SOM fractions over time (e.g. Magid et al., 1996; 6.3.5 Isolated single compounds or compound
Guggenberger et al., 1999b). Several studies have also classes often represent such a small proportion
directly linked size and density SOM fractions and of the total SOM content that the quantification
their biochemical characteristics to whole-soil C and or modelling of their dynamics may have little
N dynamics (e.g. Gregorich et al., 1989; Janzen et al., relation to the dynamics of SOM as a whole
1992; Gregorich et al., 2006). For example, physically As suggested above, compound-specific analyses have
uncomplexed SOM has been suggested to immobilize some promise in furthering our understanding of SOM
mineralized N because of its C/N ratio being relatively dynamics by differentiating functional fractions within
higher than that of whole soil but lower than that of the heterogeneous SOM pool. However, the small pool
its plant litter source (see Six and Jastrow, 2002; Gre- size of these specific compounds often prevents the gen-
gorich et al., 2006). Whalen et al. (2000) reported heavy eralization to the much larger whole SOM pool. For
density SOM to be a greater source of potentially min- example, amino sugars have been used as biomarkers
eralizable N than light fraction (LF) even though LF for microbial SOM (see above and Guggenberger et al.,
is considered a more labile source of mineralizable C 1999b; Amelung, 2001; Simpson et al., 2004; Glaser
and N due to its physical uncomplexed state (Hassink, et al., 2006), but total amino sugars only comprise 1–5%
1995). of total SOM. It is well known that microbial-derived
SOM is much greater than 1–5% of total SOM. Con-
6.3.4 The relationship between the dynamics of sequently, one could question how much the observed
specific SOM fractions and the dynamics of dynamics of amino sugars can be extrapolated to the
whole SOM has not often been considered whole fraction of microbial-derived SOM, which is a
heterogeneous fraction consisting of polysaccharides,
Most often, studies have isolated and focused on one lipids etc.
or two SOM fractions in terms of their characteristics The above outline of shortcomings is not meant
and dynamics (see Christensen, 1996a). However, how to nullify the methods used. Each of these methods can
their dynamics relate to overall SOM dynamics and can contribute a significant amount of information concern-
be used as a diagnostic tool for changes in the dynamics ing the composition and dynamics of SOM, but each
of whole SOM has not been fully explored. Numerous also has its limitations: no single method is sufficient or
studies have shown that POM and LF are more sen- even appropriate for any given objective. These limita-
sitive SOM fractions than whole SOM (Christensen, tions should be taken into consideration in future stud-
1992; Six and Jastrow, 2002; Gregorich et al., 2006), ies and should help us in developing new approaches to
but a change in dynamics of these fractions is difficult elucidate SOM dynamics (see below).
to translate into an expected change in whole SOM,
especially in the long term. For example, an increase in
POM or LF fractions does not automatically correspond 6.4 DIRECTIONS FOR FUTURE RESEARCH
to an increase in whole SOM in the longer term (e.g. Six AND PROMISING NEW TECHNIQUES
et al., 2001). Similarly, changes in total soil carbon are
6.4.1 Quantification of the turnover of different
not always paralleled by changes in LF or POM (e.g.
SOM fractions by isotope analysis
Leifeld and Kögel-Knabner, 2005). Yet, a few studies
have demonstrated the microaggregate-SOM fraction While in situ and compound-specific SOM characteri-
to be the most responsive fraction to management and zation techniques can provide useful information about
the composition, biochemical stability or origin of SOM, transformed into other non-lignin products, while only
no indication of the dynamics or fate of SOM con- 8% reaches the soil-protected lignin fraction. Based
stituents in the soil environment can be derived. Infor- on the model, the authors suggested that VSC-lignin
mation on the reactivity and turnover rates of SOM turnover in soils is controlled by the transfer of
constituents could greatly improve SOM models that VSC-lignin molecules from decomposing plant tissues
so far only distinguish conceptually discrete SOM com- to soil-protected fractions, rather than by chemical
partments with different pre-defined stabilities (e.g. recalcitrance per se, as originally suggested by tradi-
Jenkinson et al., 1994). Recent developments in mass tional chemical SOM characterization methods. On
spectrometry have enabled the coupling of molecular the other hand, py-GC-C-IRMS analyses have shown
marker analysis with stable isotope analysis. This tech- surprisingly long residence times for pyrolysis products
nology, also called compound-specific stable isotope derived from N-containing components (proteins,
analysis (CSSIA), appears to be promising for quanti- amino acids, peptides, chitin) and polysaccharides
fying sequestration and turnover rates of specific SOM (VanBergen et al., 1997; Gleixner et al., 1999, 2002).
