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Soil Biology & Biochemistry 32 (2000) 301±314
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Carbon transformations during decomposition of di€erent
components of plant leaves in soil
E.A. Webster a, b,*, J.A. Chudek b, D.W. Hopkins c
a

Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, Scotland, UK
b
Department of Chemistry, University of Dundee, Dundee DD1 4HN, Scotland, UK
c
Department of Environmental Science, University of Stirling, Stirling FK9 4LA, Scotland, UK
Accepted 17 August 1999

Abstract
We investigated the e€ect of lime addition to an upland organic soil on the decomposition of Lolium perenne leaves and
isolated fractions of L. perenne leaves in a laboratory experiment lasting 75 d. The L. perenne plants were grown in a 13CO2enriched environment and some leaf material was pretreated with ethanol and detergent in order to remove some cell contents
and soluble material. The ethanol- and detergent-treated leaves had less alkyl-C, as seen by solid-state 13C nuclear magnetic
spectroscopy (NMR), and a greater proportion of cellulose and hemicellulose than the untreated leaves. Solid-state 13C NMR
spectroscopy and scanning electron microscopy (SEM) were used to follow aspects of the C transformations during

decomposition. C mineralization was estimated from total CO2 production. The size and activity of the microbial community
was greater in limed than in soils without lime, and microbial respiration was less in both soils amended with ethanol- and
detergent-treated leaves compared to soils amended with untreated leaves. In both limed and unlimed soils, amendment with
untreated leaves led to additional CO2 production within 7 d of addition, whereas amendment with treated leaves led to a
smaller increase in CO2 production. The ¯ush of CO2 production was attributed to decomposition of the more accessible and
soluble plant components that, in the ethanol- and detergent-treated leaves, had been removed during the ethanol and detergent
treatment. The 13C NMR spectra recorded for plant material separated from soil 1 d after addition of ethanol- and detergenttreated leaves had larger alkyl-C (30 ppm) signals compared with spectra from untreated leaves. This was interpreted as
representing an accumulation of residues from decomposition of plant structural components. # 2000 Elsevier Science Ltd. All
rights reserved.
Keywords:

13

C solid-state NMR; Lolium perenne; Scanning electron microscopy

1. Introduction
Upland soils in temperate regions are often characterized by intense soil acidity and soil wetness. They
are also important reservoirs of terrestrial carbon
(Eswaran et al., 1993). In the 1970s and 1980s some
areas of upland pasture in northern Britain were limed

to allow more intensive use of the land for pastoral

* Corresponding author. Present address: Department of Environmental Science, University of Stirling, Stirling FK9 4LA, Scotland,
UK. Tel.: +44-1786-473171, ext. 6537; fax: +44-1786-467843.
E-mail address: e.a.webster@stir.ac.uk (E.A. Webster).

agriculture. Improvement involved removing the indigenous vegetation, applying lime, then reseeding with a
mixture of cold-tolerant grasses and clover (Floate,
1977; Newbould, 1985). Financial incentives for such
improvements are no longer available, but large areas
of limed pastures persist. Improvement generally
resulted in increased soil microbial biomass and rates
of both microbial C and N transformations (Hopkins
et al., 1990; Isabella and Hopkins, 1994; Hopkins,
1997).
Decomposition is governed by the physical and
chemical environment of the soil, the activity of soil
organisms and the resource quality of the plant litter

0038-0717/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.

PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 1 5 3 - 4

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E.A. Webster et al. / Soil Biology & Biochemistry 32 (2000) 301±314

(Swift et al., 1979). Plant material is a heterogeneous
mixture of compounds, each of which decomposes at a
characteristic rate (Swift et al., 1979). In order to investigate the carbon transformations during decomposition of di€erent components of plant material in a
limed organic soil, we modi®ed the composition of
Lolium perenne leaves by treatment with ethanol and
detergent to remove some of the plant cell contents
before incorporation into the soil (Ritchie and Larkin,
1982). We then used solid-state 13C nuclear magnetic
resonance (NMR) with magic angle spinning (MAS)
and cross-polarisation (CP) to investigate decomposition of di€erent components of plant material in soil
at the level of functional groups. Decomposition of
plant material in soil generally results in a relative accumulation of alkyl-C and a relative decrease in Oalkyl-C (as determined from 13C NMR spectra)
(KoÈgel-Knabner, 1997; Hopkins et al., 1997). Consequently, the alkyl-C-to-O-alkyl-C ratio may indicate
the degree of decomposition of soil organic matter

(Preston, 1996; Baldock et al., 1997). The provenance
of C contributing to the alkyl-C signal is not well
understood and in particular, the proportions attributable to either plant residues or microbial components
are not well known. Our aim was to provide information on the relative contribution of plant components to the alkyl-C pool in the short term.
Our speci®c objectives were to investigate carbon
transformations during decomposition of di€erent
components of plant material in soil, to investigate the
contribution of plant components to the alkyl-C pool
in soil, and to investigate the e€ects of liming the soil
on the decomposition of components of plant materials.

