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Soil Biology & Biochemistry 57 (2013) 868e875

Contents lists available at SciVerse ScienceDirect

Soil Biology & Biochemistry

journal homepage: www.elsevier.com/locate/soilbio

Can intra-aggregate pore structures affect the aggregates effectiveness in

protecting carbon?
K. Ananyeva, W. Wang, A.J.M. Smucker, M.L. Rivers, A.N. Kravchenko*
Dept. Crop and Soil Sci., Michigan State University, East Lansing, MI 48824-1325, United States

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 13 June 2012
Received in revised form
16 October 2012
Accepted 18 October 2012
Available online 15 November 2012

Aggregates are known to provide physical protection to soil organic matter shielding it from rapid
decomposition. Spatial arrangement and size distribution of intra-aggregate pores play an important role
in this process. This study examined relationships between intra-aggregate pores measured using X-ray
computed micro-tomography images and concentrations of total C in 4e6 mm macro-aggregates from
two contrasting land use and management practices, namely, conventionally tilled and managed row
crop agricultural system (CT) and native succession vegetation converted from tilled agricultural land in
1989 (NS). Previous analyses of these aggregates indicated that small (<15 mm) and large (>100 mm)
pores prevail in NS aggregates while medium (30e90 mm) pores are more abundant in CT aggregates
(Kravchenko et al., 2011; Wang et al., 2012). We hypothesized that these differences in pore size
distributions affect the ability of macro-aggregates to protect C. The results of this study supported this
hypothesis. Consistent with greater heterogeneity of pore distributions within NS aggregates we
observed higher total C and greater intra-aggregate C variability in NS as compared with CT aggregates.
Total C concentrations and intra-aggregate C standard deviations were negatively correlated with fractions of medium sized pores, indicating that presence of such pores was associated with lower but more
homogeneously distributed total C. While total C was positively correlated with presence of small and
large pores. The results suggest that because of their pore structure NS macro-aggregates provide more
effective physical protection to C than CT aggregates.
2012 Elsevier Ltd. All rights reserved.

Keywords:
X-ray computed micro-tomography
Spatial variability
Native vegetation succession
Conventionally tilled row crop agricultural
system

1. Introduction
Macro-aggregates are known to provide physical protection to
soil organic matter shielding it from rapid decomposition and thus
are regarded among the key elements enabling soil C sequestration
(Beare et al., 1994; Paustian et al., 1997; Bossuyt et al., 2002; von
Ltzow et al., 2006). Intra-aggregate physical protection is the
leading driver of C sequestration occurring when land under
intensive agricultural management is converted to conservational
land use practices (Jastrow, 1996; Grandy and Robertson, 2007). In
soils under conservational land use, e.g., grasslands or soils abandoned from agriculture, macro-aggregates tend to have higher C
concentrations and are richer in newer C than other soil fractions
(Jastrow, 1996; De Gryze et al., 2004). The newly added C often
serves as a binding agent holding the macro-aggregates together.
When intra-aggregate physical protection is eliminated by crushing
macro-aggregates, the intra-aggregate C accumulated by conservational management is easily mineralized (Beare et al., 1994;
* Corresponding author.
E-mail address: kravche1@msu.edu (A.N. Kravchenko).
0038-0717/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.soilbio.2012.10.019

Hassink, 1997). However, when macro-aggregates stay intact for

prolonged time periods in undisturbed soils of conservational
management, decomposition of organic binding agents is sufciently slow to allow for formation of micro-aggregates where
physical protection is enhanced by physicochemical and chemical
protection processes (Six et al., 2000; Denef et al., 2001; Chenu and
Plante, 2006).
One of the mechanisms of organic matter protection is heterogeneity of soil microenvironment which limits the access of
decomposing microorganisms and their enzymes to organic
material (Schmidt et al., 2011). Macro-aggregate formation
increases such heterogeneity, and thus organic matter protection,
by increasing complexity in spatial arrangement of soil matrix
pores (Baldock and Skjemstad, 2000). Pores affect water and air
uxes, spatial distributions of soil nutrients, and movement of
microorganisms through soil on scales ranging from a soil prole to
a micro-aggregate (Or et al., 2007). For example, it has been shown
that large pores of preferential ow paths contain younger soil
organic C than their surroundings and serve as hotspots for biological activity (Bundt, 2001). Greater microbial activity and organic
matter decomposition was observed in larger pores or were

