Soil Biology & Biochemistry 68 (2014) A4eA9

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Soil Biology & Biochemistry


j our nal h o m ep age: www. el sevi er . com/ l ocat e/ soi l bi o


Citation classics

Aggregate-associated soil organic matter as an ecosystem property
and a measurement tool
q


Johan Six
a,
*
, Keith Paustian
b


a
Department of Environmental Systems Science, Swiss Federal Institute of Technology, ETH-Zurich, Tannenstrasse 1, 8092 Zurich, Switzerland
b
Department of Crop and Soil Science, Natural Resource Ecology Laboratory, Colorado State University, Fort Collins, CO 80523, USA

a r t i c l e i n f o

Article history:
Received 21 June 2013
Accepted 25 June 2013
Available online 9 July 2013

Keywords:
Soil organic matter
Aggregates
Agricultural management
Modeling
Carbon cycling
Soil carbon stabilization
a b s t r a c t

Our 2000 paper Soil macroaggregate turnover and microaggregate formation: A mechanism for C seques-
tration under no-tillage agriculture had its genesis in attempts to identify and isolate soil organic matter
(SOM) fractions that reect the impacts of climate, soil physiochemical properties and physical distur-
bance on the soil organic carbon balance. A key prerequisite for the investigation was the development of
a simple device to isolate the microaggregates (53e250 mm) contained within stable (i.e., resistant to
slaking) macroaggregates (>250 mm) obtained by conventional wet-sieving. By comparing the abun-
dance and C content of micro-within-macroaggregates, the size distribution of intra-aggregate partic-
ulate organic matter (iPOM) and isotopically-based estimates of the age of the organic matter in the
different fractions, we were able to corroborate our hypothesis that the absence of tillage (i.e., in no-till
and native soils) promotes greater longevity of newly-formed macroaggregates, resulting in greater SOM
stabilization in microaggregates formed within stable macroaggregates. Follow-up research has indi-
cated that the microaggregate-within-macroaggregate fraction is 1) potentially a robust indicator for
management-induced SOC changes over decadal time scales, 2) of biological origin and therefore useful
in interpreting impacts of soil biota on soil C and N dynamics, but not in-situ CO2 and N2O uxes, 3)
useful in complimentary chemical and spectroscopic approaches to relate SOM dynamics to soil structure
and attributes of the soil pore space, and 4) a good candidate for being incorporated into models as a
measurable fraction.
2013 Elsevier Ltd. All rights reserved.



1. Introduction

During the 1980s and 90s, two topics of debate came into the
mainstream of soil organic matter SOM research, both of which
continue to be widely discussed and studied to the present day. One
was the issue of how to characterize SOM in terms of its compo-
nents in ways that better reected ecosystem-level perturbations
such as tillage, land cover change and climate change. There was a
growing consensus that the fulvic and humic acids revealed by
classical chemical extraction processes were not representative of
functional SOM fractions (Elliott et al., 1996). This realization
sparked an interest in physically-dened SOM fractions, whereby
the actual location of organic matter within the soil matrix and the
size and density of different organo-mineral complexes were



q
Dedicated to Dr. Ted Elliott who was instrumental in developing our Citation
Classic.
* Corresponding author. Tel.: 41 44 63 2 84 83.
E-mail address: Jsix@ethz.ch (J. Six).
viewed as key attributes determining the function and turnover of
SOM. The second topic arose from the recognition that soil C
sequestration might be a viable option for greenhouse gas and
climate change mitigation (Paustian et al., 1997). Hence, there was a
greater imperative to elucidate the underlying mechanisms for soil
C sequestration under conservation management practices and
develop means to better measure and predict soil C stocks changes
as a function of management and environmental drivers. In the US,
there was particular interest in the effects of no-tillage manage-
ment and land use change (e.g. from native to cropped) on SOM-C
stocks (Bruce et al. 1999; Lal et al. 1999).
It is within this framework that the research described in our
paper (Six et al., 2000a) was developed, conducted and interpreted.
We sought to elucidate how changes in aggregation were linked to
soil C sequestration under no-tillage compared to conventional
tillage management. This link between aggregate structure and
SOM dynamics had been investigated before (Elliott, 1986), but
here we developed a new method to isolate a specic fraction, i.e.,
the microaggregate-within-macroaggregate. Six et al. (1998, 1999)
hypothesized that this aggregate fraction is a fraction in which

