physical protection and biochemical quality of organic matter in mediterranean calcareous forest...

$: Physical protection and biochemical quality of organic matter in mediterranean calcareous forest soils: a density fractionation approach$
Physical protection and biochemical quality of organic matter in

mediterranean calcareous forest soils: a density fractionation approach

Pere Roviraa,*, V. Ramon Vallejob

aDepartment de BiologiaVegetal, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, Barcelona 08028, SpainbCEAM, Charles Darwin 14, Parc Tecnologic, 46980 Paterna, Valencia, Spain

Received 25 March 2002; received in revised form 30 September 2002; accepted 11 October 2002

Abstract

Physical protection is one of the most important ways for stabilization of organic carbon (OC) in soils, and in order to properly manage

soils as a sink for carbon, it is necessary to know how much OC a given soil could protect. To this end, we studied individual horizons taken

from 16 soil profiles under Quercus rotundifolia stands, all over calcareous parent materials. Horizons were subjected to a sequential

extraction using solutions of sodium polytungstate (NaPT) of increasing density: (i) NaPT d ¼ 1.6, using slight hand agitation, to obtain the

free light fraction (FL); (ii) NaPT d ¼ 1.6 and ultrasonic dispersion, to obtain the Occluded Fraction I (Ocl I); (iii) NaPT d ¼ 1.8, to obtain

the Occluded Fraction II (Ocl II); and (iv) NaPT d ¼ 2.0, to obtain the Occluded Fraction III (Ocl III). The fraction of density . 2.0 are taken

as dense fraction (DF). The free organic matter was further divided into FL . 50 (retained by a 50 mm mesh: coarse organic fragments) and

FL , 50 (non-retained: fine organic fragments). The fractions FL . 50 and FL , 50 were taken together as free organic matter. The rest of

the fractions are taken together as protected organic matter. The obtained fractions were analyzed for total OC, total N, and carbohydrate

content. The percentage of non-hydrolyzable OC and N in each fraction was taken as an indicator of OC and N recalcitrance, respectively.

For both OC and N, the fractions FL . 50 and DF are dominant; the rest of the fractions are of much lower quantitative importance. In H

horizons and in most A horizons, most of the OC and N are free, whereas in B horizons both OC and N are mostly protected. Overall, the

percentages of free OC and N are very high and are currently amongst the highest ever recorded.

Organic matter recalcitrance is lowest in the two most protected fractions (Ocl III and especially DF), and highest in the first occluded

fractions (Ocl II and especially Ocl I). The free organic matter (FL . 50 fraction) has an intermediate quality: it includes recognizable plant

fragments, but the indicators tested (recalcitrance, carbohydrate content, cellulose to total carbohydrates ratio) suggest that it is not always

the most fresh and non-decomposed fraction.

There are clear maxima for both protected OC and N, which can be approached by curve fitting. By exponential fit, the obtained maxima

are 84.1 g of OC and 7.7 g of N kg21 of mineral particles ,20 mm. These maxima are much higher than the upper limits obtained by other

authors. Differences in the sampling approach are suggested as the reason for such discrepancies.

q 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Mediterranean; Forest soils; Organic carbon; Density fractionation

1. Introduction

The capacity of soils to accumulate and stabilize organic

carbon (OC) has received great attention in recent years,

mainly in order to evaluate to what extent the increase in

atmospheric CO2 content could be compensated by policies

such as afforestation of waste areas, or by changing some

current agricultural practices. In Europe, most forests

currently act as sink of OC (Nabuurs et al., 1997), but it is

not clear how much longer this can be maintained, since the

study of chronosequences suggests that the capacity of soils

to stabilize OC in the long term is much lower than often

assumed (Schlesinger, 1990).

OC accumulates in soil when the amount lost by

microbial activity is smaller than the input from litterfall

and/or dead roots. Therefore, to properly manage the soils as

a sink of OC, it is relevant to understand the reasons by

which the losses of OC may decrease or, in other words,

why the soil OC may become stable. In addition to chemical

processes such as precipitation by Ca2þ or Fe3þ, there

are two main reasons: (a) biochemical recalcitrance, i.e.

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Soil Biology & Biochemistry 35 (2003) 245–261

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* Corresponding author. Tel.: þ34-93-402-1462; fax: þ34-93-411-2842.

E-mail address: [email protected] (P. Rovira).

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the content of organic fractions difficult to decompose

(recalcitrant OC), and (b) physical protection, which makes

the OC partly unavailable for the microflora.

The degree of physical protection and the degree of

biochemical recalcitrance are not independent properties. It

has been observed that fresh organic matter accumulates in

both coarse fractions (Bonde et al., 1992; Balesdent, 1996)

and in light fractions (Skjemstad et al., 1994). Size

fractionation studies show that, as a general trend, fresh

and relatively non-decomposed materials accumulate in the

sand-sized fractions, whereas aromatic-rich materials,

probably derived from lignin, seem mainly associated

with silts, and highly resistant compounds rich in alkyl

carbon (mainly lipids) to the clays (Baldock et al., 1992;

Skjemstad et al., 1998); i.e. the chemically recalcitrant OM

is also that physically protected. Nevertheless, the relation-

ship also works in the opposite sense, i.e. labile, easily

decomposed polymers of plant or microbial origin become

stabilized through their protection into microaggregates,

and may persist in the soil for long time (Schmidt et al.,

2000; Schmidt and Kogel-Knabner, 2002).

Even though the degree of physical protection and the

degree of biochemical recalcitrance are related, there is a

substantial difference between both types of OM stabiliz-

ation. While there is no apparent upper limit for the amount

of recalcitrant OC that a soil can accumulate, (in peatlands,

for instance, it may grow almost indefinitely), the amount of

protected OC in a given soil must be finite, because the

physical protection depends on the amount of mineral

particles able to form organomineral complexes or micro-

aggregates (fine silt, clays), and the amount of these

particles in a given soil is finite. However, the question of

how much OM a given soil could protect has been addressed

only recently (Hassink, 1995a, 1996).

The combination of both concepts (physical protection

and biochemical recalcitrance) suggests a natural approach

for soil OM fractionation: a given OM pool may or may not

be chemically recalcitrant, and may or may not be

physically protected. The idea is not new: papers in which

the biochemical characterization is based not on the whole

soil sample, but on the obtained physical fractions (either by

density or by size) have become common in recent years.

Solid-state 13C-NMR (Baldock et al., 1992; Golchin et al.,

1994a) and pyrolysis (Leinweber et al., 1992) are widely

applied, but both methods are sophisticated and expensive

and therefore not useful for the large sets of samples

commonly obtained in ecological studies. It is possible to

apply classical methods to the physical fractions, such as

humus fractionation procedures, but there are few examples

of this (Schnitzer and Schuppli, 1989), probably because of

the length of time needed for analysis. Nevertheless, this

problem could be overcome by applying simpler chemical

treatments to the physical fractions, such as the quantifi-

cation of recalcitrant OM by means of acid hydrolysis (Stout

et al., 1981; Leavitt et al., 1997; Paul et al., 1997; Rovira and

Vallejo, 2000), rather than a standard humus fractionation

procedure.

