labile and recalcitrant pools of carbon and nitrogen in organic matter decomposing at different...

Labile and recalcitrant pools of carbon and nitrogen

in organic matter decomposing at different depths in

soil: an acid hydrolysis approach

Pere Rovira a,*, V. Ramon Vallejo a,b

aDepartament de Biologia Vegetal, Facultat de Biologia, Universitat de Barcelona, Diagonal 645,

08028 Barcelona, SpainbCEAM, Parc Tecnologic, Charles Darwin 14, 46980 Paterna, Valencia, Spain

Received 19 December 2000; received in revised form 5 September 2001; accepted 19 September 2001

Abstract

The quality of soil organic matter (OM) depends on its distribution among labile and recalcitrant

pools and the quality of each pool considered. OM quality is assumed to decrease as decomposition

proceeds, but to verify this assumption it is necessary to define quality in operative terms. Here we

study the change in OM quality during decomposition of mixtures of four plant materials

(Medicago sativa whole ground plants, and ground litter of Eucalyptus globulus, Quercus ilex and

Pinus halepensis) with a mineral red earth, incubated at different depths (5, 20, and 40 cm) for 2

years. OM quality was evaluated from acid hydrolysis, considering three pools: (a) Labile Pool I,

obtained by hydrolysis with 5 N H2SO4 at 105 BC for 30 min; (b) Labile Pool II, obtained by

hydrolysis with 26 N H2SO4 at room temperature overnight, then with 2 N H2SO4 at 105 BC for 3

h; and (c) Recalcitrant Pool, the unhydrolyzed residue. In agreement with previously published

results, the recalcitrant C/total OC (RIC), and recalcitrant N/total N (RIN) ratios are regarded as

indicators of global OC and N quality. In addition, in Labile Pools I and II, the ratio carbohydrate

C/polyphenol C is used as indicator of OC quality.

The main findings obtained by applying this approach can be summarized as follows:

(1) In undecomposed plant materials, initial RIC ranged from 25% to 60% (Medicago and Pinus

mixtures, at extreme values). Throughout decomposition, RIC values increased strongly (Medicago

mixtures), slightly (Eucalyptus), or were roughly maintained (Quercus and Pinus), suggesting that

strong decreases in OC quality occur only for easily decomposable plant materials.

(2) Initial RIN values were between 15% and 30%, i.e., much lower than RIC ones. In contrast

with the behaviour of RIC, the RIN values strongly increased in all cases, or, in other words, N

0016-7061/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0016 -7061 (01 )00143 -4

Abbreviations: OM, organic matter; OC, organic carbon; RIC, recalcitrance index for carbon; RIN,

recalcitrance index for nitrogen.* Corresponding author. Fax: +34-93-411-2842.

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

www.elsevier.com/locate/geoderma

Geoderma 107 (2002) 109–141

quality clearly decreased for all plant materials, owing not only to a lower mineralization of the

recalcitrant N, but also to a net incorporation of N to this pool. The amount of incorporated N is

significantly related to the initial lignin content of the incubated plant material. Such incorporation

seems to occur during wet periods; in contrast, its relationship with temperature was hardly

detectable. No similar phenomenon was detected for recalcitrant C.

(3) The 13C-CPMAS-NMR spectra of the recalcitrant pool showed prominent peaks in the 0–45

ppm region, which corresponds to the alkyl C and accounts for up to 50% of the total

unhydrolyzable C in Quercus mixtures. In contrast, the aromatic zone, 110–160 ppm, was poorly

apparent. These features were maintained more or less intact during the 2 years of field incubation,

and suggest that lipidic polymers represent a substantial part of the recalcitrant pool.

(4) Throughout the decomposition process, the ratio Labile Pool II/Labile Pools I + II decreased

for carbohydrates, and increased for phenolic compounds. The use of these ratios is suggested to

evaluate the degree of decomposition of plant residues. In Labile Pool I the ratio carbohydrate C/

polyphenol C remained the same, whereas for Labile Pool II this ratio decreased strongly,

suggesting that the changes in quality may be restricted to a single pool.

(5) Samples incubated in upper horizons (5-cm depth) were subjected to a much drier pedoclimate

than those incubated at deep layers (20 and 40 cm), resulting in a slower mineralization of both the

labile and the recalcitrant pools of C and N. Nevertheless, on a mineralized OC basis, most indicators

of quality did not differ statistically between depths. Hence, the drought in the upper horizon retarded

the decomposition, but did not result in a different biochemical evolution.

Because of its simplicity, chemical fractionation into three pools is a useful approach to

characterize biochemical changes in C and N quality during plant residue decomposition. D 2002

Elsevier Science B.V. All rights reserved.

Keywords: Decomposition; Labile C; Recalcitrant C; Labile N; Recalcitrant N; Soil depth; Carbohydrates;

Polyphenolics; Lignin

1. Introduction

Quality ( q) is a key concept in soil organic matter (OM) studies, and may be defined

as the capacity of OM to be utilized by soil microbes as a source of energy, and/or

carbon skeletons for their own structures. The main consequence of a high q is high

microbial activity with respect to the substrate organic carbon (OC) content, which

results in rapid decomposition.

Microbial respiration may be used as an indicator of q, although strictly it is not the

quality, but a consequence of it. Quality is more a function of the chemical and biochemical

features of the substrate, and most indices which have been suggested are based on this

point of view: C/N ratio (Vallejo, 1993), lignin/N (Taylor et al., 1989), holocellulose/lignin

(McClaugherty and Berg, 1987), cellulose/lignin/N (Entry and Backman, 1995). These

indices reflect, to some degree, the proportion of labile compounds (carbohydrates,

proteins) and recalcitrant compounds (mainly lignin, but also suberins, resins, fats and

waxes). In the theoretical approach of Bosatta and Agren (1991), soil OM is considered as

the integral sum of an infinite number of pools, and the quality of the whole soil OM ( q) as

the result of the different qualities ( qi) of each pool i. Nevertheless, this ideal concept is not

useful, unless the number of pools is restricted to a finite (and, if possible, not large)

number. Berendse et al. (1985) suggested that decomposing plant material should be

P. Rovira, V.R. Vallejo / Geoderma 107 (2002) 109–141110

divided into three basic pools: (a) free carbohydrates, not masked by lignin, (b) carbohy-

drates masked by lignin, mainly cellulose, and (c) lignin itself. In practice, these pools are

expected to be more complex, since carbohydrates and lignin are not the only components

of organic substrates, but the approach is simple and highly operative.

Lignin in undecomposed plant residues is usually quantified as Klason lignin, i.e., the

residue of hydrolysis with concentrated sulfuric acid. The resistance to acid hydrolysis,

however, is not exclusive of lignin, but a common property of most recalcitrant organic

polymers (lignin, suberin, resins, waxes). Hence, an attack with strong acid could be used

as a criterion to separate labile and recalcitrant fractions, and therefore estimate q. For soil

OM, radiocarbon dating studies have repeatedly shown that unhydrolyzable C (after being

subjected to 6 N HCl or 26 N H2SO4) is much older than hydrolyzed C (Stout et al., 1981;

Balesdent, 1987; Leavitt et al., 1997; Paul et al., 1997). We observed recently (Rovira and

Vallejo, 2000b) that for several undecomposed plant residues, the percentage of C and N

resistant to hydrolysis with 26 N sulfuric acid was the best predictor of OC and N

mineralization, rather than hydrolysis with 6 N HCl, and much better than other indices

such as C/N or Lignin/N ratios.

