06) 147–154www.elsevier.com/locate/geoderma

Geoderma 137 (20

Similarity of differently sized macro-aggregatesin arable soils of different texture

Søren M. Kristiansen a, Per Schjønning a, Ingrid K. Thomsen a,⁎, Jørgen E. Olesen a,Kristian Kristensen b, Bent T. Christensen a

a Department of Agroecology, Danish Institute of Agricultural Sciences, Research Centre Foulum, PO Box 50, DK-8830 Tjele, Denmarkb Department of Genetics and Biotechnology, Danish Institute of Agricultural Sciences, Research Centre Foulum, PO Box 50, DK-8830 Tjele, Denmark

Received 21 June 2005; received in revised form 3 July 2006; accepted 15 August 2006Available online 28 September 2006

Abstract

The concept of structural and functional hierarchy prevails in studies on the turnover of organic matter (OM) in soil. We examined the degreeof structural hierarchy in arable soils sampled at six locations along a naturally occurring texture gradient (11 to 45% clay, 12.9 to 15.8 mg C g−1

soil). One set of samples was incubated with 14C labeled ryegrass for 8 months while another set remained unamended. Constituent aggregateswere isolated from the soils by fragmentation along natural planes of weakness. Eight aggregate size-classes (b0.063, 0.063–0.125, 0.125–0.25,0.25–0.50, 0.5–1.0, 1–2, 2–4 and 4–8 mm) were isolated and analysed for textural composition and contents of total-C (and 14C). The evolutionof CO2 (and of 14CO2) during a 15-week laboratory incubation was determined for four aggregate size-classes in the range 0.125 to 2 mm. Theaggregate size distributions reflected differences in whole soil texture with decreasing proportions of b1 mm aggregates and increases in N2 mmaggregates with increasing whole soil clay content. While the texture of aggregates N1 mm was similar to that of the whole soil, aggregates withinthe range 0.125 to 1 mm were enriched and those b0.125 mm depleted in sand. Macro-aggregates (N0.25 mm) showed a similar distribution ofnative and of 14C labelled OM. For micro-aggregates, concentrations of native and labelled OM increased with decreasing size. When correctedfor sand particles N63 μm, the only consistent trend in native and labelled OM across aggregate size-classes was a higher content in micro- than inmacro-aggregates. While native OM showed no consistent trend in lability, the 14C labeled OM in the 0.125–0.25 mm size-class was more labilethan that in macro-aggregates. The similarity of whole soils and differently sized macro-aggregates suggests that macro-aggregates in our soilsexperience a rapid turnover and merely represent fragments of larger soil units (peds and clods) that constitute the soil in situ.© 2006 Elsevier B.V. All rights reserved.

Keywords: Aggregate hierarchy; Macro-aggregates; Micro-aggregates; Organic carbon; Soil fragmentation; Soil texture

1. Introduction

The interactions between OM turnover and soil structureremain subject to much current research, the occurrence ofdifferently sized organomineral complexes (OMC) being acharacteristic feature of most soils (Oades, 1993; Christensen,1996; Baldock, 2002). Based on physical fractionation meth-ods, Christensen (1996, 2001) identified three levels ofstructural and functional complexity in the turnover of OM inarable soils. The basic elements of soil structure are clay-, silt-

⁎ Corresponding author. Tel.: +45 8999 1720; fax: +45 89991719.E-mail address: Ingrid.Thomsen@agrsci.dk (I.K. Thomsen).

0016-7061/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.geoderma.2006.08.005

and sand-sized primary OMC and particulate organic matter(POM) isolated from fully dispersed soils. The principalmechanisms related to OM turnover at this level of complexityare surface reactions between OM, microorganisms and soilminerals. These primary OMC are incorporated into aggregates(secondary OMC) of different size which in turn are embeddedin the clods and peds that constitute the structurally intact soil(the soil in situ). The aggregates released from structurally intactsoil by some disruptive force, are thought to represent a higherlevel of complexity than the primary OMC, with the physicalentrapment of POM within aggregates, and the creation of gas,moisture and solute gradients exemplifying emergent proper-ties. The structurally intact soil represents the highest level ofcomplexity. Emergent attributes at this level are related to the

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presence of coarse roots, resource islands, macropores andpreferential flow, the degree of pore system continuity, andbioturbation by soil fauna.

