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Soil Biology & Biochemistry 32 (2000) 793±804
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13

C NMR assessment of decomposition patterns during
composting of forest and shrub biomass

G. Almendros a,*, J. Dorado a, F.J. GonzaÂlez-Vila b, M.J. Blanco c, U. Lankes d
a

Centro de Ciencias Medioambientales (C.S.I.C.), Serrano 115 dpdo., E-28006 Madrid, Spain
Instituto de Recursos Naturales y AgrobiologõÂa (C.S.I.C.), PO Box 1052, E-41080 Sevilla, Spain
c
Instituto de EnergõÂas Renovables. DivisioÂn de Biomasa (C.I.E.M.A.T.), Avenida Complutense 22, E-28040 Madrid, Spain
d
UniversitaÈt Regensberg, D-93040 Regensburg, Germany
b

Accepted 18 October 1999

Abstract
A laboratory experiment was designed to investigate the degradation patterns of leaves from 12 forest and shrub species
typical of Mediterranean ecosystems by solid-state 13C NMR. The spectral data have been compared with those for the major
organic fractions, and elementary composition in three transformation stages (zero time, intermediated and advanced (168 d)).
The plant material in general showed a selective depletion of lipid and water-soluble products and a concentration in acidinsoluble residue (Klason lignin fraction), but the increasing percentage of total alkyl carbons (not observed in pine leaves)
suggests that recalcitrant aliphatic material accumulates in the course of the 168 d incubation, when the total weight losses were
up to 660 g kgÿ1. This contrasts with the fact that the concentration of extractable alkyl C (i.e. the lipid fraction) decreased in
all cases. The results for the di€erent plants suggested some general transformation trends simultaneous to speci®c
biodegradation patterns. The non-ameliorant, soil acidifying species (i.e. those a priori considered to favor the accumulation of
humus with low biological activity) have high initial concentrations of extractives, alkyl structures and comparatively lower
percentages of O-alkyl structures. The decay process in these species is not associated to the increase of the alkyl-to-O-alkyl
ratio, which is shown by the ameliorant species. Superimposed on these major trends, the biomass of the di€erent plants
underwent divergent paths in the course of composting, leading to, for example, (i) accumulation of recalcitrant,
nonhydrolyzable alkyl and aromatic structures (Retama, Genista ); (ii) enrichment of resistant O-alkyl structures such are stable
fractions of carbohydrate and tannins (Pinus, Calluna ); and (iii) accumulation of aliphatic extractives with the lowest
stabilization of protein in resistant forms (Arctostaphylos, Ilex ). In particular, in the acidifying species, the spectral patterns
suggest that the apparent stability of the aromatic domain is compatible with selective preservation of tannins together with
aliphatic structures. Such speci®c tendencies are also illustrated by the di€erence spectra (0 vs 168 d) which suggest that early
humi®cation processes are highly heterogeneous and distinct rather than the selective degradation of lipid and water-soluble
fractions and carbohydrates, and they may include stabilization of tannins and aliphatic (cutin- and protein-like)

macromolecules. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Pine; Oak; Heather; Biodegradation; Humi®cation; Litter

1. Introduction
The chemical composition of plant litter is tra-

* Corresponding author. Tel.: +34-91-562-5020; fax: +34-91-5640800.
E-mail address: humus@ccma.csic.es (G. Almendros).

ditionally considered to play a key role in the performance of the soil biogeochemical cycle. In fact,
rapid and complete degradation of plant residues is
connected with the productivity of the ecosystem
and the minimal output of organic leachates,
whereas accumulation of non-decomposed plant debris is associated with low biomass production in a
situation in which energy input is spent on the dia-

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 2 0 2 - 3

794

G. Almendros et al. / Soil Biology & Biochemistry 32 (2000) 793±804

genesis of biomacromolecules, the alteration of the
mineral environment and the vertical redistribution
of biogenic elements (Toutain, 1981, 1987).
The chemical characteristics of plant biomass
have also a great in¯uence on the biodegradation
rates (i.e. the irregular rhythms in the litter from
readily biodegradable plant remains and the regular
rhythms in comparatively more recalcitrant organic
matter (Toutain, 1987)). From a pedological viewpoint, traditionally a distinction is made between
acidifying and ameliorating species (Vedy and Jacquin, 1972). The de®nitions of these two classes
depends, not only on the ecophysiological characteristics of the plant (i.e. its performance in cation
recycling), but also on the intrinsic biodegradability
of the litter. The latter is traditionally considered to
depend on the concentration of lignin, the chemical
composition of plant extractives (phenols, tannins,
waxes, resins, etc.) as well as on the quality and
quantity of water-soluble sugars and nitrogen compounds.

As an alternative to classical studies on humus formation which suggest the importance of selective accumulation of aromatic biomacromolecules (Stevenson,
1982), research based on nuclear magnetic resonance
(NMR) has also shown the importance of alkyl C as a
source of stable structures contributing to the formation
of humic substances (Wilson, 1987; KoÈgel-Knabner and
Hatcher, 1989; Preston, 1996). These studies suggest the
accumulation of relatively recalcitrant aliphatic polyesters, such as cutins, suberins and other little known
carbohydrate-polyalkyl macromolecules in higher plants
(Nip et al., 1986).
Although the mechanisms a€ecting the sequestration
of organic matter in soil are complex (Oades, 1988;
Almendros and Dorado, 1999), it is clear that the
di€erent types of plant residues do not contribute to
the same extent to humus formation and that intrinsic
biodegradability is a key factor related to the microbial
activity and resistance to soil deserti®cation in Mediterranean-type climates. Contrarily to most chemical
degradation methods, 13C NMR is often considered as
a technique especially suitable to analyze the aliphatic
domain of complex macromolecular materials, leading
to a more apparent di€erentiation between alkyl

(mainly polymethylene) and O-alkyl (e.g. carbohydrate
and ether-linked) structures.
This study is a comparative analysis of the changes
of the plant biomass during biodegradation and the
purpose is two fold: (i) to carry out a biogeochemical
assessment of the litter in terms of the environmental
quality of the vegetation in spontaneous or reforested
formations; and (ii) to revisit some of the classic concepts of the changes during the early humi®cation
stages, including the importance of aromatic C, nitrogen compounds or water-soluble products in the leaves

as relevant factors to forecast organic matter evolution.

