007) 17–29www.elsevier.com/locate/geoderma

Geoderma 140 (2

Molecular-scale heterogeneity of humic acid inparticle-size fractions of two Iowa soils

Jingdong Mao a, Xiaowen Fang b, Klaus Schmidt-Rohr b, Ana M. Carmo c,Lakhwinder S. Hundal d, Michael L. Thompson e,⁎

a Old Dominion University, Chemistry & Biochemistry Department, Norfolk, VA 23529, United Statesb Iowa State University, Chemistry Department, Ames, IA 50011, United States

c ExxonMobil Exploration Company, Houston, TX 77060, United Statesd Metropolitan Water Reclamation District of Greater Chicago, Cicero, IL 60804-4112, United States

e Iowa State University, Agronomy Department, Ames, IA 50011, United States

Received 19 October 2006; received in revised form 28 February 2007; accepted 14 March 2007Available online 2 May 2007

Abstract

To explore the hypothesis that enrichment of decomposition-resistant compounds might vary in humic acid (HA) that is associated with silt andclay-size microaggregates, we used infrared spectroscopy, thermal analyses, nuclear magnetic resonance spectroscopy, and pyrolysis techniques tocharacterize the functional groups and structures of HA isolated from coarse silt and clay fractions of two Iowa Mollisols. Samples from the Ahorizons of Sparta (a sandy, mixed, mesic Entic Hapludoll) and Zook (a fine, smectitic, mesic Cumulic Vertic Endoaquoll) soils were gentlydispersed and divided into silt and clay fractions before HA was extracted by standardized techniques. The FTIR, thermal, and NMR analysesrevealed that HA in the sandy, well-drained Sparta soil and size fractions was enriched in aliphatic compounds, readily oxidizable carbohydrates,and lignin compared with HA in the poorly drained, clayey Zook soil. Moreover, pyrolysis spectra demonstrated that fatty acids in the Sparta HAshad longer chain lengths than those of the Zook HAs. Clay-fraction HA contained more readily oxidizable components and aliphatic moieties thandid silt-fraction HA. The NMR-derived observation that Zook soil HA contained more charcoal-like moieties than Sparta HAwas also consistentwith the greater amounts of combustion-resistant material observed in the thermal analyses of Zook HA. Others have shown that “whole” soilorganic matter characteristics differ with the size of the aggregates and particles that the organic matter is associated with. For these prairie-derivedsoils, our study confirms and deepens this observation specifically for HA associated with particle-size fractions.© 2007 Elsevier B.V. All rights reserved.

Keywords: Soil humic acid; Infrared spectroscopy; Thermal analysis; Solid-state nuclear magnetic; Resonance spectroscopy; Pyrolysis-methylation gaschromatography

1. Introduction

Soil organic matter is composed of compounds with a widerange in resistance to decomposition. To improve predictionsabout the long-term fate of organic carbon it is useful todistinguish among compounds with different levels of resis-tance to decomposition and to measure their abundance. Forinstance, the long-term predictions of dynamic soil organiccarbon models can be very sensitive to the initial amount of

⁎ Corresponding author. Tel.: +1 515 294 2415.E-mail address: thompsonm@iastate.edu (M.L. Thompson).

0016-7061/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.geoderma.2007.03.014

carbon assumed to be in resistant fractions and on the rates atwhich carbon is added to those fractions. In 1981, Van Veen andPaul conducted a sensitivity analysis of their carbon dynamicsmodel [on which the current Century model is based; Partonet al. (1992)] and reported that the most important regulator oflong-term soil organic carbon content was how fast carbonmoved into or out of their “passive” pool of soil organic matter.To test the validity and reproducibility of such models, effectivemethods of assessing resistant and non-resistant organiccompounds in soils are required.

Some compounds in soil organic matter resist decompositionbecause of their chemical composition: they are poor in reactive

Table 1Particle size, chemical, and mineral characteristics of the unfractionatedsoilmaterials

Soil Clay(b0.002mm)

Silt(0.002–0.05 mm)

Sand(0.05–2 mm)

Cationexchangecapacity

pH Dominantclay mineral

g kg−1 cmol(+)kg−1

Sparta 150 320 530 9.8 6.2 SmectiteZook 430 420 150 38.1 5.9 Smectite

18 J. Mao et al. / Geoderma 140 (2007) 17–29

functional groups that can be readily cleaved by enzymes. Soillipids, for example, are largely derived from plant polymers(cutin and suberin) that are composed of interesterified hydroxyand epoxy–hydroxy fatty acids and cross-linked polyaromaticand polyaliphatic compounds (Bernards and Lewis, 1998;Buchanan et al., 2000; Nawrath, 2002). The aliphatic portionsof cutin and suberin are particularly resistant to decomposition.Huang et al. (1999), for example, have shown that decompo-sition-resistant n-alkanes in the organic matter of some Britishpaleosols had radiocarbon ages of ∼13,000 years. Hu et al.(2000) demonstrated that polyethylene-like and presumablyhighly stable crystals of polymethylene are present in varioushumic substances. Lignin is another example of a plant polymerthat resists rapid decomposition. It is composed of highly linkedphenols that are typically rich in methoxy groups. Typically, soilorganic matter also contains charcoal (incompletely combustedplant residue that consists largely of fused aromatic moieties)that strongly resists further decomposition (Schmidt et al.,2002). Charcoal (and other types of black carbon) may be anespecially important component of decomposition-resistant soilorganic matter. Skjemstad et al. (2002) studied five agriculturalsoils in the U.S. and reported that charcoal made up 10 to 35%of the total organic carbon in the samples.

