Scholarly article on topic 'Evaluation of structural chemistry and isotopic signatures of refractory soil organic carbon fraction isolated by wet oxidation methods'

Evaluation of structural chemistry and isotopic signatures of refractory soil organic carbon fraction isolated by wet oxidation methods Academic research paper on "Agricultural biotechnology"

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Academic research paper on topic "Evaluation of structural chemistry and isotopic signatures of refractory soil organic carbon fraction isolated by wet oxidation methods"

Biogeochemistry (2010) 98:29-44 DOI 10.1007/s10533-009-9374-0

Evaluation of structural chemistry and isotopic signatures of refractory soil organic carbon fraction isolated by wet oxidation methods

Sindhu Jagadamma • Rattan Lal • David A. N. Ussiri • Susan E. Trumbore • Silvia Mestelan

Received: 25 August 2008/Accepted: 7 September 2009/Published online: 22 September 2009 © Springer Science+Business Media B.V. 2009

Abstract Accurate quantification of different soil organic carbon (SOC) fractions is needed to understand their relative importance in the global C cycle. Among the chemical methods of SOC fractionation, oxidative degradation is considered more promising because of its ability to mimic the natural microbial oxidative processes in soil. This study focuses on detailed understanding of changes in structural chemistry and isotopic signatures of SOC upon different oxidative treatments for assessing the ability of these chemicals to selectively isolate a refractory fraction of SOC. Replicated sampling (to ~ 1 m depth) of pedons classified as Typic Fragiudalf was conducted under four land uses (woodlot, grassland, no-till and con-ventional-till continuous corn [Zea mays L.]) at Wooster, OH. Soil samples (<2 mm) were treated with three oxidizing agents (hydrogen peroxide (H2O2), disodium peroxodisulfate (Na2S2O8) and

S. Jagadamma (El) • R. Lal • D. A. N. Ussiri Carbon Management and Sequestration Center, School of Environment and Natural Resources, The Ohio State University, 210 Kottman Hall, 2021 Coffey Road, Columbus, OH 43210, USA e-mail:

S. E. Trumbore

Earth System Science, University of California, Irvine, CA 92697, USA

S. Mestelan

College of Agriculture, Universidad Nacional, Centro de la Provincia de Buenos Aires, Azul, Argentina

sodium hypochlorite (NaOCl)). Oxidation resistant residues and the bulk soil from A1/Ap1 horizons of each land use were further analyzed by solid-state 13C nuclear magnetic resonance (NMR) spectroscopy and accelerator mass spectrometry to determine structural chemistry and 14C activity, respectively. Results indicated that, oxidation with NaOCl removed significantly less SOC compared to Na2S2O8 and H2O2. The NMR spectra revealed that NaOCl oxidation preferentially removed lignin-derived compounds at 56 ppm and at 110-160 ppm. On the other hand, the SOC resistant to Na2S2O8 and H2O2 oxidation were enriched with alkyl C groups, which dominate in recalcitrant macromolecules. This finding was corroborated by the 14C activity of residual material, which ranged from -542 to -259% for Na2S2O8 resistant SOC and -475 to -182% for H2O2 resistant SOC as compared to relatively greater 14C activity of NaOCl resistant residues (-47 to 61%). Additionally, H2O2 treatment on soils after light fraction removal was more effective in isolating the oldest (14C activity of -725 to -469%) SOC fraction. The D14C signature of SOC removed by different oxidizing agents, calculated by mass balance, was more or less similar irrespective of the difference in labile SOC removal efficiency. This suggests that SOC isolated by many fractionation methods is still a mixture of much younger and older material and therefore it is very important that the labile SOC should be completely removed before measuring the turnover time of stable and refractory pools of SOC.

Keywords 13C NMR • 14C age • Labile carbon • Land uses • Stable carbon • Wet oxidation


ANOVA Analysis of variance

CT Conventional-till

CPMAS Cross polarization magic angle spinning

Na2S2O8 Disodium peroxodisulfate

HCl Hydrochloric acid

HF Hydrofluoric acid

H2O2 Hydrogen peroxide

LSD Least significant difference

NT No-till

NMR Nuclear magnetic resonance

OM Organic matter

SPIDER Saturation pulse induced dipolar

exchange with recoupling

NaHCO3 Sodium bicarbonate

NaCl Sodium chloride

NaOCl Sodium hypochlorite

SOC Soil organic carbon


Soil organic carbon (SOC) is the third largest global C pool comprising 1,550 PgC in the top 1 m of soil (Batjes 1996; Lal 2007). Transferring atmospheric CO2 into the SOC pool is an important option to help mitigate global warming. The SOC exists as a heterogeneous mixture of different fractions with turnover times ranging from minutes to millennia. The biological stability of SOC is influenced by several factors including chemical structure of SOC, soil environmental factors including temperature, moisture and aeration, and soil physical and chemical properties (Krull et al. 2003). Mechanisms of SOC stabilization can be summarized as chemical recalcitrance of the organic matter (OM), physical protection as a result of inaccessibility to microbes and/or enzymes due to encapsulation of OM into aggregates, and increased stability due to interaction of SOC with mineral matrices (Sollins et al. 1996; Baldock and Skjemstad 2000). These mechanisms, combined with the input-loss ratio of C, determine the turnover rates and subsequent pools of SOC in soils. The total SOC

pool can be compartmentalized into different fractions or pools of varying stability. Unfortunately, different authors use different terminology to describe these SOC pools (Krull et al. 2003). In this study, the term labile pool refers to easily mineral-izable fraction with rapid turnover time ranging from months to years (Krull et al. 2003). The term stable pool is used to describe an intermediate fraction with turnover rates in the range of decadal to centennial time scale (Bruun et al. 2007; Baisden and Amundson 2003). The refractory pool refers to a fraction that persists in soil for long time and has turnover time of thousands of years (Krull et al. 2003). Global estimates of rapidly cycling SOC pools range from 250 to 530 Pg C (Trumbore 1997), suggesting that major part of the global SOC pool is assigned to the stable and refractory fractions. Quantification of different SOC pools is important for understanding C dynamics and for increasing the accuracy of model prediction. The turnover rates of different SOC fractions have been successfully calculated by radiocarbon technique, if samples at various times before and after the peak atmospheric nuclear weapon testing are available (Trumbore 1997; Baisden et al. 2002). The d13C values of soil organic matter (SOM) can also be used as a tracer to study the SOC dynamics in areas where C3 vegetation was replaced by C4 plants or vice versa (e.g., Balesdent et al., 1987). However, there is no consensus in the literature on the appropriate chemical treatment for the experimental isolation of SOC fractions with distinct turnover rates.