Here we apply this approach to the study of OM in

several Mediterranean forest soils. We use a density

fractionation procedure to separate protected and unpro-

tected pools, and then we use acid hydrolysis to quantify

labile and recalcitrant fractions within them. We focus our

attention on the three following points:

(i) the relationship between the degree of physical

protection and the chemical recalcitrance of the various

fractions,

(ii) how physical protection and chemical recalcitrance

depend on the position in the soil profile, and

(iii) detection of upper limits for the amount of OM that a

soil can physically protect and, if found, a comparison

with those previously published (Hassink, 1995a).

2. Materials and methods

2.1. Soils

A total of 32 horizons, selected from 16 soil profiles,

were studied. All are located in dry areas of Lleida,

Catalonia (NE of Spain), within an area located between

08150E and 18150E, and between 42800N and 41800N. All the

profiles were over calcareous parent materials (limestones,

marl, and calcareous sandstones), always in culminal points,

to avoid the possibility of lateral inputs of soil materials,

which could complicate the interpretation of results.

Quercus rotundifolia was the dominant tree species.

Other tree species may be present in the stand, such as Pinus

halepensis, but all the profiles were done under a Q.

rotundifolia tree. Typical plant species in the understorey

were Lonicera etrusca, Genista scorpius, Quercus coccifera

shrubs, Teucrium chamaedrys, Rubia peregrina, Rubus

ulmifolius, Pistacia lentiscus, Dorycnium pentaphyllum,

and other typical mediterranean shrubs.

A complete list of the profiles and their most relevant

characteristics is given in Table 1. Since the physical

protection of OM is achieved mainly through its association

with the mineral matrix, only the horizons having mineral

matter were considered, i.e., organic horizons L and F were

not included in our study. For our purposes, the nomen-

clature of the horizons was simplified in the following way:

the uppermost horizon was taken as ‘H’ horizon if the OC

content is .20%. In such a case, the underlying horizon is

taken as ‘A’ horizon. The uppermost horizon is considered

to be an ‘A’ horizon if its OC content is ,20%. Any horizon

under the ‘A’ horizon is taken as ‘B’ horizon.

The sampled horizons were air-dried, passed through a

2 mm mesh, homogenized, and analyzed for their most

relevant characteristics. Texture was obtained by the pipette

method (Anderson and Ingram, 1993), OC by dichromate

oxidation in an aluminum block digestor (Nelson and

P. Rovira, V.R. Vallejo / Soil Biology & Biochemistry 35 (2003) 245–261246

Sommers, 1996), total N by dry combustion in a CARLO

ERBA CNS analyzer, carbonates by the calcimeter method

(Bonneau and Souchier, 1979), and pH in a suspension soil

water 1:2.5 (w/v).

2.2. Organic matter fractionation

The method follows the concepts of Golchin et al.

(1994a,b), which differentiates three degrees of physical

protection for OM: non-protected (free light, extractable

without sonication), occluded (extractable by sonication)

and protected (retained in the dense residue, after soni-

cation). A flow diagram of the method is given in Fig. 1.

(1). A variable amount of soil sample (5 g in H horizons,

10 g in A or B horizons) was placed in small centrifuge

bottles (about 75 ml of capacity), and 50 ml of a solution of

sodium polytungstate (NaPT) d ¼ 1.6 g cm23 was added.

The bottle was inverted and replaced again five times,

Table 1

Main characteristics of the studied soil profiles

Tra Profileb Soil typec Horizond Thick (cm) pH(H2O)e Gravel OC N CaCO3 Texture

p CS1 Lithic Torriorthent A 2 6.78 32 132.5 10.1 104 Clay loam

p B 5 7.57 71 23.1 2.6 125 Sandy loam

p CS2 Lithic Torriorthent H 3 6.9 39 204.4 15.8 157 Clay loam

p A 8 7.48 109 11.2 1.2 302 Sandy loam

p CS3 Lithic Torriorthent A 2 7.18 29 98.7 7.3 227 Clay loam

p B 5 7.69 51 27.7 3.1 323 Sandy loam

p CS4 Lithic Xerochrept A 2 6.15 13 84.8 5.5 0.0 Sandy clay loam

p B 23 6.52 24 8.2 1.3 0.0 Sandy loam

p CS5 Lithic Torriorthent A 1.5 7.29 201 122.7 8.9 223 Clay loam

p B 5.5 7.9 81 32.1 3.5 334 Sandy loam

p CS6 Lithic Torriorthent A 6 7.48 6 22.5 2.0 106 Loamy sand

p B 31 8.22 40 6.2 1.1 111 Loamy sand

p LI1 Lithic Haplarghid A 9 6.65 374 114.3 8.2 34 Silty loam

– B 13 7.62 ND 25.7 1.9 39 Silty clay

p LI2 Lithic Camborthid A 5 7.55 433 160.3 10.7 112 Silty clay loam

p B 27 7.62 558 34.7 3.6 121 Clay loam

– LI3 Lithic Camborthid H 1 6.3 8 285.7 19.4 59 Clay

p A 8 7.6 386 26.0 3.1 39 Silty clay loam

p B 25 7.93 99 16.1 2.7 113 Clay loam

– LI4 Lithic Camborthid H 2 6.35 59 238.1 22.3 109 N.D.

p A 2 7.25 397 99.5 9.0 154 silty clay loam

p B1 6 6.71 444 34.3 3.0 24 Clay loam

p B2 23 7.42 366 24.4 2.4 83 Clay loam

– LI5 Lithic Camborthid H 3 6.82 145 247.8 17.2 63 Silty loam

p A 12 7.33 535 63.7 4.1 75 Clay loam

p B 13 7.7 389 11.7 1.3 119 Clay loam

p MR 1 Lithic Torriorthent A 4 7.36 297 150.7 9.8 393 Clay

p B 12 8.05 233 17.4 1.4 698 Silty clay loam

p MR 2 Lithic Torriorthent H 6 6.73 115 209.6 14.6 252 Silty clay

– A 14 7.88 386 31.5 2.3 49.2 Clay loam

(fissural stone layer, between both horizons)

– B 14 8.45 N.D. 7.0 0.7 53.8 Silty clay loam

p MR 3 Xerollic Calciorthid A 8 6.62 616 200.1 14.8 258 Silty clay

p B1 21 7.91 354 21.7 2.7 487 Loam

– B2 16 8.14 449 10.9 1.0 646 Silty clay loam

p MR 4 Lithic Camborthid A 3 7.7 65 90.5 7.5 328 Clay loam

p B1 7 7.68 74 20.4 2.8 371 Loam

– B2 18 8.40 306 9.9 1.0 400 Loam

– C 15 8.22 552 11.9 1.2 457 Silty loam

p MR 5 Lithic Torriorthent H 2 6.9 589 258.1 18.1 198 N.D.

p A 8 7.73 530 46.8 4.0 586 Loam

ND: not determined. Gravel content is given as g kg21 of total horizon mass (excluding big stones); OC, N and CaCO3 content are given in g kg21 of fine

earth (,2 mm). pH, OC, N and CaCO3 were analyzed by duplicate; other analyses by single determinationa Treatment of the sample. (–): not analyzed. ( p ): analyzed by density fractionation.b Letters refer to the kind of parent material. CS: calcareous sandstone; LI: limestone; MR: marl.c Soil type is given following Soil Taxonomy (Soil Survey Staff, 1975).d The organic L þ F horizon (litter layer) is always present, but is not included in the table.e Proportion 10:25 (soil/water, w/v).

