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DIVISION S-2—SOIL CHEMISTRY

A Procedure for Isolating Soil Organic Matter Fractions Suitable for Modeling

^a Agriculture and Environment Div., IACR-Rothamsted, Harpenden, Herts, AL5 2JQ, UK
^b Dep. of Chemistry, Queen Mary, University of London, London, E1 4NS, UK
^c AAT Consultants, 15 Clerk Street, Edinburgh, EH8 9JH, UK
^d Dep. of Soil Science and Agricultural Chemistry, Szent István University, 2103 Gödöll, Hungary

* Corresponding author (saran.sohi{at}bbsrc.ac.uk)

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Fractions of soil organic matter (SOM) were obtained from three soils using alternative physical fractionation procedures, and evaluated against the requirements of model pools. We compared two-stage density fractionation (isolating free and intra-aggregate fractions, before and after dispersion, respectively) with particle-size separation of dispersed soil. For full comparison, the organomineral fraction residual from density fractionation was also size separated. In standardizing the density-based method, we found recovery of intra-aggregate organic matter highly sensitive to separation density as compared with the free. Recovery of the intra-aggregate was also influenced by dispersion energy. The greatest amount was obtained using a combination of the highest density (1.80 g cm^-3) and dispersion energy (1500 J g^-1). Analysis by ¹³C nuclear magnetic resonance (NMR) showed O-alkyl/alkyl-C ratios 1.38 to 2.30 times greater in intra-aggregate organic matter than in the free. Diffuse reflectance Fourier transform infrared spectroscopy (DRIFT) also indicated a greater proportion of aliphatic hydrocarbon, carboxylic anions, and aromatic C in intra-aggregate organic matter. The findings suggest this fraction comprises more decomposed and transformed organic matter relative to the free. Higher signal/noise ratios in NMR spectra of particle-size fractions (compared with their organomineral equivalents) were attributed to C in particulate SOM, not removed by prior density separation. Whilst particle-size fractions confuse particulate SOM with that attached to mineral surfaces, fractions isolated by two-stage density separation are small in number and display distinct chemical properties. We suggest they provide a sound basis for a model of SOM turnover based on measurable pools.

Abbreviations: CPMAS, cross-polarization magic angle spinning • DRIFT, diffuse reflectance Fourier transform infrared spectroscopy • NMR, nuclear magnetic resonance • SOM, soil organic matter • TOSS, total suppression of sidebands

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
ALTHOUGH THE DECOMPOSITION of plant material in soil is a complex process, relatively simple models have successfully described the long term dynamics of SOM (Jenkinson, 1990). Such models invoke SOM fractions (represented as discrete N and C pools) which are defined by their rate of decomposition. Hence fraction i is defined as obeying:

(1)

where _i is the mineralization rate of fraction i; k_i is its first order reactivity; and Y_i is its size. Cohort models (Bosatta and Ågren, 1996) are conceptually different from the approach described above. However, they are similar mathematically, and may be considered as models whose multiple pools encompass a sufficient range of reactivity (k_i).

Measurements of system-level outputs, such as total soil C, can be used to estimate the size of model pools (Y_i) by parameter optimization, and the model used to make predictions for typical systems in which Y_i has been established. Some functional characteristic (such as C/N ratio) may be used to derive transformations of N.

Parameterization against system-level outputs in this way does not guarantee accurate prediction at the process level, and the inability to test models by instantaneous measurement of Y_i makes elucidation of SOM transformation processes difficult, explaining their generally unsatisfactory simulation of short- to medium-term dynamics (de Wiligen, 1991). Further, since it is impossible to devise a procedure that reliably separates all (and only) SOM of reactivity k_i (or specific age), it may be more expedient to model the measurable than to measure the modelable (Christensen, 1996; Elliott et al., 1996; Magid et al., 1996). In a model based around measurable SOM fractions, pools are defined by the procedure used to extract them. This has the advantage that Y_i can be measured in any soil at any time. The potential drawback is that the reactivity k_i cannot be assumed constant. Subdividing SOM according to physical properties emphasises the observation that location is a key factor determining turnover (Balesdent, 1996).