Studies on aggregate dynamics are most often based on theconcept of aggregate hierarchy advanced by Tisdall and Oades(1982) and subsequently elaborated by Oades andWaters (1991),Golchin et al. (1997), and Six et al. (1999). According to thisconcept, the concentration of OM should increase with increasingaggregate-size, and macro-aggregates should be enriched inrecently added and more labile OM (Angers and Carter, 1996;Jastrow andMiller, 1997; Six et al., 2000;Mikha and Rice, 2004).

We have previously evaluated the chemical and biologicalproperties of primary OMC in relation to OM turnover(Christensen, 1996, 2001) and the mechanisms that regulatemicrobial activity in structurally intact soils under long-termarable usage (Schjønning et al., 1999, 2003; Thomsen et al.,1999, 2003). Here we examine aggregate characteristics (tex-ture and OM distribution) using arable soils of similar miner-alogical composition and subject to the same management andclimate. From two sets of soil sampled at six locations along anaturally occurring clay gradient, aggregates were isolated byfracturing the soil along natural planes of weakness. In one setof soils, aggregation had taken place recently after previousdisturbance, while the other set was structurally intact soilssampled immediately before fractionation.

2. Materials and methods

2.1. Site and soils

Bulk soil was sampled from the 0–20 cm depth (A-horizon)at six locations (termed L1, L2, L3, L4, L5, and L6) along anaturally occurring texture gradient in an arable field nearLerbjerg, Denmark (56°22′N, 9°59′E). The field is on morainaldeposits from the last glaciation (Weichselian) and has beencropped mainly to winter wheat (Triticum aestivum L. ssp. vul-gare) with pig slurry and mineral fertilizers being applied in thespring. Straw has been removed after harvest, and a conventionaltillage system with moldboard ploughing to 20–25 cm has beenemployed. Selected soil characteristics are shown in Table 1. Allsoils classify as Hapludolls or Argiudolls. Themineralogy of soilfrom the six locations is similar, the clay fraction (b2 μm) beingdominated by illite, smectite, and vermiculite, with smaller

Table 1Selected properties of the L1 to L6 soils from locations along the Lerbjerg textural

Soil code —texture class†

Clay b2 μm Silt 2–20 μm Sand 20–2000 μm

mg g−1

L1 — sl 109 76 792L2 — sl 157 103 717L3 — scl 208 112 658L4 — scl 310 130 534L5 — sc 374 146 458L6 — c 452 148 337

† sl, sandy loam; scl, sandy clay loam, sc, sandy clay, c, clay.‡ dithionite–bicarbonate–extractable Fe.§ bicarbonate-extractable P.

proportions of kaolinite (Schjønning et al., 1999). The silt (2–20 μm) and sand (20–2000 μm) fractions are both dominated byquartz with feldspars and micas contributing somewhat less.Mean annual (1961–1990) precipitation is 650 mm and meanannual temperature 7.3 °C.

