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Geoderma 179–180 (2012) 63–72
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Geoderma
journal homepage: www.elsevier.com/locate/geoderma
What is the composition of AIR? Pyrolysis-GC–MS characterization of
acid-insoluble residue from fresh litter and organic horizons under boreal
forests in southern Finland
Sari Hilli a, b,⁎, Sari Stark a, Stefan Willför c, Annika Smeds c, Markku Reunanen c, Reijo Hautajärvi b
a
b
c
Finnish Forest Research Institute, Rovaniemi Research Unit, P.O. Box 16, FI-96300, Rovaniemi, Finland
Finnish Forest Research Institute, Rovaniemi Research Unit, Salla Ofﬁce, FI-98900, Salla, Finland
Åbo Akademi University, Process Chemistry Centre, Laboratory of Wood and Paper, Chemistry, Porthansgatan 3, FI-20500 Turku, Finland
a r t i c l e
i n f o
Article history:
Received 24 October 2010
Received in revised form 1 February 2012
Accepted 2 February 2012
Available online 19 March 2012
Keywords:
AIR
Lignin
Pyrolysis
Fractionation method
a b s t r a c t
High concentrations of the acid-insoluble residue (AIR, also known as “Klason lignin”) in decomposing litter
are considered to indicate high resistance to decomposition; however, the chemical composition of AIR in different types of litter and soil organic matter is poorly understood. In the present study, we characterized samples of common litter (L) types in boreal forests (needle, coarse tree, and moss), as well as fragmented litter
(F), and humus (H) layers in two south boreal forest sites using a combination of sequential fractionation and
pyrolysis-GC–MS. The results showed that the unfractionated samples were composed of cellulose-derived
carbohydrates, guaiacyl-type lignin and other polyphenolic compounds, and that there was little variation
among samples. However, pyrolysis-GC–MS analyses of AIR demonstrated that the composition of the AIR
fraction differed among the analyzed litter materials as well as between the layers in the soil organic horizon.
In the F and H layers, the AIR fraction contained guaiacyl-type lignin and other polyphenolics, as well as lipophilic compounds, which were indicated by the pyrolysis product methyldehydroabietate and short-chain
fatty acids. In the AIR fraction, only small amounts of carbohydrate-derived compounds were detected, conﬁrming that the sequential fractionation method efﬁciently removes soluble polysaccharides. The AIR fraction was poorly soluble in all solvents. The results presented here conﬁrm that the sequential fractionation
method efﬁciently separates water-, chloroform-, and acid-soluble (72% H2SO4) compounds from acidinsoluble compounds (AIR). However, AIR was shown to be a mixture of polyphenolic (mainly ligninderived) and lipophilic (including fatty acids and resin acid) structures, and may therefore be a poor indicator
of lignin and phenolic compounds when investigating the F and H layers in the organic horizon.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
The concept of acid-insoluble residue (AIR), which is also known
as Klason lignin in wood chemistry, plays an important role in ecological studies conducted to investigate the changes in chemical composition during the decomposition of plant residues and the factors that
regulate the plant decomposition rate. Decomposition studies have
suggested that the relative proportion of AIR increases during the litter decomposition and along the vertical gradient in the organic horizon (e.g. Baldock et al., 1992; Berg, 2000; Hilli et al., 2008, 2010;
Melillo et al., 1989; Preston et al., 2009). Decomposition rates in the
soil organic matter (SOM) appear to be negatively correlated with
increasing concentrations of AIR (Shaver et al., 2006). Therefore,
high AIR concentrations in SOM indicate an advantaged stage of
⁎ Corresponding author at: Finnish Forest Research Institute, Rovaniemi Research
Unit, P.O. Box 16, FI-96300, Rovaniemi. Tel.: + 358 50 801 5284; fax: + 358 10211
4401.
E-mail address: sari.hilli@metla.ﬁ (S. Hilli).
0016-7061/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.geoderma.2012.02.010
decomposition in which more labile fractions have already degraded,
together with a high resistance of SOM against further degradation.
The concentration of AIR in SOM is largely considered to reﬂect
the concentration of lignin (e.g. Berg, 2000; Shaver et al., 2006;
Weintraub and Schimel, 2003). This, in combination with the accumulation of AIR during SOM decomposition (Baldock et al., 1992;
Berg, 2000; Melillo et al., 1989), has led to the conjecture that lignin
is the plant residue most resistant to microbial decomposition
(Berg, 2000; Weintraub and Schimel, 2003). The importance of lignin
has been incorporated into general models of decomposition (e.g.
Moorhead and Sinsabaugh, 2006), and the concentration of AIR has
been used to assess the role of the chemical composition of SOM in
the temperature-dependence of decomposition (Fierer et al., 2005).
However, other studies have suggested that lignin does not accumulate in decomposing litter (Preston et al., 2009; Sjöberg et al., 2004)
or contribute to the stable SOM fraction to a considerable extent
(Kögel-Knabner, 2000; Lützov et al., 2006). Rather, plant compounds
such as cutins, glycolipids, resinous polyterpenoids, suberins and condensed tannins have been found to accumulate in soils (de Leeuw and
64
S. Hilli et al. / Geoderma 179–180 (2012) 63–72
Table 1
The relative abundance of pyrolysis products detected in the unfractionated needle litter, tree litter, moss litter, F layer and H layer samples at a sub-xeric and a mesic south
boreal site.
Compound
Sub-xeric L
Needle
Toluene
Ethylbenzene
Pyridine
2-Methylfuran
Styrene
1-Hydroxy-2-propanone
2-Cyclopenten-1-one
3-Furaldehyde
Acetic acid
Furfural (2-furaldehyde)
Pyrrole
3-Methyl-2-cyclopenten-1-one
Benzaldehyde
5-Methylfurfural + methylpyrrole
4-Cyclopentene-1,3-dione
2-Furanmethanol (2-hydroxymethylfuran)
2-(5H)-Furanone
1,2-Cyclopentanedione
2-Cyclopenten-1-one
2-Hydroxy-3-methylhexanoic acid
2-(2-butoxyethoxy)ethyl acetate
Guaiacol
2-(2-hydroxypropoxy)-1-Propanol +
4-Methyl-5H-furan-2-one
Neophytadiene
Methylguaiacol1
Methylguaiacol2 + heptanoic acid
Maltol
Phenol
Octanoic acid
Ethylguaiacol
Cresol1
Cresol2
4-Propylguaiacol
Eugenol
Nonanoic acid
Ethylphenol
Vinylguaiacol
2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one
Isoeugenol
Decanoic acid
Diethyl phthalate
4-Vinylphenol
5-(hydroxymethyl)-2-Furaldehyde
3-Pyridinol
Vanillin
Chavicol (4-allylphenol)
Anhydrosugar1
Homovanillin
Vanillic acid methyl ester
4-Hydroxydihydro-2(3H)-furanone
Guaiacylethanone (acetoguaiacone)
Guaiacylacetone
Dibutyl phthalate1
Dibutyl phthalate2 + tetradecanoic acid
Coniferyl alcohol1
Catechol
Hexadecanoic acid
Dihydroconiferyl alcohol
4-Hydroxy-acetophenone
Methyl dehydroabietate
Abietatetraenoic acid methyl ester1
Abietatetraenoic acid methyl ester2