The goal of our research is to improve understanding of the C and N transformations associated with SOM decomposition by devising a model based on measurable pools. In this paper, we describe the development of a physical fractionation procedure intended to provide these pools. The appropriate number of pools (n) to include in the model is determined by the number of variables that can be measured.

Parameterization data from incubation experiments will include C, N, and indiscriminate tracers ¹³C and ¹⁵N in n pools so there are potentially 4n measurements. The possible number of unknown C and N fluxes is 2n(n - 1), and this figure is halved if a relationship can be found between them. With n(n - 1) unknown fluxes and 4n known differences, it should be possible to infer the reactivity k_i of up to five SOM fractions.

In addition to identifying a maximum of five fractions (including a gaseous pool that may be measured independently), a suitable fractionation procedure should isolate SOM that differs significantly in measured chemical properties between fractions. This is important since, as a first approximation, differing chemical properties suggest probable differences in reactivity.

Physical fractionation techniques employ some combination of soil dispersion and either density or particle-size separation. Applying density separation before and after soil dispersion yields two light fractions with contrasting levels of physical protection (Golchin et al., 1994). In particle-size fractionation, particulate SOM can be recovered with the sand-size fraction by wet sieving. If the soil has been dispersed, residual soil particles can be divided into silt- and clay-size fractions by sedimentation. Christensen (1992) has reviewed the various approaches in detail.

The objective of this study was to compare two-stage density fractionation and particle-size fractionation of dispersed soil, in the context of the model requirements discussed above. We used ¹³C NMR and DRIFT to examine the chemical properties of the fractions obtained using the alternative methods.

Since the recovery of organic matter by density separation is sensitive to the precise density of the separation medium and the energy used to disperse stable aggregates, we conducted an experiment to optimize the method before comparing fraction composition.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Soils
Three soils of contrasting texture were sampled from experimental farms in southeast England (Table 1). All the soils had been under continuous arable rotation for at least 10 yr. Topsoil (0–23 cm) samples were taken from Rothamsted Experimental Station, Hertfordshire (silty clay loam), and Woburn Experimental Farm, Bedfordshire (sandy loam and heavy clay) using a spade, and were passed through a 6.25-mm sieve before fractionation.

Sedimentation of heavy particles was accelerated by centrifuging the bottles at 8000 g for 30 min. Floating particles were drawn from each bottle in turn, together with NaI solution, using a cutdown 25-ml plastic pipette (Bibby Sterilin, Staffordshire, UK) attached to a vacuum flask and pump via 6-mm diam. tubing.

The free fraction from each sample was isolated by decanting the contents of the vacuum flask into a vacuum filtration unit (Millipore, Hertfordshire, UK) containing a glass fiber filter (type GF/A, 47 mm diam., 1.6 µm retention) (Whatman International, Kent, UK). The filtrate from each sample was returned to the respective centrifuge bottle. The retained material was rinsed thoroughly with deionized water using a separate collector.

The corresponding intra-aggregate fractions, compromising particulate SOM within stable aggregates, were released by resuspending the contents of the centrifuge bottles (i.e., the soil residue and replaced NaI) and sonicating for 5, 10, or 15 min. Treating the soil suspensions in the centrifuge bottles avoided transferal during fractionation, whilst permitting probe submersion comparable to sample depth, a low soil/suspension ratio, and vessel dimensions in proportion to probe diameter (i.e., optimal dispersion conditions).

In this experiment we used an MSE Soniprep 150 sonicator (Sanyo Gallenkamp PLC, Leicestershire, UK), fitted with a 9.5-mm probe submerged 15 mm into the soil suspension. The actual rate of energy transfer was 25 W (measured by temperature change in 100 mL water over 5 min; North, 1976), and this was verified after each 300 min of use. Thus our sonication treatments equated to energy inputs of 500, 1000, and 1500 J g^-1 soil. The temperature of the samples was maintained below 30°C during treatment by containing the centrifuge bottles in an ice packed 500-mL beaker.