One set of soils was sampled in 1993 and has previouslybeen used in the studies described by Schjønning et al. (1999)and Thomsen et al. (1999). Each soil was air-dried (20–30 °C)and thoroughly mixed and sieved to b2 mm to provide ahomogeneous soil. Subsequently the soils were pre-incubatedoutdoors in order to re-establish microbial biomass and activity,to deprive the soils of labile OM and to regenerate soil structure.About 6 kg of each of the six soils were placed in plastic boxes(340×250×158 mm) with perforated bottoms (6 mm open-ings). A 30 mm layer of 0.1–0.3 mm sand enclosed in a fine-mesh synthetic cloth was located at the bottom of the boxes. Thesoil was then placed on top of the sand layer and wetted bycapillary rise of water applied to a larger container housing theplastic box with the soil. The wetting procedure lasted for about2 days. The boxes were then removed from the wettingcontainer and placed in the open on top of a 100-mm layer offine sand that ensured capillary contact to the well drained soilbeneath. Thus the soils were freely drained and allowed tofollow the drying and wetting cycles determined by localweather conditions. When friable, the soils were “tilled” by asmall hand-held implement that simulated the action of a tineharrow. Irrigation to field capacity was applied in dry periods toincrease the frequency of drying–wetting cycles and the numberof tillage procedures (see Schjønning et al., 1999 for details).After 9 months of pre-incubation, the soils were slowly air-driedand gently mixed with ground (b0.5 mm) 14C-labeled ryegrassmaterial (Lolium multiflorum L.). The grass dry matter consistedof 417 mg C and 11.3 mg N g−1 and had a specific activity of69.9 kBq g−1 C. The addition rate of grass drymatter was 2.79mgg−1 soil. The soils were rewetted and returned to the open for afurther 8-month period. The soils were kept free of vegetation.This set of soils was termed MAN (manipulated soils).

To enable comparison between the MAN soils and cor-responding natural field soils, another set of soils was sampledin winter 1997 from the same six locations as in 1993. Thesesoils were termed NAT (natural soils). The NAT soils weresampled immediately before the procedure for isolation ofaggregates began.

gradient

Bulk density Fed‡ Phosphorus§ C/N pH (CaCl2)

Mg m−3 mg kg−1

1.25 745 54 9.7 6.61.30 711 89 9.2 6.71.25 803 87 9.2 7.21.33 1039 48 9.5 7.41.22 1169 51 8.4 7.51.29 1422 39 8.0 7.5

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2.2. Isolation of aggregates

The NAT and MAN samples were brought to a ventilatedroom (20–30 °C) and spread out into a thin layer. The soils weredried to a level that would allow limited mechanical stress toinduce maximum brittle failure along natural planes of weak-ness. For light- to medium-textured soils this occurs when soilwater contents are near the lower plastic limit (Dexter and Bird,2001). During this phase, soil moisture conditions wereevaluated daily. When individual soil clods had reached thedesired condition, these were manually crumbled to b20 mm.When clods were fully air-dried, these were successivelycrumbled to b8 mm using the roller method suggested byHartge (1971).

The aggregates obtained in this way were size separated by abrief sieving procedure. Portions of 300 g were transferred to anest of sieves (8, 4, 2, 1, 0.5, 0.25, 0.125 and 0.063 mm), andgently passed through the 8-mm sieve. The material residing onthe nest of sieves was then treated for 10 s on a Retsch KS1000sieving machine (Retsch GmbH., Haan, Germany) and theaggregates on the 4-mm sieve (4–8 mm) were collected. Thematerial retained on the 2-mm sieve was treated further for 15 sand the 2–4 mm aggregates collected while the material passingthis sieve was transferred to the next smaller sieve. Theprocedure was repeated with 1, 0.5, 0.25, 0.125 and 0.063-mmsieves, using sieving times of 15, 30, 45, 45 and 45 s, re-spectively. Preliminary tests showed that these sieving timeswere sufficiently short to accomplish a quantitative separationof the various aggregate size-classes while aggregate abrasionduring the sieving procedure remained negligible. The quantityof material employed at each sieving step was in accordancewith the DIN 22019/1 standard (Deutsche Norm, 1985). In thisway, eight aggregate size-classes were obtained: b0.063,0.063–0.125, 0.125–0.25, 0.25–0.50, 0.5–1.0, 1–2, 2–4 and4–8 mm.

2.3. Incubation of MAN samples

In four aggregate fractions (0.125–0.25, 0.25–0.50, 0.5–1.0,and 1–2 mm) from the MAN set of samples, sufficient soil wasavailable to allow for incubation of two or three replicate soilportions. Samples of 50 g eachwere transferred to small stainlesssteel cylinders (diam. 61 mm), placed on ceramic plates andslowly saturated with water from beneath. After 3 days, thecylinders were taken to a water potential of −100 hPa byapplying suction to the ceramic plates. Each cylinder was thenplaced on a metal mesh in sealed 2-L jars and kept in the dark at20 °C for 15 weeks. A beaker with 10 mL of water was situatedin the jar to minimize sample desiccation. Evolved CO2 (labeledand unlabeled) was trapped in another beaker with 15 mL of 1MNaOH. Every third week, the CO2 trap was replaced by a trapwith fresh NaOH.