fractions since it allows differentiating between older These components appear to comprise a preserved
and more recent molecules of SOM fractions (for a SOM fraction of predominant microbial origin (Kiem
review, see Glaser, 2005). It could also reveal informa- and Kögel-Knabner, 2003), stabilized by microbial
tion about the plant origin of soil organic substances recycling or by chemical and physical protection
(e.g. C3 vs. C4) or about a soil’s biological quality in mechanisms through interactions with the soil mineral
natural or managed agricultural systems by differentiat- matrix rather than by biochemical recalcitrance.
ing chemically similar but isotopically different organic Stable isotope (13 C) analysis of individual amino
substances preserved or transformed by the microbial sugars by GC-C-IRMS was recently used to discrim-
population (e.g. Ostle et al., 1999). inate between old and new microbial-derived carbon
Curie-point pyrolysis coupled to gas- (Glaser and Gross, 2005) and could serve as a tool to
chromatography–combustion–isotope ratio mass determine formation, stabilization and turnover rates
spectrometry (py-GC-C-IRMS) allows simultaneous of microbial SOM. Applying this technique in a free-
characterization of the chemical structure of SOM and air carbon dioxide enrichment (FACE) experimental
analysis of the isotopic composition of the different site exposed to 13 C-depleted CO2 , Glaser et al. (2006)
pyrolysis products. Using this technique, a study on found a complete turnover of individual amino sug-
cultivated soils after a vegetation change from C3 wheat ars after seven years of elevated CO2 . However, the
to C4 maize indicated a lack of lignin-derived phenols GC-C-IRMS technology requires substantial derivati-
with a C3-signal (Gleixner et al., 1999, 2002). The zation (e.g. acetylation) of amino sugars to increase their
authors suggested that lignin must be severely biode- volatility necessary for GC separation. Necessary 13 C
graded and greatly transformed from its original form corrections due to carbon addition upon derivatization
in cultivated soils. Other studies have also suggested significantly reduce the sensitivity of CSSIA of amino
low stability of lignin in soils, based on lignin-phenol sugars, which can be problematic for natural abundance
distributions and degree of alteration across soil depths, studies (Glaser and Gross, 2005). As an alternative, He
particle size or density fractions (Rumpel et al., 2002; et al. (2006) applied a conventional GC/MS approach
Kiem and Kögel-Knabner, 2003; Rumpel et al., 2004a; to trace 15 N or 13 C isotope changes in glucosamine,
Sollins et al., 2006; Nierop and Filley, 2007), and galactosamine and muramic acid. Though also limited
13
C signatures of lignin-phenols by GC-C-IRMS to isotopic enrichment studies, this technology allows
(Dignac et al., 2005; Heim and Schmidt, 2007). This the simultaneous analyses of 13 C and 15 N. A recently
low stability is further supported by Rasse et al. developed analytical technology, i.e. high performance
(2006) who developed a two-pool model, calibrated liquid chromatography-combustion-IRMS (HPLC-C-
on lignin-specific 13 C isotopic analyses, and estimated IRMS), has enabled the high-precision determination
turnover rates faster than one year for the plant-residue of the 13 C/12 C isotope ratio of polar and thermo-labile
lignin pool and 0.05 y−1 for the soil-protected lignin compounds that can be chromatographically separated
pool. Furthermore, they suggested that 92% of the in aqueous phase (Krummen et al., 2004). By avoid-
plant-residue lignin pool is mineralized as CO2 or ing the requirement for derivatization, this technology,
