土壤结构与碳循环

Aust. J. Soil Res., 1994, 32, 1043-68 Soil Biology and Biochemistry Soil Structure and Carbon Cycling A. G ~ l c h i n , ~ J. M. O a d e ~ , ~ J. 0. SkjemstadB and P. ClarkeA A Department of Soil Science, Waite Agricultural Research Institute, The University of Adelaide, Glen Osmond, S.A. 5064. Division of Soil, CSIRO, Glen Osmond, S.A. 5064. Abstract Samples from the surface horizons of six virgin soils were collected and separated into density fractions. Based on the spatial distribution of organic materials within the mineral matrix of soil, the soil organic matter (SOM) contained in various density fractions was classified as: (a) free particulate OM, (b) occluded particulate OM, and (c) colloidal or clay-associated OM. The compositional differences noted among these three components of SOM were used to describe the changes that OM undergoes during decomposition when it enters the soil, is enveloped in aggregates and eventually is incorporated into microbial biomass and metabolites and associated with clay minerals. The occluded organic materials, released as a result of aggregate disruption, were in various stages of decomposition and had different degrees of association with mineral particles. Changes in the degree of association of occluded organic materials and mineral particles with decomposition are discussed and form the basis of a model which illustrates the simultaneous dynamics of microaggregates and their organic cores. This model indicates a major role for carbohydrate-rich plant debris in formation and stabilization of microaggregates. Keywords: soil organic matter, soil structure, carbon cycling, l3 C CP/MAS NMR, density fractionation, scanning electron microscopy, humic acid. Introduction Soil structure, especially the spatial distribution of OM within the organo- mineral matrix of soil, is considered to be one of the dominant controls over microbially mediated decomposition processes in terrestrial ecosystems (Oades 1988; van Veen and Kuikman 1990). However, there is no clear understanding of the role of soil structure in carbon cycling, or of the dynamics of soil structure other than the simultaneous loss of larger aggregates and of OM in cultivated soils. Optical examination of the soil matrix reveals assemblages of OM and mineral particles in water-stable aggregates (Waters and Oades 1991). Water-stable aggregates are arbitrarily distinguished by size as macro- >250 pm and micro- aggregates <250 pm (Edwards and Bremner 1967). Macroaggregates are easily disrupted by wetting or low energy agitation, whereas microaggregates have high stability and, for complete dispersion, require the more energetic treatment of prolonged shaking or sonification. Recent studies indicate a major role for plant debris in the stability of microaggregates. It has been shown that many aggregates A. Golchin et al. about 100-200 pm in diameter have cores of plant debris, and encrustation of plant fragments by mineral particles was proposed as one of the mechanisms in microaggregate formation and stabilization (Waters and Oades 1991; Oades and Waters 1991). Incorporation of OM into soil aggregates protects it from rapid decomposition and is one of the determinants of stability of OM in soils. Soil organic matter (SOM) is found distributed as either well individualized particles, such as pieces of plant debris, or as amorphous OM. The free particulate OM which is located between the soil aggregates can be separated from organo-mineral particles by flotation on a liquid density d = 1 .6 Mg m-3 (Golchin et al. 1994). This fraction, referred to as the free light fraction, usually consists of only slightly decomposed plant debris. After removal of the free light fraction, the occluded particulate OM can be separated from the soil by density fractionation after the aggregates have been disrupted by ultrasonic energy. A 5 min ultrasonic treatment permits the occluded organic materials that are in different stages of decomposition to be separated because they have formed different degrees of association with mineral particles and thus have different densities. A comparison of the chemical composition of occluded organic materials, separated at different densities, provides information on the dynamics of the organic cores of aggregates and thus the whole aggregates. It also enables the compounds which are responsible for association of organics and minerals and thus the integrity of the aggregates to be detected. The amorphous OM which has originated from the decomposition of particulate OM is adsorbed by clay minerals and stabilized against further decomposition. The differences in organic structure between free particulate OM, occluded