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K. Ananyeva et al. / Soil Biology & Biochemistry 57 (2013) 868e875

associated with greater numbers of large pores (Killham et al., 1993;

Yoo et al., 2006; Ruamps et al., 2011). Soil nutrients were reported
to accumulate around pores approximately 100e200 mm in diameter within soil aggregates (Jassogne, 2008). Pore arrangements
may restrict microbial access to organic material and reduce
microbial activity by limiting oxygen supply (Sexstone et al., 1985;
Golchin et al., 1994; von Ltzow et al., 2006).
Aggregate boundaries serve as dividers between smaller intraaggregate pores inside aggregates and larger inter-aggregate pores
surrounding them. Majority of air and water uxes occur via interaggregate pores from which air, water, chemicals, and microorganisms can enter the aggregates. Comparisons of the intraaggregate soil properties at different distances from aggregate
boundaries supply ample evidence of the importance of pore sizes
for intra-aggregate biogeochemical processes including those
related to C. Within the aggregates, gradients are often observed in
terms of a variety of soil properties reecting the proximity to
aggregate boundaries, that is, to inter-aggregate pores. Among the
gradients that have been observed are gradients in oxygen levels
(Sexstone et al., 1985), Ca, Mg, K, Na, Mn, K, Al, and Fe concentrations
(Santos et al., 1997; Jasinska et al., 2006), and in composition of
organic matter (Ellerbrock and Gerke, 2004; Urbanek et al., 2007).
Differences have been found between external and internal aggregate layers in terms of microbial activities (Jasinska et al., 2006) and
microbial community compositions (Blackwood et al., 2006). These
gradients appear to be soil specic. For example, Chenu et al. (2001)
found that microbial decomposition occurs throughout the whole
aggregates of a sandy soil, while only near the surface of a clay soil
aggregates. Especially diverse are results obtained for total C where
some studies reported no intra-aggregate patterns (Ellerbrock and
Gerke, 2004; Urbanek et al., 2007), while others found such gradients in some but not other soils (Santos et al., 1997; Jasinska et al.,
2006). It is likely that not only proximity to the inter-aggregate
pore space but also abundance, size, and connectivity of intraaggregate pores play a substantial role in intra-aggregate Crelated processes and resulting intra-aggregate patterns in spatial
distributions of C.
When land is abandoned from an intensive agricultural use and
is either converted into grassland or allowed to be taken by native
vegetation, multiple factors contributing to C sequestration become
activated. Among them are year-around presence of vegetation
cover supplying fresh organic inputs different in quality from those
of intensive agriculture, increased microbial and faunal activity,
elimination of soil disturbance, and an increase in quantities and
stabilities of soil aggregates. Recently it has been shown that such
soils are not only different from conventionally managed agricultural soils in terms of aggregate size distributions or aggregate
stability but also in terms of intra-aggregate pore characteristics.
For example, Peth et al. (2008) reported prevalence of long
continuous pores in a grassland aggregate while short thin interconnected pores were more abundant in a CT aggregate.
Kravchenko et al. (2011) observed greater heterogeneity in intraaggregate pore distributions in macro-aggregates from soil under
native succession vegetation as compared to conventional agriculture. Wang et al. (2012) reported greater fractions of 30e60 mm
crack-like pores in macro-aggregates from conventionally tilled soil
while greater fractions of >90 mm pores of biological origin in
aggregates from soil abandoned from agriculture for past 18 years.
We hypothesize that in addition to the listed above factors
contributing to C sequestration in soils converted to native vegetation the changes that take place in intra-aggregate pore structures constitute yet another factor that enables enhanced C
sequestration to take place in such soils.
The goal of this study is to determine whether the intraaggregate pore characteristics of macro-aggregates from