The next step would obviously be the study of the changes in q throughout the

decomposition process. These should reflect both changes in the proportions of labile and

recalcitrant pools, and changes in the quality of each pool. Labile pools would be expected

to receive compounds released from the partial biodegradation of refractory polymers; this

would ensure the maintenance of these labile pools but may lead probably to a decrease in

their overall qualities. On the other hand, secondary compounds may join the recalcitrant

pool through, among other reactions, oxidative condensation of amino acids and peptides

with aromatic compounds such as tannins, quinones or lignin itself. These reactions,

thought to be essential to the genesis of humic substances (Stevenson, 1982), result in the

incorporation of N to the recalcitrant pool, which is important because the original

recalcitrant polymers, such as suberin, lignin, some fats and waxes, do not contain N. Both

of these opposing processes probably depend on pedoclimatic conditions. Lignin bio-

degradation, for instance, is hindered under anaerobic conditions (Kirk and Farrell, 1987),

a common result of waterlogging.

We recently reported on the decomposition of plant residues at different depths in

mineral soil for 2 years (Rovira and Vallejo, 1997, 2000a). Here, we aim to apply the

approach of Berendse et al. (1985) to characterize the changes in substrate quality during

decomposition. Our interest will focus on the following points: (i) changes in the

proportion of pools, mainly the proportion of unhydrolyzable pool relative to total; (ii)

changes in the quality of the pools; (iii) how these changes are affected by the distinct

pedoclimatic conditions at each depth (upper vs. deep layers); and (iv) the comparison of

the behaviour of OC and N under this approach, paying special attention to the possible

incorporation of N to the recalcitrant pool.

2. Materials and methods

The field incubation was designed to ascertain the effect of depth upon OM

decomposition, paying special attention to differentiate the effects of depth from

P. Rovira, V.R. Vallejo / Geoderma 107 (2002) 109–141 111

differences in the age of organic matter. The experiment consisted in the incubation

under field conditions of mixtures of plant material and mineral soil, at different depths,

and the study of the mineralization of OC and N; it was described in great detail in a

previous paper (Rovira and Vallejo, 1997) and only the most important features will be

mentioned here.

2.1. Site

The experiment was carried out at the University of Barcelona (Catalonia, NE of

Spain), at 41B22V59WN, 2B6V44WE, and 60 m above sea level. Mean annual precipitation is

614 mm, mainly in the period from October until April, although the distribution of rain

varies yearly. Mean annual temperature is 15.5 BC. The soil is a Calcic Luvisol (FAO)

(Table 1).

During the 2 years of field exposure, both precipitation (P, in mm) and air temper-

ature (T, in BC) were recorded. From these data, the climatic diagrams were obtained,

according to Walter (1973) (Fig. 1). The water deficit of a given period can be

quantitatively given as

Xn

i¼1

2Ti �Xn

i¼1

Pi ð1Þ

n being the total number of days of the period under consideration. Dry periods (which

occur when 2T >P) are given as light-shadowed areas in the graph, whereas wet periods

(2T <P) are given as dark-shadowed areas.

2.2. Soil and plant materials

Four plant materials were selected: fresh, green aerial part of lucerne (Medicago sativa

shoots and leaves; flowers and fruits excluded) and dead brown litter of eucalypt

(Eucalyptus globulus), green oak (Quercus ilex) and white pine (Pinus halepensis). All

were dried and ground to pass through a 200-Am mesh. Their most relevant characteristics

are given in Table 2.

Table 1

Main features of the soil plot and the red earth (RE) used for the mixtures with plant materials

Depth (cm) OC N pH (H2O) CaCO3 Texture

0–14 4.1 0.7 7.5 68.0 Loam

14–47 3.2 0.6 7.5 75.1 Clay loam

REa 1.8 0.5 8.3 118.2 Clay loam

Only the horizons affecting the experiments are given. OC was measured by the Mebius method, N with a

CARLO ERBA analyzer, CaCO3 by the calcimeter method, pH in a suspension of soil in water 1:2.5 w/v, texture

by the pipette method. OC, N and CaCO3 are given as g kg� 1.a RE (red earth) selected to be mixed with the plant materials.


A red earth (RE) extracted from a deep layer (180 cm) of an adjacent plot was se-

lected because of its very low OC content (Table 1); it was air-dried and passed through

a 2-mm mesh.

Fig. 1. (a) Mean monthly temperature and precipitation of Barcelona. (b) Meteorological features during the 2

years of field incubation. The light-shadowed area shows the periods of water deficit, and the dark-shadowed the

wet periods, in accordance with Walter. (c) Water pressure of the soil (MPa) at the different samplings. At the

second sampling (6 months), it was not quantified.


The four plant materials were mixed with the RE in calculated proportions to obtain

mixtures (50 g) with 60 mg OC g� 1. The proportion of plant material to RE in the

mixtures (weight:weight) was 7.06:42.94 for Medicago, 6.16:43.84 for Eucalyptus,

6.36:43.65 for Quercus, and 5.87:44.13 for Pinus. Since the OC content of the RE

was very low, almost all the OC of the mixture is plant-derived. To ensure homoge-

nization, water was gently added when mixing RE with the plant material. After

homogenization, wet samples were placed in nylon mesh bags (6.5 cm wide� 6.5 cm

length� 2 cm thick; hole: 0.5 mm) which were immediately sealed and stored at 4 BCuntil burial in the soil.

2.3. Sampling and sample treatment

In autumn 1991, bags were buried in previously made holes of 11 cm diameter (1 bag

per hole) at three depths: 5, 20 and 40 cm. Holes were refilled with the same earth

extracted from it. The soil was carefully compacted around the bags to ensure contact

between the samples and the surrounding soil. Holes were 20 cm apart.

Table 2

Initial characteristics of the plant materials, and of their mixtures with the red earth

Pool Parameter Medicago Eucalyptus Quercus Pinus

(a) Plant materials alone

Whole C (mg g� 1) 411 471 456 494

N (mg g� 1) 34 17 19 11

C:N 12 28 23 44

Lignina (mg g� 1) 173 212 302 316

Lignin:N 5.1 12.5 15.4 27.9

(b) Mixtures plant material + red earth: general characteristics

Whole OC (mg g� 1) 60 60 60 60

N (mg g� 1) 6.1 2.6 3.0 1.8

(c) Mixtures plant material + red earth: fractionation (three-pools approach)

LP I % of the total OC 45.2 (0.4) 31.6 (0.5) 28.3 (1.9) 22.6 (0.3)

Carbohydratesb (mg g� 1) 25.9 (1.7) 27.8 (0.6) 27.8 (0.5) 24.8 (1.0)

Polyphenolicsc (mg g� 1) 9.4 (0.2) 17.7 (0.5) 13.9 (0.6) 10.5 (0.5)

LP II % of the total OC 29.3 (0.7) 13.7 (0.3) 18.1 (1.5) 13.5 (0.2)