Over the years, an array of physical and chemical fractionation protocols have been used for separating the SOC fractions with varying stability. Among the chemical methods, acid hydrolysis (Trumbore and Zheng 1996; Leavitt et al. 1996; Paul et al. 1997; Silveira et al. 2008), and chemical oxidation by various oxidizing agents including hydrogen peroxide (H2O2) (Theng et al. 1999; Eusterhues et al. 2005), disodium peroxodisulphate (Na2S2O8) (Eusterhues et al. 2003; Lorenz et al. 2006), and sodium hypochlorite (NaOCl) (Kleber et al. 2005; Siregar et al. 2005; Zimmermann et al. 2007) have been used frequently. Both acid hydrolysis and chemical oxidation preferentially remove labile organic compounds such as proteins, nucleic acids, and polysaccharides and retain relatively stable SOC fraction that is enriched in alkyl and aromatic C components (Leavitt

et al. 1996; Paul et al. 2001; Mikutta et al. 2006). Ultimately, the residue remained after both acid hydrolysis and chemical oxidation is a heterogeneous mixture of different C inputs. However, chemical oxidation causes changes in SOC composition similar to that observed during natural microbial oxida-tive processes (Cuypers et al. 2002; Eusterhues et al. 2003) and is more efficient at preferentially removing young SOC inputs than acid hydrolysis (Bruun et al. 2007).

Though chemical oxidation procedures are promising methods of separating SOC fractions with distinct stability, the relative efficiency of different oxidizing agents in separating stabilized SOC is not known. Most studies determine the efficiency of oxidizing agents in separating SOC fractions based on the content of residual OC remaining after oxidation treatment. A few studies, which determined mean 14C ages of oxidation-resistant OC showed a wide variation of SOC age, ranging from modern to several thousand years (e.g. Theng et al. 1992; Balesdent 1996; Eusterhues et al. 2005; Kleber et al. 2005). In addition, several studies (e.g. Bruun et al. 2007; Helfrich et al. 2007; Favilli et al. 2008) which compared the oxidation efficiency of different chemicals in similar soil types and land uses have produced contradictory results. Bruun et al. (2007) oxidized soil samples (0-7.5 cm) from a similar soil incubated with 14C-labelled barley (Hordeum vulg-are L.) for 40 years (1964-2004) and 40 days (1 June to 11 July, 2005) and found that both Na2S2O8 and NaOCl are unlikely to isolate a stable OC fraction as they consistently removed more 40-years-old C than 40-days-old C. Helfrich et al. (2007) indicated that, among the several chemical methods tested, Na2S2O8 and H2O2 were the most efficient in isolating a refractory fraction of SOC. According to Favilli et al. (2008), H2O2 was superior to NaOCl in isolating the oldest and most refractory SOC.

Since SOM contains multiple, and simple to complex biomolecules with varying chemical nature and function, a detailed understanding of the changes in structural chemistry of SOM with different oxida-tive treatments would be valuable for assessing the ability of these oxidizing agents in selective removal of labile SOC. The 13C nuclear magnetic resonance (NMR) spectroscopy offers a unique opportunity to examine the structural chemistry of SOC by

distributing the C among classes of compounds, such as alkyl C, O/N-alkyl C, aromatic C, and carboxyl C (Ussiri and Johnson 2007). During the initial stages of SOC decomposition, O/N-alkyl C (mostly derived from carbohydrates and proteins) is selectively utilized by microbes followed by the decomposition of aromatic C (derived from lignin and phenols) (Baldock et al. 1997). As the degree of decomposition progresses, the relative proportion of alkyl C, which is dominantly present in aliphatic macromolecules such as fats, waxes, resins, cutin and suberin, increases (Baldock et al. 1992; Preston 1996). Thus, the alkyl C:O/N-alkyl C ratio, calculated from 13C NMR data, has been used as an index for estimating the biodegradability of SOC (Baldock and Preston 1995). Another useful index is the % aromaticity, which is the ratio of aromatic C (110160 ppm) to the sum of aromatic and total aliphatic C (0-110 ppm) (Frund et al. 1994).

There are a few studies that have used NMR spectroscopy to understand the structural composition of SOC resistant to chemical oxidation (Eusterhues et al. 2005; Mikutta et al. 2006). Moreover, evaluation on how different oxidizing agents affect the structural chemistry of oxidation resistant SOC in similar soil types or land use regimes is completely lacking. Therefore, the focus of this study was to isolate SOC resistant to different oxidizing agents (H2O2, Na2S2O8 and NaOCl) and evaluate the changes in structural chemistry and isotopic signatures (A C, d C) of the SOC resistant to different oxidizing agents and their relative implications as a refractory pool of SOC. The specific objectives were to: (1) quantify the loss of SOC upon oxidation in four land uses of a similar soil type, (2) compare the structural chemistry of SOC before and after oxidation using solid-state 13C-cross polarization magic angle spinning (CPMAS) NMR spectroscopy, and (3) evaluate the isotopic signatures as influenced by the oxidative treatments.

Materials and methods

Site description

This study was conducted at the Ohio Agricultural Research and Development Center in Wooster, Ohio,

USA (40°48'N, 82°00'W). The soils of this site belong to Wooster series (mixed, mesic, Typic Fragiudalf) with silt-loam texture (USDA-SCS 1984). The parent material is low-lime glacial till with a discontinuous loess mantle of up to 51 cm in thickness. These soils are well drained and contain a fragipan at a depth of 50-90 cm (USDA-SCS, 1984). The native vegetation was a continuous cover of mixed, deciduous hardwood forest. Relic forest remnants occur along creeks and as small woodlots (USDA-SCS 1984). The mean annual temperature of the site is 9.1°C with 173 frost-free days occurring each year and the mean annual precipitation is 905 mm (Dick et al. 1998). The study included woodlot, grassland and agricultural land uses. The woodlot is part of the remnant hardwood forest, which dominated the area prior to land clearing for agriculture in the mid 1830s. The agricultural site is part of a long-term tillage experiment (Triplett-van Doren plots) initiated in 1962 to study the effects of tillage and crop sequences (van Doren et al. 1976). This site was maintained under grass meadow for 6 years before starting the experiment. For the present study, no-till (NT) and conventional-till (CT) management plots under continuous corn (Zea mays L.) were sampled. Typically, NT is defined as complete elimination of pre-plant tillage. The seeds in the NT plots were sown using a planter with single coulter to cut through the crop residues and loosen the soil ahead of standard planter units. The CT consisted of one moldboard plowing to a depth of 20-25 cm in spring of each season followed by two 10-cm deep secondary operations (disking, field cultivator) prior to planting. The CT treatment resulted in a complete soil surface inversion and nearly 100% incorporation of crop residues. Fertilizer was applied in the spring of every year based on soil test recommendations, with the same rates of N, P and K applied to both the tillage treatments. The fertilizer materials were broadcasted on soil surface for NT and incorporated for CT plots. Lime was broadcasted in the fall as required to maintain pH close to 6.0 in the Ap horizon. Herbicide types and rates were slightly varied over the years. The cumulative amount of herbicide application was reported as 119 kg a.i. ha-1 for NT and 111 kg a.i. ha-1 for CT soils (Dick 1984). The woodlot and grassland areas are located very close to the Triplett-van Doren plots on the similar soil type.