2.4. Analyses

Soil pH was determined with a glass electrode in asuspension made by adding 10 g of soil to 25 cm3 of a

0.01 M CaCl2. Readily available P was extracted by a 0.5 NNaHCO3 solution (Olsen et al., 1954) and dithionite–bicarbonate–extractable Fe determined according to Mehraand Jackson (1960). The total-C content in whole-soils andaggregate size-classes was determined on ball-milled sub-samples using a LECO CNS-1000 analyzer with IR detector(LECO Corporation, St. Joseph, MI). The content of 14C inthe MAN samples was determined on another aliquot of ball-milled material with a Packard Sample Oxidizer 307 (PackardInstuments Co., Meriden, CT), the evolved CO2 being trappeddirectly in a Carbosorb/Permaflour scintillation cocktail. The14C activity was subsequently determined by liquid scintilla-tion using the Packard Tri-Carb 2250 CA. Similarly, the14CO2 trapped in NaOH was determined after mixing a 2.5mL aliquot of the NaOH with 10 mL of Hionic Flourscintillation cocktail. Total CO2 in the NaOH was determinedby titration of excess NaOH with HCl after precipitation ofCO2 with BaCl2.

The primary particle composition (texture) of whole-soilsand aggregates was determined by a combined hydrometer andwet-sieving procedure (Gee and Or, 2002). For the MANsamples, material isolated in aggregate classes b0.125 mm fromthe L3, L4, L5 and L6 soils was not sufficient to allow foranalyses of texture and total-C contents. This was also true forthe b0.063 mm and N2 mm aggregates from the L2 and L1 soilsof the MAN set, respectively.

Carbon contents were calculated for aggregate size-classesbefore and after correction for sand particles larger than 63 μm.Cut-off size limits in the range 53–200 μm have regularly beenadopted to normalize C contents in aggregates isolated fromsoils with widely different texture (e.g., Elliott, 1986; Elliott etal., 1991; Puget et al., 1995; Six et al., 2000; Bossuyt et al.,2002).

2.5. Statistical analyses

To enable testing of differences in total C contents betweenthe six soils, whole soil C contents obtained from previouslypublished and unpublished samplings at the same six locationswere included. These data were subject to a two-way analysis ofvariance taking the interaction between sampling and soil asrandom error and considering the different samplings asindependent.

Differences in native and 14C-labeled C contents in aggregatesize-classes were tested for each set of soils using an additivemodel that included sampling location and aggregate size-class.The deviations from this model (interactions) were correlated.For each set of soil, correlations were strongest between nearbyaggregate size-classes and decreased as the distance increased.These correlations were accounted for by assuming that theyfollowed an autoregressive model of order 1 (AR(1), see Boxand Jenkins, 1976). For C in sand corrected aggregates, thecorrelation parameter (ρ, “rho”) was calculated separately (datanot presented) and then used in the model.

The evolution of CO2 in the incubation study was tested by atwo-way analysis of variance for the significance of effectsrelated to aggregate size-class. We used the statistical package

Fig. 1. The relative distribution of soil dry-weight across aggregate size-classes(not corrected for sand) from the L1 to L6 soils of the manipulated (MAN) andnatural (NAT) set of samples.

