Summary. Introduction

Transcription

1 European Journal of Soil Science, December 2000, 51, 583±594 Transformation of organic matter from maize residues into labile and humic fractions of three European soils as revealed by 13 C distribution and CPMAS-NMR spectra R. SPACCINI a,a.piccolo a,g.haberhauer b &M.H.GERZABEK b a Dipartimento di Scienze Chimico Agrarie, UniversitaÁ degli Studi di Napoli `Federico II', Via UniversitaÁ 100, Portici, Italy, and b Austrian Research Centre Seibersdorf, Department of Environmental Research, 2444 Seibersdorf, Austria Summary The dynamics of incorporation of fresh organic residues into the various fractions of soil organic matter have yet to be clari ed in terms of chemical structures and mechanisms involved. We studied by 13 C- dilution analysis and CPMAS- 13 C-NMR spectroscopy the distribution of organic carbon from mixed or mulched maize residues into speci c de ned fractions such as carbohydrates and humic fractions isolated by selective extractants in a year-long incubation of three European soils. The contents of carbohydrates in soil particle size fractions and relative 13 C values showed no retention of carbohydrates from maize but rather decomposition of those from native organic matter in the soil. By contrast, CPMAS- 13 C-NMR spectra of humic (HA) and fulvic acids (FA) extracted by alkaline solution generally indicated the transfer of maize C (mostly carbohydrates and peptides) into humic materials, whereas spectra of organic matter extracted with an acetone solution (HE) indicated solubilization of an aliphatic-rich, hydrophobic fraction that seemed not to contain any C from maize. The abundance of 13 C showed that all humic fractions behaved as a sink for C from maize residues but the FA fraction was related to the turnover of fresh organic matter more than the HA. Removal of hydrophobic components from incubated soils by acetone solution allowed a subsequent extraction of HA and, especially, FA still containing much C from maize. The combination of isotopic measurements and NMR spectra indicated that while hydrophilic compounds from maize were retained in HA and FA, hydrophobic components in the HE fraction had chemical features similar to those of humin. Our results show that the organic compounds released in soils by mineralization of fresh plant residues are stored mainly in the hydrophilic fraction of humic substances which are, in turn, stabilized against microbial degradation by the most hydrophobic humic matter. Our ndings suggest that native soil humic substances contribute to the accumulation of new organic matter in soils. Introduction Soil organic matter (SOM) is a basic component of the agroecosystem and acts as an essential link among the various chemical, physical and biological properties of soil. It helps to prevent erosion and deserti cation (Piccolo, 1996) and is a driving variable in environmental changes since it acts as both a source and a reservoir for carbon (Schlesinger, 1997). The soil organic matter comprises several compartments of different biochemical composition, biological stability and carbon turnover (Paustian et al., 1992). Both labile and stable pools of SOM play an important role in the stabilization and dynamics of organic C in the soil. Carbohydrates represent up Correspondence: A. Piccolo. alpiccol@unina.it Received 1 November 1999; revised version accepted 12 May 2000 to 25% of SOM and constitute most of the rapidly changing pool of C (Stevenson, 1994). They provide energy for the soil biological activity (Insam, 1996) and contribute to the shortterm physical stability of soils (Piccolo & Mbagwu, 1999). Humic matter is the most important component of the passive highly stable SOM pool, and it is the key factor in stabilization, accumulation and dynamics of organic C in the soil (Andreux, 1996). The strong association of humic substances with the inorganic soil components is regarded as a means by which this C is protected against microbial degradation, thereby conferring on humic substances mean residence times in soil which vary from tens to several hundreds of years (Paustian et al., 1992). Such stable humic fractions, however, are also increasingly believed to interact with the more rapidly changing organic matter from litter decomposition and microbial biomass (Insam, 1996) and # 2000 Blackwell Science Ltd 583

2 584 R. Spaccini et al. Table 1 Some physical and chemical properties of the soils Soil Sand /% Silt /% Clay /% Organic C /% ph(h 2 O) Denmark German Italian contribute to the accumulation of organic C in soils (Buurman, 1994; Piccolo et al., 1999b). In recent years, naturally 13 C-enriched plants, such as those employing a C4 photosynthetic pathway (Zea mays L.), or synthetically 13 C-labelled compounds have been used to follow carbon distribution in the different fractions of SOM (Baldock et al., 1989; Lichtfouse et al., 1994, 1995; Arrouays et al., 1995; Andreux, 1996). Most of this research did not produce conclusive results because of the dif culty in separating de ned chemical structures in different pools and of poor information on the mechanisms that stabilize organic matter in soil. While analysis of the labile fraction of SOM, mainly polyand monosaccharides, has been increasingly related to different soil management practices (Guggenberger et