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
da
Centro de Ciencias Medioambientales (C.S.I.C.), Serrano 115 dpdo., E-28006 Madrid, Spain
b
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
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-state13C 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 acid-insoluble 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 dierent 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 dierent 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 resistantO-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 dierence 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.72000 Elsevier Science Ltd. All rights reserved.
Keywords:Pine; Oak; Heather; Biodegradation; Humi®cation; Litter
1. Introduction
The chemical composition of plant litter is
tra-ditionally considered to play a key role in the per-formance 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 deb-ris is associated with low biomass production in a situation in which energy input is spent on the
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* Corresponding author. Tel.: +34-91-562-5020; fax: +34-91-564-0800.
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 view-point, traditionally a distinction is made between
acidifying and ameliorating species (Vedy and Jac-quin, 1972). The de®nitions of these two classes depends, not only on the ecophysiological character-istics 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 com-pounds.
As an alternative to classical studies on humus for-mation which suggest the importance of selective ac-cumulation 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 polye-sters, such as cutins, suberins and other little known carbohydrate-polyalkyl macromolecules in higher plants (Nip et al., 1986).
Although the mechanisms aecting the sequestration of organic matter in soil are complex (Oades, 1988; Almendros and Dorado, 1999), it is clear that the dierent 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 Medi-terranean-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 dierentiation between alkyl (mainly polymethylene) andO-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 con-cepts of the changes during the early humi®cation stages, including the importance of aromatic C, nitro-gen compounds or water-soluble products in the leaves
as relevant factors to forecast organic matter evol-ution.
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), Mon-terey 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 arboreaL. (ERI), tree heath;Genista scor-piusDC. (GEN), scorpion broom andRetama sphaero-carpa(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 dis-carded, 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 at-mospheric 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 at-mosphere at 288C and 65% relative air humidity. The piles were mixed with a spatula every 14 d and homo-geneous samples composed by 5 subsamples of ca. 50 g (wet weight) were taken.
2.3. Chemical analyses
Total losses of substrate weight during the incu-bation experiment were estimated indirectly from the increase of the percentage of ash (determined after combustion in an electric mue at 6508C) in compari-son to the ash content at time zero.
homogen-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 evap-orated under N2. The water-soluble fraction was
removed from the lipid-extracted residue in the same apparatus (TAPPI, 1999a), and the acid-insoluble resi-due (i.e. the Klason lignin fraction) was determined in 1 g of the extractive-free residue after Saeman's hy-drolysis (TAPPI, 1999b). Holocellulose was calculated by dierence, 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 dierence and the percentages were calculated on ash-free basis. The gravimetric data were corrected taking into account the hygroscopic moisture content.
2.4. NMR acquisition conditions
The13C-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 spec-tral 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=glucopyra-nosyde-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/dihy-droxys of tannins; ca. 153=ether-linked (syringyl (S) lignins)/tannins); 160±200 ppm=carbonyl (172=car-boxyl/amide, 198=ketone/aldehyde) (Wilson, 1984; Wilson et al., 1988; Preston, 1992; Preston et al., 1997; Huang et al., 1998).
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 anities between samples and the descrip-tors responsible for their variability. The program out-put 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 syn-thetic 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 STA-TITCF package (ITCF, 1988).
3. Results and discussion
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 ammo-nia. 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 tis-sues: the amount of water-solubles do not decrease sig-ni®cantly in ILE, RET and even increase in other species (i.e. GEN). On the other hand, the relative con-centration of acid-insoluble residue increased as expected from a humi®cation process.
The trend of the atomic O-to-C ratio to decrease could mean that the selective degradation of the carbo-hydrate 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 42214%) should be considered high enough for such a tendency to be observed.
