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Published online 28 June 2005
Published in Soil Sci Soc Am J 69:1248-1255 (2005)
DOI: 10.2136/sssaj2004.0262
© 2005 Soil Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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Soil Biology & Biochemistry

Investigating the Chemical Characteristics of Soil Organic Matter Fractions Suitable for Modeling

Saran P. Sohia,*, Nathalie Mahieub, David S. Powlsona, Beatá Madaric, Rienk H. Smittenbergd and John L. Gaunta

a Agriculture and the Environment Division, Rothamsted Research, Harpenden, Herts., AL5 2JQ, UK
b Dep. of Chemistry, Queen Mary, Univ. of London, London, E1 4NS, UK
c Embrapa Solos, Rua Jardim Botanico 1024 22460-000, Rio de Janeiro RJ, Brazil
d Dep. of Earth, Atmospheric, and Planetary Sciences, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Current models of soil organic matter (SOM) turnover tend to invoke pools that are defined by their contrasting first-order reactivity constants but which cannot be directly measured. New models may be based around fractions defined by procedures that can be used to isolate them experimentally. The drawback of such fractions is that they may display properties that are not sufficiently distinct or which vary in time or space. In this study the properties of two fractions from soils of contrasting geographical origin and under different nutrient management were examined using 13C nuclear magnetic resonance (NMR) spectroscopy. The fractions were free SOM (FR-SOM, discrete organic particles located between stable aggregates), and intra-aggregate SOM (IA-SOM, discrete organic particles within stable aggregates). The composition of both fractions was highly consistent across soil types and environments, but the fractions differed significantly in the proportion of C present in five of the seven functional C groups identified by NMR (P < 0.05). The results confirmed that IA-SOM contains a greater proportion of microbial products and more resistant C as compared with the FR-SOM. Nutrient management affected fraction composition in four functional groups most abundant in plant material or farmyard manure (P < 0.05). The effects were considerably less pronounced when the analysis was restricted to plots receiving inorganic (or zero) N. Overall the results supported the view that free and intra-aggregate organic matter occupy contrasting positions in the decomposition sequence, and are likely to display reactivities sufficiently distinct to operate as discrete pools in new SOM models.

Abbreviations: CPMAS, cross-polarization magic angle spinning • FR-SOM, free soil organic matter • FYM, farmyard manure • IA-SOM, intra-aggregate soil organic matter • NMR, nuclear magnetic resonance • REML, residual maximum likelihood • SOM, soil organic matter


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
ALL ASPECTS of agroecosystem function, through the demand for nutrients such as N, ultimately depend on the turnover of SOM. Models that describe the interlinked, microbially mediated processes that control SOM turnover usually invoke conceptual SOM fractions (as discrete N and C pools), defined by contrasting first-order reactivity constants (Sohi et al., 2001). Since it is impossible to reliably and completely isolate fractions of SOM that display specific and predefined levels of reactivity, future models that are predictive and testable should, perhaps, be based around fractions defined by the experimental procedure used to isolate and measure them, that is, operationally defined fractions (Arah and Gaunt, 2001; Christensen, 1996). The drawback of employing such fractions is that, integrated over time or through changes in soil management, their properties may not sufficiently or consistently correspond to a particular level of reactivity.

Since the contrasts and consistency of reactivity between and within SOM fractions may be proximate to the differences and variation in chemical composition, this aspect can be assessed using non-invasive characterization techniques. The NMR technique has been widely applied to ascertain the distribution of 13C (and thus C) between labile and more recalcitrant functional groups in whole soils, usually according to the proportion of peak area assigned to particular regions of the NMR spectrum (Preston, 1996). Although quantitation may be compromised for whole soils by the presence of paramagnetic materials and a low concentration of C (Kinchesh et al., 1995), these limitations are less significant when applied to fractions essentially freed from the mineral matrix (Sohi et al., 2001).

A number of studies have employed 13C NMR to demonstrate the effects of land-use on the composition of SOM (Preston, 1996), but the sensitivity to nutrient management in the arable situation has not been conclusively shown (Randall et al., 1995). However, the predictions of practical SOM models will be sensitive to agronomic decisions, so it is reasonable that the composition of measurable fractions that correspond to pools should vary, providing general differences in reactivity are maintained. Sohi et al. (2001) used a two-stage density procedure to define SOM fractions suitable for modeling, showing consistent differences between the compositions of two of the fractions (free and intra-aggregate organic matter) using the 13C NMR technique.

