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Soil Biology & Biochemistry 32 (2000) 779±792
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Litter decomposition and humus characteristics in Canadian and
German spruce ecosystems: information from tannin analysis and
13
C CPMAS NMR
Klaus Lorenz a,*, Caroline M. Preston b, Stephan Raspe a, Ian K. Morrison c,
Karl Heinz Feger a, 1
a

Institut fuÈr Bodenkunde und WaldernaÈhrungslehre, UniversitaÈt Freiburg, Bertoldstr. 17, 79085 Freiburg, Germany
b
Paci®c Forestry Centre, Natural Resources Canada, 506 West Burnside Rd., Victoria, BC, Canada V8Z 1M5
c
Great Lakes Forestry Centre, Natural Resources Canada, 1219 Queen St. E., Sault Ste. Marie, Ont., Canada P6A 5M7
Accepted 18 October 1999

Abstract
In¯uences of litter and site characteristics were investigated during the decomposition of black spruce (Picea mariana (Mill.)
B.S.P.) and Norway spruce (Picea abies (L.) Karst.) needle litter in litterbags in two black spruce sites in Canada (6 and 12

months) and two Norway spruce sites in Germany (6 and 10 months). Mass losses were greater for black spruce litter (mean
25.2%) than for Norway spruce (20.8%), despite lower quality of black spruce litter in terms of lower N (10.1 versus 17.1 mg
gÿ1), higher C-to-N ratio (49.0 versus 30.3) and higher content of alkyl C (surface waxes and cutin), indicated by CPMAS 13C
NMR spectroscopy. However, Norway spruce litter was higher in condensed tannins than black spruce (37.8 and 25.3 mg gÿ1,
respectively). Tannins were lost rapidly from both species, especially in the ®rst 6 months, with losses in 10±12 months of 75±
89% of the fraction extractable in acetone/water and 40±70% of the residual fraction. Losses were greater in the German sites
(mean 75.2%, 10 months, versus 68.4%, 12 months), which had earthworms present and higher temperature, precipitation and
catalase activity, the latter being positively correlated with tannin loss. There was a much larger contrast in the organic layers;
with the Canadian sites having lower C-to-N ratios and higher N concentrations (C-to-N, 20.3 and 29.7; N, 26.0 and 13.8 mg
gÿ1 for Canadian and German sites, respectively). The 13C NMR spectra showed that they were poorly decomposed and
unusually high in condensed tannins (consistent with chemical analysis of 28.7 and 37.6 mg gÿ1, Canada; and 3.5 and 5.0 mg
gÿ1, Germany), with depletion of lignin structures. Di€erences in other inputs (bark, wood, roots, understorey vegetation) and
in site properties (climate, decomposer community, earthworm activity) may be responsible for the considerable di€erences in
humus properties, which would not be expected from di€erences in the chemical composition and short-term decomposition of
needle litter. The tannin accumulation, lignin depletion and N sequestration in the black spruce sites may be related to
accumulation of unavailable N and associated forest management problems in these ecosystems. 7 2000 Elsevier Science Ltd.
All rights reserved.
Keywords: Condensed tannins;

13

C CPMAS NMR; Litter decomposition; Black spruce; Norway spruce; Catalase; Forest ¯oor

1. Introduction
* Corresponding author. Present address: Institut fuÈr Bodenkunde
und Standortslehre, UniversitaÈt Hohenheim, Emil-Wol€-Str. 27,
70593 Stuttgart, Germany. Tel.: +49-711-459-4066; fax: +49-711459-4067.
E-mail address: lorenzk@uni-hohenheim.de (K. Lorenz).
1
Present address: Institut fuÈr Bodenkunde und Standortslehre,
UniversitaÈt Hohenheim, Emil-Wol€-Str. 27, 70593 Stuttgart,
Germany

After climate, decomposition of leaf litter is in¯uenced by substrate quality factors, primarily nutrient
contents and organic composition (Moore et al., 1999).
Consideration of the latter is usually based on the
results of proximate analysis, especially the acid-sol-

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 2 0 1 - 1

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K. Lorenz et al. / Soil Biology & Biochemistry 32 (2000) 779±792

uble fraction (mainly re¯ecting cellulose and hemicellulose content) and the acid-insoluble residue (commonly referred to as lignin or Klason lignin ).
However, most tree and shrub litters also contain
considerable proportions of two other biopolymers,
cutin and tannin (Northup et al., 1995a; Ayres et al.,
1997; Preston et al., 1997; Preston, 1999). In addition
to constituting a signi®cant fraction of litter mass,
tannins may a€ect several aspects of ecosystem functioning. They can reduce the palatability and digestibility of plants to insects and herbivores (Schultz et
al., 1992) and inhibit microbial activity and N availability, possibly because of their protein-binding
properties (Horner et al., 1988; Kuiters, 1990;
Howard and Howard, 1993; Schimel et al., 1996,
1998). Recent studies on N release from litter of
many Mediterranean and tropical species have
emphasized the need for incorporating a measure of
polyphenol content or protein-binding into litter quality indices (Gallardo and Merino, 1993; Handayanto
et al., 1997; Mafongoya et al., 1998). It has also been

proposed that high tannin or polyphenolic content in
plants can provide a mechanism to conserve N in a
thick humus layer in nutrient-limited environments
(Northup et al., 1995a,b). However, despite great
interest in the role of condensed tannins in litter decomposition and ecosystem function, there have been
few studies providing unequivocal identi®cation and
quanti®cation of condensed tannins, or making full
use of the information available from solid-state 13C
nuclear magnetic resonance (NMR) spectroscopy.
In addition to litter quality and climate, decomposition in forest ecosystems is also in¯uenced by other

site factors including humus type, parent material and
faunal activity (Cortez, 1998). We studied decomposition of needle litter from two species, black spruce
(Picea mariana (Mill.) B.S.P.) and Norway spruce
(Picea abies (L.) Karst.). The litterbag study was carried out at two black spruce sites in Canada (6 and 12
months) and two Norway spruce sites in Germany (6
and 10 months). This design allowed comparison of
the e€ects of litter quality and site properties over two
periods, although scheduling problems did not allow
exact comparison of 1 year results. With increasing

stand age, northern black spruce forests develop nutrient limitation, at the same time as N is increasingly
sequestered in the forest ¯oor (Pastor et al., 1987;
Smith et al., 1998). In this study, in addition to
changes in litter mass, C and N, we characterized litter
and forest ¯oor samples using speci®c analyses for
condensed tannins, and solid-state 13C nuclear magnetic resonance spectroscopy with cross-polarization
and magic-angle spinning (CPMAS NMR).