Soil sampling and processing

Three replicates of horizonwise samples were collected from the pits dug down to the glacial till in each of the four land uses during summer 2005. For this study, five horizons (Ap1, Ap2, Ap3, Bt1 and Btx1) were selected for all the land uses except woodlot, where horizonation was slightly different and samples of A1, A2, E and Bt1 horizons were selected. No Btx horizon was present in the soil profile of woodlot. In addition, woodlot profile contained an E horizon, instead of Ap3 horizon in other land uses (Table 1). The samples were air dried, gently ground with a wooden roller, and sieved through a 2-mm sieve. Triplicate samples per treatment for initial C and N analyses and chemical oxidation were roller ground to pass through a 250-im sieve.

Soil analyses

Soil pH was determined in 1:2 soil weight to water volume ratio suspension using pH meter (Thomas 1996) and proportion of clay was calculated from the textural analysis conducted by the hydrometer method (Gee and Or 2002). The procedure for H2O2 oxidation was slightly modified from Helfrich et al. (2007). Briefly, 1 g of soil was wetted with 10 ml distilled water for 10 min. Then, 30 ml of 10% H2O2 was added and at the end of sometimes intense initial reaction at room temperature, the oxidation was performed at 50°C in a water bath. After 2-3 days when the frothing subsided completely, the suspension was centrifuged at 2500 xg for 15 min and the supernatant was decanted. Oxidation with H2O2 was repeated two more times. The samples were then washed three times with 40 ml deionised water and freeze-dried.

For Na2S2O8 oxidation, 0.5 g of soil was first dispersed in 250 ml distilled water. Then, 20 g Na2S2O8 buffered with 22 g sodium bicarbonate (NaHCO3) was added and allowed to react for 2 days at 80°C in a water bath. After oxidation, the suspension was centrifuged at 2500x g for 15 min and the supernatant was decanted. The samples were washed three times with 40 ml deionised water and freeze dried (Helfrich et al. 2007). The Na2S2O8 oxidized samples were then acid washed to remove the traces of carbonate remaining from the NaHCO3

Table 1 Basic parameters of soils used in the experiment

Land use Horizon Depth (cm) pH 1:1 soil water Clay (g kg-1) SOC (g kg-1) C:N ratio

Woodlot A1 0-4 5.9 (0.3)a 140 (25) 37 (0.8) 13.6 (0.03)

A2 4-13 5.3 (0.1) 129 (16) 28 (0.3) 13.3 (0.1)

E 13-28 4.2 (0.1) 89 (8) 11 (0.1) 10.2 (0.2)

Bt1 41-62 4.0 (0.2) 208 (25) 2.7 (0.2) 4.7 (0.2)

Grassland Ap1 0-4 5.4 (0.2) 158 (18) 33 (0.5) 10.1 (0.2)

Ap2 4-10 5.4 (0.2) 159 (30) 25 (0.1) 9.8 (0.1)

Ap3 10-20 5.6 (0.1) 160 (12) 15 (0.1) 8.7 (0.04)

Bt1 20-33 6.0 (0.04) 196 (18) 7.1 (0.3) 7.4 (0.5)

Btx1 76-109 4.8 (0.1) 221 (21) 1.8 (0.1) 3.5 (0.1)

No-till corn Ap1 0-3 5.4 (0.4) 138 (17) 40 (1.2) 11.2 (0.5)

Ap2 3-10 5.4 (0.2) 158 (25) 26 (1.6) 9.5 (0.7)

Ap3 10-22 5.8 (0.2) 150 (20) 14 (0.3) 8.9 (0.2)

Bt1 33-48 6.2 (0.1) 218 (9) 2.8 (0.1) 4.4 (0.3)

Btx1 77-88 4.5 (0.2) 210 (24) 1.9 (0.1) 3.5 (0.1)

Conventional-till corn Ap1 0-3 5.8 (0.3) 135 (16) 10 (0.5) 10.6 (0.4)

Ap2 3-17 6.2 (0.1) 138 (9) 10 (0.4) 10.3 (0.6)

Ap3 17-31 6.4 (0.1) 130 (13) 10 (0.2) 8.3 (0.04)

Bt1 31-44 6.6 (0.2) 183 (9) 3.6 (0.2) 5.4 (0.3)

Btx1 70-92 4.6 (0.03) 224 (17) 1.9 (0.1) 3.5 (0.1)

Mean values (N = 3) followed by standard deviation in parentheses

added during the oxidation process. Briefly, 3 g of finely ground (<250 im), Na2S2O8 resistant residue were treated with 30 ml of 1 mol l-1 HCl for 24 h with occasional stirring (Ussiri and Lal 2008). After removing the supernatant by centrifugation, the residue was washed with deionized water six times and freeze dried.

Oxidation with NaOCl was achieved by treating 5 g of soil with 50 ml of 6% NaOCl, adjusted to pH 8 with concentrated hydrochloric acid (HCl). After 6 h at 25 °C, the suspension was centrifuged and the supernatant was decanted. The extraction with NaO-Cl was repeated two more times. Afterwards, the samples were washed twice with 50 ml of 1 M sodium chloride (NaCl), three times with deionised water, and freeze-dried (Helfrich et al. 2007).

Concentrations of total C and N in soil samples before and after oxidation were measured by the dry combustion method (Vario Max CN Analyzer, Elementar GmbH, Hanau, Germany). The SOC was assumed to be equal to the total C since inorganic C concentrations were negligible and soil pH was <6.6.