P. Rovira, V.R. Vallejo / Soil Biology & Biochemistry 35 (2003) 245–261 247

avoiding strong shaking movements. Then the bottle was

left to stand overnight. The floating material was recovered

by centrifugation, and decanted onto a sieve (50 mm mesh).

When the amount of floating materials was very high, it was

necessary to previously remove part of this material with a

spatula, and place this removed material directly on the

mesh. The material retained in the mesh was washed with

deionized water, the washing added to the non-retained

material. The non-retained material (,50 mm size) was

recovered by filtration on a pre-weighed glass–fiber filter

Whatman GF/A, using a Buchner funnel. The process was

repeated once, and the obtained fractions added to the

previous ones. The extracted, coarse material (.50 mm)

was transferred to a pre-weighed crucible, dried at 60 8C to

constant weight, and taken as Free light fraction . 50 mm

(FL . 50). The glass–fiber filters were dried at 60 8C to

constant weight, and its content taken as Free light fraction

,50 mm (FL , 50).

In the next steps, since little or no coarse materials were

extracted, the recovery of the suspended OM was done in a

different way.

(2). NaPT (50 ml) d ¼ 1.6 g cm23 was added to the

residue remaining in the bottle. The contents of the bottle

were homogenized by shaking. Then the bottle was placed

in an ice bath and sonicated using a probe-type ultrasonic

disintegrator BRANSON 250, for 15 min, at a nominal

power release of 100 W. The bottle was left to stand for 4 h,

then centrifuged. The liquid, including the floating material,

was decanted into a centrifuge bottle, to which 50 ml of

distilled water was added: this causes a strong decrease in

the density of the solution, and the sedimentation of the

suspended organic matter. The bottle was centrifuged, and

the liquid extracted by siphonation. The process (without

sonication) was repeated, and the obtained fraction added to

the previous one. The sediment was transferred to a pre-

weighed crucible, dried at 60 8C to constant weight, and

taken as Occluded fraction I (Ocl I).

(3). To the residue remaining after step 2, 50 ml of NaPT

d ¼ 1.8 was added, and step 2 was repeated, without

sonication. The extracted material was taken as Occluded

fraction II (Ocl II).

(4). To the residue remaining after step 3, 50 ml of NaPT

d ¼ 2.0 was added, and step 2 was repeated, without

sonication. The extracted material was taken as Occluded

fraction III (Ocl III).

(5). The residue remaining after step 4 was washed with

distilled water, transferred to a pre-weighed crucible, dried

to constant weight, and taken as dense fraction (DF).

The sum of fractions FL . 50 and FL , 50 was taken as

free organic matter. The sum of the rest (Ocl I, Ocl II, Ocl III

and DF) was taken as protected organic matter, i.e. the need

for sonication is taken as the main criterion to separate free

from protected organic matter.

The C and N of the fraction FL , 50 were analyzed with

a CARLO ERBA analyzer, directly taking small represen-

tative portions of the filter, and the result was referred to the

total mass of fraction on the filter. Separate trials were done

to detect carbonates in this fraction; since they were not

detected, the total C is taken as OC. In some cases, (some B

horizons very poor in OM), it was not possible to proceed as

explained, because the amount of material retained on the

filter was too scarce and too irregularly placed on the filter to

take representative subsamples. In these cases, N was not

quantified, and OC was analyzed by dichromate oxidation,

taking the whole filter as the sample.

The rest of the fractions (FL . 50, Ocl I, Ocl II, Ocl III

and DF) were ground in an agatha mortar, and analyzed for

OC by dichromate oxidation, and for N in a CARLO ERBA

CNS analyzer.

2.3. Biochemical quality of the fractions

and of the whole OM

To quantify OM quality in the fractions, an acid

hydrolysis approach was applied (Rovira and Vallejo,

2000). This analysis was carried out on all the fractions

except the FL , 50 ones, because they were too entrapped

in the filter for any further analysis. The protocol follows

Fig. 1. Flow diagram of the fractionation method. NaPT, sodium

polytungstate.


that recommended by Oades et al. (1970) for a maximum

release of carbohydrates.

The ground, solid samples (500 mg for the DF, 50–

100 mg for the rest of the fractions) were hydrolyzed with

20 ml of 2.5 M H2SO4, 30 min at 105 8C in centrifugable,

sealed Pyrex tubes, in an aluminum block digestor. In

samples having carbonates, enough time was left to destroy

them by excess acid, before closing the tubes and placing

them in the digestor. The hydrolysate was recovered by

centrifugation and siphonation. The residue was washed

with distilled water; the washing was recovered by

centrifugation and added to the hydrolysate. This first

hydrolysate was taken as Labile Pool I (LP I). The

unhydrolyzed residue was dried at 60 8C. Then, 2 ml of

13 M H2SO4 was added to the tube. The tube was left

overnight in an end-over-end shaker. Then, water was added

to dilute the acid to 1 M, and a second hydrolysis was done

for 3 h, at 105 8C. This second hydrolysate was recovered as

explained above, and taken as Labile Pool II (LP II). The

residue was transferred to a pre-weighed crucible, dried at

60 8C to constant weight, and taken as Recalcitrant fraction.

The recalcitrant fraction was ground and analyzed for C

and N using a CARLO ERBA CNS analyzer. Then, the

‘recalcitrancy indices’ (RI) were calculated (Rovira and

Vallejo, 2000, 2002)

RIC ¼ ðRecalcitrant C=Total OCÞ £ 100

RIN ¼ ðRecalcitrant N=Total NÞ £ 100

The Labile Pools I and II were analyzed for total

carbohydrates, by the phenol–sulfuric method (Dubois

et al., 1956). To avoid interferences from released Fe3þ ions

(Martens and Frankenberger, 1993), an aliquot of the

hydrolysate was previously neutralized with anhydrous

Na2CO3 then centrifuged, to precipitate iron oxides.

Then, the ratio II/I þ II (as%) was calculated. Since

the carbohydrates of the Labile Pool II correspond to the

cellulose of the sample (Oades et al., 1970), the ratio II/

(I þ II) equals the cellulose to total carbohydrates ratio.