3.2. Changes during composting in the13C NMR spectral patterns
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 speciesain dierent transformation stages
Sample Weight loss
(g kgÿ1)
C
(g kgÿ1)
N
(g kgÿ1)
Atomic ratios C-to-N Lipid
(g kgÿ1)
Water-soluble
(g kgÿ1)
Acid-insoluble (lignin)
(g kgÿ1)
Holocellulose
(g kgÿ1)
H-to-C O-to-C
PIN0 0 485 13 1.58 0.73 37 160 180 252 378
PIN98 210 478 17 1.49 0.73 28 45 100 388 429
PIN168 286 475 19 1.49 0.73 24 20 95 413 430
JCO0 0 484 11 1.58 0.71 45 150 160 265 378
JCO98 321 468 19 1.62 0.71 24 60 100 355 415
JCO168 472 433 28 1.59 0.78 16 30 95 397 387
JTH0 0 452 12 1.60 0.75 39 135 200 242 338
JTH98 384 426 18 1.63 0.73 23 55 95 389 322
JTH168 454 431 24 1.57 0.68 18 20 90 411 323
ILE0 0 492 23 1.46 0.66 22 160 215 188 386
ILE98 609 462 26 1.55 0.62 17 130 190 311 241
ILE168 662 453 25 1.56 0.62 18 90 205 335 222
JUG0 0 410 8 1.65 0.86 48 110 170 179 431
JUG98 382 382 14 1.52 0.84 27 45 130 188 459
JUG168 427 372 18 1.52 0.84 21 20 90 322 371
QUE0 0 472 15 1.53 0.76 32 115 195 208 448
QUE98 393 457 23 1.52 0.76 20 15 80 424 425
QUE168 485 454 25 1.46 0.75 18 20 110 455 349
ERI0 0 544 7 1.58 0.59 79 160 170 309 338
ERI98 206 513 6 1.60 0.66 84 120 50 463 340
ERI168 207 542 8 1.55 0.58 64 110 60 458 343
CAL0 0 502 9 1.56 0.65 54 220 175 287 266
CAL98 249 498 14 1.55 0.63 34 100 100 419 311
CAL168 358 473 22 1.56 0.67 22 59 79 418 362
ARC0 0 493 28 1.48 0.68 17 230 250 224 262
ARC98 126 499 9 1.51 0.68 54 170 305 267 219
ARC168 368 490 7 1.51 0.69 67 115 195 392 244
CIS0 0 469 16 1.61 0.73 30 215 245 171 310
CIS98 262 480 24 1.55 0.66 20 135 140 300 353
CIS168 250 478 27 1.54 0.65 18 85 130 368 334
GEN0 0 475 12 1.61 0.78 38 70 70 212 627
GEN98 403 482 20 1.55 0.72 24 15 55 398 497
GEN168 427 484 22 1.54 0.71 22 15 80 388 480
RET0 0 470 25 1.65 0.75 19 145 165 171 485
RET98 487 480 39 1.58 0.65 12 40 115 337 442
RET168 632 466 35 1.57 0.66 13 20 145 321 422
a
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-sub-stituted 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 aro-matic peak at 135 ppm is also produced by C-1 and
C-4 in S units and C-1 in G units (Haw et al., 1984). There is a considerable overlap of the major lignin sig-nals at ca. 155 and 145 ppm with those of tannins, as proved in leaf biomass by Preston et al. (1997). In par-ticular part of plant tannins are biodegradation-recalci-trant compounds which are selectively preserved in the course of the humi®cation (Wilson et al., 1988). Such extractive compounds readily turn into macromolecu-lar 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 contri-bution 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
(except in the case of PIN) is attributable to the ac-cumulation of non-substituted aromatic carbons, whereas the signal intensity at about 153 ppm for O-or 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 for-est 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 aro-matic 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 (prob-ably in part laboratory artifacts) including a substan-tial 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 as-sociated 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,
Table 2
Integration values for the major C-types in the13C 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)
Aromatic-to-aliphatic
S-to-G
Methoxyl-to-aryl
O,N-aromatic-to-H-aromatic
Cain aminoacid
and OCH3
(65±45)
Alkyl-to-O-alkyl
PIN0 10.5 14.0 50.4 25.3 0.19 1.34 0.43 0.36 9.8 0.50
PIN98 6.5 17.3 57.9 18.5 0.23 1.06 0.25 0.57 7.7 0.32
PIN168 6.9 16.7 62.0 14.5 0.22 1.41 0.31 0.80 8.4 0.23
JCO0 5.1 17.0 60.3 17.7 0.22 1.38 0.22 1.00 5.9 0.29
JCO98 7.6 14.6 53.7 24.3 0.19 0.87 0.28 0.50 7.1 0.45
JCO168 7.2 13.3 62.0 17.7 0.17 1.33 0.35 0.48 7.6 0.29
JTH0 6.4 17.3 60.3 16.2 0.23 1.07 0.21 0.56 6.2 0.27
JTH98 6.5 14.1 55.7 23.9 0.18 0.83 0.34 0.43 8.2 0.43
JTH168 6.4 16.4 57.3 20.2 0.21 0.92 0.35 0.39 9.5 0.35
ILE0 8.2 9.2 48.6 34.2 0.11 0.50 0.39 0.22 6.7 0.70
ILE98 8.2 12.6 33.0 46.5 0.16 1.23 0.36 0.41 8.9 1.41
ILE168 8.5 8.7 30.2 52.8 0.10 1.19 0.45 0.27 7.3 1.75
JUG0 10.8 15.4 59.7 14.3 0.21 1.21 0.22 0.63 5.5 0.24
JUG98 9.8 15.9 55.5 18.9 0.21 1.22 0.28 0.46 7.5 0.34
JUG168 9.8 15.0 51.6 23.8 0.20 1.29 0.36 0.35 8.9 0.46
QUE0 7.8 16.3 58.4 17.8 0.21 0.94 0.25 0.66 6.5 0.30