Some soil organic matter is not easily decomposed because itis physically protected from microbial enzymes (Christensen,1996; Baldock and Skjemstad, 2000; Balesdent et al., 2000;Christensen, 2001). Such organic matter has been studied byfractionating soil aggregates and characterizing the compoundsassociated with various size fractions. A number of investiga-tors have reported that organic matter in clay fractions iscompositionally distinct from that in sand and silt fractions. Forexample, Zhang et al. (1988) investigated organic matter inpairs of cultivated and uncultivated prairie- and forest-derivedsoils in Iowa. They found that the C:N ratio of “whole” soilorganic matter decreased as microaggregate sizes decreasedfrom coarse silt to fine clay, suggesting that readily decompos-able compounds (e.g., carbohydrates) were less likely to occurin finer aggregates. Baldock et al. (1992) studied organic matterin two Mollisols, two Oxisols, and an Andisol by using 13CNMR spectroscopy. They found higher proportions of alkylcarbon in clay-fraction organic matter than in that of other sizefractions and attributed the observation to selective preservationof recalcitrant compounds. Mahieu et al. (1999) surveyed thepublished literature up to that time and also concluded organicmatter associated with clay fractions tended to be more aliphaticthan whole-soil organic matter. Similarly, in a study of particlesize fractions of four subalpine soils, Chen and Chiu (2003)reported that the abundance of alkyl C in soil organic matterincreased systematically with decreasing particle size.

Humic acid (HA), which is defined by a standardizedprocedure used to extract organic compounds from the soil (e.g.,Swift, 1996), is likely to include a significant proportion ofdecomposition-resistant compounds. Measurements of themean residence time of radiocarbon in HA suggest enrichmentin compounds that resist complete decomposition (Andersonand Paul, 1984). In their review of the literature to 1999,Mahieu et al. (1999) reported the mean aromaticity (ratio of

aromatic C to alkyl plus O-alkyl carbon) of soil HA (asdetermined by NMR studies) was ∼60%, twice the aromaticityof unfractionated, “whole-soil” organic carbon. Mao andSchmidt-Rohr (2004a, 2005) have developed a reliable protocolfor the quantitative analysis of natural organic matter by solid-state 13C NMR. By employing these methods in studies of astandard peat HA, they found that 64% of the aromatic C in theHA was nonprotonated and therefore part of fused aromaticrings that may form a physical and chemical “backbone” forHA.

In the present study, we used a variety of techniques toexplore the hypothesis that enrichment of decomposition-resistant compounds might vary in HA that is associated withsilt and clay-size microaggregates. To test the hypothesis, weused infrared spectroscopy, thermal analyses, nuclear magneticresonance spectroscopy, and pyrolysis to characterize thefunctional groups and structures of HA isolated from coarsesilt and clay fractions of two Iowa Mollisols. The two soilsdiffer from one another by textural class and drainage class.

2. Materials and methods

2.1. Soil samples

The A horizons of a Sparta soil (a sandy, mixed, mesic EnticHapludoll) and a Zook soil (a fine, smectitic, mesic CumulicVertic Endoaquoll) and their respective clay (b2 μm) and coarsesilt (20–50 μm) particle-size fractions were investigated. Bothsoils had been sampled in Story County, Iowa, USA (furtherdetails about sampling locations are available from thecorresponding author). The particle size distribution of thesoil samples was determined by the pipette method (Gee andBauder, 1986), cation exchange capacity was determined by theNa acetate method (Thomas, 1982), pH was determined at22 °C using a 1:1 soil:water suspension, and mineralogy wasdetermined by x-ray diffraction analysis, following proceduressimilar to those of Whittig and Allardice (1986). Characteristicsof the soil materials are reported in Table 1.

The soil materials were sieved in their field-moist state toremove particles N2 mm before particle-size fractionation. Thesamples were gently dispersed either by shaking overnight indistilled water (Zook) or by 5-min sonification with a probe-type sonifier at 100 W (Sparta). After wet-sieving the N53-μmparticles, the samples were fractionated into one clay (b2 μm)and two silt fractions (2–20 and 20–50 μm) by gravitysedimentation in distilled water. The clay fraction was

19J. Mao et al. / Geoderma 140 (2007) 17–29

coagulated using CaCl2 and excess electrolyte was removed bydialysis against distilled water. Clay and silt fractions werefreeze-dried, whereas unfractionated materials were air-dried.Total carbon and nitrogen were determined by high-temperaturecombustion using a LECO elemental analyzer (Nelson andSommers, 1996).

2.2. Humic acid extraction and elemental analysis

To extract humic substances, the procedure of Swift (1996)was followed. About 20 g of material was first equilibratedwith 20 mL of distilled–deionized (DD) water that had beenadjusted to a pH between 1 and 2 with HCl. The slurry wasshaken for 1 h. The liquid phase was discarded after cen-trifugation. A small amount of DD water was added to create aslurry, and the slurry pH was adjusted to 7 using 1 M NaOH.Finally, sufficient 0.1 M NaOH was added to result in a liquid:soil ratio of 10:1, and the suspension was shaken under N2

atmosphere for at least 4 h. After centrifugation, the pH of thesupernatant was adjusted to 1 using 6 M HCl. The extract wasagain centrifuged and the supernatant was removed. Theprecipitate (HA) was redissolved using a minimum amount of0.1M KOH under N2. Sufficient KCl was added to the mixtureto attain a concentration of ∼0.3 M [K+], then it was cen-trifuged to remove suspended solids. Humic acid was againprecipitated by adjusting the pH to 1 using 6 M HCl. Themixture was centrifuged and the liquid fraction was discarded.To remove inorganic impurities, the precipitate was suspendedthree times in 0.1 M HCl/0.3 M HF and centrifuged. Thepurified HA was dialyzed against distilled water in thepresence of a H+-cation exchange resin placed in an adjacentdialysis bag.