Coniferylaldehyde
Coniferyl alcohol2
Anhydrosugar2
Vanillic acid
Dehydroabietic acid
Docosanoic acid
0.7
0.1
0.2
0.1
0.1
0.9
0.3
0.5
1.8
1
0.2
Mesic L
Tree
Moss
0.7
0.1
0.2
0.2
0.2
1.2
0.2
0.6
2.4
1.8
0.2
1.8
0.3
0.4
0.4
0.2
3
0.7
1.4
7.8
3.6
Needle
Sub-xeric
Mesic
Sub-xeric
Mesic
F
F
H
H
Tree
Moss
1.6
0.3
0.4
0.3
0.4
1.8
0.6
0.9
5.5
2.1
0.3
1.1
0.2
0.3
0.2
0.2
1.3
0.4
0.7
3.4
1.8
0.4
2.7
0.3
0.5
0.4
0.4
3.1
0.8
1.5
11.3
2.8
1.1
1.1
0.2
0.4
0.2
0.2
1.2
0.3
0.8
2.8
2.7
0.2
1.9
0.3
0.7
0.5
0.2
1.2
0.6
0.8
4.2
2.2
0.6
1.9
0.3
0.8
0.2
0.2
0.6
0.6
1.1
2
2.7
0.5
1.5
0.3
0.8
0.2
0.3
0.6
0.5
0.8
2.5
2.5
0.5
0.2
0.9
0.5
0.4
0.5
1.5
1
3.3
0.6
0.2
0.4
0.3
0.6
0.4
0.8
0.3
0.3
0.5
0.7
0.6
0.9
0.2
0.9
0.3
0.2
0.1
0.4
0.3
0.9
2.3
2.6
2
0.5
1.8
2.9
2.3
2.5
2.1
0.3
1.3
0.4
2.4
0.3
2.3
0.4
1.9
0.9
1.5
0.4
0.3
0.9
0.4
1.7
0.5
0.1
0.6
0.3
1.3
0.5
0.3
1.4
0.4
0.7
0.5
0.1
3.6
0.8
1.7
0.3
2.8
0.5
1.3
0.1
2.7
0.5
1.3
0.1
2.3
0.6
1
2.6
1.5
2
0.3
2.7
0.5
2.1
4
1.5
0.6
1.9
0.4
3.4
1.9
1.2
0.3
2.6
1.6
2
1.2
0.6
1.7
0.3
0.7
0.3
1.8
0.4
0.7
1.2
0.3
0.4
0.3
0.1
0.2
0.7
0.4
0.3
0.2
0.3
0.6
0.6
1.3
0.4
0.4
0.9
1.4
1.8
0.5
0.3
0.4
0.5
1.1
0.9
0.4
0.2
0.3
0.4
0.7
1.3
2.5
2.3
0.5
3.9
0.8
0.5
1.4
2.4
0.3
2.4
2.8
0.5
1.5
3
2.5
3
0.4
0.8
0.7
0.2
0.8
0.3
1.3
0.3
0.5
0.3
0.2
0.5
1.1
0.8
0.6
0.4
1.1
1.9
2.2
1
1
0.3
3.4
0.7
2.4
0.6
0.9
0.5
3.1
0.6
1.7
0.2
3.5
0.3
1.1
1.5
0.6
0.5
2.4
0.8
0.5
0.6
0.5
0.2
5
1.4
2.8
0.5
0.5
0.5
2
0.3
2
0.6
1.5
0.6
0.4
0.8
1.1
1.3
0.8
0.3
1
0.5
3.4
0.7
2
0.3
3.5
0.5
2.4
1.4
5
2.1
0.5
2.4
0.5
1.9
1.9
1.8
0.4
3.3
0.2
1
1.3
1.4
1.8
1.5
0.7
2.1
0.9
0.8
0.4
0.7
0.4
0.8
0.4
0.4
2.6
1.4
2.1
0.8
1
2.3
1.1
0.6
0.3
1.7
1.6
0.8
0.8
1.7
0.5
0.8
1.2
0.8
1.1
1.3
0.4
0.8
0.4
2.8
0.2
0.9
0.7
2.5
3.8
1.2
0.9
1
5.1
1
3.6
2.6
12.7
2
2.9
0.6
2.2
4.5
1.9
0.6
0.6
0.6
0.4
0.4
1.5
0.6
1
1.1
0.6
1
4.8
0.4
1
1.6
1.1
2.6
1.3
1.8
2
11.1
1.7
3.5
2.9
12.4
2.7
1
15.8
0.2
0.2
1.1
0.6
0.8
1.9
3.6
1.1
0.6
10.5
0.6
0.4
0.4
0.6
0.7
0.7
0.5
0.6
0.8
0.8
1.4
0.9
1.3
0.9
1.7
1.2
0.7
1.3
1.4
18.8
2
1.5
1.3
14.5
2.6
0.8
0.8
19
1.9
1.4
1.9
25.5
2.8
Origin/compound
class
Ref.
U (Ar)
Lp (Ar)
N
C
U(Ar)
U (Hs)
C
C
C
C
N
C
Lp (Ar)
C/N
C
C
C
C
C
Lp (FA)
U
Lg
U
C
Lp (Hs)
Lg
Lg/Lp (FA)
C
Lg, C
Lp (FA)
Lg
Lg (Hs)
Lg (Hs)
Lg
Lg
Lp (FA)
Lg (Hs)
Lg
C
Lg
Lp (FA)
ART
Lg
C
N
Lg
Lg
C
Lg
Lg
C
Lg
Lg
ART
Lp (Ar), Hs/Lp (FA)
Lg
Lg
Lp (FA)
Lg
Lp (Ar)
Lp (RA)
Lp (RA)
Lp (RA)
Lg
Lg
C
Lg
Lp (RA)
Lp (FA)
a, b
b
a, b
a, b
b
c
b, d
b
a, b
a, b, c, d
a, b
b
d
a, b/e
c
c, f
c
d
b
–
–
d, g
–
–
h
a, b, g
a, b, g/a, b
–
a, g, f
–
a, b, g
e
e
g
g
–
e
a, b, g
i
g
–
e
a, b, g
f
–
g
g
l
g
c
i
g
a, b, g
e
e/b
g
g
a, b
g
–
–
–
–
g
g
l
a
–
b
S. Hilli et al. / Geoderma 179–180 (2012) 63–72
Largeau, 1993; de Leeuw et al., 2006; Kögel-Knabner, 2002). Cutin
and suberin are plant cell components that contain both aliphatic
and phenolic structures (Bernards, 2002; Kögel-Knabner, 2002;
Winkler et al., 2005). Previous studies conducted using 13C NMR
have shown that, in addition to lignin, these slowly decomposable
compounds are also found in AIR (Preston et al., 1997, 2009).
To use the concentration of AIR as a tool in ecological studies,
more information is required regarding its chemical composition in
different types of organic materials. The sequential fractionation
method, or proximate analysis, provides a close approximation of
the lignin content for materials such as wood containing low levels
of tannin (e.g. Preston et al., 2006a, 2006b, 2009); however, it is less
clear what the composition of AIR is in other materials. Owing to
the insolubility of AIR in common solvents and the composition of
predominantly macromolecular material, chemical characterization
of this fraction is challenging. Nevertheless, pyrolysis-GC–MS or
solid state 13C NMR spectroscopy have been found to be suitable
methods for characterizing insoluble organic compounds in litter
and soil organic matter (Kögel-Knabner, 2000; Preston et al., 1997).
Boreal forest soils are an important reservoir of carbon at the global scale, but the mechanisms for accumulation of the SOM in soil are
still poorly understood (Goodale et al., 2002). To increase the understanding of the chemical composition of litters and SOM in boreal
forests and to test the relevance of using AIR in soil ecological studies,
we conducted detailed characterization of common litter types
(needles, coarse tree, and moss litter) and the F and the H layers in
two southern boreal sites in Finland using a combination of sequential fractionation and pyrolysis-GC–MS. Based on studies showing
that lignin does not accumulate in SOM (Sjöberg et al., 2004), but
that the relative proportion of AIR increases during decomposition
(Berg, 2000), we hypothesized that the AIR fraction in the needle
and the coarse tree litter should be largely composed of ligninderived compounds, while in the F and the H layers, polyphenolics
and cutin-derived compounds should constitute a larger proportion
of AIR. By comparing the AIR fractions among coarse tree litter (containing high concentrations of lignin), needle litter (containing moderate concentrations of lignin), and moss litter (containing no lignin),
we also tested whether the composition of AIR differed among different types of litter materials and consequently inﬂuenced the conclusions that have been made regarding the identity of compounds
that regulate the overall decomposition rates. Furthermore, we hypothesized that if the sequential fractionation efﬁciently removes
carbohydrates from the soil, the AIR fraction should contain no carbohydrates, while if carbohydrates form an important component of
SOM, they should form a considerable proportion of the unfractionated SOM.
2. Materials and methods
2.1. Study sites and sampling
We studied two south boreal forest sites, a mesic and a sub-xeric
site, located in Juupajoki. The mesic site (61°51′N) is dominated by
65
Norway spruce (Picea abies) and the ground vegetation is dominated
by the dwarf shrub, Vaccinium myrtillus, and the mosses Dicranum sp,
Pleurozium schreberi and Hylocomium splendens. The sub-xeric site
(61°52′N) is dominated by Scots pine (Pinus sylvestris), and the
ground vegetation is dominated by the dwarf shrub Vaccinium vitisidea and the mosses P. schreberi and Dicranum sp. Further details regarding the study sites are given in Hilli et al. (2008). We removed
complete organic layers (L, F, and H layers) in small square areas
(30 cm × 30 cm, n = 28) in 2002. The samples were sorted into the
following components: 1) needle litter, 2) coarse tree litter
(branches, bark, cones, stem wood), 3) moss litter, 4) F layer, and
5) H layer, and then weighed after drying (60 °C, 48 h). We removed
all detectable roots from the samples so that the detectable root biomass did not contribute to the analyses of the F and H layers. The
dried samples were then milled to pass through a 1 mm sieve. We
formed a composite sample of the 28 ﬁeld replicates of each sample
type.