2. Material and methods
2.1. Sampling
At the beginning of autumn (end of September,
October), plant biomass from forest and brushwood
formations representative of undisturbed and degraded
continental Mediterranean ecosystems was collected in
Madrid (central Spain).
The tree species (labeled hereafter as indicated in

brackets) were Ilex aquifolium L. (ILE), holly; Juglans
regia L. (JUG), common walnut; Juniperus thurifera L.
(JTH), Spanish juniper; Juniperus communis L. (JCO),
common juniper; Pinus radiata D. Don (PIN), Monterey pine and Quercus ilex ssp. ballota (Desf) Samp
(=Quercus rotundifolia Lam.) (QUE), evergreen oak.
The shrub species were Arctostaphylos uva-ursi (L.)
Spreng (ARC), bearberry; Calluna vulgaris Hull
(CAL), Scotch heather; Cistus ladanifer L. (CIS), gum
cistus; Erica arborea L. (ERI), tree heath; Genista scorpius DC. (GEN), scorpion broom and Retama sphaerocarpa (L.) Boiss (RET), broom.
2.2. Composting experiment
The plant material (either leaves or stems with
branches) was air-dried in the laboratory and crushed
with a wooden cylinder on a table. The stems were discarded, except in the case of RET and GEN, where
the entire vegetative biomass was used. The samples
were homogenized to 10 mm by sieving.
Piles of plant material, approximately 2 kg were
composted on polyethylene trays. The samples were
moistened (60% of the water holding capacity at atmospheric pressure) and maintained at this moisture
content by spraying the pile with distilled water. No
additives or external N compounds were used in the

experiment, but the piles were kept under an air atmosphere at 288C and 65% relative air humidity. The
piles were mixed with a spatula every 14 d and homogeneous samples composed by 5 subsamples of ca. 50
g (wet weight) were taken.
2.3. Chemical analyses
Total losses of substrate weight during the incubation experiment were estimated indirectly from the
increase of the percentage of ash (determined after
combustion in an electric mu‚e at 6508C) in comparison to the ash content at time zero.
The major organic fractions were gravimetrically
determined by sequential treatments of 2 g homogen-

G. Almendros et al. / Soil Biology & Biochemistry 32 (2000) 793±804

ized sample (500 mm). The total lipid was extracted
with benzene±ethanol 2:1 (v:v) in a Soxhlet for 12 h.
The extract was dehydrated with anhydrous Na2SO4,
the solvent was concentrated at 608C and ®nally evaporated under N2. The water-soluble fraction was
removed from the lipid-extracted residue in the same
apparatus (TAPPI, 1999a), and the acid-insoluble residue (i.e. the Klason lignin fraction) was determined in
1 g of the extractive-free residue after Saeman's hydrolysis (TAPPI, 1999b). Holocellulose was calculated
by di€erence, also considering the ash content.

The determination of C, N and H was carried out
with a Carlo Erba CHNS-O-EA1108 microanalyzer,
using ca. 7 mg sample. The oxygen was determined by
di€erence and the percentages were calculated on ashfree basis. The gravimetric data were corrected taking
into account the hygroscopic moisture content.

795

2.5. Statistical data treatments
Due to the large data matrix obtained in this study
(wet chemical analyses and peak area values of the
major regions in the 13C NMR spectra of 12 plants at
three transformation stages), a statistical approach is
required for identifying transformation patterns. Apart
from basic statistics (simple correlation and analysis of
variance), correspondence analysis was applied to
examine the anities between samples and the descriptors responsible for their variability. The program output draws samples and variables as points in the two
dimensional space de®ned by axes calculated as linear
functions of the original set of variables; these synthetic axes accounted for a considerable portion of the
total variance (inertia) of the whole set of variables.

The data treatments were carried out with the STATITCF package (ITCF, 1988).

2.4. NMR acquisition conditions

3. Results and discussion

The 13C-NMR spectra were obtained in solid state
under the same conditions optimized for quantitative
comparisons between spectra of lignocellulosic and
humic substances (FruÈnd and LuÈdemann, 1989; Preston,
1996). The spectrometer used was a Bruker MSL 100
(2.35 T) operating at 25.1 MHz for 13C. Magic angle
spinning was performed at 4 kHz with 7 mm zirconium
dioxide rotors in a commercial double bearing probe.
Spinning side band intensities were rather small and
occurred about 160 ppm at the left and right hand of the
main peaks. The recycle delay of the common CPMAS
pulse sequence was set to 3 s. Cross polarization contact
time was 1 ms. The spectral width was 125 kHz and the
acquisition time 12.3 ms. A total of about 5000 scans

were accumulated for each spectrum. An exponential
function with 25 Hz line broadening was multiplied with
the free induction decay. After Fourier transformation,
a zero order phase correction and a baseline correction
were applied to process the spectra. The chemical shift
was calibrated to tetramethylsilane (=0 ppm). For spectral interpretation the following ranges and preliminary
assignations were considered: 0±46 ppm=alkyl
(13=methyl, 21=acetate, 30=polymethylene), 46±110
ppm=O-alkyl (56=methoxyl/a-amino, 73=glucopyranosyde-derived, 103=anomeric C in carbohydrate,
105=quaternary aromatic carbons in tannins); 110±160
ppm=aromatic/unsaturated (ca. 135=unsubstituted,
ca. 145=heterosubstituted: guaiacyl (G) lignins/dihydroxys of tannins; ca. 153=ether-linked (syringyl (S)
lignins)/tannins); 160±200 ppm=carbonyl (172=carboxyl/amide, 198=ketone/aldehyde) (Wilson, 1984;
Wilson et al., 1988; Preston, 1992; Preston et al., 1997;
Huang et al., 1998).