108 K. DENEF et al.
though currently still limited to 13 C analysis, has greatly served as carbon substrates for soil micro-organisms. In
broadened the types of biomarkers that can be analyzed addition, a growing number of papers are being pub-
in conjunction with stable isotope analysis. The use of lished on 13 C-enrichment or depletion experiments in
HPLC-C-IRMS for CSSIA of biomarkers in soil sci- combination with 13 C-PLFA analysis for purposes of
ence should be explored in the future. linking microbial communities to specific biogeochem-
Compound-specific stable isotope analysis has also ical processes (Boschker et al., 1998) or distinguishing
recently been applied to individual amino acids for the microbial groups actively involved in the assimilation of
purposes of determining soil organic N dynamics with carbon derived from different quality substrates (But-
respect to changes in land use, management or vegeta- ler et al., 2003; Murase et al., 2006; Williams et al.,
tion (e.g. Ostle et al., 1999). For example, 15 N enrich- 2006), often in relation to management (e.g. liming,
ment in amino acids relative to bulk soil in managed Treonis et al., 2004) or global change (e.g. elevated
grassland (N fertilized) and arable land (tilled and N CO2 effects, Phillips et al., 2002; Billings and Ziegler,
fertilized), while being the same for unmanaged natu- 2005).
ral grassland, suggested enhanced SOM turnover due A major drawback of PLFA analysis is that it does
to management (Ostle et al., 1999). In a later study, not allow for specific detection of individual species of
Bol et al. (2004) found decreasing concentrations and the microbial communities. It is possible that the activ-
concomitant decreasing 15 N signatures in phenylala- ity of different microbial species in processing SOM
nine upon conversion from arable land to fallow, indi- differs or is differently affected by land-use or envi-
cating the potential of this amino acid as a biomarker ronmental conditions. Culture-independent molecu-
for land-use change. More studies are needed in this lar approaches are emerging that enable the linking
field to confirm the potential of the use of CSSIA in of microbial species identity with a measure of func-
amino acids as a tool to assess changes in soil biological tional contribution to SOM dynamics. Stable-isotope
quality, and SOM dynamics in particular, in different probing (SIP) of nucleic acids (DNA, rRNA) is one of
ecosystems. those molecular methods capable of identifying mem-
bers of microbial populations that are specifically active
in metabolizing and circulating SOC derived from a
6.4.2 Relating SOM quality and dynamics 13
C-labelled substrate (Radajewski et al., 2000; Mane-
to microbial functioning
field et al., 2002; Lueders et al., 2004). However, nucleic
Some promising new developments in soil ecology are acid-based SIP methodologies have a number of short-
currently being explored with the purpose of linking comings and their use for linking metabolic function
SOM dynamics to microbial functioning, and to char- with taxonomic identity is debated (see also Kutsch
acterize SOM quality in terms of its biological reac- et al., Chapter 9). Molecular analyses of ‘functional
tivity (see also Kutsch et al., Chapter 9). Compound- genes’ that encode enzymes that catalyze key steps in
specific isotope analyses have been done on microbial biogeochemical pathways could be an alternative means
biomarker phospholipid fatty acids (PLFAs) by GC- to study the molecular mechanisms regulating SOM
C-IRMS (for 13 C-PLFA) and accelerator mass spec- dynamics (Zak et al., 2006).
trometry (for 14 C-PLFA) to identify the sources (e.g.