al., 1994; Zech & Guggenberger, 1996), the relation of isolated humic fractions to the dynamics of SOM is less certain. The most polar humic fraction, the fulvic acids, has been used to relate changes of organic matter to soil management (Wander & Traina, 1996; Zalba & Quiroga, 1999), but other humic fractions may be equally, if not more, important in controlling the rapid turnover of organic matter in agricultural soils. Piccolo et al. (1998) showed that apolar humic fractions, rich in aliphatic carbon, extracted from soils by an aqueous acetone solution were scarcely associated with the inorganic soil components and contributed largely to the adsorption of a weakly polar molecule such as atrazine. Almendros et al. (1998) used apolar organic solvents in multi-step extraction procedures and isolated hydrophobic humic fractions which they believed to be involved in the long-term stabilization of soil humus. Piccolo (1996) thought that hydrophobic humic components in soil exerted hydrophobic protection towards easily degradable compounds. He postulated that associations of apolar molecules deriving from plant degradation and microbial activity incorporate more polar molecules, thereby preventing their otherwise rapid microbial degradation and enhancing their persistence in soil. This view accords with results which suggest that humic matter is composed of polar microdomains surrounded by more hydrophobic components (Engebretson & von Wandruszka, 1994) and that the macropolymeric conformation of humic substances is only apparent since they really are supramolecular associations stabilized by weak but multiple dispersive (hydrophobic) forces (Conte & Piccolo, 1999; Piccolo et al., 1999a). We have investigated the changes in the labile (polysaccharides) and in the stable (humic fractions isolated with different extractants) pools of soil organic matter following a one-year laboratory incubation of maize residues in three European soils. Addition of maize residues simulated both mulching and ploughed-under management techniques, while qualitatively and quantitatively changes of SOM were assessed both by NMR spectroscopy and by determination of 12 C/ 13 C ratios. Material and methods Soils and experimental design Surface samples (0±20 cm) of three European agricultural soils along a north±south gradient were air-dried and sieved through a 2-mm sieve. The soils were (FAO Soil Classi cation) as follows: 1 a Haplic Luvisol from Roskilde, Denmark (mean annual temperature 8.4 C); 2 a Haplic Luvisol from MuÈnchen, Germany (mean annual temperature 7.0 C); 3 a Eutric Regosol from Caserta, Italy (mean annual temperature 15.1 C). Their particle size distribution, organic content and ph are listed in Table 1. The set-up for the maize incubation experiment was reported elsewhere in detail (Stemmer et al., 1999). Brie y, soil samples were amended with 13.3 mg g ±1 of air-dried and chopped maize straw residues (previously passed through a 2- cm sieve), corresponding to 6 mg C g ±1 of soil. The treatment plan was as follows. 1 Control soil, with no maize addition. 2 Mulched, maize straw just placed on the soil surface. 3 Mixed, maize straw thoroughly mixed with the soil. All treatments were in triplicate and incubated in the laboratory for one year at 14 C and with moisture content kept throughout at 40% of water-holding capacity by adding distilled water as necessary. Soil replicates were extracted for carbohydrates and humic substances before (t 0 ) and after (t 1 ) the year of incubation. Particle size fractionation Size fractionation was based on the method described in detail by Stemmer et al. (1998). To minimize destruction of labile particulate organic matter the soil water suspension was dispersed using low-energy sonication (0.2 kj g ±1 output energy). Moist samples equivalent to 35 g of air-dry weight were placed in 150 ml plastic beakers each with 100 ml of distilled water and dispersed with a probe-type ultrasonical disaggregator (50 J s ±1 for 120 s). Sieves were used to obtain larger particles, while centrifuging at 150 g and 3900 g yielded particle sizes of less than 63 and 2 m, respectively. Particle

3 Transformation of maize residues into soil 585 size fractions were obtained in the following ranges: 2000± 200 m, coarse and medium sand (CS); 200±63 m, ne sand (FS); 63±2 m, silt (S); 2±0.1 m, clay (C). The fraction < 0.1 m consisted mainly of dissolved organic matter and was discarded. Fraction samples were freeze-dried and stored for subsequent analysis. In this work, the alkaline extraction was done in each soil sample either before (HA1 and FA1) or after (HA2 and FA2) a preliminary extraction of the hydrophobic humic fraction (HE) by the acetone solution. Yields were calculated on triplicate extractions and in all cases the relative standard deviation was less than 6%. Carbohydrate content Carbohydrates were extracted from size fractions as described by Piccolo et al. (1996). A soil sample (1 g) was rst hydrolysed with 10 ml of 0.25 M H 2 SO 4 for 16 h in a rotatory shaker, the extracting solution was puri ed from interfering ions by passing it through anion and cation exchange resins, and the carbohydrate content in the hydrolysates was determined colorimetrically as glucose equivalent using the phenol±sulphuric acid method of Dubois et al. (1956). The hydrolysates were then freeze-dried and stored for isotopic 13 C analyses. Humic substances Speci c humic fractions (Piccolo et al., 1998) were isolated from soil samples with selected solutions as follows. 