3.1. General analytical characteristics
Table 1 shows some general characteristics of the
samples in the three transformation stages. The weight
losses in the course of the experiment ranged from

20.7% in ERI to 66.2% in ILE leaves.
The relative concentration of N is expected to
increase with composting time (Stevenson, 1982).
However, this tendency was not apparent in some
species traditionally considered as soil acidifying
(ARC, ILE, ERI) in which N was not transformed
into stable forms, but was presumably lost as ammonia. The analysis of the extractive fractions indicated
no general tendency. A wide range in the changes due
to decomposition was observed, with several species
losing less than half of the lipid (e.g. ILE, ERI, ARC)
whereas great losses were found in other cases. In the
course of leaf composting, the water-soluble fraction
may consist of readily biodegradable components, but
also of a pool of degradation products from plant tissues: the amount of water-solubles do not decrease signi®cantly in ILE, RET and even increase in other
species (i.e. GEN). On the other hand, the relative concentration of acid-insoluble residue increased as
expected from a humi®cation process.
The changes in the H-to-C and O-to-C atomic ratios
(Table 1) do not correspond systematically to the progressive degradation of carbohydrate which should be
re¯ected by the more or less intense decrease of both
ratios (van Krevelen, 1950). In particular, the H-to-C
ratio (an index inversely related to the aromaticity of
the samples) generally decreased, except in JTH, ILE
and ericaceae: ERI, CAL, ARC. This suggests that the
increase in aromaticity is not the dominant process.

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G. Almendros et al. / Soil Biology & Biochemistry 32 (2000) 793±804

The trend of the atomic O-to-C ratio to decrease could
mean that the selective degradation of the carbohydrate moieties have a greater in¯uence on this ratio
than the incorporation of carboxyl groups expected
from a typical humi®cation process.
The above results from the conventional chemical
analyses suggest that transformations of plant litter
during the composting experiment are not necessarily
parallel to those traditionally described for the humi®cation of soil organic matter. This could be interpreted
as the samples studied are representative only of the
early humi®cation stages. Nevertheless, the weight loss
(on average about 42 2 14%) should be considered
high enough for such a tendency to be observed.

3.2. Changes during composting in the
spectral patterns

13

C NMR

Fig. 1 shows the 13C CPMAS NMR spectra of the
leaves at the initial (zero time) and ®nal stages of
transformation (168 d). The relative content of the
major C-types, calculated by integrating the spectral
pro®le according to standard chemical shift ranges (as
a percentage of the total spectral area) is shown in
Table 2.
The carbonyl region (200±160 ppm) is dominated by
a peak with a maximum ca. 174 ppm, traditionally
attributed to carboxyl groups. In the uncomposted
leaves, however, this peak has a similar intensity, if

Table 1
Main analytical characteristics of leaves from forest and shrub speciesa in di€erent transformation stages
Sample

PIN0
PIN98
PIN168
JCO0
JCO98
JCO168
JTH0
JTH98
JTH168
ILE0
ILE98
ILE168
JUG0
JUG98
JUG168
QUE0
QUE98
QUE168
ERI0
ERI98
ERI168
CAL0
CAL98
CAL168
ARC0
ARC98
ARC168
CIS0
CIS98
CIS168
GEN0
GEN98
GEN168
RET0
RET98
RET168
a

Weight loss
(g kgÿ1)

0
210
286
0
321
472
0
384
454
0
609
662
0
382
427
0
393
485
0
206
207
0
249
358
0
126
368
0
262
250
0
403
427
0
487
632

C
(g kgÿ1)

485
478
475
484
468
433
452
426
431
492
462
453
410
382
372
472
457
454
544
513
542
502
498
473
493
499
490
469
480
478
475
482
484
470
480
466

N
(g kgÿ1)

13
17
19
11
19
28
12
18
24
23
26
25
8
14
18
15
23
25
7
6
8
9
14
22
28
9
7
16
24
27
12
20
22
25
39
35

Atomic ratios

H-to-C

O-to-C

1.58
1.49
1.49
1.58
1.62
1.59
1.60
1.63
1.57
1.46
1.55
1.56
1.65
1.52
1.52
1.53
1.52
1.46
1.58
1.60
1.55
1.56
1.55
1.56
1.48
1.51
1.51
1.61
1.55
1.54
1.61
1.55
1.54
1.65
1.58
1.57

0.73
0.73
0.73
0.71
0.71
0.78
0.75
0.73
0.68
0.66
0.62
0.62
0.86
0.84
0.84
0.76
0.76
0.75
0.59
0.66
0.58
0.65
0.63
0.67
0.68
0.68
0.69
0.73
0.66
0.65
0.78
0.72
0.71
0.75
0.65
0.66

C-to-N

Lipid
(g kgÿ1)

Water-soluble
(g kgÿ1)

Acid-insoluble
(lignin)
(g kgÿ1)

Holocellulose
(g kgÿ1)

37
28
24
45
24
16
39
23
18
22
17
18
48
27
21
32
20
18
79
84
64
54
34
22
17
54
67
30
20
18
38
24
22
19
12
13

160
45
20
150
60
30
135
55
20
160
130
90
110
45
20
115
15
20
160
120
110
220
100
59
230
170
115
215
135
85
70
15
15
145
40
20

180
100
95
160
100
95
200
95
90
215
190
205
170
130
90
195
80
110
170
50
60
175
100
79
250
305
195
245
140
130
70
55
80
165
115
145

252
388
413
265
355
397
242
389
411
188
311
335
179
188
322
208
424
455
309
463
458
287
419
418
224
267
392
171
300
368
212
398
388
171
337
321

378
429
430
378
415
387
338
322
323
386
241
222
431
459
371
448
425
349
338
340
343
266
311
362
262
219
244
310
353
334
627
497
480
485
442
422

PIN=Pinus radiata, JCO=Juniperus communis, JTH=Juniperus thurifera, ILE=Ilex aquifolium, JUG=Juglans regia, QUE=Quercus rotundifolia, ERI=Erica arborea, CAL=Calluna vulgaris, ARC=Arctostaphylos uva-ursi, CIS=Cistus ladanifer, GEN=Genista scorpius, RET=Retama sphaerocarpa. The trailing ®gures refer to the composting time in days.