2. Materials and methods
2.1. Soils
The soils were sampled from 0±15 cm depths of two
experimental plots at the Redesdale Experimental Husbandry Farm, Northumberland, UK (latitude 56813'N
longitude 2816 'W; national grid reference NY828924).
The plots are part of an experiment established in
1981 to determine the amount of lime application
required to improve the quality of the pasture for grazing (Dampney, 1985). One of the plots sampled for
our study received 20 t CaCO3 haÿ1 in 1981 and had

pH 5.2 when sampled in 1995, and the other received
no CaCO3 and had pH 3.5 when sampled. Both plots
had pH 3.9 before lime application (Isabella and Hopkins, 1994). Soils from these plots are hereafter
referred to as limed and unlimed, respectively. The
other treatments applied in 1981 were fertiliser N, P
and K additions and reseeding with a seed mixture

(predominantly L. perenne, Phleum pratense and Trifolium repens ). The establishment of the sown species on
the unlimed plot was poor and the vegetation quickly
reverted to a sward dominated by Molinia caerulea,
which is typical of the land adjacent to the experiment
(Dampney, 1985). After collection, the soils were
sieved (4 mm mesh) in the ®eld-moist state and stored
at 48C for up to 6 weeks before use.
2.2. Determination of soil microbial biomass
Before addition of plant material, microbial biomass
was determined from glucose-induced respiration rates
(Anderson and Domsch, 1978). CO2 production from
glucose amended soil (0 to 5 mg glucose gÿ1 soil with
talc as the inert carrier) was determined in triplicate by

gas chromatography (thermal conductivity detector)
over 0±6 h incubation at 228C in miniaturized respirometric devices (Heilmann and Beese, 1992; Hopkins
and Ferguson, 1994).
2.3. 13C-enriched L. perenne and ethanol and detergent
treatment
L. perenne plants were grown from seed on a 1:1
vermiculite±vermiperle mixture with nutrient solution
for 50 d in airtight growth chambers under a 16 h
light/8 h dark regime in a 13CO2-containing atmosphere as described by Hopkins et al. (1997). Approximately half of the leaves harvested after this time were
treated with ethanol and detergent to permeabilize the
cell membranes and remove some of the cell contents
including chlorophyll, soluble sugars and amino acids
(Ritchie and Larkin, 1982). This involved mixing 10 g
of fresh leaves with 200 ml 100% ethanol for 12 h at
158C, discarding the ethanol and adding 200 ml 0.5%
Triton-X solution for 12 h then discarding the TritonX solution. This procedure was repeated 3 times before
rinsing 10 times with distilled water. Untreated leaves
and ethanol- and detergent-treated leaves were dried in
an oven (408C) then chopped into approximately 5
mm lengths. The untreated and ethanol- and detergent-treated leaves had 2.00 and 1.97 at% 13C, respectively, as determined by mass spectrometry (see below).

2.4. Chemical analysis
The cellulose, hemicellulose and lignin contents of
leaves and ethanol- and detergent-treated leaves were
determined using the proximate analysis methods of
Allen et al. (1974). Duplicate chopped subsamples of
untreated leaves and ethanol- and detergent-treated
leaves (0.2 g dry wt) were mixed with 150 mg sodium
chlorite and 0.5 ml 10% ethanoic acid in 15 ml distilled water and maintained at 758C for 2 h with occasional shaking. After 1 h a further 150 mg sodium

E.A. Webster et al. / Soil Biology & Biochemistry 32 (2000) 301±314
Table 1
Assignment of C functional groups to shift ranges for solid-state

13

303

C NMR spectra (adapted from Wilson, 1987; Baldock et al., 1991)

Shift range (ppm)

Assignment

Main classes of compounds included

0±45
45±60
60±90
90±110
110±160
160±200

methyl- and alkyl-C
methoxyl- and N-alkyl-C
O-alkyl-C
acetal- and ketal-C
aromatic-C
carbonyl-C

aliphatic compounds, lipids, waxes

lignin substituents, amino acids, amino sugars
carbohydrates, lignin propyl side chains
carbohydrates
phenyl-propylene subunits of lignin
organic acids and peptides

chlorite and 0.5 ml 10% ethanoic acid were added to
the mixtures. The mixtures were cooled on ice, vacuum-®ltered through sintered glass funnels (pore size
40±100 mm) and rinsed 10 times with ice-cold distilled
water, once with 100% acetone and ®nally once with
diethyl ether. The resulting holocellulose fraction was
dried at 1058C for 30 min. To fractionate the holocellulose into hemicellulose and cellulose, a known mass
of holocellulose was added to 20 ml 24% KOH (w/v)
and maintained at 208C for 2 h. The mixture was vacuum-®ltered through sintered glass funnels and washed
with distilled water then acetone and ®nally diethyl
ether as before. The resulting cellulose fraction was
dried at 1058C for 30 min. The ®ltrate was collected
into reservoirs containing 8 ml ethanoic acid, and sucient ethanol was then added to give a ®nal volume of
3.5 times that of the original ®ltrate, and allowed to
stand for approximately 16 h during which time the

hemicellulose fraction precipitated. The hemicellulose
fraction was vacuum-®ltered through sintered glass
funnels, rinsed in ethanol and acetone then dried for
30 min at 1058C The ®nal mass of holocellulose, cellulose and hemicellulose was corrected for crude protein
content. All nitrogen and carbon determinations were
made using a Carlo-Erba CHN analyser.
2.5. Experimental
Soils were mixed with plant materials at the rate of
0.5 g untreated leaf material gÿ1 soil (dry wt) or 0.29 g
ethanol- and detergent-treated leaves gÿ1 soil (dry wt),
this being equivalent to 0.5 g of untreated leaves gÿ1
soil. Water was added to restore the soil to 60% water
holding capacity, and samples of the soil and plant
material mixtures (equivalent to 0.5 g dry wt) were
weighed into glass vials, which were then put into miniaturized respirometric devices for CO2 determination
(as above). The soil and plant material mixtures were
incubated at 158C for up to 75 d. Two parallel sets of
vials were used, one for NMR analysis and a second
for CO2 determinations. For each sampling occasion
for NMR analyses there were three replicates of each

combination of soil and plant material. CO2 measurements were determined, by gas chromatography (Hopkins and Ferguson, 1994) every 2 or 3 d on the