The intra-aggregate fractions were recovered after centrifugation, using the same procedure described for the free. The amount of organic matter recovered in the free and intra-aggregate fractions was estimated by loss-on-ignition with the glass fiber filters (500°C for 16 h). Results were expressed on an oven dry soil basis, and analyzed using Genstat 5 software (Version 4.1) (NAG, Oxford, UK). For each density–sonication treatment, three replicates of the residual organomineral fraction were subdivided by particle size. This was to enable full comparison of density fractions with those obtained by the alternative method (below), and to examine the interaction of sonication, solution density, and level of soil dispersion.

Particle-Size Fractionation
Particle-size fractionation was applied to (i) soils without prior density separation and (ii) organomineral fractions residual from density separation. For whole soil, three 15-g subsamples were predispersed using the equipment, dispersion medium, and containers as the optimized density–based method.

Sand-size fractions (25–2000 mm diam.) were removed using an Analysette 3E electromagnetic wet sieving machine (Fritsch, Idar-Oberstein, Germany). Silt- (2–25 mm) and clay-size (<2 mm) fractions in the residual suspensions were separated by sedimentation in the dark at constant temperature (25°C). Each suspension was added to a plastic cylinder (35-cm height, 6-cm diam.) and the volume made up to 1200 mL.

After settling for 17.25 h, the top 25 cm of each suspension (containing clay-size particles only) was siphoned into a 1 L centrifuge bottle using a hooked perspex tube. The contents of each bottle were flocculated with 2.5 mL of 1 M CaCl₂ and centrifuged to sediment particles >0.2-mm diam. (2500 g, 35 min; Tanner and Jackson, 1947). The supernatants were discarded, and the pellets retained in the centrifuge bottles and stored at 4°C. The cylinders were topped up to the original depth with deionized water, resuspended, and the sedimentation and centrifugation procedure repeated until no further clay-size particles were recovered. The silt-size fraction remaining in the cylinders were isolated by centrifugation at 2500 x g for 15 min. The accumulated clay pellets and silt-sized fractions were washed into preweighed petri dishes and evaporated to dryness at 50°C.

Chemical Characterization
Samples of free and intra-aggregate fraction, organomineral subfractions, and sand, silt and clay particle-size fractions were ground in a disc mill for 3 min. Free and intra-aggregate fractions were milled with the glass fiber filters since it was not possible to isolate fine embedded particles (particularly of the intra-aggregate fraction).

To acquire ¹³C cross-polarization magic angle spinning (CPMAS) NMR spectra, subsamples were packed into cylindrical zirconia rotors (internal dimension 5.6 by 17 mm), sealed with Kel-F caps and analyzed using a Bruker MSL 300 spectrometer (Bruker UK, Coventry, UK). Experimental parameters were as follows: spectrometer frequency 75.5 MHz, contact time 1 ms, relaxation time 0.5 s, and spinning speed 4.0 to 4.6 kHz. Between 24000 (3.3 h) and 113000 (15.7 h) scans were accumulated for free and intra-aggregate fractions, between 139000 (19.3 h) and 305000 (42.4 h) for whole soils, and between 109000 (15.1 h) and 449000 (62.1 h) for organomineral and particle-size fractions. Spinning sidebands were eliminated using the Total suppression of sidebands (TOSS) sequence (Dixon, 1982).

Paramagnetic centers shorten the relaxation time of nearby protons, interfering with cross polarization. Therefore, the proportion of ¹³C detected in a sample is strongly influenced by Fe as well as C content. The Fe content of the samples was determined by Aqua Regia extraction followed by inductively coupled plasma emission spectrometry (Accuris ICP-ES; Applied Research Laboratories, Vallaire, Switzerland). Carbon was determined by combustion analyzer (Integra-CN; Europa Scientific Instruments, Cheshire, UK). Magnetic susceptibility was also measured directly using a magnetic susceptibility balance housing a suspended magnet (Johnson Matthey Chemicals, Hertfordshire, UK), and mercury tetrathiocyanatocobaltate as a calibration standard (Figgis and Nyholm, 1958).