G. Almendros et al. / Soil Biology & Biochemistry 32 (2000) 793±804

not greater, than in transformed substrates. This is
probably due to aliphatic esters, such as those found
in cutins or in hemicellulose esters (Kolodziejski et al.,
1982). In addition, the amides (see N values in Table 1)
may also render a major contribution to this signal
intensity. In most samples, a sharp resonance is
observed at ca. 168 ppm which coincides with the
chemical shift of oxalates (Pacchiano et al., 1993).
The aromatic region (160±110 ppm) could be
divided into the region at between 160±140 ppm for
aromatic carbons linked to O or N and that at
between 140±110 ppm for non-substituted and C-substituted aromatic carbons. In lignins, the maximum at
ca. 153 ppm corresponds to C-3 and C-5 in S units in
etheri®ed structures, but also to C-3 and C-4 in G
units (LuÈdemann and Nimz, 1973). Commonly, the
signal intensity in the 159±141 ppm range is assigned
to phenolic carbons in lignin units (de Montigny et al.,
1993). The 145 ppm peak is more characteristic of C-3
and C-4 in etheri®ed structures; the prominent aromatic peak at 135 ppm is also produced by C-1 and

797

C-4 in S units and C-1 in G units (Haw et al., 1984).
There is a considerable overlap of the major lignin signals at ca. 155 and 145 ppm with those of tannins, as
proved in leaf biomass by Preston et al. (1997). In particular part of plant tannins are biodegradation-recalcitrant compounds which are selectively preserved in the
course of the humi®cation (Wilson et al., 1988). Such
extractive compounds readily turn into macromolecular fractions, or incorporate through covalent bonding
with other fractions in the decaying substrate. Then,
assignation to tannins of considerable portion of the
intensity of the above aromatic bands is even plausible
in 168 d transformed samples, where a certain contribution of the quaternary aromatic carbons is also
possible in the signal ca. 105 ppm (Skene et al., 1997).
In particular dipolar dephasing experiments have
shown that many of the peaks previously thought to
be due to anomeric carbon around 103 ppm (Wilson,
1984) in fact are non-protonated carbon arising from
tannins (Wilson et al., 1988).
The increase of the signal intensity at ca. 135 ppm

Fig. 1. 13C NMR spectra of plant material (uncomposted leaves: 0 d) and after 168 d of composting, without additives, in a controlled environment chamber. Vertical bars indicate the ranges for the major C-types (carbonyl, aromatic, O-alkyl and alkyl; Table 2).

798

G. Almendros et al. / Soil Biology & Biochemistry 32 (2000) 793±804

Table 2
Integration values for the major C-types in the 13C NMR spectra of original and composted leaves from tree and shrub speciesa (chemical shift
range in ppm). Ratios between speci®c spectral ranges
Sample

Carbonyl
(200±160)

Aromatic
(160±110)

O-alkyl
(110±46)

Alkyl
(46±0)

Aromaticto-aliphatic

S-to-G

Methoxylto-aryl

O,N-aromatic-toH-aromatic

Ca in aminoacid
and OCH3
(65±45)

Alkyl-toO-alkyl

PIN0
PIN98
PIN168
JCO0
JCO98
JCO168
JTH0
JTH98
JTH168
ILE0
ILE98
ILE168
JUG0
JUG98
JUG168
QUE0
QUE98
QUE168
ERI0
ERI98
ERI168
CAL0
CAL98
CAL168
ARC0
ARC98
ARC168
CIS0
CIS98
CIS168
GEN0
GEN98
GEN168
RET0
RET98
RET168

10.5
6.5
6.9
5.1
7.6
7.2
6.4
6.5
6.4
8.2
8.2
8.5
10.8
9.8
9.8
7.8
9.3
8.6
6.0
4.9
5.1
5.2
6.3
6.5
4.4
5.1
5.1
8.0
7.4
8.5
5.8
5.9
5.6
7.4
5.8
7.5

14.0
17.3
16.7
17.0
14.6
13.3
17.3
14.1
16.4
9.2
12.6
8.7
15.4
15.9
15.0
16.3
18.0
15.4
17.8
15.0
15.9
15.5
14.0
15.4
23.1
22.5
22.5
14.4
14.3
16.7
13.1
15.9
14.9
13.4
12.6
16.4

50.4
57.9
62.0
60.3
53.7
62.0
60.3
55.7
57.3
48.6
33.0
30.2
59.7
55.5
51.6
58.4
53.5
56.7
45.3
45.4
45.6
51.2
47.9
54.2
57.2
52.4
49.6
54.2
51.2
51.0
72.0
63.5
65.5
64.7
58.4
51.3

25.3
18.5
14.5
17.7
24.3
17.7
16.2
23.9
20.2
34.2
46.5
52.8
14.3
18.9
23.8
17.8
19.5
19.4
31.1
34.9
33.6
28.2
32.0
24.2
15.7
20.3
23.2
23.6
27.3
23.9
9.4
14.9
14.2
14.7
23.4
25.0

0.19
0.23
0.22
0.22
0.19
0.17
0.23
0.18
0.21
0.11
0.16
0.10
0.21
0.21
0.20
0.21
0.25
0.20
0.23
0.19
0.20
0.19
0.17
0.20
0.32
0.31
0.31
0.19
0.18
0.22
0.16
0.20
0.19
0.17
0.15
0.22

1.34
1.06
1.41
1.38
0.87
1.33
1.07
0.83
0.92
0.50
1.23
1.19
1.21
1.22
1.29
0.94
1.01
1.32
1.46
1.02
1.20
1.12
1.13
1.06
0.77
0.71
0.80
0.81
1.26
1.33
1.92
1.53
1.78
1.77
1.75
1.55

0.43
0.25
0.31
0.22
0.28
0.35
0.21
0.34
0.35
0.39
0.36
0.45
0.22
0.28
0.36
0.25
0.24
0.33
0.19
0.32
0.30
0.25
0.31
0.31
0.16
0.15
0.15
0.31
0.34
0.29
0.39
0.37
0.43
0.34
0.51
0.39

0.36
0.57
0.80
1.00
0.50
0.48
0.56
0.43
0.39
0.22
0.41
0.27
0.63
0.46
0.35
0.66
0.45
0.57
1.02
0.58
0.61
0.60
0.54
0.57
0.63
0.57
0.60
0.56
0.54
0.57
0.70
0.48
0.65
0.55
0.44
0.51

9.8
7.7
8.4
5.9
7.1
7.6
6.2
8.2
9.5
6.7
8.9
7.3
5.5
7.5
8.9
6.5
7.4
7.9
6.2
8.8
9.0
7.5
8.2
8.3
6.8
6.5
6.6
7.4
8.8
8.6
7.9
9.2
9.5
7.8
10.5
10.3

0.50
0.32
0.23
0.29
0.45
0.29
0.27
0.43
0.35
0.70
1.41
1.75
0.24
0.34
0.46
0.30
0.36
0.34
0.69
0.77
0.74
0.55
0.67
0.45
0.27
0.39
0.47
0.44
0.53
0.47
0.13
0.23
0.22
0.23
0.40
0.49

a

Sample labels refer to Table 1.

