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Degradation of hemicellulose, cellulose and lignin in decomposing
spruce needle litter in relation to N
G. Sjoberga,*, S.I. Nilssona, T. Perssonb, P. Karlssonb
aDepartment of Soil Sciences, Swedish University of Agricultural Sciences, P.O. Box 7014, SE-750 07 Uppsala, SwedenbDepartment of Ecology and Environmental Research, Swedish University of Agricultural Sciences, P.O. Box 7072, SE-750 07 Uppsala, Sweden
Received 7 July 2003; received in revised form 16 March 2004; accepted 29 March 2004
Abstract
Decomposing needles from a Norway spruce forest in southern Sweden were studied for 559 days under laboratory conditions. Falling
needles were collected in control (Co) plots and plots that had received 100 kg N ha21 yr21 as (NH4)2SO4 for 9 years under field
conditions. One of the aims was to determine whether the previously documented low decomposition rate of the N fertilized (NS)
needles could be explained by a lower degradation degree of lignin. The lignin content was studied using the alkaline CuO oxidation
method, the Klason lignin method and CPMAS 13C NMR spectroscopy. The amounts of cellulose and hemicellulose were also
determined.
The fertilized needle litters initially decomposed faster than the unfertilized, but later this reaction reversed, so that at the end the
mass loss was 45% initial C in the control and 35% initial C in NS. Klason lignin decreased with time in both treatments and overall,
the change of Klason lignin mirrored the litter mass loss. No major difference as regards the decomposition of hemicellulose occurred
between the treatments, whereas significantly lower concentrations of cellulose were found in NS needles throughout the incubation. The
CuO derived compounds (VSC) were somewhat lower in NS needles throughout the decomposition time. Initially, VSC increased
slightly in both treatments, which contradicts the Klason lignin data. There was a weak positive relationship ðp . 0:05Þ between VSC
and Klason lignin. Both vanillyls compounds (V) and cinnamyl compounds (Ci) increased slightly during decomposition, whereas
syringyl compounds (S) vanished entirely. The lignin degradation degree, i.e. the acid-to-aldehyde ratio of the vanillyl compounds
expressed as (Ac/Al)v, showed no significant effect of treatment. The 13C NMR analyses of the combined samples showed increased
content of aromatic C with increasing decomposition time. The carbohydrate content (O–alkyl C) was lower in the fertilized needle
litter throughout the incubation time. The alkyl C content tended to increase with decomposition time and N fertilization. The alkyl
C/O–alkyl C ratios increased in both treatments during the incubation. The NMR results were not tested statistically.
In conclusion, no major difference concerning lignin degradation could be found between the unfertilized and N fertilized needle
litter. Thus, the study contradicts the hypothesis that higher amounts of N reduce lignin degradation. The reduced biological activity is
probably due to direct N effects on the microorganisms and their decomposing ability.
q 2004 Elsevier Ltd. All rights reserved.
Keywords: Norway spruce; Needle litter; Lignin; Cellulose; CuO; Klason lignin; 13C NMR
1. Introduction
Studies concerning long-term effects of nitrogen (N)
additions to forests soils have shown that there are reduced
decomposition rates in N-fertilized litter and mor humus
(Nohrstedt et al., 1989; Persson et al., 2000, 2001; Michel
and Matzner, 2002; Sjoberg et al., 2003). There are several
explanations for this reduction of decomposition rates in
the presence of N:
1. Formation of more recalcitrant forest floor material after
complexation between N and polyphenols (Nommik and
Vahtras, 1982; Kelley and Stevenson, 1996).
2. Reduced lignin degradation activity by white-rot fungi
(Keyser et al., 1978; Carreiro et al., 2000).
3. Effects on the diversity among microbial species
(Lilleskov et al., 2001) and a change towards more
efficient N-assimilating microorganisms (Agren et al.,
2001).
0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.soilbio.2004.03.010
Soil Biology & Biochemistry 36 (2004) 1761–1768
www.elsevier.com/locate/soilbio
* Corresponding author. Tel.: þ46-18-67-1220; fax: þ46-18-67-2795.
E-mail address: [email protected] (G. Sjoberg).
