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 13C distribution and CPMAS-NMR spectra
R. S PACCINIa , A . PICCOLOa , G . H ABERHAUERb & M . H . G ERZABEKb
a
Dipartimento di Scienze Chimico Agrarie, UniversitaÁ degli Studi di Napoli `Federico II', Via UniversitaÁ 100, 80055 Portici, Italy, and
Austrian Research Centre Seibersdorf, Department of Environmental Research, 2444 Seibersdorf, Austria
b
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 13Cdilution analysis and CPMAS-13C-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 13C values showed no retention of carbohydrates from maize
but rather decomposition of those from native organic matter in the soil. By contrast, CPMAS-13C-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 13C 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. E-mail: [email protected]
Received 1 November 1999; revised version accepted 12 May 2000
#
2000 Blackwell Science Ltd
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
583
584 R. Spaccini et al.
Table 1 Some physical and chemical properties of the soils
Soil
Denmark
German
Italian
Sand /%
Silt /%
56.0
17.0
42.5
31.7
61.0
29.8
Clay /% Organic C /%
12.3
22.0
27.7
1.4
1.3
1.2
pH(H2O)
6.5
5.9
7.2
contribute to the accumulation of organic C in soils (Buurman,
1994; Piccolo et al., 1999b).
In recent years, naturally 13C-enriched plants, such as those
employing a C4 photosynthetic pathway (Zea mays L.), or
synthetically 13C-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).
#
2000 Blackwell Science Ltd, European Journal of Soil Science, 51, 583±594
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 12C/13C
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 2cm 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 (t0) and after
(t1) 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
Transformation of maize residues into soil 585
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%.
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.
NMR spectroscopy
Carbohydrate content
The 13C-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 100.625 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:
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 H2SO4 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 13C
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 Na4P2O7 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 12C/13C 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 12C/13C analysis.
HB/HI = [(0 ± 40) + (110 ± 145)]/[(40 ± 110) + (145 + 190)].
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 (13C/12C ratio)
The isotopic abundance (13C) 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 13C/12C ratios are expressed in the relative scale
(½) according to the following equation:
13C½ = (Rsample/Rstandard ± 1) 3 103,
where R = 13C/12C 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:
(%-Cmaize) = ( Cc ± C0)/( Cm ± C0) 3 100,
where Cc is the isotopic value of the sample, C0 is the
isotopic value of the control sample, and Cm is the
isotopic composition of the maize residue (±12.5½),
respectively.
#
2000 Blackwell Science Ltd, European Journal of Soil Science, 51, 583±594
586 R. Spaccini et al.
Table 2 Amount and distribution of carbohydrates in particle size fractions before (t0) and after (t1) soil incubation with maize residues
(standard error in parentheses; n = 3)
Soil
Danish
German
Sample
Particle sizes
/g g±1
Control (t0)
Coarse sand
Fine sand
Silt
Clay
763
1076
2195
2469
(28.3)
(21.4)
(43.3)
(18.5)
11.7
16.5
33.8
38.0
1723
1396
1699
2177
(43.9)
(37.5)
(56.6)
(58.9)
24.6
20.0
24.3
31.1
1423
1547
2028
4127
(27.7)
(47.9)
(31.2)
(45.0)
15.6
17.0
22.2
45.2
Control (t1)
Coarse sand
Fine sand
Silt
Clay
337
489
564
614
(31.8)
(40.4)
(13.3)
(24.2)
16.8
24.4
28.2
30.2
667
590
655
851
(48.5)
(41.6)
(43.9)
(91.2)
24.1
21.4
23.7
30.8
756
740
890
615
(112)
(19.1)
(81.4)
(20.2)
25.2
24.6
29.7
20.5
Mulched (t1)
Coarse sand
Fine sand
Silt
Clay
254
264
421
1034
(30.6)
(16.2)
(30.6)
(4)
12.9
13.4
21.3
52.4
936
862
772
1001
(145)
(41.6)
(38)
(63)
26.2
24.1
21.6
28.1
600
600
677
566
(35.8)
(39.3)
(62.4)
(46.8)
24.6
24.6
27.7
23.1
Mixed (t1)
Coarse sand
Fine sand
Silt
Clay
419
456
774
1366
(30.6)
(24.2)
(79)
(30)
13.9
15.1
25.7
45.3
1127
1185
1400
1154
(47.3)
(24.4)
(39.3)
(16.2)
23.1
24.4
28.8
23.7
659
559
525
529
(104.5)
(28.3)
(13.3)
(7.5)
29.0
24.6
23.1
23.3
/%
Results and discussion
Carbohydrates
Total content. Table 2 shows the content of total carbohydrates
in particle size fractions of different soils before (t0) and after
(t1) 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 (t0). More carbohydrate was found after
incubation (t1) for both maize-treated samples of the German
soil and for the maize-mixed sample of the Danish soil than in
the control samples (t1). 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 (t0) indicated that the
organic matter in these soils originated from C3 plant species.