C3 vs. C4 plant material) of the organic substrate used
6.4.3 Exploration of new avenues to characterize
by the active microbial community in various ecosys-
whole-soil and fraction SOM quality
tems (Burke et al., 2003; Bouillon et al., 2004) and
under different environmental conditions (Waldrop and Thermal analysis of whole-soil and isolated fraction
Firestone, 2004). Based on 13 C-PLFA analyses, Wal- samples by thermogravimetry–differential scanning
drop and Firestone (2004) were able to relate warming- calorimetry (TG-DSC) appears to hold some promise in
induced alterations in carbon resource utilization pat- characterizing SOM, should the link between thermal
terns to changes in microbial community composition. stability and biological stability be firmly established.
By analyzing both 13 C and 14 C content of PLFAs, This method is of great interest since it is applicable
Kramer and Gleixner (2006) were able to determine the to whole-soil samples without the requirement of time-
degree to which recent plant material vs. older SOM consuming pre-treatments (e.g. chemical extraction) as
Figure 6.5 Thermogravimetry–differential scanning calorimetry (TG–DSC) thermograms for (a) cellulose, (b) lignin, (c) cellulose
and lignin mixed (50:50 w/w), and (d) free light fraction soil organic matter (FLF-SOM) and intra-aggregate light fraction soil
organic matter (IALF–SOM) from arable soil (from Lopez-Capel et al., 2005b).
well as to isolated fractions. Through the continuous of clay-associated SOM from thermally labile to ther-
and simultaneous measurement of weight loss (TG) and mally resistant fractions with increasing time under
energy change (DSC) during heating, the method dif- cultivation. This indicated that thermal properties of
ferentiates between a thermally labile organic fraction SOM components appear to be linked to their biologi-
lost in a first exothermic region (approx. 300–350 ◦ C) cal decomposability as it is generally believed that SOM
and a more thermally resistant organic fraction lost decomposition induces a shift to more biologically resis-
in a second exothermic region (approx. 400–450 ◦ C) tant fractions (Plante et al., 2005).
(Fig. 6.5). Comparisons with 13 C-NMR analysis by The TG–DSC technique has also recently been
Lopez-Capel et al. (2005a) revealed close correlations coupled to isotope ratio mass spectrometry (IRMS)
of the thermally labile organic fraction with O-alkyl C and quadrupole mass spectrometry (QMS), enabling
and the thermally resistant organic fraction with aro- the determination of the isotope ratios as well as the
matic carbon, suggesting that thermal analysis can pro- composition of the evolved gases during a single heat-
vide an index of SOM quality. Several studies have ing experiment on one intact soil sample (Lopez-Capel
applied TG-DSC in an attempt to compare propor- et al., 2005b; Manning et al., 2005; Lopez-Capel et al.,
tions of labile (aliphatic) vs. more resistant (aromatic) 2006). In addition to the biochemical information pro-
carbon components in whole-soil SOM, physical SOM vided by the conventional TG–DSC analysis system,
fractions, plant tissue, dung and compost (Dell’Abate QMS results could provide further information about
et al., 2000; Lopez-Capel et al., 2005a, 2005b), to quan- the chemical stability of the SOM sources of C as well as
tify thermally stable black carbon in soils (Leifeld, 2007) N within a soil sample, while the isotope signatures of
and to characterize chemical changes in SOM fractions the thermally labile and recalcitrant components could
under contrasting land uses (Lopez-Capel et al., 2005a; give further insights into the dynamics of these SOM
Plante et al., 2005). In the study of Plante et al. (2005), components. In one of the first application studies,
TG–DSC was applied to clay-sized fractions of soils Lopez-Capel et al. (2005b) were able to demonstrate
ranging from forest to cultivated and long-term bare that N in pasture soil samples was mostly associated
fallow soils. A shift was observed in the distribution with a thermally resistant aromatic organic fraction.