2.2. Sample pretreatment
One subsample of each composite sample was subjected to
pyrolysis-GC–MS analysis and another subsample was subjected to
the sequential fractionation method described by Ryan et al. (1990)
prior to the pyrolysis-GC–MS analysis. We selected pyrolysis-GC–
MS because this method is able to identify speciﬁc compounds
produced after thermal and chemical degradation of the original
samples (Kögel-Knabner, 2000). In pyrolysis, certain sample types
break down into predictable compounds that can indicate source
material, as well as acting as a tool for assessment of the relative
degradation state along a litter decomposition continuum (Grandy
and Neff, 2008 and references therein). Sequential fractionation
deﬁnes operational fractions based on their extractability or
hydrolysis, thereby separating samples into the following organic
fractions: non-polar extractives (NPE; extracted with chloroform),
water-soluble extractives (WSE; extracted with hot water), acidsoluble fraction (AS; hydrolyzed with sulfuric acid combined with
autoclaving), and acid-insoluble residue (AIR; also referred to as
“Klason lignin”). For details regarding the fractionation methods, see
Hilli et al. (2008). After fractionation, both the WSE and AIR fraction
were analyzed by pyrolysis-GC–MS. The solubility of the AIR fraction
in dimethyl sulfoxide (DMSO) and tetrahydrofuran (THF) was investigated by preparing solutions with concentrations of 1 mg/ml and
trying to dissolve the solid material by warming, magnetic stirring,
and keeping the solution in an ultrasonicbath.
2.3. Pyrolysis-GC–MS analyses
Pyrolysis-GC–MS analyses were conducted on a ﬁlament pulse
Pyrola 85 pyrolyzer (Pyrol AB, Lund, Sweden) connected to a GC–
MS (HP 6890–5973 instrument). The system used an Innowax,
15 m × 0.25 mm column with a ﬁlm thickness 0.5 μm (Agilent Technologies), and He applied at a ﬂow rate of 0.8 ml/min as the carrier
gas. The oven temperature increased from 50 °C to 300 °C at
Notes to Table 1:
Lp = lipophilics; Ar = aromatics; FA = fatty acid; RA = resin acid; U = unclassiﬁed; Hs = humic substances; Lg = lignin or other polyphenols; C = carbohydrates; N = nitrogencontaining compounds (e.g., proteins).ART = artifact.
a Buurman et al. (2005).
b Buurman et al. (2007).
c Łucejko et al. (2009).
d Mészáros et al. (2007).
e Gadel and Bruchet (1987).
f Fabbri and Chiavari (2001).
g In Methods in Lignin Chemistry, Lin and Dence (1992).
h Fabbri et al. (1998).
i Lu et al. (2009).
j Räisänen et al. (2003).
l Nierop et al. (1999).
66
S. Hilli et al. / Geoderma 179–180 (2012) 63–72
3. Results
8 °C/min. The injector temperature was maintained at 260 °C with a
split ﬂow of 20 ml/min. The detector transfer line was kept at
280 °C. The samples were pyrolyzed at 650 °C for 2 s. Identiﬁcation
of the eluted compounds was accomplished by comparing the spectra
with spectral libraries (Wiley 275 and NIST 98, as well our laboratory's libraries). The components were semi-quantiﬁed by calculating
the percentage of the total peak area that each of the identiﬁed compounds accounted for.
The pyrolysis products were subdivided according to probable
origin into a number of source groups, such as carbohydrate-derived
compounds, lipophilics (further grouped into aromatics, fatty acids,
and resin acids), lignin or other polyphenols. Some compounds
could not be classiﬁed. Our goal was to compare the composition of
the unfractionated samples and the AIR fractions; therefore, the
quantitative results were not analyzed statistically.
3.1. Unfractionated litter and F and H layer samples
We identiﬁed 71 pyrolysis products in the unfractionated litter
samples (needles, coarse trees, and moss), the F layer, and the H
layer samples (Table 1). Because of the similarity in the composition
of the unfractionated samples, only pyrograms of the F and H layer
samples of the mesic site are shown (Fig. 1). Lignin- or other
polyphenol-derived substances were the most abundant compounds
detected in all unfractionated samples except for moss litter. These
were followed by pyrolysis products of carbohydrates, especially
anhydrosugars (Table 2).
In the needle and coarse tree litter in both the Scots pine and Norway spruce dominated forests, the pyrolysis products detected were
Abudance
5000000
4500000
4000000
49
3500000
3000000
2500000
9
32
28
2000000
18
10
1500000
36
21
19
23
1
31
38
34
30
1000000
12
500000
Time
2 3
7
4 5
11
8
13 14
24 26 29
20 22
25 27
16
15 17
44 46
42
40
41
3739
6
33
43 45
50
48
35
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
Abudance
9000000
8500000
8000000
7500000
7000000
6500000
6000000
5500000
5000000
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
49
10
1
Time 2
28
18
9
19 21
12
2
3
4 5
4
6
7
6
8
11 13
14
8
10
14
16
18
36
34 38
31
30
20 22 242627 29
151617
12
32
23
33
40
39
35 37
20
22
42
24
44 4647
26
48
28
50
30
32
34
Fig. 1. Pyrograms of an unfractionated sample of the F layer (above) and H layer (below ) of a Norway spruce-dominated mesic site in Juupajoki (1 = toluene, 2 = ethylbenzene,
3 = pyridine, 4 = 2-methylfuran, 5 = styrene, 6 = hydroxyacetone, 7 = 2-cyclopenten-1-one, 8 = 3-furaldehyde, 9 = acetic acid, 10 = furfural, 11 = pyrrole, 12 =
5-methylfurfural, methylpyrrole, 13 = 2-cyclopentene-1,4-dione, 14 = 2-furanmethanol, 15 = 2(5H)-furanone, 16 = 1,2-cyclopentanedione, 17 = 2-hydroxy-3-methyl-2-cyclopenten-1-one, 18 = guaiacol, 19 = methylguaiacol, 20 = maltol, 21 = phenol, 22 = ethylguaiacol, 23 = cresol1, 24 = cresol2, 25 = 4-propylguaiacol, 26 = eugenol, 27 = ethylphenol, 28 = vinylguaiacol, 29 = 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one, 30 = isoeugenol, 31 = diethyl phthalate, 32 = 4-vinylphenol, 33 = 3-pyridinol, 34 =
5-(hydroxymethyl)-2-furancarboxaldehyde, 35 = chavicol, 36 = vanillin, 37 = homovanillin + vanillic acid methyl ester, 38 = guaiacylethanone, 39 = guaiacylacetone, 40 =
dibutyl phthalate + tetradecanoic acid,41 = catechol, 42 = hexadecanoic acid, 43 = dihydroconiferyl alcohol, 44 = methyldehydroabietate, 45 = coniferyl alcohol +
abietatetraenoic acid (methyl ester), 46 = coniferylaldehyde, 47 = anhydrosugar, 48 = coniferyl alcohol, 49 = anhydrosugar, 50 = vanillic acid).
S. Hilli et al. / Geoderma 179–180 (2012) 63–72
67
Table 2
Sum of pyrolysis products derived from carbohydrates, lignin and other polyphenols and of lipophilics detected in pyrolysis-GC–MS of the unfractionated needle litter, tree litter,
moss litter, F layer and H layer samples at a sub-xeric and a mesic south boreal site.
Sub-xeric L
Carbohydrate-derived comp.
Lignin- or other polyphenols
Lipohilics:
Aromatic compounds
Fatty acids
Resin acids
Sub-xeric
Mesic
Sub-xeric
Mesic
Needle
Tree
Moss
Mesic L
Needle
Tree
Moss
F
F
H
H
13.7
22.1
4.5
0.9
0.4
3.2
22.6
35.0
10.9
2.8
0.2
7.9
41.8
4.8
8.6
2.5
0.6
5.5
27.5
34.1
5.4
1.6
1.1
2.7
23.5
43.6
4.8
0.5
0.4
3.9
37.2
8.4
14.5
2.2
7.6
4.7
28.5
28.6
4.8
0.2
2.3
2.3
26.9
30.6
4.2
1.3
0.7
2.2
29.9
23.2
3.6
0.1
0.6
2.9
36.4
21.8
3.4
0.9
0.8
0.7
essentially the same (Table 1). In needle litter from both the sub-xeric
and the mesic site, lignin-derived components such as coniferyl alcohol, diconiferyl alcohol, 4-vinylphenol, guaiacols and carbohydratederived components such as anhydrosugars and acetic acid were
abundant (Table 1). In the coarse tree litter, an anhydrosugar 2,
4-vinylguaiacol and vanillin were the most abundant compounds.
The most abundant compounds in the moss litter were acetic acid, an
anhydrosugar and furfural; however, it also contained some woodderived compounds, such as methyldehydroabietate (Table 1). The
compounds found in the litter samples were also detected in the F
and the H layer samples (Table 1, Fig. 1).
from the column used in this study. In addition, toluene, styrene
and some other compounds classiﬁed as non-speciﬁc compounds
are not included in the summary in Tables 2 and 4.