1 Humic (HA) and fulvic acids (FA) were extracted by a 1 M NaOH and 1 M Na 4 P 2 O 7 solution (50:50 by volume). 2 Humic extracts (HE) were extracted by a 0.6 M acetone± HCl solution (8:2 by volume). Soil samples were placed in rubber-stoppered polyethylene bottles with the extracting solutions (200 g l ±1 ) and shaken for 24 h on a mechanical shaker. The mixture was centrifuged, and the supernatant containing alkaline (HA and FA) or acetone (HE) extracts were ltered through glass-wool and a quartz lter, respectively. The soil residue was further extracted twice more with the same extracting solutions. For the alkaline extraction, the suspensions holding HA and FA, once ltered, were combined and rapidly brought to ph 1 with concentrated HCl, and the HA was allowed to settle for 24 h at 20 C. The precipitated humic acids were puri ed from inorganic impurities through repeated (three times) dissolution and re-precipitation in 0.5 M NaOH and 0.5 M HCl solutions, respectively. The soluble fulvic acids were puri ed by passing through non-ionic polymeric resins (Amberlite XAD-8, Sigma Chemicals) to remove the non-humi ed low-molecular-weight organic compounds (mainly polysaccharides). The eluted fulvic acids were brought to ph 5, dialysed against distilled water until free of Cl, freeze-dried and stored for NMR and 12 C/ 13 C analysis. For the acetone procedure, the fractions from subsequent extractions were combined, rotoevaporated and dialysed against water to eliminate the solvent completely. Both HA and HE were then puri ed by shaking in polyethylene bottles for 24 h with 0.5% HCl±HF solution, dialysed against water until free of Cl, freeze-dried and stored for NMR and 12 C/ 13 C analysis. NMR spectroscopy The 13 C-NMR spectra of humic extracts from soils were recorded by the Cross Polarization Magic Angle Spinning (CPMAS) technique. The analyses were done with a Bruker AMX400 at MHz using a rotating speed of 4500 Hz, a contact time of 1 ms, a recycle time of 1 s and an acquisition time of 13 ms. All runs were made with Variable Contact Time (VCT) pulse sequence in order to nd the Optimum Contact Time (OCT) for each sample and to minimize the error on the evaluation of the peak areas (Conte et al., 1997). The OCT ranged between 0.8 and 1.0 ms. The line broadening for FID (Free Induction Decay) transformation was xed at 50 Hz. The following resonance intervals were associated with the different carbons: 0±40 p.p.m. = aliphatic-alkyl C; 40±110 p.p.m. = C±N and C±O in polypetidic and carbohydratic compounds; 110±140 p.p.m. = aromatic C; 140±160 p.p.m. = phenolic-related C; 160±190 p.p.m. = carboxylic C. The areas relative to these resonance intervals were used to evaluate a degree of hydrophobicity (HB/HI) for the humic matter extracted with the alkaline solution: HB/HI = [(0 ± 40) + (110 ± 145)]/[(40 ± 110) + ( )]. The spinning side band interfering with aromatic C signal was accounted for by automatically subtracting the area under the 190±230 p.p.m. interval from that under the 110±140 p.p.m. region. Isotopic analysis ( 13 C/ 12 C ratio) The isotopic abundance ( 13 C) in the freeze-dried hydrolysates of soil and in the isolated humic extracts was measured by continuous- ow isotope ratio-mass spectrometry (Carlo Erba N Analyzer 1500 coupled with Finnigan MAT 251). The results of 13 C/ 12 C ratios are expressed in the relative scale (½) according to the following equation: 13 C½ = (R sample /R standard ± 1) , where R = 13 C/ 12 C and the standard is the Pee Dee Belemnite (PDB). The content of organic C derived from maize residues, expressed as per cent of total C, was determined as follows: (%-C maize ) = ( C c ± C 0 )/( C m ± C 0 ) 3 100, where C c is the isotopic value of the sample, C 0 is the isotopic value of the control sample, and C m is the isotopic composition of the maize residue (±12.5½), respectively.

4 586 R. Spaccini et al. Table 2 Amount and distribution of carbohydrates in particle size fractions before (t 0 ) and after (t 1 ) soil incubation with maize residues (standard error in parentheses; n = 3) Soil Danish German Italian Sample Particle sizes /g g ±1 /% /g g ±1 /% /g g ±1 /% Control (t 0 ) Coarse sand 763 (28.3) (43.9) (27.7) 15.6 Fine sand 1076 (21.4) (37.5) (47.9) 17.0 Silt 2195 (43.3) (56.6) (31.2) 22.2 Clay 2469 (18.5) (58.9) (45.0) 45.2 Control (t 1 ) Coarse sand 337 (31.8) (48.5) (112) 25.2 Fine sand 489 (40.4) (41.6) (19.1) 24.6 Silt 564 (13.3) (43.9) (81.4) 29.7 Clay 614 (24.2) (91.2) (20.2) 20.5 Mulched (t 1 ) Coarse sand 254 (30.6) (145) (35.8) 24.6 Fine sand 264 (16.2) (41.6) (39.3) 24.6 Silt 421 (30.6) (38) (62.4) 27.7 Clay 1034 (4) (63) (46.8) 23.1 Mixed (t 1 ) Coarse sand 419 (30.6) (47.3) (104.5) 29.0 Fine sand 456 (24.2) (24.4) (28.3) 24.6 Silt 774 (79) (39.3) (13.3) 23.1 Clay 1366 (30) (16.2) (7.5) 23.3 Results and discussion Carbohydrates Total content. Table 2 shows the content of total carbohydrates in particle size fractions of different soils before (t 0 ) and after (t 1 ) a one-year incubation with maize residues. At the end of incubation, total carbohydrates generally decreased in all particle size fractions for