Concentrations of C, H, and N in the extracted HA weredetermined by a high-temperature, dry combustion CHNanalyzer (Nelson and Sommers, 1996). The precision of thesedeterminations was 0.1%. Inductively coupled plasma spec-trometry was used to determine concentrations of Al, Ca, Fe, K,Mg, P, S, and Si in the HA samples. Oxygen content wascalculated as the difference between the sum of major elementalconcentrations and 100%.

2.3. Infrared spectroscopy

Diffuse-reflectance, Fourier-transformed infrared (DRIFT)spectra of a mixture of each HA and spectroscopic grade KBr(Aldrich Chemical Co.) (∼1:250, w/w) were collected by usinga Nicolet Magna-IR 560 spectrometer (Nicolet InstrumentCorp., Madison, WI). The sample chamber was continuouslyflushed with CO2-free dry air. DRIFT spectra were collectedfrom 4000 to 650 cm−1 and averaged over 328 scans(resolution±4 cm−1).

2.4. Thermal analysis

Each of the extracted HAs was analyzed by thermogravi-metry. Samples ranging from 3 mg to 20 mg were heated at10 °C min−1 from 25 °C to 110 °C and held at 110 °C for 60 min

to insure evaporation of water. Then the samples were heatedfrom 110 °C to 800 °C at 10 °C min−1, and mass loss wasrecorded over time. The heating chamber was continuouslyflushed with air at 200 mL min−1.

2.5. Solid-state nuclear magnetic resonance

All the experiments were performed using a Bruker DSX400spectrometer at 100 MHz for 13C. Except for the high-speedquantitative 13C NMR, which was run using a 4-mm double-resonance probe, all experiments were performed with 7-mmsample rotors on a double-resonance probe.

2.5.1. 13C CP/TOSS13C CP/TOSS (cross polarization/total suppression of side-

bands) experiments were performed at a spinning speed of5051 kHz and a CP time of 1 ms, with 1H 90° pulse-lengths of4 μs. Experiments combining 13C CP/TOSS with 40-μs dipolardephasing were also run to generate subspectra of unprotonatedcarbons and mobile groups like CH3. CP detection-efficiencyfactors were calculated from the ratios of the DP and CPintensities of the soil and Sparta clay HAs and applied to correctthe intensities in the CP spectra for quantitative analysis.

A 13C T2 (spin–spin relaxation time) filter experiment with arotation-synchronized Hahn echo was run with a filter time of3.96 ms (Mao et al., 2001).

2.5.2. High-speed quantitative 13C NMRQuantitative 13C DP/MAS (direct polarization / magic-angle

spinning) spectra, without and with 80-μs of recoupled dipolardephasing, were acquired at a spinning speed of 14 kHz. The90° 13C pulse-length was 3.4 μs. Recycle delays were tested bythe CP/T1-TOSS technique to make sure that the magnetizationof all carbons was relaxed to more than 90% of its equilibriumvalue (Mao et al., 2000). Recycle delays of 100 s were used forthe soil HAs, and of 20 s for the Sparta clay HA. A dipolardephasing time of 68 μs was inserted and centered on the 13C180° pulse to obtain quantitative subspectra with unprotonatedcarbons and mobile groups like CH3. The details of thistechnique, which is based on the concept of dipolar recouplingand avoids the shortcomings of standard gated decoupling athigh spinning frequencies, have been described elsewhere (Maoand Schmidt-Rohr, 2004a).

2.5.3. 13C CSA filterA 13C chemical shift anisotropy (CSA) filter (Mao and

Schmidt-Rohr, 2004b) was used to separate the signals ofanomeric carbons from those of aromatic carbons. The 1H 90°pulse-length was 4 μs, the contact time 1 ms, and the CSA filtertime in the three-pulse sequence (i.e. with one 180° pulse)70 μs, at a spinning frequency of 5 kHz. Four-pulse totalsuppression of sidebands (TOSS) (Dixon, 1982) was employedbefore detection. During detection, two-pulse phase modulation(TPPM) decoupling was applied. As part of the CSA filterscheme, the z-period is incremented in four steps of tr/4, whichprovides the “γ-integral” that suppresses sidebands up to thefourth order (deAzevedo et al., 2000).

Table 2Elemental analyses of the organic matter and extracted humic acid

Bulk sample Extracted humic acid a

Soil material C N C/N C H N O C/N

g kg−1 g kg−1

Sparta bulk soil 21 2 12 532 45 30 382 18Sparta coarse silt 61 4 17 497 50 23 419 21Sparta clay 93 7 14 486 51 27 417 18Zook bulk soil 25 2 13 526 44 23 400 23Zook coarse silt 17 2 11 522 42 18 408 30Zook clay 41 3 14 547 44 23 386 23a Other elements measured in the HA samples included trace amounts of Al,

Ca, Fe, K, Mg, P, S, and Si. The total concentration of these elements accountedfor less than 10 g kg−1 in all samples except HA associated with Sparta clay. Inthat sample, the total of trace elements accounted for 19 g kg−1 of the HA.Oxygen content was calculated as the difference between the sum of majorelemental concentrations and 100%.

20 J. Mao et al. / Geoderma 140 (2007) 17–29

2.5.4. 1H–13C heteronuclear correlation NMR (HetCor)Two-dimensional (2D) HetCor NMR experiments (Mao

et al., 2001) were performed at a spinning speed of 5051 kHz.Frequency-switched Lee–Goldburg homonuclear decouplingwas applied during the evolution period t1. Lee–Goldburgcross polarization of 0.5 ms was used to suppress 1H–1Hspin diffusion during polarization transfer and to show mostlyone- and two-bond 1H–13C connectivities. The number of scanswas 800, and the number of t1 increments was 64.