0038-0717/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.soilbio.2013.06.014
J. Six, K. Paustian / Soil Biology & Biochemistry 68 (2014) A4eA9 A5 A5 J. Six, K. Paustian / Soil Biology & Biochemistry 68 (2014) A4eA9


soil C is preferentially stabilized in no-tillage soils. The hypothesis
was tested and corroborated across four different sites, allowing us
to generalize, the proposed mechanism for soil C sequestration
with no-tillage management.

2. Background

The theoretical and methodological framework for our paper
stems most directly from the Elliott (1986) paper on aggregate
structure and organic matter in native and cultivated soils in the US
Great Plains. Elliott had long been interested in the physical
structure of the soil as it relates to SOM and the dynamics of the soil
decomposer community, as evidenced by his seminal paper on
habitable pore space and trophic interactions in soil (Elliott et al.,
1980). Later, while working on the National Science Foundation
Great Plains Project (one of the rst regional-scale agroecosystem
studies), Elliott was inspired by the conceptual model of soil
aggregate hierarchy presented by Tisdall and Oades (1982) and its
potential applicability to soils in the Great Plains, where the
generally negative effects of land use change from prairie to annual
cropland were a main focus. He found patterns consistent with the
Tisdall and Oades (1982) model, including higher C contents in
macroaggregates compared to microaggregates, but that the
additional C was subject to rapid mineralization following aggre-
gate disruption. Subsequent papers together with Cambardella
(Elliott and Cambardella, 1991; Cambardella and Elliott, 1992,
1993a,b, 1994) dened and investigated physically-based fractions
(using size and density fractionation), including particulate organic
matter (POM) and a mineral-associated fraction, termed the
enriched labile fraction (ELF). These fractions were postulated to
represent SOM pools with intermediate turnover times (i.e.,
several years to a few decades) and, in fact, the bulk of the SOM
impacted by land use and management.
It was September 1995, when Six joined Elliott and Paustian at
the Natural Resource Ecology Laboratory (NREL) in Fort Collins,
Colorado to start his PhD research within a US National Science
Foundation project titled Environmental and Management Con-
trols on Soil Structure and Organic Matter Dynamics. The global
objective of the project was to understand the controls on these
intermediate SOM fractions and to quantify their role in organic
matter and nutrient dynamics in agroecosystems e particularly
with respect to soil disturbance (e.g., tillage) and its impact on soil
structure and SOM dynamics. It must have been in one of the rst
weekly meetings between student and advisors that Elliott told Six:
In the beginning of your PhD work on this project, I will teach you
a lot, but your goal is to teach me something new by the end of your
PhD!. This statement was a continuous driving force throughout
Sixs PhD.
In the rst few experiments (Six et al., 1998, 1999, 2000c), soils
from four different sites with a no-tillage, conventional tillage, and
native vegetation treatment were fractionated into different
aggregate size classes and aggregate-associated SOM fractions
(POM and ELF) with a methodology similar to that used by
Cambardella and Elliott (1993a,b, 1994) and Jastrow (1996). The
main modication in the methodology was that intra-aggregate
POM (iPOM) was split in two different fractions: coarse iPOM and
ne iPOM. This division was the result of wanting to have two
different size classes of sand (with which the two different size
classes of iPOM are associated) to develop what was called the
Normalized Stability Index (Six et al., 2000b). But once this sepa-
ration was done, it was thought that the POM-C associated with the
coarse and ne sand fractions could as well be measured instead of
having, as originally intended, one combined fraction of iPOM. The
expedient decision to separately analyze the two iPOM fractions
turned out to be a lucky move because it formed the basis for using
the ne to coarse iPOM ratio as an indicator for macroaggregate
turnover and its inuence on soil C stabilization. It was the higher
ratio of ne to coarse iPOM in no-tillage compared to conventional
tillage soils that suggested a slower macroaggregate turnover in no-
tillage, leading to greater stabilization of C in microaggregates-
within-macroaggregates (Six et al., 1999). However, to really test
the preferential stabilization of C in microaggregates-within-
macroaggregates when macroaggregate turnover is slower due to
less soil disturbance, a method was needed to isolate the micro-
aggregates occluded within macroaggregates. One morning, some
discarded materials (e.g. mesh, a funnel, plexiglass tubing, regular
tubing, etc.) lying around in the Natural Resource Ecology Labora-
tory were used to construct the microaggregate isolator (Six et al.,
2000a) and a few days later microaggregates contained within
macroaggregates were isolated from all four soils. Once their C was
measured, the preferential stabilization of C in microaggregates in
no-tillage soils was corroborated and formed the foundation for
what was to become the Soil Biology & Biochemistry Citation Classic
paper. Upon seeing the results Elliott acknowledged that he had
unsuccessfully tried for many years to isolate microaggregates-
within-macroaggregates, but now that it was nally accom-
plished it corroborated some long-standing hypotheses. He said:
Youve done what I have been trying to do for the last 15 years!.
And there was a sense of the student having taught something to
the advisor.