Carbohydrates (mg g� 1) 28.1 ( < 0.1) 11.6 (0.7) 18.1 (0.7) 18.5 (0.6)

Polyphenolics (mg g� 1) 2.4 (0.1) 2.5 (0.1) 2.4 (0.1) 2.1 ( < 0.1)

RP % of the total OC ( =RIC) 25.5 (1.6) 54.7 (3.1) 53.6 (1.2) 63.9 (1.5)

% of the total N ( =RIN) 14.0 (0.8) 31.6 (2.9) 32.2 (0.9) 33.8 (0.3)

For the biochemical fractionation (three-pools approach), the reproducibility is given: the data are the means of

three replicates, values in parentheses are standard deviations. LP I: Labile Pool I (first hydrolysate); LP II: Labile

Pool II (second hydrolysate); RP: recalcitrant pool (unhydrolyzed residue).a Klason lignin, obtained by hydrolysis with 26 N sulfuric acid in ground plant materials previously extracted

with ethanol–benzene. The method is given in detail in Rovira and Vallejo (1997).b Carbohydrates given as glucose equivalents.c Polyphenolics given as tannic acid equivalents.


The incubation lasted 2 years, from September 1991 until September 1993. Throughout

the experiment, weed seedlings were removed from the plot surface by hand, as soon as

they appeared. No herbicide was applied.

Three bags per experimental condition (plant material and depth) were collected every

3 months. At the same time, the surrounding soil at each depth (5, 20 and 40 cm) was

sampled to evaluate its water content, by desiccation overnight at 105 BC. Once at the

laboratory, samples were removed from the nylon bags. The external part of all samples

(up to 5 mm thick), which could be affected by material from the surrounding soil, was

discarded. Only the internal part of the sample was used for chemical analyses. Field-moist

subsamples were taken to analyze the inorganic N and to measure the water content. The

rest was spread on a dish, air-dried and homogenized. A subsample was finely ground, and

analyzed for OC by the modified Mebius method (following the conditions given in Soon

and Abboud, 1991) and for total N with a CARLO ERBA elemental analyzer.

For the surrounding soil, the data of water content were corrected for gravel (>2 mm)

content. To convert data of water content (%) to water potential (MPa), the surrounding

soil was characterized previously by a water retention curve, using the ceramic plate

method (Klute, 1986).

2.4. Acid hydrolysis

The protocol follows the two-step method recommended by Oades et al. (1970) for a

maximum release of carbohydrates from soil samples.

(i) About 500 mg of ground soil sample was hydrolyzed with 20 ml of 5 N H2SO4 for

30 min at 105 BC in sealed Pyrex tubes. The hydrolysate was recovered by centrifugation

and decantation. The residue was washed with 20 ml of water and the washing added to

the hydrolysate. This hydrolysate was taken as Labile Pool I. The residue was dried at

60 BC.(ii) The remaining residue was hydrolyzed with 2 ml of 26 N H2SO4 overnight at room

temperature, under continuous shaking. Thereafter, water was added to dilute the acid to 2

N and the sample was hydrolyzed for 3 h at 105 BC with occasional shaking. The

hydrolysate was recovered by centrifugation and decantation. The residue was washed

with 20 ml of water, and the washing added to the hydrolysate. This hydrolysate was taken

as Labile Pool II.

(iii) The remaining residue was washed twice with water, transferred to a pre-weighed

crucible, and dried at 60 BC. This fraction was taken as Recalcitrant Pool.

2.5. Analysis of labile (hydrolyzable) pools

The Labile Pools I and II were analyzed:

bFor total C, with a Shimadzu TOC analyzer.

bFor total sugars, following the phenol–sulfuric method (Dubois et al., 1956), using

glucose as standard, after elimination of Fe3+ by passing aliquots of the solution through a

column of Amberlite IRA-20 cationic resin to avoid interferences (Martens and Frank-

enberger, 1993). To obtain the carbohydrate C, we assumed that the ratio weight/C was

that of glucose, i.e., 2.5.


bFor total phenolics, following the Folin–Denis method (Ribereau-Gayon, 1968),

using tannic acid as standard. To obtain the polyphenolic C, a weight/C ratio of 1.86 was

assumed. This value was obtained from the formula for tannic acid (C76H52O46) given in

Weast (1964).

The carbohydrate C/polyphenolic C ratio was then calculated, as an estimation of the

quality of the hydrolyzable C. This ratio is meant to reflect the balance between easily

biodegradable compounds (of which carbohydrates are the most typical) and refractory

compounds (of which polyphenolics are the most representative).

Whereas TOC was analyzed in all the hydrolysates, sugars and polyphenolics were

analyzed only in those of initial samples (Table 2), and for the samplings corresponding to

3, 6, 12, 18 and 24 months of incubation.

2.6. Characterization of the recalcitrant pool

The C and N of the recalcitrant pool were analyzed with a Carlo Erba CNS analyzer.

From these data, we obtained the following recalcitrancy indices (RI):

RIC ð%Þ ¼ ðUnhydrolyzed C=Total OCÞ � 100 ð2Þ

RIN ð%Þ ¼ ðUnhydrolyzed N=Total NÞ � 100 ð3Þ

Recalcitrant C forms were characterized by solid-state 13C-CPMAS-NMR spectro-

scopy, in a Varian 500 Spectrometer, at 75 MHz, using a single contact time of 1 ms and a

recycle time of 100 ms. The conditions and the assignment of resonances (Table 3) follow

Frund et al. (1994). About 106 accumulated transients were needed to obtain a reasonably

clear spectrum. The aromaticity index was calculated using the integrated areas:

Aromatic C ð110� 160Þ=½Aromatic Cþ Aliphatic C ð0� 110Þ� ð4Þ

The 160–210 ppm area was not considered in Eq. (4), since the carboxyl groups can be

assigned to both types of C structures (Schnitzer, 1990; Frund et al., 1994).

For NMR analysis, the intact samples were previously treated with HF to reduce the

amount of Fe3 + , which could obscure reasonably clean, well-resolved spectra (Skjemstad

Table 3

Assignment of the resonances in NMR

Shift (ppm) Forms of carbon

0–45 Aliphatic carbon

45–60 Methoxyl groups and C-6 of some polysaccharides, C-a of amino acids

60–80 Carbohydrates (C-2 to C-5 of hexoses), Proteinaceous material, Higher alcohols,

Aliphatic part of lignin structures, (sp3 CO/CN)

80–110 C-1 of carbohydrates, C-2, C-6 of syringyl units

110–140 Aromatic C–H carbons

140–160 Aromatic CO–R groups

160–210 Carboxyl/carbonyl groups, amides


et al., 1994). The recalcitrant fraction was not treated in this form, since previous trials

showed that most of the iron was extracted during the two consecutive hydrolyses with

sulfuric acid. Since obtaining a single spectrum is laborious, only a few selected samples

were studied.

2.7. Statistical analysis

For each plant material type, data were compared by two-way ANOVA, taking the

sampling date as the first factor (eight levels: the initial value was not considered) and

incubation depth as the second factor (three levels: 5, 20 and 40 cm). In addition, data were

compared by analysis of covariance (ANCOVA), taking depth as a factor (three levels: 5,

20 and 40 cm) and remaining OC as covariate. In both analyses, since most data were

percentages, they were all transformed by arcsines of square root (Neter et al., 1990). The

effect of a factor was considered significant when (P>F)V 0.05.

