Carbon transformations during decomposition of dierent
components of plant leaves in soil
E.A. Webster
a, b,*, J.A. Chudek
b, D.W. Hopkins
ca
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 eect of lime addition to an upland organic soil on the decomposition of Lolium perenne leaves and isolated fractions ofL. perenne leaves in a laboratory experiment lasting 75 d. The L. perenne plants were grown in a 13CO
2 -enriched 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 CO2production. 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. The13C NMR spectra recorded for plant material separated from soil 1 d after addition of ethanol- and detergent-treated leaves had larger alkyl-C (30 ppm) signals compared with spectra from undetergent-treated leaves. This was interpreted as representing an accumulation of residues from decomposition of plant structural components.# 2000 Elsevier Science Ltd. All rights reserved.
Keywords:13C solid-state NMR;Lolium perenne; Scanning electron microscopy
1. Introduction
Upland soils in temperate regions are often charac-terized 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
agriculture. Improvement involved removing the indi-genous 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
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* Corresponding author. Present address: Department of Environ-mental Science, University of Stirling, Stirling FK9 4LA, Scotland, UK. Tel.: +44-1786-473171, ext. 6537; fax: +44-1786-467843.
(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 in-vestigate the carbon transformations during decompo-sition of dierent 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 decompo-sition of dierent components of plant material in soil at the level of functional groups. Decomposition of plant material in soil generally results in a relative ac-cumulation of alkyl-C and a relative decrease in O-alkyl-C (as determined from 13C NMR spectra) (KoÈgel-Knabner, 1997; Hopkins et al., 1997). Conse-quently, 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 attribu-table to either plant residues or microbial components are not well known. Our aim was to provide infor-mation on the relative contribution of plant com-ponents to the alkyl-C pool in the short term.
Our speci®c objectives were to investigate carbon transformations during decomposition of dierent components of plant material in soil, to investigate the contribution of plant components to the alkyl-C pool in soil, and to investigate the eects of liming the soil on the decomposition of components of plant ma-terials.
2. Materials and methods
2.1. Soils
The soils were sampled from 0±15 cm depths of two experimental plots at the Redesdale Experimental Hus-bandry 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 graz-ing (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 Hop-kins, 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 pratenseand Trifo-lium 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 respiro-metric 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
atmos-phere as described by Hopkins et al. (1997). Approxi-mately 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% X solution for 12 h then discarding the Triton-X 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 deter-gent-treated leaves had 2.00 and 1.97 at% 13C, respect-ively, as determined by mass spectrometry (see below).
2.4. Chemical analysis
chlorite and 0.5 ml 10% ethanoic acid were added to the mixtures. The mixtures were cooled on ice, vac-uum-®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 holocel-lulose into hemicelholocel-lulose and celholocel-lulose, a known mass of holocellulose was added to 20 ml 24% KOH (w/v) and maintained at 208C for 2 h. The mixture was vac-uum-®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, cellu-lose and hemicellucellu-lose 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 gÿ1soil (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 min-iaturized 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
measure-ments were determined, by gas chromatography (Hop-kins 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 23 factorial with four replicates in a
random-ised 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 oc-casion (0, 1, 3, 7, 14, 28 and 75 d) and were freeze-dried. The contents of one vial (comprising soil and plant material) was ground and used for NMR analy-sis. 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 suciently large for NMR analysis. Plant material that had not been added to soil, and that, therefore remained uncontami-nated with soil microorganisms, was used as the con-trol 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 dierent regions of the spectra (Table 1) were determined using an area meter (Analytical Development Company, Hoddeston, Hertfordshire, UK) and expressed as a Table 1
Assignment of C functional groups to shift ranges for solid-state13C NMR spectra (adapted from Wilson, 1987; Baldock et al., 1991)
Shift range (ppm) Assignment Main classes of compounds included
0±45 methyl- and alkyl-C aliphatic compounds, lipids, waxes
45±60 methoxyl- and N-alkyl-C lignin substituents, amino acids, amino sugars
60±90 O-alkyl-C carbohydrates, lignin propyl side chains
90±110 acetal- and ketal-C carbohydrates
110±160 aromatic-C phenyl-propylene subunits of lignin
percentage of the area under the spectrum between 0 and 200 ppm. The areas under spinning side bands were included in the resonances representing carbonyl-C.