3. Opportunities and pitfalls

In the years following the publication of our Soil Biology &
Biochemistry Citation Classic paper, several advances have been
made. As a result, it has become clear what the strengths and
weaknesses were of both the original methodology and its impli-
cations for mechanistic understanding of soil C stabilization.

3.1. A diagnostic fraction for management effects on carbon storage

As indicated above, the results from four temperate soils under
both long-term no-tillage and conventional tillage management
indicated that greater amounts of C were protected inside the
microaggregate-within-macroaggregate fraction in no-tillage soils.
In follow-up work involving three no-tillage and conventional tillage
experiments in widely varying soil types (Mollisol, Alsol, Oxisol)
and environments (i.e. Nebraska, Kentucky, Brazil), we obtained
further support for this preferential C stabilization by nding that
over 90% of the difference in total SOC between no-tillage and con-
ventional tillage could be accounted for by C associated with the
microaggregates-within-macroaggregates (Denef et al., 2004)
(Table 1). In a fourth experiment (also in Brazil) on a highly weath-
ered Oxisol, we found that after ve years of no-tillage there was no
signicant increase in microaggregate-within-macroaggregate C
relative to conventional tillage, nor was there any difference in total
SOC levels between no-tillage and conventional tillage (Denef et al.,
2007). These initial ndings suggested that this microaggregate-
within-macroaggregate C fraction may be a diagnostic fraction for
changes in total SOC in response to changes in tillage management
practices. In more recent studies (Table 1), we have further corrob-
orated that microaggregate-within-macroaggregate C is a general
diagnostic for management-induced changes in SOC levels in agro-
ecosystems on a wide range of soil types and under drastically
different climates (See Table 1). Hence, it represents an indicator of
SOC storage capacity of best management practices as affected by
factors, such as climate, soil type and management history.
Furthermore, this fraction could provide a focal point for further
elucidating mechanisms of SOM stabilization and turnover in soil,
help explain the variability in the capacity of different soils to
J. Six, K. Paustian / Soil Biology & Biochemistry 68 (2014) A4eA9 A6 A6 J. Six, K. Paustian / Soil Biology & Biochemistry 68 (2014) A4eA9



Table 1
Agricultural management effects on total soil organic C (TOC) and microaggregate-within-macroaggregate C (mM-C) stocks.

Soil type Location Soil classication Texture/dominant
clay mineralogy
Management
change
Depth
(cm)
Change in TOC
(g C m
2
)
Contribution
of mM-C (%)
b

Reference
Mollisol Sidney, NE (USA) Pachic Haplustoll Loam/2:1
a
CT to NT 0e20 431 14 91 6 Denef et al. (2004)
Aridisol Penaor, Spain Xerollic calciorthid Loam/2:1 CT to NT 0e20 465 49 49 20 Alvaro-Fuentes
et al. (2009)
Entisol/Alsol Davis, CA (USA) Typic Xerothent/
Mollic Haploxeralf
Silt loam/Silty
clay loam/2:1
RWC to RWL
c
0e15 267 12 108 33 Kong et al. (2005)