2.5.5. CH and CH2 spectral editingFor methane (CH) selection, the dipolar distortionless

enhancement by polarization transfer (DEPT) method basedon C–H multiple-quantum coherence was used (Schmidt-Rohrand Mao, 2002). CH-group multiple-quantum coherence is notdephased by the spin-pair CH dipolar coupling, while CH2

group coherence is dephased by dipolar coupling of the carbonsto the two protons. The first of a pair of spectra recordedcontains signals of CH, as well as residual quaternary-carbonand CH3 peaks. The latter two are removed by taking thedifference with a second spectrum acquired with the same pulsesequence except for additional 40-μs dipolar dephasing beforedetection. The total number of scans was 12,000, and thespinning speed 5762 Hz with a recycle delay of 0.5 s. CH2

spectral editing was achieved by three-spin coherence selection(Mao and Schmidt-Rohr, 2005) under the same conditions asCH selection.

2.6. Pyrolysis–methylation-GC/MS

Approximately 0.5 mg of HAwas placed in a quartz sampleholder and wetted with 5 μL of a solution of 25%tetramethylammonium hydroxide (TMAH) in methanol. Themixture was immediately dried under a stream of N2 followedby pyrolysis. The pyrolysis unit consisted of a CDS AnalyticalPyroprobe 2000 heated platinum filament pyrolyzer coupled toa Varian 3400 gas chromatograph (GC) via a CDS AnalyticalInterface 1500. The GC was interfaced with a Finnigan INCOSXL mass spectrometer. Pyrolysis of the HA was carried outunder He at 700°C for 10 s inside the Pyroprobe platinum coil.The pyrolyzates were carried to the GC by a stream of He andseparated using a HP−1 capillary column (25 m×0.32 mm i.d.×0.52 μm film thickness). The GC oven temperature wasprogrammed to increase from 30 °C to 300 °C at a rate of 4°Cmin−1 and was held at 300 °C for 15 min. Ionization in the massspectrometer was done by electron impact at 70 eV. Massspectral data were collected in full scan mode from 50 to450 amu at a rate of 0.8 scan s−1. Compounds were identifiedby comparison with the National Institute of Standards andTechnology mass spectral library and with published massspectra (Klap et al., 1998). Ion chromatograms were obtainedfor HA extracted from the whole Sparta soil and from clay- andsilt-size fractions of both soils. Insufficient HA was extractedfrom the fine-textured Zook whole soil for pyrolysis studies.

The presence of a derivatizing agent such as TMAHincreases the yield and volatility of pyrolysis products ofpolar macromolecules such as humic substances, preventing

extensive decarboxylation reactions by converting carboxylicacids to methyl esters; hydroxyls are converted to methyl ethers(Challinor, 1989; De Leeuw and Baas, 1993). TMAHderivatization during pyrolysis has been previously used tocharacterize fulvic acids, humic acid, and humin, as well aswhole-soil organic matter (Grasset et al., 2002; Naafs and VanBerger, 2002).

3. Results and discussion

3.1. Bulk soil properties and elemental composition of humicacids

Sparta and Zook are cultivated soils that occur commonly incentral Iowa. The well-drained Sparta soils formed in sand dunes,and the poorly drained Zook soils formed in fine-textured, recentalluvium on floodplains. At the time of sampling, the Sparta soilwas in a corn/soybean (Zea mays/Glycine max) crop rotation. TheZook site was under a 5-year-old stand of hybrid poplar trees(Populus euramerican), but it had been previously cropped tocorn and soybeans for many years. Consistent with their parent-material properties, the Sparta sample had a high sand content,whereas the Zook sample had a high clay content (Table 1). TheC/Nmass ratios of the unfractionated soil samples were 12 and 13(Table 2). A small portion of the total nitrogen content may havederived from clay-fixed NH4

+, but the C/N mass ratios mainlyreflect the composition of organic matter and the degree ofmicrobial decomposition and humification of plant-derivedorganic matter (Stevenson, 1994). When compared to both theunfractionated samples and to the silt fractions, clay fractionscontained the highest concentrations of organic carbon andnitrogen (Table 2). Humic acids separated from the bulk soil andfrom the size fractions had elemental compositions similar tothose reported by others (e.g., Stevenson, 1994), with C/N massratios ranging from ∼18 to ∼30 (Table 2).

3.2. FTIR analyses

The FTIR patterns of HA extracted from the Sparta and Zooksoil samples and their particle size fractions provided an overall

21J. Mao et al. / Geoderma 140 (2007) 17–29

picture of the functional groups present (Fig. 1). These weresimilar to those reported by others for HA (e.g., Stevenson, 1994).We assigned bands at ∼3300 cm−1 to O–H stretching. Carboxyland carboxylate groups were indicated by bands at ∼1725 cm−1

(C=O stretch of COOH), ∼1415 cm−1 (symmetric stretch ofCOO−), and ∼1230 cm−1 (C–O stretch and OH deformation ofCOOH). The diffuse band in the region of 1040–1095 cm−1 wasassigned to the C–O stretch of aliphatic and aromatic hydroxyls.Other aromatic features included aromatic C–H stretch at∼3080 cm−1, aromatic C_C stretch at ∼1615 cm−1, andaromatic C–H out-of-plane bending at ∼770 cm−1.

