13C NMR assessment of decomposition patterns during composting of forest and shrub biomass

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: [email protected] (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

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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.

2.4. NMR acquisition conditions 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).

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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). 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 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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3.2. Changes during composting in the spectral patterns

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.

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

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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).

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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,

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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-

((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

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 ÿ43 0 25 54 66 9 8 ÿ14 48 1 51 70 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

23 3 ÿ5 ÿ38 ÿ14 ÿ3 1 6 ÿ13 ÿ6 ÿ9 ÿ21

21 30 56 63 73 84 101 105 115 135 145 153 174 ALK-to-OALK Na-toHa ME-toAR 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

ÿ15 27 54 8 62 22 45 11 ÿ3 17 20 33

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

800

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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