second, parallel set of vials for which there were three
replicates. The headspaces inside the vessels were
¯ushed out and replaced with fresh air every time CO2
was measured. The experimental design was, therefore,
as a 2  3 factorial with four replicates in a randomised design. There were 2 levels of liming (0 and 20 t
haÿ1) and 3 levels of plant composition (no addition,
addition of L. perenne or treated L. perenne ). Time
was an additional factor, and was incorporated into
the design as a repeated measure with 24 occasions.
The CO2 production data was analysed according to
this design (Genstat, 1993, 1997).
Three vials were taken at each NMR sampling occasion (0, 1, 3, 7, 14, 28 and 75 d) and were freezedried. The contents of one vial (comprising soil and
plant material) was ground and used for NMR analysis. The contents of the remaining two vials were used
for NMR analysis of plant material only. Fragments
of plant material identi®able under a microscope (10
magni®cation) were separated from the soil then
washed in distilled water before freeze-drying for
NMR analysis. The plant fragments from two vials
were combined to provide samples suciently large for
NMR analysis. Plant material that had not been
added to soil, and that, therefore remained uncontaminated with soil microorganisms, was used as the control for both NMR spectroscopy and scanning
electron microscopy.
2.6. NMR
Solid-state cross-polarization magic angle spinning
(CP MAS) 13C NMR spectra were recorded using a
Chemagnetics CMX LITE 300 MHz spectrometer (1H,
300.63 MHz; 13C, 75.46 MHz). The NMR operating
parameters were 4 kHz MAS, 1 ms contact time and 2
s relaxation delay. Tetramethylsilane was used as an
external reference. The rotor contained 0.4 g soil. The
assignments of the 13C NMR signals to functional
groups are shown in Table 1. The NMR spectra were
recorded in duplicate and standard deviations quoted
relate to analytical error. The areas under di€erent
regions of the spectra (Table 1) were determined using
an area meter (Analytical Development Company,
Hoddeston, Hertfordshire, UK) and expressed as a

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E.A. Webster et al. / Soil Biology & Biochemistry 32 (2000) 301±314

Table 2
Basal respiration over 28 d, biomass-C for unlimed and limed soil without plant material amendment (all values are the mean of three replicates
with standard deviations shown in brackets)

Basal respiration (mmol CO2 gÿ1 soil hÿ1)
Biomass-C (mg C gÿ1 soil)
C content (mg C gÿ1 soil)
N content (mg N gÿ1 soil)
C-to-N ratio

Unlimed soil

Limed soil

0.17 (0.076)
0.06 (0.002)
473.1 (7.64)
17.2 (0.25)
27.5

0.20 (0.090)
0.93 (0.002)
479.4 (1.12)
20.9 (2.07)
22.9

percentage of the area under the spectrum between 0
and 200 ppm. The areas under spinning side bands
were included in the resonances representing carbonylC.

3. Results

2.7. Scanning electron microscopy

The soils from the unlimed and the limed plots contained similar amounts of C (47 and 48% by weight,
respectively) and the C-to-N ratios were 23 and 28, respectively (Table 2). For the unamended soils, the respiration rate over 28 d and biomass-C were
signi®cantly greater (P < 0.01) for the limed than for
the unlimed soil (Table 2). The increase in respiration

Subsamples of plant material separated from the soil
after a 28 d incubation were mounted on aluminium
stubs, sputter-coated with a gold and palladium mixture for 5 min before viewing using a Jeol JSM-35
scanning electron microscope.

3.1. E€ect of liming on basal respiration and microbial
biomass

Fig. 1. Scanning electron micrographs for (a) untreated L. perenne leaves and (b) ethanol- and detergent-treated L. perenne leaves before incubation in soil. The scale bar represents 10 mm.

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E.A. Webster et al. / Soil Biology & Biochemistry 32 (2000) 301±314

Table 3
Chemical characteristics of untreated leaves and ethanol- and detergent-treated leaves expressed per mass of leaf fraction remaining after ethanol
and detergent treatment and per mass of original leaf material (all values are the means of two replicates with standard deviations shown in
brackets)

Proximate analysis
C content
N content
C-to-N ratio
Cellulose
Hemicellulose
Lignin
L-to-CH ratiod
NMR analysis
Alkyl-C
O-alkyl-C
Acetal-C
Aromatic-C
Carbonyl-C

Untreated leaves

Ethanol-/detergent-treated leaves

Ethanol-/detergent-treated leaves

396 (1)a
47 (2)
8.4
246 (2.8)
121 (0.3)
84 (8.1)
0.23

444 (1)b
40 (1)
11.1
326 (4.1)
183 (10.1)
52 (0.5)
0.10

266 (1)c
24 (1)
11.1
195 (2.5)
110 (6.0)
31 (0.3)
0.10

17
37
9
21
15

(2.1)e
(1.8)
(0.7)
(4.6)
(1.4)

13
43
10
17
14

(2.2)e
(3.9)
(0.7)
(4.7)
(0.8)