2. Materials and methods
2.1. Plant materials
In Germany, litter decomposition was studied in two
experimental areas, Schluchsee and Villingen, located
at higher altitudes of the Black Forest (SW Germany)
(Project ARINUS; Feger et al., 1990; Armbruster,
1998; Raspe et al., 1998). The Canadian study areas,
Black Sturgeon and Lake Nipigon Forests, are located
at lower altitudes in northern Ontario (Foster et al.,

Table 1
Site properties of the four study areas

Canada

Latitude
Longitude
Elevation (m asl)
Mean annual temperature (8C)
Annual precipitation (mm)
Bedrock
Soil pro®le (FAO)
Humus form
O-layer
pH (H2O)
pH (CaCl2)
Dominant tree species
Stand age (yr)
a
b

Germany

Black Sturgeon

Lake Nipigon

Schluchsee

Villingen

49812'N
88843'W
300
0.2a
784a (43% snow)
Precambrian red sandstone
Ferro-humic podzol
Mor

49826'N
87848'W
450

0.2a
784a (43% snow)
Granite outcrop
Ferro-humic podzol
Mor

47849'N
886'E
1150±1250
4.5b
1910b (30% snow)
`BaÈrhalde' granite
Haplic podzol
Mull/moder

48849 'N
8822 'E
870±945
6.3b
1330b (25% snow)

Quartz-sandstone
Dystric cambisol
Mor

3.8
3.4
Black spruce
50

4.5
3.7
Black spruce
120

3.5
2.8
Norway spruce
50

3.4

2.7
Norway spruce
100

Climate data are 30-year normals from the nearby weather station at Beardmore, Ont. (49837'N, 87857'W; 305 m asl).
Climate data are 8-year normals (Armbruster, 1998).

K. Lorenz et al. / Soil Biology & Biochemistry 32 (2000) 779±792

1986; Scaratt, 1996). Table 1 shows general features of
the sites.
2.2. Sample collection and processing
In the summer of 1995, brown needles of logged
black spruce trees were collected at the Black Sturgeon
Forest study area in Canada. Meanwhile, brown needles of logged Norway spruce trees were collected at
the German site Schluchsee. After oven-drying (308C)
and sterilizing (fumigation for 72 h with 240 g methyl
bromide mÿ3), 10 g portions of needle litters were
placed in polyester net litterbags (12  12 cm; 1 mm
mesh). In November 1995, 40 litterbags were exposed

in four blocks (1 m2) at each of the Schluchsee, Villingen, Black Sturgeon and Lake Nipigon sites. In spring
and fall (after 6 and 10 months), 20 litterbags were collected at each German site. In Canada, after 6 months
20 litterbags were collected at each site and 12 litterbags after 12 months. Due to scheduling problems, incubation time at the German sites was shorter and in
the fall a smaller number of litterbags was collected at
each Canadian site. Litterbags were handsorted to
remove debris and mesofauna. Litter aliquots from
each bag were dried at 708C to constant mass and
weighed for calculation of mass loss. For the
proanthocyanidin assay and for chemical analysis,
dried composite samples from each site and for each
date of collection were ground in a Wiley mill to pass
through a 0.85 mm sieve. In autumn 1995, samples of
the organic layer were collected at all sites and were
treated in a manner similar to the litter samples. Also,
for each site and date of collection, ®eld-moist litter
was pooled into a composite sample to measure microbial activity (for consistency, moisture was adjusted
to 60% of water capacity).

781

balsam ®r (Abies balsamea (L.) Mill.) (Preston et al.,
1997). Samples of litter and humus were analysed in
two stages to determine extractable and residual tannins. For extraction, samples were weighed into centrifuge tubes using 25 mg for fresh litter and 50 mg for
decomposed litter and organic layers. After adding 20
ml of acetone/water (70:30) (v/v), the tubes were shaken for 1.5 h. After centrifugation at 7000 rpm for 20
min, the extracts were decanted into 50 ml volumetric
¯asks followed by a second extraction and centrifugation. The extracts were combined and acetone/water
added to 50 ml. The insoluble residue was air dried for
analysis of residual tannins.
For the assay, 2 ml aliquots of extract were added
to screw-cap test tubes and dried at room temperature
with a stream of air, because tests showed that ovendrying aqueous acetone extracts at 708C seriously
degraded the response. This was not a problem with
solutions of puri®ed tannin in methanol, for which aliquots of 0.05 to 0.25 mg were dried at 708C. The
extraction residues were also transferred into screwcap test tubes for analysis.
For hydrolysis, 5 ml of reagent was added to all
samples (extracts, residues, standards, blank), which
were brie¯y stirred with a vortex mixer, then heated in
a water bath at 958C for 1 h. After cooling, solutions
were transferred into disposable cuvettes. Tubes for
residue analysis were centrifuged before transferring
the solutions. Solutions were scanned from 430 to 750
nm and absorbances were determined at the peak
maximum, at 555 2 2 nm, with allowance for sloping
baseline. If necessary, samples were diluted with reagent to bring the absorbance into the usable range.
Sample contents were determined from the calibration
curve established for balsam ®r tannin. Total condensed tannin content was calculated as the sum of
extractable and residue tannins.