Magill and Aber (1998) studied long-term N effects using
litterbags in Harvard Forest, Massachusetts. They showed
that N-treated litter had significantly higher remaining mass
content as well as lignin content than untreated litter after 6
years of decomposition. Furthermore, Sjoberg et al. (2004)
found that there was a significantly higher amount of
phenolic carbon (C) in the litter layer at the Skogaby site in
southern Sweden that had received N. Whether this is due to
a chemical stabilization of C compounds present and/or a
biological effect on decomposers still needs further
investigation.
Reactions between N and phenolic compounds have been
shown to occur, since NH3–N (Nommik and Vahtras, 1982)
and amino acids (Kelley and Stevenson, 1996) could be
bound to aromatic ring structures. However, since such
phenolic N compounds have not been detected using
CPMAS 15N NMR (Knicker and Ludemann, 1995; Sjoberg,
unpublished thesis; Sjoberg et al., 2004) they do not seem to
be of major importance for the recalcitrance of soil organic
matter in forest ecosystems. Several studies have shown that
white-rot fungi are suppressed by high additions of N (Keyser
et al., 1978; Carreiro et al., 2000). For instance, Keyser et al.
(1978) found a reduced lignolytic activity when studying the
wood-decaying white-rot fungus, Phanerochaete chrysos-
porium, in the presence of NH4þ. In contrast, a study on
another wood-decaying white-rot fungus, Bjerkandera
adusta, showed an enhanced lignin degrading ability in the
presence of NH4þ (Kaal et al., 1995). However, concerning
needle litter decomposition, other species than wood-rotting
fungi are more important (Steffen et al., 2000; Steffen,
unpublished thesis). For instance, Marasmius androsaceus
(Cox et al., 2001) and Mycena galopus are litter-decompos-
ing fungi present in newly fallen needle litter and litter layers
(Frankland, 1998). However, what effect N has on these
litter-decomposing fungi is still unknown.
By combining theory and results obtained through litter
decomposition experiments with fertilization of N, Agren
et al. (2001) came to the conclusion that the explanation for
a reduced decomposition rate after long-term additions of N
could be a change in the microbial composition towards
more N-demanding and efficient N-assimilating microor-
ganisms. Therefore, less energy would be used for
assimilating N, which may explain a lower soil respiration.
Karlsson (unpublished thesis) found significantly lower
C mineralization (CO2 evolution) in laboratory incubated
N-rich needles than in N-poor needles on a long-term basis.
The aim of our study was to test whether the reduced
evolution of CO2 in the N-rich needles was accompanied by
an increased content of lignin and thus a reduced
degradation degree of lignin. To test this, we determined
CuO derived phenolic compounds (Kogel, 1986; Sjoberg
et al., 2004), Klason lignin and also the carbohydrates
hemicellulose and cellulose (Theander and Westerlund,
1986). The organic C chemistry was further characterized
by solid-state CPMAS 13C NMR spectroscopy (Kogel et al.,
1988; Sjoberg et al., 2004).
2. Materials and methods
2.1. Study area
In May 1997, needle litter was collected from control
(Co) plots and fertilized (NS) plots that had received annual
doses of 100 kg N ha21 as (NH4)2SO4 since 1988 in the
Skogaby experiment. This experiment is situated in south-
western Sweden (568330N;138130E) in an area with Norway
spruce (Picea abies (L.) Karst.) planted in 1966. The plots
are randomized within four blocks ðn ¼ 4Þ and each plot has
a size of 45 £ 45 m2 (Bergholm et al., 1995). When the
needle litter was collected, the NS plots had received in total
900 kg N ha21.
2.2. Laboratory incubated needle litter
In this study, we examined further needle litter
materials originating from an incubation study by
Karlsson (unpublished thesis). Needle litter was collected
on 5 m £ 10 m sheets of fibre cloth placed on the ground
for a period of 2 weeks in May 1997 in the Co and NS
plots. Immediately after the collection, the needles were
bulked into one Co and one NS sample. The needle
litter was then air-dried and cleaned of twigs, cones and
green needles. The bulked samples were divided into
pseudo-replicated subsamples that were placed in plastic
jars (50 cm2 surface area, 466 cm3 volume) with air-
exchanging lids. For each sampling event, three replicate
jars were prepared. Totally, there were 18 jars. Because of
this procedure involving the use of pseudo-replicates, no
field variation could be taken into consideration. Previous
studies (Nilsson and Wiklund, 1994; Sjoberg et al., 2004)
have shown, however, that there is only a minor variation
concerning C-to-N ratios in needle litter within the Co and
NS plots at Skogaby.