The relative change in C:N ratios before and after oxidative treatments was calculated as a percentage (Schmidt and Gleixner 2005):

Relative change in C:N ratio

= 100 X (C:Nafter oxidation

C: Nbefore oxidation)/(C:Nbefore oxidation) •

Solid-state 13C-CPMAS-NMR analysis

A composite sample from the three replications of surface soil (A1/Ap1 horizon) before and after the oxidative degradation was analyzed for chemical composition using solid-state 13C-CPMAS-NMR spectroscopy. Prior to the analysis, the oxidized samples were treated with 10% hydrofluoric acid (HF) to remove paramagnetic materials and to concentrate OC through dissolution of the mineral matrix (Schmidt et al. 1997; Kiem et al. 2000). This was achieved by treating 10 g of oxidized soil with 50 ml of 10% HF in a polyethylene bottle. The suspension was shaken horizontally for approximately 12 h. After

centrifugation, the supernatant was removed. The HF treatment was repeated four times. Finally, the residue was washed three times with deionised water and freeze dried.

Solid-state 13C-CPMAS-NMR analyses were conducted at the Department of Chemistry, Ohio State University with a Bruker DSX 300 spectrometer operating at a 13C resonance frequency of 75.48 MHz. Samples were packed into a 4 mm (outside diameter) zirconium rotor with a Kel-F® cap and spun at a frequency of 10 kHz using a contact time of 1.5 ms and a recycle delay time of 1 s. In order to obtain the NMR spectra with an acceptable signal to noise ratio, the unoxidized samples and NaOCl resistant residues required approximately 65,000 scans for a period of 18 h, and the H2O2 and Na2S2O8 resistant residues required approximately 1,46,600 scans for 42 h. The spectra were divided into four major chemical shift regions, representing alkyl C (0-45 ppm), O/N-alkyl C (45-110 ppm), aromatic C (110-160 ppm) and car-boxyl C (160-220 ppm). The relative intensities of the chemical shift regions were determined by the integration of 13C-CPMAS-NMR spectra over given chemical shift ranges after phase and baseline corrections. Chemical shift assignments were externally referenced to the adamantine resonance at 29.5 ppm.

Isotopic measurements

The 14C activity of a composite sample from three replicates of surface soil (A1/Ap1 horizon) before and after oxidation was measured at W.M. Keck Carbon Cycle Accelerator Mass Spectrometer facility at University of California-Irvine. The untreated and oxidized samples containing approximately 1 mg of C were combusted at 900°C to produce CO2 and the evolved CO2 was cryogenically purified, reduced to graphite using sealed zinc tube reduction and analyzed for radiocarbon content (Xu et al. 2007; Nowinski et al. 2008). Radiocarbon data are reported as A14C, which is the fractional deviation, in parts per thousand (%), of the 14C/12C ratio of sample from that of a standard of fixed isotopic composition (0.95 times the activity of an oxalic acid standard (OX1) in 1950, which is normalized to a d13C of -19% (Stuiver and Polach 1977):

A14C = (F14C x exp (-k14C - 1) x 1000


(y - 1950))

12c + 13C


" 14C '

12C + 13C OXI

k14C, radioactive decay constant for 14C, equal to 1/8,267 year- ; y = year of C measurement.

The A14C values are corrected to a d13C value of -25% to account for the assumption that plants discriminate twice as much against C as they do against 13C (Stuiver and Polach 1977; Castanha et al. 2008). Negative A14C values indicate the predominance of SOC old enough for radioactive decay of 14C (half-life = 5,730 years) to have occurred and positive A14C values indicate the presence of bomb C produced by atomic weapons testing during 1960s.

The radiocarbon age of the sample was calculated from the F C and Libby mean life of radiocarbon (8,033 years) by the expression,

Radiocarbon age (years BP) = -8, 033 ln (F14C)

BP, means years before 1950, as the activity of OX1 standard is defined for 1950.

An aliquot of the purified CO2 obtained from combustion samples used for the 14C analyses was placed in a He-filled vial and analyzed for 13C using a Gas Bench II interface coupled to a Delta Plus continuous flow isotope ratio mass spectrometer (Xu et al. 2007). The 13C data are expressed as d13C (%):

d C = 13C/12 Csample C/ Cstandard^ =

13 C/ Cstandard) x 1°3

Mass balance calculations

The percent of the original SOC that is retained after oxidation was calculated based on the following equation:

% SOC in residue

(Residue wt.)(% SOCresidUe) (Original soil wt.) (% SOCsoil)

The radiocarbon signature of SOC removed by oxidation is derived from the mass balance of SOC and C:

A Coxidized

A Coriginal soc — % SOC in residue x A14CresidueSOC (100% - % SOC in residue)

A similar equation was used to calculate the d C of SOC removed by oxidation.

Density fractionation

The bulk soil samples from the surface horizon (A1/Ap1) of four land uses were subjected to a density fractionation to separate the light fraction (LF) from heavy fraction (HF) (Golchin et al. 1994; John et al. 2005). Ten grams of soil was placed in a centrifugation tube and 40 ml of sodium polytungstate (SPT) solution with a density of 1.6 g cm-3 (Sometu, Berlin, Germany) was added. The tubes containing the soil-SPT mixture were shaken in a reciprocating shaker at 300 rpm for 15 min. After sedimentation for 30 min, the solution was centrifuged at 5100 xg for 30 min. The supernatant with floating LF was filtered using Millipore AP20 glass-fiber filter under vacuum. Both LF and the HF were washed thoroughly with deionised water and oven dried at 50°C. Subsamples of LF and HF from each land use were separately subjected to chemical oxidation process.

Statistical analysis

The analysis of variance (ANOVA) for testing the difference among the chemical oxidation methods for SOC concentration within a horizon of each land use was computed using PROC GLM (fixed effects model) of SAS (SAS Institute Inc. 2002). The statistical significance was evaluated at the P < 0.05 level and the mean effects were separated using the F protected least significant difference (LSD) test.