The objective of the current study is to confirm the suitability of these fractions as model pools by examining the influence of geographic origin and soil management on the extent of these differences, and since re-equilibration of SOM is may occur over decades (Christensen and Johnston, 1997), the soils used were selected from field experiments under consistent long-term management.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Sites and Soils
During 1997, samples of soil from eight long-term experiments worldwide were assembled. Three were located in Europe, three in Asia, and one each in North America and the Middle East (Table 1). At each site three categories of nutrient management were considered: (i) plots receiving no fertilization, (ii) plots receiving one or more rates of inorganic N, and (iii) plots receiving farmyard manure (FYM). At four sites samples were taken from plots receiving two different rates of inorganic N fertilizer (Table 2). These were designated high N or low N, according to local practices. For the experiments with only one inorganic N treatment, plots receiving ≤100 kg N ha–1 crop–1 were placed in the low N category. Inorganic fertilizer treatments included balanced inputs of P and K, and at some sites the manure treatments received supplementary mineral (inorganic) N. All plots were in cereal rotations, including (at three sites) rice grown under flooded conditions (Table 2). At the English site, samples of FYM and wheat straw were also obtained to establish the composition of organic matter inputs and SOM under alternative nutrient management. Information on soil texture and soil classification, plow depth, and the status of the basic climatic variables at each site can be found in Table 2.


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  		Table 1. The location of long-term sites and experiments sampled for characterization of soil organic matter fractions under contrasting nutrient management.

 

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  		Table 2. Information on soils, climate and cropping pattern for plots sampled at each of the long-term sites, including details of the N amendments in each category of nutrient management.{dagger}{dagger}

 
The soils at all sites were conventionally tilled or, in the case of rice soils, puddled. Bulked samples representative of the respective plow layers were taken (randomly) within individual plots by gouge auger, shortly after 1997 summer harvest. With one exception, the experiments were based on the randomized-block design with four (Germany, Hungary, Philippines), three (Bangladesh, Nepal, Syria), or two (U.S.) replicates. The Broadbalk experiment (in England) has no replication, so we sampled in four sections (two alternate phases of two crop rotations) instead. Equal amounts of bulked soil samples from individual plots were combined to provide a single sample per treatment per site for fractionation.

The samples were imported to the UK air-dry and preground to pass a 2-mm mesh. Since the fractionation procedure was developed for application to fresh soil, the soils were capillary rewetted before fractionation: 60 g of each soil was placed in a 75-mm diam. plastic core (lined with nylon mesh), and stood on a bed of silica flour saturated with deionized water until equilibrium weight was achieved (approximately 36 h).

Density Fractionation
The soils were fractionated using a density-based procedure proposed by Sohi et al. (2001). The method distinguishes light organic particles (<1.80 g cm–3) between aggregates (FR-SOM) from light organic particles (<1.80 g cm–3) within aggregates (IA-SOM), and a residual heavy (>1.80 g cm–3) organomineral fraction. Typically the two light fractions account for 2 to 5% of total soil organic C. Briefly, six 16-g samples of rewetted soil were weighed into separate 250-mL polycarbonate centrifuge bottles, and 90 mL of NaI solution added to each. In this study the NaI was prepared to a density of 1.83 g mL–1 to account for the higher moisture content of rewetted soils. After shaking gently by hand for 30 s the bottles were centrifuged at 8000 x g for 30 min and floating FR-SOM fraction removed, together with NaI supernatant, using a pipette attached to a vacuum flask and pump. The FR-SOM was isolated from the contents of the flask using 42-mm vacuum filtration units and in this study, a single polycarbonate filter of 5-µm retention (Millipore, Watford, UK). Using deionized water, the sample was first washed under vacuum (using a separate collector), and then rinsed from the filter into a Petri dish and dried at 50°C. The density of the sodium iodide filtrate was assessed to confirm that separation of FR-SOM had occurred at 1.80 g cm–3 (± 0.005 g cm–3), before being returned to the centrifuge bottles. The pellets were resuspended and the stable aggregates dispersed using an ultrasonic probe. In this study, we used a XL2020 µLtrasonic generator fitted with a dual horn and twin 19-mm diam. probes (Misonix, Farmingdale, NY). The submergence depth for the probes was increased to 19 mm appropriate to the greater probe diameter, and dispersion energy of 750 J g–1 soil applied at a rate of 58.8 W (assessed by temperature change in cold water). After further centrifugation (as above), the IA-SOM fraction released by sonication was removed and isolated as per FR-SOM. In this study the residual organomineral fraction was not analyzed, and the dried FR-SOM and IA-SOM samples prepared for 13C NMR analysis using a pestle and mortar. The removal of fraction from filter was not quantitatively reliable, but the objective of the study was qualitative and the separation avoided dilution of the organic matter in the sample (the C content of the fractions is typically 250–350 mg C g–1).