In a few cases (Fig. 4: remaining recalcitrant N in Eucalyptus and Quercus; Fig. 9:

remaining carbohydrates in Eucalyptus and Quercus) the relationship of the response

variable was clearly non-linear, and it was not possible to linearize it through mathe-

matical transformations. In these cases, ANCOVA was not appropriate, and a different

strategy was followed. After transformation by arcsines of square root, the resulting sets

of points were fitted to polynomial curves, and the obtained curves for the several depths

were compared following Cuadras (1979). For a given pair of polynomial curves, after

verifying the equality of their residual variances, the differences were tested through the

statistic:

F ¼ ðR21 � R2

0ð1Þ � R20ð2ÞÞ=ðmþ 1Þ

ðR20ð1Þ � R2

0ð2ÞÞ=ðn1 þ n2 � 2m� 2Þ ð5Þ

in which R12 is the residual sum of squares of the global curve (including both series of

points), R02 (1) and R0

2 (2) the residual sum of squares of the first and the second curve,

respectively, m is the degree of the polynomial (which must be the same for all curves),

and n1 and n2 the number of points of the first and the second curve, respectively. The

statistic F follows a F distribution with (m + 1) and (n1 + n2� 2m� 2) degrees of

freedom. The equality of curves was accepted when (P>F)V 0.05. In this case, both

sets of data were pooled, and a single polynomial for both was considered. The

comparison between data from different depths was done in the order: (i) polynomial

curves of 20 and 40 cm depth were compared first; (ii) if the differences were non-

significant, then both sets were pooled, and the single curve for both was taken; then (iii)

this curve was compared with that of 5-cm depth. This sequence was followed because

for most analyses no differences were observed between 20 and 40 cm, whereas the

differences, when they appeared, were almost always between 5 cm and the other two

depths (20 and 40).

To test whether RIC and RIN were reliable predictors of OC and N mineralization, we

used a multiple regression analysis, in which the OC or N mineralized during a given

period of time (either 6 months or 1 year) was the dependent variable, and the independent

(external) variables were the RIC or RIN at the beginning of the period, the total


precipitation in mm, and the mean air temperature during the period. The experimental

mineralization rates were calculated as follows:

Rate ð%Þ ¼ ½ðXt � XtþuÞ=Xt� � 100 ð6Þ

where Xt is the remaining OC or N at time t, and Xt + u is the remaining OC or N at time

t+ u. Xt and Xt� u are the average of three replicates. For the regression analysis, (i) the

four types of plant material were pooled, since a single type of plant material often covers

a range of RIC or RIN too narrow to detect any significant relationship with mineralization

rate, at least under field conditions, where the variability may be high; and (ii) the

correlations for each incubation depth were calculated separately, to avoid including in the

same analysis samples submitted to different pedoclimates. The multiple regression

analysis was performed by stepwise elimination, i.e., the complete equation including

all the terms was obtained first, the non-significant terms were then eliminated and the

equation recalculated at each step, until only the significant terms remained.

Statistical analysis was performed using SPSS for Windows, v. 6.0, except for the

comparison of polynomial curves, which was done manually, using a worksheet.

3. Results

3.1. Climatic constraints (Fig. 1)

The distribution of precipitation greatly differed during the 2 years of the experiment,

and deviated strongly from the 15-year average (Fig. 1a,b). Such irregularities are typical

of the Mediterranean climate. Total precipitation and mean temperature were 660 mm and

16.1 BC during the first year, and 596 mm and 15.5 BC during the second year.

Although neither of the two summers were particularly dry, water content of the

surrounding soil was very low, especially at 5-cm depth, where the water pressure (MPa)

dropped far below the wilting point. In summer the percentage of water was often less than

5%, close to the typical values reached after air-drying treatments, and the mixtures

became severely crusted. This crusting was reversible, since it disappeared at the next

sampling. These are evidences of stronger drying–rewetting cycles at upper layers. At 20

and 40 cm depth, the fluctuations in the water content were less; moisture did not decrease

to the wilting point and the mixtures were never crusted.

3.2. Mineralization of whole OC and N

The mineralization of whole OC and N was studied in detail elsewhere (Rovira and

Vallejo, 1997, 2000a), and only the most relevant facts will be mentioned here. In

Medicago mixtures the kinetics of mineralization of OC and N was of double-exponential

type, typical of fresh plant materials, with a strong initial decay and a low mineralization

rate thereafter. In the other plant materials, both OC and N mineralization proceeded much

more gradually. For both OC and N, mineralization was usually slower at 5-cm depth,


whereas no differences were observed between mineralization at 20 and 40 cm. The

percents of remaining OC and N after 2 years of field exposure are given in Table 4.

3.3. Recalcitrant C and N

The initial RIC, between 25% and 65%, followed the order Medicago<Quercus <Eu-

Eucalyptus <Pinus. RIC increased in Medicago and Eucalyptus, slightly increased in

Quercus and did not increase in Pinus mixtures (Fig. 2).

In contrast, RIN increased in all cases (Fig. 2). At the beginning of the experiment, the

recalcitrance ratios of N were lower than those of C in all the plant mixtures, while at the

end the opposite was found for Medicago and Pinus, where the increase was particularly

strong. ANOVA analysis detected that depth influenced only Eucalyptus mixtures, in

which the RIN was significantly higher at 5 cm, whereas no differences were detected

between 20 and 40 cm. This was clearly due to the high RIN found at 5 cm during the

second year.

The behaviour of recalcitrant C and N differed (Fig. 3). Recalcitrant C always

decreased, while recalcitrant N alternated between periods of slow decrease and periods

with sequestration of N into the recalcitrant fraction. After 2 years, in some cases the total

recalcitrant N was higher than in the beginning. At 5-cm depth, the amount of recalcitrant

C and N was usually higher than at 20 and 40 cm; no differences were detected between 20

and 40 cm.

According to the ANCOVA analysis, the effect of depth on the retention of

recalcitrant C or N during incubation was usually not significant (Fig. 4), i.e., for a

given remaining total OC, the remaining recalcitrant C or N was generally not affected

by depth.

3.4. Relationship between recalcitrance and mineralization rate

Regression analysis was performed with the RIC and RIN and the mineralization rates

for OC and N (Table 5). Significant correlations were observed in all cases, either

considering periods of 6 months (0–6 months, 3–9, 6–12, etc.) or 1 year (0–12 months,

3–15, 6–18, etc.).

Table 4

Percent of remaining OC and N after 2 years of field incubation

Species OC N

5 cm 20 cm 40 cm 5 cm 20 cm 40 cm

Medicago 28.0 a1 (0.9) 19.9 b1 (0.8) 18.8 b1 (1.2) 17.6 a1 (3.1) 12.2 b1 (0.9) 11.3 b1 (1.0)

Eucalyptus 70.2 a2 (1.4) 43.2 b2 (5.6) 45.2 b2 (5.3) 76.0 a2 (1.3) 63.8 a2 (10.5) 61.9 a2 (4.9)

Quercus 65.5 a2 (6.7) 41.4 b2 (2.5) 48.5 b2 (7.5) 76.9 a2 (4.2) 53.1 b2 (5.4) 60.3 b2 (7.6)

Pinus 73.7 a2 (4.2) 56.2 a2 (15.6) 55.5 a2 (5.9) 67.2 a3 (2.8) 54.4 b2 (0.5) 57.8 b2 (4.7)

Data are means of n= 3; values in parentheses are standard deviations. For a given element, within a row, values

followed by the same letter do not differ, at P= 0.05; within a column, values followed by the same number do not

differ, at P= 0.05.