(except in the case of PIN) is attributable to the accumulation of non-substituted aromatic carbons,
whereas the signal intensity at about 153 ppm for Oor N-substituted aromatic C (i.e. phenolic C5) did not
show the systematic decrease with the humi®cation
found in the case of the humic acid fraction from forest soils (KoÈgel-Knabner et al., 1991). In general,
except in PIN and RET and, to a lesser extent, in CIS
and GEN, the concentration with time of total aromatic carbons does not account for the progressive
aromatization that should be characteristic of a humi®cation process, as reported by Wilson et al. (1983)
who found that the percentage of aromatic carbon in
pine leaves increased with ageing, whereas the results
of other deciduous species were not sharply de®ned.
To a large extent, this apparent contradiction between

gravimetric data in Table 1 and NMR peak areas may
correspond to the fact that Klason lignin consists of a
heterogeneous mixture of recalcitrant materials (probably in part laboratory artifacts) including a substantial nonhydrolyzable alkyl moiety (Preston et al.,
1997). It is also neccesary to take into account that the
typical pattern for a S±G mixture is seen in the trophic
stems of the two broom species (Fig. 1), which have a
peak at 153 ppm with a shoulder at ca. 145 ppm,
whereas several species show a weak 56 ppm signal associated to the comparatively sharp signals typical of
condensed tannins in the phenolic region (Preston et
al., 1997) most striking for ERI, CAL, JTH, ARC,
CIS, JUG and QUE.
The most prominent NMR signal corresponds to
carbohydrate, i.e. the simultaneous resonance of C-2,

G. Almendros et al. / Soil Biology & Biochemistry 32 (2000) 793±804

C-3 and C-5 of pyranoside rings in cellulose and hemicellulose (73 ppm). The ring carbon at the C-6 position
produced the peak or shoulder at ca. 63 ppm, whereas
C-1 produced the sharp 103 ppm signal. A shoulder at
84 ppm may correspond to C-4 in amorphous cellulose
(Kolodziejski et al., 1982).
The alkyl region (46±0 ppm) showed the maximum
at ca. 30 ppm, for polyethylene carbons in lipids and
lipid polymers; the chemical shifts and properties
found by Pacchiano et al. (1993) in puri®ed cutin preparations correspond to that in the aliphatic region of
the spectra, and support large contribution by lipid
polyesters in leaves even at advanced decayed stages.
A small peak or shoulder at ca. 21 ppm is frequently
attributed to acetate groups in hemicellulose (Kolodziejski et al., 1982). It should be pointed that, when
studying the correlation indices between NMR peak
intensities and gravimetric data, the best ®t for the
holocellulose was found with the signal intensity at 63
ppm …r ˆ 0:780, P < 0.01).
The protein (in the samples studied accounting for
up to 200 g kgÿ1, based on the N concentration in
Table 1) does not produce diagnostic NMR signals in
the 13C spectra. When analyzing the spectra, it should
be take into account that aminoacids contribute to the
intensity of the alkyl and carbonyl signals and more
characteristically in the 60±45 ppm range (Ca in amino
acids), the intensity of which (Table 2) has been found
to correlate with the nitrogen concentration in humictype samples (Knicker et al., 1996). With composting
time, the signal intensity in the 60±45 ppm range tends
to increase in most of the species studied, whereas
demethoxylation (i.e. a decrease in the intensity of the
56 ppm signal) is expected in the early transformation
stages of lignin. Similar results were obtained by
Huang et al. (1998) from NMR spectra of CAL litter
in which the 57 ppm peak persisted after a 23 y natural degradation process.
To some extent, the overlapping of the Ca signal
with that of the methoxyl may be a source of interference with the above-indicated change in the methoxyl
content. In fact, the intensity signal of these overlapping spectral regions was, as expected, signi®cantly
correlated …r ˆ 0:933, P < 0.01). In this study, no de®nite trend of demethoxylation was observed. The same
was observed when the S-to-G ratio is calculated as
the ratio between the area of the NMR regions with
maxima at 153 and 145 ppm respectively (Manders,
1987). Such ratio as well as the methoxyl-to-aryl ratio
(Table 2) showed no systematic tendency expected for
preferential degradation of the less condensed (S-type)
lignin fractions (Almendros et al., 1992). This could be
due to high tannin content rather than lignin, in the
corresponding cases suggested by a weak methoxyl signal, less than expected according to the expected number of methoxyl C per aromatic ring (Preston et al.,