particulate OM and amorphous OM represent the changes that OM undergoes during decomposition when it enters the soil, is enveloped in aggregates, and eventually is incorporated into microbial biomass and products and adsorbed to clay minerals. In this study, six virgin mineral soils were separated into different density fractions and electron microscopy and l3 C CP/MAS NMR spectroscopy were employed to study the following: (1) differences in the morphology and chemistry of SOM contained in different density fractions; (2) variations in the chemical structure of SOM in density fractions in different soils; (3) comparisons of the structure of the humic acid fraction and physically separated fractions from the same soil; (4) relationship of SOM turnover and dynamics of soil structure as a conceptual model. Materials and Methods Soils The soils used in this study are the same as those described by Golchin et al. (1994) with the addition of a second grey clay from Victoria. A representative sample from the A horizon (0-10 cm; 2.37% organic C; 0.28% total N; 4.5% CaC03; 50% clay; pH 8.5, soillwater ratio 1:5) of a Victoria grey clay under permanent pasture was used. The soil sampling procedure and soil sample preparation for density fractionation were described previously (Golchin et al. 1994). Soil Structure and Carbon Cycling Density Fractionation of Soils The soils were separated into fractions with densities <1.6 (free and occluded particulate OM), 1.6-1.8, 1.8-2 .O and >2.O Mg m-3. Methods for separation of the <1.6 Mg m-3 free and occluded particulate OM were described in detail in our previous work (Golchin et al. 1994). After removal of these two fractions, further density fractionation of soil was carried out by introducing the residual soil to sodium polytungstate solution of higher densities (i.e., 1 . 8 and 2.0 Mg m-3). The bottle was shaken intermittently by hand to completely resuspend the residue in the separating medium and fractions were isolated by recentrifuging. After separation of the 1.8-2 Mg m-3 fraction, the remaining soil (>2.O Mg m-3 fraction) was washed with water three times; if dispersion occurred during this stage, the sample was flocculated with Ca2+, centrifuged and dried at 60°C. With drying, this fraction separated into two layers, i.e., a dark, finer fraction dried as a 'skin' over a coarser light coloured fraction. The skin was lifted off, ground, HF treated and used for NMR analysis. An unground subsample of the skin was also used for electron microscopic studies. For carbon and nitrogen analysis, however, the whole sample was used. Since the >2.0 Mg m-3 fraction contained a large proportion of the soil sample, the dry weight of this fraction was calculated by the difference of the weights of the soil sample and of the light (<2.0 Mg m-3) fractions. SOM fractionation was replicated at least four times for each soil sample, and the replicates of each fraction were then combined for analysis. The variability of replicates was low and the mean yields of the fractions are reported in Table 1. Sample Preparation for NMR Analysis The 13c CP/MAS NMR results for the >2.0 Mg m-3 fractions were limited by poor signal-to-noise ratio and lack of resolution, possibly due to low carbon content and line- broadening by iron. To prepare this fraction suitably for NMR analysis, an HF treatment similar to that recommended by Skjemstad et al. (1994) was used. Finely ground samples of 3 g were placed in 50 mL centrifuge tubes and 50 mL of 2% HF solution was added. The tubes were shaken end-over-end for 1 h and the supernatant was discarded after centrifugation (2000,, 10 min). This process was repeated five times and followed by three more successive extractions (2x16 h and 1x64 h). After the final extraction, the residues were washed with water three times; if dispersion occurred during this stage, the sample was flocculated with ~ l ~ + , centrifuged and dried at 60°C. Extraction of Humic and Fulvic Acids The procedure for extraction of humic acids has been described in detail by Schnitzer (1982). In this study the method included extraction of soil with 0 .5 M NaOH under Nz at a soil to extractant ratio of 1:lO. The soil suspension was shaken at room temperature for 24 h and the alkaline supernatant was separated from the soil by centrifugation. The extract was then acidified with 3 M H2S04 to pH 2 and allowed to stand a t room temperature for 24 h. The fulvic acid (supernatant) was separated from humic acid (coagulate) by centrifugation and flocculated with ~ l ~ + at pH 4.3. Both fulvic and humic acids were washed with water and dried at 40°C. Chemical Analyses Whole soils and density fractions were analysed