#
2000 Blackwell Science Ltd, European Journal of Soil Science, 51, 583±594
/g g±1
Italian
/%
/g g±1
/%
The 13C 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 (13C) of soil hydrolysates from
maize-treated samples for all soils showed (Table 3) that no
carbon from maize residues (13C = ±12.5½) was transferred
into the carbohydrates still present in soils after incubation (t1).
Soil incubation with or without maize residues resulted in a
small decrease of 13C content in soil hydrolysates (larger
negative 13C values in Table 3) from all size fractions when
compared with the initial values (t0) of control samples. This
may be explained by a preferential microbial decomposition of
isotopically heavier carbohydrate fractions, thus shifting the
overall 13C 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 t1 showed an overall
decrease of carbohydrate content ranging from 55 to 85% of
the initial amount at t0. 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
Transformation of maize residues into soil 587
Table 3 Isotopic abundance (13C, ½) in soil hydrolysates from size
fractions of control and maize-treated soils before (t0) and after (t1)
incubation (standard error was less than 0.06½ for all values; n = 3)
Table 4 Extraction yields (% by weight of soil sample) of humic
fractions isolated from different treatments before (t0) and after (t1)
soil incubation
Sample
Coarse sand
Fine sand
Silt
Clay
Sample
Danish soil
Control (t0)
Control (t1)
Mulched (t1)
Mixed (t1)
±27.1
±28.9
±29.0
±29.1
±27.0
±28.9
±28.9
±28.9
±26.8
±28.8
±28.8
±28.7
±26.6
±28.8
±28.8
±28.6
German soil
Control (t0)
Control (t1)
Mulched (t1)
Mixed (t1)
Danish soil
HA1a
FA1a
HEb
HA2c
FA2c
0.45
0.14
0.26
0.45
0.15
0.60
0.28
0.30
0.54
0.30
0.51
0.20
0.43
0.60
0.22
0.81
0.28
0.31
0.75
0.33
±27.1
±28.8
±28.9
±28.9
±27.1
±28.7
±28.9
±28.9
±26.8
±28.7
±28.8
±28.9
±27.1
±28.5
±28.7
±28.6
Italian soil
Control (t0)
Control (t1)
Mulched (t1)
Mixed (t1)
±26.9
±28.4
±28.7
±28.7
±26.8
±28.7
±28.7
±28.8
±26.8
±28.5
±28.8
±28.8
±26.9
±28.6
±28.6
±28.5
German soil
HA1
FA1
HE
HA2
FA2
0.34
0.15
0.25
0.30
0.15
0.48
0.20
0.30
0.50
0.20
0.45
0.13
0.36
0.40
0.18
0.47
0.19
0.28
0.50
0.22
Italian soil
HA1
FA1
HE
HA2
FA2
0.22
0.14
0.18
0.27
0.15
0.39
0.19
0.21
0.35
0.20
0.42
0.16
0.28
0.37
0.15
0.55
0.19
0.21
0.63
0.21
incubation are shown in Table 4. Incubation (t1) signi®cantly
increased the extraction yields of all humic fractions with
respect to those obtained from control samples before
incubation (t0). 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.
Control (t0)
Control (t1) Mulched (t1) Mixed (t1)
a
Humic (HA1) and fulvic (FA1) acids extracted by alkaline solution.