This method was applied to all the obtained fractions

except the FL , 50, which often was too scarce to carry out

the analysis, and, in addition, was impossible to remove

from the filters.

2.4. Statistical analysis

The amounts of OC, N, OC/N, and carbohydrates of the

various fractions were compared by standard ANOVA

procedures: (a) to compare the characteristics of the several

fractions, overall or within a given horizon type (either H, A

or B); (b) to compare horizon types, for a given fraction. The

data were previously transformed by arcsine of squared root

if they were percentages (Neter et al., 1990); the remainder

was log-transformed.

Curve-fitting analyses were carried out by using the

algorithm of Marquardt–Levenberg to approach par-

ameters, using the SigmaPlot v. 4.0 routines (Jandel

Scientific).

3. Results

3.1. Distribution of OC and N among fractions

For both OC and N, the fractions FL . 50 and DF were

dominant (Table 2). The rest of the fractions were

quantitatively of minor relevance. From H to B horizons,

we observed a decrease in the importance of the fraction

FL . 50, and an increase in the importance of DF. This

effect was more pronounced for nitrogen. The protected OC

Table 2

Distribution of OC and N among the obtained fractions (as% of the total OC or N)

Parameter Horizon n Fractions

FL . 50 mm FL , 50 mm Ocl I Ocl II Ocl III DF

OC H 4 87.54 1.68 1.98 0.72 2.06 6.01

(5.39) (0.74) (1.61) (0.43) (1.04) (2.95)

A 15 55.87 1.94 7.35 2.63 6.84 25.37

(19.38) (0.68) (6.49) (3.23) (4.63) (16.16)

B 13 24.78 1.29 7.47 2.85 6.85 56.77

(9.11) (0.37) (4.51) (2.12) (3.60) (10.42)

N H 4 83.09 2.09 2.02 1.07 2.57 9.15

(5.99) (0.79) (1.49) (0.52) (1.15) (3.89)

A 15 47.95 1.94 6.33 2.47 4.95 36.35

(22.64) (0.60) (5.83) (2.59) (2.34) (21.33)

B 13 12.88 1.03a 4.46 1.81 4.96 75.08

(6.49) (0.31) (3.63) (1.35) (3.17) (10.03)

Data are averages; values in parentheses are standard deviations.a Average of only four samples; in the other cases the amount of recovered matter was too low for analysis.


was dominant only in B horizons. By comparison with OC,

N tends to become protected more easily (Fig. 2).

The OC/N ratio is always highest in the FL . 50

fraction, whereas the lowest value is that of the DF fraction

(Table 3). The occluded fractions have intermediate values;

the differences between them were significant only in H

horizons. Within the FL fraction, the coarse fragments

(FL . 50) had a OC/N ratio considerably higher than that of

fine particles (FL , 50).

For the OC/N ratio of any given fraction, ANOVA failed

to detect significant differences between horizons, except in

the DF fraction (Table 3), for which a decrease with

increasing depth (from H to B horizons) is observed.

3.2. Accumulation of OC and N in the fractions

As the OC in the whole horizon accumulates, the amount

of OC in each fraction increases, but for some fractions the

increase is inconsistent and data dispersion results in a lack

of significance (Fig. 3A,B). The accumulation of OC is clear

and consistent in the free fractions (both FL . 50 and

FL , 50); is less clear for the occluded fractions I and III, in

which data dispersion results in a decrease of r 2 and also of

its significance; and is unclear for occluded fraction II and

dense fraction, for which the correlation was good for B

horizons, but not significant in the H and A horizons (Fig.

3B), i.e. the accumulation of OC in the whole horizon is not

related to any increase in the amount of OC in these

fractions. Of course, this statistical result is unrealistic, but

illustrates the highly random incorporation of OC into these

fractions. It must be noted, however, that correlations

become positive and significant if B and A horizons are

pooled (not shown).

Data for H horizons must be taken with caution, since

few points are available, but overall they suggest that in

these horizons the coarse free fraction (FL . 50) becomes

increasingly important and is the main (if not the only)

cause of any net increase of total OC in the horizon. The

accumulation of OC in the rest of the fractions seems stable;

often the amount of OC in these fractions is about equal to

or even lower than that of A horizons (all the occluded

Table 3

OC/N ratios of the whole horizons and of the obtained fractions

Horizon n Whole Fractions

FL . 50 mm FL , 50 mm Ocl I Ocl II Ocl III DF

H 4 13.75 20.13 A1 15.14 B1 16.39 B1 11.75 C1 14.89 B1 11.98 C1

(0.59) (2.69) (0.94) (2.34) (1.68) (1.58) (0.90)

A 15 12.87 19.40 A1 14.40 B1 16.02 B1 13.43 B1 16.66 B1 10.04 C1,2

(2.13) (4.15) (1.70) (3.82) (2.13) (4.52) (1.87)

B 13 8.68 26.23 A1 16.75a B1 21.82 AB1 15.44 B1 16.61 B1 9.53 C2

(1.96) (7.39) (1.36) (6.55) (5.42) (6.04) (2.10)

All 32 11.28 22.14 14.99 18.42 13.99 16.41 10.08

(2.90) (6.41) (1.77) (5.73) (3.94) (4.93) (2.03)

Data are averages; values in parentheses are standard deviations. For ANOVA analysis, data were previously log-transformed. Means were compared by

the Duncan’s test. Comparison between fractions: data in the same row followed by the same letter do not differ, at P ¼ 0.05. Comparison between horizons:

data in the same column followed by the same number do not differ, at P ¼ 0.05.a Average of only four samples; in the other cases the amount of recovered matter was too low for N analysis.

Fig. 2. Percent of free carbon and nitrogen, in soil samples with different

OC contents. Vertical dashed line marks the percentage of total OC at

which the amount of free OC or N is 50%.


fractions), or continues the trend but does not increase

anymore (as in FL , 50).

For N, the results were similar to those of OC, i.e.

the accumulation of total N was closely related to the

accumulation in the free fractions, whereas for the

protected fractions the relationship was much less clear

(Fig. 4A,B). In contrast with that observed for OC, in H

horizons the accumulation of N in the fine free fraction

(FL , 50) seems to continue. In the occluded fractions

and in the dense fraction, in contrast, the accumulation of

N seems stable.

Overall, the free fraction and especially the coarse

subfraction (FL . 50) seems to be the main cause of the

accumulation of both OC and N in the soil samples in H and

A horizons (Fig. 5). For this fraction the correlations were

always high and significant and no signs of any upper limit

are seen, either for OC or for N accumulation. The

accumulation of OC and N in the protected fractions

(taken together) seems much slower, and signs of saturation

are visible.