for organic carbon (C) and total nitrogen (N). Total C was determined by dry combustion (Merry and Spouncer 1988) in an induction furnace (Leco CR-12 Automatic Carbon Analyser) and data were corrected for inorganic C (carbonate) content measured using the volumetric calcimeter method of Allison and Moodie (1965). Total N was determined by Kjeldahl digestion. Carbon-13 Nuclear Magnetic Resonance (I3 C NMR) Spectroscopy Solid-state 13C NMR spectra, with cross-polarization and magic-angle spinning (CPIMAS), of density fractions and humic and fulvic acids were obtained at 50.3 MHz on a Varian Unity 200 Spectrometer as described by Golchin et al. (1994). Ta bl e 1. Se pa ra te d fr ac tio ns a n d th ei r pr op er tie s G re at S oi l Fr ac tio n % so il w ei gh t % C in % N i n C in f ra ct io n, N in f ra ct io n, C: N o f G ro up in fr ac tio n fr ac tio n fr ac tio n % o f so il O C % o f to t. s o il N fr ac tio n So lo di c N on -c al ci c br ow n- ea rth Qu ee ns lan d gr ey c la y R ed - b ro w n e a rt h B la ck e a rt h V ic to ria gr ey c la y 1. 6- 1. 8 1. 8- 2. 0 to ta l < 2. 0 fr ac tio ns A > 2. 0 1. 6- 1. 8 1. 8- 2. 0 to ta l < 2. 0 fr ac tio ns A > 2. 0 1. 6- 1. 8 1. 8- 2. 0 to ta l < 2. 0 fr ac tio ns A > 2. 0 1. 6- 1. 8 1. 8- 2. 0 to ta l of < 2. 0 fr ac tio ns A > 2. 0 1. 6- 1. 8 1. 8- 2- 0 to ta l of < 2. 0 fr ac tio ns A > 2. 0 < 1. 6 (fr ee ) < I. 6 (o cc lu de d) 1. 6- 1. 8 1. 8- 2. 0 to ta l of < 2. 0 fr ac tio ns > 2. 0 A I nc lu de s < 1- 6 M g rn p3 (f ree a n d o c c lu de d) fr ac tio ns (G olc hin e t a l. 19 94 ). n .d ., n o t de te rm in ed . Soil Structure and Carbon Cycling Scanning Electron Microscopy Electron micrographs for 1 .6-1.8 and 1.8-2.0 Mg m-3 fractions of the red-brown earth were obtained on a Cambridge Stereoscan S250 electron microscope using oven dried (60°C), unground samples (Golchin et al. 1994). For the >2.0 Mg m-3 fraction, however, subsamples of the skin were suspended in water and drops of the suspensions were mounted on aluminium stubs, dried, and coated with gold for examination in the microscope. Results Carbon and Nitrogen Distribution in Fractions Analytical data for the 2.0 Mg m-3, by contrast, comprised most of the soil mass. The C and N contents of the fractions decreased with increasing density. In the soils studied, the highest and lowest C and N concentrations occurred in the fractions <1.6 Mg m-3 (occluded) and >2.0 Mg m-3 respectively. The C:N ratios of SOM in density fractions declined with increasing density, suggesting the nitrogen enrichment of organic components of heavy (>2 Mg m-3) fractions. The C:N ratios of the >2.0 Mg m-3 fractions were lower than those of the whole soil, while the C:N ratios of the <1.6 (free and occluded) fractions were higher. Electron Microscopy of Density Fractions Microscopic investigation of different density fractions allowed characterization of intact microaggregates as well as particulate OM and its association with mineral particles (Figs 1 a-c). Ultrasonic treatment destroyed all macroaggregates and also unstable microaggregates. The plant debris freed from inorganic components as a result of aggregate disruption was concentrated in fractions with densities <1.6 (occluded) and 1.6-1.8 Mg m-3. The debris represented fragments of roots and stems in the 10-100 pm size classes showing distinct cellular anatomy. The occluded particulate OM separated with different densities showed different degrees of association with mineral particles. The plant fragments in the <1.6 Mg m-3 (occluded) fraction separated as clean organic particles (Golchin et al. 1994). However, the plant debris in the 1.6-1.8 Mg m-3 fraction was partly encrusted with mineral particles (Fig. l a ) . The organic particles in the < 1 . 6 Mg m-3 (occluded) fraction had less recognizable cellular morphology and were assumed to be more decomposed than the 1 .6-1.8 Mg m-3 fraction. Microaggregates which resisted the destructive effects of ultrasonic energy were concentrated in the fraction with density 1.8-2.0 Mg m-% Fig. l b shows a general view of this fraction from the red-brown earth. In addition to aggregates of various morphology and size (30-100 pm), there were numerous phytoliths. It was possible to observe a nucleus of plant debris in some of these aggregates. A. Golchin et al. Fig. 1. ( a ) Organic particles of the 1 .6-1.8 Mg rnp3 fraction from the red-brown earth, showing plant debris partly encrusted with mineral particles. ( b ) A general view of the microaggregates contained in the 1.8-2.0 Mg m-3 fraction of the red-brown earth. ( c ) Microaggregates of the >2.0 Mg m-3 fraction separated from the red-brown earth. Soil Structure and Carbon Cycling The >2.0 Mg m-3 fraction contained microaggregates of <30 pm diameter (Fig. l c ) . We were not able to detect cores of plant debris or biological entities in these aggregates. 