control and maize-treated (mulched and mixed) samples in comparison with the soils at the onset of incubation (t 0 ). More carbohydrate was found after incubation (t 1 ) for both maize-treated samples of the German soil and for the maize-mixed sample of the Danish soil than in the control samples (t 1 ). Conversely, in the particle sizes of the Italian soil, the addition of maize residues caused a larger decrease of carbohydrates in both the maize-treated (mulched and mixed) samples than for the control (Table 2). These differences suggest a more intense microbial activity in the Italian soil under a warmer climate whereby addition of fresh organic matter may have promoted a sort of priming effect (Insam, 1996) and caused a larger degradation of the labile pool for treated soils than for the control. 13 C content. The results of isotopic analyses in hydrolysates from particle size fractions separated before and after soil incubation with maize residues are shown in Table 3. The data from control samples before incubation (t 0 ) indicated that the organic matter in these soils originated from C3 plant species. The 13 C values found in soil hydrolysates are well within the range of isotopic values characteristic of carbohydrates derived from plants with a C3 pathway (Balesdent & Mariotti, 1996). The isotopic abundance ( 13 C) of soil hydrolysates from maize-treated samples for all soils showed (Table 3) that no carbon from maize residues ( 13 C = ±12.5½) was transferred into the carbohydrates still present in soils after incubation (t 1 ). Soil incubation with or without maize residues resulted in a small decrease of 13 C content in soil hydrolysates (larger negative 13 C values in Table 3) from all size fractions when compared with the initial values (t 0 ) of control samples. This may be explained by a preferential microbial decomposition of isotopically heavier carbohydrate fractions, thus shifting the overall 13 C of carbohydrates to more negative values. The changes in total carbohydrates observed with incubation (Table 3) were similarly explained by an enhanced microbial degradation of native SOM from C3 plants in both control and maize-treated soils. Control soils at t 1 showed an overall decrease of carbohydrate content ranging from 55 to 85% of the initial amount at t 0. Treatment with maize appears to have somewhat limited the decrease of carbohydrate content, with the exception of the Italian soil (Table 3). Humic substances Extraction yields. The yields of extraction of humic fractions (HA1, FA1, HE, HA2, FA2) from each soil before and after

5 Transformation of maize residues into soil 587 Table 3 Isotopic abundance ( 13 C, ½) in soil hydrolysates from size fractions of control and maize-treated soils before (t 0 ) and after (t 1 ) incubation (standard error was less than 0.06½ for all values; n = 3) Sample Coarse sand Fine sand Silt Clay Danish soil Control (t 0 ) ±27.1 ±27.0 ±26.8 ±26.6 Control (t 1 ) ±28.9 ±28.9 ±28.8 ±28.8 Mulched (t 1 ) ±29.0 ±28.9 ±28.8 ±28.8 Mixed (t 1 ) ±29.1 ±28.9 ±28.7 ±28.6 German soil Control (t 0 ) ±27.1 ±27.1 ±26.8 ±27.1 Control (t 1 ) ±28.8 ±28.7 ±28.7 ±28.5 Mulched (t 1 ) ±28.9 ±28.9 ±28.8 ±28.7 Mixed (t 1 ) ±28.9 ±28.9 ±28.9 ±28.6 Italian soil Control (t 0 ) ±26.9 ±26.8 ±26.8 ±26.9 Control (t 1 ) ±28.4 ±28.7 ±28.5 ±28.6 Mulched (t 1 ) ±28.7 ±28.7 ±28.8 ±28.6 Mixed (t 1 ) ±28.7 ±28.8 ±28.8 ±28.5 incubation are shown in Table 4. Incubation (t 1 ) signi cantly increased the extraction yields of all humic fractions with respect to those obtained from control samples before incubation (t 0 ). This suggests that drying and sieving the soil and subsequent microbial activity during incubation altered the original soil associations between organic matter and clay and made the humic matter more accessible to extracting solutions. Moreover, there were differences between the samples that had been incubated with maize straw and those that had not. While the aqueous acetone solution always extracted more humic material from the mulched samples than from either the mixed or control samples after incubation, the yields of HA1 and FA1 were generally larger than the control in the mixed samples rather than in the mulched samples. Similarly, HA2 and FA2, which were extracted by an alkaline solution but only after soil extraction with the aqueous acetone solution, showed the largest yields from the mixed samples, with the exception of the German soil that gave yields similar to those of the control samples. These results indicate that the organic matter derived from the maize degradation distributed itself differently into the various humic fractions according to the degree of straw incorporation into soil (mulching against mixing) and to the consequent availability of surface areas of soil particles for adsorption of organic compounds released from maize. NMR spectra. Solid-state 13 C-NMR spectra of alkaline (HA1 and FA1) and acetone (HE) humic extracts from the three soils before and after incubation were recorded. The distributions of the measured carbon in the different p.p.m. intervals are shown in Table 5 for HA1 and FA1 extracts from all soils, and HA1 and HE spectra for all three soils before incubation (t 0 ) are Table 4 Extraction yields (% by weight of soil sample) of humic fractions isolated from different treatments