2.3. C and N analysis
2.5.
Total C and N contents of litters and organic layers
from the German sites were analyzed by ¯ash combustion using a Carlo Erba CNS Analyser Model NA
1500/AS 200. For samples from the Canadian sites, C
and N contents were detected by automatic combustion using a Leco model CR12 Carbon Analyser and a
LECO FP-228 N Analyser.
2.4. Proanthocyanidin (PA) assay
The procedure was modi®ed slightly from those
described by Preston et al. (1997) and Preston (1999).
The reagent, prepared each day, was 5% concentrated
HCl in n-butanol (v/v), with a total water content of
5% v/v and 200 mg lÿ1 of Fe2+ as FeSO4
7H2O. A
standard solution (0.5 mg mlÿ1 in methanol) was prepared using a puri®ed condensed tannin from tips of

13

C CPMAS NMR spectroscopy

Solid-state 13C NMR spectra of litter and organic
layers with cross-polarization and magic-angle spinning (CPMAS NMR) were obtained using a Bruker
MSL 300 spectrometer (Bruker Instruments, Karlsruhe, Germany) operating at 75.47 MHz. Dry, powdered samples were spun at 4.7 kHz in a 7 mm OD
rotor. Spectra were acquired with 1 ms contact time, 2
s recycle time and 6000 scans and were processed
using 30±40 Hz line-broadening and baseline correction. Chemical shifts are reported relative to tetramethylsilane (TMS) at 0 ppm, with the reference
frequency set using adamantane. Dipolar dephased
(DD) spectra were generated by inserting a delay
period of 40±50 ms without 1H decoupling between the
cross-polarization and acquisition portions of the
CPMAS pulse sequence. All DD spectra were obtained

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K. Lorenz et al. / Soil Biology & Biochemistry 32 (2000) 779±792

Table 2
Mean values of mass loss (2standard deviation) during needle litter decomposition at the Canadian sites and the German sitesa
Species

Site

Six-month …N ˆ 20† (%)

Ten-month …N ˆ 20† (%)

Twelve month …N ˆ 12† (%)

Black spruce

Schluchsee
Villingen
Black Sturgeon
Lake Nipigon
Schluchsee
Villingen
Black Sturgeon
Lake Nipigon

16.222.1a
18.221.5b
16.222.9a
19.623.6b
10.323.0
13.021.7c
14.222.6c,d
15.823.5d

23.122.0
26.622.3
ND
ND
19.524.3
22.522.4
ND
ND

NDb
ND
28.124.3
23.022.1
ND
ND
21.624.4
19.422.5

Norway spruce

a
b

Six-month means of mass loss with the same letter are not signi®cantly di€erent at P < 0.05 (Mann±Whitney-U-test).
ND ˆ no determination.

using the TOSS sequence for Total Suppression of
Spinning Sidebands.
The NMR spectra of litters were divided into chemical shift regions as follows: 0±50 ppm, alkyl C; 50±60
ppm, methoxyl C; 60±93 ppm, O-alkyl C; 93±112
ppm, di-O-alkyl C and some aromatics; 112±140 ppm,
aromatic C; 140±165 ppm, phenolic C; and 165±190
ppm, carboxyl C. The chemical shift regions for spectra of organic layers were as follows: 0±48 ppm, alkyl
C; 48±90 ppm, methoxyl and O-alkyl C; 90±110 ppm,
di-O-alkyl C; 110±137 ppm, aromatic C±C and C±H;
137±160 ppm, aromatic C±O and C±N; 160±187 ppm,
carboxyl, amide and ester C; 187±210 ppm, aldehyde
and ketone C. Areas of the chemical shift regions were
determined after integration and expressed as percentages of total area (`relative intensity'). There are limitations in the quantitative reliability of CPMAS
spectra, but it is appropriate to use them to compare
intensity distributions among similar samples and to
use DD spectra to point out structural features (Preston et al., 1994, 1997).
2.6. Determination of catalase activity
For characterization of microbial activity during decomposition, we measured activity of the enzyme cata-

lase, which in litter originates predominantly from
microorganisms. This activity is expressed by the percentage of O2 generated relative to the maximum
volume that can be split from a given volume of H2O2
during 3 min (Beck, 1971).
2.7. Statistical analysis
Results of 6-month mass loss were based on arithmetic means (2standard deviation) of 20 samples at
the Canadian sites and 20 samples at the German
sites. For statistical validity of the results the nonparametric Mann±Whitney-U-test (Sachs, 1978) was
applied to compare two means. The signi®cance level
of 95% was used. Comparison of mean values was
performed with the statistical program package
SPSS. Due to scheduling problems, total incubation
time at the German sites was shorter (10 months
compared to 12 months at the Canadian sites). For
this reason, statistical analysis of mass loss was only
done for the 6-month mass data. The 10 and 12month data are not directly comparable, but provide
a guide to changes close to 1 year of decomposition.
The tannin analyses were carried out on composite
samples from each site and are interpreted qualitatively.

Table 3
Signi®cant di€erences in mean values of mass loss after decomposition at the Canadian and the German sites (Mann±Whitney-U-test;
0.001; 0.001 < P < 0.01; 0.01 < P < 0.05)

After 6 months
Black spruce
Norway spruce
After 10 or 12 months
Black spruce
Norway spruce
a

NS ˆ not significant.

Schluchsee
Villingen

Black Sturgeon
Lake Nipigon

Schluchsee
Black Sturgeon







NS







NS



P<

Schluchsee
Lake Nipigon

Villingen
Black Sturgeon

Villingen
Lake Nipigon

NSa





NS





NS



783

K. Lorenz et al. / Soil Biology & Biochemistry 32 (2000) 779±792

Fig. 1. Tannin loss and catalase activity after decomposition of
spruce needle litter (10 months in Germany, 12 months in Canada).

3. Results
3.1. Mass, chemical and biochemical changes
Litter mass losses after 6 and 10 or 12 months of decomposition are shown in Table 2 and signi®cant
di€erences after 6 months and after 10 or 12 months
in Table 3. Mass losses after 6 months ranged from
10.3 to 19.6% and were higher for black spruce at all
sites, with the greatest di€erence being at Schluchsee
(10.3% for black spruce and 16.2% for Norway

spruce). This species e€ect persisted for the second
sampling, when mass losses for black spruce were
higher than those for Norway spruce litter within each
site. Due to the di€erent ®eld exposure times, losses
from Canadian versus German sites cannot be compared directly, but they are similar, especially for Norway spruce.
Some signi®cant site e€ects were seen at both decomposition times. At 6 months, for black spruce,
mass losses were lower (highly signi®cant, P < 0.001)
at Schluchsee than at Villingen and Lake Nipigon, and
signi®cantly (0.001 < P < 0.01) lower at Black Sturgeon than at Lake Nipigon. For Norway spruce,
highly signi®cant di€erences in mass losses were found
between Schluchsee and both Black Sturgeon and
Lake Nipigon sites, and mass loss at Schluchsee was
also signi®cantly lower than at Villingen. At 10
months, losses at Schluchsee remained lower than at
Villingen for black spruce (highly signi®cant) and for
Norway spruce (signi®cant). After 12 months, mass
losses for Norway spruce did not di€er signi®cantly
between the two Canadian sites. For black spruce, the
di€erence between sites was only at the 0.01 < P <
0.05 level. For the German sites, mass losses at
Schluchsee were always less than at Villingen for both
species and sampling times. For the Canadian sites,
however, site e€ects were more variable; mass losses
for black spruce were larger after 6 months at Lake