ÿ24f
+16
+11
ÿ19
ÿ6

a

mg gÿ1 leaf.
mg gÿ1 treated leaf.
c
mg gÿ1 original leaf.
d
L-to-CH ratio is the lignin to cellulose plus hemicellulose ratio.
e
The values for alkyl-C, O-alkyl-C, acetal-C, aromatic-C and carbonyl-C for treated and untreated leaves are % of total intensity of
NMR spectra between 0 and 200 ppm.
f
Relative change (% of untreated leaf); the value shown represents the mass loss during the ethanol and detergent treatment.
b

rate due to liming was smaller than that for biomass
C, so the respiration rate per unit of biomass (qCO2)
was substantially smaller in the limed (0.22 mmol CO2
mgÿ1 biomass C hÿ1) than in the unlimed soil (2.8
mmol CO2 mgÿ1 biomass C hÿ1).
3.2. E€ect of ethanol and detergent treatment on plant
material
The ethanol- and detergent-treated L. perenne leaves
were similar in appearance to the untreated L. perenne
leaves, except that the treated leaves were strawcoloured, whereas the untreated leaves remained green.
Scanning electron micrographs showed that the
untreated L. perenne immediately after treatment (Fig.
1a) and treated plant materials (Fig. 1b) were super®cially similar, with the epidermis appearing intact in
both cases. However, the chemical composition of the
material was altered (Table 3). The ethanol- and detergent-treatment reduced the mass of the plant material
by 410 mg gÿ1 leaf. The ethanol and detergent treatment also reduced the C and the N contents of the
plant material by 33 and 51%, respectively (Table 3).
The greater C-to-N ratio of the ethanol-extracted
leaves indicated preferential extraction of N-containing
over C-containing components. The fractional cellulose, hemicellulose and lignin contents of the ethanoland detergent-treated leaves were signi®cantly greater
(P < 0.05) than the untreated leaves (Table 3). The

13

C

ratio of lignin-to-cellulose-plus-hemicellulose was smaller for ethanol- and detergent-treated leaves (Table 3)
indicating a reduction in the degree of protection of
polysaccharide-C. The mass loss attributable to the
predominantly structural components, cellulose, hemicellulose and lignin was 115 mg gÿ1 (the sum of the
di€erences between the respective values for cellulose,
hemicellulose and lignin in columns 2 and 4 in Table
3), and it can, therefore, be deduced that the mass loss
due to the removal of other mostly nonstructural plant
components was 295 mg gÿ1 leaf.
The CP MAS 13C NMR spectra for the ethanoland detergent-treated and untreated leaves (Fig. 2)
show that proportionately more alkyl-C was removed
during the ethanol and detergent treatment because
the fractions of total spectra in the alkyl-C range were
smaller, compared to the fraction of the spectra in the
O-alkyl-C range, following the ethanol and detergent
treatment (Fig. 2 and Table 3). This is consistent with
preferential extraction of CH2 and CH groups from
compounds such as soluble sugars and amino acids in
the plant sap and plant cytoplasm, extraction of alkylrich components from the waxy cuticle, and relative
enrichment in O-alkyl-C and acetal-C in structural
components.
The solid-state 13C NMR spectra for cellulose and
for holocellulose, from both untreated leaves and treated leaves were similar (Fig. 2). Since the holocellulose
fraction is a composite of hemicellulose and cellulose,

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E.A. Webster et al. / Soil Biology & Biochemistry 32 (2000) 301±314

Fig. 2. Solid-state 13C CP/MAS NMR spectra for (a) untreated L. perenne leaves, cellulose and holocellulose extracted from untreated L. perenne
leaves and (b) ethanol- and detergent-treated L. perenne leaves and cellulose and holocellulose extracted from ethanol- and detergent-treated L.
perenne leaves, and (c) integrated regions representing functional groups and alkyl-C-to-O-alkyl-C ratios calculated from the spectra for L. perenne leaves (L), cellulose (C) and hemicellulose (H) from L. perenne leaves and (d) ethanol- and detergent-treated L. perenne leaves (TL) and cellulose (CT) and hemicellulose (HT) from treated L. perenne leaves.

this suggests that at the level detected by NMR, the
ethanol and detergent treatment did not modify the
composition of either the cellulose or hemicellulose.
3.3. Decomposition of plant materials
Repeated measures analysis of the data showed that
the composition of the plant material had the greatest

e€ect on CO2 production (Fig. 3 and Table 4) whilst
the e€ect of liming was also signi®cant (Table 4). In
addition the interaction between the composition of
plant material and liming was signi®cant (Table 4).
Following amendment with L. perenne or ethanol- and
detergent-treated L. perenne the rates of CO2 production were signi®cantly greater for both the unlimed
(Fig. 3a) and limed (Fig. 3b) soils compared with the

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E.A. Webster et al. / Soil Biology & Biochemistry 32 (2000) 301±314

Fig. 3. The amount of CO2 produced by unlimed and limed soil amended with untreated L. perenne leaves (.) or ethanol- and detergent-treated
L. perenne leaves (w), or the amount of CO2 produced by soil only (Q). Each symbol is the mean of three replicates. For statistical analysis see
Table 4.

2.0 mmol CO2 for unlimed and 1.3 mmol CO2 for
limed soil (Figs. 3a,b). This suggests that 12% (in
unlimed soil) or 8% (in limed soil) of CO2 production
can be attributed to the components of L. perenne
extracted by the ethanol and detergent treatment.

corresponding unamended soils. There was a temporal
correlation with CO2 production during d 0±7 for
both soils amended with untreated or treated plant
material. The rates of CO2 production were greater for
soils that had received the untreated leaves compared
with the corresponding soils that had received the treated leaves for the period 0±20 d incubation for the
unlimed soil and 0±40 d incubation for the limed soil
(Fig. 3). After these periods (20 and 40 d respectively),
the rates of CO2 production by the soils amended with
ethanol- and detergent-treated leaves were not signi®cantly di€erent from those of the corresponding unamended soils. The period of increased CO2 production
was longer for soils amended with untreated leaves
than for soils amended with ethanol- and detergenttreated leaves (0±40 d for the unlimed and at least 0±
50 d for limed soils). The di€erence in CO2 production
between treated leaves and the untreated leaves was

3.4.