The litter material in each jar was moistened with 8 ml
of distilled water and preincubated at 5 8C for 1 week.
Thereafter, a suspension was prepared by shaking 10 g fw
of litter, mor humus and 1 l of distilled water and from this
suspension 5-ml portions were extracted and added to each
jar in order to inoculate bacteria and fungi to the needle
samples. The incubation was then carried out at 15 8C for
559 days, corresponding to about 4 years of decomposition
in the field (Persson et al., 2000). The evolution of CO2 was
repeatedly analyzed during the incubation by gas chroma-
tography according to Persson and Wiren (1993). The mass
of C evolved per jar and hour was calculated after taking
into account the pH-dependent solubility of CO2 in the soil
water (Persson et al., 1989). Total C and N contents and
inorganic N (NH4þ and NO3
2 after extraction in 1 M KCl)
were analyzed on needle samples from the days 0, 179 and
559 of the incubation. After each sampling event, the
needle samples were immersed in liquid N, freeze-dried
and stored in a freezer at 220 8C prior to further analyses
(see below).
G. Sjoberg et al. / Soil Biology & Biochemistry 36 (2004) 1761–17681762
2.3. Wet chemical and 13C NMR techniques
The pseudo-replicated subsamples from days 0, 179 and
559 were analyzed by using wet chemical techniques for the
determination of cellulose, hemicellulose, Klason lignin and
CuO derived lignin components. Bulked samples (i.e. a
mixture of the three pseudo-replicates) were characterized
by CPMAS 13C NMR spectroscopy.
The CuO oxidation method was performed according to
Kogel (1986), and the derived lignin products were
separated and determined with HPLC (Fig. 4) according
to Sjoberg et al. (2004). The sum of the CuO oxidation
products was expressed as VSC, i.e. Vanillyl þ Syringyl þ
Cinnamyl (Kogel, 1986). The vanillyl compounds (V) are
vanillic acid and vanillin, the syringyl compounds (S) are
syringic acid and syringaldehyde and the cinnamyl
compounds (Ci) are p-coumaric acid and ferulic acid.
Therefore, the other derived CuO compounds benzoic acid,
benzaldehyde, acetophenone, acetovanillone, acetosyrin-
gone and ethyl vanillin shown in Fig. 4 were not included
in VSC (Kogel, 1986; Ziegler et al., 1986; Sjoberg
et al., 2004). Within the vanillyl compounds, the ratio
between carboxylic acid (Ac) and aldehyde (Al) side
groups, i.e. (Ac/Al)V, was used as an index of the
degradation degree of lignin (Kogel-Knabner et al., 1988)
in the needle litter. The vanillin is oxidized to vanillic acid
during degradation, resulting in an increasing ratio with
time. For internal standards, 250 mg l21 vanillin was
dissolved in 2 M NaOH and 0.01 M HCl, respectively
(Sjoberg et al., 2004). Losses of vanillin during the CuO
oxidation procedure and the following HPLC analysis were
3–8% for vanillin dissolved in NaOH and 3–6% for
vanillin dissolved in HCl. The HPLC analysis is further
described in Sjoberg et al. (2004).
Hemicellulose, cellulose and Klason lignin were deter-
mined by the Swedish Pulp and Paper Research Institute
(STFI) using a method described by Theander and
Westerlund (1986). By using acid hydrolysis with 72%
H2SO4, dilution and refluxing with dilute acid (Bethge et al.,
1971; Berg et al., 1982) an insoluble organic residue
(Klason lignin) was obtained. Hemicellulose was deter-
mined as the sum of arabinose, xylose, mannose and
galactose, whereas cellulose was determined as the total
amount of glucose.