The AIR fraction of the litter layer samples was insoluble in DMSO,
and only partially soluble in THF. Because of the poor solubility in
THF, the molecular weight proﬁle of the AIR could not be determined
by size-exclusion chromatography. The pyrolysis-GC–MS analysis of
the WSE fraction (data not shown) indicated that the tree litter and
the moss litter samples from the mesic site contained lignin-derived
guaiacol, methylguaiacol and vinylguaiacol. No other compounds
were identiﬁed by pyrolysis-GC–MS.
3.2. AIR fraction of litter and F and H layer samples
4. Discussion
The AIR fraction comprised 30.0% and 32.5% of the sample dry
weight in the pine and spruce needle litter, respectively, 35.0% and
44.7% of the sample dry weight in the pine and spruce coarse tree
litter, respectively, and 23.8% and 26.2% of the moss litter in the
pine and spruce site, respectively. In the F layer, AIR constituted
52.3% and 51.6% of the pine and spruce site, respectively, while in
the H layer it comprised 75.6% and 71.3% in the pine and spruce
site, respectively.
A smaller number of pyrolysis products were detected in AIR fractions than in the corresponding unfractionated samples. In the needle
and coarse tree litter, the AIR fractions were primarily composed of
lignin- or polyphenol-derived compounds, such as guaiacol and phenol derivatives (Table 3). The compounds found in the AIR fraction of
the moss litter differed; however, the most abundant compounds
were toluene, phenol, cresol and 4-vinylphenol (Table 3). Several
compounds, primarily derived from carbohydrates (e.g., furfural)
were found in small amounts in the litter layer samples, but they
were absent from the F and H layers (Table 3).
The compounds found in the AIR fraction of the F and H layers differed from those found in the litter materials. A resin acid, methyldehydroabietate, was the most abundant compound in the mesic site,
followed by the lignin and tannin derived compound catechol and
the carbohydrate-derived compound acetic acid (Table 3, Fig. 2).
The pyrograms of the F and H layers of the sub-xeric site were similar
to those of the mesic site (Table 3, ﬁgure not shown). Methyldehydroabietate, methylguaiacol, and guaiacol were the most abundant
pyrolysis products in both the F and H layers, but only the H layer
had a high abundance of catechol.
Several low-molecular-weight carboxylic acids abundant in the F
and H layer samples were not detected in the litter samples
(Table 3). Some substances, such as methyldehydroabietate, vanillic
acid, coniferyl alcohol and abietatetraenoic acid were found in the
AIR fraction of the F and H layer samples, but not in the AIR fraction
of the litter samples, even though these compounds were found
in the unfractionated litter samples (Table 1). The proportion of
carbohydrate-derived compounds was very low in all samples. The
proportions of different compound groups of the AIR fraction are
listed in Table 4. Phthalates are not included in the summary presented in Tables 2 and 4 because they are assumed to be an artifact
4.1. Composition of litter and SOM of unfractionated samples
Polyphenols and carbohydrate-derived compounds formed the
major components of the needle and coarse tree litter and the F and
the H layers in both the pine and spruce-dominated south boreal sites.
Conversely, moss litter was primarily composed of carbohydratederived compounds, especially acetic acid and anhydrosugar 2, which
agrees with studies showing that mosses are an important source of
carbohydrates (Boberg, 2009; Funazukuri et al., 2000; Pouwels et al.,
1987). Anhydrosugars in unfractionated samples indicates the presence
of cellulose and hemicelluloses. Their high abundance, especially that
of anhydrosugar 2, is expected because cellulose and hemicelluloses
form the most abundant component in plant materials (KögelKnabner, 2002 and references therein). Cellulose may also persist in
soils, because a major part of cellulose in trees is protected by lignin,
which only becomes available for microbial degradation after lignin
degradation (Ekschmitt et al., 2005). However, the absence of anhydrosugars in the AIR fraction, conﬁrms with the results of earlier studies
showing that acid hydrolysis during sequential fractionation efﬁciently
removes all carbohydrates from the AS fraction (Cowie and Hedges,
1984; Preston et al., 1997; Schnitzer and Preston, 1983; Stark et al.,
2012).
A high abundance of phenolic compounds was also expected
because boreal forest trees and the ericaceous understorey dwarf
shrubs are rich in both lignin and phenolic secondary metabolites
(Hilli et al., 2008; Hobbie, 1996; Kanerva et al., 2008; Kraus et al.,
2004; Vargas et al., 2006; Wardle et al., 2003). Phenol, methylphenol,
catechol, 4-methylcatecol and ethylcatecol are typically non-ligninderived polyphenolic pyrolysis products (Nierop et al., 2001).
Catechol is a pyrolysis product that may be derived from condensed
tannins, proanthocyanidines and prodelphinidines (Galletti and
Reeves, 1992; Kögel-Knabner, 2002; Ohara et al., 2003). The high
catechol concentrations observed in our study agree with the results
of previous studies in which high concentrations of tannins were
observed in soil organic horizons in both Norway spruce and Scots
pine dominated forests in Finland (Adamczyk et al., 2008; Kanerva
et al., 2008) as well as spruce- and pine dominated sites elsewhere
(Gallet and Lebreton, 1995; Preston et al., 2006a, 2006b, 2009). In addition to coniferous tree species, the understorey Vaccinium species
68
S. Hilli et al. / Geoderma 179–180 (2012) 63–72
Table 3
The relative abundance of pyrolysis products in AIR fraction of the needle litter, tree litter, moss litter, F layer and H layer samples at a sub-xeric and a mesic south boreal site.
Sub-xeric L
Compound
Toluene
Ethylbenzene
Ethylcyclopentene
Dimethyl sulfate
2-Methylfuran
Xylene
Styrene
2-Cyclopentene-1-one
4-Cyclopentene-1,3-dione
Acetic acid
Furfural
Methylfurfural
Pyrrole
Propanoic acid
Butanoic acid
Pentanoic acid
Hexanoic acid1
Propylbenzene
Propenylbenze
Methylfurfural + methylpyrrole
2(5H)-Furanone + 3-methyl-1,2-cyclopentanedione
2-Acetylfuran
Heptanoic acid
Guaiacol
Ethylguaiacol
Methylguaiacol1
Methylguaiacol2
Naphthalene
Phenol
Hexanoic acid2
Biphenyl
4-Oxopentanoic acid methyl ester = methyl levulinate
Benzofuran
2-Methylbenzofuran
1,3-Benzodioxol-2-one
1-Decene
1-Dodecene
Tridecene
Octanoic acid1
Indene
Octanoic acid2
Octenoic acid
Cresol1
Cresol2
Salicylaldehyde
Nonanoic acid1
Eugenol
Nonanoic acid2
Dimethylphenol
x-Ethylphenol
4-Ethylphenol
4-Vinylphenol
Vinylguaiacol
Isoeugenol
Dimethyl phthalate
Diethyl phthalate1
Dibutyl phthalate1 + tetradecanoic acid
Di-isobutyl phthalate
Decanoic acid
Benzoic acid
Dodecanoic acid
2-Furancarboxylic acid
2,2′-Bifuran
Vanillin
Propiovanillone + dibutylphthalate2
4-Propylguaiacol + vanillic acid methyl ester
Anhydrosugar
Homovanillin
Guaiacylethanone
Guaiacylacetone
4-Hydroxyacetylguaiacol
Catechol
Methylcatechol1
Ethylcatechol
Needle
1.8
0.6
Tree
1
Mesic L
Moss
Needle
9
2.3
Tree
2.7
Moss
Sub-xeric
Mesic
Sub-xeric
Mesic
F
F
H
H
8.3
1.7
0.5
0.6
0.9
0.8
0.8
1.6
3.9
0.6
2.1
1.1
1.4
3.7
0.5
0.4
0.8
0.4
0.3
0.3
1.8
2.3
2
0.4
0.5
0.4
0.3
0.8
0.6
0.4
1.3
2.4
5.6
1.1
6.8
1.3
6.6
4.4
0.5
1.1
2.8
4.4
11.2
5.2
1.5
0.7
0.2
0.2
0.9
0.4
0.3
3.9
0.9
5.2
5.2
1.4
0.4
7.6
4.3
3.2
3.3
0.2
6
0
0.4
0.4
0.8
0.5
0.9
0.5
0.5
1
4.7
1.8
6.4
1.3
3.2
2
4
1.7
6.5
2.3
1.5
0.5
0.2
1.7
1.1
4.6
1.5
5.9
4.4
1.2
0.7
4.8
0.8
7.6
3.7
3.6
2.8
0.8
0.5
2
0.3
1
0.4
0.8
0.7
1
0.5
0.6
2.2
0.8
0.7
1.7
0.7
1.5
4.7
0.9
1.5
0.7
1.9
1.3
3
3.8
1.6
2.2
3.3
0.5
2.3
7.9
0.4
2.3
3.6
2.2
1.4
7
0.3
2
8.1
2.8
5
1.3
2.7
3.2
1.1
0.7
3.2
1.2
7.1
5
1.3
2.4
8
0.4
4.7
1
0.8
0.5
3.3
0.7
2.5
1
1.2
2.3
1.3
6.2
1.2
1.2
0.6
0.9
1
2.8
2
1.9
1.4
1.5
0.6
15.1
4.8
2.5
0.5
2.9
1.5
1.9
1.9
2.8
0.7
0.3
6.1
2.3
1.5
1.1
0.8
18.8
4.8
1.3
0.9
1.1
1.4
1.5
0.6
0.8
3
3
1
0.6
2.8
0.5
4.8
3
1
0.9
6.9
2.3
1.9
0.9
1.6
0.4
1.1
3.2
3
1.2
1.3
0.5
1.1
3.8
2.1
0.3
5.5
1.4
0.6
0.5
1
0.1
0.2
0.4
0.9
12.3
2.3
0.5
0.5
0.3
0.8
3.4
7.5
0.9
2.9
2.6
0.8
3.9
2.4
1
2
0.6
3
1.3
0.7
11.5
4.2
17.6
2.3
1.2
Origin
Ref.