869

a conservational land use system make such aggregates more

effective in C protection as compared to those of conventional
intensive agriculture. We examined soils from two highly contrasting land uses: a heavily disturbed soil in a conventionally
plowed row crop agricultural system and an undisturbed soil under
native vegetation. During past 18 years the rst system was losing C
(Senthilkumar et al., 2009) while the second system has been
shown to rapidly accumulate it (Grandy and Robertson, 2007). The
objectives of the study are to assess spatial patterns of C distribution within the aggregates from the two systems and to examine
the relationships between intra-aggregate C and spatial distributions of intra-aggregate pores obtained via X-ray computed microtomography.
2. Materials and methods
2.1. Soil sampling
Soil samples were collected from Long Term Ecological Research
(LTER) site at the W. Kellogg Biological Station in southwest
Michigan. The soil is Kalamazoo loam (ne-loamy, mixed, mesic,
Typic Hapludalf), developed on glacial outwash. The LTER experiment was established in 1988 (for details on site description,
experimental design and research protocols see http://lter.kbs.msu.
edu) (KBS, 2011). Prior to 1988 the entire experimental site was in
conventionally plowed row crop agricultural management for at
least past 100 years. The two most contrasting treatments in terms
of soil management were used in this study, namely, conventional
tillage (chisel-plowed) corn-soybean-wheat rotation with conventional chemical inputs (CT) and native succession grassland,
abandoned from agricultural use after spring plowing in 1989 (NS).
Sampling was conducted in 2008 from two adjacent 1 ha plots,
one plot per treatment. Within each plot three randomly selected
sampling locations were identied and at each location a soil block
approximately 15  15  15 cm in size was extracted by spade from
the soil surface. Soil blocks were packed into closed-lid plastic
containers and transported to the lab, where they were gently
manually crushed to ensure breakage along the natural planes of
weakness. Then crushed soil was air-dried and dry-sieved to obtain
macro-aggregates 4e6.3 mm in size. In the rest of the manuscript
we will refer to them simply as aggregates. Grandy and Robertson
(2007) reported that based on wet-sieving results 2e8 mm
aggregates constituted around 15% and 50% of the aggregates in
CT and NS treatments, respectively. The aggregate size was chosen
as a compromise between the need to have aggregates large
enough to provide sufcient amount of soil material for multiple
intra-aggregate measurements of soil C and the need for aggregates
to be sufciently small to enable ne resolution in X-ray computed
micro-tomography scanning. Air-dried aggregates were stored in
airtight containers at the room temperature until used for analyses.
Six replicated aggregates from each treatment, i.e., two aggregates
from each sampling site, were used in further analyses.
2.2. Image collection and analyses
The aggregate images were obtained at the Advanced Photon
Source of Argonne National Laboratory (station BMD-13) using Xray micro-tomography. Image resolution was equal to approximately 15 mm in x, y, and z directions. To obtain information on
intra-aggregate pores, the images were segmented by classifying
each image voxels as either a pore or a solid material. Segmentation
was conducted using indicator kriging method (Oh and Lindquist,
1999; Wang et al., 2011). The pore characteristics that were
studied are image-based porosity, that is the percent of pores
visible at image resolution (>15 mm), and size distributions of such

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K. Ananyeva et al. / Soil Biology & Biochemistry 57 (2013) 868e875

pores. Pore size distributions were assessed using burn number

distribution approach implemented in 3DMA-Rock software
(Lindquist et al., 2000). A burn number represents the shortest
distance from the center of the pore to the pores wall and can be
regarded as the measure of pore diameter. For clarity purposes, in
this study we will refer to the pores with different burn numbers by
converting the burn number values into ranges of pore diameter
values. That is, pores with burn number equal to 1 will be referred
to as 15e37.5 mm pores, those with burn number of 2 will be called
37.5e67.5 mm pores, those with burn number of 3 67.5e97.5 mm
pores, etc. The aggregates used in this study are a subset of the
aggregates analyzed for pore spatial patterns by Kravchenko et al.
(2011) and Wang et al. (2012) where detailed descriptions of the
image collection, pre-processing, and processing procedures are
supplied. Here we only provide a summary of the pore characteristics for the 12 aggregates that were used for C measurements of
this study to facilitate the discussion of C results.