Solid state CPMAS 13C NMR spectroscopy was
performed on bulked samples of the Co and NS needle
litter at the Technical University in Munich, Germany
(Sjoberg et al., 2004). The spectra were obtained on a
Bruker DSX 200 operating at a frequency of 50.3 MHz
using zirconium rotors of 7 mm OD with KEL-F-caps. The
CPMAS technique was applied during magic-angle spin-
ning of the rotor at 6.8 kHz. A ramped 1H-pulse of 908 width
and of 5.3 ms was used during the contact time (1 ms). The13C chemical shifts were calibrated relative to tetramethyl-
silane and glycine.
Results of CuO derived lignin, Klason lignin, cellulose
and hemicellulose (Tables 1 and 2) are given in mg g21
initial C, which we have assumed to be two times the value
expressed as mg g21 organic matter.
2.4. Statistical analysis
Before the incubation started (Karlsson, unpublished
thesis), the needle litters from Co and NS plots were bulked
Table 1
Mean (^SD) concentrations of hemicellulose, cellulose, Klason lignin and
a residual fraction (mg dry matter g21 initial C) in incubated unfertilized
(Co) and fertilized (NS) needle litter
Time Treatment Hemicellulose Cellulose Klason
lignin
Residual
fractiona
0 Co 338 ^ 0a 463 ^ 6a 783 ^ 6a 353 ^ 1a
NS 315 ^ 0a 390 ^ 21b 802 ^ 4a 401 ^ 18a
179 Co 261 ^ 1a 303 ^ 1a 767 ^ 3a 152 ^ 10a
NS 222 ^ 18a 230 ^ 24b 727 ^ 11b 134 ^ 14a
559 Co 148 ^ 1a 166 ^ 3a 631 ^ 20a 183 ^ 1a
NS 152 ^ 7a 145 ^ 3b 698 ^ 38a 247 ^ 39a
Incubation times were 0, 179 and 559 days. Means ðn ¼ 2Þ with
different letters (a and b) within each column and day indicate a significant
difference ðp , 0:05Þ:a The remaining fraction containing ash and other organic compounds.
Table 2
Mean (^SD) concentrations of the sum of CuO oxidation products (VSC), vanillyl compounds (V), syringyl compounds (S), cinnamyl compounds (Ci) and
degradation degree of vanillyl compounds (Ac/Al)v) in unfertilized (Co) and fertilized (NS) needle litter (mg dry matter g21 initial C) during incubation on
days 0, 179 and 559
Time Treatment VSC V S Ci (Ac/Al)v
0 Co 57.87 ^ 0.52a 41.77 ^ 0.30a 15.47 ^ 0.90a 0.63 ^ 0.11a 0.21 ^ 0.01a
NS 56.53 ^ 0.56a 42.89 ^ 1.09a 13.01 ^ 1.28b 0.63 ^ 0.24a 0.21 ^ 0.00a
179 Co 61.67 ^ 5.58a 49.72 ^ 0.72a 9.58 ^ 8.30 2.37 ^ 2.35a 0.22 ^ 0.01a
NS 57.13 ^ 1.65a 52.40 ^ 1.71a – 4.73 ^ 0.10a 0.21 ^ 0.00a
559 Co 54.30 ^ 1.50a 49.93 ^ 1.30a – 4.37 ^ 0.25a 0.23 ^ 0.01a
NS 53.78 ^ 1.05a 48.98 ^ 0.82a 0.14 ^ 0.25 4.66 ^ 0.37a 0.22 ^ 0.02a
Means ðn ¼ 3Þ with different letters (a and b) indicate a significant difference ðp , 0:05Þ: The limit for significance ðn ¼ 3Þ is set at p , 0:05: Non-detected
CuO oxidation products are marked with-signs.
G. Sjoberg et al. / Soil Biology & Biochemistry 36 (2004) 1761–1768 1763
to one composite Co sample and one NS sample followed by
division into pseudo-replicated subsamples (n ¼ 3 jars).
The variables investigated were statistically analyzed by
one-way analysis of variance (SAS, 1989) with treatments
(Co and NS) as factors. The limit for statistical significance
was set at p , 0:05: The analysis of the CuO oxidation
products (VSC) was based on the three pseudo-replicated
samples per treatment and sampling event, whereas the
analyses of Klason lignin, hemicellulose and cellulose were
based on two of the three pseudo-replicated subsamples.