Table 4
Tannin contents and tannin loss (% of initial) during decomposition of spruce needles and tannin contents of site organic layers
Sample

Decomposition (months)

Black spruce

0
6
10, 12
0
6
10, 12

Norway spruce

Organic layer
Black spruce

Norway spruce

0
6
10, 12
0
6
10, 12

Organic layer
Black spruce

Norway spruce

Organic layer
a
b

Six and 10 months.
Six and 12 months.

0
6
10, 12
0
6
10, 12

Schluchseea
Extract (mg gÿ1)
11.3
2.1 (81.0)
1.8 (84.0)
26.2
6.0 (77.1)
3.8 (85.5)
1.5
Residue (mg gÿ1)
14.0
5.9 (57.6)
5.2 (62.5)
11.6
8.7 (25.6)
6.7 (42.7)
2.0
Total (mg gÿ1)
25.3
8.0 (68.1)
7.0 (72.1)
37.8
14.7 (61.0)
10.4 (72.6)
3.5

Villingena

Black Sturgeonb

Lake Nipigonb

1.7 (84.7)
1.4 (87.2)

5.0 (55.3)
2.9 (74.5)

4.5 (60.0)
2.7 (76.0)

4.8 (81.7)
2.8 (89.3)
2.1

10.1 (61.5)
5.7 (78.2)
14.2

8.3 (68.3)
4.5 (82.8)
23.6

6.3 (54.8)
4.2 (69.8)

6.7 (52.3)
5.0 (64.6)

6.7 (51.8)
6.0 (57.2)

7.0 (40.2)
5.3 (54.7)
2.9

8.4 (28.2)
7.0 (40.2)
14.5

8.3 (29.1)
5.9 (49.6)
14.0

8.0 (68.1)
5.6 (77.6)

11.7 (53.6)
7.9 (69.0)

11.2 (55.5)
8.7 (65.5)

11.9 (68.6)
8.2 (78.5)
5.0

18.5 (51.1)
12.7 (66.4)
28.7

16.6 (56.2)
10.3 (72.7)
37.6

784

K. Lorenz et al. / Soil Biology & Biochemistry 32 (2000) 779±792

Table 5
Carbon and N content and C-to-N ratio of spruce needle litter (decomposed 10 months in Germany, 12 months in Canada) and of organic
layers
Sample

Intact litter

Schluchsee

Villingen

Black Sturgeon

Lake Nipigon

514.5
512.4
474.5

507.6
503.8
538.6

507.3
502.5
515.0

10.5
16.9
15.0

10.4
14.7
28.8

10.7
15.2
23.2

49.0
30.3
31.6

48.8
34.3
18.7

47.4
33.1
22.2

C (mg gÿ1)
Black spruce litter
Norway spruce litter
Organic layer

519.1
515.7

516.5
515.4
342.9
N (mg gÿ1)

Black spruce litter
Norway spruce litter
Organic layer

9.2
13.6

10.1
17.1
12.5

Black spruce litter
Norway spruce litter
Organic layer

56.3
38.1

51.1
30.1
27.4

C-to-N

Nipigon, but after 12 months, were greater at Black
Sturgeon. Norway spruce showed the same trend, but
the di€erences were not signi®cant.
Table 4 summarizes data for contents of extractable,
residual and total condensed tannins in organic layers,
and in needle litters before and after two periods of
decomposition. Norway spruce litter was higher in
total and extractable condensed tannin than was litter
from black spruce (total tannin, 37.8 and 25.3 mg gÿ1,
respectively), although both had similar contents of residual tannin. For both species and for all sites, concentrations of both extractable and residual tannins
decrease.
For the extractable fraction, losses after the second
sampling period were over 80% at the German sites
and over 70% at the Canadian sites. Percentage losses
of the residual tannin fraction were less, but also
tended to be higher at the German sites, as was the
loss of total tannins. Compared to the di€erences
between Canadian versus German sites, species di€erences were small and inconsistent.
Fig. 1 shows tannin loss (as percentage of original
concentration) plotted against catalase activities determined at the end of the ®eld exposure periods. Values
for catalase activity were higher at the German sites,
with the maximum found for Norway spruce litter at
Villingen (18.4% gÿ1). This site also had the highest
mass loss for Norway spruce, although the range of
mass loss for Norway spruce was not large. There was
no signi®cant relationship between catalase activity
and mass loss or between tannin loss and mass loss.
There was, however, a strong positive correlation
between catalase activity and tannin loss, with the
greater losses of tannins and highest catalase activities
generally observed at the German sites. However, the
high tannin loss for Norway spruce at Lake Nipigon
(72.7%) was also associated with the highest catalase
activity at a Canadian site (9.2% gÿ1).
Both species had similar total C contents in fresh lit-

ter (Table 5) and showed only small decreases at both
sites. In contrast, N contents increased during decomposition. The biggest increases were found for
Norway spruce at the German sites while smaller
changes were observed at all sites for black spruce.
The latter had a lower N content (9.2 mg gÿ1) than
Norway spruce (13.6 mg gÿ1) and a higher C-to-N
ratio. During decomposition, C-to-N ratio decreased
for both species and all sites, but always remained
higher for black spruce litter than for Norway spruce.
There were large di€erences between the organic
layers of the German and Canadian sites. The latter
receive black spruce needle litter of C-to-N 56.3, but
had C-to-N ratios around 20 as a consequence of high
N content in the organic layer. The German sites
receive Norway spruce litter with C-to-N of 38.1 and
especially at Schluchsee have lower contents of both C
and N in the organic layer. Subsequently the German
sites had C-to-N ratios of 27.4 (Schluchsee) and 31.6
(Villingen). Schluchsee has abundant earthworms and
a mull/moder organic layer, resulting in the lower total
C. As previously noted, catalase activity associated
with needle litter was greater at the German sites,
despite their lower pH values. Remarkable di€erences
were found for tannin contents in the organic layers
(Table 4), which were nearly 8-fold greater at the
Canadian sites.
3.2.