799

1997). This is certainly the case for the leaf samples
under study, especially when the protein contribution
is considered and it would explain the fact that the Sto-G ratio was unrelated to the area of the 56 ppm signal.
The above observations show that the classical
assumption that lignin transformation is accompanied
by demethoxylation and carboxylation are not systematically re¯ected by the 13C NMR spectra at least
during the early humi®cation stages of leaf material.
To some extent this may also be due to the abovedescribed interfering e€ect of protein and to a signi®cant contribution by recalcitrant tannins both through
their selective preservation and through their condensation reactions controlling the decomposition rates of
the aliphatic biomacromolecules. Furthermore, the
data shown in Table 2 illustrate that the aromatic-toaliphatic ratio did not show a systematic increase
during the composting. The breakdown of esters and
the concentration of N-compounds (protein or chitin)
and high molecular weight alkyl material are probably
the more conspicuous processes. In fact, when comparing the 13C NMR results with those from the wet
chemical methods the major di€erences observed correspond to the fact that the concentration of the acidinsoluble residue (which, in the case of leaves, does not
clearly correspond to the molecular concept of lignin,
but to a residual mixture from altered nonhydrolyzable
domains of recalcitrant plant macromolecules) frequently increased up to 100%, but the concentration
of aromatic carbon in the total composting substrate
did not re¯ect a systematic increase, as stated in the
study by Zech et al. (1987). The present results also coincide with the suggestions of Hemp¯ing et al. (1987),
who considered that the hypothesis of increasing aromaticity during humi®cation in soils was questionable.
In this study, polymethylene carbon also accumulated
during the biodegradation and humi®cation of beech
and spruce litter, that was not recorded by using the
petroleum ether extract, which was also considered as
result of the selective preservation and microbial synthesis of the stable aliphatic compounds in the course
of decomposition and humi®cation.
A more sensitive index of the progress of the humi®cation could be the alkyl-to-O-alkyl ratio. In most of
the typical Mediterranean species this ratio tends to
increase, as stated by Baldock et al., (1997) which is
interpreted as the comparatively high degradation rate
of carbohydrate regarding that of lipid biomacromolecules and newly-formed insoluble alkyl structures. This
may cause the relative initial increase in polymethylene
resonances (NordeÂn and Berg, 1990) observed mainly
in ILE, JUG, QUE, GEN, RET and the ericaceae
ARC. Nevertheless, the tendency is not de®ned with
the acidifying species (or even is clearly opposed in the
case of PIN). This could correspond to a lower resist-

800

((0 d)ÿ (168 d)100))/0 d. Sample labels refer to Table 1.
Variable labels: H-to-C, O-to-C=atomic ratios; LIP=lipid, HID=water-soluble, LIG=Acid-insoluble residue (Klason lignin); HCEL=holocellulose. The following variables correspond to
13
C NMR integration data: COOH=carbonyl carbons; ARO=aromatic carbons; OALK=O-alkyl carbons; ALK=alkyl carbons; AR-to-AL=aromatic-to-aliphatic ratio; ME-toAR=methoxyl-to-aryl ratio; Na-to-Ha=ratio between O-or N-substituted aromatic carbons and non-substituted aromatic carbons. The following columns refer to signal intensities of the most
prominent spectral peaks; Caaa=area in the 60±45 ppm range (a-amino and OCH3-C).

a

b

ÿ15
27
54
8
62
22
45
11
ÿ3
17
20
33
ÿ43
9
47
52
58
8
ÿ7
5
33
ÿ24
10
49
ÿ41
ÿ10
20
74
64
13
7
ÿ16
50
10
76
91
ÿ16
26
57
11
62
27
41
20
ÿ8
9
26
39
8
3
21
ÿ29
ÿ2
ÿ7
13
27
ÿ24
ÿ14
ÿ9
ÿ7
22
ÿ6
ÿ12
ÿ45
ÿ22
ÿ11
ÿ15
2
ÿ17
ÿ24
ÿ18
ÿ33
52
15
ÿ22
ÿ45
ÿ15
5
7
ÿ6
ÿ3
6
ÿ14
ÿ32
61
ÿ27
ÿ28
ÿ69
ÿ42
ÿ4
2
10
ÿ19
20
ÿ4
ÿ17
39
10
ÿ5
ÿ35
ÿ21
0
ÿ11
13
ÿ8
3
ÿ19
ÿ20
23
ÿ21
ÿ7
ÿ35
ÿ16
6
13
ÿ6
ÿ6
54
11
12
ÿ19
10
7
24
38
6
6
2
4
23
34
30
71
ÿ45
ÿ13
ÿ36
ÿ29
ÿ35
ÿ23
3
ÿ4
ÿ24
33
36
79
ÿ47
ÿ25
52
ÿ24
ÿ9
ÿ37
ÿ3
ÿ1
25
23
19
ÿ32
54
14
2
13
19
ÿ21
14
42
ÿ8
19
ÿ3
ÿ54
0
30
150
92
13
7
ÿ18
74
7
69
113
122
ÿ52
ÿ30
23
ÿ44
ÿ14
ÿ40
ÿ5
ÿ5
2
ÿ7
ÿ7
ÿ28
59
67
15
64
32
58
24
ÿ6
ÿ6
10
15
16
ÿ23
ÿ9
ÿ9
ÿ5
ÿ5
ÿ13
5
ÿ3
16
19
29
23
3
ÿ5
ÿ38
ÿ14
ÿ3
1
6
ÿ13
ÿ6
ÿ9
ÿ21
19
ÿ22
ÿ5
ÿ5
ÿ3
ÿ6
ÿ11
ÿ1
ÿ3
16
14
22
ÿ6
1
ÿ2
7
ÿ8
ÿ5
ÿ2
0
2
ÿ7
ÿ4
ÿ5
PIN
JCO
JTH
ILE
JUG
QUE
ERI
CAL
ARC
CIS
GEN
RET

2
11
ÿ11
ÿ9
ÿ1
ÿ1
0
4
2
ÿ11
ÿ10
ÿ14

ÿ35
ÿ66
ÿ53
ÿ17
ÿ56
ÿ44
ÿ19
ÿ60
288
ÿ41
ÿ43
ÿ30

ÿ88
ÿ80
ÿ85
ÿ44
ÿ82
ÿ83
ÿ31
ÿ73
ÿ50
ÿ60
ÿ79
ÿ86

ÿ47
ÿ41
ÿ55
ÿ5
ÿ44
ÿ44
ÿ65
ÿ55
ÿ22
ÿ45
14
ÿ12

64
50
70
78
80
119
48
46
75
115
83
88

14
3
ÿ4
ÿ42
ÿ14
ÿ22
2
36
ÿ7
8
ÿ23
ÿ13

ÿ34
41
0
4
ÿ9
10
ÿ15
25
16
6
ÿ3
1

ÿ43
0
25
54
66
9
8
ÿ14
48
1
51
70

AR-toAL
ALK
OALK
ARO
COOH
HCEL
LIG
HID
LIP
C-to-N
O-to-C
H-to-C
Sample

Table 3
Relative extent of the changesa during composting of plant leaves in the 0±168 d periodb