3. Results
Klason lignin, hemicellulose and cellulose fractions did
not add up to 100% of the total mass. The remaining fraction
(Table 1) originated from ash and organic compounds not
accounted for hemicellulose and cellulose decreased during
the incubation (Table 1). Significantly lower ðp , 0:05Þ
concentrations of cellulose were found in the fertilized
needles than in the unfertilized needles throughout the
incubation. Both mass loss and the change in Klason lignin
concentration were initially higher in the fertilized needles
than in the unfertilized (Table 1, Fig. 1). Later, the opposite
trend occurred since the highest losses were obtained in the
unfertilized needles. The change in lignin with time can also
be seen in Fig. 2, by comparing CuO derived lignin (VSC)
and Klason lignin. During the first part of the incubation,
VSC increased slightly in both treatments whereas a decline
occurred after day 179 (Table 2, Fig. 2). In contrast, no such
increase in lignin could be seen when using the Klason
lignin method (Table 1, Fig. 2). Furthermore, Klason lignin
and VSC were not significantly correlated to each other
(Fig. 3).
VSC tended to be lower in the fertilized needles compared
to the control needles throughout the decomposition time
(Table 2). When taking the separate CuO derived compounds
into consideration, the cinnamyl compounds (Ci) as well as
the vanillyl compounds (V) increased with increasing
decomposition time (Table 2). Within the vanillyl com-
pounds, vanillin dominated both initially and by the end of
the incubation. The cinnamyl compounds were only based on
p-coumaric acid, since ferulic acid could not be detected
(Fig. 4). The syringyl compounds (S) were quickly decom-
posed during the incubation and vanished almost entirely
(Table 2). Initially, significantly lower ðp , 0:05Þ concen-
trations of syringyl compounds were obtained in the NS
needles than in the control needles. Within the syringyl
compounds, syringic acid could not be detected whereas
syringaldehyde was only detectable initially (Fig. 4). The
ratio between vanillic acid and vanillin, expressed as
(Ac/Al)v, showed little difference (Table 2), but tended to
increase at a higher rate in the unfertilized needles than in the
fertilized ones throughout the incubation time ðp . 0:05Þ:
The 13C NMR spectra showed a tendency for increased
aromatic C with increased decomposition time (Table 3).
Fig. 2. Remaining amount (% of initial) of CuO derived products (VSC) and
the amount of Klason lignin in unfertilized (Co) and fertilized (NS) needle
litter during an incubation time of 559 days (n ¼ 2 for Klason lignin; n ¼ 3
for CuO oxidation).
Fig. 1. Remaining amount of litter (Karlsson, unpublished thesis) and
Klason lignin expressed as % of initial C in unfertilized (Co) and fertilized
(NS) needle litter ðn ¼ 2Þ during an incubation time of 559 days.
Fig. 3. Correlation between mean values of Klason lignin and VSC (mg g21
initial C) in unfertilized (Co) and fertilized (NS) needle litter during a 559-
day incubation (n ¼ 2 for Klason lignin; n ¼ 3 for CuO oxidation).
G. Sjoberg et al. / Soil Biology & Biochemistry 36 (2004) 1761–17681764
However, the phenolic C did not increase at all. The content
of carbohydrates (O– alkyl C) decreased during the
decomposition and the content was less in the fertilized
needle litter. The alkyl C content tended to increase with
decomposition time and N fertilization. The alkyl/O–alkyl
ratios increased during incubation from 0.27 to 0.38 and
from 0.28 to 0.46 in the control and fertilized needles,
respectively.
4. Discussion
The (Ac/Al)v ratio is commonly used as a degradation
index and a value of 0.2 is typical for lignin derived from
undecomposed vascular plant material (Ertel and Hedges,
1984). The ratio did not change much with time in either the
Co or the NS treatment (Table 2). Consequently, the CuO
oxidation method showed no major lignin degradation
during the incubation and no reduction in lignin degradation
could be detected as a consequence of the elevated N
concentration.
We estimated the VSC value to be 58 mg g21 initial C
(29 mg g21 organic matter), which is higher than the VSC
value reported by Johansson et al. (1986) for spruce needle
litter in a litterbag study (24 mg g21 organic matter).