before (t 0 ) and after (t 1 ) soil incubation Sample Control (t 0 ) Control (t 1 ) Mulched (t 1 ) Mixed (t 1 ) Danish soil a HA a FA HE b c HA FA 2 c German soil HA FA HE HA FA Italian soil HA FA HE HA FA a Humic (HA1) and fulvic (FA1) acids extracted by alkaline solution. b Humic extract (HE) solubilized by the aqueous acetone solution. c Humic (HA2) and fulvic (FA2) acids extracted by alkaline solution following soil treatment with the aqueous acetone extractant. shown in Figures 1 and 2, respectively. The NMR spectra of HA1 (Italian soil) and FA1 (Danish soil), representative of humic fractions after incubation (t 1 ) with or without maizestraw additions, are shown in Figures 3 and 4, respectively. Despite the quantitation error in the solid-state NMR technique, which we kept small by applying the VCT technique, spectra of HA1 and FA1 from t 0 samples showed the structure of humic matter to vary between colder and warmer climates (Table 5, Figure 1). A progressive decrease of the alkyl region (0±40 p.p.m.) was revealed in NMR spectra of HA1 (Figure 1) from the Danish and German (41%, 39%, respectively) to the Italian (27%) soil. Concomitantly, a signi cant increase in carboxylic carbons (160±190 p.p.m.) was noted with increasing mean annual temperature (Danish = 15%, German = 14%, Italian = 27%). This increase in carboxyl C may be attributed to the side-chain oxidation of plant-derived lignin-phenolic compounds which is likely to increase in warm climates (Guggenberger et al., 1995; Zech & Guggenberger, 1996). Signals relative to phenol carbons (140± 160 p.p.m.) are present in HA1 spectra of the two northern soils but absent in that of the Italian soil, thereby suggesting a progressive oxidation of phenolic carbon with decreasing latitude. Moreover, an enhancement of aromatic carbon (110± 140 p.p.m.) in warmer climate was observed (Danish and German» 2%; Italian» 10%). In general, the shift from

6 588 R. Spaccini et al. Sample 0±40 40± ± ± ±190 HB/HI a HA1 Danish soil Control (t 0 ) Control (t 1 ) Mulched (t 1 ) Mixed (t 1 ) Table 5 Relative carbon distribution (%) in different regions of chemical shift (p.p.m.) in CPMAS- 13 C-NMR spectra of humic and fulvic acids before (t 0 ) and after (t 1 ) incubation with maize residues German soil Control (t 0 ) Control (t 1 ) Mulched (t 1 ) Mixed (t 1 ) Italian soil Control (t 0 ) Control (t 1 ) Mulched (t 1 ) Mixed (t 1 ) FA1 Danish soil Control (t 0 ) Control (t 1 ) Mulched (t 1 ) Mixed (t 1 ) German soil Control (t 0 ) Control (t 1 ) Mulched (t 1 ) Mixed (t 1 ) Italian soil Control (t 0 ) Control (t 1 ) Mulched (t 1 ) Mixed (t 1 ) a HB/HI = [(0 ± 40) + (110 ± 145)]/[(40 ± 110) + ( )]. northern towards southern soils produces HA generally more hydrophilic in character as is shown by their decreasing order of hydrophobicity (Table 5): Danish > German > Italian (0.81 > 0.74 > 0.59). The degree of hydrophobicity calculated from NMR spectra of FA1 extracts (Table 4) indicated that these had a more hydrophilic character than HA1 samples (Danish = 0.50, German = 0.53, Italian = 0.60), as is expected because of the highly oxidized nature of FAs. In comparison with HA1, FA1 spectra of Danish and German soils at t 0 were characterized by smaller aliphatic and aromatic content, absence of phenolic structures, more intense signals in the O-, N-alkyl region (40± 110 p.p.m.), including those of anomeric carbon in carbohydrates (98±110 p.p.m.), and larger content of carboxyl carbon (170 p.p.m.). Compared with the northern soils, FA1 from the Italian soil contained more aliphatic and aromatic carbon and somewhat less carbohydratic and peptidic carbon (Danish and German» 48% against Italian» 39%). The persisting differences in chemical composition between HA1 and FA1 of the Italian soil and that of soils of higher latitudes seems to indicate an in uence of climate on humic characteristics. The humic extracts (HE) isolated by an acetone±hcl solution produced NMR spectra for all soils at time t 0 (Figure 2) that suggested a highly hydrophobic character of these fractions. The HE spectra showed essentially a strong signal in the alkyl region (0±40 p.p.m.) centred at around 25 p.p.m., which can be attributed to CH 2 and CH 3 groups in polymethylene chains (Piccolo et al., 1998). For the German

7 Transformation of maize residues into soil 589 Figure 1 CPMAS- 13 C-NMR spectra of humic substances extracted by alkaline solution (HA1) from control soils before incubation (t 0 ). and Italian soils, HE spectra also showed other but less intense signals at 55, 72 (N- and O-alkyl), 127 (ole nic or aromatic C=C) and 170 (C=O bonds) p.p.m. The alkyl structures extracted by the acetone±hcl solution may well represent the most persistent fraction of stable organic matter in soils. Oades et al. (1987) and Baldock et al. (1992) showed that aliphatic compounds constitute the basic component of the recalcitrant organic matter strictly associated with the nest (< 2 m) soil particles. Moreover, the HE in our study appear very similar to the chemical characteristics of the most resistant organic matter fraction isolated from surface horizons of forest soils after sequential extraction and vigorous hydrolysis of the resulting humin residues, as reported by KoÈgel-Knabner et al. (1992) and Augris et al. (1998). These authors suggested that the