Results and discussion

Depth distribution of soil organic carbon and C:N ratio

The SOC concentration within the selected horizons ranged from 2.7 to 37 g kg- in woodlot, 1.8 to

33 g kg-1 in grassland, 1.9 to 40 g kg-1 in NT corn and 1.9 to 10 g kg- in CT corn (Table 1). Among the horizons, the SOC concentration was the highest in A1/Ap1 horizon for all land uses, where C addition from plant litter was the highest (Jobbagy and Jackson 2000). A sharp decrease in SOC concentration with depth was observed in all land uses except CT corn. The uniform SOC concentration from Ap1 to Ap3 horizons of CT corn is probably due to the incorporation of crop residues by tillage operations. Soil C:N ratios were also varied among different horizons, ranging from 4.7 to 13.6 in woodlot, 3.5 to 10.1 in grassland, 3.5 to 11.2 in NT corn and 3.5 to 10.6 in CT corn (Table 1). Narrowing of C:N ratios with depth, consistent among land uses, provided an indication that SOM in subsoil is probably more stabilized than that in surface soil (Kramer et al. 2003).

Soil organic carbon resistant to chemical oxidation

The amount of SOC resistant to oxidation significantly differed among oxidizing agents in all horizons of the four land uses (Table 2). Among the land uses, SOC resistant to NaOCl ranged from 38 to 50% of total SOC in A1/Ap1 horizon, 42 to 54% in A2/ Ap2 horizon, 41 to 49% in E/Ap3 horizon, 48 to 66% in Bt1 horizon and 64 to 79% in Btx1 horizon; SOC resistant to Na2S2O8 ranged from 4 to 10% in A1/Ap1 horizon, 5 to 11% in A2/Ap2 horizon, 8 to 12% in E/ Ap3 horizon, 14 to 38% in Bt1 horizon and 37 to 43% in Btx1 horizon; and SOC resistant to H2O2 ranged from 4 to 8% in A1/Ap1 horizon, 6 to 10% in A2/Ap2 horizon, 10 to 11% in E/Ap3 horizon, 16 to 26% in Bt1 horizon and 36 to 46% in Btx1 horizon. These results indicate that, irrespective of soil horizons and land uses, oxidation with NaOCl removed significantly less SOC compared to Na2S2O8 and H2O2. The SOC removal efficiency of Na2S2O8 and H2O2 were generally similar except in Bt1 horizons of woodlot soils, where H2O2 removed significantly higher amount of SOC than Na2S2O8. In addition, the

Table 2 Effect of wet oxidation methods on soil organic carbon removal in different horizons

Horizon Oxidants Woodlot Grassland No-till corn Conventional-till corn

SOC resistant to oxidation (g kg-1) % of total SOC SOC resistant to oxidation (g kg-1) % of total SOC SOC resistant to oxidation (g kg-1) % of total SOC SOC resistant to oxidation (g kg-1) % of total SOC

A1/Ap1 NaOCl 14 (1.7) a 38 15 (0.6) a 45 20 (0.7) a 50 4.0 (0.05) a 40

Na2S2O8 1.6 (0.05) b 4 1.4 (0.3) b 4 1.6 (0.1) b 4 1.0 (0.1) b 10

H2O2 2.5 (0.1) b 7 1.3 (0.2) b 4 1.5 (0.1) b 4 0.8 (0.1) b 8

A2/Ap2 NaOCl 12.8 (0.2) a 45 13.2 (0.8) a 54 12.6 (0.5) a 49 4.9 (0.2) a 42

Na2S2O8 1.6 (0.05) b 6 1.3 (0.2) b 5 1.4 (0.1) b 5 1.3 (0.1) b 11

H2O2 2.0 (0.1) b 7 1.4 (0.1) b 6 1.4 (0.1) b 6 1.2 (0.07) b 10

E/Ap3 NaOCl 5.2 (0.1) a 49 7.4 (0.3) a 49 5.9 (0.3) a 42 4.8 (0.4) a 41

Na2S2O8 1.3 (0.03) b 12 1.4 (0.0) b 9 1.1 (0.06) b 8 1.4 (0.2) b 11

H2O2 1.0 (0.2) b 10 1.6 (0.2) b 11 1.3 (0.05) b 10 1.3 (0.01) b 11

Bt1 NaOCl 1.8 (0.1) a 66 3.4 (0.2) a 48 1.7 (0.1) a 58 2.0 (0.2) a 55

Na2S2O8 1.0 (0.01) b 38 1.0 (0.1) b 14 0.93 (0.02) b 33 0.97 (0.05) b 27

H2O2 0.70 (0.01) c 26 1.2 (0.1) b 16 0.72 (0.05) c 25 0.87 (0.1) b 24

Btx1 NaOCl NA NA 1.4 (0.3) a 79 1.4 (0.2) a 72 1.3 (0.06) a 64

Na2S2O8 0.78 (0.03) b 43 0.76 (0.02) b 40 0.72 (0.04) b 37

H2O2 0.82 (0.2) b 46 0.76 (0.08) b 40 0.70 (0.09) b 36

N = 3; Standard deviation in brackets; Values in a column within a horizon followed by different letters are significantly different at P < 0.05 using Fisher's protected LSD

proportion of total SOC resistant to Na2S2O8 and H2O2 increased progressively with increase in soil depth in all land uses, in contrast to no clear patterns between SOC resistant to NaOCl and soil depth. In general, the SOC protective or loading capacity of mineral particles increases with decreasing total SOC concentration from surface to subsurface soils. At the higher SOC loading, where most mineral bonding sites are already occupied with organic compounds, the SOC may be associated relatively weakly to the mineral surfaces and therefore may be more susceptible to chemical oxidation. On the other hand, at lower SOC loading, organic compounds occupy a relatively larger portion of mineral surfaces with more direct bondings with minerals (Kaiser and Guggenberger 2003; Mikutta et al. 2005). Therefore, the increase in proportion of Na2S2O8 and H2O2 resistant SOC with depth may partly be attributed to a more intimate association of SOC with mineral surfaces at lower SOC loadings (Kaiser and Guggenberger 2003; Eusterhues et al. 2003, 2005). Helfrich et al. (2007) reported that, contrary to the treatment with H2O2 or Na2S2O8, the proportion of NaOCl-resistant SOC in the Rotthalmiinster soils of Germany

did not increase with progressive decrease in SOC loading. These results suggest that, as compared to NaOCl, the treatment with Na2S2O8 and H2O2 were able to isolate a SOC fraction which is more refractory in nature. Bruun et al. (2007), who compared several methods for the isolation of a stable SOC on two soils incubated with 14C-labelled barley straw for either 40 days or 40 years, found that the SOC resistant to both NaOCl and Na2S2O8 contained a larger proportion of 40-days-old C than of 40-years old C and therefore they are unlikely to isolate a stable fraction of SOC.