QUE98 9.3 18.0 53.5 19.5 0.25 1.01 0.24 0.45 7.4 0.36
QUE168 8.6 15.4 56.7 19.4 0.20 1.32 0.33 0.57 7.9 0.34
ERI0 6.0 17.8 45.3 31.1 0.23 1.46 0.19 1.02 6.2 0.69
ERI98 4.9 15.0 45.4 34.9 0.19 1.02 0.32 0.58 8.8 0.77
ERI168 5.1 15.9 45.6 33.6 0.20 1.20 0.30 0.61 9.0 0.74
CAL0 5.2 15.5 51.2 28.2 0.19 1.12 0.25 0.60 7.5 0.55
CAL98 6.3 14.0 47.9 32.0 0.17 1.13 0.31 0.54 8.2 0.67
CAL168 6.5 15.4 54.2 24.2 0.20 1.06 0.31 0.57 8.3 0.45
ARC0 4.4 23.1 57.2 15.7 0.32 0.77 0.16 0.63 6.8 0.27
ARC98 5.1 22.5 52.4 20.3 0.31 0.71 0.15 0.57 6.5 0.39
ARC168 5.1 22.5 49.6 23.2 0.31 0.80 0.15 0.60 6.6 0.47
CIS0 8.0 14.4 54.2 23.6 0.19 0.81 0.31 0.56 7.4 0.44
CIS98 7.4 14.3 51.2 27.3 0.18 1.26 0.34 0.54 8.8 0.53
CIS168 8.5 16.7 51.0 23.9 0.22 1.33 0.29 0.57 8.6 0.47
GEN0 5.8 13.1 72.0 9.4 0.16 1.92 0.39 0.70 7.9 0.13
GEN98 5.9 15.9 63.5 14.9 0.20 1.53 0.37 0.48 9.2 0.23
GEN168 5.6 14.9 65.5 14.2 0.19 1.78 0.43 0.65 9.5 0.22
RET0 7.4 13.4 64.7 14.7 0.17 1.77 0.34 0.55 7.8 0.23
RET98 5.8 12.6 58.4 23.4 0.15 1.75 0.51 0.44 10.5 0.40
RET168 7.5 16.4 51.3 25.0 0.22 1.55 0.39 0.51 10.3 0.49
C-3 and C-5 of pyranoside rings in cellulose and hemi-cellulose (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 prep-arations 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 (Kolod-ziejski 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 r0:780,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 the13C 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 (Cain amino
acids), the intensity of which (Table 2) has been found to correlate with the nitrogen concentration in humic-type 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 natu-ral degradation process.
To some extent, the overlapping of the Ca signal
with that of the methoxyl may be a source of interfer-ence with the above-indicated change in the methoxyl content. In fact, the intensity signal of these overlap-ping spectral regions was, as expected, signi®cantly correlated r0:933,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 sig-nal, less than expected according to the expected num-ber of methoxyl C per aromatic ring (Preston et al.,
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 S-to-G ratio was unrelated to the area of the 56 ppm sig-nal.
The above observations show that the classical assumption that lignin transformation is accompanied by demethoxylation and carboxylation are not system-atically 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 above-described interfering eect of protein and to a signi®-cant contribution by recalcitrant tannins both through their selective preservation and through their conden-sation reactions controlling the decomposition rates of the aliphatic biomacromolecules. Furthermore, the data shown in Table 2 illustrate that the aromatic-to-aliphatic 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 compar-ing the 13C NMR results with those from the wet chemical methods the major dierences observed cor-respond to the fact that the concentration of the acid-insoluble 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) fre-quently 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 co-incide with the suggestions of Hemp¯ing et al. (1987), who considered that the hypothesis of increasing aro-maticity 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 syn-thesis of the stable aliphatic compounds in the course of decomposition and humi®cation.
resist-ance to degradation of cutin-like material in these species or to the above-indicated eect of tannins in the selective preservation ofO-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 dierent 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 aromati-city (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 ma-terials 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 concen-tration of carbohydrate carbons observed in several species does not necessarily correspond to the preser-vation of native polysaccharides, but, e.g. to diageneti-cally altered substances not readily recognized by enzymes. Such a fraction, where the quantitative con-tribution and protecting role of tannins should not be neglected, could include domains with anhydrosugar-or Maillard-like-structures, which cannot be thoroughly distinguished from pyranoside signals in the13C NMR spectra (Almendros et al., 1997).