13

C CPMAS NMR spectroscopy

NMR spectra were interpreted based on previous
studies of litter and humus (Wilson et al., 1983;
Zech et al., 1987, 1990, 1992; KoÈgel et al., 1988;
NordeÂn and Berg, 1990; KoÈgel-Knabner et al.,
1992; de Montigny et al., 1993; Preston et al.,
1994, 1997; Baldock and Preston, 1995; Trofymow
et al., 1995; Wachendorf, 1998; Preston, 1999). The
spectra of fresh needle litters are shown in Fig. 2
for black spruce and in Fig. 3 for Norway spruce.

K. Lorenz et al. / Soil Biology & Biochemistry 32 (2000) 779±792

785

Distinct features for lignin and tannin can also
be distinguished in the DD spectra. In this experiment, signals are rapidly lost from carbons with
attached hydrogens, except those for which the
dipolar interaction is weakened by molecular
motion. Therefore, the peaks remaining in the DD
spectra represent either groups with quaternary carbons or those with some motion in the solid state,
such as methyl, methoxyl, acetate and CH2 in long
chains. Condensed tannins give a characteristic broad
peak at 105 ppm in the DD spectrum, a region which
is otherwise generally free of interferences (Wilson and
Hatcher, 1988; Preston et al., 1997). The DD spectra
of both litters show this peak, as well as a sharp feature for methoxyl of lignin at 56 ppm. In the phenolic
region, tannin features are more distinct than in normal spectra, due to di€erential dephasing of tannin
and lignin carbons. Norway spruce has a larger splitting of the peaks at 145.0 and 154.4 ppm consistent
with its higher tannin content, with a shoulder at 148
ppm (lignin). For black spruce, the sharp peak at
145.3 ppm has a weak shoulder at 148.3 ppm and a

Fig. 2. Normal and DD-TOSS 13C CPMAS NMR spectra of fresh
and decomposed black spruce needle litter.

For the litter spectra, the alkyl intensity (0±50 ppm)
comes mainly from surface waxes and cutin and the
sharp O- and di-O-alkyl peaks at 72 and 105 ppm
from carbohydrates. Tannins and lignins are the
main contributors in the aromatic and phenolic
regions. Most peaks for condensed tannins are coincident with those from other biopolymers. However, they have a characteristic split peak at 144
and 154 ppm in the phenolic region, compared to
guaiacyl lignin which has a peak at 147 ppm with
a shoulder at 153 ppm and a methoxyl signal at
55±57 ppm. Both litters have a methoxyl signal at
56 ppm, while the partially split phenolic region is
characteristic of a mixture of lignin and tannin.
Norway spruce has two broad peaks at 145 and
154 ppm, while for black spruce the phenolic region
has a peak at 145.6 ppm with a shoulder at 152.7
ppm. For Norway spruce litter, the more distinct
splitting in the phenolic region and the chemical
shift of 154 ppm compared to the shoulder at 152.7
ppm for black spruce is consistent with its higher
tannin content.

Fig. 3. Normal and DD-TOSS 13C CPMAS NMR spectra of fresh
and decomposed Norway spruce needle litter.

786

K. Lorenz et al. / Soil Biology & Biochemistry 32 (2000) 779±792

more distinct shoulder at 153 ppm. Di€erences in the
phenolic region have been emphasized to highlight features arising from tannin versus lignin structures,
though in general, the spectra of both species were
very similar. The spectra were also similar to those
reported for needle litter by Zech et al. (1987), KoÈgel
et al. (1988), NordeÂn and Berg (1990), KoÈgel-Knabner
et al. (1992) and Preston et al. (1994, 1997).
The relative intensities and alkyl-to-O-alkyl ratios
of the 13C CPMAS NMR litter spectra are shown in
Table 6. For fresh litter, the main di€erences were the
higher alkyl C in black spruce, while Norway spruce
was higher in O-alkyl C with a lower alkyl-to-O-alkyl
ratio. During decomposition, an increase of intensity
in the alkyl region was observed for both species with
the greatest changes at Schluchsee. However, a
decrease of O-alkyl C commonly associated with decomposition was only seen for Norway spruce litter
with strongest changes at the German site Villingen.
For black spruce needle litter, the relative intensity of
the O-alkyl region remained essentially unchanged.
Therefore the increase in the alkyl-to-O-alkyl ratio was
mainly due to a relative increase in alkyl C. Sitedependent di€erences in changes of the relative intensities in the other regions of the 13C CPMAS NMR
spectra were small and inconsistent, although the relative intensity of the phenolic and carboxyl regions
usually decreased.
The main features of the spectra (peak position, lineshape) were also generally unchanged by 10 or 12
months of decomposition, as shown by the representative spectra in Figs. 2 and 3. There was an increase in
the relative intensity of the 33 ppm peak for black
spruce litter at Lake Nipigon. This peak comes from

carbon in long CH2 chains of a more rigid nature,
while those at 30 ppm are more mobile (KoÈgel-Knabner et al., 1992). However, it was not associated with a
large increase of alkyl C and constitutes a very small
proportion of the total alkyl C area. For Norway
spruce, the splitting of the phenolic region became less
distinct, with loss of the intensity due to tannins at 154
ppm.
NMR spectra of the organic layers are shown in
Fig. 4 and relative areas are given in Table 7. Spectra
from the German sites, especially from Villingen, are
similar to the spectra of the decomposed Norway
spruce litter (Fig. 3) but with the features much broadened. By contrast, spectra of the organic layers from
the two Canadian sites have higher resolution than the
fresh litters, especially the peaks for long-chain CH2 at
33 ppm and for tannins at 145 and 155 ppm. All organic layers have peaks at both 30 and 33 ppm, for
more mobile and more rigid CH2, but for the Canadian sites, the peak at 33 ppm is much higher than
that at 30 ppm. The Canadian samples also have a
more intense and well-resolved peak at 130 ppm in the
aromatic region. All sites have a partially resolved
peak or shoulder at 55±57 ppm.
The DD spectra are consistent with tannin structures
in all four organic layers (broad peak at 105 ppm).
However, the Canadian samples are remarkable in
having almost no methoxyl signal in the DD spectrum,
whereas it is present in the German samples. For the
Canadian sites, the lack of the methoxyl signal, the
large splitting of the phenolic region and the relatively
sharp peak at 130 ppm in both the normal and DD
spectrum indicate a high tannin content (consistent
with the chemical assay), and almost no lignin. For