U (Ar)
Lp (Ar)
C
–
C
Lp (Ar)
U (Ar)
C
C
C
C
C
N
Hs
FA
FA
FA
Lp (Ar)
Lp (Ar)
C/N, Hs
C
C
Lp (FA)
Lg
Lg
Lg
Lg
Lp (Ar)
Lg, C
Lp (FA)
Lp (Ar)
U
Lp (Ar)
C
Lp (Ar)
Lp (FA)
Lp (FA)
Lp (FA)
Lp (FA)
Lp (Ar)
Lp (FA)
Lp (FA)
Lg (Hs)
Lg (Hs)
Lg (Ar)
Lp (FA)
Lg
Lp (FA)
Lg
Lg
Lg (Hs)
Lg
Lg
Lg
ART
ART
Lp (Ar), Hs/FA
ART
Lp (FA)
Lg (Ar)
Lp (FA)
C?
C
Lg
Lg/Lp (Ar), (Hs)
Lg
C
Lg
Lg
Lg
a, b
b
–
–
a, b
–
b
b, d
c
a, b
a, b, c, d
a, b
a, b
e
–
–
–
b
b
a, b/e
c/j
b
–
d, g
a, b, g
a, b, g
a, b, g
b
a, g, f
–
k
–
–
b
–
b
b, k
b, k
–
b, k
–
–
e
e
–
–
g
–
g
–
h
g
g
g
–
e
e/a, b
–
–
–
b
–
–
g
g/e
g/c
l
g
g
a, b, g
Lg
Lg
Lg
g
g
–
S. Hilli et al. / Geoderma 179–180 (2012) 63–72
69
Table 3 (continued)
Sub-xeric L
Compound
Methylcatechol2
Catechol monoacetate
Hexadecanoic acid
4-Hydroxy-acetophenone
Oxacycloheptadec-8-en-2-one
Methylisopimarate
Methylabieta-7,9(11)-dien-18-oate
Methyldehydroabietate
Coniferyl alcohol + abietatetraneoic
Acid methyl ester
Resorcinol
3,5-Dihydroxytoluene
Coniferylaldehyde
Vanillic acid methyl ester
Vanillic acid
Sesquiterpene
Triphenylphosphine oxide
Diethyl phthalate2
Bis(ethylhexyl)phthalate
Mataresinol
Levoglucosan
Pentadecane
Stigmastan-3,5-diene
Campestadiene
Stigmastan-triene
Methyl hexadecanoate
Methyl eicosanoate
Docosanoic acid
Needle
Tree
Mesic L
Moss
11.3
Needle
3.4
Tree
Moss
Sub-xeric
Mesic
Sub-xeric
Mesic
F
F
H
H
7.7
1.3
1.5
3.6
1.5
1
0.3
2.7
Origin
Ref.
Lg
Lg
Lp (FA)
Lg (Ar)
g
–
a, b
–
–
–
–
–
g/–
4.2
10.6
16.8
2.1
6.9
1.5
0.9
1.1
19.6
2.3
1.5
0.7
0.5
1.2
1
0.1
1
0.8
4.1
20.2
1.4
2.1
4.8
0.5
2.2
4
1.5
1.1
1.1
0.7
0.8
0.8
0.2
0.1
0.8
1.5
1
1.1
0.8
0.1
Lp (RA)
Lp (RA)
Lp (RA)
Lg/Lp (RA)
Lp (Ar)
Lp (Ar)
Lg
Lg
Lg
–
–
g
c
a
–
Lp (Ar),Hs
ART
Lg
C
Lp (FA)
Lp (Ph)
Lp (Ph)
Lp (Ph)
Lp (FA)
Lp (FA)
Lp (FA)
e
–
–
a, b
b
d
–
d
d
d
a
Ar = aromatics; FA = fatty acid; RA = resin acid; Hs = humic substances; Lg = lignin or other polyphenols; C = carbohydrates; N = nitrogen-containing compounds (e.g.,
proteins); Ph = phytosterol, ART = artifact.
a Buurman et al. (2005).
b Buurman et al. (2007).
c Łucejko et al. (2009).
d Mészáros et al. (2007).
e Gadel and Bruchet (1987).
f Fabbri and Chiavari (2001).
g In Methods in Lignin Chemistry, Lin and Dence (1992).
h Fabbri et al. (1998).
i Lu et al. (2009).
j Räisänen et al. (2003).
k Buurman et al. (2008).
l Nierop et al. (1999).
make a signiﬁcant contribution to tannins and other phenolics found
in forest soils (Adamczyk et al., 2008; Kanerva et al., 2008; Preston
et al., 2009). For example, in the leaf litter of V. myrtillus, catechol
forms the main phenolic acid together with caffeic acid (Gallet and
Lebreton, 1995). Catechol is also an important pyrolysis product of
root-derived polyphenols (Nierop et al., 2001). The abundance of
roots and rhizomes of Vaccinium species may cause higher catechol
abundance and total phenolic concentration in the spruce than
pine-dominated sites (Kanerva et al., 2008).
Previous investigations have often used 13C NMR as a method for
characterization of pine and spruce litter (Kögel-Knabner, 2002;
Preston et al., 2009). When compared with the 13C NMR studies conducted by Kögel-Knabner (2002) and Preston et al. (2009), in the present study, pyrolysis-GC–MS showed a lower relative proportion of
carbohydrate-derived compounds (anhydrosugars, acetic acid, furans
and reduced furans) in the needle and coarse tree litter but higher
concentrations of polyphenols.
4.2. Composition of AIR and its implications for SOM decomposition in
boreal forests
In accordance with the notion of AIR being primarily composed of
lignin in the SOM (Berg, 2000; Schimel et al., 1994; Shaver et al.,
2006), the AIR fraction contained several pyrolysis products originating from lignin and other polyphenols (Buurman et al., 2005; Otto
and Simpson, 2006; Rumpel et al., 2004). Comparison of the AIR
fractions of the needle, coarse tree and moss litter with the AIR fraction of pure lignin showed that the AIR in pure lignin contained mostly vanillin, guaiacols, coniferyl alcohol and coniferyl aldehyde, while
the concentrations of phenol, cresol and catechol were lower than
in our litter samples. Vanillin and guaiacols are typical pyrolysis
products of angiosperm lignin (Nierop et al., 2001; Preston et al.,
1994; van der Heijden and Boon, 1994), which were abundant in
the lignin-rich needle and coarse tree litter, but were not found
in great proportions in the moss litter. 4-Vinylguaiacol, 4methylguaiacol, guaiacol, vanillin and vanillic acid indicate lignin
and oxidative degradation of the side chains of the original coniferyl
alcohol structures of lignin (Dijkstra et al., 1998; Lima et al., 2008;
Preston et al., 1994). Soil micro-organisms may also catabolize vanillic acid to vanillate and further convert it to guaiacol (Crawford and
Olson, 1978).