For OC, the effect of precipitation was significant in all the cases but in mixtures

incubated at 20 cm (6-month intervals). This was also the only case in which the effect of

temperature on mineralization rate was significant. For N, in contrast, the effect of

precipitation or temperature on the mineralization rates was rarely detectable.

Fig. 2. Changes in RIC and RIN during the 2 years of the experiment. Points are the means of three replicates;

vertical bars are standard deviations. The degree of significance of depth effect is given.


3.5. Seasonal cycles in RIN values

RIN ratios showed a quite regular pattern, with two maxima at 9 and 24 months. This

fluctuation was not exactly seasonal, since the first maximum coincided with the end of

Fig. 3. Remaining recalcitrant C and N during the 2 years of the experiment. Points are the means of three

replicates; vertical bars are standard deviations. The degree of significance of depth effect is given. Same legends

as in Fig. 2.


the first spring, while the second coincided with the end of the second summer. Changes in

RIN values were related to climatic factors (Table 6, Fig. 5). Precipitation was the main

constraint, whereas mean temperature was not significantly correlated with RIN changes.

Fig. 4. Remaining recalcitrant C and N, related to remaining OC (for ANCOVA analysis, except in the recalcitrant

N of Eucalyptus and Quercus, for which the comparison of polynomials was applied). Analysis is performed with

all the points (not with the means). The degree of significance of depth effect is given. Same legends as in Fig. 2.


The combination of both into a single index, water deficit (Eq. (1)), usually did not

increase r values. When correlations were calculated for each depth separately, they

seldom were significant. However, they were significant when all the depths were

considered (n = 24).

The increase in RIN was due to a decrease in the amount of labile N, and to a net

increase in the amount of recalcitrant N (Fig. 6), both related to precipitation. As for

Table 6

Correlation (r) between climatic parameters, and percent of increase/decrease of RIN during the time between two

samplings (3 months), calculated from meansa

Parameter Depth Medicago Eucalyptus Quercus Pinus

T mean (BC) 5 0.013 0.001 0.131 0.107

20 0.035 0.003 0.368 0.135

40 0.098 0.008 0.263 0.456

All 0.043* 0.004 0.247* 0.221*

Precipitation (mm) 5 0.941** 0.226 0.562 0.838*

20 0.911* 0.518 0.511 0.542

40 0.582* 0.232 0.483 0.474

All 0.796*** 0.321** 0.508*** 0.574***

Water deficit 5 0.960** 0.233 0.520 0.803*

20 0.912** 0.534 0.428 0.500*

40 0.550* 0.233 0.417 0.382

All 0.789*** 0.329** 0.443*** 0.512***

Without asterisk: non-significant; *: significant at PV 0.05; **: at PV 0.01; ***: at PV 0.001.a For a given sampling n, the relative change in RIN is given by [(RIN n�RIN n� 1)/RIN n� 1]� 100.

Table 5

Multiple regression analysis

Time span Depth B1 B2 B3 B4 r2

(a) Results for OC

6 months 5 35.6** � 0.84*** 0.07* 0.575

20 52.5*** � 1.05*** 1.35** 0.555

40 31.0 � 0.83** 0.11* 0.459

12 months 5 23.2 � 1.16*** 0.09*** 0.866

20 53.0* � 1.03*** 0.05* 0.691

40 37.2 � 0.97*** 0.07* 0.658

(b) Results for N

6 months 5 48.1*** � 0.97*** 0.401

20 61.8*** � 1.18*** 0.434

40 63.2*** � 1.30*** 0.410

12 months 5 17.2 � 1.19*** 0.07* 0.741

20 79.8*** � 1.34*** 0.492

40 78.6*** � 1.43*** 0.461

The coefficients Bi are given for the equation: Mineralization rate =B1 +B2RIC [or RIN] +B3PREC+B4TEMP +

error, where PREC is the total precipitation in mm and TEMP is the mean air temperature during the period

considered. Without asterisk: non-significant; *: significant at PV 0.05; **: at PV 0.01; ***: at PV 0.001. Blank

values are assumed to be zero (i.e., not included in the final equation).


changes in RIN, correlations were usually highly significant when all the depths were

considered, whereas they were not for each depth separately. Temperature was much less

relevant; the incorporation of N into the recalcitrant pool was significantly related to

temperature only for Pinus mixtures (not shown).

3.6. Recalcitrant C (NMR)

Representative spectra of samples are shown in Fig. 7. Initial samples showed the

highest peaks at 45–80 and 80–110 ppm, which correspond mainly to carbohydrates,

amino acids, and proteinaceous materials (Table 3). In contrast, the aromatic zone (110–

160 ppm) was poorly visible. At the end of the experiment, the initially high peaks were

less marked, and the 0–45 ppm zone became more prominent.

The residue of acid hydrolysis had a different distribution of the C forms: the 0–45

ppm zone, which in the intact sample was visible but not dominant, became the main form

of C (more than half the total C of the residue) after acid hydrolysis. This zone includes

aliphatic C, but not the aliphatic part of lignin structures, which is found in the 60–80 ppm

zone. In spite of the increase in the aromaticity of the C after acid hydrolysis, aromatic C

was only about 10% of the residue C. The spectra of the residues of samples incubated for

2 years changed little.

Fig. 5. Changes in RIN related to precipitation (mm), given as the percentage of RIN increase in a given sampling,

i.e. for a sampling n: [(RINn�RINn� 1)/RINn� 1]� 100. Same legends as in Fig. 2.


3.7. Labile pools of OC

For each plant material, remaining labile C was in the order 5 cm>20 cm= 40 cm. The

proportion between Labile Pool II and total labile C (II/I + II) suffered little changes: it

Fig. 6. Changes in labile N and recalcitrant N related to precipitation, given as percent. For a given sampling n,

the % of change of labile or recalcitrant N (X) is given by [(Xn�Xn� 1)/Xn� 1]� 100. Only the global regression

line is given. Same legends as in Fig. 2.


Fig. 7. Solid-state 13C-CPMAS-NMR spectra of selected samples. (1) Initial Quercus mixtures, whole sample. (2)

Quercus mixtures, whole sample, after 2 years of field exposure at 5-cm depth. (3) Initial Quercus mixtures:

recalcitrant fraction. (4) Quercus mixtures incubated at 5-cm depth, at the end of the experiment (2 years):

recalcitrant fraction. (5) Quercus mixtures incubated at 40-cm depth, at the end of the experiment (2 years):

recalcitrant fraction.


tended to decrease in Medicago mixtures, tended to increase in Eucalyptus mixtures, and

did not change in Quercus or Pinus mixtures (Fig. 8).