Three differences among the HA FTIR spectra were subtlebut notable. First, HA associated with the Zook coarse siltfraction included some kaolinite that had not been removed ordestroyed by the pretreatments. Kaolinite was indicated bydiagnostic peaks at 3697 cm−1 (O–H stretch of inner surfacehydroxyl), 3622 cm−1 (O–H stretch of inner hydroxyl),1125 cm−1 (Si–O stretch), 1050 cm−1 (Si–O stretch), and920 cm−1 (O–H stretch of inner surface hydroxyl). Second, theoccurrence of bands at ∼1690 cm−1 and ∼1540 cm−1 (N–Hand C_O stretching) suggested that HA associated with thecoarse silt and clay fractions of the Sparta soil were enriched inamide moieties compared with those of the Zook fractions.Finally, judged from the relative intensity of the band at∼2935 cm−1 (asymmetric stretch of –CH2–), HA from theSparta coarse silt and clay fractions was also enriched inaliphatic compounds compared with the Zook HA.

3.3. Thermal analyses

Thermal analyses have long been used to provide insight intothe types of functional groups present in humic substances and

Fig. 1. Diffuse reflectance infrared spectra of humic acids extracted from unfraction

their sensitivity to combustion (Turner and Schnitzer, 1962;Peuravuori et al., 1999; Fernandez et al., 2001; Pietro and Paola,2004; Francioso et al., 2005). Plante et al. (2005) (and others)have argued that thermal stability might be used as a proxy for“resistance of organic matter to decomposition by microbes andtheir associated enzymes.” After water is evaporated at 110 °C,the functional groups of organic matter that are susceptible tocombustion include carboxyl, hydroxyl, methyl, and methylenegroups, as well as alicyclic carbon. Compounds with thesemoieties (e.g., carbohydrates and amino acids) are largely lostover the temperature range from 110 to 220 °C. In the range 220to ∼350 °C, oxidation of lignin and labile lipids occurs. Attemperatures from ∼350 to 600 °C, it is expected that carbon inmore resistant lipids and aromatic compounds will be oxidized.

Black carbon in soils, i.e., charcoal and other incompletelycombusted organic matter, is contained largely in fusedaromatic rings, and one technique proposed to estimate itsabundance in soil or sediment samples is to find the differencebetween total carbon contents (determined by high-temperaturecombustion at ∼900 °C) before and after heating a sample to375 °C for 24 h (Gustafsson et al., 1997). If all carbon other thancharcoal carbon has been oxidized at 375 °C, then thatdifference would be an index of black carbon. Other researchershave noted the potential for artifacts to be created in thisprocedure because readily combusted compounds may becondensed into fused rings that are subsequently oxidized athigher temperatures and are mistaken for black carbon. Suchartifacts are likely to increase with slow heating rates, largesample sizes that slow the removal of oxidation products fromthe sample, and incomplete purging of evolved CO2.

The differential thermal gravimetric spectra of the HAsamples are presented in Fig. 2, and the distribution of

ated samples and from coarse silt and clay fractions of Zook and Sparta soils.

Fig. 2. Differential thermal gravimetric patterns of humic acids extracted fromunfractionated samples and from coarse silt and clay fractions of Zook andSparta soils.

22 J. Mao et al. / Geoderma 140 (2007) 17–29

components is given in Table 3. Readily oxidizable compo-nents, those combusted between 220 and 350 °C and indicatedby the broad “hump” in the DTG pattern from about 4500 s toabout 5200 s, were a minor proportion of the HA from both soils(20–35% of the combustible mass). This observation isconsistent with the view that HA is enriched in compoundsthat resist microbially mediated degradation (Stevenson, 1994).Still, HA of Zook size fractions and bulk soil was consistentlymore resistant to oxidation than that of the Sparta soil. Within

Table 3Distribution of thermally decomposable components in the humic substances

220–350° 350–600° Incombustible ash WaterSoil material

% of combustible mass % of freeze-dried massSparta bulk soil HA 24 76 NDa 10Sparta coarse silt HA 33 67 4 9Sparta clay HA 35 65 ND 11Zook bulk soil HA 20 80 6 10Zook coarse silt HA 24 76 4 9Zook clay HA 30 71 5 NDa ND — none detected.

the context of each soil sample, readily oxidizable componentswere more abundant in HA extracted from the clay fraction(Table 3). The consistently greater proportion of readilyoxidizable material in the clay-fraction HA compared with thesilt-fraction HA could mean that association of organic matterwith fine particles had helped to protect those components fromdecomposition by microorganisms. From the thermal dataalone, these compounds cannot be distinguished from oneanother, but we speculate that they include some aliphaticcompounds as well as some labile components of lignin.

Except for the Sparta whole-soil HA, sharp peaks in thethermal spectra at temperatures N350 °C suggest that thecomponents of HA oxidized were fairly similar to one anotherwith regard to ease of combustion, but the exact combustiontemperature varied. For example, HA associated with the clayfractions reached combustion temperature more quickly thandid the HA from the silt fractions or the whole soil samples(Fig. 2). The exact timing and temperature of combustion of HAcomponents reflects not only chemical characteristics of thecomponents but also the uniformity of packing and thermalconductivity of the entire sample in the sample holder. Thusdetailed analyses of the thermal patterns, beyond the trendsnoted in Table 3, are probably not warranted.

3.4. NMR analyses

3.4.1. Identification of functional groupsFigs. 3–5 display 13C NMR spectra of the six HAs. Fig. 3

shows quantitative 13C spectra obtained by DP/MAS of threesamples that include the extremes of aromaticity: Sparta soil

Fig. 3. Quantitative 13C NMR spectra of (a) Sparta soil HA, (b) Sparta clay HA,and (c) Zook soil HA, obtained by direct polarization NMR at 14-kHz MASwith (a, c) 100-s and (b) 20-s recycle delays. Corresponding spectra ofnonprotonated C and methyl groups, obtained with recoupled dipolar dephasing,are overlaid as thin lines. Total measuring time: 40 h.