3.3. Selective depletion of the dierent C-types
Fig. 2 shows the dierence spectra obtained by digi-tal subtraction of the spectra at zero time and those at 168 d, the latter corrected by the losses of carbon (cal-culated from the weight loss and the elementary com-position). In some species the dierence spectra (i.e. the spectra of the material that were lost by biodegra-dation) were unexpectedly similar to the spectra at zero time. In these cases it indicated that the degra-dation occurred similarly in all C types, what could be a characteristic of the early humi®cation stages (Pre-ston et al., 1998). From the spectral pro®les, it is evi-dent that no general tendency of the selective accumulation of aromatic carbon is observed in the composting period. Similar data were reported by
Wilson et al. (1983) who also found that the trends in wet chemical analyses saw a marked loss in carbo-hydrates and an increase in residual lignin, whereas the NMR changes were much smaller. The dierence spec-tra indicate that the preferential degradation of carbon dier 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 diering in the greater loss of aro-matic carbons (tannins) in the former. The dierence spectra of some species (PIN and, to some extent, ILE) with an intense loss of alkyl and carboxyl car-bons might be interpreted as active degradation of cu-ticular polyester material. The degradation of tannins is also betrayed by the sharp bands in the dierence
spectra of most of the species, i.e., the heathers, cupressaceae and the angiosperms with leaves.
The dierence 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 biode-gradability (weight loss), there were small possibilities to obtain generalizations due to the large statistical dispersion of the data analyzed in addition to the eect of several outliers forcing most of the correlation indi-ces to be signi®cant. For example, it is often
con-Fig. 2. Dierence 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 dierent 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
sidered that the initial lignin (and nitrogen) concen-tration 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 r0:49,P< 0.05) and the
acid-insolu-ble-residue:r ÿ0:50,P< 0.05) was, in fact, most
sig-ni®cant between zero time samples (plots not shown). Total aromatic carbon correlates with the weight loss at r 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 dierent types of for-est 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) dierence with regards to the weight loss underwent by the acidi-fying plants in the course of composting. This prelimi-nary study suggested the lack of a single major limiting factor for biodegradability of the leaves stu-died. 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 acidify-ing, 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 dierent 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 rou-tine variables in relation to the major organic frac-tions) 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 hetero-geneous 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 dierences between species which are more or less favorable from a biogeochem-ical 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 con-centration of carbohydrate and the low proportion of extractives led to a rapid accumulation of resistant bio-polymers. The other cluster (from ILE to CAL) con-sisted of samples with comparatively large concentrations of low molecular weight products. Table 1 shows that, whereas operationally-de®ned lig-nin 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 eect of the quantitative contribution and reactivity of leaf tannins.
3.4.3. Speci®c transformation of dierent types of plant litter during composting
The degradation patterns in terms of the most diag-nostic variables selected after the above statistical treatments are summarized in Fig. 5. The points corre-sponding 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 preferen-tial degradation of carbohydrate as regards alkyl struc-tures. 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 trans-formation was the accumulation of O-alkyl carbons, suggesting concentration of preserved or altered het-eropolysaccharides. The ARC leaves are characterized
by a low preservation of N and a low degradation of lipid (the highest lipid value at zero time), probably re-lated to the fact that the H-to-C ratio and concen-tration of carbonyl carbons remained high after composting. Finally, the patterns of CIS and JTH re-sembled those of the most ameliorating species.
It must also be considered that the in¯uence of a dominant mineral matrix, not present in the exper-imental 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 sub-strate (i.e. neat composting) largely depends on the chemical composition of the leaves from the dierent species. The individual degradation patterns
summar-ized in Table 3 and Fig. 5 overlap with a generic eect of composting time. The most systematic generaliz-ations consist of the preferential depletion (or conden-sation) of extractives, the accumulation of nonhydrolyzable fractions and the initial increase in the alkyl-to-O-alkyl ratio that, in the most ameliorat-ing species, progresses even after the ®rst 98 incubation days.
4. Conclusions
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 Callunaleaves, alkyl structures concentrated, their insoluble character being suggested by the fact that the plant material showed progressive depletion of extrac-tive 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 carbo-hydrate does not systematically decrease in all species and carboxyl groups do not accumulate in the com-posting substrate.
There were some characteristic features associated to the species considered either as ameliorant or as acidi-fying. The latter had high initial amounts of extrac-tives, alkyl structures and comparatively lower percentages of O-alkyl structures. On the other hand, the ameliorating species showed a tendency to prefer-ential 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 dicult to recognize systematic ten-dencies for the dierent plants. The results point that early transformation processes prior to the incorpor-ation of leave fragments into soil mineral substrate are highly species-dependent and may include intense mi-crobial 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.
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