13C-NMR Analysis
The experimental parameters for obtaining 13C cross-polarization magic angle spinning (CPMAS) NMR spectra were the same as those described in Sohi et al. (2001): spectrometer frequency 75.5 MHz, contact time 1 ms, relaxation time 500 ms, and spinning speed around 4.5 kHz, elimination of spinning side-bands using the total suppression of sidebands (TOSS) sequence (Dixon, 1982), and line broadening 100 Hz. However, the samples of FR-SOM and IA-SOM were generally smaller than the rotor capacity (approximately 200 mg) and were therefore packed into the center of the rotor using spacers. The separation of the fractions from their collection filters resulted in shorter acquisition periods than reported by Sohi et al. (2001); the average accumulation for the samples in this study was 76 000 scans (10.6 h). Peak areas were divided into seven regions according to the following chemical shift limits (based on those defined by Randall et al. [1995], and indicated in Fig. 1–3) : 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), 165 to 220 ppm (carbonyl and carboxyl C). The samples of FYM and straw were also analyzed by 13C NMR using the same experimental parameters, except that a relaxation time of 1 s was used.



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  		Fig. 1. Carbon-13 cross-polarization magic angle spinning nuclear magnetic resonance (CPMAS NMR) spectra for contrasting organic inputs received by plots of the Broadbalk wheat experiment in England.

 


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  		Fig. 3. Carbon-13 cross-polarization magic angle spinning nuclear magnetic resonance (CPMAS NMR) spectra for intra-aggregate organic matter (IA-SOM) isolated from four Broadbalk plots under contrasting nutrient management.

 


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  		Fig. 2. Carbon-13 cross-polarization magic angle spinning nuclear magnetic resonance (CPMAS NMR) spectra for free organic matter (FR-SOM) isolated from four Broadbalk plots under contrasting nutrient management

 
Statistical Analysis
The peak area data from 13C NMR were statistically analyzed for two factors (fraction and nutrient management) using Genstat 5 Version 4.1 (NAG Ltd., Oxford), fitting a variance components model using the residual maximum likelihood (REML) procedure (Payne et al., 1993). This procedure is for analysis of data from experiments of unbalanced design, that is, where not all treatments are available at all sites.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Fraction Composition in the Broadbalk Plots
The 13C NMR spectra for the typical organic matter inputs on the Broadbalk plots, that is, for FYM and for wheat straw, is shown in Fig. 1. The major peaks in the straw spectrum indicated O-alkyl and acetal C, and also the presence of N-alkyl C. This reflects the abundance of cellulose, hemicellulose and protein in fresh plant material. The composition of FYM was closer to that of the SOM fractions (see below), with a substantial proportion of total peak area attributable to alkyl, N-alkyl, aromatic, phenolic, and carbonyl groups, presumably reflecting the extent of its prior degradation. The effect of the digestion of plant carbohydrates in cattle rumen on the composition of FYM has previously been highlighted using infrared spectroscopy (Russell and Fraser, 1988).