13

C NMR of the combined soil and plant material

The e€ect of addition of either untreated leaves or
ethanol- and detergent-treated leaves was initially to
increase the relative intensity of the O-alkyl-C resonance in the NMR spectra of both soils (Fig. 4). The
increase in this signal intensity was due to relative
enrichment of polysaccharide-C in the added plant material (Table 3). After a 28 d incubation, the relative
intensity of the O-alkyl-C signal had declined and the
relative intensity of the alkyl-C and methyl-C signal
increased. After a 75 d incubation there was little

Table 4
Repeated measures analysis of variance of CO2 production data, the analysis was carried out using GENSTAT statistical package (1993) (all
sources of variation in the subject stratum and in the subject time stratum were signi®cant at P < 0.01)
Source of variation
Subject stratum
Liming
Plant composition
Liming.plant composition
Residual
Subject time stratum
Time
Time.liming
Time.plant composition
Time.liming.plant composition
Residual

Revised degrees of freedom

1a
1
2
2
14

Degrees of freedom

s.s.

m.s.

Variance ratio

1
2
2
12

2.145  106
1.640  106
2.988  106
7.676  106

2.145  106
8.198  107
1.494  106
6.397  106

33.53
1281.55
23.35
25.85

23
23
46
46
276

8.443  107
2.481  106
3.585  107
1.733  106
6.829  105

3.671  106
1.079  105
7.793  105
3.767  104
2.474  103

1483.67
43.60
315.00
15.23

a
The degrees of freedom for the subject time stratum were multiplied by 0.0533 to obtain the revised degrees of freedom values before obtaining F values (Greenhouse and Geisser, 1959).

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E.A. Webster et al. / Soil Biology & Biochemistry 32 (2000) 301±314

Fig. 4. (a) Solid-state 13C CP MAS NMR spectra for unlimed soil or limed soil amended with L. perenne leaves or ethanol- and detergent-treated
L. perenne leaves after incubation for 0 or 75 d and for unlimed and limed soil at d 0; (b) integrated regions representing functional groups from
spectra for soils amended with L. perenne leaves or ethanol- and detergent-treated L. perenne leaves after incubation for 1, 28 and 75 d.

E.A. Webster et al. / Soil Biology & Biochemistry 32 (2000) 301±314

309

Fig. 5. (a) Solid-state 13C CP/MAS NMR spectra for untreated L. perenne leaves or ethanol- and detergent-treated L. perenne leaves removed
from unlimed or limed soil after incubation for 1 or 75 d and (b) integrated regions representing functional groups from spectra for L. perenne
leaves or ethanol- and detergent-treated L. perenne leaves separated from unlimed and limed soil after incubation for 1, 28 and 75 d.

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E.A. Webster et al. / Soil Biology & Biochemistry 32 (2000) 301±314

Table 5
Alkyl-C-to-O-alkyl-C ratios for combined plant material and soil
samples for limed or unlimed soil amended with untreated leaves or
ethanol- and detergent-treated leaves

Unlimed soil
d1
d 28
d 75
Limed soil
d1
d 28
d 75

Treated leaves

Untreated leaves

0.67
0.94
0.92

0.58
0.83
0.88

0.64
0.99
0.93

0.67
1.00
0.90

di€erence between the NMR spectra from the
amended and unamended soils, presumably, in part,
because the loss of added 13C as CO2 had diminished
the NMR signal from the added material.
3.5.

13

tra (i.e. the alkyl-C-to-O-alkyl-C ratios), for soils
amended with either untreated or ethanol- and detergent-treated leaves was greater after 28 d than at the
outset (Table 5). There were no further changes in the
alkyl-C-to-O-alkyl-C ratios after 28 d. In the spectra
for plant material before incorporation into the soil
the alkyl-C-to-O-alkyl-C ratios of the ethanol- and
detergent-treated leaves were signi®cantly (P < 0.05)
less than those of the untreated leaves (Table 6). However, the alkyl-C-to-O-alkyl-C ratios of both types of
plant material from both soils increased signi®cantly
(P < 0.05) at some stage between 1 and 28 d incubation, with no further change in this ratio between 28
and 75 d incubation (Table 6). The alkyl-C-to-O-alkylC ratios representing either plant material separated
from the soil or the plant material incorporated into
the soil changed in a similar manner, that is, increased
during the period of increased microbial activity (28 d)
compared with unamended soils.

C NMR of plant material recovered from soil
3.6. Scanning electron microscopy

Ethanol- and detergent-treated leaves recovered
from both soils after incubation for 1 d had more
intense signals at 30 ppm relative to spectra for plant
materials that had not been added to the soil (Fig. 5).
There were no corresponding changes in the spectra
for untreated leaves. The signal at 30 ppm for the
untreated leaves did not increase until 7 d after the addition of plant material, indicating slower initial modi®cation of the untreated leaves.
The ratios of the intensities of the alkyl-C and the
O-alkyl-C signals from the solid-state 13C NMR spec-

Both untreated (Fig. 6a) and treated leaves (Fig. 6b)
retrieved from the limed soil after a 28 d incubation
appeared more degraded than the corresponding materials from the unlimed soil (Figs. 6c,d). The evidence
for this was fragmentation of the epidermis revealing
what is presumed to be the ligni®ed coils of xylem
vessels and the sieve elements. Microorganisms were
visible on untreated and ethanol- and detergent-treated
leaves after a 28 d incubation in unlimed or limed soil.
There were bacterial colonies on and in folds of the