non-hydrolysable highly aliphatic organic residue may consist of preserved macromolecular materials directly inherited from higher plants. However, they proposed no hypothesis for the mechanism of preservation. As evidence for the stability of such hydrophobic fractions, the NMR spectra (not shown) of HE isolated after incubation (t 1 ) with maize straw were not signi cantly altered compared with their spectra at time t 0. The NMR spectra of HA1 from the Danish and German soils after incubation (t 1 ) showed no substantial differences between control and maize-treated samples (Table 5), thereby failing to support any evidence of incorporation of maizederived material into the humic acid fraction. In contrast to the northern European soils, HA1 spectra for the Italian soil (Figure 3) showed a signi cant increase of the 0±40 and 40± 110 p.p.m. regions in the maize-mixed sample as compared Figure 2 CPMAS- 13 C-NMR spectra of humic substances extracted by acetone±hcl solution (HE) from control soils before incubation (t 0 ). with the mulched and control (t 1 ) samples and an enhancement of the phenolic carbon at about 160 p.p.m. These changes suggest that some of the organic matter (mainly carbohydratic and peptidic residues but also some lignin) released by maize straw during incubation become part of the humic acid fraction of the Italian soil. Other works (Baldock et al., 1992; Golchin et al., 1994) have already pointed out that an increase in the NMR regions attributable to methoxyl groups of lignin-like material and anomeric carbon of polysaccharides indicates an accumulation in humic materials of recently deposited organic matter from plants. In the Italian soil, mixing maize with soil seemed to be more effective than simple mulching in stabilizing the released carbohydrates and peptides in the HA fraction. Table 5 and Figure 4 (for the Danish soil) showed that NMR spectra of FA1 extracted from soils after incubation (t 1 ) were signi cantly different from those of the fulvic extracts before incubation (t 0 ). The main difference was that incubation enhanced resonances in the 40±110 p.p.m. region and especially the signal at around 70 p.p.m. of C±O groups in carbohydrate structures and that at about 100 p.p.m. attributed to the anomeric carbon in the same carbohydrates. These ndings suggest that the fulvic acid fraction was modi ed substantially by incubation either with or without maize and

8 590 R. Spaccini et al. Figure 3 CPMAS- 13 C-NMR spectra of humic substances extracted by alkaline solution (HA1) from the Italian soil before (t 0 ) and after incubation (t 1 ) with (mulched and mixed treatments) and without maize residues. Figure 4 CPMAS- 13 C-NMR spectra of humic substances extracted by alkaline solution (FA1) from the Danish soil before (t 0 ) and after incubation (t 1 ) with (mulched and mixed treatments) and without maize residues. contributes, in particular, in retaining carbohydrates in the FA structure. Incorporation of carbohydrates in FAs presumably reduced mineralization of such rapidly degradable compounds. This result accords with recent studies by Wander & Traina (1996) and Zalba & Quiroga (1999) which suggested that fulvic acids are related to the short-term changes of soil management. 13 C content in humic fractions. The 13 C (½) values found for the bulk soil and for the various humic extracts before (t 0 ) and after (t 1 ) soil incubation with maize residues are reported in Table 6. The isotopic abundance in the bulk soil before incubation (t 0 ) con rmed that SOM in these European soils originated mainly from C3 plant species (Balesdent & Mariotti, 1996). A slightly less negative 13 C value (±24.20) for the bulk Italian soil suggested a past history of C4 plants grown in this soil. In all three soils, 13 C values showed differences between HA and FA fractions extracted from control soils before (t 0 ) and after (t 1 ) incubation (Table 6). Humic acids extracted either before (HA1) or after (HA2) soil treatment with acetone solution were more depleted in 13 C (larger negative values) than the respective fulvic acid fractions (FA1 and FA2). This may be explained by the different chemical composition of HA and FA which controls the selective incorporation in soil of the various organic compounds released from decomposing vegetal tissues. Apolar components from plants such as lipids and lignin materials, which are naturally depleted in 13 C content (Balesdent & Mariotti, 1996), preferentially accumulate in the more hydrophobic humic acids, thereby diminishing their 13 C isotopic composition. By contrast, the naturally 13 C- enriched polar compounds such as carbohydrates and amino acids are incorporated for mutual af nity in the more hydrophilic fulvic acids, which progressively decrease in 13 C values (larger 13 C content). For all soils, the bulk samples incubated with maize straw (t 1 ) contained more 13 C than control samples before incubation (t 0 ), thereby showing incorporation of the 13 C-enriched maize residues. In contrast to the labile pool (Table 2), 13 C from maize straw was stabilized into the humic fractions, although with differences between the mulching and mixing techniques. The isotopic dilution (Table 6) indicated that only samples mixed with maize retained the new 13 C-labelled carbon in all