Whereas the behaviour of TOC was similar for both Labile Pools, that of

carbohydrates and polyphenols differed greatly. As expected, the remaining carbohy-

Fig. 8. Remaining labile C (Pools I + II), and Pool II/Pools I + II ratio. Points are the means of three replicates;

vertical bars are standard deviations. The degree of significance of depth effect is given. Same legends as in Fig. 2.


drate content on both pools decreased continuously (not shown). The effect of depth

was highly significant in all cases. According to the Duncan’s test, the amount of

remaining carbohydrates was always higher at 5 cm depth, whereas no differences

Fig. 9. Total carbohydrates (Pools I + II), and Pool II/Pools I + II ratio, related to remaining total OC (for

ANCOVA analysis, except for the remaining carbohydrates of Eucalyptus and Quercus mixtures, for which the

comparison of polynomials was applied). All the points have been included. The degree of significance of depth

effect is given. Same legends as in Fig. 2.


were found between samples incubated at 20 and 40 cm. When ANCOVA analysis

was applied, the differences between depths were never significant (Fig. 9); thus the

differences between samples incubated at 5 cm and those incubated at 20 or 40 cm

Fig. 10. Total polyphenolics (Pools I + II), and Pool II/Pools I + II ratio, vs. remaining total OC (for ANCOVA

analysis). All the points have been included. The degree of significance of depth effect is given. Same legends as

in Fig. 2.


merely reflect the differences in the overall decomposition rate. Carbohydrates fell

down to 20–30% of the initial content, and in Eucalyptus and Quercus mixtures

leveled at that point. The Pool II/I + II ratio for carbohydrates (i.e., cellulose/total

Fig. 11. Total polyphenolics of Labile Pools I and II. Points are the means of three replicates; vertical bars are

standard deviations. The degree of significance of depth effect is given. Same legends as in Fig. 2.


carbohydrates) changed dramatically, first increasing and then decreasing. At the

lowest remaining OC (about 40% of the initial content), only 20% or less of the total

carbohydrates was in Labile Pool II.

Fig. 12. Quality of C (Carbohydrate C/Polyphenolic C) of Labile Pools I and II. Points are the means of three

replicates; vertical bars are standard deviations. The degree of significance of depth effect is given. Same legends

as in Fig. 2.


The total amount of hydrolyzable polyphenolics continuously decreased, but the

proportion Labile Pools II/I + II behaved in the opposite way to that of carbohydrates,

i.e., as decomposition proceeded, the ratio increased (Fig. 10). This reflects the contrasted

evolution of both pools: in Labile Pool I, the amount of polyphenolics decreased

continuously and, in most cases, the effect of depth was not significant. In contrast, the

amount of remaining polyphenolics in Pool II roughly maintained, and the effect of depth

was significant in most cases (Fig. 11). In Pool II, polyphenolics showed a seasonal

pattern very similar to that of recalcitrant N, and they had increased at the end of the

experiment in all cases.

During the 2 years of field decomposition, C quality (carbohydrate C/polyphenolic C

ratio) was roughly maintained in Labile Pool I, but strongly decreased in Labile Pool II

(Fig. 12). When two-way ANOVA was applied, the differences in quality between

depths were significant in all cases, except for the Labile Pool II of Pinus mixtures.

When analyzed by ANCOVA, with remaining total OC as covariate and depth as a

factor, the effect of depth was also significant in most cases for Labile Pool I, but never

for Labile Pool II (not shown). When the effect of depth was significant, for a given

value of remaining OC the highest C quality was that of samples incubated at 5-cm

depth.

4. Discussion

4.1. Significance of the fractions obtained

Oades et al. (1970) identified the monosaccharides of each hydrolysate, and concluded

that the first pool comprised non-cellulosic polysaccharides, either of plant or microbial

origin, whereas the second pool only consisted of cellulose. The second hydrolysate was a

well-defined biochemical fraction, mainly (if not exclusively) of plant origin. Here, the

first hydrolysate was of plant origin (starch, hemicelluloses, soluble sugars) only at the

beginning of the experiment, whereas as decomposition proceeded an increasing propor-

tion of its carbohydrates would be presumably of microbial origin.

The method of hydrolysis is analogous to that for Klason lignin. Thus, lignin should be

a major component of the Recalcitrant Pool. However, the low proportion of aromatic C in

the residue, together with the dominance of aliphatic C in the 0–45 ppm zone (NMR),

strongly suggests that polymers of lipidic nature are important in this pool. Fats, waxes,

resins and suberins are resistant to acid hydrolysis, and, together with lignin, dominate the

resistant fraction. All of these compounds are highly resistant to biodegradation (Mind-

erman, 1968).

The proportion of aromatic C in the recalcitrant fraction may have been underestimated.

Even small amounts of metal ions like Fe3 + or Cu2 + may appreciably affect the clearness

of the obtained spectra, and the effect is not equally distributed among the several types of

C: the signal of O-alkyl C is the less affected, whereas that of aromatic C is the most

strongly affected (Smernik and Oades, 2000a,b). Even though the strong acid hydrolysis

removes most of the metal ions of the sample, the remains may be enough to partially

catch the signal of aromatic C.


N is a common component of humic substances, but not a constituent of any of the

compounds of the Recalcitrant Pool mentioned above. Therefore, in unaltered plant-

derived compounds N should be absent in the unhydrolyzable residue. Its presence in the

initial samples may be due to the association of proteins with the lignocellulosic complex,

which can result in their physical protection against the acid attack. In addition, the time of

hydrolysis may have been too short to achieve a complete release of hydrolyzable N. In

analysis of amino acids, for instance, hydrolysis times of 12 to 24 h are common

(Stevenson, 1982), and the hydrolysis is performed with 6 N HCl, which is probably

more efficient than sulfuric acid to hydrolyze peptidic bonds. Finally, the presence of

artifacts due to the chemical condensation of N with other organic compounds cannot be

avoided.

4.2. Labile and recalcitrant pools of OC

In the initial mixtures, OC was much more resistant to acid hydrolysis than N. From our

results (Fig. 2), a RIC of 60–65% seems to be the maximum possible; when this value is

reached, the RIC ratio does not increase any more. When plant materials start at 60–65%

RIC, values do not increase, or even decrease slightly (as in Pinus case). A RIC of 60–65%

is also the maximum obtained for samples taken from forest soil profiles (Rovira and

Vallejo, unpublished). With regard to evolution of OC quality, it decreases (Medicago,

Eucalyptus), remains more or less constant (Quercus), or increases slightly, at least during

the first stages of decomposition (Pinus).

A transfer of compounds between pools is expectable. Carbohydrate dynamics

illustrates this assumption. Among carbohydrates, cellulose is the most resistant to

biodegradation (Minderman, 1968; Haider, 1992). In contrast, in our experiment the

cellulose/total carbohydrates ratio (Pool II/Pools I + II) decreased as decomposition

proceeded, except in the first stages for Eucalyptus mixtures. Transfer of carbohydrates

from Labile Pool II to Labile Pool I, probably monosaccharides or oligosaccharides

resulting from the degradation of lignocellulose, would account for that decrease, and

for the maintenance of the C quality (carbohydrate C/polyphenol C ratio) in Labile

Pool I, despite the biodegradability of carbohydrates, much higher than that of poly-

phenols.