ME-toAR

Na-toHa

ALK-to-OALK

174

153

145

135

115

105

101

84

73

63

56

30

21

Caaa

G. Almendros et al. / Soil Biology & Biochemistry 32 (2000) 793±804

ance to degradation of cutin-like material in these
species or to the above-indicated e€ect of tannins in
the selective preservation of O-alkyl structures.
The possibilities of detecting general processes in
terms of composting time were extremely limited even
with the number of samples in this study, due to the
speci®c trends of the di€erent plants. These patterns
are summarized in Table 3, in which the data are
shown as a percentage increase (or decrease) of the
variables monitored in the experiment (0±168 d). In
Table 3, ®gures with the same sign within a column
are not frequent. For example, the increase in aromaticity (aromatic-to-aliphatic ratio) is observed only in
PIN, CAL, CIS, GEN and RET. As indicated above,
alkyl material accumulates in all species except PIN
and CAL. Similar NMR studies with 13C-enriched
grass have also shown increases in methyl and alkyl C
in the early phases of decomposition (Hopkins and
Chudek, 1997). Such an accumulation of alkyl material
has also been described in highly decomposed materials and it is considered to be due not to selective
preservation, but rather to an increase in cross-linking
of the long-chain alkyl material occurring during
humi®cation (Skjemstad et al., 1997). On the other
hand, carbohydrate does not systematically decrease in
all species: O-alkyl C remained constant or increased
in PIN, ERI, CAL and the holocellulose concentration
(gravimetric) also increased in PIN, JTH, ERI, CAL
and CIS. It may be assumed that the virtual concentration of carbohydrate carbons observed in several
species does not necessarily correspond to the preservation of native polysaccharides, but, e.g. to diagenetically altered substances not readily recognized by
enzymes. Such a fraction, where the quantitative contribution and protecting role of tannins should not be
neglected, could include domains with anhydrosugaror
Maillard-like-structures,
which
cannot
be
thoroughly distinguished from pyranoside signals in
the 13C NMR spectra (Almendros et al., 1997).
3.3. Selective depletion of the di€erent C-types
Fig. 2 shows the di€erence spectra obtained by digital subtraction of the spectra at zero time and those at
168 d, the latter corrected by the losses of carbon (calculated from the weight loss and the elementary composition). In some species the di€erence spectra (i.e.
the spectra of the material that were lost by biodegradation) were unexpectedly similar to the spectra at
zero time. In these cases it indicated that the degradation occurred similarly in all C types, what could be
a characteristic of the early humi®cation stages (Preston et al., 1998). From the spectral pro®les, it is evident that no general tendency of the selective
accumulation of aromatic carbon is observed in the
composting period. Similar data were reported by

G. Almendros et al. / Soil Biology & Biochemistry 32 (2000) 793±804

Wilson et al. (1983) who also found that the trends in
wet chemical analyses saw a marked loss in carbohydrates and an increase in residual lignin, whereas the
NMR changes were much smaller. The di€erence spectra indicate that the preferential degradation of carbon
di€er greatly from one species to the next. For
instance, ILE leaves lost up to 70.6% of the aromatic
carbons in 168 d, whereas PIN litter lost up to 60.0%
of the alkyl carbon during composting. In GEN and
RET the plots suggest preferential biodegradation of
carbohydrate, whereas in heathers (ERI, CAL), PIN
and ILE considerable amounts of alkyl C are lost. The
leaves from JCO and JTH showed similar depletion
patterns, mainly di€ering in the greater loss of aromatic carbons (tannins) in the former. The di€erence
spectra of some species (PIN and, to some extent,
ILE) with an intense loss of alkyl and carboxyl carbons might be interpreted as active degradation of cuticular polyester material. The degradation of tannins
is also betrayed by the sharp bands in the di€erence

801

spectra of most of the species, i.e., the heathers,
cupressaceae and the angiosperms with leaves.
The di€erence spectra illustrated the above-indicated
fact that the most ameliorant species undergo the most
selective microbial reworking of the aliphatic moiety:
i.e., the preferential loss of O-alkyl carbons with
regard to alkyl carbons, which is evident for the
species in the last row of spectra shown in Fig. 2.
3.4. Data analyses
3.4.1. Statistical analyses based on zero-time material
When exclusively considering the characteristics of
the sample subset corresponding to zero time in order
to correlate their chemical characteristics with biodegradability (weight loss), there were small possibilities
to obtain generalizations due to the large statistical
dispersion of the data analyzed in addition to the e€ect
of several outliers forcing most of the correlation indices to be signi®cant. For example, it is often con-

Fig. 2. Di€erence spectra (13C NMR pro®les obtained by digital subtraction of the spectrum from plant biomass minus the spectrum from the
168 d degraded sample, after correction of the carbon percentages and weight losses), showing the extent to which the di€erent C-types are
degraded in leaves from forest and brushwood plants. Vertical bars indicate the ranges for the major C-types (carbonyl, aromatic, O-alkyl and
alkyl; Table 2).