Vanillic acid was present in the F and H layers, but not in the litter
samples, which agrees with previous ﬁndings showing an increase in
vanillic acid during the course of decomposition in needle litter
(Isidorov et al., 2010). The fact that we did not detect cinnamyls
and syringyls, which are typical compounds in herbs, grasses and
angiosperms, supports earlier studies indicating that they decompose
relatively quickly when compared to the conifer lignin (Sjöberg et al.,
2004). It is also possible that syringyl units are converted to guaiacyl
units by demethoxylation during the degradation process. Lignin in
the SOM can primarily be found in a degraded state Bahri et al.,
2008; Boberg, 2009; Lima et al., 2008; Martínez et al., 2010;
70
S. Hilli et al. / Geoderma 179–180 (2012) 63–72
Abudance
4500000
4000000
3500000
48
3000000
2500000
2000000
1500000
30
15
1000000
31
14
20
16
500000
25
1
0
Time 4
4
2
5
7
6
8
9
9
10
13
1718212223
24
19
45
35 3739
3
2829
32
43
46
34 36 3840
49
51
53
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Abudance
4000000
3800000
3600000
3400000
3200000
3000000
2800000
2600000
2400000
2200000
2000000
1800000
1600000
1400000
1200000
1000000
800000
600000
400000
200000
0
Time
48
41
30
3
16
14
1
4
56 7
2
5
6
7
8
9
8 9
10
11
13
2021
242627 28
15 1718
25
39
323334353637
42
434447
49
50
52
51
54
53
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Fig. 2. Pyrograms of an AIR (acid insoluble) fraction of the F layer (above) and the H layer (below) of a Norway spruce-dominated mesic site in Juupajoki (1 = styrene, 2 = 2cyclopenten-1-one, 3 = acetic acid, 4 = pyrrole, 5 = benzofuran, 6 = ethylcyclopentene, 7 = propanoic acid, 8 = 2-methylbenzofuran, 9 = butanoic acid, 10 = pentanoic
acid, 11 = naphthalene, 12 = 1,3-benzodioxol-2-one, 13 = hexanoic acid1, 14 = guaiacol, 15 = methylguaiacol1, 16 = phenol, 17 = ethylguaiacol, 18 = octanoic acid2, 19 =
dimethylphenol, 20 = cresol1, 21 = cresol2, 22 = octenoic acid, 23 = nonanoic acid1, 24 = 4-ethylphenol, 25 = vinylguaiacol, 26 = acid 16:0, 27 = 2-furancarboxylic acid,
28 = dimethyl phthalate, 29 = isoeugenol, 30 = diethyl phthalate1, 31 = 4-vinylphenol, 32 = benzoic acid, 33 = 2,2′-bifuran, 34 = di-isobutyl phthalate, 35 = vanillin, 36 =
4-propylguaiacol + vanillic acid methyl ester, 37 = guaiacylethanone, 38 = guaiacylacetone, 39 = dibutyl phthalate, 40 = propiovanillone, 41 = catechol, 42 = methylcatechol1,
43 = hexadecanoic acid, 44 = methylisopimarate, 45 = oxacycloheptadec-8-en-2-one, 46 = 4-hydroxy-acetophenone, 47 = methylabieta-7,9(11)-dien- 18-oate, 48 = methyldehydroabietate, 49 = abietatetraenoic acid methyl ester, 50 = resorcinol, 51 = bis(ethylhexyl)phthalate, 52 = 3,5-dihydroxytoluene, 53 = vanillic acid, 54 = stigmastan3,5-diene).
(Rosenberg et al., 2003; Sjöberg, 2003), which results from the fact
that brown-rot fungi predominantly utilize hemicelluloses and cellulose, but leave behind a chemically modiﬁed lignin residue (Martínez
et al., 2010; Niemenmaa, 2008; Preston et al., 2006a, 2006b). Both
lignin and lignin-like polyphenolics are thought to decrease the
decomposability of litter (Grandy and Neff, 2008).
Table 4
Sum of pyrolysis products derived from carbohydrates, lignin and other polyphenols and of lipophilics detected in pyrolysis-GC–MS of the AIR fraction of needle litter, tree litter,
moss litter, F layer and H layer samples at a sub-xeric and a mesic south boreal site.
Sub-xeric L
Carbohydrate-derived comp.
Lignin- or other polyphenols
Lipohilics:
Aromatic compounds
Fatty acids
Resin acids
Sub-xeric
Mesic
Sub-xeric
Mesic
Needle
Tree
Moss
Mesic L
Needle
Tree
Moss
F
F
H
H
0.0
43.6
27.4
22.3
5.1
0.0
6.4
68.1
1.3
1.3
0.0
0.0
3.0
34.5
7.9
3.8
4.1
0.0
1.9
34.5
5.6
1.4
4.2
0.0
10.8
66.4
3.5
1.8
1.7
0.0
0.0
37.9
10.1
8.0
2.1
0.0
2.2
47.6
28.9
12.7
5.6
10.6
0.4
40.2
24.4
0.0
7.6
16.8
2.3
57.2
17.1
2.8
7.4
6.9
13.0
38.8
28.6
5.9
1.1
21.6
S. Hilli et al. / Geoderma 179–180 (2012) 63–72
In the AIR fraction of the F and H layers, several other pyrolysis
products were also detected, including methyldehydroabietate,
which is a resin acid found also in Norway spruce wood. A previous
study indicated that methyldehydroabietate may be formed as a
degradation product of microbial oxidation of dehydroabietic and
isopimaric acids (Vorob'ev et al., 2000). This would support earlier
studies that revealed the accumulation of dehydroabietic acid with
respect to other lipophilic compounds during litter decomposition
(Kainulainen and Holopainen, 2002). These results would also agree
with those of a study conducted by Kanerva et al. (2008), who
found that dehydroabietic acid, pinifolic and isopimaric acids were
the most abundant resin acids in the L layer of boreal Norway spruce
and Scots pine forests in Finland. Several low-molecular weight compounds that may be produced by brown-rot and white-rot fungi
during the breakdown of lignin and cellulose were also detected in
the F and H layers (Hatakka, 2001).
Our ﬁnding that the AIR in the H layer, but not in the litter layer,
contains lipophilic compounds may have implications regarding the
conclusions drawn about the role of phenolics in SOM accumulation.
In an earlier investigation, the concentration of AIR was found to
increase along the decomposition gradient of the organic horizon,
and this increase was stronger in southern than northern boreal
forests (Hilli et al., 2008). However, this ﬁnding does not necessarily
indicate the accumulation of phenolics alone along the vertical soil
gradient, if the proportion of lipophilic compounds in the AIR fraction
increases along the same gradient. Our results support the earlier
ﬁndings by Preston et al. (2009), who used 13C nuclear magnetic resonance (NMR) spectroscopy of wood and foliar litter to demonstrate
that lignin was not selectively preserved in the AIR fraction during
litter decomposition. In contrast, increases in resistant structures
derived from lignin, tannins, and cutin collectively accounted for the
increased proportion of AIR during decomposition.
The presence of resin and fatty acids in the AIR fraction observed
in the present study supports the assumption that, along with
lignin- and other polyphenol-derived compounds, lipids also play an
important role in SOM formation (Dijkstra et al., 1998; Nierop et al.,
2001; Preston et al., 2009). Some earlier studies have indicated that
compounds, especially hydroxy- and epoxy fatty acids derived from
cutin and suberin, may accumulate in the AIR fraction (Preston
et al., 1997) and soils (Bernards, 2002), whereas others have suggested that hydroxy and epoxy fatty acids from the cutin of leaves
are relatively easily decomposed (Otto and Simpson, 2006). For
example, a study conducted by Winkler et al. (2005) used 13C NMR
and pyrolysis-GC to demonstrate that cutin and suberin-derived
alkyl C decreased from the L layer to the H layer. We did not ﬁnd compounds originating from these plant constituents, which suggest that
such compounds would actually decompose. However, it is also
possible that hydroxy acids present in the samples were not eluted
from the GC column owing to their nonvolatility, as no derivatization
agents were used (see Kögel-Knabner, 2000; Naafs et al., 2004; Otto
and Simpson, 2007).
4.3. Implications for the methods used in the decomposition studies
The AIR fraction contained only negligible amounts of pyrolysis
products derived from carbohydrates, such as furfural (a pyrolysis
product of polysaccharides; Marinari et al., 2007), other furans,
which are associated with cellulose degradation (Alén et al., 1996),
and acetic acid, which may reﬂect a high concentration of microbial
sugars (Buurman et al., 2005) or intact cellulose (Pouwels et al.,
1987). Sequential fractionation efﬁciently removes the soluble carbohydrates to the WSE fraction and the cellulose-derived compounds
to the AS fraction. The WSE fraction of the litter samples contained
only small amounts of lignin-derived compounds, and lignin was
not found in the F and the H layers, which is explained by the
fact that lignin is not easily extracted with water. Accordingly,
71
sequential fractionation seems to be a reliable means for separating
carbohydrate-derived compounds from the insoluble compounds,
which are also slowly decomposable. Our data does not enable assessment of the absolute amount of the nonphenolic compounds in the
AIR of SOM, but does demonstrate that the concentration of AIR
may be a poor indicator of lignin and phenolic compounds when investigating the F and H layers in the organic horizon. However,
given that the composition of AIR appears to be a mixture of different
compounds, as already suggested by Preston et al. (1997), pyrolysisGC–MS analysis is a suitable method for detailed characterization of
the AIR fraction.
Acknowledgments
We are grateful to Ville Hautajärvi and all members of the staff at
the FFRI Salla Ofﬁce for the laborious pre-treatment of the samples,
and for Sirkka Aakkonen for assistance in the laboratory. We also
thank Dr. Maija Salemaa, Dr. Leena Hamberg, Dr. Annamari Markkola,
and Raimo Pikkupeura for their help. We thank Prof. Emeritus Caroline
Preston for valuable comments that have helped improve this paper.