The trough in carbohydrate biodegradation at 20% of the initial content (Fig. 9:

Eucalyptus and Quercus) may be explained by several factors (Cheshire, 1977, 1985):

(a) stabilization in microaggregates and/or adsorption to clay surfaces, resulting in

much lower exposure to enzymes,

(b) formation of stable complexes with polyphenolics from lignin biodegradation or

with newly formed humic substances, and

(c) formation of complexes with metal ions. Calcium is the main exchangeable cation in

mediterranean soils, and its role in OM stabilization is well known (Oades, 1988).

An additional question would be why this did not occur in Medicago mixtures. The

ability of polysaccharides to establish bonds with clays varies greatly from one carbohy-

drate fraction to another, and depends mainly on the stereochemistry of the macro-


molecule, which determines the number of chemical bonds that a given polysaccharide

may establish with the clay surface (Hayes and Swift, 1978). Medicago polysaccharides

may differ from those of the other three plant materials in conformational aspects, because

of either the influence of species or type of plant material, as Medicago was also the only

green and fresh plant material studied.

In contrast with carbohydrates, the transfer of polyphenolic compounds between the

labile pools seems more limited. Increase in the total amount of polyphenolics in Labile

Pool II, probably as a result of the release of lignin fragments during the biodegradation of

lignocellulose, did not apparently affect the behaviour of Labile Pool I, in which the

polyphenol content decreased continuously.

The behaviour of carbohydrates and polyphenols suggests that Pools I and II

correspond to clearly distinguishable fractions: as decomposition proceeds, the Pool II/

Pool I + II ratio decreases for carbohydrates, whereas it tends to increase for polyphenolics.

Such ratios may be a useful tool for distinguishing fresh undecomposed residues from

more biodegraded organic matter, even though further studies are needed to verify whether

this behaviour is maintained beyond the time span of our experiment.

4.3. Incorporation of N to the recalcitrant pool

The behaviour of unhydrolyzable N and that of unhydrolyzable C differ; the

tendency of RIN to increase with time was much stronger than that of RIC. This result

agrees with those of other authors. Olson and Lowe (1989), for instance, observed a

strong increase in the proportion of unhydrolyzable N at the end of 40 years of

cultivation, whereas the proportion of OC as unhydrolyzable C did not suffer significant

changes. From our data, the reason for such a difference is a net input of N to the

unhydrolyzable pool, at least in some stages of the field incubation. Such a net in-

corporation has been observed sometimes. The sequestration of N into the ‘Klason

lignin’ fraction is assumed to be one of the reasons of the strong stabilization of both,

in decomposing scots pine litter (Berg and Eckbohm, 1991). Azam et al. (1989), in a

study of non-symbiotic fixation of N2 by soils, observed that about 10% of the N2

incorporated to the soil organic matter became resistant to hydrolysis with 6 N HCl.

Pare et al. (1998), using hydrolysis with 6 N HCl, also observed a net increase in the

amount of unhydrolyzable N during composting of animal manure, whereas the amount

of unhydrolyzable C did not change.

The mechanisms by which such incorporation could occur are poorly understood.

Abiotic reactions have been long regarded as the main way of generation of new

unhydrolyzable N, mainly those reactions in which lignin biodegradation products are

involved: abiotic fixation of ammonium by phenolic compounds or lignin itself

(Nommik and Vathras, 1982), or the reaction of aminoacids with phenolic compounds

or quinones, mainly from lignin biodegradation, have been postulated as mechanisms of

incorporation into the unhydrolyzable fraction, and in fact these reactions are still

assumed to be essential for the generation of humic substances (Stevenson, 1982; Kelley

and Stevenson, 1996), which are resistant to acid hydrolysis at least partly. Other

reactions have been suggested, such as the Maillard reaction, i.e., condensation of

carbohydrates and amino acids to form brown nitrogenous polymers (critically reviewed


by Saiz-Jimenez, 1996). Many of these reactions result in heterocyclic N, which has

been sometimes supposed to be the main N form in the unhydrolyzable OM (Flaig,

1983).

These chemical reactions are possible under laboratory conditions (Thorn and Mikita,

1992), but it is not clear whether they do really occur under real soil conditions. The

results of the most of 15N-NMR studies do not agree with this possibility. Usually, these

studies involve the incubation, under laboratory conditions, of organic substrates (litter or

similar) in the presence of 15N compounds (either organic or inorganic), and the analysis

of the end product of the incubation by 15N-NMR (Clinton et al., 1995; Knicker et al.,

1993, 1997; Bedrock et al., 1998). A common result of these studies is that the

incorporation of 15N occurs mainly, if not exclusively, through biotic processes, since

signals of peptide or protein N are always dominant in the obtained 15N-NMR spectra. In

contrast, signals of heterocyclic N are absent or extremely small. This result is not

incompatible with the formation of unhydrolyzable N, since the presence of peptides and

proteins in the residue of acid hydrolysis has also been reported (Leinweber and Schulten,

1998), but suggests that the chemical reactions of nitrogenous compounds with lignin

derivatives and other aromatic compounds resulting in heterocyclic N, as a way to

chemically stabilize N, are quantitatively irrelevant.

Such a conclusion contrasts with the findings of Leinweber and Schulten (1998), who

found in the unhydrolyzable residue of soil OM a wide variety of compounds with

heterocyclic N. Schulten and Schnitzer (1998) pointed out that the presence of heterocyclic

N in soil OM is to be expected. They also suggest that the 15N-NMR as a way to detect

heterocyclic N could be poorly sensitive. The problem must be considered as not yet

solved.

The unhydrolyzable residue has been often identified with lignin, neglecting the fact

that other polymers of either plant or microbial origin are included. From our results, the

net input of N into the nonhydrolyzable pool was significantly related to the initial lignin

content of the incubated plant materials (Table 7). Therefore, a role of lignin or lignin-

derived compounds should be assumed. However, the correlation is much higher when the

total recalcitrant C (RIC) is considered, suggesting that other components of the

recalcitrant pool could also be able to incorporate N.

Table 7

Correlation (r) between initial lignin content (%) or initial recalcitrant C (RIC), and remaining recalcitrant N (as %

of initial content)

Independent variable Depth (cm) Incubation time

6 months 12 months 18 months 24 months

Initial lignin 5 0.579* 0.711** 0.688* 0.723**

20 0.628* 0.803** 0.833*** 0.779**

40 0.649* 0.753** 0.753** 0.795**

Initial RIC 5 0.835*** 0.870*** 0.841*** 0.900***

20 0.767** 0.938**** 0.910**** 0.918****

40 0.852*** 0.853*** 0.929**** 0.948****

Correlation has been calculated with all the values (not with the means). Without asterisk: non-significant;

*: significant at PV 0.05; **: at PV 0.01; ***: at PV 0.001; ****: at PV 0.0001.


4.4. Depth effects

During the 2 years of field incubation we observed strong differences in the water

content of soil between the depths studied. At 5-cm depth, soil was drier. These contrasting

pedoclimates resulted in a lower microbial activity at 5 cm. Both OC and N mineralization

were slower at 5-cm depth, compared with 20- and 40-cm depths (Rovira and Vallejo,

1997, 2000a). These results agree with those of Donnelly et al. (1990), who reported that

humidity was a major limiting factor for the biodegradation of cellulose and lignin. These

observations indicate that pedoclimate, in Mediterranean-type conditions, tends to favour

microbial activity at deep horizons.