Table 6
Relative intensities (percent of total area) of the 13C CPMAS NMR spectra of black and Norway spruce needle litter: intact litter and after decomposition (10 months in Germany, 12 months in Canada); alkyl/O-alkyl ratios
Range (ppm)

Alkyl/O-alkyl

0±50

50±60

60±93

Black spruce
Norway spruce

22.5
17.5

5.0
5.4

39.0
42.6

Schluchsee
Villingen
Black Sturgeon
Lake Nipigon

26.9
26.0
26.7
24.3

6.9
6.1
5.1
5.7

39.9
38.9
37.9
37.8

Schluchsee
Villingen
Black Sturgeon
Lake Nipigon

25.1
20.8
19.0
21.2

6.9
5.7
5.4
6.4

39.5
37.7
38.8
40.8

93±112

112±140

Intact litter
10.4
10.8
12.5
10.5
Black spruce decomposed
9.9
8.5
9.6
9.3
10.0
10.0
10.3
10.9
Norway spruce decomposed
9.9
9.0
11.1
11.3
11.3
11.9
11.5
10.7

140±165

165±190

6.9
7.3

5.4
4.2

0.58
0.41

4.5
5.8
5.7
6.0

3.4
4.3
4.6
5.0

0.67
0.67
0.70
0.64

5.3
7.1
7.4
5.7

4.3
6.3
6.2
3.7

0.64
0.55
0.49
0.52

787

K. Lorenz et al. / Soil Biology & Biochemistry 32 (2000) 779±792

even though there is little di€erence in relative areas.
The German sites are higher in O- and di-O-alkyl C
and lower in alkyl C, but the di€erences are small.

4. Discussion
4.1. Comparison of spruce litter chemistry

Fig. 4. Normal and DD-TOSS
layers.

13

C CPMAS NMR spectra of organic

the German organic layers, there is a methoxyl peak in
the DD spectra and the phenolic region is consistent
with a mixture of lignin and tannin. The German
samples also have a broad peak at 105 ppm in DD
spectrum, indicating tannin, although the chemical
assay of the tannin content is much lower (0.35 and
0.5%). Spectra from the German and Canadian sites
indicate considerable di€erences in humus quality,

Table 7
Relative intensities (percent of total area) of the

Litter species di€er in several properties that have
been related to litter quality and readiness to decompose (Tian et al., 1995; Trofymow et al., 1995; Handayanto et al., 1997; Mafongoya et al., 1998; Moore et
al., 1999). On the positive side, the Norway spruce litter had a higher N content and lower C-to-N ratio
and its NMR spectrum indicated that it was higher in
carbohydrates and lower in cutin and surface waxes
than was the black spruce. On the other hand, it was
higher in total and extractable tannins, and the latter
has been linked to protein-binding and inhibition of N
mineralization (Handayanto et al., 1997).
Several factors need to be considered in interpreting
foliar (and humus) tannin analyses. First, foliage tannin contents are a€ected by environmental factors and
tend to increase with stresses such as nutrient limitation and insect attack (Ricklefs and Matthews, 1982;
Tiarks et al., 1988; Northup et al., 1995a). The
sampling site for Norway spruce litter is a typical Mgde®cient site (Raspe et al., 1998). Therefore, the tannin
contents of the study litters should not be regarded as
standard values for these species. Second, a variety of
methods and standards have been used to extract and
assay foliar tannins or total (poly)phenolics. We used
a highly ecient extractant for condensed tannins
(acetone/water) and the PA assay which is speci®c for
condensed tannins and was calibrated against a wellcharacterized condensed tannin extracted from balsam®r tips. However, the response in the PA assay is
dependent on tannin structure (chain-length and
branching) and foliar tannins may comprise a range of
sizes and solubilities, from monomers (e.g. catechin
and epicatechin, not detected in the PA assay) to structures that are insoluble due to either high molecular
weight or cross-linkages to other biopolymers. How-

13

C CPMAS NMR spectra of organic layers

Range (ppm)