110 K. DENEF et al.
Non-destructive techniques to assess SOM quan- spectroscopy has also been shown to have the poten-
tity and quality provide exciting new avenues to increase tial to determine litter and organic residue quality and
the speed, number, quality and relevance of SOM related decomposition rates (Joffre et al., 2001; Shep-
analyses (Janik et al., 1998) and allow the construc- herd et al., 2003, 2005), as well as SOM quality and com-
tion of large databases to explore general relationships position (Terhoeven-Urselmans et al., 2006). Leifeld
between SOM quality and dynamics (Shepherd and (2006) successfully calibrated DRIFT with NMR data
Walsh, 2002). Infrared spectroscopy has long been obtained from whole soil and fraction SOM. Capriel
used to investigate forages and other organic agri- et al. (1995) also used DRIFT to estimate alkyl-C as a
cultural products. Until recently, infrared analyses of measure for the degree of hydrophobicity of SOM. It is
soils were limited to qualitative and semi-quantitative furthermore very encouraging that MIR and NIR spec-
studies. Technological advances, particularly diffuse troscopy have also been successfully used to estimate
reflectance infrared Fourier-transform (DRIFT) meth- soil C and N mineralization rates (Fystro, 2002; Mutuo
ods, and advances in multivariate statistics are making et al., 2006), respiration rates (Palmborg and Nord-
the infrared method better suited to widespread use for gren, 1993) and enzyme activity (Mimmo et al., 2002).
SOM analysis. Infrared spectral signatures of the object These studies indicate the potential for MIR and NIR
under consideration are defined by the reflectance or spectroscopy to directly relate SOM quality to SOM
absorbance, as a function of wavelength in the elec- dynamics and microbial functioning across a large set
tromagnetic spectrum. Fundamental features related to of samples.
various components of the object occur at energy lev- Another rapid spectroscopic technique that has
els that allow molecules to rise to higher vibrational recently been suggested for SOM study is the laser-
states. These features generally occur for SOM in the induced breakdown spectroscopy (LIBS) (Bublitz et al.,
mid-infrared (MIR) range (2500–25 000 nm), which is 2001; Harmon et al., 2006). However, the technique has
dominated by intense vibration fundamentals, and the only been developed to determine whole-soil carbon
near-infrared (NIR) region (700–2500 nm), which is contents (Cremers et al., 2001; Ebinger et al., 2003).
dominated by much weaker and broader signals from On the other hand, there are several x-ray based tech-
vibration overtones and combination bands (McCarty niques such as near edge x-ray absorption fine struc-
et al., 2002; Shepherd and Walsh, 2002). Estimates of ture (NEXAFS), energy dispersive x-ray (EDX), scan-
soil properties of interest are generated from spectral ning transmission x-ray microscopy (STXM) and x-
information by calibration using multivariate statisti- ray photoelectron spectroscopy (XPS), which can yield
cal procedures such as principal components regres- great detail about not only SOM quality, but also its
sion (PCR), partial least squares regression (PLS), or microscale location within the soil matrix (Amelung
step-wise multiple linear regression (SMLR) and others et al., 2002; Jokic et al., 2003; Brodowski et al., 2005a;
(Chang et al., 2001; Shepherd and Walsh, 2002; Leifeld, Lehmann et al., 2005; Solomon et al., 2005). These tech-
2006; Janik et al., 2007). A significant shortcoming of niques are, however, quite laborious, require significant
this approach is that it is difficult to generate a ‘uni- infrastructure and are therefore not currently suitable
versal’ calibration model that will accurately determine for rapid assessments.
a particular property for all soils (Madari et al., 2005).
However, several strategies for real-time selection of
6.5 CONCLUSIONS
individual calibration sets have been developed.