sections and 4e8 interior sections. After cutting, each section of the
aggregate was oven-dried for 24 h at 104  C, weighed, and subjected to C measurement. Total C measurements were performed
on soil aggregate sections using the elemental combustion system
ECS 4010 (Costech Analytical, USA).
The variables that were analyzed in this study consisted of
individual C values from each section; the C values from sections
combined into aggregate exterior and interior groups; and
summary statistics for each aggregate calculated from individual
section values including aggregates minimum and maximum C
values, and aggregates C standard deviations.
Virtual cutting of aggregate images was performed to match
physical cutting (Fig. 1a). It yielded regions in the 3D tomographic
images that corresponded to the physically cut sections. Imagebased porosity and pore size distributions were determined in
each virtual section of each aggregate.
2.4. Statistical analysis

2.3. Aggregate cutting and intra-aggregate C measurements

To assess intra-aggregate spatial variability of soil C and to relate
C content to intra-aggregate pore characteristics, each aggregate
was cut into 11e20 sections. In order to facilitate cutting, the
aggregates were wetted to ll approximately 30% of their pore
volume with distilled water. Then cutting was performed using
a sharp #11 scalpel and a 24 magnifying glass. The relative position of each aggregate section was recorded. Depending on the size
and shape of the aggregate we were able to cut 6e12 exterior

The inuence of land management and position within the

aggregate, i.e., exterior vs. interior, on total C values was analyzed
using individual aggregate section data in PROC MIXED in SAS 9.2
(SAS Institute, 2009). The statistical model included land management, position, and interaction between them as xed factors.
Multiple runs of C measurements on ECS 4010 systems were
included as a random blocking factor. Aggregates nested within
land use and the interaction between aggregates and positions
were the random factors used as error terms for testing land use

Fig. 1. 3D X-ray computed micro-tomography image of one of the studied aggregates with a schematic representation of the sections that the aggregate was cut into (a) and 2D
images of representative NS (b) and CT (c) aggregates. Examples of the areas with no pores visible at the studied resolution, i.e., pores >15 mm, are marked by rectangles. Arrows are
pointing to large (>100 mm) pores of biological origin on the NS image and to medium (30e90 mm) pores on the CT image.

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K. Ananyeva et al. / Soil Biology & Biochemistry 57 (2013) 868e875

18.0

16.0

*
CT
NS

14.0

Total C, g/kg

and position effects, respectively. Analysis of land use effects on

minimum and maximum intra-aggregate C values and on intraaggregate standard deviations was conducted using the statistical
model with land use as the xed factor. In all analyses the normality
assumption was checked using normal probability plots and the
data were log-transformed in cases of right-skewed distributions.
Equality of variances was checked using visual examination of box
plots and, where applicable, analysis with unequal variances was
conducted. The differences were declared to be statistically significant at 0.05 level.
Analyses of the relationships of C values in aggregate sections
along with C aggregate means, minimums, maximums and standard deviations with pore characteristics in the aggregate sections
were performed in SAS 9.2 using PROC REG. Regression analyses
were conducted for the entire data set and for individual data sets
of each land use.

871

12.0

10.0

8.0

6.0

4.0

minimum

mean

maximum

Summary statistics of intra-aggregate sections

3. Results
3.1. Intra-aggregate C variability
Overall, the intra-aggregate C variability in NS aggregates was
much higher than that in CT. Standard deviations of C in sections of
NS aggregates were equal to 2.2 g/kg, which was twice as high as
that in CT aggregates, 1.1 g/kg (p < 0.05). In all but one CT aggregate
the range of C values among the aggregate sections was substantially lower than that in NS aggregates (Fig. 2). In CT aggregates the
intra-aggregate range of C values was w3.7 g/kg, while in NS it was
w7.7 g/kg and in some NS aggregates the difference between the
lowest and the highest C values was as high as w9 g/kg.
Across both CT and NS aggregates the lowest C concentrations
observed within the aggregate sections were equal to 5e6 g/kg
while the highest were around 20 g/kg. The minimum C values
in aggregate sections were equal to 7.8 and 8.7 g/kg in CT and NS
treatments, respectively, and were not signicantly different from
each other (p < 0.05) (Fig. 3). A much larger difference was
observed between maximum C values which were 1.5 times
higher in NS than in CT aggregates (Fig. 3). Along with sections
relatively rich in C, NS aggregates contained sections with C
concentrations as low as 5e7 g/kg, that is, the values similar to
those of CT aggregates.
In both CT and NS the aggregate sections located in the aggregate interiors tended to have lower C levels than the aggregate