Schluchsee
Villingen
Black Sturgeon
Lake Nipigon

0±48

48±90

90±110

110±137

137±160

160±187

187±210

16.9
18.3
19.5
22.5

39.5
42.9
38.8
37.7

12.5
12.4
10.8
10.4

12.6
11.9
13.4
12.6

8.1
6.4
7.7
7.5

7.5
6.7
8.0
7.6

2.9
1.4
1.8
1.7

788

K. Lorenz et al. / Soil Biology & Biochemistry 32 (2000) 779±792

ever, preliminary analysis of tannin extracted from
black spruce foliage (unpublished) showed similarity in
chain length and PA color development to the balsam®r tannin and the NMR spectra were consistent with
the higher tannin contents measured in Norway spruce
litter. Therefore, in the interpretation of the results,
comparisons within species are more reliable than
comparison between species and the absolute numbers
should be used with caution, especially in relation to
other studies.
The PA assay of senescent needles yielded 25.3 mg
gÿ1 condensed tannins for black spruce needles from
the Black Sturgeon site (Canada) and 37.8 mg gÿ1
from Norway spruce needles from Schluchsee
(Germany) (Table 4), with the Norway spruce needles
having a higher proportion of extractable tannin.
These results may be compared to other studies of
senescent needles, with the aforementioned cautions.
Brown Norway spruce needles from two high-elevation
sites in France had 18 and 24 mg gÿ1 total phenolics,
based on the Folin±Ciocalteau assay of aqueous
extracts (Gallet and Lebreton, 1995). The Folin±Dennis assay of hot water extracts yielded 1.2±4.5% total
phenolics for litter from cedar (Thuja plicata Donn.)
and hemlock (Tsuga heterophylla (Raf.) Sarg.)/®r
(Abies amabilis (Dougl.) Forbes) mixtures from northern Vancouver Island (Keenan et al., 1996). Yavitt and
Fahey (1986) found 4% total phenolics in lodgepole
pine (Pinus contorta Dougl.) litter using aqueous
methanol and the Folin assay. Tiarks et al. (1992)
measured 5.7% extractable and 1.7% residual condensed tannins in shortleaf pine (Pinus echinata Mill.)
using similar methods to this study and a standard
from the same species. Even with the great variety of
analytical approaches, tannin contents in the senescent
foliage of both species in this study are similar to
those reported elsewhere for Norway spruce and other
conifers. While the black spruce litter had lower quality based in its total N, C-to-N ratio and NMR spectra, it was lower in tannins and particularly in
extractable tannins than the Norway spruce. Therefore, it is unlikely that the elevated tannin contents in
the black spruce humus derive from unusually high
tannin content in foliar litter.
4.2. Spruce litter decomposition
At the end of this short-term decomposition study,
there was a loss of 19.4±28.1% of litter mass, 66±79%
of total tannins and 76±89% of total tannins. The
rapid loss of condensed tannins within 1 year, especially within the ®rst 6 months, is consistent with
other reports. Again, direct comparison of results is
dicult, because of the wide variety of methods,
including assays of total phenolics, as well as more
speci®c tests for protein-binding activity and con-

densed and hydrolyzable tannins. However, despite
this variety, losses are generally in the range of 80%
within one year for litter of both deciduous species
(Wilson et al., 1983; Baldwin and Schultz, 1984; Nikolai, 1988; Racon et al., 1988; Scho®eld et al., 1998)
and conifers (Wilson et al., 1983; Yavitt and Fahey,
1986; Tiarks et al., 1992; Keenan et al., 1996).
Both site and species e€ects were found in this
study. After decomposition for 10 months (Germany)
and 12 months (Canada), Norway spruce litter always
had less mass loss than black spruce litter, and higher
concentrations of total and extractable tannins. Concentrations of residual tannin in Norway spruce were
also higher at all sites except for Lake Nipigon, where
the values for the two species were very close. Since
other factors indicated higher litter quality for Norway
spruce, it is possible that its higher initial tannin content (especially of extractable tannins) inhibited decomposition. Another possible in¯uence would be
di€erences in needle toughness and permeability (Gallardo and Merino, 1993); however, the NMR spectra
indicate a lower content of cutin and surface waxes in
Norway spruce.
In the comparison of sites, the highest tannin losses
for both species at both 6 and 10/12 months were
found at the German site Villingen. Compared to the
Canadian sites, the German sites had higher losses of
total and extractable tannins, but losses of residual
tannins were similar (tannin losses were still less at the
Canadian sites, despite the longer second sampling
period). Mass losses within Germany were consistently
higher at Villingen than at Schluchsee. At the Canadian sites, mass losses tended to be larger at Lake
Nipigon after 6 months, but larger at Black Sturgeon
after 12 months.
There are large di€erences in the conditions for decomposition among the four sites. According to Cortez
(1998), litter decomposition is controlled by earthworm
activity as well as by climatic conditions and their
e€ects on soil moisture and temperature. Rates of tannin loss were also dependent on site conditions,
although these were not speci®ed (Baldwin and
Schultz, 1984). Compared to Villingen, Schluchsee has
a slightly lower mean annual temperature but higher
precipitation. Also, at Schluchsee Lumbricidae are
abundant (Lamparski, 1985), whereas at Villingen this
is the case only to a minor extent. The mesh size of 1
mm used in this litter bag study allows penetration of
small earthworms into the bags. However, the main
reason for the slightly lower mass and tannin losses at
Schluchsee compared to Villingen was probably the
unusually low precipitation at Schluchsee until the ®rst
sampling in Spring 1996 (only 63% of the annual wintertime mean; Armbruster, 1998). Probably, the fast
initial loss of tannins at Villingen then makes the
litter more attractive to decomposers (Harrison, 1971;

K. Lorenz et al. / Soil Biology & Biochemistry 32 (2000) 779±792

SÏlapokas and Granhall, 1991; Keenan et al., 1996),
which is also indicated by the highest catalase activity
of all sites (Fig. 1). The inhibition of catalase by tannins was also observed by Ladd and Butler (1975).
Compared to the German sites, the Canadian sites
are colder and drier, with no earthworms and lower
catalase activity, although the pH is higher. The percentage losses of tannins were lower at the Canadian
sites, for both species, tannin fractions and decomposition times (despite the slightly longer ®eld exposure
time for the Canadian sites). The more favorable climatic conditions and presence of earthworms at the
German sites is probably responsible for the higher
tannin losses and catalase activities. Satchell and Lowe
(1967) showed that tannin degradation by microorganisms is associated with an increase in the earthworm population in the litter and Tian et al. (1995)
found that millipedes and earthworms were important
to the breakdown of plant residues of low quality
(high C-to-N ratio, lignin and polyphenol contents).
In the NMR spectra, decomposition resulted in a
relative increase of alkyl versus O-alkyl C, while aromatic, phenolic and carboxyl C content showed only
small and inconsistent changes. This pattern is similar
to results from other litterbag studies of needle litter
(Wilson et al., 1983; Zech et al., 1987; NordeÂn and
Berg, 1990). At the end of the decomposition experiment, the highest relative content of alkyl C for both
species was at Schluchsee. There were only small
changes in the relative intensity of the O-alkyl region
(60±93 ppm); black spruce showed essentially no
decline, while the greatest decrease was found for Norway spruce at Villingen (42.6 to 37.7%).
4.3. Organic layers and decomposition processes
Large di€erences in the properties of the organic
layers all suggest development of a pattern of restricted
decomposition at the Canadian sites. Humus at both
Canadian sites had higher contents of C and N and
lower C-to-N ratios than the German sites. The latter
would normally be associated with a greater degree of
decomposition, but in this case probably indicates low
nutrient availability. With increasing stand age, northern black spruce forests tend to show declining nutrient availability at the same time as N is increasingly
sequestered in the forest ¯oor. Several factors may
contribute to this, including the cold climate, small
annual litter inputs of low N content, lack of earthworm activity and an increasing sequestration of N in
recalcitrant organic structures and in understorey
feathermoss (Pastor et al., 1987; Smith et al., 1998).
However, it does not appear to arise from any unusually recalcitrant organic composition of black spruce
litter. In this 1-year litterbag study, black spruce lost
more mass than Norway spruce at each site and com-