Fig. 4. Cross-polarization 13C NMR spectra of (left column) Sparta HAs and (right column) Zook HAs. (a, f, h, k, o, q) CP/TOSS spectra, highlighted in bold, areshown scaled to equal area. The corresponding dipolar-dephased spectra of nonprotonated C and mobile segments are overlain as thin lines. (b, g, j, l, p, r) 4-ms T2C

filtered spectra, shown in the correct ratio to the CP/TOSS spectra.

23J. Mao et al. / Geoderma 140 (2007) 17–29

HA, Sparta clay HA, and Zook soil HA. DP/MAS spectra afterdipolar dephasing, which give quantitative information aboutnonprotonated carbons and residual CH3 signals, are also shown(thin lines). We acquired CP/TOSS, dipolar dephased, and 13CT2-filtered

13C NMR spectra for all six samples (Fig. 4).Furthermore, we measured CH-only and 13C CSA-filteredspectra of both soil HAs, the CH2-only spectrum of Sparta soilHA, and the 13C CSA filtered spectrum of Sparta clay HA(Fig. 4). Fig. 5 shows the 2D 1H–13C HetCor spectra of Spartasoil HA, Sparta clay HA, and Zook soil HA. We also acquired2D HetCor spectra of the three other samples, but since theyexhibit features similar to their corresponding series from thesame soil, we do not show them here. From the spectra ofFigs. 3 and 4, we obtained the quantitative amounts of varioustypes of functional groups (Table 4).

The NMR-identifiable components in these HAs wereprimarily aromatics, COO groups, peptides, carbohydrateresidues, and nonpolar aliphatics. All spectra exhibitedpredominant aromatic signals between 160 and 100 ppm,

accounting for as much as 51% to 60% of all carbon atoms(Table 4). This included a significant fraction of aromatic C–Ocarbons, which resonate between 145 and 160 ppm. Thequantitative spectra of Fig. 3 showed that 75–80% of thearomatic carbon atoms were not protonated, indicating fusedaromatic rings characteristic of charcoal residues. Fig. 4 showsthat 13C T2 (spin–spin relaxation time) filtering primarily selectsignals of aromatics and of COO groups resonating at 169 ppm,which together resemble the spectra of oxidized charcoaldomains.

The strong peak around 169 ppm in the DP and 172 ppm inthe CP spectra was assigned to COO (carboxylic acid and ester)and NC_O (amide or peptide) moieties. In Sparta HAs, theyaccounted for 17–22% of all C; in Zook HAs, for 17–19%. Themaximum concentration of NC_O groups can be calculatedfrom the inverse of the atomic C:N ratio, which was 21–25 forSparta HAs and 27–35 for Zook HAs. If every N was part of anNC_O group, these groups would account for 4–5% of all C inSparta HAs and 3–4% of all C in Zook HAs. This proves that

Fig. 5. 1H–13C heteronuclear correlation (HetCor) NMR spectra of (a) Spartasoil HA, (b) Sparta clay HA, and (c) Zook soil HA.

24 J. Mao et al. / Geoderma 140 (2007) 17–29

the majority (2/3 to 3/4) of the signal around 172 ppm was fromCOO groups.

The fraction of C bonded to N can be compared with thefraction of carbon resonating in the NCH band around 55 ppm,after subtraction of the OCH3 signal identified by dipolardephasing and scaled up by the inverse of its dipolar dephasingfactor, 1/0.57. It accounted for ∼4% of all C in Sparta HAs, and

Table 4NMR assessment of carbon in functional groups in the humic acid samples

HumicAcid

Carbonyl COO andamides

AromaticC–O

Nonpolar aromaticC

% 210–185 ppm

185–160 ppm 160–145 ppm

145–100 ppm

Sparta soil 2.2 22 9.0 45Sparta silt 3.0 18 10 44Sparta clay 1.6 17 8.5 42Zook soil 2.6 19 8.3 51Zook silt 2.5 18 8.7 51Zook clay 2.3 17 7.7 51

The intensity reported for functional groups with overlapping ppm ranges (e.g., of OCediting. Fractions exceeding 10% are highlighted in bold.a Aromaticity index=(Aromatic C–O+nonpolar aromatic C) / (all aromatic C+al

for ∼3% in Zook HAs. This was in good agreement with the 4–5% of all C bonded to N in the former, and 3–4% in the lattersamples. In other words, peptides (CH–NH–CO–CH)accounted for N75% of all N in these samples.

Peptide C_O has a chemical shift around 172 ppm and crosspeaks with NCH protons at ∼4 ppm in the 1H dimension ofHetCor spectra. Such a peak was indeed seen in the HetCorspectra of Fig. 5, in particular for Sparta soil and clay HAs. Incontrast, much of the COO resonated near 169 ppm and had astrong cross peak with aromatic 1H near 7 ppm, as seen mostprominently in the HetCor spectrum of Zook soil HA, Fig. 5c.The reduced intensity of these sites in CP compared to DPspectra indicated that they are in an environment with few 1H,just like a large fraction of the aromatics. In other words, manyof the COO groups were bonded to the fused aromatic rings thatare probably oxidized charcoal residues.

Carbohydrate residues were identified from OCH peaks at70 ppm and the anomeric peaks around 100–105 ppm (O–C–O) clearly seen after CSA filtering (Fig. 4). They were identifiedas O–CH–O in the CH-only and the HetCor spectra (cross peakat 5.5 ppm 1H and 105 ppm 13C). The CH2–OH band around62 ppm expected for carbohydrates was revealed by CH2

spectral editing (Fig. 4e). Aliphatic carbon signals resonatearound 45–0 ppm, with a sharp polymethylene peak at 33 ppm(Figs. 4 and 5). There were no detectable signals ofnonprotonated alkyl carbons.

Small signals of lignin residues were identified with thepeaks of OCH3 carbons at 60–45 ppm and aromatic C–Oaround 150 ppm (Figs. 4 and 5).