Table 2Carbon concentration in the L1 to L6 soils of the natural (NAT) and manipulated(MAN) sets of soil sampled from the Lerbjerg textural gradient

Soil MAN NAT Other samplings MeantotalC†

14C retained# Total C Total C

% of added† mg g−1

L1 33.2a 13.3‡ 14.2 13.3¶ 10.9§ 12.9aL2 34.7bc 13.4 16.6 15.1 16.7 15.4bL3 34.2ab 12.8 15.9 13.3 15.5 14.4abL4 34.2ab 15.5 15.9 14.5 17.1 15.8bL5 36.0c 13.0 14.5 13.3 15.5 14.1abL6 39.6d 13.4 16.2 14.5 17.4 15.4bMean† 13.6b 15.6a 14.0b 15.5a 14.7

Results from two other samplings at the same locations are included.†Means followed by the same letter within a row or column are not significantlydifferent at p≤0.05.# After field incubation (Thomsen et al., 1999).‡ Schjønning et al. (1999).¶ Initial soil sampling 1993. Unpublished results.§ Kjaergaard et al. (2004).

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SAS (SAS Institute, 1999) for all analyses, and a significancelevel of p≤0.05 throughout.

3. Results

The clay content in whole soils ranged from 10.9% in the L1soil to 45.2% in the L6 soil (Table 1), whereas the content of silt(2–20 μm) averaged 12%. Thus sand-sized particles accountedfor a substantial proportion in all soils. The concentration of soilC did not vary systematically among the texturally contrastingsoils retrieved from the six locations (Table 2), suggesting thelevel of C in these soils was constrained by C input rather thanby their capacity to stabilize C. Despite the wide range in claycontent, the MAN samples retained rather similar portions ofthe 14C added in ryegrass 8 months earlier (33.2–36.0%;Table 2). Only the L6 soil was found to contain significantlymore 14C (39.6%) than the other soils.

The distribution of soil among the eight aggregate size-classes differed widely (Fig. 1) and reflected differences in soiltexture. The proportion of soil recovered in b1 mm aggregatesdecreased and that in N2 mm aggregates increased with in-creasing whole soil clay content. Except for the L1 soil, lessthan 15% of the soil was associated with micro-aggregates(b0.25 mm) or present as similarly sized “loose” primary par-ticles. The proportion of soil in the medium sized macro-aggregates (1–2 mm) was apparently not related to soil texture.The MAN set of samples had been subject to mixing andsieving to b2 mm, but the incubations in the open had clearlypromoted the regeneration of soil structure; more than 45% ofthe L5 and L6 soil material had become incorporated intoN2 mm aggregates when sampled for analyses. Compared withthe NAT samples, however, the MAN samples appeared not tohave regained their original state of aggregation, the NATsamples retaining a larger proportion of soil in the 4–8 mmaggregates.

Fig. 2 shows the relative distribution of primary particles(texture) in aggregate size-classes and whole soils. Except for

L6, the texture of the N1mm aggregates was similar to that of thewhole soil from which they were isolated. Aggregate size-classes within the range 0.125 to 1 mm showed accumulation ofsand particles, especially in the NATsamples. For these samples,and for the L1 and L2 soils of the MAN set of samples,aggregates b0.125 mm were depleted in sand and enriched inclay, reflecting that sand particles N125 μm by definition areexcluded from aggregates smaller than 0.125mm.Differences insilt were limited to a slight enrichment of the b0.063 mmfraction of the NAT samples (except for L6).

Fig. 3 shows the concentrations of native and 14C-labeledOM in uncorrected and sand-corrected aggregates after 8monthsof incubation of the MAN soils in the open. For aggregates notcorrected for sand, concentrations of 14C-labeled OM differedlittle among macro-aggregates (N0.25 mm) of different size andfrom differently textured soils. This was also true for native OMfor bothMAN and NATsoils. For micro-aggregates (b0.25 mm),concentrations of native and 14C-labeled OM increased withdecreasing size.When aggregate fractionswere corrected for sandparticles N63 ìm, the concentrations of native OM in aggregatesdiffered among soils whereas differently sized aggregatesN0.063 mm isolated from a particular soil remained compara-tively similar in OM (except for L1). Table 3 presents the meanconcentration of native and 14C-labeled OM in individual

Fig. 3. The concentration of native and 14C-labeled OM in aggregate size-classesfrom the L1 to L6 soils of the manipulated (MAN) and natural (NAT) set ofsamples. Results refer to uncorrected aggregates and aggregates corrected forsand particles N63 μm.