9 Transformation of maize residues into soil 591 Table 6 Isotopic abundance ( 13 C, ½) and maize-derived organic carbon (OC, %) in bulk soil and humic fractions of control and maize-treated samples before (t 0 ) and after (t 1 ) incubation (standard error was less than 0.06½ for all values; n = 3) Control (t 0 ) Control (t 1 ) Mulched (t 1 ) Mixed (t 1 ) Sample 13 C /½ 13 C /½ 13 C /½ OC maize /% 13 C /½ OC maize /% Danish soil Bulk ±26.5 ±26.6 ±24.6 ±24.5 HA 1 ±27.6 ±27.8 ±27.3 NS ± FA 1 ±27.2 ±26.6 ±26.5 NS ± HE ±29.4 ±29.4 ± ± HA 2 ±27.7 ±27.5 ± ± FA 2 ±27.0 ±26.2 ± ± German soil Bulk ±26.0 ±26.1 ±23.8 ±23.1 HA 1 ±26.9 ±27.5 ±27.3 NS ± FA 1 ±26.6 ±26.1 ±26.1 NS ± HE ±28.4 ±28.4 ±28.2 NS ± HA 2 ±27.4 ±27.3 ±27.2 NS ± FA 2 ±26.8 ±25.5 ± ± Italian soil Bulk ±24.2 ±24.2 ±22.0 ±21.7 HA 1 ±25.3 ±25.3 ±25.2 NS ± FA 1 ±24.4 ±23.6 ±23.8 NS ± HE ±26.8 ±26.1 ±25.9 NS ± HA 2 ±25.2 ±25.6 ±25.1 NS ±25.4 NS FA 2 ±24.4 ±24.0 ± ± NS, not signi cant. humic fractions. Carbon from maize incorporated in mixed samples amounted to 9.7, 12.3 and 13.5% in HA1, 9.5, 11.3 and 11.8% in FA1, and 13.9, 13.2 and 12.7% in HE, for the Danish, German and Italian soils, respectively. Conversely, in the mulched samples only the HE from Danish soil showed a small increase of 13 C content, whereas no incorporation of carbon from maize straw was evident either in HA1 or in FA1 of all soils (Table 6). It was only after removal of the hydrophobic HE fraction from the mulched samples that incorporation of carbon from maize became evident in the FA2 fraction for all soils and in the HA2 fraction for the Danish soil. The overall small incorporation of carbon from maize in humic substances from the mulching treatment indicates that maize straw placed on to the surface of soil samples underwent a more rapid and complete mineralization than in the samples thoroughly mixed with maize. Stemmer et al. (1999) studied the turnover of SOM in the same soil samples and also found that placing maize straw residues on the soil surface, as in the mulched samples, caused a rapid mineralization to be favoured in the early weeks of incubation, thereby preventing interaction of the compounds slowly released from maize with the inorganic and organic soil fractions. In mixed samples, incorporation of carbon from maize determined a less negative 13 C value in FA than in HA fractions (Table 6). This may again be interpreted with a preferential incorporation of 13 C-enriched hydrophilic material (carbohydrates and peptides) in hydrophilic fulvic acids as compared with that of 13 C-depleted lipidic compounds in more hydrophobic humic acids. Effect of selective extraction of humic fractions. The humic fraction isolated with the acetone solution (HE) had a smaller 13 C value than the alkaline extracts, HA1 and FA1, in both control samples (t 0 and t 1 ) of all three soils (Table 6). It is interesting to note that humic materials obtained by alkaline extraction after treatment with acetone (HA2 and FA2) showed isotopic dilutions reaching again the values of the original alkaline extracts (HA1 and FA1). These 13 C measurements of HE accord with previous results which indicated that an aqueous acidic solution of a dipolar aprotic solvent (acetone) can selectively extract a speci c humic fraction that is mainly apolar (Piccolo et al., 1998). Hence, the small 13 C content of HE (Table 6) and its predominant hydrophobic character (see NMR spectra of Figure 2) suggest that the 13 C-depleted alkyl compounds released by vegetal tissues are selectively incorporated in this highly hydrophobic HE fraction. The 13 C values of humic fractions (HA2 and FA2) isolated by alkaline extraction after the removal of the hydrophobic fraction with the aqueous acetone solution (Table 6) provide

10 592 R. Spaccini et al. information on the relative interaction between hydrophilic and hydrophobic components in the stable humic pool. The removal from the Italian mixed sample of the HE fraction (C from maize = 12.7%) resulted in the disappearance of any carbon from maize in the HA2 fraction and its increase in FA2 (17.5% vs. 11.8% in FA1). This suggests that almost exclusively hydrophobic alkyl-c compounds from maize were retained in HA1 of this soil and were selectively removed by the aqueous acetone solution. The hydrophilic C from maize remaining in the soil was measured only in the FA2 fraction. In the Danish and German soils, the incorporation of carbon from maize in the native humic components (Table 6) occurred with less chemical selectivity than for the Italian soil. In fact, a signi cantly larger proportion of 13 C from maize in HE fractions (13.9 and 13.2%, respectively, as compared with 12.7 in the Italian soil) did not prevent a large presence of carbon from maize in HA2 (12 and 10.7%, respectively), which suggests that organic compounds of both hydrophilic and hydrophobic characteristics became incorporated in the HA fraction of the northern soils. A large content of 13 C from maize was also found in the FA2 extract of both the Danish and German soils, indicating that 13 C released from maize had accumulated in the most hydrophilic FA fraction and could be solubilized with fulvic acids only after removal of the HE fraction. A general interpretation of these results may be related to the process of accumulation of organic compounds in soils that is controlled