C:N ratio of oxidation resistant residues

The C:N ratios of residues remaining after oxidative treatments differed from that of the untreated soil in all the land uses (Fig. 1). Oxidation using NaOCl resulted in increased C:N ratios in all horizons, as revealed by the positive relative change (7-53%, Fig. 1). Increased C:N ratio suggests a preferential oxidation of N-rich OM fractions by NaOCl. Mikutta et al. (2005) also reported higher C:N ratios for NaOCl oxidized residues as compared to untreated



rc r 40

Soil horizons

C -40 re

■jj -60 ¡u -80

jç-100 <U IX

Ap2 Ap3 Bt1

Soil horizons

Ap2 Ap3 Bt1

Soil horizons

<U D1 n

40 20 0 -20 -40 -60

Ap2 Ap3 Bt1

Soil horizons

Fig. 1 Changes in C:N ratios of oxidation resistant residues relative to untreated soils: a woodlot, b grassland, c no-till corn, d conventional-till corn

soils, and attributed this to lesser stability of N-containing compounds of SOM to NaOCl oxidation than C-containing compounds.

Oxidation using Na2S2O8 and H2O2 in general decreased the C:N ratios (Fig. 1). This result was in accordance with Leifeld and Kogel-Knabner (2001) and Plante et al. (2004), who also observed lower C:N ratios for H2O2 oxidized residues compared to untreated soils. The lower removal of N than C and consequent decreases in the C:N ratio of residues after H2O2 oxidation was suggested to result from interaction of N with mineral surfaces, protection within microaggregates, or fixation of NH4? to minerals (Helfrich et al. 2007). Cheshire et al. (2000) conducted an analysis of the nature and proportion of SOM functional groups resistant to H2O2 oxidation and identified significant amounts of amino acids in the residual clay fraction after oxidation in a Scottish Podsol. These findings also supported the stability of N-compounds to H2O2 oxidation. According to Lorenz et al. (2006), the relative change in C:N ratios of Na2S2O8 oxidized

residues were variable, but mostly negative. The C:N ratio data was supplemented with data from NMR and radiocarbon analyses for evaluating the efficacy of oxidizing agents to isolate a refractory SOC fraction.

Structural composition of SOC before and after oxidation as revealed by NMR analysis

In general, the NMR spectra of the oxidized and untreated surface soils (A1/Ap1 horizon) possessed peaks in the resonance areas of alkyl C (0-45 ppm), O/N-alkyl C (45-110 ppm), aromatic C (110160 ppm), and carboxyl C (160-220 ppm) (Fig. 2). In the untreated whole soil samples, major peaks in the alkyl C region were found in the vicinity of 24, 30 and 33 ppm (Fig. 2a), which are ascribed to long chain methine and methylene type C structures present in fatty acids, waxes and resins (Malcolm 1989). The O/N-alkyl C region was characterized by O and N substituted aliphatic C and is dominated by signals near 56, 62, 72 and 104 ppm. The resonance

Fig. 2 13C NMR spectra of soil organic carbon before and after oxidation: a untreated, b NaOCl treated, c Na2S2O8 treated, d H2O2 treated soils of Wooster, OH

2 0 0 -2 0 ppm

220 200 180 160 140 120

20 0 -20

Chemical shift (ppm) No-till corn

Chemical shift (ppm) Conventional-till corn

Chemical shift (ppm)

Chemical shift (ppm)

at 56 ppm is mostly due to methoxyl C (Hatcher 1987) associated with lignin. The peaks at 62, 72 and 104 ppm generally arise from carbohydrates and are dominated by O-substituted C, ring C and anomeric C, respectively. The aromatic C region was dominated by peaks near 129 ppm (C- or H-substituted aromatic C) and 153 ppm (O-substituted aromatic C such as in phenols), and the carboxylic C region was characterized by a single dominant signal near 173 ppm from carboxylic, amide or ester C (Malcolm 1989).

The relative signal intensities of different C groups showed that the untreated soils of all land uses were

dominated by O/N-alkyl C, with values ranging from 46% (CT) to 50% (grassland and NT) (Fig. 3). The untreated soils of different land uses also contained 20-24% alkyl C, 15 to 21% aromatic C and 11-12% carboxylic C. Among the land uses, the alkyl C:O/N-alkyl C ratio of untreated soils followed the order, 0.40 (NT corn) < 0.45 (CT corn) < 0.48 (grassland) < 0.49 (woodlot) (Table 3). The higher alkyl C:O/N-alkyl C ratio for CT corn than NT corn and for woodlot than grassland were likely driven by differences in quality of plant residues. The grasses were enriched in carbohydrates, which are dominated by O/N-alkyl C, where as the forest litter had significant

О 40 -

С 30 о

О 20 О

Ш Untreated soi]

□ NaOC]

■ NaiSiOs


^ 50 -

О 40 -О

С 30 о

Alkyl C O/N-Alkyl C Aromatic C Carboxyl C

Alkyl C O/N-Alkyl C Aromatic C Carboxyl C

60 (c)

О 40 -О

| 30! 20-o

Alkyl C O/N-Alkyl C Aromatic C Carboxyl C

О 40 -О

.2 30 +3

Alkyl C O/N-Alkyl C Aromatic C Carboxyl C

Fig. 3 Relative distributions of carbon functional groups based on solid-state 13C NMR analysis: a woodlot, b grassland, c no-till corn, d conventional-till corn

Table 3 Comparison of alkyl C:O/N-alkyl C ratios and % aromaticity derived from solid-state C NMR integration data

Land uses Alkyl C Aromaticity (%)

O/N-Alkyl C

Untreated NaOCl N2S2O8 H2O2 Untreated NaOCl Na2S2O8 H2O

Woodlot 0.49 1.03 1.18 2.37 0.31 0.16 0.29 0.31

Grassland 0.48 1.02 0.6 1.51 0.26 0.16 0.32 0.35

No-till corn 0.40 0.74 0.47 0.91 0.30 0.18 0.28 0.32

Conventional-till corn 0.45 0.9 1.16 1.22 0.33 0.21 0.42 0.38

amounts of recalcitrant macromolecules such as tannins, waxes, fats and lignin, which are dominated by alkyl C (Marin-Spiotta et al. 2008).