This study was carried out with co-funding provided within the framework of the EU/Forest Focus programme (Regulation (EC) No 2152/
2003).
References
Adamczyk, B., Kitunen, V., Smolander, A., 2008. Protein precipitation by tannins in soil
organic horizon and vegetation in relation to tree species. Biology and Fertility of
Soils 45, 55–64.
Alén, R., Kuoppala, E., Oesch, P., 1996. Formation of the main degradation compound
groups from wood and its components during pyrolysis. Journal of Analytical and
Applied Pyrolysis 36, 137–148.
Bahri, H., Rasse, D.P., Rumpel, C., Dignac, M.-F., Bardoux, G., Mariotti, A., 2008. Lignin
degradation during a laboratory incubation followed by 13C isotope analysis. Soil
Biology and Biochemistry 40, 1916–1922.
Baldock, J.A., Oades, J.M., Waters, A.G., Peng, X., Vassallo, A.M., Wilson, M.A., 1992.
Aspects of the chemical structure of soil organic materials as revealed by solidstate 13C NMR spectroscopy. Biogeochemstry 16, 1–42.
Berg, B., 2000. Litter decomposition and organic matter turnover in northern forest
soils. Forest Ecology and Management 133, 13–22.
Bernards, M.A., 2002. Demystifying suberin. Canadian Journal of Botany 80, 227–240.
Boberg, J., 2009. Litter Decomposing Fungi in Boreal Forests. Doctoral Thesis Swedish
University of Agricultural Sciences. Faculty of Natural Resources and Agricultural
Sciences Department of Forest Mycology and Pathology Uppsala. pp. 67.
Buurman, P., van Bergen, P.F., Jongmans, A.G., Meijer, E.L., Duran, B., van Lagen, B., 2005.
Spatial and temporal variation in podozol organic matter studied by pyrolysis-gas
chromatography/mass spectrometry and micromorphology. European Journal of
Soil Science 56, 253–270.
Buurman, P., Peterse, F., Almendros, M., 2007. Soil organic matter chemistry in allophonic soils: a pyrolysis-GC/MS study of a Costa Rican Andosol catena. European
Journal of Soil Science 58, 1330–1347.
Buurman, B., Macias, F., Boluda, R., Otero, X.L., 2008. Pyrolysis-gas chromatography/
mass spectrometry of soil organic matter extracted from a Brazilian Mangrove
and Spanish salt marshes. Soil Science Society of America Journal 73, 841–851.
Cowie, G.L., Hedges, J.H., 1984. Determination of nuutral sugars in plankton, sediments,
and wood by capillary gas chromatography of dquilibrated isomeric mixtures.
Analytical Chemistry 56, 497–504.
Crawford, R.L., Olson, P.P., 1978. Microbial catabolism of vanillate: decarboxylation to
guaiacol. Applied and Environment Microbiology 36, 539–543.
de Leeuw, J.W., Largeau, C., 1993. In: Engel, M.H., Macko, S.A. (Eds.), Organic Geochemistry.
Plenum Press, New York-London (str. 23).
de Leeuw, J.W., Versteegh, J.M., van Bergen, P.F., 2006. Biomacromolecules of algae and
plants and their fossil analogues. Plant Ecology 182, 209–233.
Dijkstra, E.F., Boon, J.J., Van Mourik, J.M., 1998. Analytical pyrolysis of a soil proﬁle
under Scots pine. European Journal of Soil Science 49, 295–304.
Ekschmitt, K., Liu, M., Vetter, S., Fox, O., Wolters, V., 2005. Strategies used by soil biota
to overcome soil organic matter stability — why is dead organic matter left over in
the soils? Geoderma 128, 167–176.
Fabbri, F., Chiavari, G., 2001. Analytical pyrolysis of carbohydrates in the presence of
hexamethyldisilazane. Analytica Chimica Acta 449, 271–278.
Fabbri, D., Mongardi, M., Montanari, L., Galletti, G.C., Chiavari, G., et al., 1998. Comparison
between CP/MAS 13C-NMR and pyrolysis-GC/MS in the structural characterization
of humins and humic acids of soil and sediments. Fresenius' Journal of Analytical
Chemistry 362, 299–306.
Fierer, N., Craine, J.M., McLauchlan, K., Schimel, J.P., 2005. Litter quality and the temperature sensitivity of decomposition. Ecology 86, 320–326.
72
S. Hilli et al. / Geoderma 179–180 (2012) 63–72
Funazukuri, T., Hirota, M., Nagatake, T., Goto, M., 2000. The effects of additives on
hydrolysis of cellulose with water under pressures. Progress in Biotechnology 16,
181–185.
Gadel, F., Bruchet, A., 1987. Application of pyrolysis-gas chromatography–mass
spectrometry to the characterization of humic substances resulting from decay of
aquatic plants in sediments and waters. Water Research 21, 1195–1206.
Gallet, C., Lebreton, P., 1995. Evolution of phenolic patterns in plants and associated litters and humus of a mountain forest ecosystem. Soil Biology and Biochemistry 27,
157–165.
Galletti, G.C., Reeves, J.B., 1992. Pyrolysis/gas chromatography/ion-trap detection of
polyphenols (vegetable tannins): preliminary results. Organic Mass Spectrometry
27, 226–232.
Goodale, C.L., Apps, M.J., Birdsey, R.A., Feild, C.B., Heath, L.S., Houghton, R.A., Jenkins,
J.C., Kohlmaier, G.H., Kurz, W., Liu, S., Nabuurs, G.-J., Nilsson, S., Shvidenko, A.,
2002. Forest carbon sinks in the northern hemisphere. Ecological Applications 12,
891–899.
Grandy, A.S., Neff, J.C., 2008. Molecular C dynamics downstream: the biochemical decomposition sequence and its impact on soil organic matter structure and function.
Science of the Total Environment 404, 297–307.
Hatakka, A.I., 2001. Biodegradation of lignin. In: Hofrichter, M., Steinbüchel, A. (Eds.),
Biopolymers. Lignin, Humic Substances and Coal, 1. Wiley-VCH, Weinheim,
Germany, pp. 129–180.
Hilli, S., Stark, S., Derome, J., 2008. Carbon quality and stocks in organic horizons in
boreal forest soils. Ecosystems 11, 270–282.
Hilli, S., Stark, S., Derome, J., 2010. Litter decomposition rates in relation to litter stocks
in boreal coniferous forests along climatic and soil fertility gradient. Applied Soil
Ecology 46, 200–208.
Hobbie, S.E., 1996. Temperature and plant species control over litter decomposition in
Alaska tundra. Ecology Monograph 66, 503–522.
Isidorov, V.A., Smolewska, M., Purzy'nska-Pugacewicz, A., Tyszkiewicz, Z., 2010. Chemical
composition of volatile and extractive compounds of pine and spruce leaf litter in the
initial stages of decomposition. Biogeosciences 7, 2785–2794.
Kainulainen, P., Holopainen, K., 2002. Concentrations of secondary compounds in Scots
pine needles at different stages of decomposition. Soil Biology and Biochemistry
34, 37–42.
Kanerva, S., Kitunen, V., Loponen, J., Smolander, A., 2008. Phenolic compounds and
terpenes in soil organic horizon layers under silver birch, Norway spruce and
Scots pine. Biology and Fertility of Soils 44, 547–556.
Kögel-Knabner, I., 2000. Analytical approaches for characterizing soil organic matter.
Organic Geochemistry 31, 609–625.
Kögel-Knabner, I., 2002. The macromolecular organic composition of plant end microbial residues as input to soil organic matter. Soil Biology and Biochemistry 34,
139–162.
Kraus, T.E.C., Zasoski, R.J., Dahlgren, R.A., Horwath, W.R., Preston, C.M., 2004. Carbon
and nitrogen dynamics in a forest soil amended with puriﬁed tannins from different
plant species. Soil Biology and Biochemistry 36, 309–321.
Lima, C.F., Barbosa, L.C.A., Marcelo, C.R., Silvério, F.O., Colodette, J.L., 2008. Comparison
between analytical pyrolysis and nitrobenzene oxidation for determination of
syringyl/guaiacyl ratio in Eucalyptus spp. lignin. Biology Resources 3, 701–712.
Lin, S.Y., Dence, C.W. (Eds.), 1992. Methods in Lignin Chemistry. Springer-Verlag, Berlin
Heidelberg, Germany.
Lu, Q., Xiong, W.-M., Li, W.-Z., Guo, Q.-X., Zhu, X.-F., 2009. Catalytic pyrolysis of cellulose with sulfated metal oxides: a promising method for obtaining high yield of
light furan compounds. Bioresource Technology 100, 4871–4876.
Łucejko, J.J., Modugno, F., Ribechini, E., del Río, J.C., 2009. Characterisation of archaeological waterlogged wood by pyrolytic and mass spectrometric techniques.