802

G. Almendros et al. / Soil Biology & Biochemistry 32 (2000) 793±804

sidered that the initial lignin (and nitrogen) concentration in leaf litter has a great in¯uence on the rate of
decomposition (Laishram and Yadava, 1988). In this
study the correlation of the weight loss with the N
concentration …r ˆ 0:49, P < 0.05) and the acid-insoluble-residue: r ˆ ÿ0:50, P < 0.05) was, in fact, most signi®cant between zero time samples (plots not shown).
Total aromatic carbon correlates with the weight loss
at r ˆ ÿ0:45 (P < 0.05), whereas total weight of lipid
and water-soluble concentrations were unrelated to
biodegradability. This coincided with the results
obtained by Bridson (1985) with di€erent types of forest litter. A relevant detail at this point is that,
although most ameliorant species (QUE, JUG, RET,
GEN) generally show weight losses greater than
40.0%, there was no signi®cant (P < 0.5) di€erence
with regards to the weight loss underwent by the acidifying plants in the course of composting. This preliminary study suggested the lack of a single major
limiting factor for biodegradability of the leaves studied. Leaf evolution probably depends on a pattern of
connected factors.
In order to validate the rough aprioristic classi®cation of the plants studied as ameliorating or acidifying, a correspondence analysis was carried out with
the samples at zero time (uncomposted leaves), where
the standard analytical data and the signal intensity in
the four major NMR region were used as descriptors.
The scatterplot obtained (Fig. 3) did not show sharp
clusters for the di€erent samples, but there was a series
of samples which could be included into the de®nition
of ameliorating species (GEN, RET, JUG and QUE)
characterized by the highest loading factors for the
descriptors of carbohydrate. The scores in the plane
for the remaining leaves (ERI, CAL, ARC, ILE, PIN,
CIS, JCO, JTH) suggested dominance of extractives
and acid-insoluble residue, as well as a comparatively
low N concentration.
3.4.2. Chemical changes during composting
Fig. 4 (correspondence analysis, plot based on routine variables in relation to the major organic fractions) clearly suggests a humi®cation gradient de®ned
by decreasing values on both axes, which is due to a
progressive depletion of extractive fractions (lipid and
water-soluble) and a further decrease in carbohydrate.
The sample points corresponding to the most
advanced stages tend to cluster in the region with the
greatest eigenvalues for the acid-insoluble residue and
the atomic H-to-C ratio, suggesting the accumulation
of recalcitrant material not exclusively aromatic in
nature. This is consistent with the above consideration
that the acid-insoluble residue consists of a too heterogeneous mixture of nonhydrolizable material, that may
include lignin in addition to a variable portion of lipid
biomacromolecules and tannins. The graph also

re¯ects the above-indicated di€erences between species
which are more or less favorable from a biogeochemical viewpoint. This gradient is mainly de®ned by the
information accounted for axis II. When exclusively
considering the sample points at zero time, there was a
cluster (from GEN to QUE) in which the large concentration of carbohydrate and the low proportion of
extractives led to a rapid accumulation of resistant biopolymers. The other cluster (from ILE to CAL) consisted of samples with comparatively large
concentrations of low molecular weight products.
Table 1 shows that, whereas operationally-de®ned lignin concentrations increase during the humi®cation of
all the species, the carbohydrate is not selectively
removed from the leaves in this second cluster. This
could be interpreted as an e€ect of the quantitative
contribution and reactivity of leaf tannins.
3.4.3. Speci®c transformation of di€erent types of plant
litter during composting
The degradation patterns in terms of the most diagnostic variables selected after the above statistical
treatments are summarized in Fig. 5. The points corresponding to acidifying species tend to concentrate in a
region of the plot de®ned by the lowest loading factors
for the increase in aromaticity. The ameliorating
species showed the above-indicated trend to preferential degradation of carbohydrate as regards alkyl structures. Superimposed to these major trends, a series of
species-dependent tendencies are more or less de®ned:
the most diagnostic feature of PIN and CAL transformation was the accumulation of O-alkyl carbons,
suggesting concentration of preserved or altered heteropolysaccharides. The ARC leaves are characterized

Fig. 3. Correspondence analysis showing the di€erent characteristics
of the plant biomass (uncomposted leaves) and suggesting two fairly
di€erent compositions of the ameliorating vs. acidifying species
(dashed boundary lines). Sample labels (encircled) refer to Table 1.
Variable labels correspond to Table 3.

G. Almendros et al. / Soil Biology & Biochemistry 32 (2000) 793±804

803

ized in Table 3 and Fig. 5 overlap with a generic e€ect
of composting time. The most systematic generalizations consist of the preferential depletion (or condensation) of extractives, the accumulation of
nonhydrolyzable fractions and the initial increase in
the alkyl-to-O-alkyl ratio that, in the most ameliorating species, progresses even after the ®rst 98 incubation
days.

4. Conclusions

Fig. 4. Plant biomass changes during composting as re¯ected by the
parameters related to the concentration of major organic fractions.
Sample labels (encircled) refer to Table 1. Variable labels correspond
to Table 3.

by a low preservation of N and a low degradation of
lipid (the highest lipid value at zero time), probably related to the fact that the H-to-C ratio and concentration of carbonyl carbons remained high after
composting. Finally, the patterns of CIS and JTH resembled those of the most ameliorating species.
It must also be considered that the in¯uence of a
dominant mineral matrix, not present in the experimental design, may have a great importance for
further transformation and the selective stabilization
of most structures which characteristically accumulate
in active humus types. These results suggest that humi®cation in the absence of a predominant mineral substrate (i.e. neat composting) largely depends on the
chemical composition of the leaves from the di€erent
species. The individual degradation patterns summar-

The 13C NMR analysis of the early humi®cation
stages of forest and brushwood litter shows that the
degradation occurred similarly with all carbon types,
suggesting accumulation of recalcitrant material not
exclusively aromatic in nature. In fact, except in Pinus
and Calluna leaves, alkyl structures concentrated, their
insoluble character being suggested by the fact that the
plant material showed progressive depletion of extractive fractions (lipid and water-soluble).
The 13C NMR spectra indicate that the selective
preservation of tannins may control the decomposition
of other plant macromolecules. Thus, identical carbohydrate does not systematically decrease in all species
and carboxyl groups do not accumulate in the composting substrate.
There were some characteristic features associated to
the species considered either as ameliorant or as acidifying. The latter had high initial amounts of extractives, alkyl structures and comparatively lower
percentages of O-alkyl structures. On the other hand,
the ameliorating species showed a tendency to preferential degradation of carbohydrate compared to alkyl
structures. Superimposed on the above poorly-de®ned
general transformation patterns, a series of speci®c
trends makes it dicult to recognize systematic tendencies for the di€erent plants. The results point that
early transformation processes prior to the incorporation of leave fragments into soil mineral substrate are
highly species-dependent and may include intense microbial reworking and stabilization of extractives and
aliphatic recalcitrant fractions.

Acknowledgements
The authors wish to thank Mr E. Barbero (IQOG,
CSIC) for the elementary analysis. This research has
been funded by the Spanish CICyT.
Fig. 5. Correspondence analysis illustrating the transformation paths
of di€erent types of plant leaves during composting. The descriptors
used correspond to the relative extent of the change of the most
diagnostic parameters selected in previous treatments (Table 3).
Sample labels (encircled) correspond to Table 1.

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804

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