Peak areas were calculated for spectra where (on a weight basis) sample C/Fe > 1 (Arshad et al., 1988). The distribution of C between functional groups was estimated according to the following chemical shift limits: 0 to 45 ppm (alkyl C), 45 to 65 ppm (N-alkyl C), 65 to 95 ppm (O-alkyl C), 95 to 108 ppm (acetal C), 108 to 145 ppm (aromatic C), 145 to 160 ppm (phenolic C), 160 to 185 ppm (carboxyl C), 185 to 220 ppm (carbonyl C) (Randall et al., 1995).

For DRIFT spectroscopy 2 to 4 mg of sample was mixed with 200 mg of KCl and powered in an agate mill. The samples were analyzed using a BIO-RAD FTS 165 FT-IR spectrometer fitted with a DTGS-KBr detector (Bio-Rad Laboratories, Cambridge, MA). Sixty-four scans were made with a single single-sided beam and a spectral range of 400 to 4000 cm^-1. Resolution was set at 8 cm^-1, and GRAMS version 2.01 software used to generate the spectra. Peak assignments were based on Baes and Bloom (1989), Bloom and Leenheer (1989), and Stevenson (1994).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Optimization of Density Fractionation
Analysis of variance showed separation density was a significant factor (F < 0.05) in recovery of free organic matter only in the sandy loam soil, with a larger yield at a density of 1.80 g cm^-3 than at 1.60 or 1.70 g cm^-3 (Fig. 1a). In the other two soils the quantity of organic matter recovered in this fraction was the same at all three densities. This relative insensitivity to separation density probably reflects a loose association between free organic matter and heavy organomineral particles. Since centrifugation expels entrapped air, buoyancy conferred by intra-cellular space in fresh plant debris is not a probable explanation (Magid et al., 1995).

View larger version (34K): [in this window] [in a new window]   Fig. 1. Organic matter recovered in (a) free and (b) intra-aggregate fractions at different separation densities and duration of ultrasonic dispersion (s.e.d. is the standard error in differences of means).

The duration of sonication was also a significant factor for recovery of intra-aggregate organic matter from sandy loam and heavy clay soils. At the highest separation density, the longest treatment time resulted in greatest recovery of intra-aggregate organic matter. In the heavy clay soil (but not the others) the proportion of clay-size particles present in the organomineral clay-size fraction was affected by the density of the NaI in which the soil was dispersed (Fig. 2). This could be because of a reduction in the zone around the sonicator probe tip in which particles are exposed to strong disruptive force. Although the yield of organomineral clay-size fraction was lower after 5 and 10 min sonication in NaI solution of density 1.80 g cm^-3 relative to 1.60 g cm^-3, the amount recovered after 15 min (1500 J g^-1 soil) was equal (Fig. 2). Our tests suggested that efficient recovery of free and intra-aggregate organic matter requires separation at a density of 1.80 g cm^-3. Our optimized procedure for density fractionation (Fig. 3) also specifies the greater duration of sonication (15 min, energy transmitted 1500 J g^-1) to maximize the breakdown of aggregates during soil dispersion.

View larger version (51K): [in this window] [in a new window]   Fig. 2. The effect of NaI density on yield of organomineral clay from heavy clay soil, with increasing duration of ultrasonic dispersion. Bars indicate the standard error.

View larger version (35K): [in this window] [in a new window]   Fig. 3. A standard protocol for isolation of organic matter fractions suitable for modeling.

View larger version (73K): [in this window] [in a new window]   Fig. 4. Contrasting appearance of (a) free and (b) intra-aggregate fractions isolated on glass fiber filters.

View larger version (94K): [in this window] [in a new window]   Fig. 5. Carbon-13 nuclear magnetic resonance (NMR) spectra for fractions obtained by density–size versus size-only fractionation from (a) sandy loam (b) silty clay loam and (c) heavy clay soils.