3.4.2. Comparison of the six HAsThe 13C NMR spectra of the six HAs in Fig. 4 all exhibited

the signals of the five major components discussed above but invarying proportions. The spectra showed a clear decrease in thearomaticity with particle size in the Sparta HAs, which explainsthe trend of decreasing C content observed in the elementalcomposition. As a result of this trend, the Sparta clay HA hadNMR characteristics rather different from the other HAs,including Zook clay HA. The Sparta clay HA had the largestaliphatic and smallest long-T2 component. Interestingly, in theSparta series the intensity of the COO/NCO peak did not vary

–O–CHn –OCH3 NCH Aliphatic (nonpolar alkyl)C

Aromaticityindex a

110–60 ppm

60–50 ppm

60–45 ppm

45–0 ppm

9.6 0.8 3.8 7.3 0.7110.5 1.0 4.4 9.1 0.6815 0.8 4.6 11.0 0.629.5 0.6 3.1 5.8 0.769.5 0.7 3.1 7.0 0.75

10.3 0.3 3.4 7.6 0.73

H3 and NCH) is only from the specified functional groups, separated by spectral

l alkyl C).

25J. Mao et al. / Geoderma 140 (2007) 17–29

along with that of the aromatic band. Sparta silt HAs stood outas having the most pronounced lignin (150- and 55-ppmsignals) and C=O (ketone or quinone) peaks of all six samples.In the Zook-HA series, the silt fraction also contained therelatively largest signals of lignin-derived residues.

There was no pronounced change in aromaticity in the ZookHA series, consistent with their similar elemental compositions.However, the fraction of long-T2, sp

2-hybridized carbons withthe spectral characteristics of oxidized charcoal was particularlylarge in Zook soil HA. The alkyl carbon signals were slightlyincreased in Zook clay HA. The most pronounced trend,relatively speaking, in the Zook HA series was a decrease of theCOO/NCO signal with decreasing particle size (Fig. 4, Table 4).The origin of the reduced N content in Zook silt HA was notobvious from the NMR spectra.

While the elemental composition suggested that the HAsextracted from the bulk soil samples were similar, all the NMRspectra showed consistently lower aromaticity in Sparta soil HAthan in Zook soil HA. All methods agreed that the HAs of the sizefractions of Sparta soil had clearly reduced aromaticities compared

Fig. 6. Ion chromatograms of pyrolysis–methylation gas chromatography/mass sidentifications.

to those of Zook soil, and the comparison was similar for the HAsextracted from the unfractionated Sparta and Zook soils.

In summary, Sparta HA samples contained more aliphatic C,lignin residues, peptides, and carbohydrates and less aromatic Cthan did Zook HA samples (Table 4). Furthermore, Sparta HAsshowed clearer polymethylene peaks than did Zook HAs. 2DHetCor spectra indicate that the strongest correlation peak forCOO/CONH is OCH or NCH for Sparta HAs versus aromaticprotons for Zook HAs. For HAs of both soils, the percentages ofaliphatics and carbohydrates were higher in clay-fraction HAs,although this trend was weak in the Zook HA series (Table 4).All of these observations were consistent with the FTIR patternsand the thermal data presented in Table 3, but they give usconsiderably deeper insight into the nature of the compounds inthe extracted HAs.

3.5. Py-TMAH-GC/MS analyses

Pyrolysis techniques provide a unique perspective on thecomposition of some important components of soil organic

pectrometry of HA samples methylated with TMAH. See Table 5 for peak

Table 5Compounds identified in the pyrolyzates of the humic acid samples

# Compound

1 Pyridine2 Toluene3 Methylpyridine4 N,N-dimethylacetamide5 Ethylbenzene6 M+p dimethylbenzene7 Styrene8 Methoxybenzene9 Trimethylphosphate10 Phenol11 P+o methylanisole12 Dimethylsuccinate13 1-methyl-2,5-pyrrolindinedione14 2-methoxyphenol (guiacol)15 Benzoic acid MEa

16 Methylphenol17 Indole18 C8:0 FAMEb

19 4-Methoxystyrene20 Naphthalene+unknown21 2-Methoxy-5-methylphenol22 Methylveratrole23 C9:0 FAME24 Methylindole25 2,3-Dihydrobenzofuran26 Methylnaphthalene27 Vinylguiacol28 N-methylpyroglutamic acid, ME29 3-Methoxybenzoic acid, ME30 C10:1 FAME31 C10:0 FAME32 Syringol (2,6-dimethoxyphenol)33 4-Methoxybenzoic acid, ME34 N-methylphthalamide35 1,4-Benzenedicarboxylic acid ,ME36 1,2-Benzenedicarboxylic acid ,ME37 4-Hydroxy-3-methoxybenzoic acid, ME38 C12:0 FAME39 3,4-Dimethoxyacetophenone40 3,4-Dimethoxybenzoic acid ME41 n-C21:142 Anysilpropenoic acid, ME43 5-Acetyl-1,2,3-trimethoxybenzene44 3,4,5-Trimethoxybenzoic acid, ME45 Acetylseringol46 C14:0 FAME47 Syringic acid, ME48 C15:0 FAME49 Veratrylpropenoic acid, ME50 Dialkylpthalate51 n-C25:152 C16:1 FAME53 C16:0 FAME54 C18:1 FAME55 C18:0 FAME56 C20:0 FAME57 C22:0 FAME58 C24:0 FAME59 C26:0 FAME60 C28:0 FAME61 C30:0 FAME62 C32:0 FAME

Compare with Figs. 6 and 7.a Me, methyl ester.