Davis, CA (USA) Typic Xerothent/
Mollic Haploxeralf
Silt loam/2:1 RWL to OMT
c
0e15 323 24 61 30 Kong et al. (2005)

Davis, CA (USA) Typic Xerothent/
Mollic Haploxeralf
Silt loam/2:1 CMT to OMT
c
0e15 512 24 NS Kong et al. (2005)
Alsol Wooster, OH (USA) Typic Fragiudalf Silt loam/2:1 CT to NT 0e20 2050 121 75 4
c
Six et al. (unpublished)

Lexington, KY (USA) Typic Paleudalf Silt/mixed CT to NT (0 N) 0e20 617 95 81 7 Chung et al. (2008)

2:1 & 1:1 CT to NT (84 N) 0e20 607 72 82 9 Chung et al. (2008)

CT to NT (84 N) 0e20 968 104 93 8 Denef et al. (2004)

CT to NT (168 N) 0e20 678 85 109 13 Chung et al. (2008)

CT to NT (336 N) 0e20 745 179 112 32 Chung et al. (2008)

Lexington, KY (USA) Typic Paleudalf Silt/mixed
2:1 & 1:1
0 N to 168 N
(CT only)
0e20 385 53 NS Chung et al. (2008)

Lexington, KY (USA) Typic Paleudalf Silt/mixed
2:1 & 1:1
0 N to 168 N
(NT only)
0e20 446 85 49 12
c
Chung et al. (2008)
Ultisol Horseshoe Bend, GA
(USA)
Rhodic
Kanhapludult
Sandy loam/1:1 CT to NT 0e20 578 91 95 12 Simpson et al. (2004)
Oxisol Passo Fundo (Brazil) Typic Haplorthox Clay/1:1 CT to NT 0e20 382 134 99 9 Denef et al. (2004)
CT conventional tillage; NT no-tillage; NS no signicant difference in mM-C found for treatment comparison after t-test (P < 0.05), while signicant difference was found
for TOC.
RWC unfertilized rainfed wheat/fallow; RWL unfertilized rainfed wheat/rainfed legume cover crop; CMT conventional fertilized irrigated Maize/conventional fertilized
irrigated tomato; OMT organic fertilized irrigated maize/organic fertilized irrigated tomato.
a
Lattice structure of dominant clay minerals (2:1 2:1 lattice structure, e.g. illite; 1:1 1:1 lattice structure, e.g. kaolinite).
b
Proportion of the total soil organic C response to management change that is accounted for by the change in microaggregate-within-macroaggregate C (mM-C) due to
management.
c
Data for microaggregate-within-macroaggregate C obtained from microaggregates occluded within small macroaggregates (250e2000 mm) only, due to insufcient
amounts of large macroaggregates (>2000 mm).

sequester C with conversion to best management practices, and
provide a measurable and functionally meaningful fraction that can
be incorporated into simulation models of SOM (Segoli et al., 2013).