2.7. Scanning electron microscopy
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 mix-ture for 5 min before viewing using a Jeol JSM-35 scanning electron microscope.
3. Results
3.1. Eect of liming on basal respiration and microbial biomass
The soils from the unlimed and the limed plots con-tained similar amounts of C (47 and 48% by weight, respectively) and the C-to-N ratios were 23 and 28, re-spectively (Table 2). For the unamended soils, the res-piration 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 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)
Unlimed soil Limed soil
Basal respiration (mmol CO2gÿ1soil hÿ1) 0.17 (0.076) 0.20 (0.090)
Biomass-C (mg C gÿ1soil) 0.06 (0.002) 0.93 (0.002)
C content (mg C gÿ1soil) 473.1 (7.64) 479.4 (1.12)
N content (mg N gÿ1soil) 17.2 (0.25) 20.9 (2.07)
C-to-N ratio 27.5 22.9
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 CO2mgÿ1biomass C hÿ1).
3.2. Eect of ethanol and detergent treatment on plant material
The ethanol- and detergent-treated L. perenne leaves were similar in appearance to the untreatedL. perenne
leaves, except that the treated leaves were straw-coloured, whereas the untreated leaves remained green. Scanning electron micrographs showed that the untreated L. perenneimmediately 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 deter-gent-treatment reduced the mass of the plant material by 410 mg gÿ1 leaf. The ethanol and detergent
treat-ment 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 cellu-lose, hemicellulose and lignin contents of the ethanol-and detergent-treated leaves were signi®cantly greater (P < 0.05) than the untreated leaves (Table 3). The
ratio of lignin-to-cellulose-plus-hemicellulose was smal-ler 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, hemi-cellulose and lignin was 115 mg gÿ1 (the sum of the
dierences 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 ethanol-and detergent-treated ethanol-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 alkyl-rich 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 trea-ted leaves were similar (Fig. 2). Since the holocellulose fraction is a composite of hemicellulose and cellulose, 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)
Untreated leaves Ethanol-/detergent-treated leaves Ethanol-/detergent-treated leaves
Proximate analysis
C content 396 (1)a 444 (1)b 266 (1)c
N content 47 (2) 40 (1) 24 (1)
C-to-N ratio 8.4 11.1 11.1
Cellulose 246 (2.8) 326 (4.1) 195 (2.5)
Hemicellulose 121 (0.3) 183 (10.1) 110 (6.0)
Lignin 84 (8.1) 52 (0.5) 31 (0.3)
L-to-CH ratiod 0.23 0.10 0.10
NMR analysis
mg gÿ1treated leaf. c
mg gÿ1original 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 of13C NMR spectra between 0 and 200 ppm.
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
eect on CO2 production (Fig. 3 and Table 4) whilst
the eect 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. perenneor ethanol- and detergent-treated L. perenne the rates of CO2
pro-duction were signi®cantly greater for both the unlimed (Fig. 3a) and limed (Fig. 3b) soils compared with the Fig. 2. Solid-state13C CP/MAS NMR spectra for (a) untreatedL. perenneleaves, cellulose and holocellulose extracted from untreatedL. perenne
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 CO2production were greater for
soils that had received the untreated leaves compared with the corresponding soils that had received the trea-ted 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 CO2production by the soils amended with
ethanol- and detergent-treated leaves were not signi®-cantly dierent from those of the corresponding una-mended soils. The period of increased CO2production
was longer for soils amended with untreated leaves than for soils amended with ethanol- and detergent-treated leaves (0±40 d for the unlimed and at least 0± 50 d for limed soils). The dierence in CO2production
between treated leaves and the untreated leaves was
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 CO2production
can be attributed to the components of L. perenne
extracted by the ethanol and detergent treatment.