Fig. 3. Minimum, mean, and maximum total C values in sections of the studied
aggregates from CT and NS treatments. Bars represent standard errors. The difference
between CT and NS aggregates was statistically signicant for means and maximums
(p < 0.05) (marked with *).

exteriors (Fig. 4). The difference between interior and exterior

positions was statistically signicant in NS aggregates (p < 0.05).
Overall the NS aggregates had higher mean C than CT aggregates, equal to 12.3 and 9.3 g/kg in NS and CT aggregates, respectively (Fig. 3). However, the results of comparisons between CT and
NS were different in aggregate exteriors vs. interiors (Fig. 4). In
aggregate exteriors the difference between NS and CT was equal to
2.7 g/kg; it was signicantly greater than zero (p < 0.05). However,
in the aggregate interiors the difference was smaller, 1.7 g/kg, and
not signicantly different from zero.
3.2. Relationships between C and intra-aggregate pore
characteristics
Across all aggregates from both treatments there was a weak but
statistically signicant negative correlation between total C and
image-based porosity, i.e., percent of pores >15 mm (Fig. 5a). That is,
the aggregate sections with higher percent of pores visible on the
images tended to have lower C levels. However, as can be observed
from Fig. 5a, the variability in C values from individual sections was

22.0

14.0

20.0

18.0

Total C, g/kg

Total C, g/kg

12.0
16.0
14.0
12.0
10.0

Exterior
Interior

10.0

8.0

8.0
6.0
4.0
CT6 CT7 CT8 CT9 CT10 CT12 NS6 NS7 NS8 NS9 NS10 NS11

CT aggregates

NS aggregates

Fig. 2. Total C concentrations in the sections of the studied aggregates. Aggregate ID

numbers are shown on the x-axis.

6.0

CT

NS

Fig. 4. Total C concentrations in interior and exterior sections of the CT and NS

aggregates. Bars represent standard errors. The difference between the positions was
statistically signicant in NS (p < 0.05) (marked with *).

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K. Ananyeva et al. / Soil Biology & Biochemistry 57 (2013) 868e875

image visible pores, i.e., pores 15e37.5 mm in size (Fig. 5b), and
a signicant negative correlation between C and medium sized
(37.5e67.5 mm) pores (Fig. 5c). Regression equation predicted total
C to be around only 8 g/kg when 15e37.5 mm pores constituted 30%
of all the image-based pores, while it increased to 13 g/kg when
15e37.5 mm pores constituted 90% of the image based pores.
Regression equation predicted total C to be around only 9 g/kg in
sections with >25% of 37.5e67.5 mm pores, while it was as high as
15 g/kg in sections with only w5% of 37.5e67.5 mm pores. Weak
negative correlation was also observed between C and 67.5e
97.5 mm pores. In NS no signicant correlation was observed
between C and larger size pores, while in CT intra-aggregate total C
was not correlated with pores of any sizes.

25

R2=0.11

Total C, g/kg

20

15

10

0
0

10

15

4. Discussion

20

Image-based porosity, %

25.0

R 2=0.10

Total C, g/kg

20.0

15.0

10.0

5.0

0.0
0.2

0.4

0.6

Fraction of 15-37.5

0.8

1.0

m pores

25.0
2

R =0.17

Total C, g/kg

20.0

15.0

0.05

0.30
10.0

0.0
0.00

0.05

0.10

0.15

Fraction of 37.5-67.5

0.20

0.25

0.30

m pores

Fig. 5. Correlations between total C in aggregate sections with a) image-based

porosity, i.e., pores > 15 mm for the entire data set; and with fractions of 15e
37.5 mm pores (b) and 37.5e67.5 mm pores (c) in NS aggregates.