The structural chemistry of SOC resistant to different oxidizing agents as revealed by solid-state 13C-CPMAS-NMR analysis varied considerably (Figs. 2, 3). Major changes observed in NaOCl oxidation residues include complete removal of signals near 56 ppm which arises from lignin (Fig. 2b), 36-48% reduction in signals at 110160 ppm among land uses (Fig. 2b), and 36-48%

reduction in aromaticity (Table 3) compared to the untreated whole soils. These findings indicated that NaOCl oxidation selectively removed lignin-derived compounds. In order to remove the interference of lignin and other aromatic structures from the black C determination, Simpson and Hatcher (2004) employed NaOCl oxidation of soils and subsequent NMR analysis. Similar to our results, they also observed that lignin moieties were successfully removed by NaOCl oxidation, which facilitated accurate estimation of black C by NMR analysis.

Mikutta et al. (2006) also reported that lignin compounds of SOM were preferentially removed by NaOCl when compared with the losses of bulk OC.

Loss of lignin moieties in this study corroborates the observed increase in C:N ratios of NaOCl oxidized residues compared to unoxidized soils (Fig. 1), indicating that N-containing compounds are more vulnerable to NaOCl oxidation than C-containing compounds (Fig. 1). These N compounds could be directly bonded to aromatic rings in lignin residues. Although aromatic binding of N has been demonstrated under laboratory conditions, the field detection attempts using NMR techniques were unsuccessful until when Schmidt-Rohr et al. (2004) developed saturation-pulse-induced dipolar exchange with recoupling (SPIDER), an NMR technique involving MAS for selective detection of C that are bonded to N. With this advanced technique, significant amounts of N-bonded aromatic C around 134 ppm (N-Carom) and N-bonded aliphatic C (N-CH) around 56 ppm were detected (Schmidt-Rohr et al. 2004; Olk et al. 2006). From the observed signal removal at 56 ppm and signal intensity reduction at 110-160 ppm upon NaOCl oxidation (Figs. 2, 3), it can be deduced that the widening of relative C:N ratio after NaOCl oxidation could be due to the preferential losses of N bonded to the aromatic (134 ppm) and aliphatic C (56 ppm). Thus, the NMR data showed that oxidation with NaOCl resulted changes in specific functional groups of SOM and these changes do not support evidence for an increasing degree of SOC decomposition.

The NMR spectra of the residues resistant to Na2S2O8 and H2O2 showed a relative increase in alkyl and aromatic C, and a relative decrease in O/N-alkyl and carboxyl C than the control spectra (Figs. 2, 3). These trends indicate that changes in structural chemistry of SOC as a result of Na2S2O8 and H2O2 oxidation were in agreement with the selective preservation of alkyl compounds present in recalcitrant macromolecules (e.g. fatty acids, waxes and resins) with increasing degree of humification (Kogel-Knabner et al. 1988; Baldock et al. 1997; Cuypers et al. 2002). These results further support the notion that Na2S2O8 and H2O2 oxidation preferentially removed mostly labile SOC (Bruun et al. 2007). A comparison between Na2S2O8 and H2O2 oxidized spectra across the land uses revealed that the proportion of alkyl C showed an increasing trend

for H2O2 resistant SOC (32-49%) than for Na2S2O8 resistant SOC (23-39%). Concomitantly, the proportion of O/N-alkyl C was generally lower for H2O2 (21-36%) than for Na2S2O8 (27-49%) resistant SOC (Figs. 2c, d, 3). The proportion of aromatic and carboxyl C were comparable between these two treatments. The relative enrichment of alkyl C upon oxidation with H2O2 was also evident from the alkyl C:O/N-alkyl C ratio, which was 1.1 to 2.5 times higher for SOC resistant to H2O2 than that resistant to Na2S2O8 (Table 3). The observed increase in resonance at alkyl region of H2O2 resistant C, however, cannot be directly related to the chemical stability of SOC because some of the fresh aliphatic plant residues dominated by labile compounds can be resistant to H2O2 oxidation (von Lutzow et al. 2007; Helfrich et al. 2007). It was proved when the LF was exposed to chemical oxidation. Among the land uses, the H2O2 oxidation left behind a fraction of bleached LF with 30-56% of total OC. However, the LF remaining after Na2S2O8 oxidation was too low for any reliable measurement of C. A similar test conducted by Eusterhues et al. (2005) on forest litter layers reported that 26-79% of total OC in L and Of layers were resistant to H2O2 treatment. For the Oh horizons, the chemically resistant fraction was smaller, but still in the range of 8-19% of total OC. This result, which has been confirmed elsewhere (von Lutzow et al. 2007), suggests that horizons with large amounts of relatively fresh plant material contain considerable amounts of H2O2-resistant organic matter.

Isotopic analyses of SOC before and after oxidation

The untreated A1/Ap1 soils of all the land uses were characterized by a higher 14C activity (39-103%) (Table 4), indicating that these soils contain 14C produced due to atmospheric thermonuclear weapon testing during 1960s (bomb 14C), and a significant portions of the SOM are exchanging C with atmospheric CO2 on decadal or shorter time scales (Trumbore 1997). Among the oxidizing agents, the 14C activity of SOC resistant to NaOCl was distinctly different from that resistant to Na2S2O8 and H2O2 (Table 4). The 14C activity of NaOCl resistant SOC was only slightly lower than the untreated whole soils. These results, along with the lower C removal

Table 4 The 14C activity of soil organic carbon susceptible and resistant to oxidation in the surface soil

Land uses Untreated NaOCl oxidation Na2S2O8 oxidation H2O2 oxidation H2O2 oxidation after LF

soils removal

D14C of D14C of D14C of D14C of D14C of D14C of D14C of D14C of D14C of SOC (%) retained oxidized retained oxidized retained oxidized retained oxidized SOC (%) SOC (%) SOC (%) SOC (%) SOC (%) SOC (%) SOC (%) SOC (%)

Woodlot 1G3 б1 129 -259 11б -182 124 -4б9 119

Grassland 75 49 9б -378 91 -288 9G -55G 88

No-till corn б5 34 9б -3бб 87 -32б 8G -б14 79

Conventional- 39 -47 97 -542 8G -475 84 -725 97

till corn

efficiency (Table 2) and preferential removal of aromatic compounds (Fig. 2) by NaOCl oxidation indicate that NaOCl oxidation is not effective in removing all young SOC from soils. Based on SOC recovery efficiency and C ages, Favilli et al. (2008) reported that NaOCl is a weak reagent to oxidize all labile OM in soil. On the contrary, the Na2S2O8 and H2O2 resistant residues were depleted of 14C activity as revealed by the negative values of A14C (Table 4), indicating that these chemicals are more efficient in isolating a refractory SOC fraction enriched with aliphatic C functional groups. Since a portion of LF was resistant to H2O2 oxidation, the C activity of the heavy fraction resistant to H2O2 was measured. It showed remarkably lower 14C activity than the activity of H2O2 resistant SOC containing plant residues (Table 4). These results indicate that removing LF, which consists of mainly labile SOC, increases the turnover time of the H2O2 resistant SOC fraction. Among the four land uses, the relative decrease in activity of H2O2 resistant SOC upon LF

removal was slightly greater for soils from woodlot and NT corn, where the proportion of LF was relatively higher.