Many studies have shown the capability of MIR A large number of techniques have been developed
and NIR spectroscopy for determining several soil for the characterization of SOM. It is apparent that
properties, including total soil C and N contents, tex- a suite of techniques combining physical or chemi-
ture, aggregation and CEC (Chang et al., 2001; Brown cal fractionation, analytical pyrolysis or NMR for bio-
et al., 2006; Madari et al., 2006; Vagen et al., 2006). chemical characterization, and isotopic analysis tech-
Infrared analyses of bulk soil samples without fraction- niques for an assessment of dynamics, is necessary for
ation have also been well correlated to several SOM an exhaustive examination of the nature and dynamics
fractions such as POM, silt- and clay-associated SOM, of SOM. Soil organic matter fractionation and charac-
and black carbon (Zimmermann et al., 2007b). Infrared terization approaches have led to major achievements in
the understanding of carbon stabilization and destabi- to be made by observing ‘microbial life at the micro-
lization mechanisms in the short and long term. From bial scale’ (Schimel, 2007) and our understanding of
these results it has become clear that carbon cycling SOM characteristics may be informed by application of
and stabilization in soils is a function of both the min- advances in the field of nanotechnology.
eral and structural properties of the soil as well as the Most experimental methodologies that fractionate
biochemical characteristics of the SOM. While SOM or characterize SOM destroy to some degree (depend-
fractionation schemes have revealed a few diagnos- ing on methodology) the natural state of SOM, caus-
tic SOM fractions in terms of their responsiveness to ing information about its stabilization mechanisms to be
land-use or management practices (e.g. uncomplexed lost. Therefore, future SOM research could greatly ben-
SOM, microaggregate-SOM), they mostly fail to sep- efit from non-destructive characterization techniques
arate truly homogeneous SOM fractions in terms of that provide information on in situ chemical and bio-
their turnover times or biochemical properties, and logical characteristics as well as physical distribution
they usually do not link SOM fractions to specific of SOM fractions. Promising non-destructive tech-
stabilization mechanisms unequivocally. A quantita- niques (such as MIR, NIR and DRIFT spectroscopy)
tive assessment of the relative contributions of different continue to evolve and should make important con-
(and possibly co-occurring) stabilization mechanisms to tributions to rapidly assess SOM quantity and qual-
the stability of specific SOM fractions requires further ity, and directly relate SOM quality to SOM dynam-
investigation. ics and microbial functioning across a large set of
The use of molecular biomarkers and compound- samples.
specific isotope analysis in SOM research could reveal In conclusion, to better understand SOM stabi-
the dynamics of specific, meaningful SOM compo- lization processes and to make better predictions of
nents within heterogeneous SOM fractions and clar- SOM dynamics, the following will be essential: (1) SOM
ify SOM transformations. In particular, the concept of research that combines existing, but more standardized,
microbial ‘recycling’ or ‘re-synthesis’ of specific molec- methodologies to characterize the molecular composi-
ular components needs further investigation since it tion of meaningful SOM fractions and provides mea-
appears to be an important process controlling the sures of SOM stability (e.g. age, turnover times) and
longevity of SOM components in the soil. In addi- microbial functioning; (2) collaborative research to max-
tion, SOM research could greatly benefit from recent imize the potential of highly specific and costly tech-
technological developments in microbial ecology that niques requiring highly skilled expertise in the method-
allow identifying the microbial communities or species ologies, instrumentation and data analysis; (3) compara-
involved in SOM decomposition. In this way, SOM tive studies that allow a generalization of findings across
dynamics under changing environmental conditions soil types, soil layers and environments; and (4) more
(e.g. climate change) or human activities could be better long-term field manipulation experiments, preferably
understood and potentially foreseen through the struc- with C3–C4 conversions or other isotopic tracers to
tural and activity-related responses of the microbial obtain better estimates of turnover rates of specific SOM
community. fractions.
Studies on SOM characteristics are mostly limited
to the surface layers of soils and need to be extended
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6 r Characterization of soil organic matter

Karolien Denef, Alain F. Plante and Johan Six

Bulk soil (intact)

Light fraction (LF) Organo-mineral SOM fractions

> 250 µm: macroaggregates

sand occluded POM (in aggregates)

Primary organo-mineral Secondary organo-mineral

6.2.1.2 Physical fractionation particles. However, it is difficult to isolate either com-

Hymatomelanic extract with (precipitated) (not precipitated)

Table 6.1 Chemical shift assignments in CPMAS

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