ANOVA and ANCOVA were used mainly to compare the results on the basis of time

(physical clock) and of mineralized OC (biological clock). The effect of depth, when

significant, was always in the sense of an increased abundance of recalcitrant C or N at

upper layers. In both cases the differences between samples incubated at 20 and 40 cm

were usually not significant, that is, there are two distinct types of samples: those

incubated at 5 cm, and those incubated at 20 and 40. Nevertheless, in ANCOVA analyses

the effect of depth was rarely significant; the only exception was the dynamics of

polyphenolics in Labile Pools I and II. Thus, on the whole the drought at 5 cm delayed

biodegradation, but its effect on the biochemical changes of the decomposing plant

material was not very important.

Plant residues decomposing exposed to repeated drying/wetting cycles under laboratory

conditions have been reported to retain more N than those decomposing under continu-

ously wet conditions, for a given amount of mineralized OC (Franzluebbers et al., 1994).

This is consistent with field experiments (Holland and Coleman, 1987; Varco et al., 1993),

with some exceptions (Brown and Dickey, 1970; Wilson and Hargrove, 1986). As for the

comparison between depths, little information is available. Our previous results (Rovira

and Vallejo, 1997, 2000a) showed a lower N mineralization at upper layers, but when the

remaining N was plotted against the remaining OC, no significant effect of depth is

detected; as the differences merely reflected the faster decomposition of organic residues at

deep layers.

Regarding the possible mechanisms of this higher N retention, Franzluebbers et al.

(1994) suggested that drying/wetting cycles enhanced the sequestration of N by recalci-

trant compounds. However, sequestration of N in the recalcitrant fractions was higher in

buried than in surface-placed canola residues (Brassica campestris L.) on a residue

remaining basis, but equal on a lignin remaining basis (Franzluebbers et al., 1996), a result

which did not agree with the hypothesis of a higher sequestration of N into recalcitrant

compounds due to more intense drying/wetting. Our results point out that it all depends on

the basis considered: the retention of N in the recalcitrant pool is usually higher at 5 cm at

a given time, but not for a given remaining OC, in any case.

How drying/wetting could result in higher retention or incorporation of N in the

recalcitrant pool remains unclear, because little information is available about the effect of

drying/wetting on the chemical reactions involved in the process. Only for NH4+ fixation

to clays is there evidence of enhancement by drought (Nommik and Vathras, 1982). On the

other hand, our results indicate that the incorporation of N to the recalcitrant pool occurs

mainly in wet periods. Dry periods may be required to release nitrogenous compounds to


the soil solution, a previous step to their incorporation to the recalcitrant pool during the

following wet period.

4.5. C and N quality: a global view

The ideal quantitative index of q should take into account two components: first, the

distribution of OM (or, in our case, OC and N) among the pools considered, and

second, the quality of each pool considered ( qi). Regarding the former, our results

suggest that the relative abundance of the unhydrolyzable pool, or in other words, the

RIC and RIN, are simple and useful indicators of OC and N quality. The main feature

of our approach is that, whereas OM quality is usually described as a combination of

OC fractions and N abundance (e.g., the C/N, Lignin/N or Lignin/Cellulose/N ratios),

we have regarded OC quality and N quality as two distinct and, to a certain extent,

independent properties. In a previous paper (Rovira and Vallejo, 2000b), the initial RICand RIN ratios were found to be reliable predictors of OC and N mineralization after 1

or 2 years. Here, we show that OC and N mineralization could be predicted considering

both quality indicators and climatic constraints, even though some multiple regression

analysis provide unexpected results, such as the lack of significance of temperature in

most cases, which can result from the long survey periods considered (6 months and

especially 1 year). The lack of detectable effect of precipitation on N mineralization

may be associated with its effect on the redistribution of N between labile and

recalcitrant pools: wet periods, which are expected to result in high microbial activity

and so in high N mineralization, are also periods of input of N to the recalcitrant pool,

and so of decreases in N quality.

The quality of the pools ( qi) cannot be included in an index of quality for whole OC

or N, unless a method to evaluate the quality in all the pools considered is available. For

soluble (or solubilizable, or hydrolyzable) fractions we have used the carbohydrate C/

polyphenol C ratio, but other chemical indicators are possible, such as the amount of OC

retained by a column of resin such as PVP or XAD-8. The retained fraction corresponds

to more or less hydrophobic compounds including phenolics and soluble humic

substances, and is much less biodegradable than the non-retained fraction (Namour

and Muller, 1998). Nevertheless, there is no clear method to evaluate the quality of the

recalcitrant pool. Solid state 13C-CPMAS-NMR is useful for the rough characterization

of the unhydrolyzable residue, but not for the evaluation of quality, since large changes

in the biodegradability may depend on subtle chemical changes, hardly detectable in a

typical NMR spectrum. For N the problem is more severe, since 15N-NMR is only pos-

sible for labeled substrates. Other methods such as Pyrolysis-GC or Pyrolysis-MS could

be helpful.

A summary of the main results of our experiment, and their interpretation in terms

of changes in quality, is shown in Fig. 13. Some details are to be remarked: (i) N

quality decreased more severely than OC quality; (ii) the decrease in the global

quality of OC did not affect the quality of all the pools, since a decrease in the car-

bohydrate C/polyphenol C ratio was detected only in Labile Pool II, probably because

of the accumulation of phenolic compounds resulting from the fragmentation of the

lignin build-up; (iii) the quality of Labile Pool I did not change; since this pool, of


the three considered, is presumably the most available for the microflora, this may

have long-term implications for microbial activity, and for the whole decomposition

process.

Acknowledgements

The authors are indebted to the EC for funding this research (Environment and Climate

Program, VAMOS Project no. EV5CT920141). The help of Isidre Casals and Francisco

Cardenas, from the Serveis Cientıfico-Tecnics of the University of Barcelona, is greatly

acknowledged.

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Bedrock, C.N., Cheshire, M.V., Williams, B.L., Solntseva, I., Chapman, S.J., Chudek, J.A., Goodman, B.A.,

Fig. 13. Summary of the biochemical changes during decomposition, and their translation to quality ( q) terms. (1)

RIC increases with time; (1V) first alternative dynamics of RIC; if the initial value is high, it hardly increases; (1W)second alternative dynamics of RIC; if the initial value is very high, it decreases slightly, at least during the first

stages (Pinus); (2) RIN increases, more than RIC; (3) carbohydrate C/polyphenol C ratio for Labile Pool I, which

suffers little changes or not changes at all; (4) carbohydrate C/polyphenol C ratio for Labile Pool II, decreases. (a)

global quality of C, qC; (aV) alternative dynamics of qC: if the initial value is low, it decreases slightly; (aW):second alternative dynamics of qC: if the initial value is very low, it may increase slightly, at least during the first

stages; (b) global quality of N, qN, decreases; (c) quality of the Labile Pool I, qL1, roughly maintained; (d) quality

of the Labile Pool II, qL2, decreases.


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