3.3. Biochemical characteristics of the fractions

Overall, the RIC values were considerably higher than

RIN ones (Table 4). Few differences were detected

between horizon types for a given fraction: only in the

Ocl I fraction for carbon, and in the DF for nitrogen. In

contrast, the differences between fractions within a given

horizon were relevant, for both OC and N. In H horizons

no significant differences between fractions were

observed; in A and B horizons a clear increase of RIC

and RIN is detectable from FL . 50 to Ocl I; then, from

Fig. 3. (A) Accumulation of OC in the various fractions of B horizons, vs. total OC in the horizon (both in g kg21 of total mineral matter in the horizon). Linear

correlations are given: ns: not significant; (*) significant at P ¼ 0.05; (**) significant at P ¼ 0.01. (B) Accumulation of OC in the various fractions of A and H

horizons, vs. total OC in the horizon (both in g kg21 of total mineral matter in the horizon). Linear correlations are given: ns: not significant; (*) significant at

P ¼ 0.05; (**) significant at P ¼ 0.01.


Ocl I to DF the indices drop again. The lowest indices

were for DF, except in the case of RIN, which was

highest in the DF of B horizons.

Carbohydrate C was between 10 and 22% of the total OC

in the fractions (Table 5). Its distribution among fractions

depended on the horizon. In H horizons the ratio

Carbohydrate C/Total OC was similar for all fractions, but

in A and B horizons the occluded fractions seemed depleted

in carbohydrates by comparison with the FL . 50 and the

DF fractions. In some fractions the effect of horizon type

was significant, but in the reverse sense: whereas for the

FL . 50 fraction the carbohydrate content was lowest in

the H horizons, for Ocl II and Ocl III it was highest. This

was also observed for the Ocl II fraction, even though the

effect was not significant.

In the FL . 50 fraction, the proportion of cellulose to

total carbohydrates (LP II/total) tended to increase with

depth, from H to B horizons. In the other fractions,

the differences between horizons were not significant

(Table 5).

3.4. Detection of upper limits for protected OC and N

Detection was done by means of curve-fitting pro-

cedures, pooling all the protected fractions (Figs. 5 and 6).

In the fitting process, data corresponding to H horizons were

not taken into account for reasons discussed later. Never-

theless, parallel trials were carried out to see whether

considering H horizons in the fitting process would result in

important changes in the obtained graphs. For exponential-

type curves, differences were small (not shown).

In B horizons, the amount of protected OC (or N)

increases linearly with increasing total OC (or N) in the

horizon (Fig. 5). The correlations are very high and

significant (Table 6a). In these horizons, incorporation of

both OC and N into the protected fractions seems intense, as

Fig. 3 (continued )


evidenced by the strong slopes of the regression lines

(Fig. 6). These slopes are not maintained in A horizons, in

which signs of saturation of the protective capacity are

visible. The global dynamics can be fitted to exponential

curves (Fig. 6, Table 6b). For OC, the upper limit for the

protective capacity (about 52 g kg21 of mineral matter) is

reached when the total OC has reached about 150 g kg21 of

mineral matter. For N, the limit (about 4–5 g, i.e. one order

of magnitude smaller) is reached at about 7–8 g of total

N kg21 of mineral matter.

In the case of OC, the fit is improved if data are expressed

on a fine-particles basis, i.e. as g OC kg21 of OM-free fine

silt plus clay (,20 mm), or coarse silt plus fine silt plus clay

(,50 mm), but not if only clays are considered (,2 mm)

(Fig. 6, Table 6b). Note that when data are expressed on a

fine-particles basis, for H horizons only three points are

available, because in one of the H horizons the amount

of OM was too high to obtain a reliable textural analysis

(Table 1).

Irrespective of the fraction chosen as a basis, the data for

H horizons fall always below the saturation line. In fact,

they are often smaller than many of the data corresponding

to A horizons, i.e. the protective capacity of fine particles is

less saturated. For N, this trend (decreased saturation of fine

particles as the whole horizon becomes richer in N) is

clearer, and seems to appear even in the N-richest A

horizons.

To check for the possibility that curves other than those

of exponential type were more appropriate to fit the data,

polynomial regression was also applied. If data are fitted to

polynomial curves, it is possible to account for the decrease

in the amount of protected OC in H horizons (Fig. 6), and

the fitting improves (Table 6c). In these cases, the estimates

of maximum protective capacity for OC and N are slightly

Fig. 4. (A) Accumulation of N in the various fractions of B horizons, vs. total N in the horizon (both in g kg21 of total mineral matter in the horizon). Linear

correlations are given: ns: not significant; (*) significant at P ¼ 0.05; (**) significant at P ¼ 0.01. (B) Accumulation of N in the various fractions of A and H

horizons, vs. total N in the horizon (both in g kg21 of total mineral matter in the horizon). Linear correlations are given: ns: not significant; (*) significant at

P ¼ 0.05; (**) significant at P ¼ 0.01.


higher than for exponential-type curves, but the differences

are not substantial. The main difference is that, in the

polynomial regression, the maximum values are obtained

not in (hypothetical) H horizons very high in OM, but in A

horizons.

4. Discussion

4.1. Methodological constraints

The recovery of OC in the fractions was about

91.9 ^ 13.7% (mean, SD), whereas that of N was

74.3 ^ 15.1%. The high losses of N suggest that N-rich

compounds are relatively labile and may be lost by

ultrasonic treatment. Since the losses of OC and N during

sonication and/or repeated fractionation and washing could

not be ascribed to any of the obtained fractions, the OC and

especially the N content may be underestimated for some of

the fractions, perhaps for all.

An additional constraint is the reaction of NaPT to

form Ca-polytungstate (CaPT), which is highly insoluble.

In some of the obtained fractions, a fine white powder

was visible; since all the soils were over calcareous

parent materials, and some of them were highly rich in

carbonates, the generation of CaPT during the fraction-

ation is to be expected. It was not possible to remove it

by repeated washing with distilled water (centrifugation

and decantation), hence this white powder was included

in the fraction, increasing its weight and, as a result,

decreasing its OC concentration. The effect is of little

relevance when the amount of the fraction is high, as in

FL . 50 and DF fractions, but for some occluded

fractions, which were often obtained in very low

amounts, OC concentration may be underestimated.

Since the amount of CaPT included finally in each

fraction is highly random, this is a source of variation,

Fig. 4 (continued )


and may explain the high variability observed in the OC

concentration of these fractions. To our knowledge, the

problem of CaPT formation when working with calcar-

eous soils is still unresolved.

4.2. Distribution of OC and N amongst density fractions

In many of the studied horizons, most of the OC is free,

and can be extracted directly, without sonication. The sum

Fig. 5. Accumulation of free (FL . 50 þ FL , 50) and protected (Ocl I þ Ocl II þ Ocl III þ DF) OC and N, vs. total OC or N in the horizon; in all cases in

g kg21 of total mineral matter in the horizon. Data for protected OC and N have been fitted to an exponential curve: y ¼ maxð1 2 expð2kxÞÞ: Values for the

parameters are given in Table 6b (total mineral). Data for free OC and N have been fitted to y ¼ total 2 maxð1 2 expð2kxÞÞ: For the protected OC and N in B

horizons, the linear fit was very good (Table 6a), and is also given in this figure.