The 13C isotopic signatures of oxidation resistant SOC are useful tracers for evaluating the effectiveness of different oxidizing agents for the separation of SOC fractions with distinct stability. In woodlot and grassland, where the native plant species are C3 type and there was no shift to C4 vegetation occurred over time, an enrichment of 13C (less negative d13C values) was observed after oxidative treatments (Table 5). Compared to the d C values of untreated samples, the d C values of residues after oxidation in both the woodlot and grassland increased by 0.9 and 0.33%, respectively, upon NaOCl oxidation, 0.66 and 1.63% upon Na2S2O8 oxidation, 3.5 and 5.3% upon H2O2 oxidation and 5.4 and 6.8% upon H2O2 oxidation after LF removal. The largest enrichment of 13C was therefore associated with the SOC present in the H2O2 resistant residue after LF removal. These results are in accord with other studies that

Table 5 The 13C signatures of soil organic carbon susceptible and resistant to oxidation in the surface soil

Land uses

Untreated soils

dl3C of SOC (/ )

NaOCl oxidation

d13C of d13C of retained oxidized SOC (%o) SOC (%o)

Na2S2O8 oxidation

H2O2 oxidation

dl3C of retained SOC (/ )

dl3C of oxidizedSOC (/ )

d13C of d13C of retained oxidized SOC (%o) SOC (%o)

H2O2 oxidation after LF removal

d13C of d13C of retained oxidized SOC (%o) SOC (%o)

Woodlot -27.3 -2б.4 -27.9 -2б.7 -27.4 -23.8 -27.7 -21.9 -27.4

Grassland -27.8 -27.5 -28.1 -2б.2 -27.9 -22.5 -28.1 -21.G -27.9

No-till corn -18.2 -18.5 -17.8 -22.б -18.G -22.4 -18.3 -25.G -18.1

Conventional- -19.4 -19.7 -19.2 -23.7 -18.9 -23.б -19.2 -26.G -19.3

till corn

documented enrichment of heavier 13C compared to lighter 12C with increasing degree of SOM decomposition (Nadelhoffer and Fry 1988; Balesdent et al. 1993; Bernoux et al. 1998). Such a trend in d13C signatures was not observed in soils from cultivated sites, where the d C signatures of contemporary plant inputs are a mixture of both C3 native species (average d13C of -27%) and C4 corn (average d13C of -12%) (Table 5). The mean d13C signatures in two cultivated sites are as follows: -18.8% for unoxidized soils, -19.1% for SOC resistant to NaOCl oxidation, -23.2% for SOC resistant to Na2S2O8 oxidation, -23.0% for SOC resistant to H2O2 oxidation and -25.5% for SOC resistant to H2O2 oxidation after LF removal. These data showed that the significant amount of young, corn-derived SOC that entered the soil over decadal time scales were most susceptible to oxidation with H2O2 (after LF removal) than with Na2S2O8 and NaOCl.

Mass balance calculations

The A14C and d13C values of the SOC removed by oxidation were estimated by mass balance calculations (Tables 4, 5). The data revealed that, irrespective of the differences in labile SOC removal efficiency, the isotopic signatures of the SOC removed by different oxidizing agents remained more or less similar. The A C activity of removed SOC ranged from 116 to 129% for woodlots, 88 to 96% for grassland, 79 to 96% for NT corn and 80 to 97% for CT corn (Table 4), suggesting that many of the millennial ages reported for organic matter indeed represent mixtures of much younger and much older material. Incomplete removal of labile SOC can thus lead to an underestimation of the turnover time of more stable fraction of SOC. Similarly the d C data of removed SOC, estimated by mass balance, ranged from -27.4 to -27.9% for woodlots, -27.9 to -28.1% for grassland, -17.8 to -18.3% for NT corn and -18.9 to -19.3% for CT corn (Table 5). Lower than expected d13C values (-12%) of removed SOC from NT and PT corn suggests that some relic C (C3 SOC) was also removed by the oxidizing agents. Nonetheless, these results reiterate that the isotopic signature of the rapidly cycling labile SOC pool is influenced by the plant species from which it was derived.


The data presented support the conclusion that different chemicals commonly used for wet oxidation of SOC (Na2S2O8, H2O2, NaOCl) react differently on total SOC. The NaOCl oxidation resulted in a lower SOC removal efficiency and left behind a residue with wider C:N ratios than the untreated soils. On the other hand, Na2S2O8 and H2O2 oxidized substantial amounts of SOC and the residue remained had narrower relative C:N ratios. The solid-state 13C NMR analysis was useful in evaluating the chemical transformations of SOC during the wet oxidation processes. The NaOCl oxidation preferentially removed the aromatic functional groups of SOM. However, the SOC resistant to Na2S2O8 and H2O2 oxidation were enriched with alkyl C groups, which dominate in recalcitrant macromolecules. The isoto-pic analyses not only confirmed that Na2S2O8 and H2O2 were more efficient than NaOCl in isolating a refractory fraction of SOC, but also provided evidence that H2O2 treatment after LF removal is more effective in isolating the oldest SOC.

Results obtained from this study will strengthen the current knowledge on the usefulness of different oxidizing agents on identifying the SOC fractions with homogeneous reactivity. Such information is important for accurate quantification of the effects of land use or management changes on longer term sequestration of SOC. The experimental isolation and quantification of SOC pool with homogeneous stability is also useful for validating the conceptual pools of the SOM prediction models for improving their process-based simulation accuracy.

Acknowledgments We would like to thank Prof. W.A. Dick for rendering access to the Triplett-van Doren experiment at Wooster, OH. We greatly appreciate the help from Mr. Sandy Jones and Dr. K. Lorenz for laboratory analyses, and from Dr. T. Young for NMR analysis. The senior author is thankful to Ohio Agricultural Research and Development Center of the Ohio State University for partially supporting this study through a graduate student research grant.


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