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.
b
shown in Figures 1 and 2, respectively. The NMR spectra of
HA1 (Italian soil) and FA1 (Danish soil), representative of
humic fractions after incubation (t1) 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 t0 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
NMR spectra. Solid-state 13C-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 (t0) are
#
2000 Blackwell Science Ltd, European Journal of Soil Science, 51, 583±594
588 R. Spaccini et al.
160±190
HB/HIa
4.6
2.1
3.2
3.1
14.9
19.4
19.1
17.1
0.8
0.8
0.9
0.8
2.7
0.7
1.2
2.7
4.7
1.9
2.8
2.7
15.2
16.0
15.7
15.1
0.7
0.8
0.7
0.8
35.2
33.7
28.6
39.2
10.0
10.3
6.1
3.5
0
0
0
2.2
27.8
31.9
36.7
23.2
0.6
0.5
0.5
0.6
33.5
30.2
32.1
31.8
47.6
50.0
47.4
55.1
0
0
0
0
0
0
0
0
18.9
19.8
20.5
13.1
0.5
0.4
0.5
0.5
German soil
Control (t0)
Control (t1)
Mulched (t1)
Mixed (t1)
33.9
30.1
34.3
26.2
47.8
54.2
48.5
60.3
1.6
0
0
0
0
0
0
0
18.7
15.7
17.2
13.5
0.5
0.4
0.5
0.3
Italian soil
Control (t0)
Control (t1)
Mulched (t1)
Mixed (t1)
36.0
31.3
35.7
30.0
39.4
47.0
44.6
51.0
2.9
0
0
0
0
0
0
0
24.7
21.7
19.7
19.0
0.6
0.4
0.5
0.4
Sample
0±40
40±110
110±145
HA1
Danish soil
Control (t0)
Control (t1)
Mulched (t1)
Mixed (t1)
41.9
45.8
44.6
42.7
35.6
32.6
29.9
36.5
2.6
0
3.2
0.6
German soil
Control (t0)
Control (t1)
Mulched (t1)
Mixed (t1)
39.7
43.6
38.8
41.1
37.7
37.8
41.6
38.4
Italian soil
Control (t0)
Control (t1)
Mulched (t1)
Mixed (t1)
27.0
24.1
28.6
33.3
FA1
Danish soil
Control (t0)
Control (t1)
Mulched (t1)
Mixed (t1)
145±160
Table 5 Relative carbon distribution (%) in
different regions of chemical shift (p.p.m.)
in CPMAS-13C-NMR spectra of humic and
fulvic acids before (t0) and after (t1)
incubation with maize residues
a
HB/HI = [(0 ± 40) + (110 ± 145)]/[(40 ± 110) + (145 + 190)].
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 t0 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
#
2000 Blackwell Science Ltd, European Journal of Soil Science, 51, 583±594
(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 t0
(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 CH2 and CH3 groups in
polymethylene chains (Piccolo et al., 1998). For the German
Transformation of maize residues into soil 589
Figure 1 CPMAS-13C-NMR spectra of humic substances extracted
by alkaline solution (HA1) from control soils before incubation (t0).
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 (t1)
with maize straw were not signi®cantly altered compared with
their spectra at time t0.
The NMR spectra of HA1 from the Danish and German
soils after incubation (t1) 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-13C-NMR spectra of humic substances extracted
by acetone±HCl solution (HE) from control soils before incubation
(t0).
with the mulched and control (t1) 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 (t1) were
signi®cantly different from those of the fulvic extracts before
incubation (t0). 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
#
2000 Blackwell Science Ltd, European Journal of Soil Science, 51, 583±594
590 R. Spaccini et al.
Figure 3 CPMAS-13C-NMR spectra of humic substances extracted
by alkaline solution (HA1) from the Italian soil before (t0) and after
incubation (t1) 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 13C (½) values found for
the bulk soil and for the various humic extracts before (t0) and
after (t1) soil incubation with maize residues are reported in
Table 6. The isotopic abundance in the bulk soil before
incubation (t0) con®rmed that SOM in these European soils
originated mainly from C3 plant species (Balesdent &
Mariotti, 1996). A slightly less negative 13C value (±24.20)
for the bulk Italian soil suggested a past history of C4 plants
grown in this soil.