Analytica Chimica Acta 654, 26–34.
Lützov, M.V., Kögel-Knabner, I., Ekschmitt, K., Matzner, E., Guggenberger, G., Marschner,
B., Flessa, H., 2006. Stabilization of organic matter in temperate soils: mechanisms
and their relevance under different soil conditions - a review. European Journal of
Soil Science 57, 426–445.
Marinari, S., Masciandaro, G., Ceccanti, B., Grego, S., 2007. Evolution of soil organic matter changes using pyrolysis and metabolic indices: a comparison between organic
and mineral fertilization. Bioresource Technology 98, 2495–2502.
Martínez, A.T., Rencoret, J., Nieto, L., Jiménez-Barbero, J., Gutiérrez, A., Del Río, J.C., 2010.
Selective lignin and polysaccharide removal in natural fungal decay of wood as evidenced by in situ structural analyses. Environmental Microbiology. doi:10.1111/
j.1462-2920.2010.02312.x (Article ﬁrst published online: 1 AUG 2010).
Melillo, J.M., Aber, J.D., Linkins, A.E., Ricca, A., Fry, B., Nadelhoffer, K.J., 1989. Carbon and
nitrogen dynamics along the decay continuum: plant litter to soil organic matter.
Plant and Soil 115, 189–198.
Mészáros, E., Jakab, E., Várhegyi, G., 2007. TG/MS, Py-GC/MS and THM-GC/MS study
of the composition and thermal behavior of extractive components of Robinia
pseudoacacia. Journal of Analytical and Applied Pyrolysis 79, 61–70.
Moorhead, D.L., Sinsabaugh, R.L., 2006. A theoretical model of litter decay and microbial
interactions. Ecology Monograph 76, 151–174.
Naafs, D.F.W., van Bergen, P.F., Boogert, S.J., de Leeuw, J.W., 2004. Solvent-extractable
lipids in an acid andic forest soil; variations with depth and season. Soil Biology
and Biochemistry 36, 297–308.
Niemenmaa, O., 2008. Monitoring of fungal growth and degradation of wood. Division
of Microbiology Department of Applied Chemistry and Microbiology. Viikki
Biocenter, University of Helsinki Finland, Yliopistopaino, Helsinki, Finland.
Nierop, K.G.J., Buurman, P., de Leeuw, J.W., 1999. Effect of vegetation on chemical composition of H horizons in incipient podzols as characterized by 13C NMR and pyrolysis-GC/MS. Geoderma 90, 111–129.
Nierop, K.G.J., van Lagen, B., Buurman, P., 2001. Composition of plant tissues and soil
organic matter in the ﬁrst stages of a vegetation succession. Geoderma 100, 1–24.
Ohara, S., Yasuta, Y., Ohi, H., 2003. Structure elucidation of condensed tannins from
barks by pyrolysis/ gas chromatography. Holzforschung 57, 145–149.
Otto, A., Simpson, M.J., 2006. Evaluation of CuO oxidation parameters for determining
the source and stage of lignin degradation in soil. Biochemistry 80, 121–142.
Otto, A., Simpson, M.J., 2007. Analysis of soil organic matter biomarkers by sequential
chemical degradation and gas chromatography–mass spectrometry. Journal of
Separation Science 30, 272–282.
Pouwels, A.D., Tom, A., Eijkel, G.B., Boon, J.J., 1987. Characterisation of beech wood and
its holocellulose and xylan fractions by pyrolysis-gas chromatography–mass
spectrometry. Journal of Analytical and Applied Pyrolysis 11, 417–436.
Preston, C.M., Hempﬂing, R., Schulten, H.-R., Schnitzer, M., Trofymow, J.A., Axelson,
D.E., 1994. Characterization of organic matter in a forest soil of coastal British
Columbia by NMR and pyrolysis-ﬁeld ionization mass spectrometry. Plant and
Soil 158, 69–82.
Preston, C.M., Trofymow, J.A., Sayer, B.G., Niu, J., 1997. 13C nuclear magnetic resonance
spectroscopy with cross-polarization and magic-angle spinning investigation of
the proximate-analysis fractions used to assess litter quality in decomposition
studies. Canadian Journal of Botany 75, 160–1613.
Preston, C.M., Bhatti, J.S., Flanagan, L.B., Norris, C., 2006a. Stocks, chemistry, and sensitivity to climate change of dead organic matter along the Canadian Boreal Forest
Transect Case Study. Climatic Change 74, 223–251.
Preston, C.M., Trofymow, J.A., Flanagan, P.W., 2006b. Decomposition, 13C, and the
“lignin paradox”. Canadian Journal of Soil Science 86, 235–245.
Preston, C.M., Nault, J.R., Trofymow, J.A., 2009. Chemical changes during 6 years of
decomposition of 11 litters in some Canadian forest sites. Part 2. 13C abundance,
solid-State 13C NMR spectroscopy and the meaning of “lignin”. Ecosystems 12,
1078–1102.
Räisänen, U., Pitkänen, I., Halttunen, H., Hurtta, M., 2003. Formation of the main degradation compounds from arabinose, xylose, mannose and arabinitol during pyrolysis. Journal of Thermal Analysis and Calorimetry 72, 481–488.
Rosenberg, W., Nierop, K.G.J., Knicker, H., de Jager, P.A., Kreutzer, K., Weiß, T., 2003.
Liming effects on the chemical composition of the organic surface layer of a mature
Norway spruce stand (Picea abies [L.] Karst.). Soil Biology and Biochemistry 35,
155–165.
Rumpel, C., Eusterhues, K., Kögel-Knabner, I., 2004. Location and chemical composition
of stabilized organic carbon in topsoil and subsoil horizons of two acid forest soils.
Soil Biology and Biochemistry 36, 177–190.
Ryan, M.G., Melillo, J., Ricca, A., 1990. A comparison of methods for determining
proximate carbon fractions of forest litter. Canadian Journal of Forest Research
20, 166–171.
Schimel, D.S., Braswell, B.H., Holland, E.A., McKeown, R., Ojima, D.S., Painter, T.H.,
Parton, W.J., Townsend, A.R., 1994. Climate, edaphic and biotic controls over storage and turnover of carbon in soils. Global Biogeochemical Cycles 8, 279–293.
Schnitzer, M., Preston, C.M., 1983. Effects of acid hydrolysis on the 13C NMR spectra of
humic substances. Plant and Soil 75, 201–211.
Shaver, G.R., Giblin, A.E., Nadelhoffer, K.J., Thieler, K.K., Downs, M.R., Laundre, J.A.,
Rastetter, E.B., 2006. Carbon turnover in Alaskan tundra soils: effects of organic
matter quality, temperature, moisture and fertilizer. Journal of Ecology 94,
740–753.
Sjöberg, G., 2003. Long-term effects of nitrogen addition on decomposition of forest
soil organic matter. Doctoral thesis, Swedish University of Agricultural Sciences.
Uppsala. Acta Universitatis Agriculturae. Agraria, 419, 46.
Sjöberg, G., Nilsson, S.I., Persson, T., Karlsson, P., 2004. Degradation of hemicelluloses,
cellulose and lignin in decomposing spruce needle litter in relation to N. Soil Biology
and Biochemistry 36, 1661–1768.
Stark, S., Hilli, S., Willför, S., Smeds, A.I., Reunanen, M., Penttinen, M., Hautajärvi, R.,
2012. Composition of lipophilic compounds and carbohydrates in the accumulated
plant litter and soil organic matter in boreal forests. European Journal of Soil
Science 63, 65–74.
van der Heijden, E., Boon, J.J., 1994. A combined pyrolysis mass spectrometric and light
microscopic study of peatiﬁed Calluna wood isolated from raised bog peat
deposits. Organic Geochemistry 22, 903–919.
Vargas, D.N., Bertiller, M.B., Ares, J.O., Carrera, A.L., Sain, C.L., 2006. Soil C and N dynamic
induced by leaf-litter decomposition of dwarf shrubs and perennial grasses of the
Patagonian Monte. Soil Biology and Biochemistry 37, 1–13.
Vorob'ev, A.V., Grishko, V.V., Ivshina, I.B., Schmidt, E.N., Pokrovskii, L.M., Kuyukina, M.S.,
Tolstikov, G.A., 2000. Microbial transformations of diterpene acids. Mendeleev
Communications 11, 72–73.
Wardle, D.A., Nilsson, M.-C., Zackrisson, O., Gallet, C., 2003. Determinants of litter mixing
effects in a Swedish boreal forest. Soil Biology and Biochemistry 35, 827–835.
Weintraub, M.N., Schimel, J.P., 2003. Interactions between carbon and nitrogen mineralization and soil organic matter chemistry in arctic tundra soils. Ecosystems 6,
129–143.
Winkler, A., Haumaier, L., Zech, W., 2005. Insoluble alkyl carbon components in soils
derive mainly from cutin and suberin. Organic Geochemistry 36, 519–529.