3.2. Aggregates and soil biota

Earthworm and fungal activities have been linked to not only
macroaggregate formation but also microaggregate formation
(Shipitalo and Protz, 1989; Barois et al., 1993) and more specically the
formation of microaggregates-within-macroaggregates with
concomitant C stabilization within this fraction (Bossuyt et al. 2004,
2005; Simpson et al., 2004; Pulleman et al., 2005a,b; Fonte et al.,
2007, 2010). Thin sections of the earthworm gut, casts and bulk soil
from earthworm microcosms show that during gut transit, organic
materials and minerals are intimately mixed and become encrusted
with mucus (i.e. extracellular polysaccharides derived from gut
microora) to form new microaggregates (Shipitalo and Protz, 1988,
1989; Barois et al., 1993). Also, based on thin section observations,
biogenic macroaggregates in pasture soils containing many earth-
worms had a four times greater proportion (w20%) of micro-
aggregates than the equivalent macroaggregates of an arable soil
(w5%) with few earthworms (Jongmans et al., 2001; Pulleman et al.,
2005a). Furthermore, more ne POM was occluded in the micro-
aggregates within biogenic macroaggregates than in microaggregates
occluded within physicogenic macroaggregates (Pulleman et al.,
2005a). After only 12 days of incubation with
13
C-labeled plant ma-
terial, Bossuyt et al. (2004, 2005) found a four-fold greater mass of
microaggregates-within-macroaggregates (i.e., 25% versus 6% of the
macroaggregate mass consisted of microaggregates-within-
macroaggregates) containing more new and protected C inside
them when earthworms were present versus when they were absent.
When comparing agroforestry and slash-and-burn systems with
secondary forest from which they were derived, Fonte et al. (2010)
observed that the microaggregate-within-macroaggregate fraction
contained as much as 3.0 g C kg
1
soil less than in the secondary forest
and this was mostly attributed to a loss in earthworm abundance with
cultivation because the lower earthworm abundance leads to less
formation of microaggregates-within-macroaggregates (Bossuyt
et al., 2004). Fonte et al. (2007) found that earthworms increased
the accumulation of newly added residue C into the microaggregate-
within-macroaggregate fraction by 35% in low-input agroecosystems.
Simpson et al. (2004), on the other hand, showed that microbial-
derived C was more stabilized under no-tillage than conventional
tillage management, mostly because fungal hyphae remain intact in
no-tillage soils and hence improve soil structural stability with con-
current deposition of fungal-derived C (indicated by glucosamine
content) in microaggregates-within-macroaggregates. These results
indicate clearly that earthworms and fungi have a direct and rapid
impact on microaggregate formation and the stabilization of newly
generated C, thereby indicating the biological origins of this fraction
and its potential value in understanding the effect of soil management
via biota on C stabilization.

3.3. Aggregates versus pores

In our 2000 paper we relied on the isolation of aggregate
structures to understand generation, survival and cycling of soil C,
but we know that organic matter decomposition and microbial
activities, in fact, occur within the soil pores. Hence, in some sense,
we are looking at the walls of a house to understand what is
happening in the living-room. Another parallel that can be drawn is
between inverse modeling and using aggregates to understand
SOM dynamics, i.e., we are estimating C uxes/losses by quanti-
fying what is stabilized within aggregates. This inverse relationship
between aggregates and pores has been described by many (Elliott
and Coleman, 1988; Dexter, 1988; Kay, 1990). Elliott and Coleman
J. Six, K. Paustian / Soil Biology & Biochemistry 68 (2014) A4eA9 A7 A7 J. Six, K. Paustian / Soil Biology & Biochemistry 68 (2014) A4eA9


(1988) postulated a pore hierarchy analogous to the aggregate hi-
erarchy concept of Tisdall and Oades (1982). They proposed that
there are four hierarchical pore categories: (i) macro-pores; (ii)
pore space between macroaggregates; (iii) pores between
microaggregates-within-macroaggregates; and (iv) pores within
microaggregates. Dexter (1988), on the other hand, formulated the
porosity exclusion principle, which argues that aggregates of a
lower hierarchical order exclude the pore spaces between the
building blocks of aggregates of a higher hierarchical order. Finally,
Kay (1990) argued that inorganic and organic stabilizing com-
pounds (e.g. oxides, polysaccharides) with their general small di-
mensions will predominantly stabilize microaggregates, but roots
and hyphae will bind particles separated by greater pores e as
earlier postulated by Tisdall and Oades (1982). Hence, the aggregate
hierarchy concept and the pore exclusion principle both lead to the
same hierarchical order of soil structure and associated binding
agents. Even though this link has been clearly outlined, SOM dy-
namic studies have focused much more on aggregates than on
pores. One exceptional series of studies is, however, by Strong et al.
(1998, 1999a,b,c) in which pore system measurements and ma-
nipulations were used to elucidate the inuence of the soil matrix
on N mineralization and nitrication. These studies clearly indicate
the potential of a pore network approach to complement the
aggregate-based approach to understand SOM dynamics.