CT
NS

0.25

0.20
0.03
0.15
0.02
0.10
0.01

0.05

0.00

0.00

37.5-67.5

very high, especially in the sections with lower image-based

porosity.
The relationships between C and pores differed between the
two studied treatments. In NS there was a statistically signicant
positive correlation between total C and presence of the smallest

0.04

Fraction (pores >127.5 m)

5.0

Fraction (pores 37.5-67.5 m)

Image analysis indicated substantial differences in intraaggregate pore size distributions of NS and CT systems
(Kravchenko et al., 2011; Wang et al., 2012). Kravchenko et al.
(2011) observed greater uniformity of pore distributions within
CT as opposed to NS aggregates, while Wang et al. (2012) reported
that NS aggregates had more large pores (>97.5 mm) and more
small pores (<15 mm) than CT aggregates; however, medium size
pores (37.5e97.5 mm) were more abundant in CT aggregates. An
example of two representative 2D images from CT and NS aggregates is shown in Fig. 1b and c. NS aggregates contained multiple
areas of soil material with very few pores visible at the image
resolution, that is > 15 mm pores (Fig. 1b marked by rectangles). At
the same time, there were multiple large round pores (>100 mm) in
NS aggregates that likely originated from either roots or animal
borrowing activities (Fig. 1b marked by arrows). For the aggregates
used in this study such pores were 5 times more abundant in NS
than in CT soil (Fig. 6). Conversely, aggregates from CT possessed
a uniformly distributed highly interconnected network of moderately sized pores (37.5e67.5 mm) (Fig. 1c marked by arrows). Such
pores were present in NS aggregates as well, though in fewer
numbers. In the studied aggregates the fraction of such pores was
equal to 0.23 in CT as opposed to 0.16 in the NS aggregates (Fig. 6).
As has been noted by Wang et al. (2012) these pores were often
cracks of non-biological origin observed to evenly permeate most
CT aggregates.
We hypothesized that these differences in intra-aggregate pore
characteristics could lead to more effective physical protection of

>127.5

Pore size, m
Fig. 6. Fractions of 37.5e67.5 mm and >127.5 mm pores in the studied CT and NS
aggregates. Bars represent standard errors. Left y-axis corresponds to pores 37.5e
67.5 mm; right y-axis corresponds to pores >127.5 mm. In both pore size classes the
difference between CT and NS treatments was statistically signicant (p < 0.05).

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K. Ananyeva et al. / Soil Biology & Biochemistry 57 (2013) 868e875

a
Average intra-aggregate total C, g/kg

18.0
16.0

R2=0.45

14.0
12.0
10.0
8.0
6.0
4.0

-5

-4

-3

-2

ln(Maximum intra-aggregate fraction of >127.5 m pores)

Standard deviation for intra-aggregate total C, g/kg

b
3.5
3.0

R2=0.42

2.5
2.0
1.5
1.0
0.5
0.0

-5

-4

-3

-2

ln(Maximum intra-aggregate relative frequency of >127.5 m pores)