Comparing the NMR spectra for the FR-SOM fraction under contrasting soil nutrient management, composition appeared sensitive to the rate as well as type of fertilizer application. Under the high N treatment the composition of FR-SOM (Fig. 2) was more similar to that of straw (Fig. 1), with a large proportion of peak area attributable to O-alkyl C (34%) and C in acetal (13%) groups. With lower rates of inorganic N application, the proportion of resistant (lignin-derived) degradation products increased: in FR-SOM isolated from the zero-N plot 36% of C was aromatic and only 21% located in O-alkyl groups. These differences presumably reflect the contrast in the amount of organic matter returned to the soil after harvest, with less ‘dilution’ of the more stable components of FR-SOM by fresh straw-derived material in the zero-N plot: Assuming that the amounts of C returned after harvest are roughly equal to the amounts of straw removed, the high N plot receives approximately five times the level of fresh inputs postharvest as the zero-N plot (Anon, 1998). It is also likely that greater lignification of wheat straw resulting from low N availability, and possibly a corresponding accumulation of polyphenolics (Handayanto et al., 1995), contributed to the observations. Significantly, although the FYM plot yields the same amount of grain and straw as the high inorganic N plot, the FR-SOM fraction displayed aromatic as well as alkyl C contents more similar to those of the zero N. This appeared to rule out the possibility that black C (charcoal) could be a significant factor in explaining the characteristics of SOM fractions in this soil, which also has no history of significant burning (other than stubble). The sample from the FYM plot also displayed a higher proportion of N-alkyl C and lesser proportions of O-alkyl C, and the lowest proportion of acetal C. The amount of C added to the plot as FYM is at least twice that removed in straw and thus fresh plant material returned to the soil. We hypothesize that in this plot the FR-SOM fraction includes material derived from FYM as well as that derived directly from plants. This hypothesis is supported by the sharp NMR peak at 57 ppm (N-alkyl C), which also distinguished FYM and straw input materials (Fig. 1).

For the Broadbalk plots that did not receive FYM the composition of IA-SOM was relatively consistent (Fig. 3.). In each case aromatic C was more abundant than O-alkyl, the difference being most marked in the zero-N treatment, where only 14% of peak area was in the O-alkyl region. These samples displayed prominent alkyl peaks centered at 33 ppm. The spectrum for IA-SOM from the FYM plot was distinct in that it displayed not only a prominent aromatic peak, but also the greatest proportion of C in N-alkyl groups and 15% of peak area in the alkyl C region.

Overall, the differences in the composition between the FR-SOM and IA-SOM fractions at Broadbalk were most marked in the plots that receive inorganic N or no fertilizer, and mainly reflected in the greater proportion of C present in aromatic and alkyl groups in IA-SOM. The general differences in the composition of these fractions were consistent with those seen for the three soils described by Sohi et al. (2001). These pronounced differences in composition between the FR-SOM and IA-SOM fractions under contrasting nutrient management were not detectable by 13C NMR analysis of the corresponding whole soils (Randall et al., 1995).

General Difference in Fraction Composition across Sites
Qualitative analysis of NMR spectra for SOM fractions isolated from the Broadbalk plots suggested that rate of inorganic N fertilizer application had a discernible affect on the composition of FR-SOM, and FYM incorporation a more appreciable effect. The observations also held for IA-SOM, although the differences between treatments were smaller. In addition, there were certain key differences between the FR-SOM and IA-SOM fractions apparent for all treatments. The overall objective of this study was to establish whether FR-SOM and IA-SOM can be considered distinct in composition (and thus reactivity) irrespective of nutrient management regime and various factors relating to geographical location—climatic conditions, soil type, cropping pattern, and so on. To address this we made a statistical analysis of 13C NMR peak area data for FR-SOM and IA-SOM from all sites and plots using the REML procedure (Payne et al., 1993); results of the analysis are in Table 3. Since comparison of the fractions from the English (Broadbalk) plots suggested a particular effect of FYM we made three analyses: (a) all results; (b) all results, but with inorganic N and control treatments considered together and compared with those from the FYM; and (c) results from the FYM treatment excluded.


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  		Table 3. Significance of variance components in the composition of soil organic matter fractions isolated from 25 plots of eight long-term experiments where different combinations of nutrient management treatments are compared.