Fig. 2. The content of primary particles (clay, b2 μm; silt, 2–20 μm; sand, 20–2000 μm) in aggregate size-classes and whole soil from the L1 to L6 soils of themanipulated (MAN) and natural (NAT) set of samples.

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aggregate size-classes from NAT and MAN samples. Whencorrected for particles N63 μm, the only consistent trend acrossthe different aggregate size-classes in terms of native and 14C-

Table 3Mean contents of native and 14C-labeled C in uncorrected and sand-correctedaggregate size-classes from the natural (NAT) and manipulated (MAN) sets ofsamples

Aggregatesize-class(mm)

NAT† MAN† MAN†

Native C 14C-labelled C

mg g−1 fraction μg g−1 fraction

Uncorrectedb0.063‡ 27.7(0.7) 32.0 843(30)0.063–0.125 24.4(0.8)a 21.2(0.7)a 837(33)a0.125–0.25 17.7(0.8)b 14.5(0.5)b 583(19)b0.25–0.5 12.5(0.8)c 11.8(0.5)c 422(19)c0.5–1 14.1(0.8)d 12.3(0.5)c 390(19)cd1–2 15.9(0.8)e 13.2(0.5)de 417(19)cd2–4 16.4(0.8)be 14.4(0.5)b 480(21)e4–8 15.1(0.8)d 13.9(0.5)be 496(22)e

Sand corrected0.063–0.125 43.9(1.3)a 38.2(1.6)a 1404(96)a0.125–0.25 44.4(1.3)a 37.8(0.9)ab 1468(55)a0.25–0.5 39.9(1.3)b 34.7(0.9)ac 1197(55)b0.5–1 42.0(1.4)abc 33.1(0.9)c 1016(55)c1–2 40.0(1.3)bc 34.0(0.9)c 1041(55)c2–4 39.4(1.3)bc 35.2(1.0)abc 1080(60)bc4–8 36.6(1.3)b 35.1(1.0)abc 1152(61)bc

Standard errors of the means are shown in brackets.† Means followed by the same letter within a column are not significantlydifferent at p≤0.05.‡ The residual fraction b0.063 mm was not included in the statistical analyses.

Fig. 4. The proportion of native and 14C-labeled C that was evolved as CO2

during laboratory incubation of four aggregate size-classes from the manipulated(MAN) set of samples. The soils had been amended with 14C-labeled ryegrassand incubated outdoors for 8 months before aggregates were isolated and subjectto the 15 weeks of incubation in the laboratory.

152 S.M. Kristiansen et al. / Geoderma 137 (2006) 147–154

labeled OM was a higher concentration in micro-aggregates thanin macro-aggregates.

The lability of native and 14C-labeled OM in the 0.125–2 mm aggregate size-classes from the MAN set of samples isillustrated in Fig. 4. During the 15 weeks of incubation in thelaboratory, 2.5 to 5% of the native C was evolved as CO2. Noconsistent trend was observed across aggregate size-classes,although significant differences in CO2 evolution were ob-served between individual size-classes within a soil. Averagedacross all size-classes, aggregates isolated from the most clayeysoils (L5 and L6) appeared to respire slightly more C. For 14C-labeled OM, 9 to 20% was respired during incubation, and the14C in 0.125–0.25 mm micro-aggregates was more accessibleto decomposition than that in the 0.25–2 mmmacro-aggregates.The 14C in macro-aggregates from the L1, L2, L3 and L6 soilsshowed similar lability, whereas the lability of 14C in macro-aggregates from the L4 and L5 soils decreased with increasingaggregate size.

4. Discussion

It has become widely accepted that soil structure exerts adominant control on the turnover of organic matter (OM) andthat the nature of the OM, in turn, plays an important role in the

formation of soil structure (Ladd et al., 1996). Through its effectson porosity, soil structure influences gas exchange, watercontent and the transport of solutes — soil properties that arecrucial to microbial growth and activity (Stotzky, 1997). Sorp-tion onto mineral surfaces and incorporation into soil aggregatescan render OM inaccessible to decomposition for shorter orlonger periods of time.