c
Standard deviation for intra-aggregate total C, g/kg

soil C within NS as compared to CT aggregates. Note that here we

are specically referring to 4e6 mm macro-aggregates, while
aggregates of different size classes can possess different pore
characteristics. It is likely that absence of sizeable pathways might
restrict access of microorganisms and microbial enzymes into areas
of NS aggregates with low abundance of >15 mm pores, while
limited air ow might reduce microbial activity, thus protecting
organic matter there from decomposition. Small pores (<4 mm)
indeed have been reported to be positively correlated with C
accumulation (Strong et al., 2004).
On the other hand, root-originated large pores could serve as
sources of incoming C within the aggregates by either exudates
from roots and microorganisms located within them (Alami et al.,
1999; Hinsinger et al., 2005; Watteau et al., 2006) or by dissolved
organic matter transported through such pores (Park et al., 2007).
Air regime in such pores is benecial for aerobic bacterial activity.
Other conditions however might render these pores to be a harsher
environment for bacterial survival. For example, bacteria there
might be more susceptible to predation, while special water regime
of such pores, e.g., fast convective water ow for short periods of
time and lack of water for most of the time, might limit movement
of bacteria and transport of microbial enzymes (Or et al., 2007).
Nunan et al. (2003) observed lower bacterial densities in direct
proximity of larger pores while the densities increased at >20 mm
distances from pore boundaries.
Consistent with the above considerations, the aggregates of this
study where small (15e37.5 mm) and large (>127.5 mm) pores were
more abundant indeed appeared to be more effective in C protection by exhibiting higher C levels and greater intra-aggregate C
variability. In the NS aggregates the intra-aggregate sections with
higher percent of 15e37.5 mm pores tended to have higher C
(Fig. 5b). Across both treatments the aggregates with high fraction
of >127.5 mm pores tended to have higher C levels (Fig. 7a). Greater
intra-aggregate C variability can be interpreted as a potential
presence of intra-aggregate areas where conditions are especially
benecial for C protection as opposed to the main body of the
aggregate. Majority of aggregates from NS, that is, from the land use
that intensively sequesters C (Grandy and Robertson, 2007),
demonstrated such variability with almost every aggregate containing sections with very low as well as very high C levels (Fig. 2).
The overall higher mean C levels of NS aggregates appear to be not
due to a uniform increase in C but due to presence of such intraaggregate maximum C spots (Fig. 3). The importance of large
pores in this process is further highlighted by positive correlation
between >127.5 mm pores and standard deviations of intraaggregate C (Fig. 7b). The same statistically signicant trend was
present when the relationship between >127.5 mm pores and intraaggregate C standard deviations was examined in the CT and NS
aggregates separately (data not shown).
These results are consistent with those reported in literature.
Strong et al. (2004) reported faster decomposition of plant residues
in 15e60 mm pores, but lower C decomposition in soils with higher
percent of very small and very large pores. Chenu et al. (2001)
showed that bacterial growth and decomposition of added
substrate was limited mainly to the surface of clayey aggregates
with ne pores, while sandy aggregates with frequent medium and
large pores permitted easy transport of substrate and bacteria into
their interiors, and active substrate decomposition there. Strong
et al. (2004) reported a negative correlation between total C and
15e60 mm pores as well as greater microbial activity in such pores.
Our results also point to the negative role of medium sized pores
in C protection. A negative correlation between C in the aggregate
sections and the fractions of 37.5e67.5 mm pores was observed for
the entire data set (data not shown) and for the NS aggregates
(Fig. 5c). Greater presence of such pores not only resulted in overall

873

3.5
3.0

R2=0.26

2.5
2.0
1.5
1.0
0.5
0.0
0.10

0.15

0.20

0.25

0.30

Average intra-aggregate relative frequency of 37.5-67.5 m pores

Fig. 7. Relationships between maximum intra-aggregate fractions of >127.5 mm pores
(log-transformed) and total C concentration within the aggregates (a) and standard
deviation of total C concentration within the aggregates (b); and (c) between standard
deviation of total C concentration within the aggregates and the intra-aggregate
averages in fractions of 37.5e67.5 mm pores.

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K. Ananyeva et al. / Soil Biology & Biochemistry 57 (2013) 868e875