Table 4

RIC and RIN values for the obtained density fractions


FL . 50 mm Ocl I Ocl II Ocl III DF

RIC H 4 55.26 1A 61.03 1A 59.63 1A 51.73 1A 42.30 1A

(13.98) (1.06) (12.41) (7.81) (2.34)

A 15 52.52 1A 60.89 1B 55.80 1AB 43.94 1C 40.97 1C

(4.91) (7.62) (5.60) (7.92) (5.36)

B 13 59.67 1A 72.25 2B 61.38 1A 50.38 1C 44.20 1C

(4.10) (5.98) (5.43) (9.40) (12.36)

All 32 55.58A 65.44 B 58.10A 47.32 C 42.31 D

(7.58) (8.68) (7.36) (9.16) (8.48)

RIN H 4 39.99 1A 53.62 1A 34.49 1A 35.75 1A 31.55 1A

(7.09) (18.28) (8.05) (10.60) (3.88)

A 15 31.80 1A 43.92 1B 36.45 1AB 39.37 1AB 39.13 1AB

(6.80) (15.22) (7.50) (9.08) (10.91)

B 13 38.51 1A 52.57 1 AB 38.49 1A 42.44 1A 59.44 2B

(11.56) (14.66) (3.93) (7.90) (25.86)

All 32 35.70A 48.20 B 36.83A 40.04A 45.30 B

(9.77) (16.02) (6.79) (9.13) (20.45)

The free fraction ,50 mm was not analyzed. Data are averages; values in parentheses are standard deviations. For ANOVA analysis, data were previously

transformed by arc sine of square root. Means were compared by the Duncan’s test. Comparison between fractions: data in the same row followed by the same

letter do not differ, at P ¼ 0.05. Comparison between horizons: data in the same column followed by the same number do not differ, at P ¼ 0.05.


of protected fractions dominates only in samples having less

than 6% of OC. Most of the A horizons are above this limit,

and for many samples more than 70% of the total OC is

found in the free, light fraction (FL . 50 þ FL , 50).

As expected, these values are much higher than those

reported for agricultural soils, in which the light fraction

usually accounts for up to only 20% of total OC, usually

much less (Janzen et al., 1992; Biederbeck et al., 1994;

Wander and Traina, 1996; Alvarez et al., 1998). However,

they are also much higher than values reported for forest

soils. Boone (1994), for instance, reported that the light

fraction accounts for up to 21% of the organic matter of the

soil, with seasonal fluctuations. It must be noted that, in all

the references mentioned, the light fraction was recovered

after dispersion (partial or total) of the structure, either by

sonication or by a chemical dispersing agent; then the ‘light

fraction’ of these authors is equivalent to the sum of the free

fraction plus at least one of the occluded fractions of our

work. Currently, to our knowledge, the values obtained for

our horizons are among the highest ever recorded.

The data of Golchin et al. (1994a, 1995) are of special

interest for comparison with our data, since they were

obtained by applying the same fractionation scheme.

According to these authors, in forest soils the free light

fraction, extractable without ultrasonic treatment, accounts

for up to 31% of the total OC, depending on soil type and

soil use, i.e. much less than in our horizons. The only similar

value we know is that given by Molloy and Speir (1977) for

a grassland soil in New Zealand, in which the light fraction

accounted for an 84% of the total OC. Nevertheless, this was

an exceptional value; in all the other soils studied by these

authors, the light fraction accounted only for 20–40% of

total OC.

At least in part, these differences may be explained by

differences in the sampling method. Most authors analyze

soil cores, taken to a depth of 10–15 cm. If these cores are

not subdivided by layers, all the layers are pooled in a

single sample. In contrast, we have sampled each horizon

separately. Note that in many cases the upper horizons are

very thin (Table 1); by taking a core of 10 or 15 cm, the H

and A horizons, and often also part of B horizons, would

have been taken together, and the high dominance of the

free light fraction in the uppermost horizons largely

diluted.

The data from Spycher et al. (1983) are more comparable

to ours, since they also sampled soils at several depths, and

analyzed each layer separately. These authors observed that

the percentage of total OC in the light fraction abruptly

decreases, from the first 3 cm to the next 10 cm, and more

gradually thereafter, in agreement with our results. Never-

theless, even in the uppermost layer the light fraction

accounts for only 53% of the total OC, for a horizon which

is 11.67% of the OC; i.e. considerably less than in our

horizons (Fig. 2). However, in this case, the differences in

comparison with our data may be explained by the pre-

treatment of the samples. In the work of Spycher et al.

(1983), the density separation was done on ground soil (60

mesh). As shown in a previous paper (Rovira et al., 1998),

the recovery of debris in the light fractions strongly depends

on debris size, being smaller as size decreases. The grinding

of the soil material can lead therefore to an underestimation

of the light fraction. This explanation may be also applied to

Table 5

Carbohydrates in the density fractions: Total (as% of the total C in the fraction), and II/I þ II ratio


FL . 50 mm Ocl I Ocl II Ocl III DF

Total H 4 15.28 1A 18.71 1A 17.17 1A 19.70 1A 18.71 1A

(3.70) (4.97) (4.37) (2.97) (2.43)

A 15 20.87 2A 12.33 2B 13.00 1B 14.52 2B 20.73 1A

(2.52) (5.27) (4.54) (3.88) (4.79)

B 13 21.22 2A 10.95 2B 13.60 1B 13.64 2B 19.79 1A

(4.39) (3.57) (3.84) (3.05) (3.43)

All 32 20.24A 12.47 B 13.75 BC 14.91 C 20.10A

(4.09) (5.16) (4.47) (3.99) (4.10)

II/I þ II H 4 20.04 1A 22.33 1A 22.25 1A 17.78 1A 18.23 1A

(6.46) (9.86) (4.99) (9.11) (7.17)

A 15 23.68 1,2AB 30.28 1B 22.82 1AB 23.32 1AB 17.02 1A

(5.37) (14.98) (8.85) (7.86) (6.96)

B 13 29.28 2A 26.44 1AC 21.68 1BC 22.59 1BC 17.73 1B

(6.86) (6.49) (5.29) (7.36) (5.59)

All 32 25.62 AB 27.92 B 22.25A 22.24A 17.47 C

(7.07) (12.09) (7.05) (8.08) (6.49)

The free fraction ,50 mm was not analyzed. Data are averages; values in parentheses are standard deviations. For ANOVA analysis, data were previously

transformed by arc sine of square root. Means were compared by the Duncan’s test. Comparison between fractions: data in the same row followed by the same

letter do not differ, at P ¼ 0.05. Comparison between horizons: data in the same column followed by the same number do not differ, at P ¼ 0.05.


the above mentioned data of Molloy and Speir (1977), in

which the soil samples were also ground (250 mm) before

densimetric treatment.