789

parable proportions of tannin. In a multisite Canadawide long-term litterbag study, black spruce litter mass
loss after 3 years was 43.9%, the highest of ®ve conifer
species (Moore et al., 1999).
The 13C CPMAS NMR spectra of the organic layers
from the German sites are similar to those in many
reports (Zech et al., 1987, 1990, 1992; KoÈgel et al.,
1988; KoÈgel-Knabner et al., 1992; de Montigny et al.,
1993; Preston et al., 1994; Baldock and Preston, 1995;
Wachendorf, 1998; Preston, 1999). Spectra from the
Canadian sites have similar peak positions and only
slight di€erences in relative areas. However, the much
sharper features of the Canadian sites indicate less
transformation of the original biopolymers. In particular, the sharp peak at 33 ppm comes from accumulation of long-chain CH2 from cutin, suberin and plant
waxes although microbial biomass may also contribute
in this region. The NMR spectra also show unusual
accumulation of condensed tannins, and a remarkable
lack of signals due to lignin. By comparison, spectra
of organic layers from the German sites have broader
features and a normal mixture of lignin and tannin-derived structures.
Little is known about the mechanism of tannin loss
from litter and the fate of tannins in humus. There is
evidence that both tannins and simpler phenolics are
lost by leaching (Kuiters and Sarink, 1986; Yavitt and
Fahey, 1986; Gallet and Lebreton, 1995; Scho®eld et
al., 1998), which is consistent with the rapid loss of
extractable tannins in this study. There are few studies
of tannins in humus, for which comparison is again
dicult because of the variety of methods used. However, these generally agree in showing low concentrations of 1% or less in humus (Kuiters and
Denneman, 1987; Gallet and Lebreton, 1995; Preston,
1999) and Scho®eld et al. (1998) were unable to detect
or recover tannins in the mineral soil of a microcosm
experiment. In our study, organic layers from Norway
spruce sites in Germany had 3.5 and 5.0 mg tannins
gÿ1, similar to the low values reported elsewhere.
However, the Canadian black spruce sites had much
higher amounts (28.7 and 37.6 mg gÿ1) with NMR
supporting the chemical assay, despite their receiving
litter with lower tannin content.
The link between accumulation of tannins and inhibition of N availability may be through formation of
tannin±protein complexes (Howard and Howard,
1993; Northup et al., 1995b; Handayanto et al., 1997;
Mafongoya et al., 1998). However, such complexes
have not been unequivocally detected or isolated in litter or humus, while tannin-rich fractions extracted
from humus under salal (Gaultheria shallon Pursh)
were all depleted in N (Preston, 1999). Scho®eld et al.
(1998) found no evidence for the formation of tannin±
protein complexes in willow (Salix exigua Nutt.) litter.
The lack of accumulation of tannins in humus and

790

K. Lorenz et al. / Soil Biology & Biochemistry 32 (2000) 779±792

mineral soil indicates that under most circumstances
their microbial decomposition is not unduly hindered,
as would be expected for a major biopolymer component of trees and shrubs (Grant, 1976; Deschamps,
1982; Gamble et al., 1996).
It is, therefore, unlikely that the humus tannin accumulation at the Canadian sites can be attributed to
either high litter tannin content or an inherent resistance of black spruce litter to decomposition. It was
not possible in this study to determine whether the
tannin and N accumulations were linked. A high proportion of the humus tannin was extractable and,
therefore, not likely to be sequestered in insoluble tannin±protein complexes. It may be that tannins, like
alkyl C, can accumulate under certain conditions
where decomposition is hindered, as was found to a
lesser extent for humus from sites of contrasting productivity on northern Vancouver Island (de Montigny
et al., 1993). As striking as the accumulation of tannins, is the unusual depletion of lignin structures in
humus at the Canadian sites. Further studies are
necessary to ascertain the spatial and temporal extent
of these phenomena in black spruce ecosystems, and
whether even temporary accumulations of humus tannin may contribute to long-term reduction of N availability.

5. Conclusions
Condensed tannins in black and Norway spruce needle litter decreased quickly during decomposition for
up to 1 year at the Canadian and German sites.
Greater mass losses were observed for black spruce
needles despite less favourable C-to-N ratios and
higher contents of surface lipids and cutin than Norway spruce needles. The higher initial tannin contents
in Norway spruce needle litter, especially of the extractable fraction, may have been responsible for the
slower decomposition. The results also indicate a
strong in¯uence of climate on early stages of decomposition.
Humus at both Canadian sites had extremely high
tannin contents and was poorly decomposed, despite
having lower C-to-N ratios and higher N contents that
would normally indicate more advanced decomposition. Coincident with the accumulation of organic N
(typical of nutrient-limited black spruce ecosystems),
condensed tannins and long-chain CH2 structures, was
a unique depletion of structures characteristic of lignin.
Di€erences in needle litter quality, cannot account for
such di€erences in the humus.
Further investigations are needed to understand the
di€erences of decomposition pathways in the Canadian
and German sites. These may arise from di€erences in
other inputs (bark, wood, roots, understorey veg-

etation) and in site properties (climate, decomposer
community, earthworm activity). As the Canadian
sites accumulate both tannins and poorly available N,
it is important to understand whether tannins per se
are a critical factor in the development of restricted decomposition and nutrient limitation in black spruce
ecosytems.

Acknowledgements
We gratefully acknowledge the reliable technical assistance of Ulrike Benitz, Kevin McCullough, Gary
Koteles, Pamela Perreault and Daniela Wohlfahrt. We
thank the BMBF (German Ministry of Science and
Technology, Bonn) for funding and the GKSS and
DLR for coordinating visits of German scientists in
Canada. Canadian travel to Germany was funded
through The Going Global Program for Science and
Technology of the Department of Foreign A€airs and
International Trade, Canada.

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