lower intra-aggregate C levels but also made the intra-aggregate C

distribution more uniform. Standard deviation of intra-aggregate C
was negatively correlated to the abundance of 37.5e67.5 mm pores
(Fig. 7c). As mentioned earlier, such pores were found to be more
uniformly distributed through CT than through NS aggregates
(Kravchenko et al., 2011) and, consistently, the distribution of C
through CT aggregates was much more uniform than that in NS
aggregates (Figs. 2 and 3).
Another likely cause for the presence of more uniform C
distributions within CT aggregates is frequent aggregate turnover
due to tillage (Balesdent et al., 2000; Six et al., 2000) when soil
material is recurrently mixed during aggregate destruction and
reformation. However, analysis of the relationship between the
presence of intra-aggregate medium sized pores and intraaggregate C variability indicated that not only in CT but also in NS
aggregates, higher abundance of such pores was associated with
lower variability in C values (Fig. 7c).
The advantage of this study is that the aggregate samples were
collected in close spatial proximity (w25 m apart), that is, from the
soil of the same mineralogy and texture. Also until 1989 they had
similar vegetation and management history. This allows us to
unequivocally point to the lack of soil disturbance and differences
in vegetation, that is, in timings, quantities and qualities of organic
inputs, as the key factors driving the differences in both intraaggregate pore distributions and their relationships with soil C
levels. A recent study conducted at KBS-LTER demonstrated that NS
had more than twice higher microbial biomass, short-term evolved
CO2eC, and mineralizable C than those of CT treatment (Jangid
et al., 2011).
As an indication of vegetations contribution to aggregate
formation, consider that in most of the aggregates with high fraction of >127.5 mm pores, we observed root residues inside the large
pores. This suggests that these were the aggregates of root origin,
that is, the aggregates formed around relatively large living roots.
Aggregate formation around plant roots could have led to
compaction of the soil material not only directly adjacent to the
root but also throughout the entire aggregate, thus eliminating
medium sized pores (Hinsinger et al., 2005). Such compacted
regions could be the areas with very few pores visible at image
resolution (pores >15 mm) marked on Fig. 1b for the NS aggregate.
Roots are probably the leading aggregate forming factor in NS
where root systems from a variety of plant species are active during
most of the year. There were only a few aggregates of root origin,
i.e., the aggregates with clearly visible root remnants in large
round pores, among CT aggregates (Kravchenko et al., in press). The
majority of CT aggregates could have formed not around live roots
but around dead POM nuclei material or as a result of physical
effects of wetting/drying or fungal enmeshment (Park and
Smucker, 2005; Urbanek et al., 2011). We can speculate that the
inux of root originated C and the lack of medium pores are the
driving causes of the enhanced C protection in the aggregates of
root origin as compared to other aggregates. Furthermore, the local
compaction occurring within root-originated aggregates might be
particularly benecial for formation of micro-aggregates thus
further enhancing C protection (Six et al., 2000, 2002; Chenu and
Plante, 2006). However, further experimentation with aggregates
possessing different pore size distributions but of the same
management treatment would be necessary to test this hypothesis.
Another source of C inux into aggregates is from interaggregate pores that resulted in the higher C in aggregate exteriors observed in this study (Fig. 4). These observations are
consistent with the hypothesis by Park and Smucker (2005) and
Park et al. (2007) regarding movement of dissolved organic matter
from inter-aggregate pores into the aggregates as one of the
mechanisms of increasing C contents. Park and Smucker (2005)

reported that newer C is located more in the aggregate exteriors,

and diffuses into aggregate interiors during subsequent wetting
and drying. Continuous inux of dissolved organic C from outside
might be reinforcing and strengthening the existing intraaggregate bonds derived from interactions of metals and organic
ligands with mineral surfaces (Horn and Smucker, 2005). Slower
macro-aggregate turnover in NS (Six et al., 1999) would allow for
a longer duration for enrichment of C exteriors with inter-aggregate
C inuxes. Further studies are necessary to address this point.

5. Conclusions
Previously reported differences in intra-aggregate pore size
distributions between macro-aggregates of an Alsol under
a conventionally tilled agricultural system and under a long term
native succession vegetation system were associated with intraaggregate patterns in distribution of total C. These results were
consistent with the hypothesis that NS aggregates provide greater C
protection benets than CT aggregates. However, further studies
with direct measurements of C decompositions and microbial
activities in aggregates of different treatments and with different
pore size distributions are necessary to enable its full testing.
Overall higher total C and higher intra-aggregate variability in C
were observed in the aggregates with greater numbers of very
small and very large pores, while lower total C and lower variability
were found in aggregates with greater abundance of medium sized
pores. It appears that greater heterogeneity in spatial distribution
of intra-aggregate pores, characterized by multiple areas with very
few if any pores >15 mm and more biologically originated >100 mm
pores, was advantageous for C accumulation.
It can be speculated that the differences between pore structures within soil aggregates translate into different modes of biological activity, and thus different rates of organic matter
decomposition. In undisturbed soils, macro-aggregate interiors
may provide both the environments conducive to enhanced
microbial activity, e.g., vicinities of large pores, and the environments with limited oxygen/bacterial access where microbial
activity will be limited. Conversely, an increased intra-aggregate
aeration can promote rapid and uniform decomposition of C
through whole bodies of macro-aggregates in highly disturbed
soils. Further measurements are necessary to explore these
hypotheses.

Acknowledgements
The project was supported in part by the National Research
Initiative of the USDA Cooperative State Research, Education and
Extension Service, grant number 32008-35102-04567. Support for
this research was also provided by the NSF Long-Term Ecological
Research Program at the Kellogg Biological Station and by Michigan
State University AgBioResearch. Authors would like to thank H.-C.
Chun, A. Worth, M. Ladoni, J-D Munoz, and M. Mazher for their
help in sampling, CMT scanning, and aggregate processing.

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