 
The initial analysis (all results, four treatments) confirmed the generality of key differences between FR-SOM and IA-SOM that were observed in the Broadbalk plots, and previously in three field soils analyzed by Sohi et al. (2001). Across all sites and treatments, the distribution of C differed significantly between FR-SOM and IA-SOM in five of the seven NMR peak area regions: alkyl, O-alkyl and acetal C (P < 0.001), and aromatic and carbonyl C (P < 0.05) (Table 3). The mean distribution of C in the respective fractions (with standard error deviations) is indicated in Fig. 4 . The differences result from a greater abundance of more stable functional groups (alkyl, aromatic, and carbonyl) in IA-SOM, and a higher concentration of more labile forms (O-alkyl and acetal) in FR-SOM. The analysis also showed a significant direct effect of the prevailing nutrient management regime on the N-alkyl and acetal C and also O-alkyl and phenolic content of both fractions (Table 3). This is probably because plant material is abundant in O-alkyl and acetal C (indicative of hemicellulose) and FYM in N-alkyl and phenolic C (Fig. 1). The nature and level of organic matter inputs to the soil exerts an influence on the rate (and hence extent) of decomposition in all SOM fractions, although the effect will be most apparent in the more labile fractions. There was also an interaction between nutrient management regime and the extent of the differences in O-alkyl, acetal, and aromatic C content between FR-SOM and IA-SOM (Table 3). Given the prominence of O-alkyl and acetal (hemicellulose) C in straw, and the abundance of aromatic C in FYM (Fig. 1) it appeared likely that the interactive effects resulted specifically from the inclusion of FYM treatments in the study. Re-analysis of the data after grouping control and inorganic N treatments supported this view: significant differences between fractions were greater (extending to include phenolic C content, P = 0.05), and the significance level of the interactions increased (Table 3). Conversely, after removing the results for the FYM plots from the analysis, that is, considering only the control (zero N) and low and high inorganic N treatments, there were no significant interactions in any peak area region (Table 3). The mean values (with s.e.d.) plotted in Fig. 5 emphasize that in plots fertilized with inorganic N, the rate of application leads to minor differences in the composition of FR-SOM (Fig. 5a) or IA-SOM (Fig. 5b), particularly compared to the general differences between the fractions.



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  		Fig. 4. The mean distribution of C between functional groups for free and intra-aggregate SOM fractions isolated from 26 plots at eight long-term sites (receiving either no fertilizer, low or high rates of inorganic N fertilizer, or FYM) and estimated by peak area analysis of Carbon-13 cross-polarization magic angle spinning nuclear magnetic resonance (CPMAS NMR) spectra (error bars represent standard error deviation).

 


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  		Fig. 5. Distribution of C between functional groups in (a) free and (b) intra-aggregate SOM from soils receiving no fertilizer, and low and high rates of inorganic N fertilizer, and estimated by peak area analysis of Carbon-13 cross-polarization magic angle spinning nuclear magnetic resonance (CPMAS NMR) spectra (error bars represent standard error deviation for comparison of treatments for both fractions).

 
Our analyses showed that, across a wide range of geographical locations, significant differences in the composition of FR-SOM and IA-SOM could be detected in seven of the eight main peak area regions identifiable in 13C NMR. Geographical location encompasses the influence of soil type, climate (from tropical to cool temperate), cropping pattern and nutrient management. Given the consistency of differences that we observed in this study, we expect the reactivity of these SOM pools to be relatively constant between locations, and that models that simulate the dynamics of SOM based on these pools should be widely applicable.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
We found remarkable consistency in the chemical composition of free and Intra-aggregate-SOM fractions across 26 long-term plots. The differences between the fractions were highly consistent and confirmed that the intra-aggregate fraction (IA-SOM) contained a greater proportion of functional C groups indicative of recalcitrant or microbial C than the free fraction (FR-SOM). The composition of IA-SOM was less influenced by nutrient management than FR-SOM; the effect of FYM application was more pronounced than contrasting rates of inorganic N application. The results support the view that the fractions occupy different positions in the decomposition sequence and that their reactivity is likely to be sufficiently distinct as to support their separate inclusion in a SOM turnover model.


   ACKNOWLEDGMENTS
 
The authors thank colleagues who provided soil samples and information on the long-term experiments used in this study: Dr. S. Albrecht (USDA Pendleton), Dr. C. Pilbeam (ICARDA and BRRI), Mr. P. Poulton (Rothamsted Research), Dr. M. Körschens (UFZ), Dr. T. Árendás (Agricultural Research Institute, Hungary), Ms. E. Huelgas (IRRI), and Dr. S. Pandey (NARC). 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. The contribution of H. Yates in soil organic matter fractionation at Rothamsted is acknowledged. We also thank A. Todd for statistical analysis of the data. The work was funded by the UK Biotechnology and Biological Sciences Research Council (Grant no. 68/D10271), from whom Rothamsted Research also receives grant-aided support.

Received for publication August 4, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 




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