3.4. Complimentary information from the aggregate approach

The usefulness of aggregate fractionation to understand soil
functioning has been questioned with some justication (Young
et al., 2001; Young and Ritz, 2000), but here we argue that the
isolation of aggregates should be viewed as a complimentary tool to
other techniques such as tomography (De Gryze et al., 2006), thin
sectioning (Pulleman et al., 2005a,b), and synchrotron-based ana-
lyses (Jastrow, personal communication); both isolating and
viewing approaches provide complimentary information e not
surprisingly because aggregates and their pores are closely con-
nected (see Section 3.3).
Since there is a continuum in aggregate stabilities, the functions
of the isolated aggregates are very much dependent on the amount
of energy used to isolate them. It is, therefore, no surprise that
aggregate studies based on sieving have drawn some criticism: e
collected aggregates may say little about the true structure of the
soil, nor accurately simulate disaggregation processes that occur in
the eld and sieving techniques provide little information as to
how aggregates were connected in the original ped, i.e., their
context in relation to the overall soil matrix is lost. (Young et al.,
2001; Young and Ritz, 2000; Ashman et al., 2003). On the other
hand, viewing the intact soil matrix through, for example to-
mography or thin sections, does not reveal the properties and dy-
namics in which we are interested. Furthermore, the dependency of
aggregate distributions on the method used to separate them can be
used to our advantage. Several studies (Elliott, 1986; Gregorich et al.,
1989; Six et al., 2000d; Gale et al., 2000) have shown that comparing
C associated with aggregates isolated using less destructive sieving
methods (e.g. rewetting the soil by misting prior to wet sieving)
versus aggregates isolated with the slaking method (Kemper et al.,
1985) strongly supports (at least for 2:1 clay dominated mineral
soils) the important role of SOM in holding smaller (<250 mm) stable
aggregates together in larger (>250 mm) stable aggregates. This
supports the aggregate hierarchy concept (Tisdall and Oades, 1982)
and provides the framework for physical protection of soil C versus
losses of soil C due to a loss of soil structure (Elliott, 1986). Hence, it is
by comparing the results of different aggregate isolation methods
that we can learn about the functioning of different organic binding
agents (i.e. SOM compounds) at different scales in aggregates of
different stabilities. Furthermore, better assessments of aggregate
stability must rely on the measurement of different aggregate dis-
tributions due to different levels of energy imposed on the soil (Six
et al., 2000b) and can be related to different soil processes (see
Fig. 1). Nonetheless, with the viewing techniques, we can focus on
the soil morphology, which has the following advantages:

It is the ideal method to study the small-scale biogeography of
microorganisms, e.g., what does the local microhabitat for bac-
teria and fungi look like?
The inherent small-scale soil variability can be assessed.

3.5. Aggregates and CO
2
plus N
2
O uxes

Since there is the link between C and N cycling and aggregate sizes,
there is great temptation to use different aggregate size classes in
order to elucidate the spatial and temporal variability of CO
2
and N
2
O
uxes. However, the aggregate properties inuencing uxes, such as
anaerobiosis and nutrient availability, are inevitably changed upon the
isolation of the aggregates. The environmental conditions within an
aggregate that determine the microbial activity are in part conferred
by the constantly changing matrix surrounding the aggregate. For
example, it is the connectivity between the aggregate and the sur-
rounding soil matrix that will ultimately determine how anaerobic the
inner-aggregate atmosphere is, even though the pore size distribution
and connectivity within the aggregate itself is also of importance
because it inuences microbial motility (Elliott and Coleman, 1988).
Hence, measuring CO
2
and N
2
O uxes associated with different
aggregate size classes will only give, at best, a rough indication of the
potential uxes. Nevertheless, measurement of active microbial
community structures across aggregate size classes can be done and
help with elucidating C and N cycling, especially when combined with
isotopes (Kong et al., 2010, 2011; Kong and Six, 2012).

4. Future

Since the publication of our paper, much research has been con-
ducted in an attempt to further elucidate the properties, dynamics
and responses of the microaggregate-within-macroaggregate frac-
tion. However, here are a few things to consider for continued
exploration of aggregates and SOM dynamics:

1) The microaggregate-within-macroaggregate fraction as a broadly
applicable diagnostic fraction for SOM changes.