Studies addressing the relationship between OM turnover,management and aggregate formation have typically relied onsamples from differently textured soils and different locations(e.g. Christensen, 1986; Puget et al., 1995; Petersen et al., 1997;Six et al., 2000; Denef et al., 2002). Effects of texture,mineralogy, soil OM contents and differences in OM inputs aretherefore likely to be confounded. To evaluate the effect of soiltexture, we retrieved samples from six locations along a texturegradient subject to the same climate and management and withthe same mineralogical composition.

To study the relationship between aggregates and OMturnover, aggregates have to be detached from the bulk soil bysome disruptive force. Therefore the outcome of any aggregatestudy will reflect both the characteristics of the aggregate in situand the disruptive energy and abrasiveness of the fractionationprocedure. It has repeatedly been shown that the outcome ofwet-sieving depends on the specific procedure adopted,especially whether or not slaking occurs (Elliott, 1986; Beareand Bruce, 1993; Puget et al., 1995; Six et al., 2000; Ashmanet al., 2003). Moreover, wet-sieving procedures inevitably re-lease water soluble OM and dispersible colloids, causingchanges in aggregate composition (Emerson and Greenland,1990; Watts et al., 1996).

The experimental verification of aggregate hierarchyderives mainly from studies of soils subject to wet-sievingprocedures that involve aggregate slaking (e.g. Elliott, 1986;Jastrow et al., 1996; Six et al., 2000; Ashman et al., 2003;Mikha and Rice, 2004). Slaking promotes the isolation of verystable aggregates for which OM is the major binding agent.Soil fractionations that avoid slaking provide little support forthe existence of aggregate hierarchy (Christensen, 1986; Pugetet al., 1995; Petersen et al., 1997; Ashman et al., 2003). Due tothe great tensile strength of dry aggregates (e.g. Munkholmand Kay, 2002), prolonged sieving of air-dry soil tends toaccomplish aggregate abrasion rather than fragmentation. Wetherefore considered conventional wet- and dry-sievingprocedures to be less feasible for fragmenting soil into itsconstituent aggregates.

For a given soil subject to mechanical stress, an optimumwater content exists at which soil friability is maximal. Forlight- and medium textured soils, this optimum occurs at a watercontent near the lower plastic limit (Dexter and Bird, 2001).Analogues to a farmer's evaluation of soil workability, this soilcondition is readily assessed for a given soil. We fragmented oursoils into constituent aggregates when individual clods and pedsreached their state of maximum friability. Thereby we maxi-mized brittle fracturing along natural planes of weakness whileminimizing changes in aggregate composition. The physicalforces applied to fragment the soils in this state introducemainly shear stress whereas compression is minimized (Hatibu

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and Hettiaratchi, 1993). The sieving phase that followed frag-mentation was adopted only as a means of separating theaggregates according to size. The short sieving times ensuredthat further fragmentation and abrasion of aggregates wereminimized. Thus our procedure for isolation of aggregatesavoided aggregate slaking, solubilization of OM, colloid dis-persion, and soil plastic deformations, while it allowed the soilto fragment along natural planes of weakness. These method-ological considerations are of crucial importance when eval-uating the role and properties of aggregates in situ.

The distribution of native OM and that of 14C-labeled OMfrom the ryegrass addition followed the same pattern (Fig. 3 andTable 3) with increasing concentrations in micro-aggregates astheir size decreased and rather similar concentrations in macro-aggregates regardless of size. This general pattern was retainedeven when aggregate weights were corrected for their contentsof N63 μm sand particles. Although corrections for sand con-tents modified the differences between aggregate size-classes,micro-aggregates remained significantly higher in OM thanmacro-aggregates. The lability of native OM was generallysimilar for micro- and macro-aggregates, while the lability of14C-labeled OM was distinctly higher in micro-aggregates thanin macro-aggregates (Fig. 4). These observations are consistentwith studies based on dry-sieving procedures (Christensen,1986; Puget et al., 1995) or on wet-sievings that prevent aggre-gates from being slaked (Elliott, 1986; Petersen et al., 1997;Ashman et al., 2003). Studying soils from a field experimentreceiving 14C-labeled crop residues, Bossuyt et al. (2002) alsoobserved a higher concentration and lability of 14C-labeled OMin micro-aggregates compared to macro-aggregates. They sug-gested that micro-aggregates offer most of the OM protection insoil. Ashman et al. (2003) compared two wet-sieving proce-dures, one which accomplished slaking and one where slakingwas prevented. Without slaking, micro-aggregates showedhigher C concentrations and respiration rates than macro-aggregates. With slaking, the reverse pattern was observed. Thestudy of Ashman et al. (2003) and other comparative studies(e.g. Elliott, 1986; Puget et al., 1995) emphasize the intimatedependency between the biological characteristics of individualaggregate size-classes and the method by which they areisolated from the intact soil.