However, the latter authors did not include the syringyl
compounds (syringaldehyde and syringic acid) in their
study, which might explain their lower value. As regards the
development of lignin degradation with time (Fig. 1), both
the CuO oxidation and the Klason lignin methods showed
results, which were in accordance with Johansson et al.
(1986). Hypothetically, VSC derived from the NS needle
litter would reach a steady state with time as was found for
Klason lignin, which eventually might have yielded more
remaining VSC derived from the fertilized needles com-
pared to the unfertilized ones (Fig. 2). The correlation
between the CuO oxidation method and the Klason lignin
method demonstrated a weak positive relationship ðp .
0:05Þ; which was also the case in the litterbag study by
Johansson et al. (1986).
Vanillyl compounds are the dominating group within the
CuO derived compounds from spruce lignin. The increases
in vanillyl compounds and cinnamyl compounds with time
(Table 2) seemed to be the main reason for the initial
relative increase in VSC compared to the Klason lignin
(Fig. 2). The syringyl compounds (Table 2) were
initially lower in the N fertilized litter and disappeared
almost entirely during the study in both treatments,
probably because syringaldehyde became demethoxylated
(R–OCH3 ! R–H) during decomposition (H. Knicker;
pers. comm.). Demethoxylation of syringyl compounds,
which contain two OCH3 groups, could theoretically also
increase the amount of vanillyl compounds (which contain
one OCH3 group). Compared to vanillyl compounds,
syringyl compounds are more rapidly decomposed (Ander
et al., 1984; Hedges and Weliky, 1989). The vanillyl
compounds tended to increase after 179 days in our study
(Table 2). The dominance of vanillin within the vanillyl
group, both initially and by the end of the incubation (Fig. 4),
is in accordance with previous studies of gymnosperm
lignin (Kogel et al., 1988; Sanger et al., 1996, 1997; Sjoberg
et al., 2004). Hedges et al. (1988) investigated the ability of
white-rot fungi to degrade CuO derived oxidation products
Fig. 4. The distribution pattern among the CuO oxidation products (mg g21
initial C). The products are derived from unfertilized needle litter ðn ¼ 3Þ
on day 0 and 559: benzoic acid (1), vanillic acid (2), syringic acid (3),
benzaldehyde (4), acetophenone (5), vanillin (6), p-coumaric acid (7),
syringaldehyde (8), acetovanillone (9), ferulic acid (10), acetosyringone
(11) and ethyl vanillin (12). The CuO products 3, 10 and 12 were below the
detection limit.
Table 3
Organic C composition (%) analyzed with CPMAS 13C NMR in bulked ðn ¼ 3Þ samples of unfertilized (Co) and fertilized (NS) needle litter
Time Treatment Carbonyl C Aromatic C Phenol C O–alkyl C Alkyl C
220–160 ppm 160–110 ppm 160–140 ppm 110–45 ppm 45–0 ppm
0 Co 5 16 6 62 17
NS 6 17 6 60 17
179 Co 4 15 4 61 20
NS 6 18 5 54 22
559 Co 6 18 5 55 21
NS 8 19 6 50 23
The chemical shifts are given in ppm. Phenol-C (160–140 ppm) is included in aromatic-C (160–110 ppm).
G. Sjoberg et al. / Soil Biology & Biochemistry 36 (2004) 1761–1768 1765
from birch wood. These authors found an increasing trend of
vanillic acid, since the mass-normalized yields of vanillic
acid increased from 0.15 to 0.20% by weight after 12 weeks.
They interpreted the data as a result of microbial oxidation
that had no direct connection to the ring cleavage that
otherwise occurs during lignin degradation.