Table 6
Intensity of alkyl-C and O-alkyl-C regions and alkyl-C-to-O-alkyl-C ratios from 13C NMR spectra for untreated leaves and for ethanol- and
detergent-treated leaves before and after incubation in limed or unlimed soil (values shown are the means of two measurements with standard
deviations shown in brackets
Proportion of spectral area represented by alkyl- and O-alkyl C (% of total intensity)
Untreated leaves
alkyl
Before incorporation into soil
17 (2.1) 100%b
After incubation in unlimed soil
d1
18 (0.3) 105%
d 28
22 (1.9) 122%
d 75
20 (1.4) 118%
After incubation in limed soil
d1
20 (1.2) 118%
d 28
20 (2.5) 118%
d 75
21 124%

alkyl-C/O-alkyl-C ratiosa

Ethanol-/detergent-treated leaves

Untreated leaves

Ethanol-/detergent-treated leaves

O-alkyl

alkyl

O-alkyl

alkyl/O-alkyl

alkyl/O-alkyl

36 (1.8) 100%

13 (2.2) 100%

44 (3.9) 100%

0.47 (0.03)

0.30 (0.02)

46(2.3) 128%
38(2.0) 105%
30(0.0) 83%

16 (0.6) 123%
21 (2.4) 162%
23 (2.0) 177%

43 (3.7) 99%
34 (0.8) 77%
31 (0.9) 70%

0.40cc (0.03)
0.57d (0.20)
0.67d (0.05)

0.39c (0.02)
0.64d (0.06)
0.77d (0.04)

46 (0.3) 128%
36 (0.1) 100%
31 86%

16 (2.4) 123%
21 (0.5) 162%
11 (2.2) 162%

46 (0.5) 105%
33 (0.6) 75%
34 (0.9) 77%

0.43c (0.02)
0.57c (0.07)
0.67

0.36c (0.05)
0.64c,d (0.03)
0.63d (0.08)

a
The alkyl-C-to-O-alkyl-C ratios for untreated leaves and for ethanol- and detergent-treated leaves before incorporation into the soils were signi®cantly di€erent (P = 0.03; paired t-test). After incubation in the soil, there was no signi®cant di€erence between the ratios for untreated leaves
and for ethanol- and detergent-treated leaves (P > 0.05).
b
% is the relative amount of added plant material.
c
Within a column and for each soil, di€erent letters (c & d) denote ratios which are signi®cantly di€erent.

E.A. Webster et al. / Soil Biology & Biochemistry 32 (2000) 301±314
311

Fig. 6. Scanning electron micrographs for plant materials after incubation in soils for 28 d (a) untreated L. perenne leaves after incubation in unlimed soil, (b) treated L. perenne leaves after incubation in unlimed soil, (c) untreated L. perenne leaves after incubation in limed soil, (d) treated L. perenne leaves after incubation in limed soil. The bar represents 10 mm.

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E.A. Webster et al. / Soil Biology & Biochemistry 32 (2000) 301±314

epidermis, and hyphae protruding through the epidermis, and from the cut ends of the leaf fragments.

4. Discussion
The aim of the ethanol and detergent treatment of
some L. perenne leaves was to reduce the relative proportion of the cell contents and increase the relative
proportions of structural components in treated leaves
compared to the untreated leaves in order to determine
the relative contributions of these fractions of plant
material to decomposition in limed and in unlimed
soil.
4.1. E€ect of liming
The e€ect of liming the soil was to increase the size
and activity of the microbial community and this e€ect
remained detectable 15 yr after improvement. The fact
that qCO2 was smaller in the limed soil suggests that
the microbial community used less C catabolically.
Consequently, in limed soil the microorganisms were
better able to convert a larger proportion of C to biomass. The greater microbial biomass in limed soil provides additional evidence. There are problems
associated with determining microbial biomass in organic and acid soils (Powlson, 1994). The uncertainty
relates to the conversion factor used to calculate microbial biomass based on glucose-induced respiration.
However, the fact that in limed soil, glucose-induced
respiration was greater than in unlimed soil con®rms
that microbial biomass was greater.
4.2. Decomposition of soluble components
In both soils receiving untreated leaves there was a
¯ush of CO2 production within 7 d that did not occur
to the same extent in soils receiving ethanol- and detergent-treated leaves. This suggested that the components removed during the ethanol and detergent
treatment were compounds which, in untreated leaves,
were immediately accessible to the microbial community and rapidly exhausted. A similar ¯ush of CO2
production was observed during the 48 h after addition of 13C enriched alanine or 13C enriched glucose
to an organic soil (Webster et al., 1997). Marstorp
(1996a, b) attributed the ¯ush of CO2 observed 20 h
after L. perenne addition to soil, to decomposition of
water-soluble amino-acids and sugars. However, no
resonances could be attributed to the decomposition
products from soluble compounds because there was
no resonance absent from the NMR spectra of the
ethanol- and detergent-treated L. perenne leaves but
present in the spectra for L. perenne leaves during d 1
of incubation. This may be because the 13C in de-