Spectra from organomineral and particle-size fractions were characterized by generally low signal/noise ratios, attributable to paramagnetic interference and low C concentration (only one sample displayed C/Fe > 1). However, particle-size fractions (Fig. 5; right hand side) generated consistently greater signal than their organomineral counterparts (Fig. 5; left hand side), presumably because of the presence of free and intra-aggregate fractions not removed by prior density separation. It appears that this material is recovered with clay- and silt- as well as sand-size fractions, and—comprising relatively small amounts of SOM—contributes disproportionately to the NMR signal detected in these fractions. We suggest that the latter effect is a result of the greater physical separation of paramagnetic centers from cross-polarizing protons in organic particles. If this can be proved then this suggests that the NMR signal from fractions obtained without prior density separation will emanate from two sources: particulate SOM and SOM attached to mineral surfaces.

The relatively defined peaks apparent in silt and clay particle-size fractions from the silty clay loam and heavy clay soils, indicative of O-alkyl and alkyl C (Fig. 5b and 5c; right hand side), may well reflect particulate organic matter. The O-alkyl and aromatic C peaks were diminished in clay fraction relative to silt, a trend previously described by Randall et al. (1995). For the organomineral fractions, discernible peaks were only present in the heavy clay soil. For this soil, organomineral silt and clay were enriched in carboxyl C when compared to free or intra-aggregate fractions, and organomineral clay to a greater extent than silt.

DRIFT spectra for free and intra-aggregate fractions were interpretable in the wavenumber range 1430 to 4000 cm^-1 (Si–O peaks emanating from the filter material in the milled sample overlapped at higher wavenumbers) (Fig. 6). All samples showed pronounced absorption between 1620 to 1600 cm^-1, indicating aromatic C (C = C stretching) and carboxylic anions (–COO asymmetric stretching). These peaks also confirmed the consistently greater aromatic C content of the intra-aggregate fraction observed by NMR, and the generally higher proportion of aromatic C present in silty clay loam soil fractions. Less defined peaks at 1525 cm^-1 and 3030 cm^-1 indicated further aromatic C, and C–H stretching on aromatic structures. Peaks between 1460 to 1450 cm^-1 indicated aliphatic hydrocarbon (C–H stretching), and more pronounced peaks between 2940 to 2900 cm^-1 the stretching of C–H on aliphatic chains. Their markedly greater intensity in intra-aggregate organic matter (for the sandy loam and heavy clay soils) was further indication that this fraction has undergone greater microbial transformation than the free. The greater presence of alkyl C in intra-aggregate was consistent with the NMR findings. The DRIFT spectra for organomineral and particle-size fractions could not be interpreted due to the dominance of peaks from mineral components.

View larger version (22K): [in this window] [in a new window]   Fig. 6. Comparison of diffuse reflectance Fourier transform infrared (DRIFT) spectra for free and intra-aggregate organic matter fractions from (a) sandy loam (b) silty clay loam and (c) heavy clay soils.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

	ACKNOWLEDGMENTS

 
The Bruker MSL 300 NMR spectrometer was provided by the University of London Intercollegiate Research scheme, and the assistance of Dr. A. Aliev is appreciated. This publication is an output from a project funded by the UK Ministry of Agriculture, Fisheries and Food (MAFF), and by the UK Department for International Development (DFID) for the benefit of developing countries. The views expressed are not necessarily those of DFID. IACR-Rothamsted receives grant-aided support from the UK Biological and Biotechnology Research Council (BBSRC).

Received for publication January 10, 2000.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 

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This Article Abstract Figures Only Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (53) Citing Articles via Google Scholar Google Scholar Articles by Sohi, S. P. Articles by Gaunt, J. L. Search for Related Content PubMed Articles by Sohi, S. P. Articles by Gaunt, J. L. GeoRef GeoRef Citation Agricola Articles by Sohi, S. P. Articles by Gaunt, J. L. Related Collections Soil Mineralogy Soil Models Soil Chemistry