Overall, N tends to be more protected than OC (Fig. 2).

This trend is reflected by the decrease in the OC/N ratio with

increasing density of the fractions, although this decrease is

not always consistent in our study. The decrease in the OC/

N ratio with increasing density seems to be a general feature

of SOM, since it has been observed in whole-horizon

fractionations (Golchin et al., 1994b) as well as when the

fractionation is applied only to the macroorganic matter

(Hassink, 1995b; Meijboom et al., 1995).

4.3. Physical protection vs. biochemical quality

One of the aims of this paper was to study whether in

mediterranean forest soils OM quality would increase or

decrease as the degree of physical protection increases.

Looking at the obtained RIC values, the relationship

between physical protection and biochemical recalcitrance

is not simple: the first two occluded fractions (Ocl I and Ocl

II) are the most recalcitrant ones, the two most protected

fractions (Ocl III and DF) are the least recalcitrant ones, and

the free organic fragments (FL . 50) have an intermediate

degree of recalcitrance. This pattern seems very consistent,

since it was observed for all the horizon types (H, A and B).

Golchin et al. (1994a, 1995) observed that the free-light

fraction is composed mainly of plant fragments, recogniz-

able under microscopic examination, usually bigger than

100 mm. Our results agree with this, since (i) in our

fractionation, the coarse subfraction (FL . 50) is largely

dominant, the fine materials (FL , 50) being only a small

percentage of the fraction (Table 2); (ii) the OC/N ratio of

this fraction is the highest one; (iii) the relative carbohydrate

content is very high, similar to the values given by Molloy

and Speir (1977) for the light fraction; and (iv) the ratio of

cellulose to total carbohydrates is the highest. As shown in a

previous paper (Rovira and Vallejo, 2002), in small-sized

Fig. 6. Protected OC and N, vs., total OC and N. Both are given (i) in g kg21 of clay (above); (ii) in g kg21 of fine silt plus clay (centre); or (iii) in g kg21 of

coarse silt plus fine silt plus clay (below). Data for B horizons have been fitted to a linear dynamics ðy ¼ ax þ bÞ; data for B plus A horizons have been fitted to

exponential curves ðy ¼ maxð1 2 expð2kxÞÞ; or to third degree-polynomial curves. The obtained parameters are given in Table 6.


plant debris the cellulose to total carbohydrates ratio

decreases as decomposition proceeds. Hence a high

cellulose to total carbohydrates ratio is an indicator of

relatively fresh plant materials. Together, these observations

suggest that poorly decomposed debris constitute most of

the FL fraction, even though, because of its dark color, the

presence of humified organic matter is to be assumed.

The fine subfraction (FL , 50) seems quite different from

the coarse one (FL . 50), at least with regard to the OC/N

ratio which is significantly lower. It seems logical to assume

that coarse fragments are made mainly of structural plant

tissue, whereas the fine fragments come from non-structural

plant tissues, more susceptible to fragmentation and easier

to decompose and usually also richer in proteins. Because of

its low OC/N ratio and also because of its low size (high

surface/volume ratio), it is expected that the fine subfraction

will decompose fast and that its amount will always be low,

as found in our samples.

Even though the dense fraction (DF) is also relatively

rich in carbohydrates, its cellulose to total carbohydrates

ratio is always very small, much lower than in FL . 50

(Table 5). According to Golchin et al. (1994a, 1995),

microbial products are dominant in the DF, whereas plant-

derived compounds are scarce, and in particular lignin is

almost absent, as deduced from the virtual absence of signal

for aromatic C in 13C NMR spectra. The very low

percentage of unhydrolyzable C (low RIC values) for this

fraction agrees with these findings (Table 4). Overall, this

fraction should correspond to the fine, colloidal fraction

obtained in particle-size analysis, whose organic matter is

highly aliphatic and hydrolyzable and has a narrow OC/N

ratio (Anderson et al., 1981), indicative of recent microbial

products (Tiessen et al., 1984). It must be noted that in the

DF the recalcitrance of nitrogen (RIN) strongly increases

with depth, being in B horizons much higher than in H

horizons (Table 4), whereas for the RIC ratio no clear trend

Table 6

Parameters for the fitting functions for protected OC and N, given in Figs. 5 and 6

(a) Linear fit, only for B horizons: y ¼ b0 þ b1x

Element Fraction b0 b1 R 2

OC Particles ,2 mm 15.27 0.5965** 0.9523

Particles ,20 mm 7.44 0.5869** 0.8986

Particles ,50 mm 5.63* 0.5845** 0.8726

Total mineral 3.42* 0.5662** 0.9237

N Particles ,2 mm 1.49* 0.7404** 0.9761

Particles ,20 mm 0.698* 0.7312** 0.9718

Particles ,50 mm 0.495* 0.7370** 0.9658

Total mineral 0.285* 0.7329** 0.9494

(b) Exponential fit: y ¼ Maxð1 2 expð2kxÞÞ

Element Fraction Max k R 2

OC Particles ,2 mm 183.45** 0.0051* 0.4627

Particles ,20 mm 84.06** 0.0129** 0.6829

Particles ,50 mm 64.77** 0.0164** 0.7018

Total mineral 51.82** 0.0164** 0.6504

N Particles ,2 mm 17.42** 0.0748* 0.3884

Particles ,20 mm 7.69** 0.1992** 0.6057

Particles ,50 mm 5.85** 0.2579** 0.6351

Total mineral 4.43** 0.2591** 0.5823

(c) Polynomial fit (third degree): y ¼ b0 þ b1x þ b2x2 þ b3x3

Element Basis b0 b1 b2 b3 R 2

OC Particles , 2 mm 28.9297 1.1019 20.0027 ,0.0001 0.4880

Particles , 20 mm 5.3404 0.7145* 20.0016 ,0.0001 0.7247

Particles , 50 mm 5.2353 0.6610* 20.0017 ,20.0001 0.7510

Total mineral 20.1098 0.8514 20.0057 ,0.0001 0.6583

N Particles , 2 mm 22.8634 1.6622 20.0516 0.0006 0.3973

Particles , 20 mm 21.3484 1.5303** 20.0747** 0.0010 0.7814

Particles , 50 mm 20.8595 1.4584** 20.0925** 0.0008* 0.7882

Total mineral 0.3230 0.7940 20.0397 0.0001 0.6257

The data corresponding to H horizons have been included in the figures, but have not been taken into account for fitting. Significance of the parameters: (*)

at P ¼ 0.05; (**) at P ¼ 0.01. Without asterisk: non-significant. In all cases, x is total OC or N (in g kg21 of the considered fraction), and y is the protected OC

or N (in g kg21 of the considered fraction). The fraction may be the mineral particles ,2 mm (clays), ,20 mm (fine silt þ clays), ,50 mm (coarse silt þ fine

silt þ clays), or the whole mineral matter (all size fractions).


with depth was observed. The strong resistance of N to acid

hydrolysis in the DF of B horizons may be due to the

presence of fixed N (interlayers of clays), which is known to

be partly resistant to acid attack (Freney and Miller, 1970).