composition products were distributed in too large a
number of functional groups to be detected using
NMR. Given the amount of C mineralised and attributable to the plant components removed during the
ethanol and detergent treatment, it is likely that these
compounds were mineralised leaving few residues.
4.3. Decomposition of plant structural components
There were di€erences between the NMR spectra for
plant material separated from soil after incubation for
1 d. The increase in alkyl-C observed after 1 d in treated leaves is probably due to relative accumulation of
residues produced during microbial decomposition of
plant structural components. Condron and Newman
(1998) also reported that recalcitrant plant fragments
produced resonances in the alkyl-C region (at 30
ppm). The observed increase was unlikely to be due to
an increase in microbial tissue because there was no
corresponding resonance in the spectra for untreated
leaves that supported more active microbial communities. The fact that the resonance in the spectra for
treated leaves after 1 d was narrow suggests that the
compounds it represented were relatively homogeneous
(Wilson, 1987), whereas microbial compounds give
characteristic multiple resonances in NMR spectra
(Baldock et al., 1990; Golchin et al., 1996; Webster et
al., 1997). A similar increase in the alkyl-C resonance
was observed in spectra for untreated leaves, but not
until 7 d after addition. Knicker et al. (1997) also
reported a substantial increase in alkyl-C (at 32 ppm)
in L. perenne after incubation for 117 d. We suggest
therefore, that in soils amended with treated leaves,
the microorganisms exploited the structural components of L. perenne whereas in soils amended with
untreated leaves, they exploited the soluble plant components initially and decomposed the structural components only as the soluble compounds became
exhausted. KoÈgel-Knabner et al. (1992) observed that
the accumulation of alkyl-C in forest soils was due to
neither accumulation of microbial polymers, nor accumulation of nonsaponi®able plant compounds. Our
results do not necessarily contradict those of KoÈgelKnabner et al. (1992) because they analysed soil organic matter that had accumulated in forest soil,
whereas we have investigate decomposition of plant
material added to soil over a relatively short time.
4.4. Alkyl-C-to-O-alkyl-C ratios and decomposibility of
organic matter
The alkyl-C-to-O-alkyl-C ratio has been suggested
as an indicator of the decomposibility of soil organic
matter (Baldock and Preston, 1995; Baldock et al.,
1997) with a high alkyl-C-to-O-alkyl-C ratio indicating
lower resource quality of soil organic matter. In spec-

E.A. Webster et al. / Soil Biology & Biochemistry 32 (2000) 301±314

tra for the combined soil and plant materials and the
plant material separated from soil, the alkyl-C-to-Oalkyl-C ratios changed with time in a similar manner.
In both cases the period during which the ratios
increased coincided with the period of greatest microbial activity. This suggested that changes in the
alkyl-C-to-O-alkyl-C ratio were consistent with microbial alteration and utilisation of the soil and plant
material.
4.5. Litter quality
The fact that the ethanol- and detergent-treated
leaves contained proportionately less N, and had a
greater C-to-N ratio, than untreated leaves suggested
that, on the basis of chemical composition, the ethanol- and detergent-treated leaves were a poorer quality
substrate for microbial metabolism. It is likely, however, that during the ethanol and detergent treatment,
the integrity of the leaf cuticle was disrupted, reducing
the e€ectiveness of the cuticle as a barrier to microorganisms. The ethanol and detergent treatment also
reduced the lignin content, decreasing the ratio of lignin to cellulose-plus-hemicellulose compared to
untreated leaves. This suggested that, in the ethanoland detergent-treated leaves, there may have been less
physical protection and consequently greater accessibility of structural polysaccharide-C to microbial degradation. It is likely that in the limed soil the reduced N
content of the ethanol- and detergent-treated L. perenne leaves was not a limiting factor. In limed soil,
therefore, the treated leaves are the better quality
resource for microbial decomposer organisms. The microbial community in limed soil was apparently, less
constrained by the lower N-content of the ethanoland detergent-treated leaves and was better able to utilise the ethanol- and detergent-treated leaves despite
their increased C-to-N ratio.
One di€erence between the untreated leaves and the
ethanol- and detergent-treated leaves, as seen by
NMR, was that the NMR spectra for the treated
leaves had a smaller alkyl-C-to-O-alkyl-C ratio than
the spectra for the untreated leaves. The lower ligninto-cellulose ratio of the treated leaves indicated that
the treated leaves may be the better quality resource.
This suggested that the alkyl-C-to-O-alkyl-C ratio may
indicate the quality of plant material as a resource for
microorganisms. Baldock and Preston (1995) and Baldock et al. (1997) suggested that a low alkyl-C-to-Oalkyl-C ratio indicated greater resource quality of soil
organic matter.

5. Conclusions
Our objective was to determine the relative contri-

313

butions of di€erent components of plant material
towards decomposition in soil. We have shown that
around 12% (in unlimed soil) and 8 % (in limed soil)
of C mineralization can be attributed to soluble plant
components. We have also shown that in the short
term the residues produced during decomposition of
plant structural components, including cellulose and
hemicellulose, contributed towards the alkyl-C pool in
soil. Decomposition of cellular material and soluble
plant components, however, appeared not to make as
large a contribution to the alkyl-C pool. The alkyl-Cto-O-alkyl-C ratio may be an indicator of the quality
of soil organic matter as a substrate for microorganisms. Our results showed that the alkyl-C-to-O-alkyl-C
ratio also re¯ected the underlying changes in plant material during decomposition in soil, and may indicate
the quality of plant material as a substrate for microorganisms before incubation in soil.
In the limed soil, the microorganisms were collectively more metabolically versatile, and were not
restricted by the lower N content of the ethanol- and
detergent-treated plant material. The greater apparent
versatility enabled the microorganisms in the limed soil
to exploit the more accessible polysaccharide-C in the
treated plant material. The scanning electron micrographs provided additional evidence of the greater eciency of the decomposer community in limed soil.

Acknowledgements
We are grateful to the UK Natural Environment
Research Council for ®nancial support. Thanks are
also due to Dr. R. Webster for statistical advice, Margaret Gruber and Martin Kierans for assistance with
photography and electron microscopy.