In all three soils, 13C values showed differences between
HA and FA fractions extracted from control soils before (t0)
and after (t1) incubation (Table 6). Humic acids extracted
either before (HA1) or after (HA2) soil treatment with acetone
solution were more depleted in 13C (larger negative values)
#
2000 Blackwell Science Ltd, European Journal of Soil Science, 51, 583±594
Figure 4 CPMAS-13C-NMR spectra of humic substances extracted
by alkaline solution (FA1) from the Danish soil before (t0) and after
incubation (t1) with (mulched and mixed treatments) and without
maize residues.
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 13C
content (Balesdent & Mariotti, 1996), preferentially accumulate in the more hydrophobic humic acids, thereby diminishing
their 13C isotopic composition. By contrast, the naturally 13Cenriched polar compounds such as carbohydrates and amino
acids are incorporated for mutual af®nity in the more
hydrophilic fulvic acids, which progressively decrease in
13C values (larger 13C content).
For all soils, the bulk samples incubated with maize straw
(t1) contained more 13C than control samples before incubation
(t0), thereby showing incorporation of the 13C-enriched maize
residues. In contrast to the labile pool (Table 2), 13C 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 13C-labelled carbon in all
Transformation of maize residues into soil 591
Table 6 Isotopic abundance (13C, ½) and maize-derived organic carbon (OC, %) in bulk soil and humic fractions of control and maize-treated
samples before (t0) and after (t1) incubation (standard error was less than 0.06½ for all values; n = 3)
Control (t0)
13C /½
Control (t1)
13C /½
13C /½
Danish soil
Bulk
HA1
FA1
HE
HA2
FA2
±26.5
±27.6
±27.2
±29.4
±27.7
±27.0
±26.6
±27.8
±26.6
±29.4
±27.5
±26.2
±24.6
±27.3
±26.5
±28.1
±26.0
±25.2
NS
NS
7.5
10.0
7.2
±24.5
±26.2
±25.2
±27.0
±25.7
±23.5
9.7
9.5
13.9
12.0
19.3
German soil
Bulk
HA1
FA1
HE
HA2
FA2
±26.0
±26.9
±26.6
±28.4
±27.4
±26.8
±26.1
±27.5
±26.1
±28.4
±27.3
±25.5
±23.8
±27.3
±26.1
±28.2
±27.2
±24.5
NS
NS
NS
NS
7.4
±23.1
±25.6
±24.5
±26.3
±25.6
±24.0
12.3
11.3
13.2
10.7
11.4
Italian soil
Bulk
HA1
FA1
HE
HA2
FA2
±24.2
±25.3
±24.4
±26.8
±25.2
±24.4
±24.2
±25.3
±23.6
±26.1
±25.6
±24.0
±22.0
±25.2
±23.8
±25.9
±25.1
±22.5
NS
NS
NS
NS
12.7
±21.7
±23.5
±22.2
±24.3
±25.4
±21.9
13.5
11.8
12.7
NS
17.5
Sample
Mulched (t1)
OCmaize /%
13C /½
Mixed (t1)
OCmaize /%
NS, not signi®cant.
fractions (Table 6). This may again be interpreted with a
preferential incorporation of 13C-enriched hydrophilic material
(carbohydrates and peptides) in hydrophilic fulvic acids as
compared with that of 13C-depleted lipidic compounds in more
hydrophobic humic acids.
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 13C 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 13C value in FA than in HA
Effect of selective extraction of humic fractions. The humic
fraction isolated with the acetone solution (HE) had a smaller
13C value than the alkaline extracts, HA1 and FA1, in both
control samples (t0 and t1) 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 13C 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 13C content of
HE (Table 6) and its predominant hydrophobic character (see
NMR spectra of Figure 2) suggest that the 13C-depleted alkyl
compounds released by vegetal tissues are selectively
incorporated in this highly hydrophobic HE fraction.
The 13C 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
#
2000 Blackwell Science Ltd, European Journal of Soil Science, 51, 583±594
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 13C 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 13C from
maize was also found in the FA2 extract of both the Danish
and German soils, indicating that 13C 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
#
2000 Blackwell Science Ltd, European Journal of Soil Science, 51, 583±594
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 13C 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-13C-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
Transformation of maize residues into soil 593
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fraction (HE) behaved as a sink for carbon released by maize
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maize was generally shown by the FA fractions, especially
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Acknowledgement
This work was supported by the European Union project
EV5V-CT94-0434, `Decomposition of organic matter in
terrestrial ecosystems: Microbial communities in litter and
soil'.
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