Within the cinnamyl group, p-coumaric acid (4-hydro-
xycinnamic acid) was the dominating compound whereas
ferulic acid (4-hydroxy-3-methoxycinnamic acid) was
below the detection limit (Fig. 4). The tendency for an
increase in cinnamyl compounds with increasing decompo-
sition time (Table 2), contradicts the results of Johansson
et al. (1986) and Hedges and Weliky (1989). One hypothesis
concerning increasing trends of cinnamyl compounds could
hypothetically be fungal growth and melanin production
during the incubation. Melanin is a macromolecular
component built up by phenolic monomers and is found
within cell walls of various fungi and bacteria (Butler and
Day, 1998a). Melanin is believed to function as a protector
against microbial decomposition (Kogel-Knabner, 2002)
and can be degraded by white-rot fungi and their lignin-
degrading peroxidase enzymes (Butler and Day, 1998b). We
have no good explanation for the increase of cinnamyl
compounds with time in our study and the decrease with
time in the litterbag studies of Johansson et al. (1986);
Hedges and Weliky (1989). The only differing factor we can
identify is that the needle litter in our study was kept at
constantly high (15 8C) temperature and moisture con-
ditions, whereas the litter in the litterbags kept in the field
was more affected by varying temperature and moisture
conditions. The rapid decomposition of hemicellulose and
cellulose in our study (Table 1) was in accordance with the
6-year litterbag study of pine needle litter by Melillo et al.
(1989). Carreiro et al. (2000) found that cellulase activity
was stimulated by N addition.
At later decomposition stages, more resistant compounds
such as lignin are slowly decomposed (Kirk and Farrell,
1987). The Klason lignin method is believed to be more
reliable in estimating lignin for woody material than for
needle litter material due to an overestimation caused by the
contents of cutin and suberin (Zech et al., 1987) as well as
tannins (Preston et al., 1997) in the latter material.
During the 559 days of incubation, the C-to-N ratio
decreased from 55 to 34 in the unfertilized needles and
from 23 to 17 in the fertilized needles (Karlsson,
unpublished thesis). The fertilized needle litter initially
decayed faster than the unfertilized, but during the later
stages of decomposition the situation became reversed,
resulting in a higher mass loss in the unfertilized litter
than in the fertilized (Fig. 1). This is in accordance with
a previous study by Berg et al. (1982), which showed
lower mass loss rates in N-rich litter than in N-poor litter
during long-term decomposition. Karlsson (unpublished
thesis) hypothesized that the lower release of CO2 from
the fertilized needle litter than from unfertilized
litter might have been caused by high concentrations of
NH4–N and NO3–N in the former litter type. The real
cause is not entirely clear, but the drop in CO2 evolution
rate came after day 57 and coincided with the first
appearance of NO32. The pH in the NS needle litter
increased to almost eight at day 57 (due to extensive
ammonia formation) and thereafter dropped to four after
about 300 days as a consequence of nitrification. Thus,
processes associated with ammonification or nitrification
might have been responsible for the drop in microbial
activity in the later part of the incubation.
CPMAS 13C NMR spectroscopy on decomposed needle
litter in litterbags has previously been used by Zech et al.
(1987), Kogel et al. (1988), Norden and Berg (1990), Lorenz
et al. (2000) to study biochemical changes. The degree of
decomposition can be expressed as the ratio between alkyl C
and O–alkyl C (Zech et al., 1987; Baldock et al., 1997;
Lorenz et al., 2000). Zech et al. (1987) showed increased
ratios with time when studying spruce and pine litter
decomposition. Furthermore, Lorenz et al. (2000) studied
Norway spruce needle litter and found an increased ratio
with increasing soil depth and decomposition. The same
was also seen in our study where the ratio increased on
average from 0.35 to 0.50. One explanation is that during
decomposition, O–alkyl C decreases due to microbial
consumption of polysaccharides, while alkyl C represents
more recalcitrant compounds.
5. Conclusions
Cellulose decomposed faster in N-rich litter than in
control litter. There was no clear evidence of reduced lignin
degradation in the N-rich litter according to the CuO
oxidation method, the Klason lignin method or the
qualitative analysis using 13C NMR. The conclusion is
therefore that we could not find a higher amount of lignin in
the N fertilized needles than in the unfertilized. The
common hypothesis that N addition increases the recalci-
trance of soil organic matter could, therefore, not be
supported. The exact mechanisms are not clear, and further
studies are needed to reveal the factors that are causing the
reduction in biological activity.
Acknowledgements
We thank Heike Knicker for running the CPMAS 13C
NMR analyses and for useful comments concerning the
results obtained by alkaline CuO oxidation as well as
CPMAS 13C NMR. Financial support was provided by
the Swedish Research Council for the Environment,
Agricultural Science and Spatial Planning (FORMAS).
G. Sjoberg et al. / Soil Biology & Biochemistry 36 (2004) 1761–17681766
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