3.4.13C NMR of the combined soil and plant material
The eect of addition of either untreated leaves or ethanol- and detergent-treated leaves was initially to increase the relative intensity of the O-alkyl-C reson-ance 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 ma-terial (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 atP< 0.01)
Source of variation Revised degrees of freedom Degrees of freedom s.s. m.s. Variance ratio
Subject stratum
Liming 1 2.145106 2.145106 33.53
Plant composition 2 1.640106 8.198107 1281.55
Liming.plant composition 2 2.988106 1.494106 23.35
Residual 12 7.676106 6.397106 25.85
Subject time stratum
Time 1a 23 8.443
107 3.671106 1483.67
Time.liming 1 23 2.481106 1.079105 43.60
Time.plant composition 2 46 3.585107 7.793105 315.00
Time.liming.plant composition 2 46 1.733106 3.767104 15.23
Residual 14 276 6.829105 2.474103
a
The degrees of freedom for the subject time stratum were multiplied by 0.0533 to obtain the revised degrees of freedom values before obtain-ingFvalues (Greenhouse and Geisser, 1959).
Fig. 3. The amount of CO2produced by unlimed and limed soil amended with untreatedL. perenneleaves (.) or ethanol- and detergent-treated
Fig. 4. (a) Solid-state13C CP MAS NMR spectra for unlimed soil or limed soil amended withL. perenneleaves or ethanol- and detergent-treated
Fig. 5. (a) Solid-state13C CP/MAS NMR spectra for untreatedL. perenneleaves or ethanol- and detergent-treatedL. perenneleaves removed from unlimed or limed soil after incubation for 1 or 75 d and (b) integrated regions representing functional groups from spectra forL. perenne
dierence 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.
3.5.13C NMR of plant material recovered from soil
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 ad-dition 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-tra (i.e. the alkyl-C-to-O-alkyl-C ratios), for soils amended with either untreated or ethanol- and deter-gent-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). How-ever, 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 incu-bation, with no further change in this ratio between 28 and 75 d incubation (Table 6). The alkyl-C-to-O-alkyl-C 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.
3.6. Scanning electron microscopy
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 ma-terials 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 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
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) alkyl-C/O-alkyl-C ratiosa
Untreated leaves Ethanol-/detergent-treated leaves Untreated leaves Ethanol-/detergent-treated leaves
alkyl O-alkyl alkyl O-alkyl alkyl/O-alkyl alkyl/O-alkyl
Before incorporation into soil
17 (2.1) 100%b 36 (1.8) 100% 13 (2.2) 100% 44 (3.9) 100% 0.47 (0.03) 0.30 (0.02)
After incubation in unlimed soil
d 1 18 (0.3) 105% 46(2.3) 128% 16 (0.6) 123% 43 (3.7) 99% 0.40cc(0.03) 0.39c (0.02) d 28 22 (1.9) 122% 38(2.0) 105% 21 (2.4) 162% 34 (0.8) 77% 0.57d (0.20) 0.64d (0.06)
d 75 20 (1.4) 118% 30(0.0) 83% 23 (2.0) 177% 31 (0.9) 70% 0.67d (0.05) 0.77d (0.04)
After incubation in limed soil
d 1 20 (1.2) 118% 46 (0.3) 128% 16 (2.4) 123% 46 (0.5) 105% 0.43c (0.02) 0.36c (0.05) d 28 20 (2.5) 118% 36 (0.1) 100% 21 (0.5) 162% 33 (0.6) 75% 0.57c (0.07) 0.64c,d (0.03)
d 75 21 124% 31 86% 11 (2.2) 162% 34 (0.9) 77% 0.67 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 sig-ni®cantly dierent (P= 0.03; pairedt-test). After incubation in the soil, there was no signi®cant dierence 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
Fig. 6. Scanning electron micrographs for plant materials after incubation in soils for 28 d (a) untreatedL. perenneleaves after incubation in unlimed soil, (b) treatedL. perenneleaves after incu-bation in unlimed soil, (c) untreatedL. perenneleaves after incubation in limed soil, (d) treatedL. perenneleaves after incubation in limed soil. The bar represents 10mm.