13C NMR Spectroscopy of Density Fractions The 13C CP/MAS NMR spectra of different density fractions are shown in Figs. 2-7. Although all spectra exhibited similar resonances, the effects of soil types and densities of fractions were evident on the distribution of intensities of signals. For a qualitative comparison, the spectra were divided into five chemical shift regions according to the chemical types of carbon as follows: 0-46 ppm (alkyl carbon), 46-110 ppm (0-alkyl carbon), 110-164 ppm (aromatic carbon), 1 Chernlcal Shlft (pprn) Fig. 2. Solid-state 13C CP/MAS NMR spectra acquired for density fractions of the Queensland grey clay. A. Golchin et al. 164-190 pprn (carbonyl carbon). The resonances in the 0-46 pprn range arise from aliphatic structures. The sharp peaks at 30-34 pprn indicate the presence of aliphatic carbon in long chain polymethylene structures. The region at 46-110 pprn exhibits signals attributed to carbon bound to oxygen and nitrogen. The prominent signals at 72-75 pprn arise mainly from oxygenated carbon of carbohydrates. The presence of carbohydrates is confirmed by resonances centred at 102-106 pprn which originate from dioxygenated (acetal) carbon of polysaccharides. Smaller resonances centred at 62-65 pprn are also due to the C-6 carbon in carbohydrates (Preston and Schnitzer 1984) The peaks at 56-58 pprn include carbon from methoxyl groups in lignin as well as carbon in amino acids. Broad signals in the 110-164 pprn region are characteristics of aromatic carbon. The resonances at 116-118 and 129-131 pprn are due to protonated and alkyl-substituted aryl Chemical Shin (ppm) Fig. 3. Solid-state 13c CP/MAS NMR spectra acquired for density fractions of the black earth. Soil Structure and Carbon Cycling carbon. The presence of 0-substituted aromatic carbon is indicated by signals at 145-156 ppm. The distinct peaks at 173-175 represent carboxyl, amide and ester carbon. The proportion of different types of carbon present in various fractions, determined by integrating the five regions of the spectra in Figs. 2-7, are shown in Figs 10 and 11. The 13C CP/MAS NMR spectra of the <1.6 Mg m-3 (free) fractions were comprised predominantly from 0-alkyl C, indicating that polysaccharides are quantitatively the most significant organic compound in this fraction. Due to the plant-like character of this fraction (particulate OM with wide C:N ratio) the carbohydrates present were probably cellulose (Golchin et al. 1994). The < I . 6 (occluded), 1.6-1.8 and 1.8-2.0 Mg m-3 fractions also contained a considerable amount of carbohydrate, but in these fractions there was less 0-alkyl / 0 " ',. Chemical Shin (ppm) Fig. 4. Solid-state 13c CPjMAS NMR spectra acquired for density fractions of the Victoria grey clay. A. Golchin et al. C and more alkyl C compared with the <1.6 (free) fraction. A comparison of the < 1.6 (occluded) fractions with the 1.6-1.8 and 1 8-2.0 Mg m-3 fractions for the relative proportion of different types of carbon showed clear trends. A decrease in the proportion of 0-alkyl C and an increase in that of alkyl C in passing from the 1.6-1.8 and 1.8-2.0 fractions to <1.6 (occluded) fractions was evident (Figs 10 and 11). After removal of the light fractions (<2-0 Mg m-3), the organic materials contained in the heavy fractions (>2.0 Mg m-3) showed predominantly signals due to carbohydrate and aliphatic structures. The most noticeable feature of the spectra from the fractions >2.0 Mg m-3 was the absence of signals ascribable to phenolic C, indicating that lignin and tannin structures were almost absent. The relative proportion of aromatic C was lower and that of the carbonyl C was higher in this fraction than in the lighter fractions. < 1.6 Mg m.' (occluded) d - Fig. 5. Solid-state 13c CP/MAS NMR spectra acquired for density fractions of the solodic. Soil Structure and Carbon Cycling The chemical composition of SOM, contained in density fractions of each sample, varied between soils. The largest differences in the composition of organic carbon were noted among the Vertisols (Fig. 10). There was more 0-alkyl C in the density fractions of the Victoria grey clay than in those of the black earth and Queensland grey clay. The density fractions of the black earth and Queensland grey clay, however, had higher aromatic and alkyl carbon contents respectively. The differences observed in the organic structure of the SOM between the Alfisols were smaller (Fig. 11). The NMR results showed that the organic materials contained in the density fractions of the so