When our soils were allowed to break up along naturalplanes of weakness, constituent macro-aggregates of differentsize appeared to be similar with respect to content and lability ofOM. The formation of soil aggregates may follow two differentroutes (Dexter, 1988). Aggregates may be produced by thecombination of structural elements of a lower hierarchical order(e.g. primary OMC and POM). This is the original assumptionof the aggregate hierarchy concept (Tisdall and Oades, 1982).Alternatively, aggregates may result from the fragmentation ofstructural elements of a higher hierarchical order (e.g. the soil insitu). The clods and peds that make up the intact soil under fieldconditions are produced and degraded by wet/dry and freeze/thaw cycles, by tillage and by compactive forces from trafficand machinery, by the action of large root and by soilmacrofaunal activity (Six et al., 2004). This fragmentationresults in a high degree of similarity between aggregates.

The similarity of differently sized macro-aggregates ob-served here and in other studies that have avoided aggregateslaking does not align with the original concept of aggregatehierarchy. According to this concept, the primary OMC arebound into micro-aggregates (b0.25 mm) by persistent OMmainly of microbial origin. The micro-aggregates are occludedwithin macro-aggregates (N0.25 mm) by more labile OM ofmicrobial and plant origin and by temporary binding agentssuch as hyphae and fine roots. The formation of macro-aggregates involves physical protection of otherwise labilePOM derived from recent organic amendments. While micro-aggregates are considered to remain stable for long periods oftime, macro-aggregates are regarded as more transient andsusceptible to soil management (Tisdall and Oades, 1982).

A rapid turnover of macro-aggregates, as suggested by Planteet al. (2002), means that micro-aggregates and POM rapidly passthrough individual macro-aggregates, and that the composition ofmacro-aggregates simply reflects the whole soil in terms ofparticle size distribution and contents of native and recently addedOM. Similar lines of argumentation have been offered to explainthe lack of differentiation in microbial communities associatedwith differently sized macro-aggregates (Petersen et al., 1997;Schutter and Dick, 2002). Such quasi-stable pools of differentlysized macro-aggregates certainly retain a crucial role in regulatingthe physical and mechanical properties of soil, whereas their rolein the physical protection of recently added OM becomes moretransient. Thus for long-term arable soils with moderate OMinputs, physical protection of POMwithin macro-aggregates doesnot seem to represent a quantitatively significant OM stabilizingmechanism. In permanent grassland and other less physicallydisturbed soils, macro-aggregate turnover may be less dynamicand may play a greater role in the protection of POM and recentlyadded OM. Six et al. (1999) stated that under conventional tillage,there is not enough time between the formation and disruption ofmacro-aggregates to form significant numbers of new micro-aggregateswithinmacro-aggregates. Thus themore rapid turnoverof macro-aggregates in conventionally tilled soil than in no-tillsoils reduces the incorporation of recently added OM into micro-aggregates and results in less physical protection of OM.

Acknowledgement

The technical assistance of Karin Dyrberg and Stig T.Rasmussen is gratefully acknowledged. This work was finan-cially supported by The Danish Agricultural and VeterinaryResearch Council (grant no. 23-01-0133) and The Ministry ofFood, Agriculture and Fisheries (project HAR98-DJF-4).

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