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epidermis, and hyphae protruding through the epider-mis, and from the cut ends of the leaf fragments.
4. Discussion
The aim of the ethanol and detergent treatment of some L. perenneleaves was to reduce the relative pro-portion 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. Eect of liming
The eect of liming the soil was to increase the size and activity of the microbial community and this eect 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 bio-mass. The greater microbial biomass in limed soil pro-vides additional evidence. There are problems associated with determining microbial biomass in or-ganic and acid soils (Powlson, 1994). The uncertainty relates to the conversion factor used to calculate mi-crobial 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 CO2production within 7 d that did not occur
to the same extent in soils receiving ethanol- and deter-gent-treated leaves. This suggested that the com-ponents removed during the ethanol and detergent treatment were compounds which, in untreated leaves, were immediately accessible to the microbial commu-nity and rapidly exhausted. A similar ¯ush of CO2
production was observed during the 48 h after ad-dition 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. perenneleaves 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 at-tributable 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 dierences 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 trea-ted 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 commu-nities. 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 com-ponents of L. perenne whereas in soils amended with untreated leaves, they exploited the soluble plant ponents initially and decomposed the structural com-ponents 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 ac-cumulation of nonsaponi®able plant compounds. Our results do not necessarily contradict those of KoÈgel-Knabner et al. (1992) because they analysed soil or-ganic 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
spec-tra for the combined soil and plant materials and the plant material separated from soil, the alkyl-C-to-O-alkyl-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 mi-crobial activity. This suggested that changes in the alkyl-C-to-O-alkyl-C ratio were consistent with mi-crobial 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 etha-nol- and detergent-treated leaves were a poorer quality substrate for microbial metabolism. It is likely, how-ever, that during the ethanol and detergent treatment, the integrity of the leaf cuticle was disrupted, reducing the eectiveness of the cuticle as a barrier to microor-ganisms. The ethanol and detergent treatment also reduced the lignin content, decreasing the ratio of lig-nin to cellulose-plus-hemicellulose compared to untreated leaves. This suggested that, in the ethanol-and detergent-treated leaves, there may have been less physical protection and consequently greater accessibil-ity of structural polysaccharide-C to microbial degra-dation. It is likely that in the limed soil the reduced N content of the ethanol- and detergent-treated L. per-enne leaves was not a limiting factor. In limed soil, therefore, the treated leaves are the better quality resource for microbial decomposer organisms. The mi-crobial community in limed soil was apparently, less constrained by the lower N-content of the ethanol-and detergent-treated leaves ethanol-and was better able to uti-lise the ethanol- and detergent-treated leaves despite their increased C-to-N ratio.
One dierence 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 lignin-to-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 Bal-dock et al. (1997) suggested that a low alkyl-C-to-O-alkyl-C ratio indicated greater resource quality of soil organic matter.
5. Conclusions
Our objective was to determine the relative
contri-butions of dierent 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-C-to-O-alkyl-C ratio may be an indicator of the quality of soil organic matter as a substrate for microorgan-isms. Our results showed that the alkyl-C-to-O-alkyl-C ratio also re¯ected the underlying changes in plant ma-terial during decomposition in soil, and may indicate the quality of plant material as a substrate for micro-organisms before incubation in soil.
In the limed soil, the microorganisms were collec-tively 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 micro-graphs 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, Mar-garet Gruber and Martin Kierans for assistance with photography and electron microscopy.
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