26 J. Mao et al. / Geoderma 140 (2007) 17–29

matter. This analytical approach favors those compounds thatcan be pyrolyzed and derivatized so that they are amenable to gaschromatography. In this study, our pyrolysis technique did notallow absolute quantitative comparisons of all HA compo-nents, but, on a relative basis, it provided a more completecharacterization of the aliphatic components of the HA samples.The pyrolysis products obtained in this study were consistentwith those of previous reports of both pyrolysis/methylation andoffline thermochemolysis of humic substances (Fig. 6; Table 5)(e.g., Hatcher and Clifford, 1994; Saiz-Jimenez, 1994; Fabbriet al., 1996). Aliphatic structures were dominated by fatty acidmethyl esters (FAME) with C skeletons containing 8 to 32 Catoms, with strong even-over-odd carbon number predominance(N=18, 23, 30, 38, 46, 48, and 52 to 62). Both short-chainFAME (such as C14, C16, and C18 compounds) and long-chain,even-numbered FAME (NC18) have been attributed mainly toaliphatic biopolymers derived from higher plants (cutin andsuberin) (Naafs and Van Berger, 2002; Nawrath, 2002). Short-chain FAME in the HA samples may also be related to microbialsynthesis (Lichtfouse et al., 1995; Grasset et al., 2002).

Among the major aromatic structures present in the HApyrolyzates, methylated benzene carboxylic acids (compounds15, 33, 35, 36, 37, 36, 40, 49, and 50) were identified. In a fewcases, methylation of phenolic hydroxyls did not take placeduring pyrolysis with TMAH (compounds 10, 14, 16, 27, 32,37, and 47). Aromatic compounds derived from the lignin ofgrasses and vascular plants predominated (compounds 14, 27,32, 33, 37, 39, 40, 42, 44, 45, 47, and 49), confirming theimportance of lignin as a building block of humic substances(Hatcher and Clifford, 1994; Saiz-Jimenez, 1994; Fabbri et al.,1996). These compounds were methylated derivatives ofphenolic compounds (p-hydroxyphenyls), monomethoxyphe-nyls (guiacyl compounds), and dimethoxyphenyls (syringylcompounds). Whereas gymnosperm lignin is composed exclu-sively of guiacyl units, lignin derived from angiosperms andgrasses also contains syringyl and p-hydroxyphenyl units inaddition to guiacyl units (Clifford et al., 1995; Del Rio et al.,1998). The predominance of p-hydroxyphenyl derivatives(compounds 33 and 42) in addition to compound 49 (a syringylunit) in the pyrolyzates of silt-fraction HA of Zook soilsuggested a greater influence of grasses on HA in the Zook siltfraction than on HA in the Sparta silt fraction.

To compare the relative abundances of major aliphaticcompounds versus major aromatic compounds present in thepyrolyzates of the HA samples, the peak heights of selectedaromatic compounds derived from lignin (compounds 40, 44, and49) and the straight chain FAMEC16 throughC32were normalizedto 3,4-dimethoxybenzoic acid methyl ester (compound 40) forboth Sparta and Zook HAs (Fig. 7). FAME predominated in HAderived from the clay fractions compared with HA derived fromthe silt fractions, consistent with previous reports that “whole”organic matter in finer fractions of soil is enriched in aliphatics

Compare with Figs. 6 and 7.a Me, methyl ester.b Fatty acid methyl ester.

Notes to Table 5

Fig. 7. Abundance of selected aromatic compounds derived from lignin (compounds 40, 44, and 49) and the straight chain FAME (C16 through C32) relative to 3,4-dimethoxybenzoic acid methyl ester (compound 40) for both Sparta and Zook HAs.

27J. Mao et al. / Geoderma 140 (2007) 17–29

compared with that of coarser fractions (Mahieu et al., 1999;Schulten and Leinweber, 1999; Schmidt et al., 2000).

The maximum chain length of FAME in the pyrolyzates ofHA extracted from both Zook size fractions was shorter (up toC18) than that of FAME in HA from the comparable Spartasamples (up to C32) (Fig. 7). The greater abundance of short-chain FAME over aromatic compounds in the pyrolyzates ofZook clay HA could be attributed to dominance of microbialover plant origins or to selective preservation of short-chaincompounds in the higher-clay soil.

4. Conclusions

The four approaches to characterization of HA in the soils ofthis study provided consistent comparisons among the soils andthe size fractions. The FTIR, thermal, NMR analyses revealedthat HA in the sandy, well-drained Sparta soil and size fractionswas enriched in aliphatic compounds, readily combustiblecarbohydrates, and lignin compared with HA in the poorlydrained, clayey Zook soil. Moreover, pyrolysis spectra dem-onstrated that fatty acids in the Sparta HAs had longer chain

lengths than those of the Zook HAs. Clay-fraction HAcontained more readily combustible components and aliphaticmoieties than did silt-fraction HA. The NMR-derived observa-tion that Zook HA contained more charcoal-like moieties thandid Sparta HA was also consistent with the greater amounts ofcombustion-resistant material observed in the thermal analysesof Zook HA. Others have shown that “whole” soil organicmatter characteristics differ with the size of the aggregates andparticles that the organic matter is associated with. For theseprairie-derived soils, our study confirms and deepens thisobservation specifically for HA associated with particle-sizefractions. The results also suggest that particle size distributionand drainage class can have measurable impacts on the types ofhumic substances that are extractable from soils.

Acknowledgments

We gratefully acknowledge the financial support of theNational Science Foundation (grant CHE-0138117) and theIowa Agriculture and Home Economics Experiment Station,Ames, Iowa (supported by Hatch Act and State of Iowa funds).

28 J. Mao et al. / Geoderma 140 (2007) 17–29

We thank P. Leinweber, D. Laird, and E. Paul for helpfulcomments on this manuscript.

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