As the horizon becomes poorer in both OC and N, the

proportion of fixed N to total N increases (Stevenson, 1982).

The first occluded fraction (Ocl I) is the most recalcitrant

one: both RIC and RIN are highest in this fraction (even

though for the DF fraction of B horizons, the RIN value is

higher). From 13C-NMR data, Golchin et al. (1994a, 1995)

observed that this fraction is rich in aromatic carbon and

concluded that it accumulates lignin and other resistant

residues of plant origin, being very stable against microbial

attack. Isotope data (Golchin et al., 1995; Gregorich et al.,

1996) indicate that this fraction is the oldest one; in

agricultural soils it is the one that retains the highest

proportion of carbon derived from the previous crop, or

from the previous forest vegetation. This is attributed to its

physical protection, but the high RIC values found in our

study suggest that this stability may be due also to its high

recalcitrance.

The other two occluded fractions seem an intermediate

between Ocl I and DF. Within the set of protected fractions,

from Ocl I to DF (i.e. as the degree of protection increases)

several trends may be observed: (i) the relative richness in

carbohydrates increases, (ii) the cellulose to total carbo-

hydrates ratio decreases, (iii) the RIC ratio decreases. i.e. the

higher the physical protection, the higher the quality of

carbon. However, the decrease in the relative cellulose

content suggests that this trend is not due to the physical

protection of labile plant-derived fragments or compounds,

but to the association of newly formed microbial products

with these fractions.

Little differences between horizons were observed, for

a given obtained fraction. Only in the total carbohydrate

content does the H horizon behave differently from A and

B horizons. This meets the desirable aim of a

fractionation method for soil OM: the differences in the

global OM characteristics are explained mainly by

differences in the proportions of the several fractions,

rather than by differences in the chemical characteristics

of the fractions.

4.4. The capacity of soils to protect OC and N

According to Hassink (1995a, 1996), mineral fractions

,20 mm are mainly responsible for the physical protection

of organic matter, because of its occlusion into micro-

aggregates; the association with other particles is not taken

into account. Our data agree with this view. For both OC

and N, the degree of fitting of both the exponential and the

polynomial models is low if only the clays (,2 mm) are

taken into account, but it increases strongly if fine silt (2–

20 mm) is included (Table 6). In contrast, if coarse silt (20–

50 mm) is added, the degree of fitting increases only slightly,

and if sand is included (i.e. the whole mineral matter of

the horizon), the fit decreases again. This suggest that

particles ,20 mm, are responsible for most of the physical

protection of soil organic matter, whereas the effect of

coarse silt is of much lower importance.

In Hassink’s approach, the protective capacity of soils is

related to the abundance of these fractions in the mineral

matrix, in the form

Cmaxðg kg21Þ ¼ 4:09 þ 0:37X

Nmaxðg kg21Þ ¼ 0:40 þ 0:037X

where X is the percentage of mineral particles ,20 mm of

the soil. If we assume X ¼ 100%, i.e. 1 kg soil ¼ 1 kg

particles ,20 mm, then we find that 1 kg of fine silt plus

clay could protect 41.09 g of OC, and 4.1 g of N. These

values are much lower than those obtained in our approach,

which are 84.1 g of OC and 7.7 g of N kg21 of particles

,20 mm (Table 6b). If the limits suggested by Hassink are

taken, then the protective capacity of most of the horizons

studied in this paper would be strongly oversaturated

(Fig. 7).

Again, differences in soil sampling procedures may be

responsible for this difference. Hassink based their

regression equations on previously published data from

other authors, which often sampled the soils as cores, to a

depth of 10–20 cm, thereby pooling data of several layers in

a single mean value. Since the strong decrease in OC

content and degree of protection occurs within a few cm,

such a method involves a considerable loss of information.

An important detail of our approach, is the exclusion of H

horizons in the fitting process. H horizons may be viewed as

highly temporal layers through which OM is transported

downwards to the mineral soil, hence not comparable to the

A and B horizons, in which the stabilization of OC is much

longer. In forest soils of the semiarid mediterranean zone, H

horizons are usually poorly developed or absent, and they

develop strongly only when the incorporation of litter

material to the mineral soil is hampered by a stone or gravel

layer (Casals et al., 2000; Romanya et al., 2000). Our H

horizons were morphologically similar to the A horizons,

albeit darker because of their higher OC content. The

criterion of 20% OC is that usually taken to separate H from

A horizons; it is somewhat arbitrary, and then to exclude H

horizons from the analysis is also somewhat arbitrary.

Anyway, including H horizons in curve fitting resulted in

little change in the results: only in a slight decrease of the

maximum values of protective capacity, which in spite of

this were still much higher than the values given in

Hassink’s equation (not shown).

These upper limits reflect the physical protective

capacity of the fine mineral particles, but they are also the

result of an equilibrium between input and output of OC and

N coming from the free fractions. Both the protection and

the de-protection of organic matter may depend on climatic

constraints (number and intensity of drying–wetting cycles,

temperature, freezing and thawing), and as a consequence


may differ geographically. Some mineralogical properties

of the soil, such as clay type, are also expected to affect its

ability to protect organic matter. Our data have been

obtained with soils coming from a relatively small area,

under a dry mediterranean climate; and the obtained values

for the protective capacity of OC and N could be not valid

for other climates. As mentioned by Christensen (1992), the

soils of arid zones are expected to accumulate more free

organic matter than those from wet areas. Both latitudinal

and altitudinal gradients are possible.

The assumption of a big value for the protective capacity

implies that most soils could accumulate much more OC

than they actually do, since B horizons are highly OC-

unsaturated. The poor capacity of soils to act as a sink for

OC, as noted by Schlesinger (1990), may be related to low

rates of incorporation of free OC to the protected fractions

and also to the low OM inputs below the first cm of the soils,

either because root inputs concentrate in the uppermost

layers (Jackson et al., 1996), and/or because in many soils

the transport of OM down the profile could be of lower

importance than often assumed.

5. Conclusions

Most of the organic matter in the studied Mediterranean

forest soils is free and an upper limit can be detected for the

protective capacity of the soil horizons. This upper limit,

much higher than that suggested previously by other

authors, is reached only in the uppermost horizons (H, or

some A rich in organic matter), whereas the deep layers (B

horizons) are strongly undersaturated and their ability to

stabilize organic matter is largely under-used.

Acknowledgements

The authors are indebted to the European Community for

funding this research (VAMOS project). The collaboration

of the Serveis Cientıfico-Tecnics of the University of

Barcelona is greatly acknowledged.

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