We have identied the microaggregate-within-macroaggregate
fraction as a diagnostic for SOM changes induced by management
across many soil types and climate regimes (Table 1). However, there



Fig. 1. The relationship between different aggregate stability measures and soil
functions.
J. Six, K. Paustian / Soil Biology & Biochemistry 68 (2014) A4eA9 A8 A8 J. Six, K. Paustian / Soil Biology & Biochemistry 68 (2014) A4eA9


are still many soil types and environments that need to be considered
before we can state with full condence that the microaggregate-
within-macroaggregate fraction is a highly accurate and broadly
applicable diagnostic measure for total SOC changes in response to
changes in management practices in terrestrial ecosystems. In
particular, Andosols have not been examined and these soils do have
a fundamentally different soil structure; one in which the
microaggregate-within-macroaggregate fractions may not have the
same diagnostic power.
However, if the microaggregate-within-macroaggregate frac-
tion is found to be truly diagnostic across most soil types and en-
vironments, it would be of enormous signicance and lead to a
rapid and better understanding of how management impacts SOM
dynamics and C sequestration in the terrestrial biosphere.
2) Integration of physical and chemical aspects of SOM dynamics.
The chemical characterization of whole soil SOM has a long
history and has provided insights into SOM quality and dynamics,
but it has ignored the inuence of the soil matrix. At the same time,
the physical fractionation of SOM emphasizes the inuence of the
soil matrix on SOM pool sizes and dynamics, but does not make the
link with SOM chemistry. Hence, it is evident that combining the
latest state-of-the-art techniques to physically separate SOM pools
and subsequently to chemically characterize the various pools has
great potential to further elucidate SOM dynamics. This is especially
the case if isotope analyses can be supplemented to quantify
transformation rates and turnovers. Research progress of this type
is slow despite some excellent example studies (e.g., Golchin et al.,
1995; Six et al., 2001; Gleixner et al., 2002; Kolbl and Kogel-
Knabner, 2004; Carrington et al., 2012).

3) Integration of isolation and viewing of soil structures.

As indicated in Section 3.4, only a few studies (e.g., Pulleman
et al., 2005a,b; Mueller et al., 2012) have integrated the isola-
tion and viewing approaches even though it clearly has its po-
tential to help us better understand how the soil matrix inuences
SOM dynamics. Especially signicant are some of the latest syn-
chrotron, spectroscopic, and tomography based techniques which
have become much more accessible and have shown some great
promises (Solomon et al., 2009; Nunan et al., 2003; Mueller et al.,
2012).

4) The microaggregate-within-macroaggregate fraction as a model-
able pool.

The potential to use the microaggregate-within-macroaggregate
fraction as a measurable pool in SOM models has been suggested
and implemented to a limited degree (Segoli et al., 2013). However,
it is only when the previous three future endeavors are conducted
that the microaggregate-within-macroaggregate fraction will begin
to provide a measurable and functionally-meaningful fraction that
could be incorporated into simulation models of SOM. The latter
would be an enormous advance because it would allow for: 1)
incorporating more mechanistic understanding in our SOM models;
2) a true validation of the internal structure of the model; and 3)
calibrate and validate SOM models based on more than just
comparing modeled and measured total C.

5. Conclusions

The relationship between aggregate stability and SOM had been
investigated intensively, but our Soil Biology & Biochemistry Citation
Classic paper added a dynamic dimension to this relationship by
showing that it is the turnover of the macroaggregates that de-
termines the stabilization of C within microaggregates. The logical
consequence is that the microaggregate-within-macroaggregate
fraction shows promising potential for early detection of changes
in soil C arising from changes in management. However, that po-
tential will only be realized if we further focus on elucidating the
dynamics of this fraction in different soils under different climatic
conditions across different ecosystems by applying isolation and
viewing approaches and integrating the ndings into simulation
models.


Acknowledgment

We thank Dr. Karolien Denef for putting together Table 1 and Dr.
Steven De Gryze for putting together Fig. 1. We are grateful to Dr.
Mirjam Pulleman for early discussions on isolating and viewing
soil structure. We would also like to thank Dr. Richard Burns for
inviting us to evaluate the use and signicance of our work on
aggregation and soil organic matter.


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