References
Allen, S.E., Grimshaw, H.M., Parkinson, J.A., Quarmby, G., 1974.
Chemical Analysis of Ecological Materials. Blackwell Scienti®c
Publications, Oxford, pp. 245±255.
Anderson, J.P.E., Domsch, K.H., 1978. A physiological method for
the quantitative measurement of microbial biomass in soils. Soil
Biology & Biochemistry 10, 215±221.
Baldock, J.A., Currie, G.J., Oades, J.M., 1991. Organic matter as
seen by solid-state 13C nuclear magnetic resonance spectroscopy
and pyrolysis tandem mass spectrometry. In: Advances in Soil
Organic Matter Research. Royal Society of Chemistry,
Cambridge, pp. 45±60.
Baldock, J.A., Oades, J.M., Nelson, P.N., Skene, T.M., Golchin, A.,
Clarke, P., 1997. Assessing the extent of decomposition of natural
organic materials using solid-state 13C NMR spectroscopy.
Australian Journal of Soil Research 35, 1061±1083.
Baldock, J.A., Preston, C.M., 1995. Chemistry of carbon decomposition processes in forests as revealed by solid-state carbon-13

314

E.A. Webster et al. / Soil Biology & Biochemistry 32 (2000) 301±314

nuclear magnetic resonance. In: McFee, W.W., Kelly, J.M.
(Eds.), Carbon Forms and Functions in Forest Soils. Soil Society
of America, Madison.
Baldock, J.A., Oades, J.M., Vassallo, A.M., Wilson, M.A., 1990.
Solid-state CP/MAS 13C NMR analysis of bacterial and fungal
cultures isolated from a soil and incubated with glucose.
Australian Journal of Soil Research 28, 213±225.
Condron, L.M., Newman, R.H., 1998. Chemical nature of soil organic matter under grassland and recently established forest.
European Journal of Soil Science 49, 597±603.
Dampney, P.M.R., 1985. A trial to determine the lime requirement
for reseeded grassland on a peaty hill soil. Soil Use and
Management 1, 95±100.
Eswaran, H., Van den Berg, E., Reich, P., 1993. Organic carbon in
soils of the world. Soil Science Society of America Journal 57,
192±194.
Floate, M.J., 1977. British hill soil problems. Soil Science 123, 325±
331.
GENSTAT 5 Release 3 Reference Manual, 1993. Oxford University
Press, Oxford.
Payne, R.W., Morgan, G.W., Arnold, G.M. (Eds.), 1997.
GENSTAT 5 Procedure Library Manual, Release pl10. Lawes
Agricultural Trust, Harpenden.
Golchin, A., Clarke, P., Oades, J.M., 1996. The heterogeneous
nature of microbial products as shown by solid-state 13C CP/
MAS NMR spectroscopy. Biogeochemistry 34, 71±97.
Greenhouse, S.W., Geisser, S., 1959. On methods in the analysis of
pro®le data. Psychometrika 24, 95±112.
Heilmann, B., Beese, F., 1992. Miniaturized method to measure carbon dioxide production and biomass of soil microorganisms. Soil
Science Society of America Journal 56, 596±598.
Hopkins, D.W., 1997. Decomposition in a peaty soil limed for pastoral agriculture. Soil Use and Management 13, 104±106.
Hopkins, D.W., Chudek, J.A., Webster, E.A., Barraclough, D.,
1997. Following the decomposition of ryegrass labelled with 13C
and 15N in soil by solid-state nuclear magnetic resonance spectroscopy. European Journal of Soil Science 48, 623±631.
Hopkins, D.W., Ferguson, K.E., 1994. Substrate induced respiration
in soil amended with di€erent amino acid isomers. Applied Soil
Ecology 1, 75±81.

Hopkins, D.W., Shiel, R.S., O'Donnell, A.G., 1990. Yield and nitrogen utilization by Lolium perenne and Trifolium repens on limed
stagnohumic gley soil in a pot experiment. Grass Forage Science
45, 107±112.
Isabella, B.A., Hopkins, D.W., 1994. Nitrogen transformations in a
peaty soil limed for pastoral agriculture. Soil Use and
Management 10, 107±111.
Knicker, H., LuÈdemann, H.-D., Haider, K., 1997. Incorporation studuring incubation of organic residues by 15Ndies of NH+
4
CPMAS-NMR-spectroscopy. European Journal of Soil Science
48, 431±441.
KoÈgel-Knabner, I., 1997. 13C and 15N NMR spectroscopy as a tool
in soil organic matter studies. Geoderma 80, 243±270.
KoÈgel-Knabner, I., Hatcher, P.G., Tegelaar, E.W., de Leeuw, J.W.,
1992. Aliphatic components of forest soil organic matter as determined by solid-state 13C NMR and analytical pyrolysis. The
Science of the Total Environment 113, 89±106.
Marstorp, H., 1996a. In¯uence of soluble carbohydrates, free amino
acids, and protein content on the decomposition of Lolium multi¯orum shoots. Biology and Fertility of Soils 21, 257±263.
Marstorp, H., 1996b. Interactions in microbial use of soluble plant
components. Biology and Fertility of Soils 22, 45±52.
Newbould, P., 1985. Improvement of native grassland in the
uplands. Soil Use and Management 1, 43±49.
Preston, C.M., 1996. Applications of NMR to soil organic matter
analysis: history and perspectives. Soil Science 161, 144±166.
Powlson, D.S., 1994. The soil microbial biomass: before, beyond and
back. In: Ritz, K., Dighton, J., Giller, K.E. (Eds.), Beyond the
Biomass compositional and functional analysis of soil microbial
communities. Wiley, Chichester.
Ritchie, R.J., Larkin, A.W.D., 1982. Cation exchange ¯uxes of the
cell walls of Enteromorpha intestinalis (L). Journal of
Experimental Botany 132, 140±153.
Swift, M.J., Heal, O.W., Anderson, J.M., 1979. Decomposition in
Terrestrial Ecosystems. Blackwell Scienti®c Publications, Oxford.
Webster, E.A., Chudek, J.A., Hopkins, D.W., 1997. Fates of 13C
from enriched glucose and glycine in an organic soil. Biology and
Fertility of Soils 25, 389±395.
Wilson, M.A., 1987. NMR Techniques and Applicataions in
Geochemistry and Soil Chemistry. Pergamon Press, Oxford.