by their chemical af nity with the existing organic matter. Polar compounds are preferentially adsorbed by hydrophilic organic components, whereas less polar or apolar compounds are retained by hydrophobic organic surfaces. The randomness of the process and the heterogeneity of the organic molecules lead to the accumulation of organic matter in which hydrophilic associations may be contiguous with hydrophobic domains or contained in one another. Recent results (Hayes, 1997; Conte & Piccolo, 1999; Piccolo et al., 1999a) have suggested that humic substances are associations of small heterogeneous molecules held together in conformations stabilized by weak forces which are mainly dispersive±hydrophobic interactions. Moreover, Piccolo et al. (1999b) have shown that hydrophobic domains exert an enhanced protection towards incorporated polar compounds by preventing the microbial activity associated with water. It is conceivable therefore that both hydrophobic and hydrophilic components, while released by rapidly degrading organic residues, are adsorbed preferentially on soil organic materials by chemical af nity. Adsorption on soil particles of hydrophobic compounds, especially of long-chain aliphatic molecules, may be progressively able to incorporate and co-adsorb also hydrophilic compounds. This labile polar material remains con ned away from the aqueous medium and biological degradation by a process of hydrophobic protection. Removal of the hydrophobic components, as we did by acetone extraction (HE), can then either uncover underlying polar materials which then become more soluble in aqueous media or rearrange the humic conformations, thereby enhancing the solubility of the hydrophilic compounds incorporated in the humic structure. Conclusions Our results with the three European soils showed that the labile and stable pools of soil organic matter (SOM) behaved differently as sinks of decomposing fresh organic matter such as maize straw. Quantitative analyses of carbohydrates extracted by acid hydrolysis from particle size fractions of the soil and relative 13 C measurements in hydrolysates indicated that, rather than xing carbohydrate material from maize, incubation either with or without maize enhanced decomposition of this native labile component of the organic matter. Conversely, our data showed that the stable pool of SOM represented by different humic fractions could incorporate the organic C derived from decomposition of maize straw, especially when this was thoroughly mixed with soil. Incorporation of organic matter from maize into humic substances was generally con rmed by CPMAS- 13 C-NMR spectra of humic fractions extracted by alkaline solution, as KoÈgel-Knabner & Hatcher (1989), Zech et al. (1992) and Guggenberger et al. (1995) had found, which suggested progressive stabilization in humic substances of aliphatic compounds, polysaccharides and peptides of plant and microbial origin. Values of C isotopic abundance together with structural information of humic extracts obtained by NMR spectra showed that both hydrophilic and hydrophobic components from maize straw were incorporated into the humic pool of the soil. The increased carbon from maize found in humic and fulvic fractions isolated after removal from soils of a highly aliphatic organic fraction indicated that the mutual interactions of different classes of compounds in uence solubility and chemical reactivity. In particular, hydrophobic materials appear to favour the persistence of hydrophilic components incorporated in soil organic matter by precluding their contact with water and, thus, with microorganisms. This interpretation accords with other ndings that support the hypothesis that incorporation of products of degrading organic matter in the native humic material represents a basic mechanism of soil organic matter accumulation as well as the reason for its longterm stabilization (Baldock et al., 1989; Piccolo, 1996; Lichtfouse et al., 1998; Piccolo et al., 1999b). Our study also points out that humic fractions removed by mild extraction with acetone have structural features similar to the recalcitrant alkyl fraction isolated as humin in other studies (Baldock et al., 1992; Augris et al., 1998). The hydrophobic material extracted by acetone solution may then represent a rather stable fraction of organic matter in soils because of the

11 Transformation of maize residues into soil 593 entropy-driven separation of this fraction from water and microbial activity (Conte & Piccolo, 1999). Our 13 C values indicated that while the hydrophobic fraction (HE) behaved as a sink for carbon released by maize straw during incubation, a larger incorporation of carbon from maize was generally shown by the FA fractions, especially after removal of the protecting hydrophobic layer of humus by acetone. This con rms previous reports (Wander & Traina, 1996; Zalba & Quiroga, 1999) which indicated fulvic acids to be the humus fraction most sensitive to soil management. This study produced additional evidence of the important role played by native humic substances in accumulating new organic matter added to soil by chemically protecting the most labile biomolecules released during decomposition of plant tissues from immediate microbial mineralization. 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